CN103394126B - Cartilage repair material and preparation method thereof - Google Patents
Cartilage repair material and preparation method thereof Download PDFInfo
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- CN103394126B CN103394126B CN201310291932.1A CN201310291932A CN103394126B CN 103394126 B CN103394126 B CN 103394126B CN 201310291932 A CN201310291932 A CN 201310291932A CN 103394126 B CN103394126 B CN 103394126B
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Abstract
The invention relates to a cartilage repair material and a preparation method thereof. The material mainly comprises a protection layer, a stand layer and an enrichment layer, wherein the protection layer is attached to the surface of the stand layer, and the enrichment layer is arranged on the other surface of the stand layer. The protection layer is made of silk fibroin; the stand layer is of a membrane structure which is prepared by an original animal pericardium or a peritoneum and has a natural rough surface and a smooth surface; the enrichment layer is made of sol prepared by II type collagen, and a plurality of porous channels penetrating through the overall enrichment layer are arranged on the enrichment layer. Compared with the prior art, the prepared cartilage repair material is used for repairing cartilage injury by matching the stimulation of subchondral bone marrow, and can be applied to large-area cartilage injury, the repair of the cartilage injury can be completed through one operation, and the secondary operation of autologous chondrocyte transplantation technology and donor site complications can be solved and avoided; by matching the application of bone marrow stimulation technology, the curative effect can be remarkably improved through the material, so that the cartilage injury can be repaired into hyalinized cartilage tissues.
Description
Technical field
The invention belongs to tissue engineering biomaterials art, be specifically related to a kind ofly stimulate cartilage repair material of use in conjunction and preparation method thereof with subchondral bone bone marrow.
Background technology
Articular surface is covered with layer of transparent cartilage, and wherein more than 95% is cartilage matrix components, has the effect of dispersion and buffering pressure at right angle and shearing force, only has the chondrocyte of 5% to be dispersed in substrate, maintains matrix components balance.Because articular surface hyaline cartilage tissue has the functional character that impassivity, avascular anatomical features and buffering bear human pressure, thus its self-regeneration and regeneration capacity very poor.And articular cartilage defect is a class common disease, the cartilage injury that the reason such as wound, inflammation, tumor, regression causes, disappearance very common, often show as intractable pain, joint movement disorder, severe patient can lose function of joint, has become the one of the main reasons causing limbs maimed person and DB.
The Therapeutic Method of current clinical practice mainly comprises subchondral bone bone marrow stimulation (generally adopting microfrature) more, autologous cartilage block is transplanted, Autologous Chondrocyte is transplanted and the regenerating bone or cartilage technology etc. of autologous substrate induction.Microfrature utilizes joint lens by the debridement of cartilage damaged part, and the subchondral bone exposed punches, and enables mesenchymal stem cells MSCs arrive damage location and play a part to repair damage location.Its effect is remarkable rapidly, but effective short duration.Autologous cartilage block is transplanted to be needed to take from the healthy cartilage block in body non-weight bearing position, is applicable to less (the < 2cm of cartilage defect area
2) injury in treating, but to exist for district arthralgia, degeneration, joint instability Deng Gong district secondary disease.From reported first Autologous Chondrocyte graft application such as Brittberg in 1994 since clinical, through the development of more than ten years, Autologous Chondrocyte implantation technique experienced by repeatedly technological improvement.Autologous Chondrocyte first generation implantation technique needs to adopt autologous patient periosteum to close defect location, technology relative complex after chondrocyte suspension is transplanted to damage location, and operating time is longer, and there is periosteal proliferation, periosteum comes off, cell loss equivalent risk; Second-generation technology adopts collagen/hyaluronic acid membrane to replace Self periosteum to close defect surfaces, but still cannot avoid the risk that cell runs off; Third-generation technology directly plants chondrocyte on collagem membrane, cartilage defect is fixed on by Fibrin Glue, but still the following problem that Autologous Chondrocyte implantation technique exists cannot be solved: 1), need second operation, add misery and burden that patient bears; 2), autologous health tissues is drawn materials and can be caused for district's pain, for complication such as district's cartilage degradations; 3), because the chondrocyte life-span is short, the healthy cartilaginous tissue that gathers is limited, and many patients do not reach the cell quantity needed for transplanting and cannot completed treatment due to incubation in vitro.
Current third generation cartilage transplantation treatment technology is costly, is unfavorable for clinical expansion.First the regenerating bone or cartilage technology of autologous substrate induction was applied in 2003 by Behrens, and it covers one deck collagem membrane on the basis of microfrature, in order to provide cell attachment support and protective barrier.
Patent 98811734.7 discloses a kind of thin film of the reconstruction for body internal skeleton or cartilaginous tissue, this thin film comprise one deck be mainly II Collagen Type VI and have open spongy texture hypothallus and at least one deck have closed, relative impermeability structure barrier layer, hypothallus thickness 2 ~ 10mm, barrier layer 0.2 ~ 2mm.This collagem membrane relies on native cartilage cell progressively to complete reparation to hypothallus growth after implanting cartilaginous lesion site, but owing to lacking vascular tissue in cartilage, and chondrocyte is wrapped in matrix components, therefore chondrocyte cell migration difficulty, convalescence when causing thin film to be repaired for cartilage injury extends, and even can not complete reparation.In addition, because thickness during this film clinical practice is unadjustable, being applied to cartilage defect patient that is comparatively dark or that comprise subchondral bone defect can cause defect filling insufficient, the speed of growth faster fibrous connective tissue is first grown into the defect of depression, causes damage location neocartilage tissue thickness not enough and affects long-term effect; And be applied to the more shallow patient of cartilage injury and can cause filling excessively, cause damage location cambium loose.
Patent 200680031664.8 discloses a kind of sheet of collagen membrane material for meniscus tear, its side has smooth barrier face, this barrier surface T suppression cell adhere to thereon with T suppression cell from wherein passing through, and the fibrous face relative with smooth barrier face allows cell to grow thereon.This sheet of collagen membrane material main component is type i collagen, for meniscus tear, and articular chondrocytes epimatrix is mainly II Collagen Type VI, therefore this sheet of collagen membrane material implantation can cause the difference of local microenvironment, be unfavorable for chondrocyte growth, affect repairing effect, be not suitable for cartilage injury and repair.
To sum up, subchondral bone bone marrow stimulates is the method be most widely used in clinical treatment.But the method curative effect lasting time is short, have impact on its clinically continuation application.The regenerating bone or cartilage technology of autologous substrate induction improves the curative effect of subchondral bone bone marrow stimulation greatly.The method stimulates the same with subchondral bone bone marrow, and operation technique is easy, without the need to Cell culture invitro, is conducive to it in clinical extensive use.But due to existing film product Problems existing, cause the curative effect of the method unsatisfactory, await further improvement.
Summary of the invention
For existing treatment technology Problems existing, the object of this invention is to provide a kind of cartilage repair material and preparation method thereof, it coordinates the application of subchondral bone bone marrow stimulating technology to significantly improve curative effect, enables cartilage injury with the reparation of similar transparent sample cartilaginous tissue.
Cartilage repair material prepared by the present invention, comprises protective layer, shelf layer and enriched layer; Protective layer is bonded on the shiny surface of shelf layer by Fibrin Glue or collagen gel or hyaluronic acid derivatives, and enriched layer is combined with each other with the matsurface of shelf layer by solidifying of collagen solution.
Described protective layer, is made up of fibroin albumen, thickness 50 ~ 150 μm;
Described shelf layer, is animal derived de-cell pericardium or peritoneum, possesses natural matsurface and shiny surface, thickness 0.1 ~ 0.5mm;
Described enriched layer is gel prepared by II Collagen Type VI, thickness 1 ~ 4mm; There is the porous channel running through whole enriched layer, 50 ~ 100 μm, porous channel aperture, pitch of holes 100 ~ 150 μm.
The preparation method of the cartilage repair material of the present invention, is characterized in that, animal derived pericardium or peritoneum after de-cell, for the preparation of the shelf layer of cartilage repair material; Silk fibroin protein solution and epoxide and NaCI form fibroin albumen thin film at a certain temperature, adopt fibroin albumen thin film is bonded in cartilage repair material shelf layer as Fibrin Glue, collagen gel, hyaluronic acid derivatives shiny surface on prepare cartilage repair material protective layer; II Collagen Type VI adds transforminggrowthfactor-β1 (TGF-β 1), dexamethasone, insulin-like growth factor I (IFG-I) and nerve growth factor after dissolving, adopt as slip casting, curtain coating, scraper plate, injection, casting technology, cartilage repair material shelf layer matsurface prepares gel layer, solidify rear employing cheesing techniques and prepare porous channel at gel layer, thus obtain cartilage repair material enriched layer.Concrete steps comprise:
Prepared by step one, cartilage repair material shelf layer: animal derived pericardium or peritoneum are taken off cell by the method for document introduction, lyophilizing, obtains the de-cell biological film with natural light sliding surface and matsurface, thickness 0.1 ~ 0.5mm; Cartilage repair material shelf layer is obtained after oxirane (EO) or irradiation sterilization.
The method of this step is adopted to process submucous layer of small intestine, pericardium, peritoneum and amniotic membrane respectively; and carry out rabbit cartilage injury repairing effect contrast test respectively; found that: submucous layer of small intestine and amniotic material single monolayer thick are spent thin; easily curling when being applied to cartilaginous lesion site after rehydration; entirely can not fill damage location; and material is excessively thin, effective support and mechanical protection effect cannot be provided, cannot injury repairing be completed.And repair cartilage injury with multilamellar submucous layer of small intestine or amniotic material, because the compactness of material increases greatly, be unfavorable for cell moving into damage location, be unfavorable for injury repairing.After multilayer material rehydration, easily there is the phenomenon of local detachment between layers, cause the cartilaginous tissue repaired to occur crack, greatly affect repairing effect.By the preliminary identification of this step, pericardium, peritoneum can be used in cartilage injury and repair after taking off cell.The cartilage repair material adopting de-cell pericardium or peritoneum to prepare has natural double-decker, and side fiber alignment is comparatively fine and close, forms shiny surface; barrier protection can be provided for damage location; side fiber alignment is relatively loose, forms matsurface, can provide support for Growth of Cells.But cartilage repair material shelf layer reparation cartilage injury's effect of application preparation is also unsatisfactory.Subsequent step carries out reasonable reformation to the two sides of cartilage repair material shelf layer, to prepare the cartilage repair material being more suitable for this application respectively for produced problem in effectiveness preliminary identification process.
The preparation of step 2, cartilage repair material protective layer: preparing w/v according to literature method is in the silk fibroin protein solution of 6% ~ 10%, add the epoxide of 1% ~ 5% and the sodium chloride (NaCI) of 1% ~ 3%, after mix homogeneously, obtain fibroin albumen colloidal sol; Adopt as curtain coating, scraper plate or casting technology, prepare the film of thickness 50 ~ 150 μm, be obtained by reacting fibroin albumen thin film in 60 ~ 80 DEG C; Fibroin albumen thin film being embathed in distilled water to removing unreacted epoxide completely, obtaining the protective layer of cartilage repair material;
Adopt Fibrin Glue, collagen colloidal sol, hyaluronic acid colloidal sol protective layer to be adhered to the shiny surface of shelf layer, obtain the protective layer of cartilage repair material and the complex of shelf layer.
This step is the improvement for biomembrane material bad mechanical property.Fibroin albumen has good biocompatibility, nontoxic, nonirritant, and its catabolite itself has no side effect to tissue.Fibroin albumen and epoxide in the pliability silk extract gel compressive strength of 60 ~ 80 DEG C of reactions more than 100g/mm
2, prove that its compressive strength is good, the pressure that joint motion causes can be born, can be articular cartilage graft and mechanical protection is provided, avoid it to be subject to mechanical damage.Prepared protective layer has good pliability, can be that the mescenchymal stem cell transmission of pressure in articular cartilage graft stimulates, be conducive to it and break up to cartilage direction and secrete a large amount of extracellular matrixs, improve repairing effect.
The preparation of step 3, cartilage repair material enriched layer: the solution II Collagen Type VI being mixed with 4 ~ 8mg/mL, adds the TGF β 1,10 of 5 ~ 20ng/mL
-8~ 10
-6the nerve growth factor of the dexamethasone of mol/L, the insulin-like growth factor I of 5 ~ 50ng/mL and 7 ~ 12ng/mL, mix homogeneously, the shelf layer matsurface of the complex of above-mentioned preparation prepares the collagen layer that thickness is 1 ~ 4mm, and 37 DEG C of placements make collagen solution solidify; Adopt as mechanical punching, laser boring (Laser), electric spark high speed punching (EDM) or electrochemistry punching (ECM) technology are punched at collagen layer, 50 ~ 100 μm, aperture, pitch of holes 100 ~ 150 μm, porous channel runs through collagen layer, completes the preparation of enriched layer.Solidifying of enriched layer collagen makes the complex of enriched layer and above-mentioned preparation be bonded together.
This step is the improvement for damage location mescenchymal stem cell quantity not sufficient, when subchondral bone bone marrow stimulates in conjunction with existing biofilms material treatment cartilage injury, Chang Yinwei migrate to damage location mescenchymal stem cell limited amount and finally with the fibrous cartilage reparation that wearability is poor
.this step is by the preparation of enriched layer, after carrying out bone marrow stimulation, material is covered damage location, the marrow blood discharged can by abundant for the micropore filling of enriched layer, substantially increase the saturation of material to marrow blood, thus more mescenchymal stem cell can be obtained, the cytokine of simultaneously adding also is conducive to later stage mescenchymal stem cell and independently divides a word with a hyphen at the end of a line to damage location, is conducive to improving repairing effect.II Collagen Type VI used is consistent with the main component of normal chondrocyte epimatrix, thus provides damage location to have the microenvironment consistent with surrounding normal cartilage, is conducive to mescenchymal stem cell and breaks up to cartilage direction.
Compared with existing product, the advantage applies of cartilage repair material prepared by the present invention exists:
(1) by carrying out efficacy checking to human acellular amniotic membrane, submucous layer of small intestine, pericardium and peritoneum; result shows; de-cell pericardium, peritoneum can provide mechanical protection for damage location; and can as the support of the growth of mesenchymal stem cells of damage location; be conducive to promoting that mescenchymal stem cell is to the reparation of damage location, improves repairing effect.Its thickness is moderate, can fill cartilage defect preferably, make damage location neocartilage thickness and surrounding normal cartilage thickness basically identical, thus make defect obtain comparatively smooth repairing effect, be more suitable for for the preparation of cartilage repair material shelf layer;
(2) adopt the good fibroin albumen of biocompatibility to prepare protective layer, can, effectively for articular cartilage graft provides mechanical protection, avoid it to be subject to mechanical damage when joint motion; And prepared protective layer has good pliability, can be that mescenchymal stem cell in articular cartilage graft transmits mechanical stimulation, be conducive to its secretion to Chondrocyte Differentiation and extracellular matrix;
(3) there is thicker enriched layer, substantially increase the saturation to marrow blood, be conducive to obtaining more mescenchymal stem cell.Enriched layer II Collagen Type VI used is consistent with the main component of normal chondrocyte epimatrix, thus provides damage location to have the microenvironment close with surrounding normal cartilage, is conducive to mescenchymal stem cell to the differentiation of cartilage direction, thus obtains good repairing effect.
Compared with prior art, the cartilage repair material prepared by the present invention coordinates subchondral bone bone marrow to stimulate and is used for cartilage injury's reparation, can be applied to larger area (such as 2 ~ 8cm
2) cartilage injury, single operation can complete cartilage injury's reparation, solves the second operation problem of Autologous Chondrocyte implantation technique, it also avoid for district latter problem; Without the need to Cell culture invitro step, avoid because cell injuring model procedure failure causes completing successive treatment, the extrinsic factors such as serum, cytokine, culture medium in cell cultivation process can be avoided to introduce in body cause the side reactions such as irritated simultaneously.Coordinate the application of bone marrow stimulating technology to significantly improve curative effect, enable cartilage injury with the reparation of class transparent sample cartilaginous tissue.
Accompanying drawing explanation
Fig. 1 is the comparison picture that cartilage repair material shelf layer selects different raw material effect test.
Fig. 2 is that rabbit cartilage injury 3d draws materials the picture of result of the test.
Fig. 3 is that the cartilage repair material prepared of the present embodiment repairs the section picture of rabbit cartilage injury histologic examination in conjunction with bone marrow stimulation.
Fig. 4 is that the 12m of pig cartilage damage test draws materials result picture.
Detailed description of the invention
The LYJ-350 type small-scale test casting machine (monolayer is used) used in example is purchased from Beijing Oriental Taiyang Science Co., Ltd; XH-B200 type laser-beam drilling machine is purchased from Shenzhen Yong Shengfa automation equipment row; NK-CK153 type numerical control punching machine is purchased from Shanghai Jie Weifu mechanical & electronic equipment corporation, Ltd.
Cartilage repair material prepared by the present embodiment, matcoveredn, shelf layer and enriched layer; Protective layer is bonded on the shiny surface of shelf layer, and enriched layer is combined with each other with the matsurface of shelf layer by solidifying of collagen solution.
Described protective layer, is made up of fibroin albumen, thickness 50 ~ 150 μm;
Shelf layer, is animal derived de-cell pericardium or peritoneum, possesses natural matsurface and shiny surface, thickness 0.1 ~ 0.5mm;
Enriched layer is gel prepared by II Collagen Type VI, thickness 1 ~ 4mm; There is the porous channel running through whole enriched layer, 50 ~ 100 μm, porous channel aperture, pitch of holes 100 ~ 150 μm.
Embodiment 1, the raw-material selection of cartilage repair material shelf layer
The preparation reference Badylak SF of de-cell trees-Osima jacoti, Osima excavata, Lantz GC, Coffey A, Geddes LA.Small intestinal submucosa as a large diameter vascular graft in the dog.J Surg Res, 1989; 47:74 – 80 carries out, and the submucous layer of small intestine monolayer of acquisition and 4 stackedly adds bed board, lyophilizing, for subsequent use after EO sterilizing;
The cell free preparation method of people's amniotic membrane can list of references: Song Yongzhou, Cui Huixian, Wang Zhenxian etc. the preparation of human acellular amniotic membrane matrix and biocompatibility [J] thereof. and Chinese Tissue Engineering Study and clinical rehabilitation, 2008,12 (01): 51 ~ 55.Amniotic membrane monolayer and 8 ply bed boards after de-cell, lyophilizing, for subsequent use after EO sterilizing;
De-cell bovine pericardium and pig peritoneum reference literature: Gu Hanqing, Deng Fei, Zheng Jianming etc. the preparation research [J] of natural extracellular matrix. Chinese science and technology paper is online.The de-cell bovine pericardium of preparation and pig peritoneum monolayer bed board respectively, lyophilizing, for subsequent use after Co6025kGry sterilizing.
Choose the heavy new zealand rabbit of about 2kg, be divided into 4 groups, often organize 10, carry out human acellular amniotic membrane, submucous layer of small intestine, peritoneum and pericardium rabbit cartilage injury repairing effect contrast test respectively.Animal gets dorsal position, preserved skin after anesthesia, iodophor disinfection operative site.Get 2cm stringer otch inside knee joint, expose condyle and trochlear surface in femur, make diameter about 3mm at trochlear surface, the cartilage defect of deeply about 1mm.With the subchondral bone annular punching that Kirschner wire exposes in defect, about pitch of holes 0.9mm, hole depth is advisable to ooze out marrow blood.After abundant hemostasis, the human acellular amniotic membrane of above-mentioned preparation, submucous layer of small intestine, peritoneum and pericardium are trimmed to the circle of diameter 3mm, matsurface covers defect down respectively, and Fibrin Glue is fixed.Closed articular cavity, movable joint, confirm that material is not shifted, come off after each layer tissue of layer-by-layer suture.8w draws materials, and compares the effect that human acellular amniotic membrane, submucous layer of small intestine, peritoneum and pericardium coordinate microfrature to repair for cartilage injury.
The 8w that Fig. 1 shows the present embodiment draws materials the section result of test: when human acellular amniotic membrane (1A) prepared by conventional method for removing cells or submucous layer of small intestine (1B) are repaired for cartilage injury, cartilage injury's reparation can not be completed because thickness is too thin during monolayer application, poor to cartilage defect filling effect, not easily pave after rehydration, mechanical property is not good, does not complete cartilage injury's reparation; And 8 is stacked when adding application, because the compactness extent of material increases greatly, have impact on cell moves into, and causes harmful effect (1C) to repairing effect.During multilayer material superposition application, material combines not tight between layers simultaneously, and newborn cartilage tissue creates crack, has a strong impact on repairing effect (1D).And repair for cartilage injury after the de-cell of de-cell pig peritoneum (1E), bovine pericardium (1F), can obtain effect relatively preferably, reparation surface is essentially smooth smooth.
Embodiment 2, cartilage repair material repair rabbit cartilage injury
Prepared by step one, cartilage repair material shelf layer: pig peritoneum is taken off cell according to literature method, Liu Heli. the research [D] of the preparation and modification of natural extracellular matrix. Tianjin: Medical University Of Tianjin, 2007. obtain the de-cell pig peritoneum with natural light sliding surface and matsurface, and thickness is 0.1mm.The lyophilizing of cell pig peritoneum will be taken off, after irradiation sterilization, obtain cartilage repair material shelf layer.
The preparation of step 2, cartilage repair material protective layer: reference literature method (Min Sijia; Yao Juming; Zhu Liangjun; Ah portion's health time; temple intrinsic call. there is the physicochemical property of the fibroin protein gel of cross-linked structure. silkworm industry science; 1999,25 (2): 108-112) silk fibroin protein solution that w/v is 6% is prepared
;add the sorbitol polyglycidyl ether of 1% and the NaCI of 1%, after mix homogeneously, adopt LYJ-350 type small-scale test casting machine (monolayer is used) to prepare the thin film of thickness 50 μm according to flow casting molding technology, after reacting be set as the condition of 60 DEG C in casting machine temperature under, obtain fibroin albumen thin film; Fibroin albumen thin film is embathed 2 days in distilled water, removes unreacted sorbitol polyglycidyl ether.Obtain the protective layer of cartilage repair material.With Fibrin Glue, protective layer is adhered to the shiny surface of shelf layer, obtains the protective layer of cartilage repair material and the complex of shelf layer.
The preparation of step 3, cartilage repair material enriched layer: the solution II Collagen Type VI being mixed with 4mg/mL, adds the TGF β 1,10 of 5ng/mL
-8the nerve growth factor of the dexamethasone of mol/L, the insulin-like growth factor I of 5ng/mL and 7ng/mL, mix homogeneously.The complex shelf layer of above-mentioned preparation is upwards placed in 6 orifice plates, adds 0.95mL collagen solution, shelf layer matsurface is contacted with collagen solution.Light rolling plate makes collagen solution be laid on shelf layer matsurface, and 37 DEG C of placements make collagen solution solidify, then the collagen thickness after solidifying is 1mm.Adopt XH-B200 type laser-beam drilling machine to punch at collagen layer, 50 μm, aperture, pitch of holes 100 μm, hole depth 1mm, completes the preparation of enriched layer.The collagen set of enriched layer makes the complex of enriched layer and above-mentioned preparation be bonded together.
The position debridement of rabbit articular cartilage damage, the punching of Kirschner wire annular, pitch of holes is about 0.9mm, and hole depth is advisable to overflow marrow blood.By moulding according to damage location character for the cartilage repair material prepared, and according to the damage location degree of depth its enriched layer disappeared and cut, make cartilage graft shape thicknesses consistent with damage location.Cartilage graft enriched layer after moulding covers defect down, and Fibrin Glue is fixed.Closed articular cavity, movable joint, confirm that material is not shifted, come off after each layer tissue of layer-by-layer suture.Draw materials after 4w, gross examination of skeletal muscle and Histological section's result display, damage location neocartilage packed height is consistent with surrounding normal cartilage, and damage location has a large amount of cell to move into, and is conducive to obtaining good long-term repair effect (Fig. 3 A).
The shelf layer of the present embodiment cartilage graft adopts de-cell pig peritoneum substrate, and its structure is relatively loose, is conducive to cell and moves into, and adopts Co6025kGy to carry out sterilizing simultaneously, makes it degrade relatively very fast.Less for damaged area, lesion depths is more shallow, recover faster acute injury time, the degradation time of cartilage graft can match with the cartilage newborn time, contributes to obtaining good repairing effect.Area simultaneously owing to damaging is less or the degree of depth is more shallow, the mesenchymal stem cells MSCs quantity that enriched layer holds is relatively less, adds the cytokine of low concentration and can reach and promote mescenchymal stem cell to move in cartilage graft and to the effect of Chondrocyte Differentiation.
Fig. 2 shows the present embodiment rabbit cartilage injury 3d and to draw materials the picture of result of the test
Result shows, the present invention prepares the pressure that cartilage repair material well can bear joint motion generation, do not occur that cartilage repair material is collapsed by pressure or occurs the phenomenon of fold, and damage location smooth surface is smooth, be combined closely with surrounding normal cartilage, articular cartilage repair materials prepared by proof has good compressive strength and pliability, be conducive to come off (2A) that avoid later stage activity to cause, and de-cell pig peritoneum 3d prepared by conventional method is when drawing materials, demarcate obviously with surrounding tissue, and affect by joint early ambulant, there is bulge phenomenon local, easily cause coming off of repair materials, affect repairing effect (2B).
Fig. 3 shows cartilage repair material prepared by the present embodiment repairs rabbit cartilage injury histologic examination section picture in conjunction with bone marrow stimulation
Rabbit cartilage injury 4w result (3A) of drawing materials shows, disappeared by the cartilage repair material prepared the present invention and cut, make it reach good filling, defect is grown into avoid fibrous connective tissue to think, the defect surfaces of reparation is smooth.When drawing materials, visible material is not degradable, and damage location has a large amount of cell migration and enters, and contributes to obtaining good long-term repair effect.Conventional de-cell peritoneum repairs rabbit cartilage injury Histological section in conjunction with bone marrow stimulation, rabbit cartilage injury 4w result of drawing materials shows (3B), cartilaginous lesion site is repaired substantially, but the cell concentration that damage location cell migration enters is obviously less, is unfavorable for obtaining desirable repairing effect.
Embodiment 3, cartilage repair material repair pig cartilage damage
The preparation of step one, cartilage repair material shelf layer: shelf layer adopts de-cell bovine pericardium preparation, bovine pericardium takes off cell list of references and carries out: Zhang Wenbin, Wu Hanjiang, Hu Guangwei, this stack of Yao, Zhou Yiqun. bovine pericardium derives preparation and physicochemical property detection [J] of membrane material. stomatology research, 2005,21 (1): 27-30. take off cell bovine pericardium has natural shiny surface and matsurface, thickness 0.4mm.The lyophilizing of cell bovine pericardium will be taken off, after EO sterilizing, obtain cartilage repair material shelf layer.
The preparation of step 2, cartilage repair material protective layer: reference literature method prepares the silk fibroin protein solution (Min Sijia of 10%; Yao Juming; Zhu Liangjun; Ah portion's health time; temple intrinsic call. there is the physicochemical property of the fibroin protein gel of cross-linked structure. silkworm industry science; 1999,25 (2): 108-112).Add the diglycidyl ether (PGDE) of 5% and the NaCI of 3%, adopt the method for injection molding after mix homogeneously, being added to special interior height is in 150 μm of stainless steel moulds, covers upper cover and is placed on 80 DEG C of baking ovens and is obtained by reacting fibroin albumen thin film.It is embathed 5d in distilled water, removes unreacted PGDE, obtain the protective layer of cartilage repair material.The shape of the shelf layer prepared according to step one by protective layer is moulding, with the collagen colloidal sol of 8mg/mL, Silk fibroin gel is adhered to the shiny surface of acellular matrix film, obtains the protective layer of cartilage repair material and the complex of shelf layer.
The preparation of step 3, cartilage repair material enriched layer: II Collagen Type VI is mixed with the solution of 8mg/mL by the acetic acid with 1 ‰, adds the TGF β 1,10 of 20ng/mL
-6the nerve growth factor of the dexamethasone of mol/L, the insulin-like growth factor I of 50ng/mL and 12ng/mL, mix homogeneously.The complex shelf layer of above-mentioned preparation is upwards placed in the glass dish of diameter 90mm, adds 25.434mL collagen solution, the matsurface of complex shelf layer is contacted with collagen solution.Light rolling plate makes collagen solution be laid on shelf layer matsurface, and 37 DEG C of placements make collagen solution solidify, then the collagen thickness after solidifying is 4mm.According to mechanical perforation techniques, adopt the collagen layer punching that NK-CK153 type numerical control punching machine is solidifying, 100 μm, aperture, pitch of holes 150 μm, punching degree of depth 4mm, completes the preparation of enriched layer.Solidifying of enriched layer collagen makes the complex of enriched layer and above-mentioned preparation be bonded together.
With the punching of Kirschner wire annular after the position debridement of pig joint injury, pitch of holes 2 ~ 3mm, hole depth is advisable to overflow marrow blood.By moulding according to damage location character for the cartilage graft prepared, and according to the damage location degree of depth its enriched layer disappeared and cut, make cartilage graft shape thicknesses consistent with damage location.Cartilage graft enriched layer after moulding covers defect down, and Fibrin Glue is fixed.Closed articular cavity, movable joint, confirm that material is not shifted, come off after each layer tissue of layer-by-layer suture.Draw materials after 12m, gross examination of skeletal muscle injured surface is repaired smooth, integrates well with surrounding normal cartilage, without obviously demarcating.Histological section's toluidine blue result display damage location neocartilage bluish violet is obviously painted, completes class hyaline cartilage sample and repairs (Fig. 4 A).
The present embodiment cartilage graft adopts de-cell bovine pericardium substrate; and protective layer and the enriched layer of preparation are all thicker; make cartilage graft mechanical performance better; degradation time in vivo is longer; also can realize filling completely to darker cartilage injury; and have the more mescenchymal stem cell of space more fully, be more suitable for that cartilage injury's area is large, lesion depths is comparatively dark, the cartilage injury of heavy burden position and the longer outmoded injury of rehabilitation duration.
The 12m that Fig. 4 shows the pig cartilage damage test of the present embodiment draws materials result picture
Result shows, cartilage repair material prepared by the present invention, by adding TGF β 1, dexamethasone, insulin-like growth factor I, Derived from Mesenchymal Stem Cells is made to be chondrocyte, and suitably II Collagen Type VI of the mechanical stimulus hyaline cartilage cell-specific that to have made chondrocyte secrete a large amount of, makes damage location complete transparent sample repair of cartilage (Fig. 4 A).And adopt bone marrow stimulation reparation cartilaginous lesion site (4B) Toluidine blue staining result display local route repair tissue to have crack, less effective, and the specific bluish violet of II Collagen Type VI not obvious, illustrate that this reparation does not complete transparent sample repair of cartilage, anti-pressure and abrasion-proof is poor, and long-term effect is bad.
Claims (2)
1. a cartilage repair material, is characterized in that, comprises protective layer, shelf layer and enriched layer; Protective layer is bonded on the shiny surface of shelf layer by Fibrin Glue or collagen gel or hyaluronic acid derivatives, and enriched layer is combined with each other with the matsurface of shelf layer by solidifying of collagen solution;
Described protective layer, is made up of fibroin albumen, thickness 50 ~ 150 μm;
Described shelf layer, is animal derived de-cell pericardium or peritoneum, possesses natural matsurface and shiny surface, thickness 0.1 ~ 0.5mm;
Described enriched layer is gel prepared by II Collagen Type VI, the transforminggrowthfactor-β1 containing 5 ~ 20ng/mL, 10
-8~ 10
-6the nerve growth factor of the dexamethasone of mol/L, the IGF-Ⅰ of 5 ~ 50ng/mL and 7 ~ 12ng/mL; Thickness 1 ~ 4mm, has the porous channel running through whole enriched layer, 50 ~ 100 μm, porous channel aperture, pitch of holes 100 ~ 150 μm.
2. the preparation method of cartilage repair material according to claim 1, it is characterized in that, concrete steps, method comprise:
Prepared by step one, cartilage repair material shelf layer: animal derived pericardium or peritoneum are carried out de-cell, lyophilizing, obtains the de-cell biological film with natural light sliding surface and matsurface, thickness 0.1 ~ 0.5mm; Cartilage repair material shelf layer is obtained after oxirane or irradiation sterilization;
The preparation of step 2, cartilage repair material protective layer: be add the epoxide of 1 ~ 5% and the sodium chloride of 1 ~ 3% in the silk fibroin protein solution of 6 ~ 10% at w/v, obtain fibroin albumen colloidal sol after mix homogeneously; Adopt curtain coating, scraper plate or casting technology, prepare the film of thickness 50 ~ 150 μm, be obtained by reacting fibroin albumen thin film in 60 ~ 80 DEG C; Fibroin albumen thin film being embathed in distilled water to removing unreacted epoxide completely, obtaining the protective layer of cartilage repair material;
Adopt Fibrin Glue, collagen colloidal sol, hyaluronic acid colloidal sol protective layer to be adhered to the shiny surface of shelf layer, obtain the protective layer of cartilage repair material and the complex of shelf layer;
The preparation of step 3, cartilage repair material enriched layer: the solution II Collagen Type VI being mixed with 4 ~ 8mg/mL, adds the TGF β 1,10 of 5 ~ 20ng/mL
?8~ 10
?6the nerve growth factor of the dexamethasone of mol/L, the IGF-Ⅰ of 5 ~ 50ng/mL and 7 ~ 12ng/ml, mix homogeneously, the shelf layer matsurface of the complex of above-mentioned preparation prepares the collagen layer that thickness is 1 ~ 4mm, and 37 DEG C of placements make collagen solution solidify; Adopt any one in mechanical punching, laser boring (Laser), electric spark high speed punching (EDM), electrochemistry punching (ECM), punch at collagen layer, 50 ~ 100 μm, aperture, pitch of holes 100 ~ 150 μm, porous channel runs through collagen layer, completes the preparation of enriched layer; Solidifying of enriched layer collagen, makes the complex of enriched layer and above-mentioned preparation be bonded together.
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Denomination of invention: A cartilage repair material and its preparation method Effective date of registration: 20200915 Granted publication date: 20141231 Pledgee: Xianyang financing guarantee Limited by Share Ltd. Pledgor: SHAANXI BIO REGENERATION MEDICINE Co.,Ltd. Registration number: Y2020990001114 |
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Date of cancellation: 20211119 Granted publication date: 20141231 Pledgee: Xianyang financing guarantee Limited by Share Ltd. Pledgor: SHAANXI BIO REGENERATIVE MEDICINE Co.,Ltd. Registration number: Y2020990001114 |
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