WO2011150482A1 - Procédés permettant d'obtenir des composites réabsorbables, composites, membrane, échafaudage et applications associées - Google Patents

Procédés permettant d'obtenir des composites réabsorbables, composites, membrane, échafaudage et applications associées Download PDF

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WO2011150482A1
WO2011150482A1 PCT/BR2011/000169 BR2011000169W WO2011150482A1 WO 2011150482 A1 WO2011150482 A1 WO 2011150482A1 BR 2011000169 W BR2011000169 W BR 2011000169W WO 2011150482 A1 WO2011150482 A1 WO 2011150482A1
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Prior art keywords
amino acid
collagen
col
hap
composite
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PCT/BR2011/000169
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English (en)
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Sybele Saska
Reinaldo Marchetto
Younès MESSADDEQ
Sidney José de Lima RIBEIRO
Ana Maria Minarelli Gaspar
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Fundação De Amparo À Pesquisa Do Estado de São Paulo
Universidade Estadual Paulista Julio De Mesquita Filho
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Priority to PCT/BR2011/000169 priority Critical patent/WO2011150482A1/fr
Publication of WO2011150482A1 publication Critical patent/WO2011150482A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1875—Bone morphogenic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • 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
    • A61L27/3637—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 origin of the biological material other than human or animal, e.g. plant extracts, algae
    • 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
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
    • A61L27/48—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with macromolecular fillers
    • 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
    • A61L27/58—Materials at least partially resorbable by the body
    • 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
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/12—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L31/125—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
    • A61L31/129—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix containing macromolecular fillers

Definitions

  • the present invention relates to novel methods of obtaining reabsorbable composites, based on bacterial cellulose and collagen, for application in tissue repairing.
  • the present invention relates to the composites obtained by the methods described herein and their uses.
  • biomaterials are fundamentally important once it is related to an improvement in quality of life, represented by an increase in life expectancy, in health in general and in the population ' s welfare. In this way, it is observed a huge effort to produce new biomaterials devices for medical application.
  • reabsorbable membranes have been used for filling of surgical failures, bone defects or protection of the lateral wall of the maxillary sinus, being performed in order not to show any rejection signs, and for others indications such as in the wounds ' protection acting as a biological curative.
  • the vegetable cellulose has been largely used in the field of biomaterials, such as hemostatic agents (Valentine, R.; Wormald, P.J.; Sindwani, R.; Advances in absorbable biomaterials and nasal packing. Otolaryngol Clin North Am. (2009) 42: 813-28), however, its production involves well-honed extraction and purification processes.
  • the bacterial cellulose is becoming an alternative to the application in the production of biomaterials, as it has numerous characteristics that make it different from the vegetable cellulose, such as higher crystallinity, purity, traction resistance, elasticity, durability, high capacity for water absorption and retention besides ultraviolet radiation absorption (Klemm, D.; Heublein, B.; Fink, H-P.; Bonn, A. Cellulose: spectacular biopolymer and sustainable raw material. Angew. Chem. Int. Ed. (2005) 44: 3358 - 3393).
  • the collagen was incorporated into the cellulose in the production process of cellulose fibers by the bacterium, which restricts the uniformity in terms of amount of collagen incorporated into cellulose fibers, which does not occur covalently.
  • the homogeneity of the composite formed is questionable due to the fact that the amount of collagen is predisposed towards the metabolism of bacteria, making it impossible to state whether there are standardization of the collagen content in the composite formed on these methodologies. This standardization is crucial, since the ultimate goal of these products is the in vivo application.
  • the present invention provides a novel method of reabsorbable composites production based on bacterial cellulose and collagen, which promotes a more effective tissue repairing, wherein the collagen is covalently incorporated into the matrix by cross-linking and homogeneously, which allows the control of the amount of collagen incorporated and consequently the increase of this protein ' s concentration inside the cellulose matrix.
  • the methodology employed by the present invention has made it possible to design a more compact and homogeneous system with the filling pores, an important characteristic for a membrane to be employed as a mechanical barrier, because will further improve the function of cell occlusion.
  • the method comprises the following steps:
  • the method may include the further step of precipitation and incorporation of the hydroxyapatite (HAp) after step (c).
  • HAp hydroxyapatite
  • the method comprises the subsequent incorporation of peptides that induce the proliferation and/or cell differentiation.
  • Another particularly preferred object of the present invention is the reabsorbable composite obtained by the methods of the present invention.
  • Another embodiment of the present invention relates to a reabsorbable composite comprising, respectively, bacterial cellulose, collagen and hydroxyapatite, and their uses in bone tissue regeneration.
  • Figure 1 shows the primary structure of peptides OGP and OGP
  • Figure 2 is a schematic representation of the Erlenmeyer flask used to culture the bacteria Acetobacter xylinum and subsequent production of hydrated bacterial cellulose pellicle.
  • Figure 3 shows the analytical-scale chromatogram of the OGP (10-14) peptide.
  • Figure 4 shows the analytical-scale chromatogram of the OGP of the OGP peptide (a - crude b - purified).
  • Figure 5 depicts SEM micrographs showing in A - BC membrane, B - BC-COL membrane, C - cross section of the BC-COL membrane.
  • Figure 6 shows the XRD diffraction patterns of (a) BC, (b) COL, (c) BC-COL with their peaks marked.
  • Figure 7 represents the vibrational spectrum in the infrared region from samples: BC (a) pure collagen (b) BC-glycine (c) and BC-COL composite (d).
  • Figure 8 represents the vibrational spectrum in the infrared region between 1000 and 1850 cm “1 obtained from samples: BC (a) pure collagen (b) BC-glycine (c) and BC-COL composite (d) showing the displacement to the lower energy region.
  • Figure 9 depicts SEM micrographs showing: A - BC membrane, B and C - nanocomposite (BC-COL)-HAp with their respective magnifications, 2.000x and 30.000x, D and E - nanocomposite (BC-COL)-HAp in cross section with their respective magnifications 7.000x and 15.000x.
  • Figure 10 illustrates the P and Ca ions mapping of the (BC-COL)- Hap composite.
  • Figure 11 depicts the TEM image of the (BC - COL)-Hap composite.
  • Figure 12 represents the XRD diffraction patterns of (a) BC, (b) COL, (c) BC-COL and (d) (BC-COL)-HAp.
  • Figure 13 represents the vibrational spectrum in the infrared T BR2011/000169
  • Figure 14 shows the epifluorescence of the osteoblastic cells derived from rat calvaria cultured on BC (A), BC-COL (B) and BC_OGP 10 '9 M (C).
  • the green fluorescence indicates actin cytoskeleton staining. It is noted smaller cell population of cultures on BC. Scale Bar: 100 pm for A-C.
  • Figure 15 shows the epifluorescence of the osteoblastic cells derived from rat calvaria cultured on BC (A), BC/COL (B), BC_OGP 10 "9 M (C), BC_OGP 10 "6 M (D), BC_OGP (10- 14) 10 "6 M (E) and BC_OGP (10-14) 10 "9 M (F).
  • the green fluorescence indicates actin cytoskeleton staining. Scale Bar: 100 pm for A-C.
  • Figure 16 shows the epifluorescence of the osteoblastic cells derived from rat calvaria cultured on (BC-COL)-HAp (A), (D) and (G), (BC- COL)-HAp_OGP 10 "9 M (B), (E) and (H) and (BC-COL)-HAp_OGP (10-14) 10 '9 M (C), (F) and (I). Green fluorescence indicates actin cytoskeleton staining and red fluorescence indicates nuclear DNA staining.
  • Figure 17 depicts the cellular proliferation of osteoblastic cells derived from rat calvaria cultured on BC and (BC-COL)-Hap membranes associated or not with growth factors, assessed at 14 and 21 days. Data are presented as mean ⁇ standard deviation.
  • Figure 18 depicts the alkaline phosphatase (ALP) activity of osteoblastic cells derived from rat calvaria cultured on BC and BC (BC-COL)- Hap membranes associated or not with growth factors, assessed at 21 days. Data are presented as mean ⁇ standard deviation.
  • ALP alkaline phosphatase
  • the present invention relates to a method of obtaining reabsorbable composites based on bacterial cellulose and collagen, which comprises the following steps:
  • the step (a) can be made by any method for obtaining bacterial cellulose known in the prior art.
  • the bacterial cellulose of the present invention is obtained by culturing the Acetobacter xylinum bacteria in static culture medium.
  • the step (b) of the method above is accomplished by modifying the surface of the bacterial cellulose matrix by esterification of an amino acid or amino acid derivative, as described by Hilpert et al. (Hilpert, K.; Winkler, D.F.H.; Hancock, R.E.W. Peptide arrays on cellulose support: SPOT synthesis, a time and cost efficient method for synthesis of large numbers of peptides in a parallel and addressable fashion. Nature Protocols. (2007) 2: 1333-1349).
  • the present invention uses a different method of esterification, which overcomes the disadvantages described above.
  • the esterification carried out uses the cabonyldiimidazole (CDI) as carboxylate-activating agents of the amino acid or amino acid derivative, whose reactivity is similar to the acid chlorides used in such technique, however, more easily manipulated.
  • CDI cabonyldiimidazole
  • N-methylimidazole N-methylimidazole
  • the NMI along with CDI, provides very good yield and side products such as carbon dioxide and imidazoles, are relatively innocuous. Furthermore, the racemization of amino acids or amino acid derivatives also tends to be minimal, due to the mild conditions of reaction, which does not occur with other esterification mechanisms. Additionally, the reaction time employed by the esterification of the present invention is shortened (maximum of 2 hours) and does not allow the formation of oligomers as in the case of the use of DMAP, which employs a reaction time of about 3 hours.
  • the amino acid or amino acid derivative in step (b) is aliphatic and nonpolar. More particularly, the amino acid is glycine (Gly) and amino acid derivative is 9- Fluorenilmethyloxicarbonyl -glycine (Fmoc-Gly).
  • step (b) After the completion of step (b), the measurement of the degree of esterification is performed by UV spectrophotometry at 290 nm. After assessing the degree of amino acid or amino acid derivative incorporation into bacterial cellulose, the following step (c) of collagen insertion is conducted.
  • the step (c) of collagen incorporation comprises the following steps:
  • c1) deprotecting the amino group from amino acid derivative incorporated into the membrane when an amino acid derivative is used; and c2) performing a covalent bond between the amino acid or amino acid derivative covalently bound to the matrix and the collagen that will be inserted, using an aqueous solution containing collagen (1 eq) and 1-ethyl-3-(3- dimethylaminopropyl) carbodiimide (4 eq) at a temperature of 0 °C to 10 °C, preferably at 4 °C.
  • the amount of collagen in the system is measured by hydroxyproline analysis using the Woessner's method (Woessner Jr., J.F. The Determination of Hydroxyproline in Tissue and Protein Samples Containing Small Proportions of this Imino Acid. Arch Biochm Bioph. 1961 ; 93:440-447).
  • the method may further comprise the additional step of precipitation and hydroxyapatite (HAp) incorporation following the step (c).
  • additional step is accomplished by:
  • the method comprises a subsequent incorporation of peptides that induce the proliferation and/or cell differentiation, wherein the composites obtained, with HAp incorporation or not, are maintained in solution containing said peptides.
  • Such peptides are incorporated into the cellulose matrix through adsorption.
  • the potential peptides to be used may have various purposes according to their primary structure, and they may be modulators or growth factors and antimicrobials.
  • the peptides concentration in solution is in the range of 10 "6 to 10 "9 M.
  • the peptides that can be used are inducers of cellular differentiation and proliferation.
  • the peptides are OGP (osteogenic growth peptide) or OGP (10-14) (osteogenic growth peptide (10-14)).
  • the composites obtained by the method of the present invention may be subsequently:
  • the present invention relates to composites obtained by the methods described herein.
  • tissue repairing it is meant applications such healing or regeneration of any tissue type.
  • the composite of the present invention can be used to regenerate a bone tissue and repair of dermal or epidermal tissue.
  • the present invention preferably relates to use of said composite for bone tissue regeneration and tissue repair of the epidermis and dermis.
  • the present invention relates to a reabsorbable composite comprising, respectively, bacterial cellulose, collagen and hydroxyapatite, and their uses for bone tissue regeneration.
  • the bacterial cellulose contained in the composite of the invention is obtained from any bacterial source know in the art.
  • the bacterial cellulose is obtained by growing the bacteria Acetobacter xylinum in static culture medium.
  • each amino acid In the condensation step of each amino acid, it was used a molar excess compared to the initial substitution degree of 3 equivalents for each Fmoc-amino acid and 3 equivalent for each DIC and HOBT, in a DCM:DMF (1:1) mixture (DCM Synth®, and DMF Synth®). A solution of piperidine 20% (v/v) (Merck ®) in DMF was employed in the a-amino deprotecting procedure (removal of the Fmoc group).
  • the proportion of amino acids, as well as synthesis yield and peptide content of the sequences synthesized and purified were determined by amino acid analysis and mass spectrometry (ESI-MS positive mode).
  • the analytical scale high efficiency liquid chromatography was performed in a Varian ProStar instrument equipped with a C18 reverse-phase column.
  • the system comprises two model 210 pumps, ProStar 400 autosampler, ProStar 320 UV/Visible Detector model 320 and a Star integrator software, all controlled by a workstation for data manipulation and processing. All the solvents were chromatographic grade and all water used was ultrapure type.
  • the semi-preparative high efficiency liquid chromatography was performed on a Beckman System Gold equipped with a C18 reverse-phase column.
  • the system comprises two model 116 pumps, UV Visible Detector connected to a Chart Recorder from Amersham Biosciences Model: REC112. All the solvents were chromatographic grade and all water used was ultrapure type.
  • Peptides were previously weighed (approximately 1 mg) and hydrolyzed in 1 mL of 6M HCI and 160 ml_ of 5% phenol in water at 110 °C for 72 hours in a N2 atmosphere under stirring. After hydrolysis, the material was concentrated under vacuum, then dissolved in dilution buffer 0.2 M sodium citrate, pH 2.2 and filtered through Millex GV unit (0.22 pm) (Millipore ®) before injection into the HPLC system.
  • the amino acids analysis were performed by Shimadzu Liquid Chromatograph equipped with three LC-10A/C-47A pumps, one SIL 10AF autosampler, UV SPD 10A and fluorescence RF 10A detectors, employing the post-column method using ortho-phthalaldehyde (OPA).
  • OPA ortho-phthalaldehyde
  • LC/ESI liquid chromatography mass spectrometry
  • the bacterial cellulose (BC) pellicle were obtained by growing Acetobacter xylinum (overproducing strain) in static culture medium, which contained the following basic composition: 2% (w/v) glucose, 0.5% (w/v) peptone, 0.5% yeast extract, 0.27 % (w/v) and disodium phosphate anhydrous 0.115% (w/v) citric acid monohydrate.
  • the bacterial culture was performed in a 500-ml Erlenmeyer flask for 120 hours of culturing time at 28 °C (5 mm thick) ( Figure 2).
  • the cellulose was obtained in the form of a gelatinous membrane formed on the medium/surface interface.
  • the membrane has been undergone a treatment with dilute hydroxide and sodium hypochlorite solution for 30 minutes, then exhaustively washed with distilled water and stored in ultra-pure water. After several changes of ultrapure water, the membranes were autoclaved for 15 minutes at 120 °C.
  • the membranes developed were based on bacterial cellulose, type I collagen, hydroxyapatite and growth factor (Table 2).
  • BC matrix with 5 mm thickness was prepared with 13 and 25 mm diameters.
  • a solution of type I collagen from rat tail tendon (concentration 4 mg/mL) (Sigma®- Saint Louis/USA) was used for preparing highly hydrated BC matrix with 5 mm thick and 25 mm in diameter.
  • UV-VIS ultraviolet-visible spectrophotometer
  • Deprotection of the a-amino groups was performed with 20% (v/v) piperidine solution for 2 hours at room temperature was proceeded. After the deprotection-processing, successive washes with DMF were performed under a vacuum system to remove the excess of the piperidine solution, followed by a series of washes with DMF and subsequent repeated washing with ultrapure water for solvent exchange, and then the collagen incorporation to BC matrix was performed.
  • BC-COL samples thus obtained were dried in a pressed mold at 37 °C and placed in envelopes suitable to gamma radiation sterilization (25kGy sterilization dose). BC-COL samples which will be used for subsequent incorporation of growth factor were stored at 4 °C. The BC-COL samples were frozen and lyophilized for scaffolds production.
  • the collagen was initially incorporated into the BC, according to the protocols BC-COL described in item 2.2.1 , and then HAp.
  • hydroxyapatite incorporation was carried out by methods known in the art (Hutchens, A.S.; Benson, R.S; Evans, B.R.; et al. Biomimetic synthesis of calcium-deficient hydroxyapatite in a natural hydrogel. Biomaterials. 2006; 27: 4661-4670).
  • the immersion of cellulose-aa-collagen composite was performed firstly in 0.05M CaC ⁇ solution (calcium chloride) and subsequently in a 0.1 M Na2HPO4 solution (sodium hydrogen phosphate).
  • the (BC-COL)-HAp samples were dry into pressed mold at 37 °C, and the membranes thus obtained were placed in envelopes to gamma radiation sterilization (25kGy sterilization dose). The procedures for subsequent growth factors incorporation were the same as those described for BC-COL.
  • the (BC-COL)-HAp samples were frozen and lyophilized for scaffolds production.
  • the growth factor incorporation was performed by the adsorption process.
  • the peptide binds to the composite by hydrogen bonds, whose release occur with increasing ionic strength (biological conditions).
  • the concentrations used were 10 "6 and 10 "9 M.
  • Spreafico et al. Spreafico, A.; Frediani, B.; Capperucci, C; Leonini, A.; Gambera, D.; Ferrata, P.; Rosini, S.; Di Stefano, A.; Galeazzi, M.; Marcolongo, R. Osteogenic growth peptide effects on primary human osteoblast cultures: potential relevance for the treatment of glucocorticoid-induced osteoporosis. J. Cell Biochem., v. 1, p. 1007-1020, 2006), concentrations less than 10 "14 showed marked cellular proliferation, whereas the concentration of 10 "9 M improved the activity of alkaline phosphatase, and increased bone-nodule formation and mineralization.
  • samples 13 mm diameter were obtained following the protocols described above for BC, BC-COL and (BC-COL)-HAp.
  • the solutions of the respective peptides and their concentrations were prepared according to Table 3. Each sample was immersed in 5 ml_ of the respective solution for 72 hours at 10 °C.
  • the SEM micrographs were taken to verify the fine structure of the samples surface morphology.
  • the micrographs were obtained with an electron microscope model XL 30 FEG (Philips) at an accelerating voltage of 5 kV.
  • Each sample was placed in copper support, cover with a 1 nm thick layer of gold for 60 seconds, operated at 3KV and current 9.5 ⁇ . Analyses were performed at the Materials Characterization Laboratory of Department of Materials Engineering at the Federal University of Sao Carlos - SP.
  • Transmission electron microscopy analyses were performed using a FEI-Tecnai20 microscope at the CETENE-Recife/PE, operated at 200 kV.
  • the samples were previously prepared by grinding and suspending in ethanol. After homogenization of the samples, a drop of suspension was deposited on the copper support.
  • the X-rays diffraction patterns were obtained using a Kristallof lex- Siemens diffractometer with Ni filter and Cu Ka radiation at 4° to 70° to determine the crystal structure and the percentage of the crystalline fraction from samples.
  • the FTIR spectra were obtained using a 8300-SHIMADZU FT-IR spectrometer. The respective samples were previously weighed and tablets were prepared with KBr mixture (1 :100). Thirty- two scans were accumulated with a resolution of 4 cm "1 , and the results were used to identify functional groups.
  • the total collagen content in the samples was determined using the methodology described by Woessner Jr. (Woessner Jr., J.F. The Determination of Hydroxyproline in Tissue and Protein Samples Containing Small Proportions of this Imino Acid. Arch Biochm Bioph. 1961 ; 93:440-447).
  • a solution of L-hydroxyproline (Fluka ®) (5 mg in 50 mL of 0.001 M HCI) was prepared followed by several dilutions to obtain standards of concentrations between 1-5 g/2mL.
  • the OHPro oxidation in each standard was initiated by adding 1 mL of chloramine T (Sigma ®) under stirring, and the solution was left to stand for 20 minutes at room temperature.
  • the acid hydrolysis was made (6M HCI at 130 °C for 24 hours) of the respective samples: BC, BC-COL and (BC-COL)-HAp, followed by neutralization with 0.25 M NaOH in order not to exceed the concentration of 0.4 M NaCI, as this salt may inhibit the color development.
  • the OHPro content in the hydrolysed samples was determined using the protocol described above, except that after the cooling and prior to the spectrophotometric reading, the OHPro was extracted with benzene (Sigma ®). For this end, 10 ml of benzene were added to each tube, followed by stirring for 5 seconds, and the benzene layer was removed quickly. The extraction was rapidly repeated with further addition of 10ml_ benzene and centrifuged at low speed for 1 minute.
  • the absorbance of the solutions was measured with a spectrophotometer at 557nm, and a standard rate of 10% hydroxyproline (1 :10) was used to determine the total amount of collagen in the samples (Hollander, A.P.; Heathfield, T.F.; Webber, C; Iwata, Y.; Bourne, R.; Rorabeck, C; Poole, R.A. Increased damage to type II collagen in osteoarthritic articular cartilage detected by a new immunoassay. J. Clin. Invest., v. 93, p.1722-1732, 1994).
  • the cell culture selected to perform in vitro studies was a primary culture of rat calvaria osteogenic cells.
  • the following in vitro assays have been proposed as:1. Staining with DAPI-phalloidin and propidium iodide, direct fluorescence analysis that assesses cell adhesion and cell spreading on the material tested to stain the ubiquitous actin cytoskeleton and to stain the nuclear DNA, 2. Cell viability analysis evaluated by MTT colorimetric assay, 3. alkaline phosphatase (ALP) analysis.
  • ALP alkaline phosphatase
  • the statistical analysis was performed by parametric or non-parametric tests, for independent data and comparison of two or more samples (ANOVA or Kruskal-Wallis, respectively, whether or not there are normal sampling distribution and homogeneity of variances), followed by multiple comparisons test, when applicable.
  • Cells were isolated by sequential trypsin/collagenase digestion of calvarial bone from newborn (2-4 days) Wistar rats. Cells were plated on the respective membranes contained in the plates at a cell density of 2 x 10 4 cells/well, and grown for periods of 21 days in a Modified Minimum Essential Medium, with L-glutamine (a-MEM, Invitrogen ®, USA) supplemented with 10% fetal bovine serum (Invitrogen), 7 mM ⁇ -glycerophosphate (Sigma ®, USA) 50pg/mL gentamicin (Invitrogen ®), 5mg/L ascorbic acid (Sigma ®) at 37 °C in a humidified atmosphere with 5% C02.The culture medium was changed every 2- 3 days and the cell outgrowth and morphology were assessed by phase microscopy in cultures grown on polystyrene.
  • a-MEM L-glutamine
  • Invitrogen ® Invitrogen ®, USA
  • the cell adhesion and spreading were assessed by direct fluorescence using Alexa Fluor 488-conjugated phalloidin (Molecular Probes®, USA) for ubiquitous actin cytoskeleton staining; and 4',6-diamidino-2- phenylindole dihydrochloride (DAPI, Molecular Probes®) or propidium iodide for nuclear DNA staining.
  • the BC analyses were carried out for periods of 10 and 17 days, BC with respective peptides at concentrations above and BC-COL, for periods of 1 , 3 and 7 days; and BC and (BC-COL)-HAp both containing OGP or OGP (10-14) at a concentration of 10 " 9 M.
  • MTT [3-(4,5- Dimethylthiazol-2-yl)-2,5- Diphenyltetrazolium) Bromide] colorimetric assay, a tetrazolium salt that is reduced by the mitochondrial proteases, and is active only in viable cells.
  • MTT aliquots at 5mg/mL in PBS were prepared following the incubation of primary cultures with said solution at 10% in culture medium for 4 hours in a standard humidified incubator at 37°C containing 5% CO 2 /95% atmospheric air. After this period, cultures were washed with 1 ml_ of warm PBS.
  • the ALP activity was evaluated at 14 and 21 days by thymolphthalein release from thymolphthalein monophosphate, using a commercial kit following the manufacturer's instruction (Labtest Diagnostica, Belo Horizonte, MG, Brazil). Firstly, 50 ⁇ _ thymolphthalein monophosphate were mixed with 0.5 ml_ of 0.3 M diethanolamine buffer, pH 10.1 and incubated for 2 min at 37 °C.To the solution it was added to 50 ⁇ _ aliquots obtained from each well for 10 min at 37 °C. For color development, 2 ml_ of 0.09 M Na 2 C0 3 and 0.25 M NaOH were added.
  • Total protein contents were extracted from each well with 0.1 % sodium lauryl sulphate (Sigma) for 30 min and mixed 1 :1 with Lowry solution (Sigma) for 20 min at room temperature.
  • the extract was diluted in Folin and Ciocalteau's phenol reagent (Sigma) for 30 min at room temperature. Absorbance was measured at 680 nm using a spectrophotometer (Cecil CE3021 , Cambridge, UK). The total protein content was calculated based on the albumin standard curve expressed as pg/mL.
  • the growth factors have been synthesized by the solid-phase method (SPPS).
  • SPPS solid-phase method
  • the mass obtained was 720mg, which was cleaved into two equal fractions of 360mg.
  • the first was unprotected, resulting in 304mg of peptidyl-resin; the latter proceeded with the synthesis process of the OGP sequence which after deprotection resulted in 525mg of peptidyl-resin.
  • cleavage solution 94.5 % TFA, 2.5% EDT, 0.5 % Tioanisol and 2.5 % ultra-pure water
  • the precipitation of OGP and OGP (10-14) peptides from the resin was performed with cold diethyl ether (washed 6 times) and then the extraction of the respective peptides with 10% acetic acid using a 5mL polypropylene syringes systems fitted with a porous polyethylene filter attached to a vacuum system with integrated waste collection flask for each peptides extracted.
  • the crude extract of each peptide was lyophilized and the mass value obtained for OGP and OGP (10-14) were 21.7 mg and 3.7 mg, respectively.
  • the purification yield was calculated from the ratio relative to the crude peptide mass.
  • the values of purification yield for the relative purities are exemplified in Table 4.
  • the micrograph of the BC-COL composite shows that collagen filled and covered throughout the BC nanostructure surface homogeneously ( Figure 5A).
  • the methodology used to obtain the BC-COL membrane allowed to develop a more compressed system, with filling the spaces between the cellulose fibrils. This is an important characteristic for a membrane to be employed as a mechanical barrier in guided bone regeneration, because favoring even more the cell occlusion function, preventing the infiltration of mesenchymal stem cells and fibroblasts, as well as the connective tissue invagination into the defective bone more effectively.
  • Another characteristic is that the surface roughness of the membrane favors cell adhesion and spreading.
  • the X-ray diffractograms of BC, BC-COL membranes and type I collagen are depicted in Figure 6.
  • the diffraction peaks at 15° and 22.5° are characteristic of cellulose.
  • the XRD pattern for collagen resulted in a broad peak at 2 ⁇ ranging from 15° to 35°, which is a typical XRD pattern of pure collagen, showing amorphous polymer with low crystallinity (ZHANG, L.J.; FENG, X.S.; LIU, H.G.; QIAN, D.J.; ZHANG, L; YU, X.L; CUI, F.Z. Hydroxyapatite/collagen composite materials formation in simulated body fluid environment. Mater. Letters., v.
  • BC-COL membranes The typical BC crystalline phases were also observed in BC-COL membranes, but with a decrease in the intensity of the BC peaks. This decrease suggests the collagen incorporation to BC structure, conferring a more amorphous pattern to composite. Note that the BC-COL pattern does not reflect a simple mixture of BC with COL; in the XRD pattern, a change in crystal structure in the 15 th region was observed, with the emergence of a new peak around 16.8°.
  • Figure 7 shows the infrared spectra for BC samples, lyophilized pure collagen, modified bacterial cellulose and BC-COL.
  • the pure collagen presents typical bands for proteins of about
  • Figure 9 depicts the micrographs of the (BC-COL)-Hap composite.
  • the figure 9B confirms the formation of the (BC-COL)-Hap composite, indicating that the membrane surface was covered with small crystals of HAp and the collagen and HAp crystals filled and covered, homogeneously, throughout the BC porous nanostructure surface.
  • This precipitation homogeneity of HAp on the composite surface favors the improvement of cell adhesion and spreading, being an important characteristic for a membrane to be employed as a mechanical barrier in guided bone regeneration, because it has an advantage over BC-COL.
  • it promoted a better cell occlusion, preventing the infiltration of connective tissue into the defective bone.
  • the (BC- COL)-HAp system showed an increased surface roughness, promoting an improved cell adhesion and spreading of osteoblastic cells compared to BC- COL system.
  • the EDX data for (BC-COL)-Hap samples revealed a Ca/P molar ratio of about 1.33, these data regarding the octacalcium phosphate (OCP) precursor phase of biological apatite.
  • OCP octacalcium phosphate
  • the TEM images showed nanosize HAp crystals surrounding bacterial cellulose nanofibrils. It was observed crystals of about 15 to 100nm ( Figure 11). These namometric structures of HAp crystals and BC fibrils promote an improved cell adhesion and spreading. Moreover, as these crystals are namometric, they are more easily dissolved by the body and thus there is an availability of Ca 2 and P0 4 3" faster in the biomaterial/tissue interface, promoting a cell proliferation and differentiation in periods earlier than with micrometer HAp crystals with the same chemical composition of such HAp crystals of the (BC-COL)-Hap composite.
  • Figure 12 shows the XRD pattern for the (BC-COL)-Hap composite, with similar crystallinity patterns for the HAp of bone tissue. The significant peaks observed for the HAp crystals in the samples were in 26°, 29°, 32°, 40° and 46°.
  • Figure 13 shows the FT-IR (BC-COL)-Hap spectra.
  • the typical infrared bands of phosphate (PO 4 3" ) associated with the apatite structure which appeared around 1093, 1020, 570-600 cm '1 are assigned to the stretching of the (P0 4 3 ) ions (WAN, Y. Z.; HUANG, Y.; YUAN, C. D.; RAMAN, S.; ZHU, Y.;
  • Figure 16 shows the cell adhesion and proliferation at the time periods of 1 , 3 and 7 days to (BC-COL)-HAp with or without growth factor.
  • BC-COL sample of day 7
  • FIG. 16 - 1 sample of day 7 (BC-COL)-HAp_OGP(10-14) showed cells distributed throughout the surface areas forming multilayers (Fig. 16 - 1).
  • cell proliferation was quantitatively determined by the by MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5- Diphenyltetrazolium) Bromide] colorimetric assay.
  • Cell proliferation was measured by absorbance values obtained and the mean and standard deviation for each group used to construct the graph of cell proliferation ( Figure 16). The original values were tested using nonparametric Kruskal-Wallis statistical test for independent data (Table 6).
  • the ALP activity was measured by thymolphthalein release from the thymolphthalein monophosphate hydrolysis.
  • the ALP is an enzyme present in the membrane of mature osteoblasts, whose function is to hydrolyze phosphate groups present in organic molecules, thus releasing phosphate ions, which are necessary for the process of mineralization.
  • FIG 17 shows the ALP activity in the BC, BC_OGP 10 "9 M, BC_OGP (10-14) 10- 9 M, (BC-COL)-HAp, (BC-COL)-HAp _OGP Kr 9 M and (BC- COL)-HAp_OGP(10-14) 10 "9 M membranes.
  • the low levels of ALP activity in the other membranes may be related to the delay observed in cell adhesion and proliferation.
  • the ALP activity peak of cultures grown on BC, BC_OGP 10 "9 M, BC_OGP (10-14) 10 "9 M and (BC-COL)-HAp probably occurs in periods exceeding 21 days.
  • the composites showed a homogenous distribution of collagen and HAp crystals on the matrix of BC or BC-COL composites (in the case of crystals) with a homogeneous nanostructure via in situ synthesis at room temperature.
  • the composite (BC -COL)-HAp showed similar characteristics to natural bone, such as phase composition, crystal size and crystallinity.
  • the composites synthesized by biomimetic route promoted excellent cell proliferation and, therefore, the BC-COL and (BC-COL)-HAp composites of the present invention are useful biomaterials for tissue repair, particularly bone.
  • the present inventors conducted a study comparing two methods of activation: i) DIC/NMI used in the present invention, where activation occurs in situ, and ii) the traditional DIPCDI/DMAP normally used in the prior art, with pre-activation, in the absence of catalyst (formation of symmetrical anhydride) and subsequent reaction by adding the catalyst.
  • Reaction volume 8 mi- Amount of reagents: 1 equivalent relative to Fmoc-Gly. After the reaction and washing period, the two materials were analyzed for incorporation of Fmoc-Gly. Then, the spectrophotometric analysis was employed by assessing the absorption intensity at 290 nm after treatment of samples with piperidine.
  • CDI/NMI activation as used by the present invention.

Abstract

La présente invention concerne de nouveaux procédés permettant d'obtenir des composites réabsorbables, à base de cellulose bactérienne et de collagène, destinés à la réparation tissulaire. En outre, la présente invention concerne les composites obtenus par les procédés décrits ici ainsi que leurs applications.
PCT/BR2011/000169 2010-05-31 2011-05-30 Procédés permettant d'obtenir des composites réabsorbables, composites, membrane, échafaudage et applications associées WO2011150482A1 (fr)

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US8435552B2 (en) 2007-02-09 2013-05-07 Royal College Of Surgeons In Ireland Collagen/hydroxyapatite composite scaffold, and process for the production thereof
US9138483B2 (en) 2007-02-09 2015-09-22 Royal College Of Surgeons In Ireland Collagen/hydroxyapatite composite scaffold, and process for the production thereof
CN102641727A (zh) * 2012-04-25 2012-08-22 大连中汇达科学仪器有限公司 以离子液体为溶剂制备壳聚糖胶原生物吸附剂
CN102641728A (zh) * 2012-04-25 2012-08-22 大连中汇达科学仪器有限公司 以离子液体为溶剂制备纤维素/胶原生物吸附剂
EP2703015A1 (fr) 2012-08-29 2014-03-05 Straumann Holding AG Membrane biorésorbable
US9238090B1 (en) 2014-12-24 2016-01-19 Fettech, Llc Tissue-based compositions
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CN108498859A (zh) * 2018-03-30 2018-09-07 福州大学 一种抗菌性生物活性玻璃纳米纤维支架及其制备方法
CN108498859B (zh) * 2018-03-30 2020-11-10 福州大学 一种抗菌性生物活性玻璃纳米纤维支架及其制备方法

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