CN103505761B - Preparation method and application of silk bracket, and three-phase silk ligament graft and preparation method thereof - Google Patents
Preparation method and application of silk bracket, and three-phase silk ligament graft and preparation method thereof Download PDFInfo
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- CN103505761B CN103505761B CN201310454716.4A CN201310454716A CN103505761B CN 103505761 B CN103505761 B CN 103505761B CN 201310454716 A CN201310454716 A CN 201310454716A CN 103505761 B CN103505761 B CN 103505761B
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Abstract
The invention discloses a preparation method and application of a silk bracket, and a three-phase silk ligament graft and a preparation method thereof. Silks are woven into a net-shaped bracket with a macroporous structure; the bracket is divided into three areas: a ligament area, a cartilage area and a bone area; mesenchymal stem cells, mesenchymal stem cells carrying with TGF-beta gene lentiviral transfection and mesenchymal stem cells carrying with BMP-2 gene lentiviral transfection are respectively planted in the three areas; the net-shaped bracket modified by the divided areas is rolled to form a bionic ligament graft with a physiological transition structure. The method avoids the problem of stress concentration due to direct connection of the soft and hard tissues; the planting gene-modified mesenchymal stem cells have excellent directional differentiation features, so that the cultivating time and steps of planting a plurality of different cells are saved, and the problem of weak biological fixation of the ligament graft-bone combination part in the existing single-phase tissue engineering ligament and the problem of the traditional method incapable of enabling the cells to grow deeply are solved.
Description
Technical field
The invention belongs to medical tissue engineering field, be specifically related to the preparation field of the organizational project ligament of ligament-bone junction surface anatomical structure.
Background technology
Anterior cruciate ligament (ACL) is the important joint capsule internal ligament of knee joint, and moving normally to knee joint and stablize has very important effect.Cross ligament damage is knee joint common diseases.After cross ligament damage, its low-down healing ability result in most patient all needs to transplant reconstruction operations.The source of implantation of ligament thing mainly contains autologous or allogeneic implantation of ligament and organizational project ligament.
The current clinical natural ligament that adopts is transplanted more, but the source that natural ligament is transplanted is not enough, and natural ligament transplanting also can cause a lot of complication after drawing materials.1/3 partial reconstitution anterior cruciate ligament in many employing patellar ligaments at present, but after having got this part tendon, patellar ligament will become relatively weak, and a patella defect part, postoperative easy fracture, in addition, the most important thing is postoperatively have quite a few patient that pain before kneecap occurs.Another conventional graft Shi Hams, these graft two ends do not have sclerotin, and ligament-bone interface healing is poor, and in bone road, cicatrix is filled, and mechanical property can not reach the requirement of anterior cruciate ligament, and muscular strength of going down on one's knees after cutting Zi Ti hamstring can weaken.Although allosome implantation of ligament avoids district's complication of drawing materials, there is immunological rejection, aids infection virus and the deficiency such as expensive, clinical practice is restricted.
Due to autologous or allosome ligament demand limited source greatly, organizational project ligament becomes a kind of and well selects.Politef ligament (Gore-Tex ligament) and pet fiber ligament (LARS artificial ligament) is mainly contained at present for human body.
Mesenchymal stem cells MSCs has the stem cell of multi-lineage potential as the class in the non-hemopoietic tissue of bone marrow, receives much concern in stem-cell research field always.The features such as because it has easy to drawing materials, low damage, easily cultivates, many differentiation are again good genophores, so utilizing gene transfection to carry out gene therapy having wide research space and application prospect simultaneously.Mesenchymal stem cells MSCs can be divided into chondrocyte, adipose cell, myocardial cell, thymic stromal cell and fibroblast etc., just due to its there is Multidirectional Differentiation and the ability of self renewal, utilize at present Gene transfer techniques gene therapy and differentiation-inducing on application cause and pay close attention to widely.
TGF-β is the TGF superfamily belonging to one group of adjustment Growth of Cells recently found and differentiation.Discovered in recent years TGF-β has important regulating action to the growth of cell, differentiation and immunologic function.Research shows, TGF-β can not only regulate bone, chondrocyte growth to break up, and also regulates the expression of other cytokines in cartilage and effect.TGF-β has stimulation to the cell that mesenchyme originates from, and plays inhibitory action to the cell of epithelium or neuroectodermal origin.In general, the effect of TGF-β to mesenchymal stem cells MSCs be early stage and low-density time promote propagation, when late period and high density promote differentiation.Research shows that TGF-β both effectively can promote that mesenchymal stem cells MSCs is to the differentiation of cartilage direction, can promote again the synthesis of cartilage specificity substrate.
BMP is taught by Urist MR and finds in nineteen sixty-five, can induced animal or human mesenchymal's cell differentiation be bone, cartilage, ligament, tendon and nervous tissue.BMP is widely used in treatment fresh fracture, Cranial defect, bone does not connect, spinal fusion and ischemic necrosis of femoral head.In numerous factor, BMP is the local factors uniquely can osseous tissue being induced separately to be formed, and thus has obvious facilitation to bone tissue restoration.
Previously research shows to use gelatin-chondroitin sulfate-hyaluronate sodium Application of composite in repair of cartilage, after compound, mechanical property is close to normal cartilage, hyaluronic acid composition in support can suppress MMP-3 and MMP-13 to generate, and increases II collagen expression, reduces articular chondrocyte apoptosis.Hydroxyapatite has bone inductive effect, can be integrated in area of new bone in osseous tissue is rebuild.Along with the degraded and absorbed of hydroxyapatite, the growth of induction natural bone tissue, thus improve the synosteosis ability of material, and containing nano level hydroxyapatite in people's bone composition, apply its granule, just likely in the forming process of new bone, directly absorbed by area of new bone, promote formation and the growth of area of new bone further.Meanwhile, part of hydroxyl apatite dissolves, and enters osseous tissue with phosphate, Lactated form, also can slow down the generation due to aseptic inflammations such as acid excessively strong the caused swelling in local.
Current artificial ligament still has some limitations: 1. due to artificial ligament and abiotic reconstruction, and creep, fatigue, mechanics failure easily occur long term; 2. the tough belt supporting frame of phase structure (i.e. single structure) is difficult in bone road, regenerate typical ligament-bone junction surface four-layer structure (collagen fiber, fibrous cartilage, calcification fibrous cartilage and bone) at present, in most cases forms ligament collagen fiber and is connected with the direct of osseous tissue; 3. the three-phase support of a kind of PLGA of bibliographical information; its between layers in conjunction with not good enough; the mechanical property of ligament portion can't be satisfied with requirement (the Spalazzi JP of ACL reconstruction; Doty SB; Moffat KL, et al.Development ofcontrolled matrix heterogeneity on a triphasic scaffold for orthopedicinterface tissue engineering.Tissue Eng2006; 12:3497 – 508.); Although the silk stent that 4. prepared by traditional weaving method can obtain the mechanical strength being similar to ACL; MSC also can adhere on support, breed and break up (Altman GH; Horan RL; Lu HH; Moreau J; Martin I, Richmond JC, et al.Silk matrix for tissue engineered anteriorcruciate ligaments.Biomaterials2002; 23:4131 – 41), but porosity is little between the silk fiber of this weaving manner, mesenchymal stem cells MSCs has just started to stick to material surface after implanting, In vitro culture 1 week and the surperficial completely secreted extracellular matrix bag quilt of 2 weeks after-poppets, and inside does not have cell to enter.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of silk stent, application and three-phase silk ligament graft, preparation method.
For achieving the above object, present invention employs following technical scheme.
A kind of three-phase silk ligament graft, comprise the silk stent that convolution is column, mesenchymal stem cells MSCs, carry the mesenchymal stem cells MSCs of TGF-β gene slow-virus transfection and carry the mesenchymal stem cells MSCs of BMP-2 gene slow-virus transfection, described silk stent comprises the silk stent body with microcellular structure, one side surface of silk stent body is divided into First Transition district successively, ligament reconstructive center and the second transition region, First Transition district and the second transition region lay respectively at the two ends of the silk stent of column, First Transition district and the second transition region are divided into ligament reconstructive marginal zone successively along the convolution direction of silk stent, cartilage modified region and bone modified region, mesenchymal stem cells MSCs is positioned at ligament reconstructive center and ligament reconstructive marginal zone, the mesenchymal stem cells MSCs carrying TGF-β gene slow-virus transfection is positioned at cartilage modified region, the mesenchymal stem cells MSCs carrying BMP-2 gene slow-virus transfection is positioned at bone modified region.
Described silk stent body adopts netted silk stent to be prepared from.
Described ligament reconstructive center and ligament reconstructive marginal zone all adopt fibroin albumen to modify, cartilage modified region adopts gelatin, hyaluronate sodium and chondroitin sulfate to modify, and bone modified region adopts fibroin albumen and hydroxyapatite to modify successively.
A preparation method for three-phase silk ligament graft, comprises the following steps:
1) silkworm silk braider is woven into the netted silk stent that aperture is 2-4mm;
2) side surface of netted silk stent is longitudinally divided into First Transition district, ligament reconstructive center and the second transition region successively along netted silk stent, First Transition district and the second transition region are laterally divided into ligament reconstructive marginal zone, cartilage modified region and bone modified region successively along netted silk stent;
3) use Co 60 to irradiate to sterilize to netted silk stent, then mesenchymal stem cells MSCs is planted in ligament reconstructive center and ligament reconstructive marginal zone, the mesenchymal stem cells MSCs carrying TGF-β gene slow-virus transfection is planted in cartilage modified region, the mesenchymal stem cells MSCs carrying BMP-2 gene slow-virus transfection is planted in bone modified region, mesenchymal stem cells MSCs, carry TGF-β gene slow-virus transfection mesenchymal stem cells MSCs and carry BMP-2 gene slow-virus transfection mesenchymal stem cells MSCs adopt 0.8-1.5 × 10
5individual cell/cm
2concentration plant;
4) wait the cell adhesion of planting after netted silk stent, netted silk stent is laterally carried out convolution according to from ligament reconstructive marginal zone to the order of Zai Daogu modified region, cartilage modified region along it, makes pillared implantation of ligament thing.
Described silkworm silk carries out sericin removal process before braiding, or, weave, in step 2 with the silkworm silk of non-sericin removal) carry out sericin removal process by weaving the netted silk stent obtained before.
The step of described sericin removal process comprises: boiled by the aqueous sodium carbonate of mass fraction 0.2-0.3%, then according to the ratio of often liter of aqueous sodium carbonate process 2-3g silkworm silk, the silkworm silk of non-sericin removal or the netted silk stent that is woven into by the silkworm silk of non-sericin removal are placed in the aqueous sodium carbonate of boiling, then continue heated and boiled 20-30 minute.
Before described step 3), modify ligament reconstructive center, ligament reconstructive marginal zone, cartilage modified region and bone modified region, the concrete grammar of modification is:
The first step, under room temperature, service property (quality) mark is that ligament reconstructive center, ligament reconstructive marginal zone and bone modified region are soaked into by the silk fibroin protein solution of 1.8-2.0%, simultaneously, employing solute is that cartilage modified region is soaked into by the solution of gelatin, hyaluronate sodium and chondroitin sulfate, then leave standstill 1-1.5 hour by netted silk stent in-15 to-25 DEG C, after leaving standstill, in-70 to-80 DEG C leave standstill 1-1.5 hour again;
Second step, after the first step, carries out vacuum lyophilization by netted silk stent;
3rd step, after second step, 8-15min is all soaked with the methanol aqueous solution of volume fraction 90% in ligament reconstructive center, ligament reconstructive marginal zone and bone modified region, after immersion, netted silk stent is put into drying baker drying or natural air drying, then prepare hydroxyapatite coating layer in bone modified region.
The preparation method of described silk fibroin protein solution is: be dissolved in lithium bromide water solution under the condition of heating, stirring by silkworm silk and obtain solution a, deionized water is used to dialyse solution a, by the solution a after dialysis in 4 DEG C, centrifugal under the condition of 8000-10000 rev/min, get centrifugal after supernatant obtain silk fibroin protein solution.
Described solute is the preparation method of the solution of gelatin, hyaluronate sodium and chondroitin sulfate: be dissolved in 10-20mL distilled water by 0.5g gelatin powder, 0.1g chondroitin sulfate powder and 5mg hyaluronate sodium, then in distilled water, adds the EDAC that 2-3mL mass fraction is 1% again.
The condition of described vacuum lyophilization is: vacuum is 0.01-0.5mbar, and temperature is-30 to-50 DEG C, and the time is 48-72h.
The method preparation that described hydroxyapatite coating layer adopts two kinds of solution alternately to infiltrate, wherein a kind of solution be with pH value be 7.4 Tris-HCl buffer and the calcium chloride solution that configures of calcium chloride, another kind of solution is sodium hydrogen phosphate aqueous solution.
A preparation method for silk stent, comprises the following steps:
1) silkworm silk braider is woven into the netted silk stent that aperture is 2-4mm;
2) utilize separation incubator that netted silk stent is longitudinally divided into First Transition district, ligament reconstructive center and the second transition region successively along netted silk stent, and First Transition district and the second transition region are laterally divided into ligament reconstructive marginal zone, cartilage modified region and bone modified region successively along netted silk stent; Described separation incubator comprises base plate, is arranged at the sealed membrane on base plate and is arranged at the infiltrate chamber on sealed membrane, described infiltration chamber is divided into multiple independently sub-chamber, sub-chamber and ligament reconstructive center, ligament reconstructive marginal zone, cartilage modified region and bone modified region one_to_one corresponding, described sealed membrane offers and described sub-chamber through hole one to one; Netted silk stent is placed between sealed membrane and base plate, then infiltrate chamber and base plate are clamped, in clamping process, sealed membrane is pressed into netted silk stent, realizes the separation of ligament reconstructive center, ligament reconstructive marginal zone, cartilage modified region and bone modified region;
3) under room temperature, in corresponding sub-chamber, service property (quality) mark is that ligament reconstructive center, ligament reconstructive marginal zone and bone modified region are soaked into by the silk fibroin protein solution of 1.8-2.0%, simultaneously, employing solute is that cartilage modified region is soaked into by the solution of gelatin, hyaluronate sodium and chondroitin sulfate, then leave standstill 1-1.5 hour by netted silk stent in-15 to-25 DEG C, after leaving standstill, in-70 to-80 DEG C leave standstill 1-1.5 hour again;
4) after step 3), netted silk stent is carried out vacuum lyophilization;
5) after step 4), 8-15min is all soaked with the methanol aqueous solution of volume fraction 90% in ligament reconstructive center, ligament reconstructive marginal zone and bone modified region, after immersion, netted silk stent is put into drying baker drying or natural air drying, then prepare hydroxyapatite coating layer in bone modified region.
Described silkworm silk carries out sericin removal process before braiding, or, weave, in step 2 with the silkworm silk of non-sericin removal) carry out sericin removal process by weaving the netted silk stent obtained before.
The step of described sericin removal process comprises: boiled by the aqueous sodium carbonate of mass fraction 0.2-0.3%, then according to the ratio of often liter of aqueous sodium carbonate process 2-3g silkworm silk, the silkworm silk of non-sericin removal or the netted silk stent that is woven into by the silkworm silk of non-sericin removal are placed in the aqueous sodium carbonate of boiling, then continue heated and boiled 20-30 minute.
The preparation method of described silk fibroin protein solution is: be dissolved in lithium bromide water solution under the condition of heating, stirring by silkworm silk and obtain solution a, deionized water is used to dialyse solution a, by the solution a after dialysis in 4 DEG C, centrifugal under the condition of 8000-10000 rev/min, get centrifugal after supernatant obtain silk fibroin protein solution.
Described solute is the preparation method of the solution of gelatin, hyaluronate sodium and chondroitin sulfate: be dissolved in 10-20mL distilled water by 0.5g gelatin powder, 0.1g chondroitin sulfate powder and 5mg hyaluronate sodium, then in distilled water, adds the EDAC that 2-3mL mass fraction is 1% again.
The condition of described vacuum lyophilization is: vacuum is 0.01-0.5mbar, and temperature is-30 to-50 DEG C, and the time is 48-72h.
The method preparation that described hydroxyapatite coating layer adopts two kinds of solution alternately to infiltrate, wherein a kind of solution be with pH value be 7.4 Tris-HCl buffer and the calcium chloride solution that configures of calcium chloride, another kind of solution is sodium hydrogen phosphate aqueous solution.
The silk stent that the preparation method of above-mentioned silk stent prepares is preparing the application in implantation of ligament thing.
Beneficial effect of the present invention is embodied in:
Silk stent of the present invention is divided into tough zone, cartilage modified region and bone modified region, and convolution is carried out according to order after the cell that plantation is corresponding, make the ligament-bone junction surface of described three-phase silk ligament graft on cross section, have structure of dividing a word with a hyphen at the end of a line from " ligament → cartilage → bone " from inside to outside, simulate and rebuild the physiology transition structure at normal ligament-bone junction surface; Because this three-phase silk ligament graft has the organizational structure close with normal ligament-synosteosis portion, it can form area of new bone in bone graft road, and the biological fixation in implantation of ligament thing-synosteosis portion is more firm, and not easily pull-off, adds the insertion intensity of ligament; Because this three-phase silk ligament graft has the divide a word with a hyphen at the end of a line structure (ligament → cartilage → bone) close with normal ligament-synosteosis portion, effectively can avoid concentrating of stress, improve the biomechanical property of implantation of ligament thing.
Because silkworm silk is the absorbable material with excellent toughness, make implantation of ligament thing have complete braiding structure, connect closely between layers, there is the mechanical strength close with normal ligament.After transplanting, what on the one hand the slow absorption of timbering material caused mechanical strength weakens also for cell proliferation provides space to decrease allosome tissue, and the cell formative tissue on the other hand after proliferation and differentiation in turn enhances the mechanical strength of material.
Preparation due to this three-phase implantation of ligament thing have employed first cultivates seed cell on network, convolution network again, form the method for cylindric ligament, ensure that has cell to grow in cylindrical deep layer too, solves the problem that traditional ligament weaving method seed cell cannot enter ligament deep layer.
The inventive method only uses a kind of seed cell-mesenchymal stem cells MSCs, utilize slow virus carrier that the mesenchymal stem cells MSCs after TGF-β and BMP-2 gene transfection is had good differentiation capability, respectively to ligament cell, chondrocyte and osteoblast differentiation under the impact of the local microenvironment in 3rd district, be beneficial to the normal anatomical structures forming ligament and bone junction surface.
The inventive method carries the mesenchymal stem cells MSCs of BMP-2 gene and TGF-β gene in bone modified region and the inoculation of cartilage modified region, and this cell by gene transfection can continuous expressed BMP-2(bone morphogenesis protein-2 in the process of its proliferation and differentiation) and these two kinds of cytokines of TGF-β (transforming growth factor-β) can effectively break up to bone and cartilage direction by inducing bone mesenchymal stem cell.
Subregion of the present invention is modified by fibroin albumen, gelatin+hyaluronate sodium+chondroitin sulfate and fibroin albumen+hydroxyapatite respectively, micropore is not formed by means of only on the macroporous structure being modified at braided support, the surface area increasing material is convenient to growing into of mesenchymal stem cells MSCs, also build the microenvironment that applicable mesenchymal stem cells MSCs breaks up to three kinds of different cells (ligament cell, chondrocyte, osteoblast), advantageously in survival and the propagation of noble cells, efficiently avoid dedifferenting with aging of noble cells.
Accompanying drawing explanation
Fig. 1 is subregion and the convolution schematic diagram of silk stent body of the present invention; In Fig. 1, A is tough zone, and B is cartilage modified region, and C is bone modified region;
Fig. 2 is the micro-structure diagram of silk stent of the present invention;
Fig. 3 is the micro-structure diagram of graft of the present invention;
Detailed description of the invention
Below in conjunction with accompanying drawing, the invention will be further described.
A kind of desirable organizational project ligament should possess following characteristics: 1. timbering material has good biocompatibility; 2. organizational project ligament has enough mechanical strengths and anti-fatigue ability; 3. the ligament strength maintenance time must long enough, is beneficial to ligament reconstructive; 4. ligament-bone junction surface needs to regenerate typical ligament-bone junction surface four-layer structure (collagen fiber, fibrous cartilage, calcification fibrous cartilage and bone), is beneficial to the constant intensity at increase ligament-bone junction surface and avoids stress to concentrate; 5. seed cell can grow and breed on timbering material; 6. seed cell needs to be distributed in ligament nexine and skin equably.
In order to reach above requirement, it is final developing direction that the organizational project ligament be jointly made up of cell and absorbable biological material carries out biology reconstruction.Lightweight (the 1.3g/cm of silkworm silk
3), intensity high (about 4.8GPa), good springiness (elastic deformation is up to 35%), the silk stent of braiding can obtain the mechanical strength being similar to ACL.Silkworm silk has good biocompatibility, and seed cell can adhere to, breeds and break up on support.The degraded of silkworm silk is very slow, can maintain enough mechanical properties during the reconstruction of ligament.Real regeneration ligament, needs repopulating cell on support, by cell proliferation with constantly secrete substrate, forms newborn ligament, and man-made support is constantly degraded simultaneously, reaches the target of ligament regeneration.Fibroin albumen, gelatin+hyaluronate sodium+chondroitin sulfate and fibroin albumen+hydroxyapatite can be used to modify netted silk stent, be respectively fibroblast, chondrocyte and osteoblast and the distinctive microenvironment being more suitable for its growth and propagation is provided.Netted silk stent is divided into tough zone, cartilage area (i.e. cartilage modified region) He Guqu (i.e. bone modified region), different materials is used to modify respectively, and plant corresponding cell, and more netted silk stent convolution is become column implantation of ligament thing, can the structure of dividing a word with a hyphen at the end of a line at ligament-bone junction surface.Meanwhile, the netted silk stent of the even repopulating cell of convolution can make column implantation of ligament object depth layer have cell to grow into too, advantageously in the regeneration of ligament.
Accordingly, the present invention proposes the preparation method of the three-phase silk ligament graft of a kind of ligament-bone junction surface normal anatomical structures, the anatomical structure that prepared implantation of ligament thing can either simulate normal ligament-synosteosis portion is fixed with the high strength obtaining ligament-bone junction surface, stress is avoided to concentrate, there is again complete braiding structure to obtain desirable mechanical strength simultaneously, comprise the following steps:
1. the preparation of network
1.1 silkworm silk sericin removals
The aqueous sodium carbonate of mass fraction 0.2-0.3% is boiled, according to the ratio of often liter of aqueous sodium carbonate process 2-3g silkworm silk, the silkworm silk of non-sericin removal is placed in the aqueous sodium carbonate of boiling, then agitating heating constantly boiling 20-30 minute.Take out silkworm silk, with deionized water wash silkworm silk three times, dry in the shade.
The braiding of 1.2 networks
1.2.1 the silkworm silk of sericin removal is used to weave
With braider utilize the flat pin of latitude or screw needle technology to be woven into sericin removal silkworm silk netted silk stent that aperture is 2-4mm size.
1.2.2 weave with the silkworm silk not taking off sericin
With braider utilize the flat pin of latitude or screw needle technology to be woven into non-sericin removal silkworm silk netted silk stent that aperture is 2-4mm size.Again by the woven method of netted silk stent in 1.1, carry out sericin removal process, obtain the netted silk stent taking off sericin.
According to practical situation, if silkworm silk is easily contaminated in braiding process, so can select to adopt the method as described in 1.2.2.
2. the modification of netted silk stent
The subregion of 2.1 netted silk stents
Netted silk stent is divided into three regions (as Fig. 1): A district: tough zone, B district: cartilage modified region, C district: bone modified region.Netted silk stent after convolution subregion, can form columnar structure engineering ligament.The feature of this organizational project ligament is: the cross section at ligament two ends forms the transition region of the ligament → cartilage → bone from central authorities to edge.A district all uses silk fibroin protein solution (i.e. silkworm silk solution) to modify, and the a-quadrant of netted silk stent central part is ligament reconstructive district, and the a-quadrant on netted silk stent both sides can the tough region of collagen in ligament bone graft road.
The modification of 2.2 netted silk stents
2.2.1 the preparation of silk fibroin protein solution
Lithium bromide deionized water is made into the solution that concentration is 9.3mol/L, and the ratio of dissolving 1g silkworm silk according to 4mL lithium-bromide solution dissolves silkworm silk.Put into glass beaker after being weighed by the silkworm silk of required dissolving, lithium-bromide solution is watered in proportion on silkworm silk, puts into stirrer, beaker is placed on heatable magnetic stirring apparatus, gentle agitation, and temperature of liquid is heated to 60 DEG C, dissolve 3-5 hour, dissolve completely to silkworm silk, be designated as solution a.Solution a is added in bag filter (3500MWCO), uses the deionized water of 1-1.5L to dialyse according to every 10-12mL solution a, respectively at 1 hour, 4 hours, evening on the same day, the next morning, second night, the 3rd day morning upgrade deionized water.Solution a in bag filter is loaded in centrifuge tube, is under the condition of 4 DEG C in temperature, with the rotating speed of 9000 revs/min, centrifugal 30 minutes, gets supernatant, then by above-mentioned condition, supernatant is once centrifugal again, get supernatant and obtain silkworm silk solution.The silkworm silk solution prepared by 1mL is added in the container that weighed, after 60 DEG C of baking oven bone dries, again weighs, deducts the weight of original container, divided by 100mL, obtain the concentration of final silkworm silk solution, be finally made into the silkworm silk solution that mass fraction is 2%.
2.2.2 the modification of netted each subregion of silk stent
Make rectangular frame with the draw point of diameter 1.5mm, supported by netted silk stent, support network compartment diameter macropores is about 2-4mm, and with separation incubator, netted silk stent being pressed subregion shown in Fig. 1, is independently between regional.Described separation incubator comprises base plate, is arranged at the sealed membrane on base plate and is arranged at the infiltrate chamber on sealed membrane, described infiltration chamber is divided into multiple independently sub-chamber, sub-chamber and A, B, C district one_to_one corresponding, described sealed membrane offers and described sub-chamber through hole one to one; Be placed between sealed membrane and base plate by netted silk stent, then infiltrate chamber and base plate are clamped, in clamping process, sealed membrane is pressed into netted silk stent, realizes the separation in A, B, C district.
Tough zone, bone modified region use mass fraction 2.0% silk fibroin protein solution prepared fully to infiltrate.Cartilage modified region uses gelatin+hyaluronate sodium+chondroitin sulfate to modify: 0.5g gelatin powder, 0.1g chondroitin sulfate powder and 5mg hyaluronate sodium add in 10mL distilled water and are stirred to dissolving, the ethyldimethyl amine carbodiimide aqueous solution adding 2mL mass fraction 1% again obtains solution b, pour solution b into cartilage modified region, itself and netted silk stent are fully infiltrated, under room temperature, glue connection 2-3min is placed on-20 DEG C and stops crosslinked in 1 hour, be then placed in-80 DEG C 1 hour.Then whole support is carried out evacuation lyophilization (vacuum is 0.25mbar, and temperature is-40 DEG C) and complete pore-creating in 48 hours.
Then 15 minutes are soaked to tough zone and bone modified region volume fraction 90% methanol aqueous solution, then put into 60 DEG C of drying baker and dry the pore-creating of fixing institute.Hydroxyapatite coating layer is introduced in bone modified region by the method for " two kinds of solution alternately infiltrate " after fixing.Concrete steps are as follows: first configure two kinds of solution: 1. calcium solution: the calcium chloride solution with pH value being the Tris-HCl buffer configuration 200mM of 7.4; 2. phosphorus solution: 120mM sodium hydrogen phosphate aqueous solution.Then first the calcium solution that bone modified region configures is soaked, react 1 hour at 37 DEG C of calorstats.Then pour out calcium liquid, and blot residual liquid with filter paper, then soak with the phosphorus solution of configuration, react 1 hour at 37 DEG C of calorstats equally.After exhausting phosphorus solution, then soak 1 hour with calcium solution, then exhaust calcium solution, add phosphorus solution and soak 1 hour, finally exhaust phosphorus solution, after drying in the shade, hydroxyapatite coating layer can be formed around silkworm silk.See Fig. 2, around silk fiber, form hydroxyapatite coating layer, so far obtain three-phase silk stent.
3. the plantation of cell
The three-phase silk stent prepared is used Co 60 illumination-based disinfection, according to 0.8-1.5 × 10
5/ cm
2concentration respectively mesenchymal stem cells MSCs, the mesenchymal stem cells MSCs carrying TGF-β gene slow-virus transfection and the mesenchymal stem cells MSCs correspondence of carrying BMP-2 gene slow-virus transfection are inoculated in tough zone, cartilage modified region and bone modified region.
4. convolution support
Repopulating cell 24 little up to 7 days (the concrete time with cell adhesion in support, and can normal proliferative be as the criterion) after, by three-phase silk stent according to the direction convolution of X direction (direction, ligament transection face) from ligament → cartilage → bone, make column implantation of ligament thing, cell adhesion is grown on material, sees Fig. 3.
Ligament-bone the junction surface of the three-phase silk ligament graft of the ligament according to said method made-bone junction surface normal anatomical structures has the structure of dividing a word with a hyphen at the end of a line from " ligament → cartilage → bone " from inside to outside on cross section, simulates and has rebuild the physiology transition structure at normal ligament-bone junction surface.This physiology transition structure, makes this implantation of ligament thing form area of new bone in bone graft road, and the biological fixation in implantation of ligament thing-synosteosis portion is more firm, and not easily pull-off, adds the insertion intensity of ligament.Meanwhile, this transition structure also avoids the stress at ligament-bone junction surface to concentrate effectively, improves the anti-fatigue ability of implantation of ligament thing.3rd district of this three-phase silk ligament graft are modified by fibroin albumen, gelatin+hyaluronate sodium+chondroitin sulfate and fibroin albumen+hydroxyapatite respectively, have built the microenvironment being applicable to three kinds of different cells (ligament cell, chondrocyte, osteoblast).Advantageously in survival and the propagation of different cell, efficiently avoid dedifferenting with aging of noble cells.In addition, the netted silk stent of three-phase silkworm silk of this three-phase silk ligament graft has complete braiding structure, connects closely between layers, makes this organizational project ligament have the mechanical strength close with normal ligament.The preparation of this three-phase implantation of ligament thing have employed and first cultivated on network by seed cell in addition, convolution network again, form the method for cylindric ligament, ensure that has cell to grow in cylindrical deep layer too, solves the problem that traditional ligament weaving method seed cell cannot enter ligament deep layer.Finally, the method only uses a kind of seed cell-bone marrow stroma stem cell, its Multidirectional Differentiation is made by carrying out gene transfection to it, decrease complicated process and the step of a kind of graft plantation various kinds of cell, significantly will be better than adult cell as its multiplication capacity of stem cell with multi-lineage potential, this effectively can shorten healing time.
In a word, first silkworm silk is woven into the network with macroporous structure by the present invention, support is divided into three regions again: tough zone, cartilage area, bone district, tough zone uses silk fibroin protein solution to carry out modification and is beneficial to ligament regeneration, cartilage area uses gelatin+hyaluronate sodium+chondroitin sulfate to carry out modification and is beneficial to regenerating bone or cartilage, and bone district silk fibroin protein solution and hydroxyapatite modify the regeneration being beneficial to bone.Plant mesenchymal stem cells MSCs respectively in 3rd district, carry the mesenchymal stem cells MSCs of TGF-β gene slow-virus transfection and carry the mesenchymal stem cells MSCs of BMP-2 gene slow-virus transfection.The network that convolution subregion is modified, forms the bionical implantation of ligament thing with physiology transition structure (ligament → cartilage → bone).This structure not only effectively prevent hard and soft tissue and directly connects the problem that the stress that causes concentrates, and the mesenchymal stem cells MSCs of plantation genetic modification has good directed differentiation characteristic and saves the incubation time and step of planting multiple different cell, also solve implantation of ligament thing-illusive problem of synosteosis portion biological fixation that current homogeneous structure engineering ligament exists, the convolution of last support solves traditional method cell and cannot to grow into the problem of deep layer.
Claims (8)
1. a three-phase silk ligament graft, it is characterized in that: comprise the silk stent that convolution is column, mesenchymal stem cells MSCs, carry the mesenchymal stem cells MSCs of TGF-β gene slow-virus transfection and carry the mesenchymal stem cells MSCs of BMP-2 gene slow-virus transfection, described silk stent comprises the silk stent body with microcellular structure, one side surface of silk stent body is divided into First Transition district successively, ligament reconstructive center and the second transition region, First Transition district and the second transition region lay respectively at the two ends of the silk stent of column, First Transition district and the second transition region are divided into ligament reconstructive marginal zone successively along the convolution direction of silk stent, cartilage modified region and bone modified region, mesenchymal stem cells MSCs is positioned at ligament reconstructive center and ligament reconstructive marginal zone, the mesenchymal stem cells MSCs carrying TGF-β gene slow-virus transfection is positioned at cartilage modified region, the mesenchymal stem cells MSCs carrying BMP-2 gene slow-virus transfection is positioned at bone modified region, described ligament reconstructive center and ligament reconstructive marginal zone all adopt fibroin albumen to modify, cartilage modified region adopts gelatin, hyaluronate sodium and chondroitin sulfate are modified, bone modified region adopts fibroin albumen and hydroxyapatite to modify successively.
2. a kind of three-phase silk ligament graft according to claim 1, is characterized in that: described silk stent body adopts netted silk stent to be prepared from.
3. a preparation method for three-phase silk ligament graft, is characterized in that: comprise the following steps:
1) silkworm silk braider is woven into the netted silk stent that aperture is 2-4mm;
2) side surface of netted silk stent is longitudinally divided into First Transition district, ligament reconstructive center and the second transition region successively, First Transition district and the second transition region are transversely divided into ligament reconstructive marginal zone, cartilage modified region and bone modified region successively; Described ligament reconstructive center and ligament reconstructive marginal zone all adopt fibroin albumen to modify, cartilage modified region adopts gelatin, hyaluronate sodium and chondroitin sulfate to modify, and bone modified region adopts fibroin albumen and hydroxyapatite to modify successively;
3) use Co 60 to irradiate to sterilize to netted silk stent, then mesenchymal stem cells MSCs is planted in ligament reconstructive center and ligament reconstructive marginal zone, the mesenchymal stem cells MSCs carrying TGF-β gene slow-virus transfection is planted in cartilage modified region, the mesenchymal stem cells MSCs carrying BMP-2 gene slow-virus transfection is planted in bone modified region, mesenchymal stem cells MSCs, carry TGF-β gene slow-virus transfection mesenchymal stem cells MSCs and carry BMP-2 gene slow-virus transfection mesenchymal stem cells MSCs adopt 0.8-1.5 × 10
5individual cell/cm
2concentration plant;
4) wait the cell adhesion of planting after netted silk stent, netted silk stent is laterally carried out convolution according to from ligament reconstructive marginal zone to the order of Zai Daogu modified region, cartilage modified region along it, makes pillared implantation of ligament thing.
4. the preparation method of a kind of three-phase silk ligament graft according to claim 3, it is characterized in that: described silkworm silk carries out sericin removal process before braiding, or, weave, in step 2 with the silkworm silk of non-sericin removal) carry out sericin removal process by weaving the netted silk stent obtained before.
5. the preparation method of a kind of three-phase silk ligament graft according to claim 3, it is characterized in that: described step 3) before, modify ligament reconstructive center, ligament reconstructive marginal zone, cartilage modified region and bone modified region, the concrete grammar of modification is:
The first step, under room temperature, service property (quality) mark is that ligament reconstructive center, ligament reconstructive marginal zone and bone modified region are soaked into by the silk fibroin protein solution of 1.8-2.0%, simultaneously, employing solute is that cartilage modified region is soaked into by the solution of gelatin, hyaluronate sodium and chondroitin sulfate, then leave standstill 1-1.5 hour by netted silk stent in-15 to-25 DEG C, after leaving standstill, in-70 to-80 DEG C leave standstill 1-1.5 hour again;
Second step, after the first step, carries out vacuum lyophilization by netted silk stent;
3rd step, after second step, 8-15min is all soaked with the methanol aqueous solution of volume fraction 90% in ligament reconstructive center, ligament reconstructive marginal zone and bone modified region, after immersion, netted silk stent is put into drying baker drying or natural air drying, then prepare hydroxyapatite coating layer in bone modified region.
6. a preparation method for silk stent, is characterized in that: comprise the following steps:
1) silkworm silk braider is woven into the netted silk stent that aperture is 2-4mm;
2) utilize separation incubator that netted silk stent is longitudinally divided into First Transition district, ligament reconstructive center and the second transition region successively, and First Transition district and the second transition region are transversely divided into ligament reconstructive marginal zone, cartilage modified region and bone modified region successively; Described separation incubator comprises base plate, is arranged at the sealed membrane on base plate and is arranged at the infiltrate chamber on sealed membrane, described infiltration chamber is divided into multiple independently sub-chamber, sub-chamber and ligament reconstructive center, ligament reconstructive marginal zone, cartilage modified region and bone modified region one_to_one corresponding, described sealed membrane offers and described sub-chamber through hole one to one; Netted silk stent is placed between sealed membrane and base plate, then infiltrate chamber and base plate are clamped, in clamping process, sealed membrane is pressed into netted silk stent, realizes the separation of ligament reconstructive center, ligament reconstructive marginal zone, cartilage modified region and bone modified region;
3) under room temperature, in corresponding sub-chamber, service property (quality) mark is that ligament reconstructive center, ligament reconstructive marginal zone and bone modified region are soaked into by the silk fibroin protein solution of 1.8-2.0%, simultaneously, employing solute is that cartilage modified region is soaked into by the solution of gelatin, hyaluronate sodium and chondroitin sulfate, then leave standstill 1-1.5 hour by netted silk stent in-15 to-25 DEG C, after leaving standstill, in-70 to-80 DEG C leave standstill 1-1.5 hour again;
4) through step 3) after, netted silk stent is carried out vacuum lyophilization;
5) through step 4) after, 8-15min is all soaked with the methanol aqueous solution of volume fraction 90% in ligament reconstructive center, ligament reconstructive marginal zone and bone modified region, after immersion, netted silk stent is put into drying baker drying or natural air drying, then prepare hydroxyapatite coating layer in bone modified region.
7. the preparation method of a kind of silk stent according to claim 6, it is characterized in that: described silkworm silk carries out sericin removal process before braiding, or, weave, in step 2 with the silkworm silk of non-sericin removal) and carry out sericin removal process by weaving the netted silk stent obtained before.
8. the silk stent that the preparation method of a silk stent as claimed in claim 6 prepares is preparing the application in implantation of ligament thing.
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| CN110037830B (en) * | 2019-04-18 | 2021-12-10 | 东华大学 | Bionic full-molded three-phase structure artificial ligament and preparation method thereof |
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| CN103071187A (en) * | 2013-01-14 | 2013-05-01 | 西安交通大学 | Ligament-bone composite scaffold with biomimetic connecting interface and forming method thereof |
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| US20140309738A1 (en) * | 2011-06-01 | 2014-10-16 | The Board Of Trustees Of The University Of Illinois | Membrane-Scaffold Composites for Tissue Engineering Applications |
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| CN102107021A (en) * | 2010-08-10 | 2011-06-29 | 贾庆卫 | Silk ligament and preparation method thereof |
| CN103157135A (en) * | 2011-12-14 | 2013-06-19 | 西安瑞捷生物科技有限公司 | Reticular tissue engineering scaffold |
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