CN103505761B - Preparation method and application of silk bracket, and three-phase silk ligament graft and preparation method thereof - Google Patents

Abstract

The invention provides a preparation method and application of a silk stent and a three-phase silk ligament graft and a preparation method. The silk is woven into a mesh-like stent with a macroporous structure, and the stent is divided into three areas: ligament area, cartilage area, In the bone area, bone marrow mesenchymal stem cells, bone marrow mesenchymal stem cells transfected with lentivirus carrying TGF-β gene and bone marrow mesenchymal stem cells transfected with lentivirus carrying BMP-2 gene were planted in three areas respectively. The mesh scaffold forms a bionic ligament graft with a physiological transition structure. The invention not only avoids the problem of stress concentration caused by the direct connection of soft and hard tissues, but also has good directional differentiation characteristics of genetically modified bone marrow mesenchymal stem cells. It saves the culture time and steps of planting a variety of different cells, and also solves the problem of unreliable biological fixation of the ligament graft-osseocombination existing in single-phase tissue engineering ligaments and the problem that cells cannot grow deep into the traditional method.

Description

Preparation method and application of silk scaffold and three-phase silk ligament graft and preparation method

technical field

The invention belongs to the field of medical tissue engineering, in particular to the field of preparation of tissue engineering ligaments for reconstructing the anatomical structure of ligament-bone joints.

Background technique

The anterior cruciate ligament (ACL) is an important intracapsular ligament of the knee joint, which plays a very important role in the normal movement and stability of the knee joint. Anterior cruciate ligament injury is a common disease of the knee joint. The very low ability to heal after an ACL injury results in the need for graft reconstruction in most patients. The sources of ligament grafts mainly include autologous or allogeneic ligament grafts and tissue engineered ligaments.

At present, autologous ligament transplantation is mostly used clinically, but the source of autologous ligament transplantation is insufficient, and autologous ligament transplantation will cause many complications after taking materials. At present, 1/3 of the patellar ligament is usually used to reconstruct the anterior cruciate ligament, but after taking this part of the tendon, the patellar ligament will become relatively weak, and part of the patella is defective, so it is easy to fracture after operation. Prepatellar pain occurs in a considerable number of patients. Another commonly used graft is the hamstring tendon. There is no bone at both ends of the graft, the ligament-bone interface is poorly healed, the bone tunnel is filled with scars, and the mechanical properties cannot meet the requirements of the anterior cruciate ligament. Knee flexor strength will be weakened. Although the allograft ligament transplantation avoids complications in the area where the material is obtained, it has the disadvantages of immune rejection, HIV infection and high price, which limit its clinical application.

Due to the high demand and limited sources of autologous or allograft ligaments, tissue engineered ligaments have become a good choice. Currently used in the human body are mainly polytetrafluoroethylene ligament (Gore-Tex ligament) and polyethylene terephthalate fiber ligament (LARS artificial ligament).

Bone marrow mesenchymal stem cells, as a kind of stem cells with multilineage differentiation potential in bone marrow non-hematopoietic tissues, have been attracting much attention in the field of stem cell research. Because it has the characteristics of easy acquisition, low damage, easy cultivation, multi-differentiation, etc., and is a good gene carrier, it has broad research space and application prospects in gene therapy by gene transfection. Bone marrow mesenchymal stem cells can be differentiated into chondrocytes, adipocytes, cardiomyocytes, thymus stromal cells and fibroblasts. Due to their multidirectional differentiation and self-renewal ability, gene transfection technology is currently used in gene therapy and Applications to induce differentiation have attracted widespread attention.

TGF-β belongs to a group of recently discovered TGF superfamily that regulate cell growth and differentiation. In recent years, it has been found that TGF-β has an important regulatory effect on cell growth, differentiation and immune function. Studies have shown that TGF-β can not only regulate the growth and differentiation of bone and chondrocytes, but also regulate the expression and function of other cytokines in cartilage. TGF-β has a stimulatory effect on cells of mesenchymal origin and an inhibitory effect on cells of epithelial or neuroectodermal origin. Generally speaking, the effect of TGF-β on bone marrow mesenchymal stem cells is to promote proliferation at early stage and low density, and to promote differentiation at late stage and high density. Studies have shown that TGF-β can not only effectively promote the differentiation of bone marrow mesenchymal stem cells to cartilage, but also promote the synthesis of cartilage-specific matrix.

BMP was discovered by Professor Urist MR in 1965 and can induce animal or human mesenchymal cells to differentiate into bone, cartilage, ligament, tendon and nerve tissue. BMP is widely used in the treatment of fresh fractures, bone defects, nonunion, spinal fusion and avascular necrosis of the femoral head. Among many factors, BMP is the only local factor that can induce the formation of bone tissue alone, so it can obviously promote the repair of bone tissue.

Previous studies have shown that gelatin-chondroitin sulfate-sodium hyaluronate is used for cartilage repair, and the mechanical properties of the compound are close to those of normal cartilage. The hyaluronic acid in the scaffold can inhibit the production of MMP-3 and MMP-13, and increase II collagen. expression, reduce chondrocyte apoptosis. Hydroxyapatite has an osteoinductive effect and can be integrated into new bone during bone tissue reconstruction. With the degradation and absorption of hydroxyapatite, the growth of natural bone tissue is induced, thereby improving the osseointegration ability of the material, and the composition of human bone contains nano-scale hydroxyapatite, and the application of its particles may be in the new bone. During the formation process, it is directly absorbed by new bone, further promoting the formation and growth of new bone. At the same time, part of the hydroxyapatite dissolves and enters the bone tissue in the form of phosphate and lactate, which can also slow down the occurrence of aseptic inflammation such as swelling caused by local excessive acidity.

The current artificial ligament still has certain limitations: ①Because the artificial ligament is not a biological reconstruction, it is prone to creep, fatigue, and mechanical failure in the long run; ②The current single-phase structure (that is, a single structure) of the ligament scaffold is difficult to regenerate in the bone tunnel A typical four-layer structure of the ligament-bone junction (collagen fibers, fibrocartilage, calcified fibrocartilage, and bone) forms a direct connection between ligament collagen fibers and bone tissue in most cases; The combination between the layers is not good, and the mechanical properties of the ligament part cannot meet the requirements of ACL reconstruction (Spalazzi JP, Doty SB, Moffat KL, et al. Development of controlled matrix heterogeneity on a triphasic scaffold for orthopedic interface tissue engineering. Tissue Eng2006;12:3497–508.); ④ Although silk scaffolds prepared by traditional weaving methods can obtain mechanical strength similar to that of ACL, MSCs can also adhere, proliferate and differentiate on the scaffolds (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 the porosity between silk fibers in this weaving method is small, bone marrow mesenchymal stem cell implantation After incorporation, it began to adhere to the surface of the material. After 1 week and 2 weeks of in vitro culture, the surface of the scaffold was completely covered by the secreted extracellular matrix, and no cells entered the inside.

Contents of the invention

The object of the present invention is to provide a preparation method and application of a silk scaffold, a three-phase silk ligament graft and a preparation method.

In order to achieve the above object, the present invention adopts the following technical solutions.

A three-phase silk ligament graft, including a coiled silk scaffold, bone marrow mesenchymal stem cells, bone marrow mesenchymal stem cells transfected with TGF-β gene lentivirus and BMP-2 gene lentivirus transfected Bone marrow mesenchymal stem cells, the silk scaffold includes a silk scaffold body with a microporous structure, one side surface of the silk scaffold body is divided into a first transition zone, a ligament reconstruction center zone and a second transition zone in turn, the first transition zone and the The second transition zone is respectively located at the two ends of the columnar silk scaffold. The first transition zone and the second transition zone are divided into ligament reconstruction edge zone, cartilage modification zone, bone modification zone, and bone marrow mesenchyme along the rolling direction of the silk scaffold. Stem cells were located in the central area of ligament reconstruction and the edge area of ligament reconstruction, bone marrow mesenchymal stem cells transfected with TGF-β gene lentivirus were located in the cartilage modification area, and bone marrow mesenchymal stem cells transfected with BMP-2 gene lentivirus were located in the bone modification area. district.

The silk support body is prepared by using a meshed silk support.

Both the central area of the ligament reconstruction and the peripheral area of the ligament reconstruction are modified with silk fibroin, the cartilage modification area is modified with gelatin, sodium hyaluronate and chondroitin sulfate, and the bone modification area is sequentially modified with silk fibroin and hydroxyapatite. grooming.

A method for preparing a three-phase silk ligament graft, comprising the following steps:

1) Weave the silk with a weaving machine into a mesh silk support with a hole diameter of 2-4mm;

2) Divide one side surface of the meshed silk stent longitudinally along the meshed silk stent into the first transition zone, the central ligament reconstruction zone and the second transitional zone, divide the first transition zone and the second transitional zone along the meshed silk stent Horizontally, it is divided into ligament reconstruction marginal area, cartilage modification area and bone modification area;

3) Use cobalt-60 irradiation to sterilize the mesh silk scaffold, and then plant bone marrow mesenchymal stem cells in the central area and edge area of ligament reconstruction, and plant bone marrow mesenchymal stem cells transfected with TGF-β gene lentivirus in the Cartilage modification area, bone marrow mesenchymal stem cells transfected with BMP-2 gene lentivirus were planted in the bone modification area, bone marrow mesenchymal stem cells, bone marrow mesenchymal stem cells transfected with TGF-β gene lentivirus and BMP Bone marrow mesenchymal stem cells transfected with -2 gene lentivirus were planted at a concentration of 0.8-1.5× ¹⁰⁵ cells/ ^cm2 ;

4) After the cells to be planted adhere to the meshed silk scaffold, the meshed silk scaffold is folded along its transverse direction in order from the ligament reconstruction edge area to the cartilage modification area and then to the bone modification area to make a columnar ligament graft .

The silk is desericinized before weaving, or the silk without desericin is used for weaving, and the meshed silk support obtained by weaving is desericinized before step 2).

The step of desericin treatment comprises: boiling an aqueous solution of sodium carbonate with a mass fraction of 0.2-0.3%, and then according to a ratio of 2-3 g of silk per liter of aqueous solution of sodium carbonate, the silk that has not been desericinized or the silk that has not been desericated The mesh-shaped silk support woven into the glued silk is placed in boiling sodium carbonate aqueous solution, and then continue to heat and boil for 20-30 minutes.

Before the step 3), modify the central area of ligament reconstruction, the edge area of ligament reconstruction, the cartilage modification area and the bone modification area. The specific method of modification is:

The first step is to use silk fibroin solution with a mass fraction of 1.8-2.0% at room temperature to soak the central area of ligament reconstruction, the edge area of ligament reconstruction and the bone modification area. At the same time, the solutes are gelatin, sodium hyaluronate and cartilage sulfate Soak the cartilage modification area with the solution of protein, then let the mesh silk scaffold stand at -15 to -25°C for 1-1.5 hours, and then stand at -70 to -80°C for 1-1.5 hours;

In the second step, after the first step, vacuum freeze-dry the mesh silk support;

The third step, after the second step, soak the central area of the ligament reconstruction, the edge area of the ligament reconstruction, and the bone modification area with 90% methanol aqueous solution for 8-15 minutes, and then put the mesh silk scaffold into the drying oven to dry Or air dry naturally, and then prepare hydroxyapatite coating on the bone modification area.

The preparation method of the silk fibroin solution is as follows: dissolving silk in a lithium bromide aqueous solution under the condition of heating and stirring to obtain a solution a, dialyzing the solution a with deionized water, and dialysis the solution a at 4° C., 8000 ° C. Centrifuge under the condition of -10000 rpm, take the centrifuged supernatant to obtain silk fibroin solution.

Described solute is the preparation method of the solution of gelatin, sodium hyaluronate and chondroitin sulfate: 0.5g gelatin powder, 0.1g chondroitin sulfate powder and 5mg sodium hyaluronate are dissolved in 10-20mL double-distilled water, then to Add 2-3 mL of 1% EDAC to double-distilled water.

The vacuum freeze-drying conditions are as follows: a vacuum degree of 0.01-0.5 mbar, a temperature of -30 to -50° C., and a time of 48-72 hours.

The hydroxyapatite coating is prepared by alternating infiltration with two solutions, one of which is a calcium chloride solution prepared with a Tris-HCl buffer solution with a pH value of 7.4 and calcium chloride, and the other solution is Disodium hydrogen phosphate aqueous solution.

A preparation method of silk support, comprising the following steps:

1) Weave the silk with a weaving machine into a mesh silk support with a hole diameter of 2-4mm;

2) Use a partition culture device to divide the meshed silk scaffold into the first transition area, the central area of ligament reconstruction and the second transition area sequentially along the longitudinal direction of the meshed silk scaffold, and divide the first transition area and the second transition area along the meshed silk The bracket is laterally divided into ligament reconstruction edge area, cartilage modification area and bone modification area; the partition culture device includes a bottom plate, a sealing film arranged on the bottom plate, and an infiltration fluid chamber arranged on the sealing film, and the infiltration fluid chamber The interior is divided into a plurality of independent sub-chambers, and the sub-chambers correspond to the central area of ligament reconstruction, the edge area of ligament reconstruction, the cartilage modification area and the bone modification area. One corresponding through hole; place the mesh silk support between the sealing film and the bottom plate, and then clamp the infiltration fluid chamber and the bottom plate, during the clamping process, the sealing film is pressed into the mesh silk support to realize the central area of ligament reconstruction, Reconstruct the separation of marginal zone, cartilage modified zone and bone modified zone;

3) Use silk fibroin solution with a mass fraction of 1.8-2.0% in the corresponding sub-chamber at room temperature to soak the central area of ligament reconstruction, the edge area of ligament reconstruction and the bone modification area. At the same time, the solutes are gelatin and sodium hyaluronate And the solution of chondroitin sulfate to soak the cartilage modification area, and then put the mesh silk scaffold at -15 to -25°C for 1-1.5 hours, and then stand at -70 to -80°C for 1-1.5 hours ;

4) After step 3), vacuum freeze-dry the mesh silk scaffold;

5) After step 4), soak the central area of the ligament reconstruction, the edge area of the ligament reconstruction, and the bone modification area with 90% methanol aqueous solution for 8-15 minutes. After air drying, a hydroxyapatite coating was prepared on the bone modification area.

The silk is desericinized before weaving, or the silk without desericin is used for weaving, and the meshed silk support obtained by weaving is desericinized before step 2).

The step of desericin treatment comprises: boiling an aqueous solution of sodium carbonate with a mass fraction of 0.2-0.3%, and then according to a ratio of 2-3 g of silk per liter of aqueous solution of sodium carbonate, the silk that has not been desericinized or the silk that has not been desericated The mesh-shaped silk support woven into the glued silk is placed in boiling sodium carbonate aqueous solution, and then continue to heat and boil for 20-30 minutes.

The preparation method of the silk fibroin solution is as follows: dissolving silk in a lithium bromide aqueous solution under the condition of heating and stirring to obtain a solution a, dialyzing the solution a with deionized water, and dialysis the solution a at 4° C., 8000 ° C. Centrifuge under the condition of -10000 rpm, take the centrifuged supernatant to obtain silk fibroin solution.

Described solute is the preparation method of the solution of gelatin, sodium hyaluronate and chondroitin sulfate: 0.5g gelatin powder, 0.1g chondroitin sulfate powder and 5mg sodium hyaluronate are dissolved in 10-20mL double-distilled water, then to Add 2-3 mL of 1% EDAC to double-distilled water.

The vacuum freeze-drying conditions are as follows: a vacuum degree of 0.01-0.5 mbar, a temperature of -30 to -50° C., and a time of 48-72 hours.

The hydroxyapatite coating is prepared by alternating infiltration with two solutions, one of which is a calcium chloride solution prepared with a Tris-HCl buffer solution with a pH value of 7.4 and calcium chloride, and the other solution is Disodium hydrogen phosphate aqueous solution.

The application of the silk scaffold prepared by the above method for preparing the silk scaffold in the preparation of ligament grafts.

The beneficial effects of the present invention are reflected in:

The silk scaffold of the present invention is divided into a ligament area, a cartilage modification area and a bone modification area, and after the corresponding cells are planted, they are folded in order, so that the ligament-bone junction of the three-phase silk ligament graft is in the transverse direction. On the cut surface, there is a transitional structure from the inside to the outside from "ligament→cartilage→bone", simulating and reconstructing the physiological transition structure of the normal ligament-bone junction; tissue structure, which can form new bone in the graft bone canal, and the biological fixation of the ligament graft-osseointegration is more firm, not easy to pull out, and increases the insertion strength of the ligament; because the three-phase silk ligament graft has the same - The transition structure (ligament→cartilage→bone) similar to the osseointegration can effectively avoid the concentration of stress and improve the biomechanical properties of the ligament graft.

Since silk is a resorbable material with good toughness, the ligament graft has a complete weaving structure, the layers are closely connected, and the mechanical strength is similar to that of normal ligaments. After transplantation, on the one hand, the slow absorption of the scaffold material weakens the mechanical strength and provides space for cell proliferation to reduce the allogeneic tissue; on the other hand, the proliferation and differentiation of cells form tissue and enhance the mechanical strength of the material.

Since the preparation of the three-phase ligament graft adopts the method of first culturing the seed cells on the mesh support, and then rolling the mesh support to form a cylindrical ligament, it is ensured that cells also grow into the deep layer of the cylinder. It solves the problem that the seed cells cannot enter the deep layer of the ligament in the traditional ligament weaving method.

The method of the present invention only uses one kind of seed cell-bone marrow mesenchymal stem cells, and the bone marrow mesenchymal stem cells transfected with TGF-β and BMP-2 genes by lentiviral vectors have good differentiation ability, and can be used in the local microbiome of the three districts. Under the influence of the environment, they differentiate into ligament cells, chondrocytes and osteoblasts, which is conducive to the formation of the normal anatomical structure of the ligament and bone joint.

The method of the present invention inoculates bone marrow mesenchymal stem cells carrying BMP-2 gene and TGF-β gene in the bone modified area and cartilage modified area, and the cells transfected by the gene will continuously express BMP-2 in the process of proliferation and differentiation. 2 (bone morphogenetic protein-2) and TGF-β (transforming growth factor-β), two cytokines, can effectively induce bone marrow mesenchymal stem cells to differentiate into bone and cartilage.

The partitions in the present invention are respectively modified by silk fibroin, gelatin+sodium hyaluronate+chondroitin sulfate and silk fibroin+hydroxyapatite, which not only forms micropores on the macroporous structure of the braided support, but also increases the density of the material. The surface area facilitates the growth of bone marrow mesenchymal stem cells, and also creates a microenvironment suitable for the differentiation of bone marrow mesenchymal stem cells into three different types of cells (ligament cells, chondrocytes, and osteoblasts), which is more conducive to the survival and proliferation of differentiated cells , effectively avoiding the dedifferentiation and aging of differentiated cells.

Description of drawings

Fig. 1 is a schematic diagram of partitioning and folding of the silk stent body of the present invention; in Fig. 1, A is a ligament area, B is a cartilage modification area, and C is a bone modification area;

Fig. 2 is the microstructure figure of silk support of the present invention;

Fig. 3 is the microstructural figure of graft described in the present invention;

Detailed ways

The present invention will be further described below in conjunction with accompanying drawing.

An ideal tissue engineered ligament should have the following characteristics: ①The scaffold material has good biocompatibility; ②The tissue engineered ligament has sufficient mechanical strength and fatigue resistance; ③The maintenance time of the ligament strength must be long enough to benefit the ligament. Reconstruction; ④The ligament-bone junction needs to regenerate a typical four-layer structure of the ligament-bone junction (collagen fibers, fibrocartilage, calcified fibrocartilage, and bone), which is conducive to increasing the fixation strength of the ligament-bone junction and avoiding stress concentration; ⑤The seed cells can grow and proliferate on the scaffold material; ⑥The seed cells should be evenly distributed in the inner and outer layers of the ligament.

In order to meet the above requirements, biological reconstruction of tissue engineered ligaments composed of cells and absorbable biomaterials is the ultimate development direction. Silk is light in weight (1.3g/cm ³ ), high in strength (about 4.8GPa), and good in elasticity (elastic deformation up to 35%). The woven silk scaffold can obtain a mechanical strength similar to that of ACL. Silk has good biocompatibility, and seed cells can adhere, proliferate and differentiate on the scaffold. Silk degrades very slowly and maintains adequate mechanical properties during ligament reconstruction. To truly regenerate ligaments, cells need to be planted on the scaffolds to form new ligaments through cell proliferation and continuous secretion of matrix. At the same time, the artificial scaffolds are continuously degraded to achieve the goal of ligament regeneration. Silk fibroin, gelatin + sodium hyaluronate + chondroitin sulfate and silk fibroin + hydroxyapatite can be used to modify the mesh silk scaffold to provide more suitable for the growth and proliferation of fibroblasts, chondrocytes and osteoblasts, respectively. unique microenvironment. Divide the mesh silk scaffold into ligament area, cartilage area (i.e. cartilage modification area) and bone area (i.e. bone modification area), use different materials to modify respectively, and plant corresponding cells, and then roll the mesh silk scaffold into columnar ligament Grafts that can reconstruct the transitional structure of the ligament-bone junction. At the same time, the coiled mesh silk scaffold that has been evenly planted with cells can also allow cells to grow into the deep layer of the columnar ligament graft, which is more conducive to the regeneration of the ligament.

Accordingly, the present invention proposes a method for preparing a three-phase silk ligament graft for reconstructing the normal anatomical structure of the ligament-bone junction. The prepared ligament graft can simulate the anatomical structure of the normal ligament-bone junction to obtain a The high-strength fixation of the bone joint avoids stress concentration, and at the same time has a complete braided structure to obtain ideal mechanical strength, including the following steps:

1. Preparation of Mesh Scaffold

1.1 silk desilicone glue

Boil a sodium carbonate aqueous solution with a mass fraction of 0.2-0.3%, and place the silk that has not been desericinized in the boiling sodium carbonate aqueous solution according to the ratio of 2-3g silk per liter of sodium carbonate aqueous solution, and then stir and heat to continue boiling for 20- 30 minutes. Take out the silk, wash the silk three times with deionized water, and dry it in the shade.

1.2 Weaving of mesh support

1.2.1 Weaving with desericin silk

Use the knitting machine to weave the de-sericinized silk into a net-shaped silk support with an aperture of 2-4 mm by weft flat needle or threaded needle technology.

1.2.2 Weaving with silk that has not been removed from sericin

Use the weaving machine to weave the non-desilicified silk into a netted silk support with an aperture of 2-4 mm by weft flat needle or threaded needle technology. Then use the method in 1.1 to remove the sericin from the woven meshed silk stent to obtain a meshed silk stent from which the sericin has been removed.

According to the actual situation, if the silk is easily polluted during the weaving process, you can choose to adopt the method as described in 1.2.2.

2. Modification of mesh silk scaffolds

2.1 Partition of mesh silk support

Divide the mesh silk scaffold into three areas (as shown in Figure 1): Area A: ligament area, Area B: cartilage modification area, and Area C: bone modification area. The rolled and partitioned mesh silk scaffold can form a columnar tissue engineering ligament. The characteristic of the tissue engineered ligament is that the cross section of the two ends of the ligament forms a transition zone from the center to the edge of the ligament→cartilage→bone. Area A is modified with silk fibroin solution (silk solution). The A area in the central part of the mesh silk scaffold is the ligament reconstruction area, and the A area on both sides of the mesh silk scaffold can reconstruct the collagen ligament area in the bone tunnel of the ligament graft.

2.2 Modification of mesh silk scaffold

2.2.1 Preparation of silk fibroin solution

Make lithium bromide into a solution with a concentration of 9.3 mol/L with deionized water, and dissolve silk according to the ratio of 4 mL of lithium bromide solution to 1 g of silk. Weigh the silk to be dissolved and put it into a glass beaker, pour the lithium bromide solution on the silk in proportion, put in a stirring bar, place the beaker on a heatable magnetic stirrer, stir gently, and heat the liquid temperature Dissolve at 60°C for 3-5 hours until the silk is completely dissolved, which is recorded as solution a. Add solution a into a dialysis bag (3500MWCO), and use 1-1.5L of deionized water for each 10-12mL of solution a for dialysis, respectively, in 1 hour, 4 hours, the same night, the next morning, and the next night , The deionized water was updated in the morning of the third day. Put the solution a in the dialysis bag into a centrifuge tube, centrifuge at 9000 rpm for 30 minutes at a temperature of 4°C, take the supernatant, then centrifuge the supernatant once again according to the above conditions, and take The supernatant was obtained as a silk solution. Add 1mL of the prepared silk solution to the weighed container, dry it completely in an oven at 60°C, weigh it again, subtract the weight of the original container, divide it by 100mL, and obtain the final concentration of the silk solution, and finally make the mass The fraction is 2% silk solution.

2.2.2 Modification of each partition of the mesh silk scaffold

Use steel needles with a diameter of 1.5mm to make a rectangular frame, and support the meshed silk support. The diameter of the large holes between the frame grids is about 2-4mm. are independent between. The separated culture device includes a bottom plate, a sealing film arranged on the bottom plate, and an infiltration liquid chamber arranged on the sealing film, and the infiltration liquid chamber is divided into a plurality of independent sub-chambers, and the sub-chambers are connected to A and B. One-to-one correspondence between the sub-chambers and C areas, the sealing film is provided with through holes corresponding to the sub-chambers one-to-one; the mesh silk support is placed between the sealing film and the bottom plate, and then the infiltration liquid chamber and the bottom plate are clamped During the clamping process, the sealing film is pressed into the mesh silk support to realize the separation of A, B, and C areas.

The ligament area and bone modification area were fully infiltrated with the prepared 2.0% silk fibroin solution. The cartilage modification area is modified with gelatin + sodium hyaluronate + chondroitin sulfate: add 0.5g gelatin powder, 0.1g chondroitin sulfate powder and 5mg sodium hyaluronate into 10mL double distilled water and stir until dissolved, then add 2mL with a mass fraction of 1% Ethyldimethylaminopropyl carbodiimide aqueous solution to obtain solution b, pour solution b into the cartilage modification area, make it fully infiltrated with the mesh silk scaffold, glue at room temperature for 2-3min, and then place at -20°C Cross-linking was terminated for 1 hour and then placed at -80°C for 1 hour. Then the whole scaffold was subjected to vacuum freeze-drying (vacuum degree of 0.25mbar, temperature of -40°C) for 48 hours to complete the hole formation.

Then the ligament area and bone modification area were soaked in 90% methanol aqueous solution for 15 minutes, and then placed in a 60°C drying oven to dry and fix the holes made. After fixation, the hydroxyapatite coating was introduced into the modified bone area by the method of "alternative infiltration of two solutions". The specific steps are as follows: first prepare two kinds of solutions: ① Calcium solution: prepare 200 mM calcium chloride solution with Tris-HCl buffer solution with a pH value of 7.4; ② phosphorus solution: 120 mM disodium hydrogen phosphate aqueous solution. Then soak the bone modification area with the prepared calcium solution, and react in a 37°C incubator for 1 hour. Then pour out the calcium liquid, blot the remaining liquid with filter paper, then soak it with the prepared phosphorus solution, and react in a thermostat at 37°C for 1 hour. After exhausting the phosphorus solution, soak in calcium solution for 1 hour, then absorb calcium solution, add phosphorus solution and soak for 1 hour, finally absorb phosphorus solution, and form hydroxyapatite coating around silk after drying in the shade. Referring to Fig. 2, a hydroxyapatite coating is formed around the silk fiber, and a three-phase silk scaffold is obtained so far.

3. Seeding of Cells

^The prepared three-phase silk scaffold was sterilized by cobalt-60 irradiation, ^and bone marrow mesenchymal stem cells, bone marrow mesenchymal stem cells transfected with TGF-β gene lentivirus and Bone marrow mesenchymal stem cells transfected with BMP-2 gene lentivirus were correspondingly inoculated in the ligament area, cartilage modification area and bone modification area.

4. Folding bracket

After planting the cells for 24 hours to 7 days (the specific time is based on the fact that the cells adhere to the scaffold and can proliferate normally), the three-phase silk scaffold is folded in the direction of the transverse axis (the direction of the ligament cross-section) from ligament→cartilage→bone , to make a columnar ligament graft, and the cells adhere and grow on the material, as shown in Figure 3.

The ligament-bone joint of the three-phase silk ligament graft reconstructing the normal anatomical structure of the ligament-bone joint made by this method has a transitional structure from the inside to the outside on the cross-section, which simulates and The physiological transitional structure of the normal ligament-bone junction is reconstructed. This physiological transitional structure enables the ligament graft to form new bone in the grafted bone canal, and the biological fixation of the ligament graft-osseocombination is more firm and difficult to pull out, increasing the insertion strength of the ligament. At the same time, this transitional structure can effectively avoid the stress concentration of the ligament-bone joint and improve the fatigue resistance of the ligament graft. The three regions of the three-phase silk ligament graft were respectively modified by silk fibroin, gelatin + sodium hyaluronate + chondroitin sulfate and silk fibroin + hydroxyapatite, creating suitable for three different cells (ligament cells, chondrocytes) , osteoblasts) microenvironment. It is more conducive to the survival and proliferation of different cells, and effectively avoids the dedifferentiation and aging of differentiated cells. In addition, the three-phase silk mesh silk scaffold of the three-phase silk ligament graft has a complete weaving structure, and the layers are tightly connected, so that the tissue engineered ligament has a mechanical strength similar to that of a normal ligament. In addition, the preparation of the three-phase ligament graft adopts the method of first culturing the seed cells on the mesh scaffold, and then rolling the mesh scaffold to form a cylindrical ligament, which ensures that cells grow into the deep layer of the cylinder as well. It solves the problem that the seed cells cannot enter the deep layer of the ligament in the traditional ligament weaving method. Finally, this method uses only one kind of seed cell-bone marrow stromal stem cells, which can be multidirectionally differentiated by gene transfection, which reduces the complicated procedures and steps for planting a variety of cells in one transplant, as a multilineage differentiation potential. The proliferative ability of stem cells is significantly stronger than that of adult cells, which can effectively shorten the healing time.

In short, the present invention first weaves silk into a mesh-like scaffold with a macroporous structure, and then divides the scaffold into three areas: ligament area, cartilage area, and bone area. The ligament area is modified with silk fibroin solution to facilitate ligament regeneration. The cartilage area was modified with gelatin + sodium hyaluronate + chondroitin sulfate to facilitate cartilage regeneration, and the bone area was modified with silk fibroin solution and hydroxyapatite to facilitate bone regeneration. Bone marrow mesenchymal stem cells, bone marrow mesenchymal stem cells transfected with TGF-β gene lentivirus and bone marrow mesenchymal stem cells transfected with BMP-2 gene lentivirus were planted in the three areas respectively. The mesh scaffold modified by rolling and partitioning forms a bionic ligament graft with a physiological transition structure (ligament→cartilage→bone). This structure not only effectively avoids the problem of stress concentration caused by the direct connection of soft and hard tissues, but also has good directional differentiation characteristics of genetically modified bone marrow mesenchymal stem cells, which saves the cultivation time and steps of planting a variety of different cells, and also solves the problem of It solves the problem of unreliable biological fixation of the ligament graft-osseocombination existing in single-phase tissue engineering ligaments. Finally, the folding of the scaffold solves the problem that cells cannot grow deep into the traditional method.

Claims (8)

1. A three-phase silk ligament graft, characterized in that: comprising coiled silk scaffolds, bone marrow mesenchymal stem cells, bone marrow mesenchymal stem cells transfected with TGF-β gene lentivirus, and BMP-2 carrying BMP-2 Bone marrow mesenchymal stem cells transfected with gene lentivirus, the silk scaffold includes a silk scaffold body with a microporous structure, and one side surface of the silk scaffold body is divided into a first transition zone, a ligament reconstruction center zone and a second transition zone in sequence , the first transition zone and the second transition zone are respectively located at the two ends of the columnar silk scaffold, and the first transition zone and the second transition zone are divided into ligament reconstruction edge zone, cartilage modification zone and bone modification zone in turn along the rolling direction of the silk scaffold Bone marrow mesenchymal stem cells are located in the central area of ligament reconstruction and peripheral area of ligament reconstruction, bone marrow mesenchymal stem cells transfected with TGF-β gene lentivirus are located in the cartilage modification area, bone marrow mesenchymal stem cells transfected with BMP-2 gene lentivirus The mesenchymal stem cells are located in the bone modification area, the central area of the ligament reconstruction and the edge area of the ligament reconstruction are modified with silk fibroin, the cartilage modification area is modified with gelatin, sodium hyaluronate and chondroitin sulfate, and the bone modification area is sequentially modified with silk fibroin. Modified with protein and hydroxyapatite. 2. A three-phase silk ligament graft according to claim 1, characterized in that: the silk stent body is prepared from a reticular silk stent. 3. A preparation method for a three-phase silk ligament graft, characterized in that: comprising the following steps: 1) Weaving the silk into a netted silk support with an aperture of 2-4mm with a weaving machine; 2) Divide one side surface of the mesh silk stent vertically into the first transition zone, ligament reconstruction center zone and second transition zone, and divide the first transition zone and the second transition zone into ligament reconstruction edge zone in sequence , cartilage modification area and bone modification area; the ligament reconstruction central area and ligament reconstruction edge area are modified with silk fibroin, the cartilage modification area is modified with gelatin, sodium hyaluronate and chondroitin sulfate, and the bone modification area is sequentially modified with Modified with silk fibroin and hydroxyapatite; 3) Use cobalt-60 irradiation to sterilize the mesh silk scaffold, and then plant bone marrow mesenchymal stem cells in the center area and edge area of ligament reconstruction, and plant bone marrow mesenchymal stem cells transfected with TGF-β gene lentivirus in the Cartilage modification area, bone marrow mesenchymal stem cells transfected with BMP-2 gene lentivirus were planted in the bone modification area, bone marrow mesenchymal stem cells, bone marrow mesenchymal stem cells transfected with TGF-β gene lentivirus and BMP Bone marrow mesenchymal stem cells transfected with -2 gene lentivirus were planted at a concentration of 0.8-1.5× ¹⁰⁵ cells/ ^cm2 ; 4) After the cells to be planted adhere to the meshed silk scaffold, the meshed silk scaffold is folded along its transverse direction in order from the ligament reconstruction edge area to the cartilage modification area and then to the bone modification area to form a columnar ligament graft . 4. according to the preparation method of a kind of three-phase silk ligament graft described in claim 3, it is characterized in that: described silk carries out desericin treatment before weaving, or, carries out weaving with the silk that does not desericin, in step 2) Desericin treatment is performed on the meshed silk scaffold obtained by weaving. 5. according to the preparation method of a kind of three-phase silk ligament graft described in claim 3, it is characterized in that: before described step 3), carry out to ligament reconstruction center area, ligament reconstruction edge area, cartilage modification area and bone modification area Modification, the specific method of modification is: The first step is to use silk fibroin solution with a mass fraction of 1.8-2.0% at room temperature to soak the central area of ligament reconstruction, the edge area of ligament reconstruction and the bone modification area. At the same time, the solutes are gelatin, sodium hyaluronate and cartilage sulfate Soak the cartilage modification area with the solution of protein, then let the mesh silk scaffold stand at -15 to -25°C for 1-1.5 hours, and then stand at -70 to -80°C for 1-1.5 hours; In the second step, after the first step, vacuum freeze-dry the mesh silk support; The third step, after the second step, soak the central area of the ligament reconstruction, the edge area of the ligament reconstruction and the bone modification area with a methanol solution with a volume fraction of 90% for 8-15 minutes. After soaking, put the mesh silk scaffold into a drying oven to dry Or air dry naturally, and then prepare hydroxyapatite coating on the bone modification area. 6. A preparation method of silk support, characterized in that: comprising the following steps: 1) Weaving the silk into a netted silk support with an aperture of 2-4mm with a weaving machine; 2) Separate the mesh silk scaffold into the first transition zone, the central zone of ligament reconstruction and the second transition zone longitudinally and sequentially divide the first transition zone and the second transition zone into the edge of ligament reconstruction by using a partition culture device area, a cartilage modification area, and a bone modification area; the partition culture device includes a base plate, a sealing film arranged on the base plate, and an infiltration fluid chamber arranged on the sealing film, and the infiltration fluid chamber is divided into a plurality of independent sub-sections. The cavity and the sub-chamber correspond one-to-one to the central area of ligament reconstruction, the edge area of ligament reconstruction, the cartilage modification area and the bone modification area, and the through holes corresponding to the sub-chambers are opened on the sealing film; The mesh-shaped silk support is placed between the sealing film and the bottom plate, and then the infiltration fluid chamber and the bottom plate are clamped. During the clamping process, the sealing film is pressed into the mesh silk support to realize the central area of ligament reconstruction, the edge area of ligament reconstruction, the cartilage modification area and Separation of bone modification areas; 3) Use a silk fibroin solution with a mass fraction of 1.8-2.0% in the corresponding sub-chamber at room temperature to soak the central area of ligament reconstruction, the edge area of ligament reconstruction and the bone modification area. At the same time, the solutes are gelatin and sodium hyaluronate And the solution of chondroitin sulfate to soak the cartilage modification area, and then put the mesh silk scaffold at -15 to -25°C for 1-1.5 hours, and then stand at -70 to -80°C for 1-1.5 hours ; 4) After step 3), vacuum freeze-drying the mesh silk support; 5) After step 4), soak the central area of the ligament reconstruction, the edge area of the ligament reconstruction, and the bone modification area with methanol aqueous solution with a volume fraction of 90% for 8-15 minutes. After air drying, a hydroxyapatite coating was prepared on the bone modification area. 7. the preparation method of a kind of silk support according to claim 6, is characterized in that: described silk is carried out desericin treatment before weaving, or, carries out weaving with the silk that does not desericin, before step 2) will The meshed silk support obtained by weaving is subjected to desericinization treatment. 8. The application of the silk scaffold prepared by the preparation method of the silk scaffold as claimed in claim 6 in the preparation of ligament grafts.