CN104841022A - Application of nanofiber membrane in preparation of rotator cuff injury treatment material - Google Patents
Application of nanofiber membrane in preparation of rotator cuff injury treatment material Download PDFInfo
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Abstract
The present invention provides application of a nanofiber membrane in preparation of a rotator cuff injury treatment material, the nanofiber membrane is an electrospinning fiber membrane to promote cell growth, the nanofiber membrane optimization preferably includes cytokines, a pharmaceutical excipient and a biodegradable polymer material, the mass ratio of cytokines to pharmaceutical excipient is (0.05-0.5) : (1000-10000), and the mass ratio of cytokines to biodegradable polymer material is (0.5-0.9) : (500-10000). The present invention provides a new use of the nanofiber membrane, the nanofiber membrane is used for preparation of the rotator cuff injury treatment material, the nanofiber membrane has good biocompatibility and biodegradability, can promote cell adhesion and regeneration, accelerates the remodeling of tendon bone area, promotes the healing of the tendon bone, can effectively promote the regeneration of the injured rotator cuff, is non-toxic, non-immunogenicity, and small in side effects, and has great significance in clinic.
Description
Technical field
The present invention relates to the application of a kind of nano fibrous membrane in the material of preparation treatment rotator cuff injury.
Background technology
Shoulder sleeve is before being covered in shoulder joint, the general name of the muscular tissue such as subscapularis, supraspinatus, infraspinatus, teres minor at upper, rear.Be positioned at below acromion and triangular muscle, be closely connected with joint capsule.The function of shoulder sleeve makes head of humerus further to glenoid cavity direction in abduction of the upper limb process, maintains head of humerus and glenoid normal anatomical relationship.Rotator cuff injury will weaken even loses this function, has a strong impact on upper limb stretch.Rotator cuff tear is one of clinical modal damage, and the limited and muscular strength of pain, shoulder joint function can be caused to decline.
Although operation skill development in recent years rapidly, the initial constant intensity at tendon-bone place is improved and makes, and the rate of tearing again after the repairing of shoulder sleeve is still very high.The age of patient, the regression of shoulder sleeve, atrophy, lipid infiltration degree, the important risk factor that rotator cuff tear degree is torn after being all considered to repairing again.These risk factors can cause self repair ability of organizing of diseased region to decline, thus become the main cause of shoulder sleeve repairing operation failure.Therefore, the tissue repairing ability strengthening shoulder sleeve becomes recent study hotspot.
In recent years, intensity and biological factor intervention about strengthening the repairing of shoulder sleeve from amechanical angle stimulate the research of the inherent healing ability of patient to have considerable progress.Somatomedin is attached to suture surface by some researchs, or is dissolved in fibrin sealant, makes it to be released to shoulder sleeve mend.Although these methods also can play a role, because somatomedin can only keep active in a short period of time, thus limit the performance of its effect.Genetic engineering provides the approach that produces relevant growth factors, but uses the safety of viral vector still to there is dispute.
Summary of the invention
The object of the invention is to overcome above-mentioned deficiency, a kind of nano fibrous membrane is applied in the material of preparation treatment rotator cuff injury, can effectively promotes tendon rapid regeneration.
The invention provides the application of a kind of nano fibrous membrane in the material of preparation treatment rotator cuff injury, wherein, described nano fibrous membrane is the electrospun fiber membrane of Promote cell's growth.
In one preferred embodiment, described nano fibrous membrane is applied to the material prepared in rotator cuff injury and promote tendon rapid regeneration.
In one preferred embodiment, the constituent of described nano fibrous membrane comprises cytokine, pharmaceutic adjuvant and degradable high polymer material, wherein, the mass ratio of cytokine and pharmaceutic adjuvant is (0.05-0.5): (1000-10000), and the mass ratio of cytokine and degradable high polymer material is (0.5-0.9): (500-10000).
Preferably, the mass ratio of cytokine and degradable high polymer material is (0.5-0.9): 1000, is more preferably (0.6-0.8): 1000.
Preferably, the mass ratio of cytokine and pharmaceutic adjuvant is 0.2:(2000-6000), be more preferably 0.2:(4000-5000).
Wherein, described cytokine is selected from one or more in basic fibroblast growth factor (bFGF), insulin-like growth factor-i, epidermal growth factor, TGF-β1 3 and platelet-derived growth factor etc.
Wherein, described pharmaceutic adjuvant is one or more in cyclodextrin, PVA and lecithin.
Wherein, described degradable high polymer material can be one or more in polyhydroxycarboxyliacid acid, polylactic-co-glycolic acid, PLA-PEG copolymer, polylactic acid-polycaprolactone co-polymer, polycaprolactone, poly phosphate, Merlon or condensing model etc., in one preferred embodiment, described degradable high polymer material is polylactic-co-glycolic acid.
Wherein, in described nano fibrous membrane, the embedding rate of bFGF is 32-40%.
Wherein, the fibre diameter of described nano fibrous membrane is 0.6-1.2 μm.
In one preferred embodiment, described nano fibrous membrane is prepared from according to following step:
Step 1, is dissolved in buffer by cytokine and pharmaceutic adjuvant, is dissolved in by degradable high polymer material in organic solvent;
Step 2, is added drop-wise to the buffer containing cytokine and pharmaceutic adjuvant in the organic solvent containing degradable high polymer material, supersound process;
Step 3, processes step 2 emulsion obtained and is prepared into nano fibrous membrane by electrostatic spinning process.
Preferably, in the buffer after step 1 processes, the concentration of alpha-cyclodextrin is 60-100mg/mL, and in organic solvent, the concentration of PLGA is 10-15wt%.
Preferably, described buffer is phosphate buffer,
Preferably, described organic solvent is chloroform.
Nano fibrous membrane described in foregoing of the present invention load can also have other drug, the medicine, anti-inflammation drugs, antibiotic etc. of such as Promote cell's growth.
The invention provides a kind of new purposes of nano fibrous membrane, be applied to prepare in the material for the treatment of rotator cuff injury, there is good biocompatibility and biological degradability, attachment and the regeneration of cell can be promoted, accelerate reinventing of tendon bone place, promote tendon symphysis, effectively can promote the regeneration after rotator cuff injury, nontoxic, non-immunogenicity, side effect is little, significant clinically.
Accompanying drawing explanation
Fig. 1 utilizes the PLGA nano fibrous membrane of growth factor-loaded bFGF to carry out rotator cuff injury to repair schematic diagram;
The PLGA micro/nano fibrous membrane material surface scan electromicroscopic photograph of Fig. 2 to be the PLGA micro/nano fibrous membrane material surface scan electromicroscopic photograph of non-load and growth factor-loaded bFGF and the transmission electron microscope photo of bi-material: a be not growth factor-loaded bFGF, b is the PLGA micro/nano fibrous membrane material surface scan electromicroscopic photograph of growth factor-loaded bFGF, c is the transmission electron microscope photo of the PLGA micro/nano fibrous membrane material of not growth factor-loaded bFGF, and d is the transmission electron microscope photo of the PLGA micro/nano fibrous membrane material of growth factor-loaded bFGF;
Fig. 3 is the cumulative release rate curve that the electro spinning nanometer fiber membrane PLGA of load bFGF discharges bFGF in vitro;
The stereoscan photograph that Fig. 4 is HDFs after the PLGA nano fibrous membrane surface of growth factor-loaded bFGF cultivates 5 days and different time points cell counting curve: a are the stereoscan photograph of HDFs after the PLGA nano fibrous membrane surface of growth factor-loaded bFGF cultivates 5 days, b is the stereoscan photograph of HDFs after PLGA nano fibrous membrane surface cultivates 5 days, and c is different time points cell counting curve;
Fig. 5 is that different time points is drawn materials and organized HE coloration result: in Fig. 5 B, the material that arrows is not degradable; Triangular sign is shown as fibrocyte, and circle indicates blood vessel;
Fig. 6 is tendon-bone interface sarranine-fast green dyeing (A) and different dye areal analysis (B);
Fig. 7 is tendon-bone interface sirius red dyeing (A) and gray value analysis (B);
Fig. 8 is biomechanics test analysis (tendon cross-sectional area A, peak load B, rigidity C, limit stress D).
Detailed description of the invention
With reference to the accompanying drawings, the present invention is further illustrated in conjunction with specific embodiments, to understand the present invention better.
One, the preparation of nano fibrous membrane
A) material
Polylactic acid-glycollic acid is made up of lactide and Acetic acid, hydroxy-, bimol. cyclic ester ring-opening polymerization method, take stannic chloride as catalyst.Dichloromethane and chloroform are purchased from chemical reagents corporation of Chinese Medicine group; Alpha-cyclodextrin (being applicable to cell culture, >=98%) is provided by Sigma-Aldrich Co., Ltd; BFGF and bFGF Elisa assay kit is provided by R & D company.Other top grade pure chemistry reagent are all purchased from Chinese pharmaceutical agent company limited (China, Shanghai).
B) manufacture of the PLGA fibrous membrane of bFGF is loaded with
By bFGF(100 μ L, 20 μ g/ml) be dissolved in 0.6ml and contain (PBS, 50mM, pH7.4) in the phosphate buffer of 76mg/ml cyclodextrin.The solution of this 0.7ml is made an addition to (w/v, 1gPLGA add 7ml chloroform) in the solution of 12.5%PGLA/ chloroform, then under ice bath, carry out ultrasonic Treatment (VC505, Sonics & Materials Inc., Newtown, CT).Obtained emulsion is prepared into bFGF-PLGA nano fibrous membrane by electrostatic spinning process.HV generator is as the configuration of electrospinning processes, and voltage sets is in 10-25 kilovolt, and the 1ml syringe that emulsion is 0.6mm metal thin tube by then outlet diameter adds with the flow of 3ml/h, and flow micro-injection pump controls.
1gPLGA is dissolved in 4g dichloromethane (DCM) and 2g dimethyl formamide (DMF) and prepares electrostatic spinning PLGA solution, be prepared into PLGA nano fibrous membrane by electrostatic spinning process.
The equal lyophilizing of all fiber mats spends the night to remove residual solution and water, then to leave in 4 DEG C of exsiccators for future use.
Two, test and detection method
1, the physical features of nano fibrous membrane
Scanning electron microscope (FEI Quanta 200, Eindhoven, Netherl) is utilized to observe the morphological feature of nano fibrous membrane.Utilize transmission electron microscope (TEM; JEM-2100F, Joel, Japan) observe the structure of nano fibrous membrane.
Utilize Kruss GmbH DSA 100 Mk 2 clinometer to measure the water contact angle of (hamburger, Germany) nano fibrous membrane, then utilize sessile drop method to process image with DSA1.8 software.
The strip that fibrous membrane is cut to 70.0mm × 7.0mm × 0.6mm by us carrys out test mechanical strength.Uniaxial tensile force is detected by multifunctional pull tester (Instron 5567, Norwood, MA).The load-deformation curve of fibrous membrane is at 0.5mm/s(n=3) pulling force speed under record stress deformation curve and obtain.The Young's modulus of fibrous membrane, tensile strength and fracture time increasing degree all pushed away by load-deformation curve.
2, the extracorporeal releasing test of bFGF
The emulsion electrospinning PLGA fibrous membrane suspension Anker TGL-16C centrifuge being loaded with bFGF being dissolved in dichloromethane under the rotating speed of 12000rpm centrifugal five minutes, abandoning supernatant.After the dichloromethane evaporation remained, add 1ml phosphate buffer (PBS, pH7.4), utilize bFGF Elisa assay kit to detect the amount of bFGF in solution.Embedding rate (%)=(P/Pt) × 100, P are the actual albumen quality recording embedding; Pt is the albumen quality embedded in theory.
Release conditions under the physiological environment that we utilize phosphate buffer (PBS, pH7.4) to simulate in human body in vitro.Fibrous membrane is incubated with 150rpm air bath concussion case at 37 DEG C after putting into the release bottle that 1ml PBS is housed.Collect float when predetermined time interval, then add isopyknic fresh PBS.The bFGF content of electrospun fiber membrane utilizes Elisa assay kit to measure.
3, cell assay in vitro
We utilize human body dermal fibroblast (HDFs) to detect the behavior of cell on electrospinning fibre support.The separate tissue that the hypertrophic cicatrix tissue from people that fibroblast is provided by Shanghai Communications University the 9th the People's Hospital plastic surgery obtains obtains, and is then inoculated on the cell culture medium containing hyclone, penicillin (100 units/ml) and streptomycin (100 μ g/ml).Then 5%CO is contained constant
2couveuse in cultivate.Culture medium is changed once for every three days.Forth generation HDF fibroblast is adopted to evaluate.
The electrospun fiber membrane of sterilizing is cut into diameter be the discoid of 5mm and prewet 2 hours with cell culture medium.Then HDFs suspension (50ml, 3 × 10
4cell/ml) be seeded to film surface, transfer on the culture dish (Costar, Corning, NY, USA) in 96 holes.Subsequently the support inoculated is contained 5%CO at 37 DEG C
2couveuse in hatch 4 hours, then in each hole, add 100ml culture medium, and two days change once.
Stereoscan photograph is shooting in 1,3 and 5 day after inoculation.Now HDFs to be attached on film and to have started hypertrophy.The fibrous membrane obtained is rinsed 2 times under the PBS solution of 1 times of concentration, under 4 DEG C of conditions, to be then fixed in 4% glutaraldehyde 2 hours.Then dewater gradually under graded ethanol, with distilled water rinsing three times.Observe under scanning electron microscope after dry sample metal spraying plated film.
The increment situation of HDF is checked by Cell counting Kit (CCK-8, Dojindo Laboratories, Kumamoto, Japan) after starting to hatch 3,6 or 9 days.After removing culture matrix, rinse cell-scaffold sample twice with PBS.Then according to description introduction, in every increment product, 100 μ l culture mediums and 10 μ lCCK-8 reagent are added, at 37 DEG C containing 5%CO
2couveuse in preserve 2.5 hours.Subsequently, get 100 μ l sample liquid and be transferred on 96 hole culture dishs, measure the change of light absorption value at 450 nm by microplate reader.All tests all repeat three times above.
4, the foundation of chronic rotator cuff injury model
144 Sprague-Dawley(SD are taken in this experiment) rat (during acquisition, body weight is about 350-400 gram).Operation technique completes according to the rule of operation delivered.In one-stage operation, under general anesthesia state, the supraspinatus of left for rat shoulder is separated from its humerus end stop.Under lateral position, outside rat left scapuloanterior (LScA), do a vertical shape skin incision, blunt separation triangular muscle.When shoulder joint adduction, after stretch and slight inward turning, appear the stop of supraspinatus at the large joint knot of humerus.At stop nearside 3mm place with 5-0 prolene suture (Ethicon) labelling supraspinatus.Under the tractive of suture, tendon stop is cut off from the sharp property of shoulder sleeve gap infraspinatus stop leading edge.Finally, along myofibrillar direction, stringer is carried out to supraspinatus forward-and-rearward adhesion organization and loosen, make a call to three unijunctions to recognize position when repairing at the stump of tendon.After operating position muscle and skin layer-by-layer suture, rat is placed in cage, unrestricted motion.Postoperative three days every 12 hours according to rat body weight give analgesic (oral dipyrone, subcutaneous give and BUP).After performing the operation three weeks, rat is divided into three groups at random.In matched group, tendon stump is directly retightened in footprint district (wearing bone fixation) (n=48).At experimental group, while carrying out osseous tunnel repairing to 48 rats, simple PLGA film is utilized to strengthen; Strengthen with the PLGA fibrous membrane being loaded with bFGF for other 48.When the damage of reparation chronic degenerative, the scar tissue that first will be formed is cut, and exposes the suture also free tendon of labelling.Footprint district remaining soft tissue is carefully struck off and slight decorticating with #15 blade.Before and after tendon-bone stop, edge 22G syringe needle makes chiasma type tunnel.Be through suture on tendon through osseous tunnel, be strongly fixed in the metaphyseal cortex of humerus, thus complete the Anatomical Reconstruction to supraspinatus footprint district stop.The rat of matched group does not utilize fibrous membrane to strengthen.In experimental group, PLGA or bFGF-PLGA fibrous membrane is covered and repairs position, form the multi-layer compound structure of a sticking patch-tendon.Closure of wound subsequently and post surgery treatment identical with one-stage operation.
5, histologic analysis
The the 2nd, 4 and 8 week after surgery, sentence the analysis of euthanasia for tectology to often organizing eight rats respectively.After conventional postmortem, tissue samples is fixed in the neutral buffered formalin of 10%, carries out decalcification process, then use paraffin embedding with Immunocal (Decal, Congers, NewYork).Sagittal plane along the supraspinatus repaired and greater tubercle is cut into slices to tissue, and thickness is 5 microns.Hematoxylin-eosin dyeing, sarranine-fast green dyeing, sirius red dyeing are carried out to section.
We utilize to see under an optical microscope and look into sarranine-fast green dyeing to understand the newborn fibrocartilaginous situation at tendon-synosteosis place.Digital picture Lycra DFC420C photographing unit shooting (Leica, Solms).Under amplification 50 times of conditions, manually iris out the Yi Ran district of sarranine, recycling ImageJ software (National Institutes of Health, Bethesda, Maryl) calculates the area in Yi Ran district.The different dye area of each sample with μm
2for unit.
Sirius red dyeing is used for the semiquantitative determination to collagen deposition and Maturity.By the birefringence of collagen under quantitative assay polarized light to find the difference of collagen maturation degree between different tissues.The plane of polarization of assay method for a change polarized light is until the most strength of light, and for the ease of comparing, the specimen embedded section of different group will ensure identical thickness, and light intensity measures under identical lighting condition, and has once been measured by all samples.The microphotograph of clapping is imported in Image J, digitized of going forward side by side process.Image after process is dark space (gray value is set to zero) not having the position of collagen, has the position gray scale of collagen from 1-255 assignment.In tendon position random selecting ten squares (50 μm × 50 μm) region of tendon-bone junction, measure and calculate these area grayscale values.
6, biological mechanics determining
The the 2nd, 4 and 8 week after surgery, often organize the analysis of eight rats for biomechanics.The shoulder taken off with it from animal is put in freezing-80 DEG C of refrigerators immediately and at room temperature thaws in the previous day of biological mechanics determining.The supraspinatus of humerus and upper attachment thereof is carefully separated with surrounding tissue, removes scar tissue, then belly of muscle is removed from tendon.Digital calipers is utilized to measure the cross-sectional area of tendon at humerus end stop.The instrument measuring biomechanical data needs customization to form according to use, and tendon sand paper and cyanoacrylate adhesive are fixed on screw cramp, and humerus vice is fixing to avoid setting board of skeleton end fracture.The tendon of supraspinatus is connected with 45-N strain gauge, and the other end of strain gauge is connected to a linear bearing, only allows the pulling force identical with tendon direction.Humerus pincers are fixed on linear module.Distance between humerus with Tendon forceps keeps equal when each sample measures.Setting pretension is 0.1N, and draw rate is 14 μm/s, the corresponding to approximately 0.4%strain until tendon ruptures completely, peak load during record fracture and the site of fracture.The displacement miking linear module reaching 1 μm of resolution.The displacement data of linear segment is used for the calculating of rigidity.Maximum load calculates respectively according to fracture position grouping.
7, data analysis
This experiment obtains the approval of the care of animal committee of Shanghai Communications University.When tectology and biological mechanics determining, the timing node that all operator obtain for group, tissue is not all known.
Continuous data all represents with mean ± standard deviation.One factor analysis of variance is carried out to data and compares between two by Holm-Sidak method.Set p<0.05 as having significant difference.
Three, result
1, fibrous membrane characteristic
PLGA electrospun fiber membrane and bFGF-PLGA electrospun fiber membrane are successfully prepared by emulsion electrospinning.In PLGA electrospun fiber membrane and bFGF-PLGA electrospun fiber membrane, electrospinning fibre form is shown in Fig. 2.Fibre structure in all samples is not all containing Beads, and the fiber random crosslinking of size uniformity is shaped and makes fiber present smooth outward appearance.The visible Fig. 2 of the form of fiber under transmission electron microscope (c and d).Fig. 2 d shows bFGF core space and the PLGA shell district of bFGF-PLGA electrospinning fibre, then can clearly observe the fiber alignment that in PLGA electrospun fiber membrane, PLGA is simple in Fig. 2 c.The fibre diameter of PLGA electrospun fiber membrane and bFGF-PLGA electrospun fiber membrane is respectively 1.04 ± 0.25 μm and 0.92 ± 0.26 μm.
In order to illustrate the impact of emulsion electrostatic spinning technology on electrospun fibrous surface character, we determine the water contact angle of electrostatic spinning fiber film.The water contact angle of PLGA electrospun fiber membrane and bFGF-PLGA electrospun fiber membrane is respectively 126.3 ± 3.7 ° and 123.2 ± 4.1 °.
We assess the mechanical strength of bFGF-PLGA electrospun fiber membrane by stress test.The hot strength of PLGA electrospun fiber membrane and bFGF-PLGA electrospun fiber membrane is respectively 3.33 ± 0.31Mpa and 3.18 ± 0.29MPa, and stretch modulus is then respectively 46.14 ± 4.42MPa and 39.36 ± 3.69MPa.PLGA electrospun fiber membrane and the no significant difference of bFGF-PLGA electrospun fiber membrane between tensile strength and tensile modulus (p>0.05).
2, the release in vitro data of bFGF
Emulsion electro spinning technology using cyclodextrin as the inclusion of interior aqueous phase to increase viscosity and the stable emulsion of interior aqueous phase, namely as bFGF stabilising carriers and high embedding rate is provided.The albumen embedding rate of bFGF-PLGA fibrous membrane is the load capacity of 36.26 ± 7.61%, bFGF on electrospinning fibre is 0.7252 × 10-3 ± 0.07% (w/w).
The bFGF preparation of bFGF-PLGA core configuration is shown in Fig. 3.Initial detects that the burst of 15.4% discharges for 2 days, and being then the steady release of lasting 14 days (the 2nd day to the 16th day), is the slow releasing (the 16th day to the 24th day) of lasting 8 days subsequently.Be positioned at or define outburst release close to the bFGF of fiber surface, bFGF being wrapped up and significantly can slow down initial outburst release into fiber core.The bFGF preparation of bFGF-PLGA electrospun fiber membrane is 62.2%, shows that between culture period, the activity of core-shell structure copolymer fibre structure to bFGF provides good protection.
3, the bFGF of load can promote fibroblast proliferation
The proliferative conditions of HDFs after tissue culturing plate, PLGA electrospun fiber membrane and bFGF-PLGA electrospun fiber membrane upper 1 day, 3 days and 5 days compares (Fig. 4) by we.BFGF-PLGA electrospun fiber membrane has more Growth of Cells, cellular morphology be typical fusiform (Fig. 4 a), and PLGA film observes minimum Growth of Cells (Fig. 4 b).In all observation groups, the quantity of HDFs all increases with the increase of incubation time (Fig. 4 c).Wherein the increase of PLGA electrospun fiber membrane group cell quantity is more slow, the HDFs hypoproliferation on prompting PLGA electrospun fiber membrane.But, bFGF-PLGA electrospun fiber membrane group after 5 days with 1 day after or 3 days after the HDFs difference of breeding have statistical significance (p<0.05), the fiber-loaded propagation of bFGF to HDFs of prompting PLGA has facilitation effect at a specified future date.
4, gross examination of skeletal muscle
Do not observe infection evidence under the operative region naked eyes of laboratory animal, do not stick together and contracture and cause range of motion limited yet.Tendon is repaired all as seen and bone keeps seriality during all sacrifice of animal.During postmortem, the visible essence difference of naked eyes is not observed at tendon of supraspinatus muscle and healing position.The electrospinning film of the tendon attachment footmark district visible residues of postoperative 2 weeks experimental grouies, timing node afterwards does not then observe remaining electrospinning film.
5, histologic analysis
BFGF-PLGA group adopts the PLGA micro/nano fibrous membrane material of growth factor-loaded bFGF, and PLGA group adopts PLGA micro/nano fibrous membrane material, and Control group is blank.
Cell counting and host tissue reaction:
Find (Fig. 5) at low power Microscopic observation, the fibrous membrane of experimental group finds residual film in the 2nd and the 4th week after surgery, and the fibrous membrane degradation speed of bFGF-PLGA group is faster.When the 8th week after surgery, fibrous membrane is completely degraded.Postoperative second week, high power lens is observed and is found that membrane fiber has started to absorb, the visible fibroblast of fibre gap.Particularly bFGF-PLGA group, recognizable fibrous membrane and surrounding tissue are closely crosslinked together.Graft many places performance cellular infiltration, is mainly and is dispersed in original position fascia structure fibre bundle or the fibroblast of interfascicular.4th week after surgery, fibroblast distribution is medium and arrangement lacks regular, starts there is vascularization between film and host tissue.Cell arrangement and cell counting compared with the 8th week homogeneous phase to poor.In bFGF-PLGA group, the fibrocartilage tissue of tendon dead-centre position has better same tropism.Compared with experimental group, the healing ability of matched group is more weak, and reproduction speed is all slower at each timing node.
Metachromasy:
Postoperative 2nd week, PLGA and bFGF-PLGA fibrous membrane all made tendon-synosteosis place mucopolysaccharide stained area comparatively matched group increase (matched group 354503 ± 19693 μm
2, PLGA group 407041 ± 16211 μm
2, bFGF-PLGA group 430569 ± 9109 μm
2).Compared with PLGA group, the metachromasy significance of bFGF-PLGA group raises (P=0.018).Postoperative 4th week and the 8th week, experimental group is the matched group dyeing gross area larger (matched group 402729 ± 14704 and 409909 ± 14257 μm comparatively
2, PLGA group 444898 ± 17109 and 472893 ± 12230 μm
2, bFGF-PLGA group 459096 ± 13042 and 488769 ± 11389 μm
2), no difference of science of statistics (Fig. 6) between PLGA group and bFGF-PLGA group.BFGF-PLGA shows the performance of promotion tendon-synosteosis place regenerating bone or cartilage, and the effect of especially early stage bFGF release is the most obvious.
Collagen structure:
Observe the birefringence of healing place under polarized light, the experimental group finding three timing nodes all comparatively matched group collagen content has remarkable increase (18.5 ± 0.6 gray values during matched group 2 weeks, when 4 week when 29.8 ± 0.9,8 weeks 42.1 ± 1.3; During PLGA group 2 weeks when 20.5 ± 0.9,4 weeks when 31.4 ± 0.7,8 weeks 43.8 ± 1.0; During bFGF-PLGA group 2 weeks when 22.6 ± 0.7,4 weeks when 32.7 ± 0.8,8 weeks 45.4 ± 1.2).And bFGF-PLGA group comparatively PLGA group collagen birefringence has remarkable increase (Fig. 7).The above results all points out bFGF-PLGA group to promote collage synthesis.
6, Biomechanics test
Healing place tendon cross-sectional area:
Postoperative 2 weeks, matched group compared with experimental group, cross-sectional area no difference of science of statistics (matched group 8.4 ± 0.5mm
2, PLGA group 8.6 ± 0.7mm
2, bFGF-PLGA group 9.0 ± 0.9mm
2, matched group and PLGA group P=0.866, matched group and bFGF-PLGA group P=0.307).Postoperative 4 weeks, the cross-sectional area of experimental group and matched group had significant difference (matched group 14.1 ± 1.5mm
2, PLGA group 15.7 ± 1.0mm
2, bFGF-PLGA group 16.1 ± 1.1mm
2, matched group and PLGA group P=0.046, matched group and bFGF-PLGA group P=0.008), no difference of science of statistics (P=0.836) between two experimental grouies.Postoperative 8 weeks, the cross-sectional area of experimental group and matched group had significant difference (matched group 16.6 ± 0.6mm
2, PLGA group 17.8 ± 0.9mm
2, bFGF-PLGA group 18.4 ± 0.6mm
2) (Fig. 8 A).Therefore, after 4 weeks, the increase of experimental group healing place cross-sectional area is likely the thickness additionally increased owing to implanting electrospinning film.
The peak load of supraspinatus and rigidity:
Postoperative 2 weeks, the peak load at tendon of supraspinatus muscle-synosteosis place and rigidity no difference of science of statistics (matched group 8.4 ± 0.6N and 5.5 ± 0.5N/mm, PLGA group, 8.7 ± 0.9N and 5.7 ± 0.7N/mm, bFGF/PLGA9.1 ± 0.9N and 6.1 ± 0.6N/mm) between three groups.Postoperative 4 weeks, experimental group compares matched group its tendon of supraspinatus muscle-peak load at synosteosis place and the increase of rigidity has statistical significance (matched group 18.3 ± 1.0N and 8.0 ± 0.8N/mm, PLGA group 20.7 ± 1.6N and 9.4 ± 0.8N/mm, bFGF-PLGA group, 21.4 ± 1.3N and 9.7 ± 0.5N/mm).No difference of science of statistics between two experimental grouies.Postoperative 8 weeks, bFGF-PLGA group is PLGA group and matched group comparatively, its tendon of supraspinatus muscle-peak load at synosteosis place and the increase of rigidity have statistical significance (matched group 27.2 ± 1.1N and 13.2 ± 0.7N/mm, PLGA group 28.4 ± 1.2N and 13.7 ± 0.7N/mm, bFGF-PLGA group 32.7 ± 1.0N and 14.9 ± 0.3N/mm) (Fig. 8 B, Fig. 8 C).After pointing out 8 weeks, the mechanical strength through tendon of supraspinatus muscle-synosteosis place that bFGF-PLGA film is strengthened significantly increases.
The limit stress of healing process of tendons point:
Postoperative 2 weeks and 4 weeks, ultimate strength no difference of science of statistics (matched group 1.00 ± the 0.03MPa and 1.31 ± 0.08MPa healed between three groups a little, PLGA group 1.00 ± 0.02MPa and 1.32 ± 0.06MPa, bFGF-PLGA group, 1.02 ± 0.02MPa and 1.33 ± 0.05MPa).Postoperative 8 weeks, bFGF-PLGA group comparatively other two groups, the limit stress of its healing point is larger, and this change has statistical significance (matched group 1.59 ± 0.02MPa, PLGA group 1.62 ± 0.03MPa, bFGF-PLGA group 1.82 ± 0.03MPa) (Fig. 8 in the chronic rotator cuff injury kposthesis of rat, compared with repairing merely, connecting healing place at tendon-bone uses bFGF-PLGA fibrous membrane can increase the intensity of healing place, increases fibrous cartilage amount and optimizes the arrangement of collagen.The regeneration of shoulder sleeve is the very promising Therapeutic Method of one to utilize bFGF-PLGA to promote), this is consistent with the result of aforementioned peak load and rigidity.
In the chronic rotator cuff injury kposthesis of rat, compared with repairing merely, connect healing place at tendon-bone and use bFGF-PLGA fibrous membrane can increase the intensity of healing place, increase fibrous cartilage amount and optimize the arrangement of collagen.The regeneration of shoulder sleeve is the very promising Therapeutic Method of one to utilize bFGF-PLGA to promote.
Be described in detail specific embodiments of the invention above, but it is just as example, the present invention is not restricted to specific embodiment described above.To those skilled in the art, any equivalent modifications that the present invention is carried out and substituting also all among category of the present invention.Therefore, equalization conversion done without departing from the spirit and scope of the invention and amendment, all should contain within the scope of the invention.
Claims (10)
1. the application of nano fibrous membrane in the material of preparation treatment rotator cuff injury, wherein, described nano fibrous membrane is the electrospun fiber membrane of Promote cell's growth.
2. application according to claim 1, is characterized in that, described nano fibrous membrane is applied to the material prepared in rotator cuff injury and promote tendon rapid regeneration.
3. application according to claim 1 and 2, it is characterized in that, the constituent of described nano fibrous membrane comprises cytokine, pharmaceutic adjuvant and degradable high polymer material, wherein, the mass ratio of cytokine and pharmaceutic adjuvant is (0.05-0.5): (1000-10000), and the mass ratio of cytokine and degradable high polymer material is (0.5-0.9): (500-10000).
4. application according to claim 1 and 2, is characterized in that, the mass ratio of cytokine and degradable high polymer material is (0.5-0.9): 1000.
5. application according to claim 1 and 2, is characterized in that, the mass ratio of cytokine and pharmaceutic adjuvant is 0.2:(2000-6000).
6. application according to claim 1 and 2, it is characterized in that, described cytokine be selected from basic fibroblast growth factor, insulin-like growth factor-i, epidermal growth factor, TGF-β1 3 and platelet-derived growth factor one or more.
7. application according to claim 1 and 2, is characterized in that, described pharmaceutic adjuvant is one or more in cyclodextrin, PVA and lecithin.
8. application according to claim 1 and 2, it is characterized in that, described degradable high polymer material is selected from one or more in polyhydroxycarboxyliacid acid, polylactic-co-glycolic acid, PLA-PEG copolymer, polylactic acid-polycaprolactone co-polymer, polycaprolactone, poly phosphate, Merlon or condensing model.
9. application according to claim 1 and 2, is characterized in that, described nano fibrous membrane is prepared from according to following step:
Step 1, is dissolved in buffer by cytokine and pharmaceutic adjuvant, is dissolved in by degradable high polymer material in organic solvent;
Step 2, is added drop-wise to the buffer containing cytokine and pharmaceutic adjuvant in the organic solvent containing degradable high polymer material, supersound process;
Step 3, processes step 2 emulsion obtained and is prepared into nano fibrous membrane by electrostatic spinning process.
10. application according to claim 8, is characterized in that, in the buffer after step 1 processes, the concentration of alpha-cyclodextrin is 60-100mg/mL, and in organic solvent, the concentration of PLGA is 10-15wt%.
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| CN113082298A (en) * | 2019-08-31 | 2021-07-09 | 深圳市立心科学有限公司 | Artificial bone composite material |
| CN114150398A (en) * | 2021-12-06 | 2022-03-08 | 北京工商大学 | Nanofiber membrane and preparation method and application thereof |
| CN114432501A (en) * | 2022-01-30 | 2022-05-06 | 上海松力生物技术有限公司 | Rotator cuff patch and preparation method thereof |
| CN114533231A (en) * | 2022-04-27 | 2022-05-27 | 杭州锐健马斯汀医疗器材有限公司 | Balloon body and preparation method and application thereof |
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