ITMI20131377A1 - PATCHES FOR ULCERS, WOUNDS, SKIN ABRASIONS - Google Patents
PATCHES FOR ULCERS, WOUNDS, SKIN ABRASIONSInfo
- Publication number
- ITMI20131377A1 ITMI20131377A1 IT001377A ITMI20131377A ITMI20131377A1 IT MI20131377 A1 ITMI20131377 A1 IT MI20131377A1 IT 001377 A IT001377 A IT 001377A IT MI20131377 A ITMI20131377 A IT MI20131377A IT MI20131377 A1 ITMI20131377 A1 IT MI20131377A1
- Authority
- IT
- Italy
- Prior art keywords
- msc
- patch
- ulcers
- treatment
- patches
- Prior art date
Links
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
- A61L2300/414—Growth factors
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/12—Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/34—Materials or treatment for tissue regeneration for soft tissue reconstruction
Description
Descrizione di una domanda di brevetto per invenzione industriale a nome Fondazione I.R.C.C.S. Istituto Neurologico "Carlo Besta" e INNOVHUB - Stazioni Sperimentali per l'Industria Description of a patent application for an industrial invention in the name of the I.R.C.C.S. Neurological Institute "Carlo Besta" and INNOVHUB - Experimental Stations for Industry
DESCRIZIONE DESCRIPTION
La presente invenzione si riferisce ad un patch per il trattamento di ulcere in pazienti diabetici e al procedimento per il suo ottenimento. The present invention relates to a patch for the treatment of ulcers in diabetic patients and to the procedure for obtaining it.
La cicatrizzazione delle ferite è un problema clinico nei pazienti diabetici. Non esistono al momento trattamenti farmacologici definitivi di questa condizione patologica e per questo motivo spesso si ricorre all’amputazione [Siitonen OI, Niskanen LK, Laasko M, et al. Lower-extremity amputations in diabetic and nondiabetic patients. DIABETES CARE 1993;16:16-20]. Wound healing is a clinical problem in diabetic patients. There are currently no definitive pharmacological treatments for this pathological condition and for this reason amputation is often used [Siitonen OI, Niskanen LK, Laasko M, et al. Lower-extremity amputations in diabetic and nondiabetic patients. DIABETES CARE 1993; 16: 16-20].
La terapia cellulare sta lentamente guadagnando terreno nelle cure mediche di routine e, in particolare, nella cura delle ferite della pelle. Tali terapie, infatti, offrono promettenti soluzioni per la riparazione e/o sostituzione del tessuto danneggiato e il ripristino delle funzionalità perse perché possiedono molti dei criteri necessari per la guarigione delle ferite [Jackson WM, Nesti LJ, Tuan RS. A Review: Mesenchymal stem cell therapy for attenuation of scar formation during wound healing. STEM CELL RES THER. Cell therapy is slowly gaining ground in routine medical care and, in particular, skin wound care. These therapies, in fact, offer promising solutions for the repair and / or replacement of damaged tissue and the restoration of lost functionality because they possess many of the criteria necessary for wound healing [Jackson WM, Nesti LJ, Tuan RS. A Review: Mesenchymal stem cell therapy for attenuation of scar formation during wound healing. STEM CELL RES THER.
2012;31:3:20 - Wu Y, Wang J, Scott PG, et al . Bone marrowderived stem cells in wound healing: a review. WOUND REPAIR REGEN. 2007;15 (Suppl 1):S18-26]. 2012; 31: 3: 20 - Wu Y, Wang J, Scott PG, et al. Bone marrowderived stem cells in wound healing: a review. WOUND REPAIR REGEN. 2007; 15 (Suppl 1): S18-26].
Alcune recenti scoperte hanno incredibilmente suggerito che le cellule stromali mesenchimali multipotenti (d’ora in poi dette anche MSC) isolate da tessuti differenti offrono benefici in vivo come agenti tessuto-ricostituenti. Some recent discoveries have incredibly suggested that multipotent mesenchymal stromal cells (hereafter also referred to as MSCs) isolated from different tissues offer in vivo benefits as tissue-restorative agents.
Esse agiscono, infatti, sia tramite rilascio di fattori trofici sia tramite le loro proprietà di differenziative. [Wu Y, Wang J, Scott PG, et al . Bone marrow-derived stem cells in wound healing: a review. WOUND REPAIR REGEN. 2007;15 (Suppl 1):S18-26 - Li H, Fu X. Review: Mechanisms of action of mesenchymal stem cells in cutaneous wound repair and regeneration. CELL TISSUE RES. They act, in fact, both through the release of trophic factors and through their differentiating properties. [Wu Y, Wang J, Scott PG, et al. Bone marrow-derived stem cells in wound healing: a review. WOUND REPAIR REGEN. 2007; 15 (Suppl 1): S18-26 - Li H, Fu X. Review: Mechanisms of action of mesenchymal stem cells in cutaneous wound repair and regeneration. CELL TISSUE RES.
2012;348:371-377]. 2012; 348: 371-377].
In particolare, sono attualmente studiate per il trattamento di ferite della pelle. Il loro utilizzo è giustificato dal fatto che sono abbondanti, facilmente isolabili ed espandibili in vitro. [Nie C, Yang D, Xu J, et al. Locally administered adipose-derived stem cells accelerate wound healing through differentiation and vasculogenesis. CELL TRANSPLANT. 2011;20:205-216]. In particular, they are currently being studied for the treatment of skin wounds. Their use is justified by the fact that they are abundant, easily isolated and expandable in vitro. [Nie C, Yang D, Xu J, et al. Locally administered adipose-derived stem cells accelerate wound healing through differentiation and vasculogenesis. CELL TRANSPLANT. 2011; 20: 205-216].
Recentemente differenti dispositivi biosintetici, bioriassorbibili e non riassorbibili, sono stati utilizzati da soli o in combinazione con cellule per trattare le ferite [Veleirinho B, Coelho DS, Dias PF, et al. Nanofibrous poly(3-hydroxybutyrate-co-3-hydroxyvalerate)/chitosan scaffolds for skin regeneration. INT J BIOL MACROMOL. 2012;51:343-350 - Sundaramurthi D, Vasanthan KS, Kuppan P, et al. Electrospun nanostructured chitosan- poly(vinyl alcohol) scaffolds: a biomimetic extracellular matrix as dermal substitute. BIOMED MATER. 2012;7:045005 -Huang WY, Yeh CL, Lin JH, et al. Development of fibroblast culture in three-dimensional activated carbon fiber-based scaffold for wound healing. J MATER SCI MATER MED. 2012;23:1465-1478 - Ma K, Liao S, He L, et al. Effects of nanofiber/stem cell composite on wound healing in acute full-thickness skin wounds. TISSUE ENG PART A. 2011;17:1413-1424]. Recently different biosynthetic devices, bioabsorbable and non-resorbable, have been used alone or in combination with cells to treat wounds [Veleirinho B, Coelho DS, Dias PF, et al. Nanofibrous poly (3-hydroxybutyrate-co-3-hydroxyvalerate) / chitosan scaffolds for skin regeneration. INT J BIOL MACROMOL. 2012; 51: 343-350 - Sundaramurthi D, Vasanthan KS, Kuppan P, et al. Electrospun nanostructured chitosan- poly (vinyl alcohol) scaffolds: a biomimetic extracellular matrix as dermal substitute. BIOMED MATER. 2012; 7: 045005 -Huang WY, Yeh CL, Lin JH, et al. Development of fibroblast culture in three-dimensional activated carbon fiber-based scaffold for wound healing. J MATER SCI MATER MED. 2012; 23: 1465-1478 - Ma K, Liao S, He L, et al. Effects of nanofiber / stem cell composite on wound healing in acute full-thickness skin wounds. TISSUE ENG PART A. 2011; 17: 1413-1424].
Tra i differenti dispositivi, quelli comprendenti fibroina della seta (d’ora in poi SF) caricata con MSC hanno dimostrato di possedere proprietà efficaci nella riparazione sperimentale di ferite della pelle [Guan G, Bai L, Zuo B, et al. Promoted dermis healing from fullthickness skin defect by porous silk fibroin scaffolds (PSFSs).BIOMED MATER ENG. 2010;20:295-308 - Kanokpanont S, Damrongsakkul S, Ratanavaraporn J, et al. An innovative bilayered wound dressing made of silk and gelatin for accelerated wound healing. INT J PHARM. 2012;436:141- 153]. Tuttavia, l’applicazione di dispositivi caricati con cellule vive in vivo potrebbe avere alcuni rischi collegati alla possibilità di trasferire patogeni o di indurre reazioni immunologiche avverse nell’ospite, soprattutto quando per la terapia vengono utilizzate cellule allogeniche o xenogeniche [Sogal A, Tofe AJ. Risk assessment of bovine spongiform encephalopathy transmission through bone graft material derived from bovine bone used for dental applications. J PERIODONTOL. 1999;70:1053-1063 - Kurobe H, Maxfield MW, Breuer CK,et al .Concise review: tissue-engineered vascular grafts for cardiac surgery: past, present, and future. STEM CELLS TRANSL MED. 2012;1:566-571 - Leyh RG, Wilhelmi M, Walles T, et al. Acellularized porcine heart valve scaffolds for heart valve tissue engineering and the risk of crossspecies transmission of porcine endogenous retrovirus. J THORAC CARDIOVASC SURG. 2003;126:1000-1004]. Among the different devices, those including silk fibroin (hereinafter SF) loaded with MSC have been shown to possess effective properties in the experimental repair of skin wounds [Guan G, Bai L, Zuo B, et al. Promoted dermis healing from fullthickness skin defect by porous silk fibroin scaffolds (PSFSs) .BIOMED MATER ENG. 2010; 20: 295-308 - Kanokpanont S, Damrongsakkul S, Ratanavaraporn J, et al. An innovative bilayered wound dressing made of silk and gelatin for accelerated wound healing. INT J PHARM. 2012; 436: 141-153]. However, the application of devices loaded with live cells in vivo could have some risks related to the possibility of transferring pathogens or inducing adverse immunological reactions in the host, especially when allogeneic or xenogenic cells are used for therapy [Sogal A, Tofe AJ . Risk assessment of bovine spongiform encephalopathy transmission through bone graft material derived from bovine bone used for dental applications. J PERIODONTOL. 1999; 70: 1053-1063 - Kurobe H, Maxfield MW, Breuer CK, et al. Concise review: tissue-engineered vascular grafts for cardiac surgery: past, present, and future. STEM CELLS TRANSL MED. 2012; 1: 566-571 - Leyh RG, Wilhelmi M, Walles T, et al. Acellularized porcine heart valve scaffolds for heart valve tissue engineering and the risk of crossspecies transmission of porcine endogenous retrovirus. J THORAC CARDIOVASC SURG. 2003; 126: 1000-1004].
Compito tecnico della presente invenzione è quindi quello di eliminare gli inconvenienti lamentati dalla tecnica nota, in particolare fornendo un nuovo patch per il trattamento di ulcere in pazienti diabetici. The technical task of the present invention is therefore to eliminate the drawbacks complained of by the known art, in particular by providing a new patch for the treatment of ulcers in diabetic patients.
Nell’ambito di questo compito tecnico uno scopo dell’invenzione è quello di fornire un nuovo patch di facile manipolazione e preparazione per il trattamento di ulcere. As part of this technical task, an aim of the invention is to provide a new patch that is easy to handle and prepare for the treatment of ulcers.
Un altro scopo dell’invenzione è quello di fornire un nuovo patch per il trattamento di ulcere che riduca il rischio di traferire infezioni o cellule geneticamente mutate. Another purpose of the invention is to provide a new patch for the treatment of ulcers that reduces the risk of transferring infections or genetically mutated cells.
Altresì questa invenzione fornisce un nuovo patch per il trattamento di ulcere con meno reattività immunologica e infiammatoria. Also this invention provides a new patch for the treatment of ulcers with less immunological and inflammatory reactivity.
Non ultimo, scopo dell’invenzione è quello di fornire un procedimento per la preparazione di un patch semplice ed economico per il trattamento di ulcere. Last but not least, the purpose of the invention is to provide a procedure for the preparation of a simple and inexpensive patch for the treatment of ulcers.
Il compito tecnico, nonché questi ed altri scopi, secondo la presente invenzione vengono raggiunti fornendo un patch per il trattamento di ulcere in un paziente diabetico, comprendere che comprenda un supporto elettrofilato nanostrutturato in fibroina della seta supportante prodotti cellulari rilasciati da cellule stromali mesenchimali derivate da tessuto adiposo, dette cellule stromali mesenchimali essendo rimosse da detto supporto in fibroina della seta dopo il rilascio di detti prodotti cellulari. The technical task, as well as these and other objects, according to the present invention are achieved by providing a patch for the treatment of ulcers in a diabetic patient, comprising a nanostructured silk fibroin electro-spun support supporting cell products released from mesenchymal stromal cells derived from adipose tissue, said mesenchymal stromal cells being removed from said silk fibroin support after the release of said cell products.
L’invenzione rivela inoltre un procedimento per ottenere un patch per il trattamento di ulcere in un paziente diabetico, comprendente le fasi di: The invention also discloses a process for obtaining a patch for the treatment of ulcers in a diabetic patient, including the steps of:
- Preparazione di un supporto elettrofilato nanostrutturato in fibroina della seta; - Preparation of a nanostructured electro-spun support in silk fibroin;
- Semina su detto supporto in fibroina della seta di cellule stromali mesenchimali di origine adiposa; - Sowing on said fibroin support of the silk of mesenchymal stromal cells of adipose origin;
- Incubazione di detto supporto in fibroina della seta in presenza di dette cellule stromali mesenchimali per il rilascio di prodotti cellulari da dette cellule stromali mesenchimali; - Incubation of said silk fibroin support in the presence of said mesenchymal stromal cells for the release of cellular products from said mesenchymal stromal cells;
caratterizzato dal fatto di comprendere una ulteriore fase di: characterized by the fact of including a further phase of:
- rimozione di dette cellule stromali mesenchimali da detto supporto in fibroina della seta dopo il rilascio di detti prodotti cellulari. - removal of said mesenchymal stromal cells from said silk fibroin support after the release of said cell products.
Altre caratteristiche dell’invenzione sono evincibili dalle rivendicazioni dipendenti di seguito riportate. Other features of the invention can be deduced from the dependent claims set out below.
Ulteriori caratteristiche e vantaggi dell’invenzione risulteranno maggiormente evidenti dalla descrizione che segue, illustrata a titolo indicativo e non limitativo con riferimento alle figure da 1 a 15 allegate. Further features and advantages of the invention will become more evident from the following description, illustrated for indicative and non-limiting purposes with reference to the attached figures 1 to 15.
Le Figure 1 A-B mostrano immagini al microscopio elettronico a scansione (d’ora in poi SEM) a differenti ingrandimenti dei supporti elettrofilati nanostrutturati in fibroina della seta (d’ora in poi ES‐SF‐patch) prodotti utilizzando una concentrazione di polimero (Cp) = 7,5%, in peso, una differenza di potenziale (V) = 24 kV, una velocità di flusso (F) = 3 ml/h, una distanza spinneretcollettore (D) = 10 cm. Figures 1 A-B show scanning electron microscope images (hereafter SEM) at different magnifications of nanostructured silk fibroin electrospun supports (hereinafter ES ‐ SF ‐ patch) produced using a polymer concentration (Cp ) = 7.5%, by weight, a potential difference (V) = 24 kV, a flow rate (F) = 3 ml / h, a spinneret-collector distance (D) = 10 cm.
La Figura 2 mostra la variazione della banda IR della Ammide I dello spettro IR dell’ ES-SF‐ patch prima (non trattato) e dopo immersione in metanolo per diversi tempi (da 5 a 15 min). Figure 2 shows the variation of the IR band of Amide I of the IR spectrum of the ES-SF-patch before (untreated) and after immersion in methanol for different times (from 5 to 15 min).
La Figura 3 mostra l’andamento dell’indice di cristallinità calcolato come rapporto tra le intensità dei contributi dei picchi di Ammide III a 1265 cm<-1>e 1235 cm<-1>in funzione del tempo di immersione in metanolo. Figure 3 shows the trend of the crystallinity index calculated as the ratio between the intensity of the contributions of the Amide III peaks at 1265 cm <-1> and 1235 cm <-1> as a function of the time of immersion in methanol.
La Figura 4 mostra il termogramma ottenuto tramite Calorimetria Differenziale a Scansione (DSC) dell’ ES‐SF‐patch prima (nontrattate) e dopo immersione in metanolo per diversi tempi. Figure 4 shows the thermogram obtained by Differential Scanning Calorimetry (DSC) of the ES ‐ SF ‐ patch before (untreated) and after immersion in methanol for different times.
La Figura 5 mostra la curva sforzo‐deformazione rappresentativa dell’ ES‐SF‐patch. Figure 5 shows the stress-strain curve representative of the ES-SF-patch.
Le Figure 6 A-J mostrano l’aspetto morfologico dei patch di fibroina della seta (SF) in coltura con cellule stromali mesenchimali di origine adiposa (d’ora in poi Ad-MSC) prima e dopo decellularizzazione. Figures 6 A-J show the morphological appearance of the silk fibroin (SF) patches in culture with mesenchymal stromal cells of adipose origin (hereinafter Ad-MSC) before and after decellularization.
La Figura 6A è un’ immagine acquisita al microscopio elettronico a scansione. I patch di fibroina presentano la loro caratteristica struttura filamentosa. Nelle Figure 6B e 6C sono rappresentati i patch di fibroina con cellule mesenchimali dopo 2 (6B) e 7 (6C) giorni di coltura (Ad-MSC-SF). Si può notare come dopo 7 giorni le cellule creino un monostrato uniforme sul patch. Lo stesso risultato è verificabile con colorazione di ematossilina ed eosina (6D). Le cellule a forma affusolata sono evidenziate dalle frecce e i loro nuclei da asterischi. Le Figure 6E-6H sono immagini acquisite al microscopio confocale delle cellule coltivate su patch che esprimono positività ai marcatori della linea mesenchimale, CD146 (6E); CD44 (6F) e integrina CD49d (6G) e negatività al CD45 (6H), marcatore della linea ematopoietica. Le Figure 6I e 6J mostrano i patch di fibroina dopo un’ora di trattamento di decellularizzazione con acqua distillata (D-Ad-MSC-SF). Non sono evidenziabili elementi cellulari con colorazione di ematossilina ed eosina (6I) e al microscopio elettronico (6J). Nelle figure SF= fibroina della seta e c= cellule Ad-MSC. Figure 6A is an image acquired with a scanning electron microscope. Fibroin patches exhibit their characteristic filamentous structure. Figures 6B and 6C show fibroin patches with mesenchymal cells after 2 (6B) and 7 (6C) days of culture (Ad-MSC-SF). It can be seen that after 7 days the cells create a uniform monolayer on the patch. The same result is verifiable with hematoxylin and eosin staining (6D). The tapered cells are highlighted by arrows and their nuclei by asterisks. Figures 6E-6H are confocal microscopic images of cells grown on patches expressing positive mesenchymal line markers, CD146 (6E); CD44 (6F) and integrin CD49d (6G) and negativity to CD45 (6H), a marker of the hematopoietic line. Figures 6I and 6J show the fibroin patches after one hour of decellularization treatment with distilled water (D-Ad-MSC-SF). Cellular elements with hematoxylin and eosin staining (6I) and electron microscopy (6J) are not detectable. In the figures SF = silk fibroin and c = Ad-MSC cells.
Le Figure 7 A-C mostrano un incremento della rigenerazione tissutale in topi diabetici con Ad-MSC-SF e D-Ad-MSC-SF. Topi diabetici db/db sono stati trattati con patch di fibroina della seta in combinazione con AD-MSC e dopo decellularizzazione. In tutti i trapianti sono stati aggiunti 30µL di hydrogel commerciale per creare un ambiente umido che potesse evitare l’essicazione della lesione, il conseguente deterioramento dello scaffold e assicurare un’adesione prolungata alla ferita. Figures 7 A-C show an increase in tissue regeneration in diabetic mice with Ad-MSC-SF and D-Ad-MSC-SF. Diabetic db / db mice were treated with silk fibroin patches in combination with AD-MSC and after decellularization. In all transplants, 30µL of commercial hydrogel was added to create a humid environment that could avoid the drying of the lesion, the consequent deterioration of the scaffold and ensure prolonged adhesion to the wound.
In Figura 7A è rappresentata la cinetica della cicatrizzazione delle ferite in presenza di soluzione fisiologica (controllo), solo SF, Ad-MSC-SF e D-Ad-MSC-SF. In Figura 7B è mostrato un esempio della misurazione della ferita con centimetro. L’esame macroscopico delle lesioni cutanee a 0,3 e 10 giorni dall’inoculo, degli animali “controllo”, non trattati, mostravano una cicatrizzazione spontanea della lesione in 15 giorni dal trapianto. La stessa velocità di rigenerazione si osservava per gli animali trattati con solo fibroina. Al contrario, la fibroina con cellule Ad-MSC e decellularizzata, acceleravano fin dal terzo giorno del trapianto la rigenerazione della ferita (Figura 7C) con una differenza statistica (* p< 0.05, ** p<0.01 rispetto al controllo). Le Figure 8A e 8B mostrano come i patch di AD-MSC-SF e i patch di D-Ad-MSC-SF incrementano l’espressione di geni coinvolti nell’angiogenesi e nel rimodellamento della matrice extracellulare. L’ indagine molecolare è stata valutata attraverso lo studio di 84 geni (analizzati con la tecnica di RT-PCR profiler) coinvolti nei meccanismi di rigenerazione tessutale. Figure 7A shows the kinetics of wound healing in the presence of physiological solution (control), only SF, Ad-MSC-SF and D-Ad-MSC-SF. Figure 7B shows an example of the wound measurement with centimeter. Macroscopic examination of skin lesions at 0.3 and 10 days after inoculation of untreated "control" animals showed spontaneous healing of the lesion in 15 days after transplantation. The same rate of regeneration was observed for animals treated with fibroin alone. On the contrary, fibroin with Ad-MSC cells and decellularized, accelerated the regeneration of the wound from the third day of the transplant (Figure 7C) with a statistical difference (* p <0.05, ** p <0.01 compared to the control). Figures 8A and 8B show how AD-MSC-SF patches and D-Ad-MSC-SF patches increase the expression of genes involved in angiogenesis and remodeling of the extracellular matrix. The molecular investigation was evaluated through the study of 84 genes (analyzed with the RT-PCR profiler technique) involved in the mechanisms of tissue regeneration.
L’analisi a 7 giorni del profilo genico del tessuto post trapianto di AD-MSC-SF e D-Ad-MSC-SF è mostrata rispettivamente in figura 8A e figura 8B. Nei patch trattati con le cellule si evidenzia un aumento dei geni coinvolti nell’angiogenesi (Vegfa, Egf and Pdgfa, Wnt5 �, Wisp1 and Tgfβr3) e nel rimodellamento e sintesi della matrice extracellulare (Mmp2, Itgβ6, Col5 �2, Col5 �1, Col4�1 e Tagln) rispetto al controllo, la sola fibroina (Figura 8A). L’analisi sui patch D-Ad-MSC-SF mostra un andamento simile con una down modulazione del fattore infiammatorio IL6. * p<0.05; **p<0.01 versus i patch non trattati. The 7-day analysis of the gene profile of AD-MSC-SF and D-Ad-MSC-SF post transplant tissue is shown in Figure 8A and Figure 8B, respectively. In the patches treated with cells there is an increase in the genes involved in angiogenesis (Vegfa, Egf and Pdgfa, Wnt5 �, Wisp1 and Tgfβr3) and in the remodeling and synthesis of the extracellular matrix (Mmp2, Itgβ6, Col5 �2, Col5 �1 , Col4�1 and Tagln) compared to the control, fibroin alone (Figure 8A). The analysis on the D-Ad-MSC-SF patches shows a similar trend with a down modulation of the inflammatory factor IL6. * p <0.05; ** p <0.01 versus untreated patches.
Le Figure 9A, 9B, 9C, 9D, 9E, e 9F mostrano come i patch AD-MSC-SF e i patch D-Ad-MSC-SF stimolano la migrazione delle cellule endoteliali da vena di cordone ombelicale umano (HUVEC) mediante analisi con scratch test. Figures 9A, 9B, 9C, 9D, 9E, and 9F show how the AD-MSC-SF patches and the D-Ad-MSC-SF patches stimulate the migration of endothelial cells from human umbilical cord vein (HUVEC) by analysis with scratch test.
Cellule endoteliali da vena di cordone ombelicale umano (d’ora in poi HUVEC), fibroblasti del derma (d’ora in poi DF), cheratinociti (d’ora in poi KC) sono stati coltivati a confluenza in inserti ibidi. Endothelial cells from human umbilical cord vein (hereinafter HUVEC), dermal fibroblasts (hereinafter DF), keratinocytes (hereinafter KC) were cultivated at confluence in ibid inserts.
Dopo 48 ore gli inserti sono stati rimossi e nei pozzetti sono stati adagiati i supporti delle transwell da 24 pozzetti nei quali sono stati posizionati i patch SF, AD-MSC-SF e D-Ad-MSC-SF. After 48 hours the inserts were removed and the supports of the 24-well transwells were placed in the wells in which the SF, AD-MSC-SF and D-Ad-MSC-SF patches were placed.
La migrazione delle cellule endoteliali da vena di cordone ombelicale umano (HUVEC) (Figura 9A), di DF (Figura 9B) e dei cheratinociti (KC) (Figura 9C) è stata quantificata, contando il numero delle cellule migranti nel setto di separazione. I dati riportano le conte fatte in 5 campi differenti ad un microscopio ottico con obiettivo 20x a diversi tempi. Gli istogrammi riportano la media ± la deviazione standard di 3 esperimenti indipendenti con * p<0.05; **p<0.01 rispetto al controllo, il patch non trattato. Durante la migrazione cellulare sono state scattate fotografie al microscopio ottico per stabilire il tempo necessario a coprire il setto di separazione. Dalle immagini si può notare come i patch D-Ad-MSC-SF incrementino la migrazione delle HUVEC rispetto ai patch SF e ai patch AD-MSC-SF. Endothelial cell migration from human umbilical cord vein (HUVEC) (Figure 9A), DF (Figure 9B) and keratinocytes (KC) (Figure 9C) was quantified by counting the number of migrating cells in the septum. The data report the counts made in 5 different fields to an optical microscope with a 20x objective at different times. The histograms report the mean ± the standard deviation of 3 independent experiments with * p <0.05; ** p <0.01 compared to the control, the untreated patch. During cell migration, optical microscope photographs were taken to establish the time required to cover the separation septum. From the images it can be seen how the D-Ad-MSC-SF patches increase the migration of HUVECs compared to SF patches and AD-MSC-SF patches.
Le Figure 10A, 10B, 10C, 10D mostrano il rilascio di fattori angiogenici da parte di AD-MSC-SF e D-Ad-MSC-SF. Figures 10A, 10B, 10C, 10D show the release of angiogenic factors by AD-MSC-SF and D-Ad-MSC-SF.
Alla fine dello scratch test campioni di terreno sono stati raccolti e analizzati per la presenza di fattori angiogenici quali il fattore di crescita endoteliale vascolare (d’ora in poi VEGF) (Figura 10A), il fattore di crescita dei fibroblasti 2 (d’ora in poi FGF-2) (Figura 10B), il fattore di crescita trasformante beta (d’ora in poi TGF-beta) (Figura 10C) e il fattore di crescita dell’epidermide (d’ora in poi EGF) (Figura 10D). La quantificazione è stata ottenuta con kit ELISA. Il valore determinato dal terreno di controllo che non ha visto le cellule è stato sottratto ai valori ottenuti. I dati sono rappresentati dalla media ± la deviazione standard con * p<0.05; **p<0.01 rispetto al terreno condizionato dalle HUVEC in presenza di patch non trattati. At the end of the scratch test, soil samples were collected and analyzed for the presence of angiogenic factors such as vascular endothelial growth factor (hereafter VEGF) (Figure 10A), fibroblast growth factor 2 (hereafter onwards FGF-2) (Figure 10B), the transforming growth factor beta (hereafter TGF-beta) (Figure 10C) and the epidermal growth factor (hereafter EGF) (Figure 10D ). Quantification was obtained with ELISA kit. The value determined by the control medium which did not see the cells was subtracted from the values obtained. The data are represented by the mean ± the standard deviation with * p <0.05; ** p <0.01 compared to HUVEC conditioned medium in the presence of untreated patches.
Le Figure 11A e 11B mostrano i patch trattati con AD-MSC-SF e D-Ad-MSC-SF stimolano la crescita di microvasi nel test del ring di aorta. Figures 11A and 11B show patches treated with AD-MSC-SF and D-Ad-MSC-SF stimulate the growth of microvessels in the aorta ring test.
L’aorta isolata da un ratto è stata pulita e sezionata a formare degli anelli (ring) dallo spessore di 1mm. Gli anelli sono stati successivamente incubati a 37°C per 30 minuti in una soluzione gelificante e ad essi è stata aggiunta un’aliquota del terreno condizionato dai AD-MSC-SF e D-Ad-MSC-SF e SF. In Figura 11A è stata quantificata la crescita dei vasi. I dati sono stati elaborati attraverso la conta di 3 esperimenti indipendenti e la media ± la deviazione standard è stata riportata sul grafico con * p<0.05; **p<0.01 rispetto al terreno condizionato dalle HUVEC in presenza di patch non trattati. In Figura 11B sono state riportate le fotografie scattate per i diversi trattamenti: terreno di controllo (terreno che non ha mai incontrato le cellule), SF, AD-MSC-SF e D-Ad-MSC-SF. The aorta isolated from a rat was cleaned and sectioned to form rings with a thickness of 1mm. The rings were subsequently incubated at 37 ° C for 30 minutes in a gelling solution and an aliquot of the medium conditioned by AD-MSC-SF and D-Ad-MSC-SF and SF was added to them. Vessel growth was quantified in Figure 11A. The data were processed by counting 3 independent experiments and the mean ± standard deviation was reported on the graph with * p <0.05; ** p <0.01 compared to HUVEC conditioned medium in the presence of untreated patches. Figure 11B shows the photographs taken for the different treatments: control medium (medium that has never encountered the cells), SF, AD-MSC-SF and D-Ad-MSC-SF.
La Figura 12 mostra la caratterizzazione delle cellule mesenchimali stromali di origine adiposa. Le cellule in coltura hanno una morfologia fibroblastica e a diversi tempi sono state caratterizzate per verificare il loro profilo mesenchimale (CD44+, CD73+, CD90+, CD105+, CD166+) e non quello ematopoietico (CD45-). Figure 12 shows the characterization of mesenchymal stromal cells of adipose origin. Cultured cells have a fibroblastic morphology and have been characterized at different times to verify their mesenchymal profile (CD44 +, CD73 +, CD90 +, CD105 +, CD166 +) and not the hematopoietic profile (CD45-).
Le Figure 13A, 13B, 13C, 13D, 13E e 13F mostrano l’ analisi istologica della ferita cutanea dopo trattamento con SF, AD-MSC-SF e D-Ad-MSC-SF. Figures 13A, 13B, 13C, 13D, 13E and 13F show the histological analysis of the skin wound after treatment with SF, AD-MSC-SF and D-Ad-MSC-SF.
A 14 giorni dall’inoculo, i topi sono stati sacrificati, la cute a livello dell’inserzione dei patch è stata isolata e analizzata istologicamente. I patch di controllo trattati con SF mostrano una elevata ipercellularità, con un alterata ricostruzione delle fibre di collagene e segni di displasia determinati da uno stato immaturo delle cellule della cute. Nei campioni trattati con D-Ad-MSC-SF sono evidenti strutture più organizzate con derma ricco di cellule e di microvasi. L’organizzazione tissutale è migliore nei topi che hanno ricevuto i patch trattati con le cellule dove è possibile notare la formazione di tessuto epidermico multistrato (Figura 13E) e la formazione di neovasi a livello del derma (Figura 13D). At 14 days after inoculation, the mice were sacrificed, the skin at the level of the insertion of the patches was isolated and histologically analyzed. The control patches treated with SF show a high hypercellularity, with an altered reconstruction of the collagen fibers and signs of dysplasia caused by an immature state of the skin cells. In the samples treated with D-Ad-MSC-SF, more organized structures with dermis rich in cells and microvessels are evident. Tissue organization is better in mice that have received patches treated with cells where it is possible to note the formation of multilayer epidermal tissue (Figure 13E) and the formation of new vessels in the dermis (Figure 13D).
Le Figure 14A, 14B, 14C e 14D mostrano uno schema dello scratch test utilizzato per valutare l’attività dei patch SF, AD-MSC-SF e D-Ad-MSC-SF sulla migrazione cellulare di HUVEC, fibroblasti (DF) e cheratinociti (KC). Figures 14A, 14B, 14C and 14D show a scratch test scheme used to evaluate the activity of SF, AD-MSC-SF and D-Ad-MSC-SF patches on cell migration of HUVEC, fibroblasts (DF) and keratinocytes (KC).
Per il test le cellule HUVEC, DF, KC sono state piastrate in inseriti Ibidi posti in piastre multipozzetto da 24 pozzetti (Figura 14A). Quando le popolazioni cellulari sono arrivate a confluenza gli inserti sono stati tolti e 0.5 mL di terreno sono stati aggiunti. Nei pozzetti sono stati adagiati i supporti di transwell con filtro di policarbonato da 8-μm su cui erano stati messi i patch di controllo SF (Figura 14B), AD-MSC-SF (Figura 14C) e D-Ad-MSC-SF (Figura 14D). Dopo 6, 24 e 48 ore i supporti sono stati tolti ed è stata fatta la conta delle cellule che migravano nel setto di separazione in 5 campi differenti con un microscopio ottico Zeiss 20x. For the test the HUVEC, DF, KC cells were plated in inserted Ibidi placed in 24 well multiwell plates (Figure 14A). When the cell populations came to confluence the inserts were removed and 0.5 mL of medium was added. The transwell supports with 8-μm polycarbonate filter were placed in the wells on which the control patches SF (Figure 14B), AD-MSC-SF (Figure 14C) and D-Ad-MSC-SF ( Figure 14D). After 6, 24 and 48 hours the supports were removed and the count of the cells migrating in the separation septum in 5 different fields was made with a Zeiss 20x optical microscope.
La Figura 15 mostra l’espressione dei geni angiogenici nelle cellule AD-MSC in coltura su plastica e su patch di fibroina. Figure 15 shows the expression of angiogenic genes in AD-MSC cells cultured on plastic and on fibroin patches.
L’analisi in RT-PCR è stata eseguita per valutare l’espressione dei geni coinvolti nell’angiogenesi (VEGFa, FGF-2, TGF-beta, EGF) sulle cellule in coltura e sulle cellule cresciute su patch di fibroina. Il valore di riferimento è rappresentato dai geni espressi dalle cellule in coltura su plastica. Il gene housekeeping utilizzato è GAPDH. L’analisi è stata condotta in triplicato Nel confronto dell’espressione genica tra le cellule coltivate su plastica e quelle coltivate su fibroina non vi è nessuna differenza per i geni VEGF, FGF-2, TGF-beta, al contrario l’espressione del gene EGF è risultata significativamente down-modulata con *p<0.05 versus SF. I dispositivi decellularizzati hanno suscitato un interesse significativo per applicazioni tissutali ingegnerizzate. RT-PCR analysis was performed to evaluate the expression of genes involved in angiogenesis (VEGFa, FGF-2, TGF-beta, EGF) on cultured cells and cells grown on fibroin patches. The reference value is represented by the genes expressed by cells cultured on plastic. The housekeeping gene used is GAPDH. The analysis was carried out in triplicate In the comparison of gene expression between cells grown on plastic and those grown on fibroin there is no difference for the VEGF, FGF-2, TGF-beta genes, on the contrary the expression of the gene EGF was significantly down-modulated with * p <0.05 versus SF. The decellularized devices have sparked significant interest in engineered tissue applications.
È stato infatti suggerito che i dispositivi biologici decellularizzati potrebbero imitare il naturale substrato di matrice extracellulare (ECM). Inoltre, i dispositivi decellularizzati precedentemente rivestiti con cellule coltivate o dispositivi con cellule derivate dalla matrice extracellulare, una volta trapiantate in vivo, possono simulare la presenza di cellule viventi fornendo prodotti cellulari rimasti intrappolati nelle maglie del dispositivo, possibilmente mediante un processo di rilascio lento [Cheng HW, Tsui YK, Cheung KM,et al . Decellularization of chondrocyte-encapsulated collagen microspheres: a three-dimensional model to study the effects of acellular matrix on stem cell fate. TISSUE ENG PART C METHODS. 2009;15:697-706 - Hoshiba T, Lu H, Kawazoe N, et al. Decellularized matrices for tissue engineering. EXPERT OPIN BIOL THER 2010;10:1717-1728 - Chen R, Ho H, Tsai Y,et al. Process development of an acellular dermal matrix (ADM) for biomedical applications. BIOMATERIALS 2004;25:2679-2686 -Whitlock PW, Smith TL, Poehling GG, et al. A naturally derived, cytocompatible, and architecturally optimized scaffold for tendon and ligament regeneration. BIOMATERIALS 2007;28:4321-4329 -Song JJ, Ott HC. Organ engineering based on decellularized matrix scaffolds. TRENDS MOL MED. 2011;17:424-432. Review -Lu H, Hoshiba T, Kawazoe N, et al. Comparison of decellularization techniques for preparation of extracellular matrix scaffolds derived from three-dimensional cell culture. J BIOMED MATER RES A. 2012;100:2507-2516]. Inoltre, i differenti metodi per rimuovere le cellule dal dispositivo, in generale, non alterano né la nativa struttura 3D del dispositivo nè dei prodotti derivati dalle cellule (Keane TJ, Londono R, Turner NJ, et al. Consequences of ineffective decellularization of biologic scaffolds on the host response. BIOMATERIALS. 2012;33:1771-1781). Nel presente studio abbiamo voluto verificare se sia i patch di fibroina della seta cellularizzata con cellule AD-MSC (d’ora in poi Ad-MSC-SF) che i patch di fibroina della seta decellularizzati ( d’ora in poi D-Ad-MSC-SF) possono essere efficaci nel trattamento delle ferite della pelle. Per determinare l'efficacia, i patch di fibroina della seta cellularizzata o decellularizzata sono stati trapiantati in topi diabetici (<db / db>Leprdb / J). Questi topi sono portatori di una mutazione nel recettore della leptina che determina un significativo ritardo nella guarigione della ferita simile a quella osservata in pazienti diabetici [Michaels J, Churgin SS, Blechman KM, et al. db/db mice exhibit severe wound-healing impairments compared with other murine diabetic strains in a silicone-splinted excisional wound model. WOUND REPAIR REGEN. 2007;15:665-670]. Indeed, it has been suggested that decellularized biological devices could mimic the natural substrate of extracellular matrix (ECM). Furthermore, decellularized devices previously coated with cultured cells or devices with cells derived from the extracellular matrix, once transplanted in vivo, can simulate the presence of living cells by providing cell products trapped in the mesh of the device, possibly by a slow release process [ Cheng HW, Tsui YK, Cheung KM, et al. Decellularization of chondrocyte-encapsulated collagen microspheres: a three-dimensional model to study the effects of acellular matrix on stem cell fate. TISSUE ENG PART C METHODS. 2009; 15: 697-706 - Hoshiba T, Lu H, Kawazoe N, et al. Decellularized matrices for tissue engineering. EXPERT OPIN BIOL THER 2010; 10: 1717-1728 - Chen R, Ho H, Tsai Y, et al. Process development of an acellular dermal matrix (ADM) for biomedical applications. BIOMATERIALS 2004; 25: 2679-2686 - Whitlock PW, Smith TL, Poehling GG, et al. A naturally derived, cytocompatible, and architecturally optimized scaffold for tendon and ligament regeneration. BIOMATERIALS 2007; 28: 4321-4329 -Song JJ, Oct HC. Organ engineering based on decellularized matrix scaffolds. TRENDS MOL MED. 2011; 17: 424-432. Review - Lu H, Hoshiba T, Kawazoe N, et al. Comparison of decellularization techniques for preparation of extracellular matrix scaffolds derived from three-dimensional cell culture. J BIOMED MATER RES A. 2012; 100: 2507-2516]. Furthermore, the different methods for removing cells from the device, in general, do not alter either the native 3D structure of the device or the cell-derived products (Keane TJ, Londono R, Turner NJ, et al. Consequences of ineffective decellularization of biologic scaffolds on the host response. BIOMATERIALS. 2012; 33: 1771-1781). In the present study we wanted to verify whether both the fibroin patches of the cellularized silk with AD-MSC cells (hereinafter Ad-MSC-SF) and the decellularized silk fibroin patches (hereafter D-Ad- MSC-SF) can be effective in treating skin wounds. To determine efficacy, cellularized or decellularized silk fibroin patches were transplanted into diabetic mice (<db / db> Leprdb / J). These mice carry a leptin receptor mutation that results in a significant delay in wound healing similar to that seen in diabetic patients [Michaels J, Churgin SS, Blechman KM, et al. db / db mice exhibit severe wound-healing impairments compared with other murine diabetic strains in a silicone-splinted excisional wound model. WOUND REPAIR REGEN. 2007; 15: 665-670].
E’ stata creata una lesione cutanea a livello dorsale con un punch per biopsie di 5 mm di diametro. È stata valutata l'efficacia dei trattamenti misurando l'area di chiusura della ferita e dall'esame istologico. Contemporaneamente, abbiamo studiato l'espressione dei geni nella ferita dal profilo RT, nonché la capacità in vitro dei patch di fibroina della seta cellularizzati e decellularizzati di influenzare la migrazione di cellule endoteliali della vena ombelicale umana (HUVEC), cheratinociti (KC) e fibroblasti dermici (DF) e le loro proprietà angiogeniche attraverso il test del ring di aorta e valutando il rilascio di fattori angiogenici. A dorsal skin lesion was created with a 5 mm diameter biopsy punch. The effectiveness of the treatments was assessed by measuring the wound closure area and by histological examination. Simultaneously, we investigated the expression of genes in the wound from the RT profile, as well as the in vitro ability of cellularized and decellularized silk fibroin patches to influence the migration of human umbilical vein endothelial cells (HUVEC), keratinocytes (KC) and fibroblasts. dermal (DF) and their angiogenic properties by testing the aorta ring and evaluating the release of angiogenic factors.
I risultati mostrati nella presente invenzione rivelano per la prima volta che sia i patch con cellule Ad-MSC che i patch D-Ad-MSC-SF mostrano una forte capacità di migliorare la guarigione in topi diabetici, principalmente attraverso un meccanismo simile che migliora l'angiogenesi. The results shown in the present invention reveal for the first time that both the Ad-MSC cell patches and the D-Ad-MSC-SF patches show a strong ability to enhance healing in diabetic mice, primarily through a similar mechanism that improves the angiogenesis.
MATERIALI E METODI MATERIALS AND METHODS
Produzione e caratterizzazione di strutture elettrofilate nanostrutturate in fibroina della seta - Elettrofilatura di strutture planari. Production and characterization of nanostructured silk fibroin electrospun structures - Electrospinning of planar structures.
Una soluzione acquosa di fibroina è stata preparata secondo la metodologia descritta in un precedente lavoro [J Appl. Polym. Sci., 2004, 91, 2383‐2390]. La fibroina è stata sgommata in autoclave a 120°C per 40 minuti e sciolta in una soluzione acquosa 9,3 M di LiBr per 3 ore a 60°C. Successivamente la soluzione è stata posta in dialisi in acqua distillata per 3 giorni ottenendo una soluzione acquosa di fibroina (~1% in peso). Membrane di fibroina sono state poi prodotte versando 10 mL della soluzione ottenuta in piastre Petri e lasciando evaporare la componente acquosa per due giorni. Immediatamente prima di iniziare il processo di elettrofilatura, le soluzioni polimeriche da elettrofilare sono state preparate sciogliendo a differenti valori di concentrazione in acido formico le An aqueous solution of fibroin was prepared according to the methodology described in a previous work [J Appl. Polym. Sci., 2004, 91, 2383-2390]. The fibroin was degummed in an autoclave at 120 ° C for 40 minutes and dissolved in a 9.3 M LiBr aqueous solution for 3 hours at 60 ° C. Subsequently the solution was placed in dialysis in distilled water for 3 days obtaining an aqueous solution of fibroin (~ 1% by weight). Fibroin membranes were then produced by pouring 10 mL of the obtained solution into Petri dishes and allowing the aqueous component to evaporate for two days. Immediately before starting the electrospinning process, the polymeric solutions to be electrospun were prepared by dissolving at different concentration values in formic acid the
membrane precedentemente prodotte. previously produced membranes.
L’apparato di elettrofilatura è composto da due generatori di The electrospinning apparatus consists of two generators of
tensione ad alto voltaggio (F.u.G. Elektronik GmbH ‐ HCN 35- high voltage voltage (F.u.G. Elektronik GmbH - HCN 35-
12500) in grado di generare una differenza di potenziale massima 12500) capable of generating a maximum potential difference
pari a 25 kV. Lo spinneret, costituito da un capillare metallico in equal to 25 kV. The spinneret, consisting of a metal capillary in
acciaio inossidabile (diametro interno di 0,5 mm) è stato collegato stainless steel (inner diameter of 0.5mm) was connected
al polo positivo, mentre come collettore è stato utilizzato un foglio to the positive pole, while a sheet was used as the collector
di alluminio collegato al polo negativo. Una pompa a siringa aluminum connected to the negative pole. A syringe pump
(Graseby Medical ‐ MS 2000) è stata utilizzata per regolare la (Graseby Medical - MS 2000) was used to regulate the
velocità del flusso della soluzione polimerica ed un capillare in flow rate of the polymer solution and a capillary in
politetrafluoretilene per connettere la siringa allo spinneret. Come polytetrafluoroethylene to connect the syringe to the spinneret. How
illustrato nella sottostante Tabella 1, diverse combinazioni di shown in Table 1 below, different combinations of
parametri di elettrofilatura sono state testate per individuare le electrospinning parameters were tested to identify the
condizioni di processo ottimali in relazione all’uniformità delle optimal process conditions in relation to the uniformity of the
matrici ed alle dimensioni ed omogeneità delle fibre elettrofilate. matrices and the size and homogeneity of the electrospun fibers.
Tabella 1 – Parametri di processo nell’elettrofilatura. Table 1 - Process parameters in electrospinning.
Concentrazione Flusso Voltaggio Distanza Concentration Flow Voltage Distance
del polimero (% (ml/h) (kV) spinneretin peso) collettore (cm) Cp F V D of polymer (% (ml / h) (kV) spinneretin weight) collector (cm) Cp F V D
5 – 7,5 – 10 3 – 5 – 7 20 – 24 7 – 10 L’ottimizzazione del processo di elettrofilatura ha portato alla 5 - 7.5 - 10 3 - 5 - 7 20 - 24 7 - 10 The optimization of the electrospinning process has led to
definizione dei parametri sperimentali per l’ottenimento di matrici definition of the experimental parameters for obtaining matrices
planari uniformi e aventi fibre di dimensioni e distribuzione il più planar uniform and having fibers of the most size and distribution
possibile uniformi, come mostrato in Tabella 2. possible uniforms, as shown in Table 2.
Tabella 2 ‐ Parametri utilizzati nella deposizione di matrici Table 2 - Parameters used in the deposition of matrices
elettrofilate planari. planar electrospun.
Parametri sperimentali Experimental parameters
Concentrazione del polimero (Cp) 7,5% (in peso) Polymer concentration (Cp) 7.5% (by weight)
Flusso 3 ml/h Flow 3 ml / h
Voltaggio 24 kV Voltage 24 kV
Distanza spinneret-collettore 10cm Distance from spinneret to collector 10cm
Dopo elettrofilatura, le matrici elettrofilate in fibroina vengono After electrospinning, the electrospun fibroin matrices come
trattate in metanolo per 15 min per aumentarne la cristallinità e treated in methanol for 15 min to increase its crystallinity e
quindi le proprietà meccaniche e l’insolubilità in acqua. hence the mechanical properties and insolubility in water.
Caratterizzazione morfologica Morphological characterization
Microscopia Elettronica a Scansione (SEM) Scanning Electron Microscopy (SEM)
Le analisi morfologiche sono state condotte mediante microscopio Morphological analyzes were carried out using a microscope
elettronico a scansione (SEM; Jeol JSM‐6380 LV) su campioni di scanning electron (SEM; Jeol JSM ‐ 6380 LV) on samples of
strutture elettrofilate planari in fibroina della seta (ES‐SF‐patch), planar electrospun structures in silk fibroin (ES ‐ SF ‐ patch),
montati su appositi porta campioni, metallizzati in oro (Bal‐Tec Med 020) e osservati a differenti ingrandimenti (400‐11000 X) ad una tensione di 20 kV. mounted on special sample holders, metallized in gold (Bal ‐ Tec Med 020) and observed at different magnifications (400‐11000 X) at a voltage of 20 kV.
I migliori risultati sono stati ottenuti utilizzando una concentrazione polimerica al 7,5% in peso di fibroina in acido formico, un campo elettrico di 24 kV con una distanza spinneret‐collettore di 10 cm e un flusso di 3 ml/h. Come mostrato in Figura 2, gli ES‐SF‐patch prodotti con questa combinazione di parametri di processo mostrano fibre uniformi, di sezione circolare ed un diametro medio di 628±107 nm. The best results were obtained using a 7.5% by weight polymer concentration of fibroin in formic acid, an electric field of 24 kV with a spinneret-collector distance of 10 cm and a flow of 3 ml / h. As shown in Figure 2, the ES ‐ SF ‐ patches produced with this combination of process parameters show uniform fibers with a circular cross section and an average diameter of 628 ± 107 nm.
Caratterizzazione chimico ‐ fisica Physical-chemical characterization
Spettroscopia Infrarossa in Trasformata di Fourier (FT‐IR) L’analisi conformazionale degli ES‐SF‐patch è stata effettuata per mezzo di analisi di spettroscopia infrarossa (IR) utilizzando uno spettrometro (Thermo Nicolet 6700 FT‐IR Spectrometer) equipaggiato con accessorio ATR (Attenuated Total Reflectance) e dotato di cristallo di ZnSe. L’indice di cristallinità è stato calcolato come il rapporto tra le intensità dei contributi a 1265 cm<‐1>(caratteristica della struttura β‐sheet) e 1235 cm<‐1>(caratteristica della struttura random coil) del picco relativo all’Ammide III. Le analisi sono state condotte su provini ottenuti da matrici prima e dopo trattamento in metanolo. Fourier Transformed Infrared Spectroscopy (FT ‐ IR) The conformational analysis of the ES ‐ SF ‐ patches was performed by means of infrared (IR) spectroscopy analysis using a spectrometer (Thermo Nicolet 6700 FT ‐ IR Spectrometer) equipped with the ATR accessory (Attenuated Total Reflectance) and equipped with ZnSe crystal. The crystallinity index was calculated as the ratio between the intensity of the contributions at 1265 cm <‐1> (characteristic of the β ‐ sheet structure) and 1235 cm <‐1> (characteristic of the random coil structure) of the peak relative to the Amide III. The analyzes were carried out on specimens obtained from matrices before and after treatment in methanol.
Mediante la deconvoluzione della banda relativa all’ammide primaria è possibile riconoscere le strutture secondarie della fibroina della seta. Tale elaborazione dei dati è stata eseguita con la funzione Peak Fitting Module del software Origin® 7.5. By means of the deconvolution of the band relating to the primary amide, it is possible to recognize the secondary structures of silk fibroin. This data processing was performed with the Peak Fitting Module function of the Origin® 7.5 software.
La struttura secondaria della fibroina rigenerata potrebbe essere di tipo differente (β‐sheet, β‐turn type II, random coil) [Biophys J., 2000, 78 (5), 2690‐2701, Polymer, 2001, 42 (25), 9969‐9974]. In funzione delle condizioni di processo, una conformazione può prevalere sulle altre. Come noto dalla letteratura scientifica, esistono molti trattamenti, sia chimici sia fisici, che possono essere utilizzati per aumentare il grado di cristallinità della fibroina rigenerata promuovendo la transizione conformazionale da random coil a β‐sheet. Uno dei trattamenti più comunemente utilizzati per realizzare questo tipo di transizione è quello che utilizza solventi organici come gli alcoli o i loro vapori. Immergendo la fibroina rigenerata in un bagno di metanolo è possibile ottenere un notevole aumento della struttura con conformazione β‐sheet e quindi della cristallinità del materiale [Polymer, 2005, 46 (5), 1625‐1634,Biophys J., 2000, 78 (5), 2690‐2701, Macromol Biosci., 2006, 6 (4), 285‐292]. La spettroscopia infrarossa è una tecnica di notevole interesse per poter valutare i cambiamenti conformazionali della fibroina rigenerata e trattata con metanolo. La fibroina mostra, in uno spettro infrarosso, bande di assorbimento caratteristiche nelle zone delle Ammidi I, II e III rispettivamente a circa 1625 cm<‐1>, 1528 cm<‐1>e 1260-1230 cm<‐1>[Biophys J., 2000, 78 (5), 2690‐2701, Polymer, 2001, 42 (25), 9969‐9974, Macromol Biosci., 2006, 6 (4), 285‐292]. In questo studio l’attenzione è stata concentrata sull’analisi delle variazioni del picco dell’Ammide I in funzione del tempo di immersione in metanolo (Figura 2). The secondary structure of regenerated fibroin could be of a different type (β ‐ sheet, β ‐ turn type II, random coil) [Biophys J., 2000, 78 (5), 2690‐2701, Polymer, 2001, 42 (25), 9969‐9974]. Depending on the process conditions, one conformation can prevail over the others. As known from the scientific literature, there are many treatments, both chemical and physical, that can be used to increase the degree of crystallinity of regenerated fibroin by promoting the conformational transition from random coil to β-sheet. One of the most commonly used treatments to achieve this type of transition is the one that uses organic solvents such as alcohols or their vapors. By immersing the regenerated fibroin in a methanol bath it is possible to obtain a significant increase in the structure with β-sheet conformation and therefore in the crystallinity of the material [Polymer, 2005, 46 (5), 1625-1634, Biophys J., 2000, 78 ( 5), 2690‐2701, Macromol Biosci., 2006, 6 (4), 285‐292]. Infrared spectroscopy is a technique of considerable interest to be able to evaluate the conformational changes of the regenerated fibroin and treated with methanol. Fibroin shows, in an infrared spectrum, characteristic absorption bands in the Amides I, II and III zones at approximately 1625 cm <‐1>, 1528 cm <‐1> and 1260-1230 cm <‐1> respectively [Biophys J ., 2000, 78 (5), 2690‐2701, Polymer, 2001, 42 (25), 9969‐9974, Macromol Biosci., 2006, 6 (4), 285‐292]. In this study, attention was focused on the analysis of the variations of the Amide I peak as a function of the immersion time in methanol (Figure 2).
Le matrici non trattate in metanolo hanno mostrato un contributo principale a 1621 cm<‐1>attribuibile ad una conformazione β‐sheet con una serie di contributi di assorbimento a numeri d’onda maggiori (1640‐1700 cm<‐1>) attribuibili a conformazioni random coil [Biophys J., 2000, 78 (5), 2690‐2701]. La presenza di un picco di assorbanza IR caratteristica di una conformazione ordinata nella fibroina rigenerata per elettrofilatura, è da attribuire all’utilizzo, come solvente, di acido formico che è noto per promuovere la formazione di strutture β‐sheet durante l’evaporazione [Int J Biol Macromol., 2001, 29 (2), 91‐97]. The matrices not treated in methanol showed a main contribution at 1621 cm <‐1> attributable to a β ‐ sheet conformation with a series of absorption contributions at higher wavelengths (1640‐1700 cm <‐1>) attributable to random coil conformations [Biophys J., 2000, 78 (5), 2690-2701]. [Int J Biol Macromol., 2001, 29 (2), 91‐97].
Dalle misure effettuate sui campioni trattati in metanolo, è stato possibile osservare che, all’aumentare del tempo di immersione degli ES‐SF‐patch in metanolo, l’intensità relativa dei picchi a 1621 cm<‐1>aumenta mentre diminuisce quella dei contributi a numeri d’onda maggiori. È interessante notare come questo effetto sia evidente già per tempi di immersione in metanolo di 5 minuti. Pertanto, il trattamento di immersione in metanolo ha dimostrato la capacità di promuovere transizioni conformazionali da strutture amorfe e/o poco ordinate in strutture secondarie maggiormente ordinate con conformazione β‐sheet anche per nanofibre in fibroina rigenerate per elettrofilatura. La capacità del metanolo di favorire la formazione una struttura secondaria maggiormente ordinata è stata confermata anche dall’andamento dell’indice di cristallinità IR che cresce all’aumentare del tempo di immersione degli ES‐SF‐patch in metanolo (Figura 3). From the measurements carried out on the samples treated in methanol, it was possible to observe that, as the immersion time of the ES ‐ SF ‐ patches in methanol increases, the relative intensity of the peaks at 1621 cm <‐1> increases while that of the contributions decreases. at higher wave numbers. It is interesting to note that this effect is already evident for 5 minutes of immersion in methanol. Therefore, the methanol immersion treatment demonstrated the ability to promote conformational transitions from amorphous and / or poorly ordered structures into more ordered secondary structures with β-sheet conformation even for electrospin regenerated fibroin nanofibers. The ability of methanol to promote the formation of a more ordered secondary structure was also confirmed by the trend of the IR crystallinity index which increases with increasing immersion time of the ES ‐ SF ‐ patches in methanol (Figure 3).
Deconvoluzione della banda di Ammide I Deconvolution of the Amide I band
Successivamente all’ottenimento dello spettro infrarosso per i diversi provini è stata eseguita una deconvoluzione del picco relativo all’Ammide I nell’intervallo compreso tra 1600 e 1700 cm<‐1>che ha permesso di evidenziare i contributi dei principali componenti spettrali all’interno del picco considerato e di quantificarli, come mostrato in Tabella 3. After obtaining the infrared spectrum for the different specimens, a deconvolution of the peak relative to Amide I was performed in the interval between 1600 and 1700 cm <‐1> which allowed to highlight the contributions of the main spectral components within peak considered and quantify them, as shown in Table 3.
Tabella 3 ‐ Valori % dell’area dei diversi contributi spettrali, con Table 3 -% values of the area of the different spectral contributions, with
rispettiva lunghezza d’onda e conformazione molecolare, dei respective wavelength and molecular conformation, of the
campioni in fibroina elettrofilata non trattati e trattati in metanolo. untreated and methanol treated electrospun fibroin samples.
Lunghezza Area del Conformazione d’onda del picco (%) molecolare picco (cm<-1>) Area length of the waveform of the molecular peak (%) peak (cm <-1>)
ES-SF-patch 1628 20 β-sheet non trattato ES-SF-patch 1628 20 untreated β-sheet
1648 50 Random coil 1671 30 turn 1648 50 Random coil 1671 30 turn
Lunghezza Area del Conformazione d’onda del cco (%) molecolare picco (cm<-1>pi Area length of the waveform of the peak molecular cco (%) (cm <-1> pi
) )
ES-SF-patch 1626 53 β-sheet trattato in ES-SF-patch 1626 53 β-sheet treated in
metanolo per methanol for
15 min 15 min
1648 26 Random coil 1667 21 turn 1648 26 Random coil 1667 21 turn
Come atteso, si può osservare che l’area percentuale del picco As expected, it can be observed that the percentage area of the peak
caratteristico della conformazione β‐sheet, per i campioni trattati in characteristic of the β-sheet conformation, for the samples treated in
metanolo per 15 minuti è pari a oltre il 50% dell’area totale, contro methanol for 15 minutes is equal to more than 50% of the total area, against
il 20% del campione appena elettrofilato. Anche questo dato 20% of the sample just electro-spun. This data too
conferma la precedente osservazione qualitativa di un aumento di confirms the previous qualitative observation of an increase of
ordine cristallino in seguito a trattamento dell'ES-SF-patch con crystalline order following treatment of the ES-SF-patch with
metanolo. methanol.
Calorimetria Differenziale a Scansione (DSC) Differential Scanning Calorimetry (DSC)
Tramite calorimetro differenziale a scansione (DSC; Mettler Toledo DSC‐30) sono state effettuate analisi su campioni di ES‐SF‐patch prima e dopo trattamento in metanolo. Il peso dei provini era compreso fra 1,5 mg e 3,5 mg e la metodologia di prova prevedeva un riscaldamento una rampa di temperatura da 20°C fino a 500°C in atmosfera inerte di azoto (N2) con velocità di riscaldamento di 10°C/minuto. Using a differential scanning calorimeter (DSC; Mettler Toledo DSC ‐ 30), analyzes were carried out on ES ‐ SF ‐ patch samples before and after treatment in methanol. The weight of the specimens was between 1.5 mg and 3.5 mg and the test methodology involved heating a temperature ramp from 20 ° C up to 500 ° C in an inert atmosphere of nitrogen (N2) with a heating rate of 10 ° C / minute.
Le analisi DSC hanno evidenziato differenze nelle proprietà termiche degli ES‐SF‐patch prima e dopo i trattamenti in metanolo per 5, 10 e 15 minuti (Figura 4). I campioni non sottoposti a trattamento in metanolo hanno evidenziato due transizioni endotermiche a 180°C e 215°C rappresentate da due picchi di lieve intensità e forma allargata, seguiti da un picco endotermico più intenso a 280°C. Mentre questo ultimo picco è attribuibile ai processi di fusione/decomposizione di strutture β‐sheet scarsamente orientate, i primi due possono essere messi in relazione con fenomeni di mobilità termica e/o a processi di fusione nelle zone amorfe delle macromolecole della fibroina. Sebbene delle zone con conformazione β‐sheet siano già presenti all’interno delle nano fibre di fibroina anche prima dei trattamenti in metanolo, queste risultano scarsamente ordinate e orientate, riuscendo a formare solo dei cristalliti dispersi in una matrice disordinata con scarsa stabilità termica [Biophys J., 2000, 78 (5), 2690‐2701]. DSC analyzes revealed differences in the thermal properties of the ES ‐ SF ‐ patches before and after treatments in methanol for 5, 10 and 15 minutes (Figure 4). Samples not treated in methanol showed two endothermic transitions at 180 ° C and 215 ° C represented by two peaks of mild intensity and enlarged shape, followed by a more intense endothermic peak at 280 ° C. While this last peak is attributable to the fusion / decomposition processes of poorly oriented β-sheet structures, the first two can be related to thermal mobility phenomena and / or to fusion processes in the amorphous zones of the fibroin macromolecules. Although zones with β-sheet conformation are already present inside the fibroin nano fibers even before methanol treatments, these are poorly ordered and oriented, managing to form only dispersed crystallites in a disordered matrix with poor thermal stability [Biophys J., 2000, 78 (5), 2690-2701].
Osservando la Figura 4 è possibile notare come, dopo i trattamenti in metanolo, non siano stati rilevati i picchi endotermici fra i 160°C e i 220°C. In accordo con le analisi IR, dopo immersione degli ES‐SF‐patch in metanolo vi è una transizione conformazionale di parte della struttura amorfa della fibroina in struttura ordinata. Il metanolo, probabilmente, è in grado di indurre la formazione di un ordine a “lungo raggio” limitando la mobilità termodinamica delle macromolecole e, di conseguenza, le transizioni in questo intervallo di temperature non risultano essere più visibili già dopo 5 minuti di trattamento in metanolo. Analizzando l’andamento del picco di fusione/decomposizione, è possibile osservare come, all’aumentare del tempo di immersione in metanolo, il picco presente a 280°C si sposti verso temperature più alte (fino a 288°C) e si restringa sensibilmente. Questo comportamento è molto simile a quanto rilevato per materiali amorfi in fibroina rigenerata (ad esempio membrane di fibroina ottenute per solvent casting) sottoposti a trattamenti in grado stimolare la cristallizzazione della fibroina stessa [J. Polym. Sci.: Polym. Phys. Ed., 1979, 17, 515– 520]. Passando da 5 a 10 minuti di immersione in metanolo delle matrici elettrofilate, un picco secondario è stato osservato per temperature attorno ai 300°C. Questo picco è stato precedentemente osservato per altri tipi di materiali in fibroina rigenerata da acido formico ed associato alla decomposizione termica di zone con conformazione β‐sheet altamente orientate [Biophys J., 2000, 78 (5), 2690‐2701]. La presenza di questo nuovo picco negli ES‐SF‐patch, insieme agli effetti precedentemente descritti, dimostrano come l’immersione di queste matrici in bagni di metanolo sia un trattamento adeguato a promuovere la formazione di nanofibre in fibroina con una struttura cristallina regolare ed ordinata, promuovendo la transizione di regioni amorfe o scarsamente ordinate in regioni ad alto ordine strutturale. By observing Figure 4 it is possible to note that, after the treatments in methanol, the endothermic peaks between 160 ° C and 220 ° C were not detected. According to the IR analyzes, after immersion of the ES ‐ SF ‐ patches in methanol there is a conformational transition of part of the amorphous structure of the fibroin into an ordered structure. Methanol is probably able to induce the formation of a "long range" order by limiting the thermodynamic mobility of the macromolecules and, consequently, the transitions in this temperature range are no longer visible after 5 minutes of treatment in methanol. By analyzing the trend of the melting / decomposition peak, it is possible to observe how, as the time of immersion in methanol increases, the peak present at 280 ° C moves towards higher temperatures (up to 288 ° C) and shrinks significantly . This behavior is very similar to that observed for amorphous materials in regenerated fibroin (for example fibroin membranes obtained by solvent casting) subjected to treatments capable of stimulating the crystallization of the fibroin itself [J. Polym. Sci .: Polym. Phys. Ed., 1979, 17, 515–520]. Passing from 5 to 10 minutes of immersion in methanol of the electrospun matrices, a secondary peak was observed for temperatures around 300 ° C. This peak has previously been observed for other types of formic acid-regenerated fibroin materials and associated with the thermal decomposition of highly oriented β-sheet conformation zones [Biophys J., 2000, 78 (5), 2690-2701]. The presence of this new peak in the ES ‐ SF ‐ patches, together with the effects previously described, demonstrate how the immersion of these matrices in methanol baths is an adequate treatment to promote the formation of fibroin nanofibers with a regular and ordered crystalline structure. , promoting the transition of amorphous or poorly ordered regions into regions of high structural order.
Caratterizzazione Fisico-Meccanica Physical-Mechanical Characterization
Prove a trazione Tensile tests
La caratterizzazione meccanica è stata eseguita su strutture planari (ES‐SF‐patch) tramite analizzatore Dinamico‐Meccanico (DMA, TA Instruments, DMA 2980 Dynamic Mechanical Analyzer), utilizzando l’afferraggio Tension Film. Le prove a trazione sono state eseguite su 3 campioni, utilizzando i parametri sperimentali The mechanical characterization was performed on planar structures (ES ‐ SF ‐ patch) using a Dynamic ‐ Mechanical analyzer (DMA, TA Instruments, DMA 2980 Dynamic Mechanical Analyzer), using the Tension Film gripping. Tensile tests were performed on 3 samples, using the experimental parameters
riportati in Tabella 4. shown in Table 4.
Tabella 4 ‐ Parametri utilizzati per le prove di trazione eseguite Table 4 - Parameters used for the tensile tests performed
sulle strutture planari con analizzatore DMA. on planar structures with DMA analyzer.
Parametri sperimentali Experimental parameters
Precarico 0,005 N Preload 0.005 N
Rampa di forza 0,01 N/min Force ramp 0.01 N / min
Tratto utile 8 mm Useful section 8 mm
Lunghezza provino 15 mm Specimen length 15 mm
A titolo esemplificativo, è stata riportata, in Figura 5, una curva By way of example, a curve has been reported in Figure 5
sforzo‐deformazione rappresentativa per l’ES‐SF‐patch; si può representative strain-strain for the ES-SF-patch; you can
notare come l’andamento della curva sforzo‐deformazione sia note how the trend of the stress-strain curve is
sostanzialmente lineare, permettendo di concludere che la fibroina substantially linear, allowing to conclude that fibroin
elettrofilata risulta essere un materiale tendenzialmente elastico. electrospun is a basically elastic material.
In Tabella 5 sono riportati i valori medi e relativa deviazione Table 5 shows the average values and relative deviation
standard dei parametri meccanici considerati. standard of the mechanical parameters considered.
Tabella 5 ‐ Valori medi e relativa deviazione standard del modulo di Young, dello sforzo e della deformazione a rottura per l’ES‐SF‐patch. Table 5 - Average values and relative standard deviation of Young's modulus, stress and strain at break for the ES ‐ SF ‐ patch.
Modulo di Young Sforzo a rottura Deformazione a rottura (MPa) (MPa) (%) Young's modulus Stress at break Strain at break (MPa) (MPa) (%)
99,03 ± 1,13 2,28 ± 0,87 2,28 ± 0,81 99.03 ± 1.13 2.28 ± 0.87 2.28 ± 0.81
In letteratura sono riportati alcuni lavori che analizzano il comportamento meccanico di matrici planari elettrofilate in fibroina [Min B.M. et al., Macromolecular Bioscience, 2006; Ayutsede J. et al., Polymer, 2005]; tuttavia i dati di caratterizzazione meccanica vengono ricavati con metodologie di analisi e condizioni al contorno differenti. Le prove presenti in letteratura, infatti, utilizzano i campioni in condizioni di umidità al 50% e al 65%; la DMA a nostra disposizione non prevede la possibilità di effettuare tale prova a diverse condizioni di umidità. In the literature there are some papers that analyze the mechanical behavior of planar electrospun fibroin matrices [Min B.M. et al., Macromolecular Bioscience, 2006; Ayutsede J. et al., Polymer, 2005]; however the mechanical characterization data are obtained with different analysis methods and boundary conditions. The tests present in the literature, in fact, use the samples in conditions of humidity at 50% and 65%; the DMA at our disposal does not foresee the possibility of carrying out this test at different humidity conditions.
ISOLAMENTO CELLULARE E CULTURA CELL ISOLATION AND CULTURE
Lo studio è stato approvato dal consiglio di revisione locale istituzionale della Fondazione IRCCS Istituto Neurologico Carlo Besta, Milano Italia e consenso informato scritto è stato ottenuto da tutti i volontari. The study was approved by the local institutional review board of the IRCCS Carlo Besta Neurological Institute Foundation, Milan Italy and written informed consent was obtained from all volunteers.
I campioni di grasso da regioni periombelicali di quattro volontari sono stati raccolti presso la Fondazione IRCCS Istituto Neurologico C. Besta con una tecnica di biopsia periombelicale. Cellule stromali mesenchimali di origine adiposa (Ad-MSC) sono state ottenute come precedentemente descritto [Blasi A, Martino C, Balducci L, et al. Dermal fibroblasts display similar phenotypic and differentiation capacity to fat-derived mesenchymal stem cells, but differ in anti-inflammatory and angiogenic potential. VASC CELL. 2011;8:3:5]. Le cellule sono state coltivate in un mezzo di cultura (Stem Cells Medium – SCM), consistente in DMEM-F12 complementato con 10% di siero fetale di bovino (Gibco, Grand Island, NY, USA), 1% di soluzione di penicillina e streptomicina (Sigma-Aldrich, Basel, Svizzera) e ottimizzato per la crescita di cellule staminali come precedentemente descritto [Alessandri G, Pagano S, Bez A, et al.. Isolation and culture of human muscle derived stem cells able to differentiate into myogenic and neurogenic cell lineages. LANCET 2004;364:1872-1883]. Le cellule Ad-MSC sono state regolarmente seminate a 2x10<4>cellule/cm<2>in fiasche T75-cm<2>e divise settimanalmente. Per gli esperimenti, le cellule non sono state usate dopo 6 passaggi. La vitalità cellulare è stata valutata mediante colorante Trypan Blue . Fat samples from periumbilical regions of four volunteers were collected at the C. Besta Neurological Institute IRCCS Foundation with a periumbilical biopsy technique. Mesenchymal stromal cells of adipose origin (Ad-MSC) were obtained as previously described [Blasi A, Martino C, Balducci L, et al. Dermal fibroblasts display similar phenotypic and differentiation capacity to fat-derived mesenchymal stem cells, but differ in anti-inflammatory and angiogenic potential. VASC CELL. 2011; 8: 3: 5]. The cells were cultured in a culture medium (Stem Cells Medium - SCM), consisting of DMEM-F12 complemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA), 1% penicillin solution and streptomycin (Sigma-Aldrich, Basel, Switzerland) and optimized for stem cell growth as previously described [Alessandri G, Pagano S, Bez A, et al .. Isolation and culture of human muscle derived stem cells able to differentiate into myogenic and neurogenic cell lineages. LANCET 2004; 364: 1872-1883]. Ad-MSC cells were routinely seeded at 2x10 <4> cells / cm <2> in T75-cm <2> flasks and divided weekly. For the experiments, the cells were not used after 6 passages. Cell viability was assessed with Trypan Blue dye.
ANALISI DI CITOMETRIA DI FLUSSO. FLOW CYTOMETRY ANALYSIS.
La citometria a flusso (FC) è stata eseguita su Ad-MSCs per valutare i marcatori mesenchimali ed ematopoietici: CD44, CD45, CD73, CD166 (BD Pharmingen, San Jose, CA, USA), CD105 (AbDSerotec, Raleigh, NC, USA) e CD90 ( Millipore Temecula, CA, USA). Brevemente, 5 x 10<4>cellule/provetta sono state colorate con fluorocromi coniugati con anticorpi monoclonali e incubati per 30' a 4°C al buio. I campioni sono stati centrifugati a 300xg per 5', lavati due volte con PBS e fissate con paraformaldeide al 4% (Sigma Aldrich). Le analisi sono state eseguite con citofluorimetro e analizzate con software CEll Quest (BD Pharmingen). Immunoglobuline di topo isotipo-miste sono state usate come controllo. Almeno 20.000 eventi sono stati acquisiti per ogni campione. Le cellule non vitali sono state escluse dall’analisi. Flow cytometry (FC) was performed on Ad-MSCs to evaluate mesenchymal and hematopoietic markers: CD44, CD45, CD73, CD166 (BD Pharmingen, San Jose, CA, USA), CD105 (AbDSerotec, Raleigh, NC, USA ) and CD90 (Millipore Temecula, CA, USA). Briefly, 5 x 10 <4> cells / tube were stained with fluorochromes conjugated with monoclonal antibodies and incubated for 30 'at 4 ° C in the dark. The samples were centrifuged at 300xg for 5 ', washed twice with PBS and fixed with 4% paraformaldehyde (Sigma Aldrich). The analyzes were performed with a flow cytometer and analyzed with CEll Quest software (BD Pharmingen). Isotype-mixed mouse immunoglobulins were used as controls. At least 20,000 events were acquired for each sample. Non-viable cells were excluded from the analysis.
Preparazione di patch di fibroina della seta cellularizzati o decellularizzati Preparation of cellularized or decellularized silk fibroin patches
Fogli di fibroina della seta elettrofilati sono stati prodotti secondo il metodo di Marelli B et al. [Marelli B, Ghezzi CE, Alessandrino A, et al. Silk fibroin derived polypeptide-induced biomineralization of collagen.. BIOMATERIALS. 2012;33:102-108]. I patch di fibroina della seta di cinque millimetri di diametro sono stati tagliati utilizzando uno strumento sterile per biopsia di 5 mm (KLS Martin cod. 28-240-05-07 Germania). I patch sono stati quindi posti in piastre di Petri, lavati con soluzione di etanolo al 70% per 1 ora, sterilizzati ai raggi UV e poi conservati a 4°C fino all'uso. Electrospun silk fibroin sheets were produced according to the method of Marelli B et al. [Marelli B, Ghezzi CE, Alessandrino A, et al. Silk fibroin derived polypeptide-induced biomineralization of collagen .. BIOMATERIALS. 2012; 33: 102-108]. The five mm diameter silk fibroin patches were cut using a sterile 5 mm biopsy instrument (KLS Martin cod. 28-240-05-07 Germany). The patches were then placed in Petri dishes, washed with 70% ethanol solution for 1 hour, UV sterilized and then stored at 4 ° C until use.
Cellule Ad-MSC sono state applicate sui patch mettendo ogni patch in un pozzetto di una piastra multipozzetto da 96 (Corning Incorporated, Corning, NY, USA) e poi coprendo il patch con 200 µL di terreno di coltura SCM che conteneva 3x10<4>cellule. I patch seminati con cellule o con il mezzo di coltura sono stati incubati in atmosfera umidificata contenente 5% di CO2a 37°C per almeno 7 giorni. Il terreno di coltura è stato cambiato una volta. Ad-MSC cells were applied to the patches by placing each patch in a well of a 96-well multi-well plate (Corning Incorporated, Corning, NY, USA) and then covering the patch with 200 µL of SCM culture medium which contained 3x10 <4> cells. Patches seeded with cells or culture medium were incubated in a humidified atmosphere containing 5% CO2 at 37 ° C for at least 7 days. The culture medium was changed once.
La decellularizzazione dei patch di fibroina della seta è stata ottenuta rimuovendo il terreno di coltura, lavando i patch con H2O distillata sterile per due volte, seguito da due incubazioni con acqua MilliQ per 30' ciascuna a 4°C. I patch D-Ad-MSC-SF sono stati poi trasferiti in un nuovo pozzetto e conservati in frigorifero a 4°C fino al momento dell’utilizzo per i trapianti o per la caratterizzazione morfologica. I patch D-Ad-MSC-SF conservati per più di 7 giorni non sono stati utilizzati in vivo. The decellularization of the silk fibroin patches was achieved by removing the culture medium, washing the patches with sterile distilled H2O twice, followed by two incubations with MilliQ water for 30 'each at 4 ° C. The D-Ad-MSC-SF patches were then transferred to a new well and stored in the refrigerator at 4 ° C until they were used for transplants or for morphological characterization. D-Ad-MSC-SF patches stored for more than 7 days were not used in vivo.
Analisi morfologica di patch di fibroina della seta cellularizzati e decellularizzati. Morphological analysis of cellularized and decellularized silk fibroin patches.
L’analisi morfologica di patch di fibroina della seta, Ad-MSC-SF e D-Ad-MSC-SF è stata esaminata con istologia standard e con microscopio elettronico a scansione (SEM). Per l'istologia, i campioni sono stati fissati in 10% di formalina tamponata neutra, disidratati ed inclusi in paraffina. Sezioni dello spessore di 5 micrometri sono state montate su vetrini e colorate con Ematossilina eosina (H & E). Per l’analisi mediante microscopio elettronico a scansione (SEM), i patch sono stati lavati in tampone cacodilato (CB) 0,1 M a pH 7,2, fissati con glutaraldeide 2,5% (Sigma-Aldrich) in tampone cacodilato per 2 ore a 4°C e disidratati in una serie graduata di etanolo fino all’assoluto. I patch cellularizzati e decellularizzati sono stati posti su matrici in metallo, rivestiti d'oro per uno spessore di 15 nm e visualizzati con un microscopio elettronico a scansione Philips XL30 (Centro di Microscopia Elettronica - Cume, Università degli Studi di Perugia, Perugia, Italia). I patch Ad-MSC-SF sono stati studiati mediante immunofluorescenza per confermare la presenza di marcatori mesenchimali. In breve, i patch Ad-MSC-SF coltivati per 7 giorni sono stati fissati per 30' a temperatura ambiente in paraformaldeide al 4% e permeabilizzati con 0.1% Triton X-100. I seguenti anticorpi primari sono stati applicati per tutta la notte a 4°C: topo anti-CD44 umano (1:500), CD45 (1:400), CD146 (1:400) e coniglio anti CD49d umana (1:500) (tutti acquistati da Chemicon-Millipore, Temecula, CA, USA). Le cellule sono state poi incubate in presenza di anticorpi secondari coniugati Cy2 e/o Cy3 (Jackson ImmunoResearch, West Grove, PA) a temperatura ambiente per 45' e montati con Fluorsave™ (Calbiochem-Millipore, Temecula, CA, USA). Le immagini digitali 3D sono state acquisite utilizzando un microscopio a scansione laser confocale Leica TCS SP2 AOBS (Leica Microsystems). The morphological analysis of silk fibroin patches, Ad-MSC-SF and D-Ad-MSC-SF was examined with standard histology and with a scanning electron microscope (SEM). For histology, specimens were fixed in 10% neutral buffered formalin, dehydrated and embedded in paraffin. Sections 5 micrometers thick were mounted on slides and stained with Hematoxylin and Eosin (H&E). For scanning electron microscope (SEM) analysis, the patches were washed in 0.1 M cacodylate buffer (CB) at pH 7.2, fixed with 2.5% glutaraldehyde (Sigma-Aldrich) in cacodylate buffer to 2 hours at 4 ° C and dehydrated in a graduated series of ethanol until absolute. The cellularized and decellularized patches were placed on metal matrices, coated with gold to a thickness of 15 nm and visualized with a Philips XL30 scanning electron microscope (Centro di Microscopia Elettronica - Cume, University of Perugia, Perugia, Italy ). Ad-MSC-SF patches were studied by immunofluorescence to confirm the presence of mesenchymal markers. Briefly, the Ad-MSC-SF patches grown for 7 days were fixed for 30 'at room temperature in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. The following primary antibodies were applied overnight at 4 ° C: mouse anti-human CD44 (1: 500), CD45 (1: 400), CD146 (1: 400) and rabbit anti-human CD49d (1: 500) (all purchased from Chemicon-Millipore, Temecula, CA, USA). The cells were then incubated in the presence of Cy2 and / or Cy3 conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA) at room temperature for 45 'and mounted with Fluorsave ™ (Calbiochem-Millipore, Temecula, CA, USA). 3D digital images were acquired using a Leica TCS SP2 AOBS (Leica Microsystems) confocal laser scanning microscope.
Procedura chirurgica: attecchimento di patch Ad-MSC-SF e D-Ad-MSC-SF in topi diabetici (Leoi<db/db>). Surgical procedure: Ad-MSC-SF and D-Ad-MSC-SF patch engraftment in diabetic mice (Leoi <db / db>).
Le procedure che coinvolgono animali sono state condotte in conformità alle linee guida istituzionali che sono in conformità alle linee guida italiane per la cura degli animali (DL 116/92), alla Direttiva del Consiglio delle Comunità Europee (86/609/CEE) e alla Guida per la cura e l'uso di animali da laboratorio degli Istituti Nazionali di Sanità. Il protocollo per l'uso di animali da laboratorio è stato approvato dal Ministero della Salute italiano e da un comitato etico interno. The procedures involving animals have been conducted in compliance with the institutional guidelines which are in compliance with the Italian guidelines for the care of animals (DL 116/92), the Directive of the Council of the European Communities (86/609 / EEC) and the Guide for the care and use of laboratory animals of the National Institutes of Health. The protocol for the use of laboratory animals was approved by the Italian Ministry of Health and an internal ethics committee.
Topi maschi Lepr<db/db>di otto settimane (Charles River Laboratories, Calco, Lecco, Italia) sono stati alloggiati per almeno 1 settimana nelle loro gabbie ad una temperatura costante. I topi sono stati randomizzati in quattro gruppi di 6 ciascuno. I topi controllo senza innesto hanno ricevuto solo patch di soluzione salina e i topi trattati hanno ricevuto rispettivamente patch di SF, Ad-MSC-SF e D-Ad-MSC-SF. Eight-week-old male Lepr <db / db> mice (Charles River Laboratories, Calco, Lecco, Italy) were housed for at least 1 week in their cages at a constant temperature. The mice were randomized into four groups of 6 each. Ungrafted control mice received patches of saline only and treated mice received patches of SF, Ad-MSC-SF, and D-Ad-MSC-SF, respectively.
In breve, i topi sono stati anestetizzati con iniezione intraperitoneale di cloralio idrato (4%, 400 mg/kg) ed è stata effettuata una ferita nel panniculus carnosus con uno strumento da biopsia (KLS cod Martin. 28-240-05-07 Germania) sterile di 5 millimetri su ogni lato della zona posteriore della schiena del topo. I patch sono stati applicati sulle ferite e fissati alla pelle con 30 µl di idrogel (ExtracelTM-X Kit Maxi Idrogel, Glycosan BioSystems Inc. Alameda, CA, USA) per garantire l'adesione prolungata alla ferita. Briefly, the mice were anesthetized with intraperitoneal injection of chloral hydrate (4%, 400 mg / kg) and a wound was made in the panniculus carnosus with a biopsy instrument (KLS cod Martin. 28-240-05-07 Germany ) 5 mm sterile on each side of the mouse's posterior back area. The patches were applied to the wounds and fixed to the skin with 30 µl of hydrogel (ExtracelTM-X Kit Maxi Hydrogel, Glycosan BioSystems Inc. Alameda, CA, USA) to ensure sustained adhesion to the wound.
I topi sono stati posti in gabbie individuali. I topi sono stati fotografati durante il decorso postoperatorio a 3, 7, 10 giorni usando una fotocamera digitale Canon ad alta risoluzione. Le aree di chiusura della ferita sono state misurate come precedentemente descritto [Altman AM, Yan Y, Matthias N, et al. The mice were placed in individual cages. The mice were photographed during the 3, 7, 10 day postoperative course using a high resolution Canon digital camera. Wound closure areas were measured as previously described [Altman AM, Yan Y, Matthias N, et al.
IFATS collection: Human adipose-derived stem cells seeded on a silk fibroin-chitosan scaffold enhance wound repair in a murine soft tissue injury model. STEM CELLS 2009;27:250-258]. In breve, una riga metrica è stata posizionata adiacente al limite della ferita e tre misurazioni individuali fotomicrografiche sono state prese per ciascun topo. La percentuale di chiusura della ferita (contrazione) è stata calcolata con la formula: area al giorno postoperatorio 0 - area al giorno postoperatorio X / area al giorno postoperatorio 0x100. Tutte le misure sono state acquisite indipendentemente in cieco da due ricercatori. I campioni di cute di topo trattati con patch di controllo e patch di SF, Ad- MSC-SF e D-Ad-MSCs-SF sono stati raccolti dopo i trapianti. Le biopsie sono state processate per l’osservazione microscopica. I campioni sono stati sottoposti ad esame istologico della cute per i seguenti parametri principali: la riepitelizzazione, neovascolarizzazione, organizzazione della fibra cutanea. IFATS collection: Human adipose-derived stem cells seeded on a silk fibroin-chitosan scaffold enhance wound repair in a murine soft tissue injury model. STEM CELLS 2009; 27: 250-258]. Briefly, a metric line was placed adjacent to the wound boundary and three individual photomicrographic measurements were taken for each mouse. The percentage of wound closure (contraction) was calculated using the formula: area on postoperative day 0 - area on postoperative day X / area on postoperative day 0x100. All measurements were independently blinded by two investigators. Mouse skin samples treated with control patches and patches of SF, Ad-MSC-SF and D-Ad-MSCs-SF were collected after transplants. The biopsies were processed for microscopic observation. The samples were subjected to histological examination of the skin for the following main parameters: re-epithelialization, neovascularization, organization of the skin fiber.
Scratch Test. Scratch Test.
Lo scratch test è stato impostato con inserti Ibidi (ibidi GmbH, Munchen, Germania). Brevemente, inserti ibidi sono stati posti sul fondo dei pozzetti in una piastra multipozzetto da 24 rivestita con 0,2 mg/ml di soluzione di collagene di tipo I (Sigma-Aldrich). The scratch test was set up with Ibidi inserts (ibidi GmbH, Munchen, Germany). Briefly, ibid inserts were placed at the bottom of the wells in a 24-well multi-well plate coated with 0.2 mg / ml of type I collagen solution (Sigma-Aldrich).
Cheratinociti (KC) umani, fibroblasti del derma (DF) e HUVEC (tutti acquistati da Centro substrati Cellulari, ISZLER, Brescia, Italia) sono stati seminati ad una densità finale di 5.000 cellule/cm<2>in inserti ibidi in terreno di coltura (SCM). Le cellule sono state mantenute per 24 ore a 37°Ce 5% di CO2per consentire la crescita di cellule in monostrato. Successivamente gli inserti ibidi sono stato rimossi, le cellule sono state delicatamente lavate con PBS ed e sono stati aggiunti 0,5 mL di terreno di coltura (SCM) (senza ulteriori fattori di crescita). Successivamente in ogni pozzetto sono stati posizionati i supporti di transwells con filtri in policarbonato da 8 µm (Corning Life Sciences). Su ogni filtro, a seconda dello schema sperimentale, sono stati posizionati i patch SF, Ad-MSC-SF e D-Ad-MSC-SF e rapidamente bagnati con 200 µL di terreno di coltura (SCM). Dopo 6, 24, 36 o 48 ore, i supporti delle transwells sono stati rimossi e le cellule sono state osservate al microscopio Axiophot-2 Zeiss (un sistema di saggio è mostrato in Figura 15). Human keratinocytes (KC), dermal fibroblasts (DF) and HUVEC (all purchased from Cellular Substrates Center, ISZLER, Brescia, Italy) were seeded to a final density of 5,000 cells / cm <2> in hybrid inserts in culture medium (SCM). The cells were kept for 24 hours at 37 ° C and 5% CO2 to allow the growth of cells in monolayer. Subsequently the ibidic inserts were removed, the cells were gently washed with PBS and 0.5 mL of culture medium (SCM) was added (without additional growth factors). Subsequently, transwells supports with 8 µm polycarbonate filters (Corning Life Sciences) were placed in each well. On each filter, according to the experimental scheme, the patches SF, Ad-MSC-SF and D-Ad-MSC-SF were placed and quickly wetted with 200 µL of culture medium (SCM). After 6, 24, 36 or 48 hours, the supports of the transwells were removed and the cells were observed under the Axiophot-2 Zeiss microscope (an assay system is shown in Figure 15).
Per valutare gli effetti riparativi dei patch SF, le cellule migrate nel setto di separazione sono state contate al microscopio a ingrandimenti 20x in 5 campi diversi. Ogni prova di migrazione è stata eseguita in triplicato. Al termine dello scratch test, aliquote terreno di coltura condizionato (CM) sono state raccolte e FGF2 umana, EGF (Mini ELISA kit di sviluppo acquistato da Peprotech, Stati Uniti d'America), TGF-beta (Demeditec, Germania) e VEGF (Ray Biotech, Inc, GA USA) sono stati quantificati mediante ELISA secondo il protocollo fornito con il kit. Il valore di fondo (<10%) di ciascun fattore di crescita analizzato e contenuto nel terreno di controllo SCM (non coltivato con le cellule) è stato sottratto. L'assorbanza è stata misurata a 450 nm o 405 nm con un lettore di micropiastre fotometrico DV990BV4 (GDV, Italia). I dati sono stati espressi come media ± SD del fattore secreto. Il saggio è stato ripetuto due volte e ogni campione è stato analizzato in triplicato. To evaluate the reparative effects of the SF patches, the cells migrated into the separation septum were counted under the microscope at 20x magnification in 5 different fields. Each migration test was performed in triplicate. At the end of the scratch test, aliquots of conditioned culture medium (CM) were collected and human FGF2, EGF (Mini ELISA development kit purchased from Peprotech, USA), TGF-beta (Demeditec, Germany) and VEGF ( Ray Biotech, Inc, GA USA) were quantified by ELISA according to the protocol provided with the kit. The background value (<10%) of each growth factor tested and contained in the SCM control medium (not cultured with cells) was subtracted. The absorbance was measured at 450 nm or 405 nm with a DV990BV4 photometric microplate reader (GDV, Italy). Data were expressed as mean ± SD of the secreted factor. The assay was repeated twice and each sample was analyzed in triplicate.
PCR quantitativa in tempo reale (RT-PCR). Quantitative real-time PCR (RT-PCR).
RT-PCR è stata eseguita su Ad-MSCs coltivate su plastica e su patch di SF. Per l'analisi dei livelli di mRNA, 500ng di RNA totale isolato usando il kit RNeasy (Qiagen) è stato retrotrascritto utilizzando iScript cDNA Synthesis Kit (Bio-Rad Laboratories). Reazioni a catena della polimerasi in triplicato sono state effettuate su un sistema con strumentazione per Real Time PCR Biorad iCycler, iQ (Bio-Rad Laboratories). RT-PCR was performed on Ad-MSCs grown on plastic and on SF patches. For the analysis of mRNA levels, 500ng of total RNA isolated using the RNeasy kit (Qiagen) was back-transcribed using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories). Triplicate polymerase chain reactions were performed on a system with Biorad iCycler, iQ (Bio-Rad Laboratories) Real Time PCR instrumentation.
L’ espressione genica relativa è stata calcolata con un metodo comparativo (2<-ΔΔCT>) utilizzando GAPDH come gene housekeeping. Sequenze primer sono state progettate utilizzando Primer3 software. The relative gene expression was calculated with a comparative method (2 <-ΔΔCT>) using GAPDH as a housekeeping gene. Primer sequences were designed using Primer3 software.
Profilo genico. Gene profile.
Campioni di tessuto cutaneo sono stati asportati e analizzati per il profilo genico. In breve, 500 ng di RNA totale è stato utilizzato per ogni piastra ed è stato retrotrascritto utilizzando RT<2>First Strand Kit (SABioscience Corporation, Frederick, MD, USA), secondo le istruzioni del produttore. Gli RT<2>™ Profiler sono stati eseguiti in triplicato per valutare il profilo genico delle cellule coinvolte nel processo di guarigione delle ferite del topo (PAMM-121, SABioscience Corporation, Frederick, MD, USA). Le piastre sono state correre analizzate con termociclatore Applied Biosystems AB 7300 real-time PCR, secondo istruzioni del produttore. L'espressione dei geni di interesse è stata analizzata utilizzando il portale di analisi dei dati sul sito della SABioscence (SABioscience Corporation, Frederick, MD, USA). Skin tissue samples were excised and analyzed for the gene profile. Briefly, 500 ng of total RNA was used for each plate and it was back-transcribed using RT <2> First Strand Kit (SABioscience Corporation, Frederick, MD, USA), according to the manufacturer's instructions. RT <2> ™ Profilers were performed in triplicate to evaluate the gene profile of cells involved in the mouse wound healing process (PAMM-121, SABioscience Corporation, Frederick, MD, USA). The plates were run analyzed with Applied Biosystems AB 7300 real-time PCR thermal cycler, according to the manufacturer's instructions. The expression of the genes of interest was analyzed using the data analysis portal on the SABioscence website (SABioscience Corporation, Frederick, MD, USA).
Saggio dell’anello di aorta di ratto. Rat aorta ring assay.
Il saggio dell’anello di aorta di ratto è stato effettuato sui terreni condizionati dai patch di fibroina Ad-MSC-SF e D-Ad-MSC-SF per studiare il loro potenziale angiogenico. Questo test permette di testare la capacità delle molecole prodotte dai diversi trattamenti di condizionare il processo di formazione di microvasi. I terreni condizionati sono stati prelevati durante lo scratch test con le HUVEC. Il saggio dell’anello di aorta è stato eseguito come precedentemente descritto [Nicosia RF, Ottinetti A. Growth of microvessel in serum-free matrix culture of rat aorta: a quantitative assay of angiogenesis in vitro. LAB INVEST 1990;63:115-122]. Brevemente, l'aorta dorsale è stata asportata da ratti Sprague-Dawley di 6 settimane (Charles River Laboratories, Calco, Lecco, Italia). Sono stati preparati anelli di circa 1 mm di spessore. Gli anelli sono stati posti in una soluzione di collagene preparata come descritto [Elsdale T, Bard J. Collagen substrata for study of cell behavior. J CELL BIOL. 1992;54:5539- 555l]. Circa 40 µL di soluzione di collagene sono stati collocati in ciascun pozzetto e successivamente un anello di aorta per pozzetto è stato posto nella soluzione di collagene. Le piastre sono state poi incubate in incubatore umidificato a 37°C per 30' per ottenere gelificazione del collagene. Alla fine dell’incubazione, i pozzetti sono stati riempiti con 500 µL di EBM (LONZA, Walkersville, MD, USA), terreno di controllo, o con i terreni condizionati da SF, Ad-MSC-SF e D-Ad-MSC-SF. Le piastre sono state incubate per 10-12 giorni. La quantificazione di angiogenesi è stata ottenuta prendendo fotografie al microscopio Zeiss con ingrandimenti 10x ogni tre giorni e contando il numero di microvasi nascenti dagli anelli di aorta [Nicosia RF, Ottinetti A. Growth of microvessel in serum-free matrix culture of rat aorta: a quantitative assay of angiogenesis in vitro. LAB INVEST 1990;63:115-122]. The rat aorta ring assay was performed on media conditioned by the Ad-MSC-SF and D-Ad-MSC-SF fibroin patches to study their angiogenic potential. This test allows to test the ability of the molecules produced by the different treatments to condition the process of formation of microvessels. The conditioned soils were collected during the scratch test with the HUVECs. The aorta ring assay was performed as previously described [Nicosia RF, Ottinetti A. Growth of microvessel in serum-free matrix culture of rat aorta: a quantitative assay of angiogenesis in vitro. LAB INVEST 1990; 63: 115-122]. Briefly, the dorsal aorta was excised from 6-week-old Sprague-Dawley rats (Charles River Laboratories, Calco, Lecco, Italy). Rings of approximately 1 mm thick were prepared. The rings were placed in a collagen solution prepared as described [Elsdale T, Bard J. Collagen substrata for study of cell behavior. J CELL BIOL. 1992; 54: 5539-555l]. Approximately 40 µL of collagen solution was placed in each well and subsequently one aorta ring per well was placed in the collagen solution. The plates were then incubated in a humidified incubator at 37 ° C for 30 'to obtain gelation of the collagen. At the end of the incubation, the wells were filled with 500 µL of EBM (LONZA, Walkersville, MD, USA), control medium, or with the media conditioned by SF, Ad-MSC-SF and D-Ad-MSC- SF. The plates were incubated for 10-12 days. Quantification of angiogenesis was obtained by taking Zeiss microscope photographs at 10x magnification every three days and counting the number of microvessels arising from the aorta rings [Nicosia RF, Ottinetti A. Growth of microvessel in serum-free matrix culture of rat aorta: a quantitative assay of angiogenesis in vitro. LAB INVEST 1990; 63: 115-122].
Analisi statistica. Statistic analysis.
I risultati sono stati espressi come media ± DS. Test Anova a due code è stato utilizzato per tutte le analisi statistiche ed eseguito con il software Prism GraphPad, versione 4.0. Valori di P inferiori a 0,05 sono stati considerati significativi con n = 3. Results were expressed as mean ± SD. Two-tailed Anova test was used for all statistical analyzes and performed with Prism GraphPad software, version 4.0. P values below 0.05 were considered significant with n = 3.
RISULTATI RESULTS
I patch di fibroina della seta (SF) supportano l’adesione e la crescita di cellule Ad-MSC. The silk fibroin (SF) patches support the adhesion and growth of Ad-MSC cells.
La coltura di cellule Ad-MSC ha mostrato una tipica morfologia fibroblastica e, se analizzato con tramite citometria a flusso, esprimeva marcatori delle cellule stromali mesenchimali come CD44 , CD73 , CD90 , CD105 , CD166 , mentre risultava negativo per i marcatori ematopoietici quali CD45 (Figura 12). Abbiamo poi esaminato se i patch di fibroina della seta potevano supportare l’attecchimento, la proliferazione e il mantenimento del profilo fenotipico di Ad-MSC. A tal fine cellule Ad-MSC sono state seminate e coltivate su patch SF per 7 giorni ed analizzate mediante SEM, colorazione H&E e microscopia confocale (Figura 6A-J). Al SEM, i patch SF non trattati, sembravano essere costituiti da materiale filamentoso costituito da microfibre (Figura 6A) rapidamente colonizzati dalle cellule Ad-MSC seminate (Figura 6B). I patch sono stati completamente coperti da un monostrato uniforme e compatto di Ad-MSC entro 7 giorni (Figura 6C). Questo risultato è stato osservato anche tramite la colorazione H&E (Figura 6D). La microscopia ottica confocale ha ulteriormente dimostrato che le Ad-MSC coltivate su patch SF avevano mantenuto il loro profilo fenotipico; erano positive per i markers mesenchimali CD146 (Figura 6E), CD44 (Figura 6F) e per la CD49d integrina (Figura 6G). Le cellule seminate su patch hanno confermato la loro negatività per il marcatore ematopoietico CD45 (Figura 6H). Si è ulteriormente analizzata la morfologia di patch SF dopo decellularizzazione usando acqua distillata. La colorazione H&E (Figura 6I) e l’ analisi con SEM (Figura 6J) hanno dimostrato che la decellularizzazione dei patch SF utilizzando acqua per 1 ora era molto efficace nella rimozione di tutte le Ad-MSC dai patch SF. The culture of Ad-MSC cells showed a typical fibroblastic morphology and, when analyzed with flow cytometry, expressed mesenchymal stromal cell markers such as CD44, CD73, CD90, CD105, CD166, while it was negative for hematopoietic markers such as CD45 ( Figure 12). We then examined whether the silk fibroin patches could support the engraftment, proliferation and maintenance of the phenotypic profile of Ad-MSC. For this purpose Ad-MSC cells were seeded and cultured on SF patches for 7 days and analyzed by SEM, H&E staining and confocal microscopy (Figure 6A-J). At SEM, the untreated SF patches appeared to consist of filamentous material consisting of microfibers (Figure 6A) rapidly colonized by seeded Ad-MSC cells (Figure 6B). The patches were completely covered with a uniform and compact monolayer of Ad-MSC within 7 days (Figure 6C). This result was also observed via H&E staining (Figure 6D). Confocal light microscopy further demonstrated that Ad-MSCs grown on SF patches had retained their phenotypic profile; they were positive for the mesenchymal markers CD146 (Figure 6E), CD44 (Figure 6F) and for the integrin CD49d (Figure 6G). Patch seeded cells confirmed their negativity for the hematopoietic marker CD45 (Figure 6H). The morphology of SF patches after decellularization was further analyzed using distilled water. H&E staining (Figure 6I) and SEM analysis (Figure 6J) showed that the decellularization of SF patches using water for 1 hour was very effective in removing all Ad-MSCs from SF patches.
Ad-MSC-SF e D-Ad-MSC-SF migliorano la guarigione della cute in topi diabetici db/db. Ad-MSC-SF and D-Ad-MSC-SF improve skin healing in db / db diabetic mice.
Topi diabetici db/db sono stati utilizzati per esperimenti in vivo. Ciascun topo ha subito una lesione cutanea di 5 mm con uno strumento per biopsia sterile di 5 mm con una procedura chirurgica. Diabetic db / db mice were used for in vivo experiments. Each mouse sustained a 5 mm skin lesion with a sterile 5 mm biopsy instrument with a surgical procedure.
La cicatrizzazione della ferita è stata misurata mediante analisi planimetrica e ha rivelato un forte incremento di guarigione delle ferite in scaffold di fibroina della seta cellularizzati ma anche in quelli decellularizzati (Figure 7 A-C). In particolare, la cicatrizzazione delle ferite al giorno post-operatorio 3 era 9,5% ± 0,5 nel gruppo di controllo, 11,5 ± 0,8% nel gruppo di SF, 49% ± 0,8 nel gruppo D-Ad-MSC SF e 50% ± 2,4 nel gruppo Ad-MSC-SF. Wound healing was measured by planimetric analysis and revealed a strong increase in wound healing in cellularized but also decellularized silk fibroin scaffolds (Figures 7 A-C). In particular, wound healing at postoperative day 3 was 9.5% ± 0.5 in the control group, 11.5 ± 0.8% in the SF group, 49% ± 0.8 in the D- group. Ad-MSC SF and 50% ± 2.4 in the Ad-MSC-SF group.
La valutazione della riduzione della superficie della ferita postoperatoria al giorno 10 era il 50% ± 2,4 in nessun gruppo di trapianto, il 48% ± 1.9 nel gruppo SF, 94% ± 2,9 in gruppo D-Ad-MSC-SF e il 99,84% ± 1,9 nel gruppo Ad-MSC-SF gruppo (Figura 7A). L'area della ferita è stata misurata utilizzando un righello metrico che è stato posizionato adiacente alla ferita, come mostrato nella Figura 7B. In Figura 7C sono mostrate le fotografie indicanti la cinetica della guarigione delle ferite in tutti i gruppi. Assessment of postoperative wound surface reduction at day 10 was 50% ± 2.4 in no transplant group, 48% ± 1.9 in the SF group, 94% ± 2.9 in the D-Ad-MSC-SF group and 99.84% ± 1.9 in the Ad-MSC-SF group (Figure 7A). The wound area was measured using a metric ruler that was positioned adjacent to the wound as shown in Figure 7B. Photographs showing the kinetics of wound healing in all groups are shown in Figure 7C.
Una epitelizzazione quasi completa della ferita è stata raggiunta al giorno postoperatorio 10 in topi trattati sia con scaffold cellularizzati di fibroina della seta che scaffold decellularizzati di fibroina della seta, mentre erano necessari 15-17 giorni nei gruppi controllo e in topi con SF innestate. L'esame istologico ha mostrato che la guarigione delle ferite nel controllo e nei topi trattati con SF è stata ritardata rispetto ai topi trattati con Ad-MSC-SF e D-Ad-MSC-SF. Tuttavia, dopo 14 giorni di trattamento, i topi trattati con Ad-MSC-SF mostravano un grado superiore di organizzazione tissutale non solo in confronto con quelli trattati solo con SF, ma anche con quelli che hanno ricevuto patch di D-Ad-MSC-SF. Infatti, nei topi trattati con Ad-MSC-SF, l’epidermide e le fibre di collagene apparivano ben riorganizzate ed erano presenti anche nuovi follicoli piliferi. Inoltre, è stato osservato anche un incremento significativo della densità microvascolare (Figura 13). Almost complete wound epithelialization was achieved at postoperative day 10 in mice treated with both cellularized silk fibroin scaffolds and decellularized silk fibroin scaffolds, while 15-17 days were required in the control groups and in mice with grafted SF. Histological examination showed that wound healing in the control and SF-treated mice was delayed compared to Ad-MSC-SF and D-Ad-MSC-SF-treated mice. However, after 14 days of treatment, Ad-MSC-SF-treated mice exhibited a higher degree of tissue organization not only in comparison with those treated with SF only, but also with those who received patches of D-Ad-MSC- SF. In fact, in mice treated with Ad-MSC-SF, the epidermis and collagen fibers appeared well reorganized and new hair follicles were also present. In addition, a significant increase in microvascular density was also observed (Figure 13).
Ad-MSC-SF e D-Ad-MSC-SF migliorare l'espressione di geni coinvolti nella angiogenesi in vivo. Ad-MSC-SF and D-Ad-MSC-SF enhance the expression of genes involved in angiogenesis in vivo.
Durante la rigenerazione della ferita in vivo, un campione di topo è stato sacrificato, i tessuti rimossi al sito di trapianto e analizzati per determinare i geni coinvolti nell'angiogenesi, nell’adesione cellulare, nella la produzione della matrice extracellulare (ECM) e nell’infiammazione. Il profilo genico analizzato con gli Rt profiler di Ad-MSC-SF e D-Ad-MSC-SF rispetto ai trapianti di SF è mostrato in Figure 8A e 8B. In sintesi, in topi trattati con Ad-MSC-SF, abbiamo trovato una significativa up-modulazione di diversi geni coinvolti nell'angiogenesi e nella rigenerazione dei tessuti, come Vegfa, Egf e Pdgfa, Wnt5a, Wisp1 e Tgfβr3. Inoltre anche geni coinvolti nella deposizione della matrice extracellulare (ECM) e nel rimodellamento come Mmp2, Itgb6, Col5a2, Col5a1, Col4a1 and Tagln erano aumentati. Viceversa, abbiamo trovato una down-modulazione di geni coinvolti nella sintesi di collagene di tipo 1 e 3 (COL3A1), nell'adesione e organizzazione del citoscheletro (Itga6, Itgb1, Ctnnβ1, Actα2). Alcuni geni coinvolti nella immunomodulazione e infiammazione, come Il6st erano leggermente incrementati, mentre il fattore inibitore della migrazione dei macrofagi (MIF) era significativamente downmodulato (Figura 8A). È interessante notare che l'analisi comparativa tra topi trattati con D-Ad-MSC-SF e con SF hanno rivelato un profilo genico simile rispetto a quello osservato nei topi trattati con patch Ad-MSC-SF. In effetti, si è osservato un incremento di geni coinvolti nel processo di angiogenesi e nella rigenerazione dei tessuti (Wnt5a, Egf) e un down-modulazione di geni coinvolti nella sintesi del collagene, nel rimodellamento e nell'organizzazione del citoscheletro (Col4a1, Tagln, Itga6, Itgb1, Actα2). Tuttavia, nel trattamento con D-Ad-MSC-SF, i geni coinvolti nel processo infiammatorio sono ancora meno espressi rispetto a quelli trattati con Ad-MSC-SF. Analogamente a quelli osservati nel trattamento con Ad-MSC-SF, il MIF era fortemente down-modulato (Figura 8B). During in vivo wound regeneration, a mouse sample was sacrificed, tissues removed at the transplant site and analyzed to determine genes involved in angiogenesis, cell adhesion, extracellular matrix (ECM) production, and 'inflammation. The gene profile analyzed with the Ad-MSC-SF and D-Ad-MSC-SF Rt profilers compared to SF transplants is shown in Figures 8A and 8B. In summary, in mice treated with Ad-MSC-SF, we found a significant up-modulation of several genes involved in angiogenesis and tissue regeneration, such as Vegfa, Egf and Pdgfa, Wnt5a, Wisp1 and Tgfβr3. In addition, genes involved in extracellular matrix (ECM) deposition and remodeling such as Mmp2, Itgb6, Col5a2, Col5a1, Col4a1 and Tagln were also increased. Conversely, we found a down-modulation of genes involved in the synthesis of collagen type 1 and 3 (COL3A1), in the adhesion and organization of the cytoskeleton (Itga6, Itgb1, Ctnnβ1, Actα2). Some genes involved in immunomodulation and inflammation, such as Il6st, were slightly increased, while the macrophage migration inhibitory factor (MIF) was significantly downmodulated (Figure 8A). Interestingly, the comparative analysis between D-Ad-MSC-SF and SF-treated mice revealed a similar gene profile to that observed in Ad-MSC-SF patch treated mice. Indeed, there was an increase in genes involved in the process of angiogenesis and tissue regeneration (Wnt5a, Egf) and a down-modulation of genes involved in collagen synthesis, remodeling and organization of the cytoskeleton (Col4a1, Tagln , Itga6, Itgb1, Actα2). However, in the treatment with D-Ad-MSC-SF, the genes involved in the inflammatory process are even less expressed than in those treated with Ad-MSC-SF. Similarly to those observed in Ad-MSC-SF treatment, the MIF was strongly down-modulated (Figure 8B).
Ad-MSC-SF e D-Ad-MSC-SF migliorano la migrazione di HUVEC. Ad-MSC-SF and D-Ad-MSC-SF enhance HUVEC migration.
Dati in vivo hanno mostrato che D-Ad-MSC-SF possedevano una notevole capacità di migliorare la guarigione delle ferite in topi diabetici ed era quasi pari a quelli ottenuti con l'Ad-MSC-SF. Pertanto i patch di fibroina della seta hanno la capacità di funzionare come una spugna, intrappolando le molecole secrete da Ad-MSC in vitro che vengono successivamente rilasciate in vivo. Al fine di verificare se D-Ad-MSC-SF può rilasciare molecole che influenzano la guarigione, si sono quindi eseguiti esperimenti in vitro sulla migrazione delle HUVEC, dei fibroblasti del derma (DF) e dei cheratinociti (KC) utilizzando il saggio scratch test (uno schema rappresentativo del test è riportato in Figura 14). Come mostrato in Figura 9 (A-F) , la migrazione di HUVEC è stata notevolmente migliorata sotto l'influenza di patch di Ad-MSC-SF e di D-Ad-MSC-SF se comparata rispetto ai rispettivi patch non trattati SF o transwells contenenti solo il mezzo di controllo (Figura 9A). Anche se meno intenso, Ad-MSC-SF e D-Ad-MSC-SF inducevano una stimolazione della migrazione di fibroblasti del derma (DF) (Figura 9B), mentre, nessuno stimolo sulla migrazione di cheratinociti (KC) è stato osservato (Figura 9C). E’ interessante notare che i patch di D-Ad-MSC-SF sono stati i più efficaci nell'indurre una copertura completa della scratch area da HUVEC, che è stata completata in 48 ore (Figura 9D). Invece, Ad-MSC-SF e D-Ad-MSC-SF sono stati ugualmente efficaci e sono state necessarie 36 ore per completare la copertura dell’area scratch con DF (Figura 9E). I cheratinociti (KC) hanno completato la copertura dell’area scratch in 24 ore e non sono state osservate differenze tra i vari trattamenti (Figura 9F). In vivo data showed that D-Ad-MSC-SF possessed a remarkable ability to improve wound healing in diabetic mice and was nearly on par with those obtained with Ad-MSC-SF. Therefore silk fibroin patches have the ability to function like a sponge, trapping molecules secreted by Ad-MSC in vitro which are subsequently released in vivo. In order to verify whether D-Ad-MSC-SF can release molecules that affect healing, in vitro experiments were performed on the migration of HUVEC, dermal fibroblasts (DF) and keratinocytes (KC) using the scratch test assay. (a representative diagram of the test is shown in Figure 14). As shown in Figure 9 (A-F), the migration of HUVEC was significantly improved under the influence of Ad-MSC-SF and D-Ad-MSC-SF patches when compared to their respective untreated SF patches or transwells containing control medium only (Figure 9A). Although less intense, Ad-MSC-SF and D-Ad-MSC-SF induced stimulation of dermal fibroblast migration (DF) (Figure 9B), while, no stimulation of keratinocyte (KC) migration was observed ( Figure 9C). Interestingly, the D-Ad-MSC-SF patches were the most effective in inducing complete scratch area coverage from HUVEC, which was completed in 48 hours (Figure 9D). Instead, Ad-MSC-SF and D-Ad-MSC-SF were equally effective and it took 36 hours to complete the scratch area coverage with DF (Figure 9E). The keratinocytes (KC) completed the coverage of the scratch area in 24 hours and no differences were observed between the various treatments (Figure 9F).
Ad-MSC-SF e D-Ad-MSC-SF secernono molecole angiogeniche in terreno di coltura durante la migrazione delle cellule endoteliali. Ad-MSC-SF and D-Ad-MSC-SF secrete angiogenic molecules in culture medium during endothelial cell migration.
Per rilevare le molecole angiogeniche rilasciate da Ad-MSC-SF e D-Ad-MSC-SF durante la migrazione di cellule endoteliali da vena di cordone ombelicale umano (HUVEC), i terreni condizionati sono stati raccolti e analizzati mediante ELISA. In particolare, è stata valutata la presenza di VEGF, FGF2, TGF-beta e EGF, tutti fattori che hanno dimostrato di giocare un ruolo importante nell'angiogenesi della ferita [Kwon DS, Gao X, Liu YB,et al. Treatment with bone marrow-derived stromal cells accelerates wound healing in diabetic rats. INT WOUND J. 2008;5:453-463 -Liu Y, Dulchavsky DS, Gao X, et al. Wound repair by bone marrow stromal cells through growth factor production. J SURG RES. To detect angiogenic molecules released by Ad-MSC-SF and D-Ad-MSC-SF during migration of endothelial cells from human umbilical cord vein (HUVEC), conditioned media were collected and analyzed by ELISA. In particular, the presence of VEGF, FGF2, TGF-beta and EGF was evaluated, all factors that have been shown to play an important role in wound angiogenesis [Kwon DS, Gao X, Liu YB, et al. Treatment with bone marrow-derived stromal cells accelerates wound healing in diabetic rats. INT WOUND J. 2008; 5: 453-463 -Liu Y, Dulchavsky DS, Gao X, et al. Wound repair by bone marrow stromal cells through growth factor production. J SURG RES.
2006;136:336-341] (Figura 10 A-D). In sintesi, si è riscontrato un incremento significativo del VEGF nei terreni condizionati da Ad-MSC-SF, ma non da D-Ad-MSC-SF (Figura 10A). FGF2 è aumentata in quantità quasi equivalenti nei terreni condizionati sia di D-Ad-MSC-SF che di Ad-MSC-SF (Figura 10B) e la concentrazione di TGF-beta era quasi doppio nei terreni condizionati da D-Ad-MSC-SF rispetto a Ad-MSC-SF (Figura 10C). Tuttavia, EGF è stato trovato in piccole quantità nei terreni condizionati da Ad-MSC-SF e da D-Ad-MSC-SF e non era diverso dal terreno di controllo (Figura 10D). Inoltre, si è anche analizzato mediante RT-PCR l'espressione genica del VEGF, FGF2, TGFbeta e EGF in Ad-MSC coltivate su plastica e su patch di fibroina della seta per verificare se i patch di fibroina della seta possono alterare l'espressione di geni coinvolti nella stimolazione angiogenica. Rispetto a cellule Ad-MSC coltivate su plastica, cellule Ad-MSC-SF non hanno mostrato differenze significative in VEGF, FGF2 e TGF-beta, mentre solo l'espressione di EGF era significativamente down-modulata (Figura 15). Tutti insieme questi dati indicano che i patch di Ad-MSC-SF stimolano la migrazione di HUVEC preferenzialmente attraverso il rilascio di VEGFa e FGF2. Per contro, VEGF non viene rilasciato da D-Ad-MSC-SF che, invece, potrebbero indurre la migrazione di HUVEC attraverso il rilascio di quantità significative di FGF2 e TGF-beta. EGF non sembra essere coinvolto nella migrazione di HUVEC probabilmente perché la fibroina della seta significativamente down-modula la sua espressione su Ad-MSC. 2006; 136: 336-341] (Figure 10 A-D). In summary, a significant increase in VEGF was found in soils conditioned by Ad-MSC-SF, but not by D-Ad-MSC-SF (Figure 10A). FGF2 increased in nearly equivalent amounts in both D-Ad-MSC-SF and Ad-MSC-SF conditioned media (Figure 10B) and TGF-beta concentration was nearly double in D-Ad-MSC- conditioned media. SF versus Ad-MSC-SF (Figure 10C). However, EGF was found in small amounts in Ad-MSC-SF and D-Ad-MSC-SF conditioned media and was no different from the control medium (Figure 10D). Furthermore, the gene expression of VEGF, FGF2, TGFbeta and EGF in Ad-MSC grown on plastic and silk fibroin patches was also analyzed by RT-PCR to verify if silk fibroin patches can alter the expression. of genes involved in angiogenic stimulation. Compared to Ad-MSC cells grown on plastic, Ad-MSC-SF cells showed no significant differences in VEGF, FGF2 and TGF-beta, while only EGF expression was significantly down-modulated (Figure 15). Taken together, these data indicate that Ad-MSC-SF patches stimulate HUVEC migration preferentially through the release of VEGFa and FGF2. Conversely, VEGF is not released by D-Ad-MSC-SF which, on the other hand, could induce migration of HUVEC through the release of significant amounts of FGF2 and TGF-beta. EGF does not appear to be involved in HUVEC migration probably because silk fibroin significantly down-modulates its expression on Ad-MSC.
Ad-MSC-SF e D-Ad-MSC-SF stimolano la crescita di microvasi nel saggio dell’anello di aorta. Ad-MSC-SF and D-Ad-MSC-SF stimulate the growth of microvessels in the aorta ring assay.
Dal momento che Ad-MSC-SF e D-Ad-MSC-SF secernono fattori che stimolano la migrazione di cellule HUVEC, si è quindi valutato il potenziale angiogenico dei patch usando il saggio dell’ anello di aorta. Questo metodo ha portato a studiare la capacità delle molecole di influenzare la formazione ex vivo delle strutture microvascolari. A tal fine, i terreni condizionati sono stati raccolti durante co-coltura di Ad-MSC-SF e D-Ad-MSC-SF con HUVEC. Come mostrato in Figura 11 A-B, i trattamenti con i terreni condizionati da Ad-MSC-SF e, in misura minore da D-Ad-MSC-SF, hanno migliorato la crescita capillare. La massima stimolazione dell'angiogenesi è stata osservata il giorno 7. In questo periodo di osservazione, il numero di capillari in presenza dei terreni condizionati da Ad-MSC-SF era di 45 ± 5, in presenza di D-Ad-MSC-SF era 32 ± 8, mentre nel terreno di controllo e in SF era rispettivamente 12 ± 6 e 15 ± 4 (Figura 11A). La Figura 11B mostra le fotografie della crescita capillare da anelli di aorta nelle diverse condizioni di trattamento. Since Ad-MSC-SF and D-Ad-MSC-SF secrete factors that stimulate the migration of HUVEC cells, the angiogenic potential of the patches was then evaluated using the aorta ring assay. This method led to study the ability of molecules to influence the ex vivo formation of microvascular structures. To this end, conditioned media were collected during co-culture of Ad-MSC-SF and D-Ad-MSC-SF with HUVEC. As shown in Figure 11 A-B, treatments with Ad-MSC-SF and, to a lesser extent, D-Ad-MSC-SF conditioned media improved capillary growth. The maximum stimulation of angiogenesis was observed on day 7. In this observation period, the number of capillaries in the presence of the media conditioned by Ad-MSC-SF was 45 ± 5, in the presence of D-Ad-MSC-SF it was 32 ± 8, while in the control medium and in SF it was 12 ± 6 and 15 ± 4, respectively (Figure 11A). Figure 11B shows photographs of capillary growth from aortic rings under different treatment conditions.
Discussione Discussion
Non solo i patch di fibroina della seta sono facili da maneggiare, ma hanno anche grande resistenza meccanica, supportano l'adesione e la proliferazione cellulare, e permettono lo stoccaggio e il rilascio di fattori di crescita prodotti dalle cellule [Hofmann S, Knecht S, Langer R, et al. Cartilage-like tissue engineering using silk scaffolds and mesenchymal stem cells.TISSUE ENG. Not only are silk fibroin patches easy to handle, they also have great mechanical strength, support cell adhesion and proliferation, and allow for storage and release of cell-produced growth factors [Hofmann S, Knecht S, Langer R, et al. Cartilage-like tissue engineering using silk scaffolds and mesenchymal stem cells.TISSUE ENG.
2006;12:2729-2738 - Wang Y, Kim HJ, Vunjak-Novakovic G, et al. 2006; 12: 2729-2738 - Wang Y, Kim HJ, Vunjak-Novakovic G, et al.
Stem cell-based tissue engineering with silk biomaterials. BIOMATERIALS 2006;27:6064-6082 - Altman GH, Diaz F, Jakuba C, et al. Silk-based biomaterials. BIOMATERIALS. Stem cell-based tissue engineering with silk biomaterials. BIOMATERIALS 2006; 27: 6064-6082 - Altman GH, Diaz F, Jakuba C, et al. Silk-based biomaterials. BIOMATERIALS.
2003;24:401- 416 - Marelli B, Alessandrino A, Fare' S, et al. Compliant electrspun silk fibroin tube for small vessel bypass grafting. ACTA BIOMATERIALIA. 2010;6:4019-4026 - Wenk E, Merkle HP, Meinel L. Silk fibroin as a vehicle for drug delivery applications. J CONTROL RELEASE. 2011;150:128-141]. Per queste ragioni si sono utilizzati i patch di fibroina della seta cellularizzati SF con Ad-MSC per testare la loro capacità di migliorare la guarigione delle ferite nei topi diabetici. Inoltre, si sono esaminati gli effetti di D-Ad-MSC-SF al fine di determinare se la presenza di cellule viventi nel sito di trapianto è essenziale per evocare efficacia riparativa e se D-Ad-MSC-SF può conservare efficacia biologica nella riparazione della ferita. In sintesi si è trovato: 1) i patch di fibroina della seta seminati con Ad-MSC e analizzati mediante SEM, hanno mostrato la capacità di sostenere l'adesione e la crescita delle Ad-MSC senza alterare l'espressione dei marcatori delle cellule mesenchimali. 2) l’analisi mediante SEM ha anche dimostrato che la decellularizzazione di patch SF con H2O distillata è stata molto efficace per rimuovere completamente le cellule Ad-MSC senza alterare la struttura a maglia della fibroina della seta. 3) Il trapianto di Ad-MSC-SF coltivate per 7 giorni ha significativamente accelerato la guarigione della pelle rispetto ai patch di fibroina della seta o ai topi controllo senza innesto. 4) i patch D-Ad-MSC-SF sono anche efficaci per migliorare la guarigione, quasi come Ad-MSC-SF. 5) L'analisi del profilo genico con RT profiler durante la guarigione in topi trattati con Ad-MSC-SF o con D-Ad-MSC-SF ha mostrato un profilo molto simile nell'espressione dei geni, con un incremento di geni coinvolti nella stimolazione dell'angiogenesi e della rigenerazione tissutale. 6) In vitro, sia Ad-MSC-SF che D-Ad-MSC-SF stimolano in particolare la migrazione di HUVEC e una maggiore formazione capillare attraverso il rilascio di molecole angiogeniche come il VEGF, FGF2 e TGF-beta, mentre l’EGF non sembrava essere coinvolto. 2003; 24: 401-416 - Marelli B, Alessandrino A, Fare 'S, et al. Compliant electrspun silk fibroin tube for small vessel bypass grafting. ACTA BIOMATERIALIA. 2010; 6: 4019-4026 - Wenk E, Merkle HP, Meinel L. Silk fibroin as a vehicle for drug delivery applications. J CONTROL RELEASE. 2011; 150: 128-141]. For these reasons, SF cellularized silk fibroin patches with Ad-MSC were used to test their ability to improve wound healing in diabetic mice. Furthermore, the effects of D-Ad-MSC-SF were examined in order to determine whether the presence of living cells at the transplant site is essential to evoke restorative efficacy and whether D-Ad-MSC-SF can maintain biological efficacy in repair. of the wound. In summary it was found: 1) the silk fibroin patches seeded with Ad-MSC and analyzed by SEM, showed the ability to support the adhesion and growth of Ad-MSCs without altering the expression of the mesenchymal cell markers . 2) the analysis by SEM has also shown that the decellularization of SF patches with distilled H2O was very effective in completely removing the Ad-MSC cells without altering the knitted structure of the silk fibroin. 3) Transplantation of Ad-MSC-SF grown for 7 days significantly accelerated skin healing compared to silk fibroin patches or graft-free control mice. 4) D-Ad-MSC-SF patches are also effective for improving healing, almost like Ad-MSC-SF. 5) The analysis of the gene profile with RT profiler during healing in mice treated with Ad-MSC-SF or with D-Ad-MSC-SF showed a very similar profile in the expression of genes, with an increase of genes involved in the stimulation of angiogenesis and tissue regeneration. 6) In vitro, both Ad-MSC-SF and D-Ad-MSC-SF particularly stimulate the migration of HUVEC and increased capillary formation through the release of angiogenic molecules such as VEGF, FGF2 and TGF-beta, while the EGF did not appear to be involved.
Dati precedenti avevano dimostrato che le MSC sia applicate da sole [Kwon DS, Gao X, Liu YB, et al. Treatment with bone marrowderived stromal cells accelerates wound healing in diabetic rats. INT WOUND J. 2008;5:453-463] o associate a scaffold di fibroina della seta-Chitosano erano efficaci nell’ accelerare la guarigione delle ferite in un modello murino di lesioni dei tessuti molli o in ratti diabetici (Altman AM, Yan Y, Matthias N, et al. IFATS collection: Human adipose-derived stem cells seeded on a silk fibroin-chitosan scaffold enhance wound repair in a murine soft tissue injury model. STEM CELLS 2009;27:250-258 - Inpanya P, Faikrua A, Ounaroon A, et al. Effects of the blended fibroin/aloe gel film on wound healing in streptozotocin-induced diabetic rats. BIOMED MATER. 2012;7:035008]. Previous data had shown that MSCs are applied alone [Kwon DS, Gao X, Liu YB, et al. Treatment with bone marrowderived stromal cells accelerates wound healing in diabetic rats. INT WOUND J. 2008; 5: 453-463] or associated with silk fibroin-Chitosan scaffolds were effective in accelerating wound healing in a mouse model of soft tissue injury or in diabetic rats (Altman AM, Yan Y , Matthias N, et al. IFATS collection: Human adipose-derived stem cells seeded on a silk fibroin-chitosan scaffold enhance wound repair in a murine soft tissue injury model. STEM CELLS 2009; 27: 250-258 - Inpanya P, Faikrua A , Ounaroon A, et al. Effects of the blended fibroin / aloe gel film on wound healing in streptozotocin-induced diabetic rats. BIOMED MATER. 2012; 7: 035008].
I dati mostrati nella presente invenzione hanno notevolmente ampliato queste osservazioni mostrando per la prima volta che, non solo i patch di Ad-MSC-SF accelerano la riparazione delle ferite, ma, cosa più importante, che funziona anche la loro controparte di fibroina della seta (FS) decellularizzata. Inoltre, a differenza di altri studi, si sono utilizzati topi diabetici db/db, un modello animale sperimentale in cui la riparazione delle ferite è fortemente ritardata, simulando in questo modo una condizione patologica comunemente osservata nei pazienti diabetici [Michaels J, Churgin SS, Blechman KM, et al. db/db mice exhibit severe wound-healing impairments compared with other murine diabetic strains in a silicone-splinted excisional wound model. WOUND REPAIR REGEN. 2007;15:665-670 - Rosenberg CS. Wound healing in the patient with diabetes mellitus. NURS CLIN NORTH AM. 1990;25:247-261]. Il razionale per l'utilizzo di cellule stromali mesenchimali (MSC) in combinazione con i patch di fibroina della seta (SF) per migliorare la guarigione si basa su precedenti evidenze sperimentali che hanno mostrato che le cellule stromali mesenchimali e la fibroina della seta possono lavorare in sinergia [Marolt D, Augst A, Freed LE, et al. Bone and cartilage tissue constructs grown using human bone marrow stromal cells, silk scaffolds and rotating bioreactors. BIOMATERIALS. The data shown in the present invention greatly expanded these observations by showing for the first time that, not only do Ad-MSC-SF patches accelerate wound repair, but, more importantly, that their silk fibroin counterpart also works. (FS) decellularized. Furthermore, unlike other studies, diabetic db / db mice were used, an experimental animal model in which wound repair is highly delayed, thus simulating a pathological condition commonly observed in diabetic patients [Michaels J, Churgin SS, Blechman KM, et al. db / db mice exhibit severe wound-healing impairments compared with other murine diabetic strains in a silicone-splinted excisional wound model. WOUND REPAIR REGEN. 2007; 15: 665-670 - Rosenberg CS. Wound healing in the patient with diabetes mellitus. NURS CLIN NORTH AM. 1990; 25: 247-261]. The rationale for using mesenchymal stromal cells (MSCs) in combination with silk fibroin (SF) patches to enhance healing is based on previous experimental evidence that has shown that mesenchymal stromal cells and silk fibroin can work. in synergy [Marolt D, Augst A, Freed LE, et al. Bone and cartilage tissue constructs grown using human bone marrow stromal cells, silk scaffolds and rotating bioreactors. BIOMATERIALS.
2006;27:6138-6149 - Lanza Rp, Langer R, Vancanti J, editors. Priciples of tissue engineering. 2<nd>ed. San diego, Ca: Academic Press; 2000 - Ghezzi CE, Marelli B, Muja N, et al. Mesenchymal stem cell-seeded multilayered dense collagen-silk fibroin hybrid for tissue engineering applications. BIOTECHNOL J. 2006; 27: 6138-6149 - Lanza Rp, Langer R, Vancanti J, editors. Priciples of tissue engineering. 2 <nd> ed. San diego, CA: Academic Press; 2000 - Ghezzi CE, Marelli B, Muja N, et al. Mesenchymal stem cell-seeded multilayered dense collagen-silk fibroin hybrid for tissue engineering applications. BIOTECHNOL J.
2011;6:1198- 207]. Secondo queste pubblicazioni, gli scaffold di fibroina della seta, mimando una matrice naturale extracellulare, favoriscono l'adesione e la proliferazione di cellule stromali mesenchimali e, quando applicati alla ferita della cute, possono servire come substrato per le cellule stromali mesenchimali [Altman AM, Yan Y, Matthias N, et al. IFATS collection: Human adipose-derived stem cells seeded on a silk fibroin-chitosan scaffold enhance wound repair in a murine soft tissue injury model. STEM CELLS 2009;27:250-258]. D'altra parte, le MSC sono considerate cellule con capacità attiva di rilasciare fattori angiogenici e trofici critici per la corretta rigenerazione della ferita [Kwon DS, Gao X, Liu YB,et al. Treatment with bone marrowderived stromal cells accelerates wound healing in diabetic rats. INT WOUND J. 2008;5:453-463 - Liu Y, Dulchavsky DS, Gao X, et al. Wound repair by bone marrow stromal cells through growth factor production. J SURG RES. 2006;136:336-341 - Watson SL, Marcal H, Sarris M, et al. The effect of mesenchymal stem cell conditioned media on corneal stromal fibroblast wound healing activities. BR J OPHTALMOL. 2010;94:1067-1073 - Mishra PJ, Mishra PJ, Banerjee D, et al. Cell-free derivatives from mesenchymal stem cells are effective in wound therapy. WORLD J STEM CELLS. 2012;4:35-43] o in grado di differenziarsi in linee cellulari differenti [Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. SCIENCE. 1999;284:143-147] tra cui cellule del derma [Nie C, Yang D, Xu J, et al. Locally administered adipose-derived stem cells accelerate wound healing through differentiation and vasculogenesis. CELL TRANSPLANT. 2011;20:205-216 - Altman AM, Yan Y, Matthias N, et al. IFATS collection: Human adiposederived stem cells seeded on a silk fibroin-chitosan scaffold enhance wound repair in a murine soft tissue injury model. STEM CELLS 2009;27:250-258 - Wu Y, Chen L, Scott PG, et al. Mesenchymal stem cells enhance wound healing through differentiation and angiogenesis. STEM CELLS. 2007;25:2648-2659]. A questo proposito, si è chiaramente dimostrato, per la prima volta, che i patch di fibroina della seta decellularizzati ottenuti con una semplice procedura utilizzando H2O distillata sufficiente per eliminare tutte le cellule, mantenevano l’ efficacia per accelerare la guarigione. Pertanto, in accordo con le osservazioni precedenti [Watson SL, Marcal H, Sarris M, et al. The effect of mesenchymal stem cell conditioned media on corneal stromal fibroblast wound healing activities. BR J OPHTALMOL. 2011; 6: 1198-207]. According to these publications, silk fibroin scaffolds, mimicking a natural extracellular matrix, promote adhesion and proliferation of mesenchymal stromal cells and, when applied to the skin wound, can serve as a substrate for mesenchymal stromal cells [Altman AM, Yan Y, Matthias N, et al. IFATS collection: Human adipose-derived stem cells seeded on a silk fibroin-chitosan scaffold enhance wound repair in a murine soft tissue injury model. STEM CELLS 2009; 27: 250-258]. On the other hand, MSCs are considered cells with an active capacity to release angiogenic and trophic factors critical for proper wound regeneration [Kwon DS, Gao X, Liu YB, et al. Treatment with bone marrowderived stromal cells accelerates wound healing in diabetic rats. INT WOUND J. 2008; 5: 453-463 - Liu Y, Dulchavsky DS, Gao X, et al. Wound repair by bone marrow stromal cells through growth factor production. J SURG RES. 2006; 136: 336-341 - Watson SL, Marcal H, Sarris M, et al. The effect of mesenchymal stem cell conditioned media on corneal stromal fibroblast wound healing activities. BR J OPHTALMOL. 2010; 94: 1067-1073 - Mishra PJ, Mishra PJ, Banerjee D, et al. Cell-free derivatives from mesenchymal stem cells are effective in wound therapy. WORLD J STEM CELLS. 2012; 4: 35-43] or capable of differentiating into different cell lines [Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. SCIENCE. 1999; 284: 143-147] including dermal cells [Nie C, Yang D, Xu J, et al. Locally administered adipose-derived stem cells accelerate wound healing through differentiation and vasculogenesis. CELL TRANSPLANT. 2011; 20: 205-216 - Altman AM, Yan Y, Matthias N, et al. IFATS collection: Human adiposederived stem cells seeded on a silk fibroin-chitosan scaffold enhance wound repair in a murine soft tissue injury model. STEM CELLS 2009; 27: 250-258 - Wu Y, Chen L, Scott PG, et al. Mesenchymal stem cells enhance wound healing through differentiation and angiogenesis. STEM CELLS. 2007; 25: 2648-2659]. In this regard, it was clearly demonstrated, for the first time, that the decellularized silk fibroin patches obtained with a simple procedure using enough distilled H2O to eliminate all the cells, maintained their effectiveness to accelerate healing. Therefore, in agreement with the previous observations [Watson SL, Marcal H, Sarris M, et al. The effect of mesenchymal stem cell conditioned media on corneal stromal fibroblast wound healing activities. BR J OPHTALMOL.
2010;94:1067-1073 - Mishra PJ, Mishra PJ, Banerjee D, et al. Cell-free derivatives from mesenchymal stem cells are effective in wound therapy. WORLD J STEM CELLS. 2012;4:35-43], i dati della presente invenzione confermano che le cellule stromali mesenchimali (MSC) esercitano la loro efficacia nella guarigione delle ferite principalmente attraverso un meccanismo paracrino e pertanto la presenza di cellule vive sullo scaffold può non essere necessaria. I dati di Altman M et. al., suggeriscono un meccanismo di trans-differenziazione di cellule umane Ad-MSC seminate su scaffold SF-Chitosano che migliora la riparazione delle ferite [Altman AM, Yan Y, Matthias N, et al. IFATS collection: Human adipose-derived stem cells seeded on a silk fibroin-chitosan scaffold enhance wound repair in a murine soft tissue injury model. STEM CELLS 2009;27:250-258]. Tuttavia, negli esperimenti di Altman M. et. al. sono stati utilizzati topi immunodeficienti, diversi dal modello murino db/db che ha un efficiente sistema immunitario e quindi può rapidamente eliminare le cellule umane xenogeniche trapiantate. 2010; 94: 1067-1073 - Mishra PJ, Mishra PJ, Banerjee D, et al. Cell-free derivatives from mesenchymal stem cells are effective in wound therapy. WORLD J STEM CELLS. 2012; 4: 35-43], the data of the present invention confirm that mesenchymal stromal cells (MSCs) exert their efficacy in wound healing mainly through a paracrine mechanism and therefore the presence of live cells on the scaffold may not be necessary. Data from Altman M et. al., suggest a mechanism of trans-differentiation of human Ad-MSC cells seeded on SF-Chitosan scaffolds that improves wound repair [Altman AM, Yan Y, Matthias N, et al. IFATS collection: Human adipose-derived stem cells seeded on a silk fibroin-chitosan scaffold enhance wound repair in a murine soft tissue injury model. STEM CELLS 2009; 27: 250-258]. However, in the experiments of Altman M. et. to the. Immunodeficient mice were used, other than the db / db mouse model which has an efficient immune system and therefore can rapidly eliminate transplanted xenogenic human cells.
Nella presente invenzione, inoltre, il meccanismo paracrino di riparazione della ferita è stato supportata da esperimenti in vitro. In particolare, utilizzando il saggio scratch test, si è trovato che entrambi D-Ad-MSC-SF e Ad-MSC-SF sono stati in grado di stimolare la migrazione di HUVEC e, in misura minore, la migrazione DF ma non la migrazione di KC. L'analisi dei terreni condizionati prelevati dalle co-colture con HUVEC, ha rivelato che il VEGF, FGF2 e TFG-beta erano tutti significativamente aumentati nei terreni condizionati da Ad-MSC-SF, mentre, nei terreni condizionati da D-Ad-MSC-SF, FGF2 e TFG-beta sono stati rilevati, ma non VEGF. Poiché la coltura di Ad-MSC su patch SF non ha influito l’espressione di VEGF, FGF2 o TGF-beta, si è concluso che FGF2 e TGF-beta sono rimasti intrappolati all'interno dei patch di D-Ad-MSC-SF e sono stati pertanto rilasciati, mentre il VEGF non è rimasto intrappolato nello scaffold SF, o è stato intrappolato, ma non rilasciato. È ben noto che lo scaffold SF può essere caricato con farmaci o fattori di crescita attivi [Wenk E, Merkle HP, Meinel L. Silk fibroin as a vehicle for drug delivery applications. J CONTROL RELEASE. 2011;150:128-141 -Karageorgiou V, Tomkins M, Fajardo R, et al. Porous silk fibroin 3-D scaffolds for delivery of bone morphogenetic protein-2 in vitro and in vivo. J BIOMED MATER RES A. 2006;78:324- 334], tuttavia, resta ancora da chiarire se gli scaffold di fibroina della seta seminati con cellule possiedono legame capacità di legare i diversi fattori di crescita. E' anche interessante notare che si è trovato una correlazione tra il livello molto basso di EGF nei terreni condizionati e l'incapacità di Ad-MSC-SF o D-Ad-MSC-SF di stimolare la migrazione di KC. Una delle possibili spiegazioni di questo risultato può essere dedotto dalla capacità di SF di indurre una significativa down-modulazione dell'espressione genica di EGF in Ad-MSC. E' possibile che, almeno in vitro, SF possa guidare Ad-MSC verso la produzione di fattori di crescita (VEGF, TGF-beta e FGF2) che influenzano la proliferazione e la migrazione delle cellule stromali mesenchimali, piuttosto che fattori (come EGF) che influenzano maggiormente cellule neuroectodermiche come KC. Tuttavia, non è chiaro se questo è vero anche in vivo. Esperimenti effettuati usando MSC di midollo osseo, non associate a scaffold di SF, per trattare ferite in ratti diabetici hanno mostrato un aumento dell'espressione di EGF e dei fattori di crescita mesenchimali [Kwon DS, Gao X, Liu YB,et al. Treatment with bone marrow-derived stromal cells accelerates wound healing in diabetic rats. INT WOUND J. 2008;5:453-463]. In addition, in the present invention, the paracrine wound repair mechanism was supported by in vitro experiments. In particular, using the scratch test assay, both D-Ad-MSC-SF and Ad-MSC-SF were found to be able to stimulate HUVEC migration and, to a lesser extent, DF migration but not migration. by KC. Analysis of conditioned media taken from HUVEC co-cultures revealed that VEGF, FGF2 and TFG-beta were all significantly increased in Ad-MSC-SF conditioned media, while in D-Ad-MSC conditioned media. -SF, FGF2 and TFG-beta were detected, but not VEGF. Since culturing Ad-MSC on SF patches did not affect the expression of VEGF, FGF2, or TGF-beta, it was concluded that FGF2 and TGF-beta were trapped within the D-Ad-MSC-SF patches. and were therefore released, while the VEGF was not trapped in the SF scaffold, or was trapped, but not released. It is well known that the SF scaffold can be loaded with drugs or active growth factors [Wenk E, Merkle HP, Meinel L. Silk fibroin as a vehicle for drug delivery applications. J CONTROL RELEASE. 2011; 150: 128-141 - Karageorgiou V, Tomkins M, Fajardo R, et al. Porous silk fibroin 3-D scaffolds for delivery of bone morphogenetic protein-2 in vitro and in vivo. J BIOMED MATER RES A. 2006; 78: 324- 334], however, it remains to be elucidated whether cell-seeded silk fibroin scaffolds possess binding ability to bind different growth factors. It is also interesting to note that a correlation was found between the very low level of EGF in conditioned media and the inability of Ad-MSC-SF or D-Ad-MSC-SF to stimulate KC migration. One of the possible explanations for this result can be deduced from the ability of SF to induce a significant down-modulation of EGF gene expression in Ad-MSC. It is possible that, at least in vitro, SF may drive Ad-MSC towards the production of growth factors (VEGF, TGF-beta and FGF2) that influence the proliferation and migration of mesenchymal stromal cells, rather than factors (such as EGF) which mostly affect neuroectodermal cells such as KC. However, it is unclear whether this is also true in vivo. Experiments using bone marrow MSCs, not associated with SF scaffolds, to treat wounds in diabetic rats showed increased expression of EGF and mesenchymal growth factors [Kwon DS, Gao X, Liu YB, et al. Treatment with bone marrow-derived stromal cells accelerates wound healing in diabetic rats. INT WOUND J. 2008; 5: 453-463].
A questo proposito il profilo genico nei topi trattati con patch di FS cellularizzati o decellularizzati almeno parzialmente sembra sostenere l’ osservazione in vitro. In sintesi, non si sono trovate molte differenze nell'espressione genica tra topi trattati con patch di Ad-MSC-SF o patch di D-Ad-MSC-SF, suggerendo che entrambi i trattamenti possono avere un meccanismo d'azione simile che probabilmente ha comportato la stimolazione dell'angiogenesi che ha portato al miglioramento del processo di cicatrizzazione. Più in particolare, simile ai dati in vitro, un incremento significativo dell’espressione della famiglia genica di FGF e di TGF-beta è stata osservata nei topi trattati con patch di D-Ad-MSC-SF e patch di Ad-MSC-SF. Inoltre, l'analisi in vivo del profilo genico ha anche rivelato alcune differenze interessanti. In particolare, nei topi trattati con Ad-MSC-SF, i geni coinvolti nel rimodellamento di ECM (Mmp2, Col5α2, Col5α1) e, forse ancora più importante, i geni coinvolti nel processo infiammatorio (Il6st) erano aumentati. Questo potrebbe indicare che l'assenza di cellule viventi xenogeniche sui patch SF nel sito di trapianto potrebbe avere un impatto minore nello stimolare infiammazione nell'ospite. In this regard, the gene profile in mice treated with cellularized or decellularized FS patches at least partially seems to support in vitro observation. In summary, not many differences in gene expression were found between mice treated with Ad-MSC-SF patch or D-Ad-MSC-SF patch, suggesting that both treatments may have a similar mechanism of action which is likely involved the stimulation of angiogenesis which led to the improvement of the healing process. More specifically, similar to the in vitro data, a significant increase in the expression of the FGF and TGF-beta gene family was observed in mice treated with D-Ad-MSC-SF patch and Ad-MSC-SF patch. . Furthermore, the in vivo analysis of the gene profile also revealed some interesting differences. In particular, in the mice treated with Ad-MSC-SF, the genes involved in ECM remodeling (Mmp2, Col5α2, Col5α1) and, perhaps more importantly, the genes involved in the inflammatory process (Il6st) were increased. This could indicate that the absence of xenogenic living cells on the SF patches at the graft site could have a minor impact in stimulating inflammation in the host.
In conclusione, i risultati della presente invenzione dimostrano che la combinazione di Ad-MSC e patch SF è efficace nell’accelerare la riparazione della ferita in un modello di topi diabetici che ha dimostrato di avere una capacità significativamente ritardata di guarigione della ferita [Michaels J, Churgin SS, Blechman KM, et al. db/db mice exhibit severe wound-healing impairments compared with other murine diabetic strains in a silicone-splinted excisional wound model. WOUND REPAIR REGEN. 2007;15:665-670]. Ancora più importante, la presente invenzione dimostra per la prima volta che un successo quasi equivalente nella riparazione delle ferite può essere ottenuto utilizzando patch di fibroina della seta decellularizzati. Entrambi i trattamenti migliorano la guarigione attraverso un meccanismo simile che coinvolge principalmente il rilascio di molecole angiogeniche e stimolanti la deposizione di collagene. La rimozione delle cellule dai patch di SF, può avere notevoli vantaggi pratici rispetto ai patch cellularizzati, tra cui ad esempio: a) la facilità di manipolazione e preparazione: 1 ora di trattamento con H2O distillata è stato sufficiente per ottenere la decellularizzazione completa dei patch, b) la possibilità di immagazzinare i patch prima dell'uso, c) il rischio ridotto di trasferire infezioni o cellule geneticamente mutate; d) i patch decellularizzati possono avere meno reattività immunologica e infiammatoria ed infine e) un notevole risparmio utilizzando uno scaffold decellularizzato per l’ applicazione clinica [Lev-Tov H, Li CS, Dahle S, et al. Cellular versus acellular matrix devices in treatment of diabetic foot ulcers: study protocol for a comparative efficacy randomized controlled trial. TRIALS. 2013; Jan 9;14:8]. La presente invenzione può pertanto rappresentare un passo importante verso il successo nel trattamento delle ulcere in pazienti diabetici. In conclusion, the results of the present invention demonstrate that the combination of Ad-MSC and SF patch is effective in accelerating wound repair in a diabetic mouse model that has been shown to have significantly delayed wound healing ability [Michaels J , Churgin SS, Blechman KM, et al. db / db mice exhibit severe wound-healing impairments compared with other murine diabetic strains in a silicone-splinted excisional wound model. WOUND REPAIR REGEN. 2007; 15: 665-670]. More importantly, the present invention demonstrates for the first time that nearly equivalent success in wound repair can be achieved using decellularized silk fibroin patches. Both treatments enhance healing through a similar mechanism that primarily involves the release of angiogenic and collagen deposition stimulating molecules. The removal of cells from SF patches can have significant practical advantages over cellularized patches, including for example: a) ease of handling and preparation: 1 hour of treatment with distilled H2O was sufficient to achieve complete decellularization of the patches , b) the possibility of storing the patches before use, c) the reduced risk of transferring infections or genetically mutated cells; d) decellularized patches may have less immunological and inflammatory reactivity and finally e) significant savings by using a decellularized scaffold for clinical application [Lev-Tov H, Li CS, Dahle S, et al. Cellular versus acellular matrix devices in treatment of diabetic foot ulcers: study protocol for a comparative efficacy randomized controlled trial. TRIALS. 2013; Jan 9; 14: 8]. The present invention can therefore represent an important step towards success in the treatment of ulcers in diabetic patients.
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| WO2005012606A2 (en) * | 2003-04-10 | 2005-02-10 | Tufts University | Concentrated aqueous silk fibroin solution and use thereof |
| US20110293666A1 (en) * | 2007-11-28 | 2011-12-01 | Xianyan Wang | Bioengineered Tissue Constructs and Methods for Production and Use |
| WO2012040310A2 (en) * | 2010-09-22 | 2012-03-29 | Virginia Commonwealth University | Preparation rich in growth factor-based fibrous matrices for tissue engineering, growth factor delivery, and wound healing |
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| WO2005012606A2 (en) * | 2003-04-10 | 2005-02-10 | Tufts University | Concentrated aqueous silk fibroin solution and use thereof |
| US20110293666A1 (en) * | 2007-11-28 | 2011-12-01 | Xianyan Wang | Bioengineered Tissue Constructs and Methods for Production and Use |
| WO2012040310A2 (en) * | 2010-09-22 | 2012-03-29 | Virginia Commonwealth University | Preparation rich in growth factor-based fibrous matrices for tissue engineering, growth factor delivery, and wound healing |
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| HONGXU LU ET AL: "Comparison of decellularization techniques for preparation of extracellular matrix scaffolds derived from three-dimensional cell culture", JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART A, 1 January 2012 (2012-01-01), pages n/a - n/a, XP055115230, ISSN: 1549-3296, DOI: 10.1002/jbm.a.34150 * |
| MAUNEY ET AL: "Engineering adipose-like tissue in vitro and in vivo utilizing human bone marrow and adipose-derived mesenchymal stem cells with silk fibroin 3D scaffolds", BIOMATERIALS, ELSEVIER SCIENCE PUBLISHERS BV., BARKING, GB, vol. 28, no. 35, 9 October 2007 (2007-10-09), pages 5280 - 5290, XP022290568, ISSN: 0142-9612, DOI: 10.1016/J.BIOMATERIALS.2007.08.017 * |
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