CN106075575A - A kind of composite for knee osteoarthritis repair of cartilage and preparation method thereof - Google Patents

Abstract

The invention discloses a composite material for knee osteoarthritis cartilage repair, which is made of silk fibroin, collagen and hyaluronic acid, wherein the mass ratio of silk fibroin: collagen: hyaluronic acid is 30:60~70 : 2.5~10. The invention also discloses a preparation method thereof, which has simple process and easy-to-obtain and cheap raw materials. The obtained composite material can improve the disadvantages of insufficient mechanical properties and rapid degradation of collagen, and at the same time has the lubricating effect of hyaluronic acid, promotes cell proliferation and differentiation, and improves important physiological processes of joints. The composite material obtained by the present invention has the characteristics of good biocompatibility, degradability and non-immunogenicity, can effectively promote the reconstruction and regeneration of cartilage, and is a kind of better suitable for middle and early knee osteoarthritis Medical material for cartilage defect repair.

Description

Composite material for knee osteoarthritis cartilage repair and preparation method thereof

technical field

The invention belongs to the field of biomedicine, and in particular relates to a composite material for knee osteoarthritis cartilage repair and a preparation method thereof.

Background technique

Knee osteoarthritis (KOA) is a bone and joint disease characterized by inflammatory lesions such as deformation, hyperosteogeny, and necrosis of the knee articular cartilage in patients. The main pathological feature is the degeneration or even disappearance of articular cartilage. The treatment of KOA is mainly divided into surgical treatment and non-surgical treatment. The main methods of surgical treatment are: autologous or allogeneic osteochondral transplantation, tissue transplantation, joint debridement, drilling, osteotomy around the knee joint, knee arthrodesis, total knee arthroplasty (TKA), etc. Although TKA is effective in the treatment of KOA, it is mainly used for advanced patients, and there are also problems such as high cost, large surgical trauma, and wear and tear of the prosthesis. For young patients with mild articular cartilage wear in the early stage of KOA, TKA is not the best choice. Among other surgical methods, compared with drilling, arthroplasty, and microfracture, it has been clinically proven that autologous mosaic osteochondral transplantation has significant long-term clinical efficacy, but its disadvantage is at the expense of other cartilage. Therefore, it is imperative to develop a material that can effectively repair KOA cartilage defects.

Among the natural materials used in cartilage tissue engineering, silk fibroin has good biocompatibility, can maintain the normal growth state and performance of chondrocytes, and is used for cartilage and bone reconstruction and regeneration. As the most abundant and widely distributed protein in animals, collagen has the characteristics of unique triple helix structure, low antigenicity, non-toxicity, good biocompatibility and biodegradability, and has been widely used in tissue engineering scaffolds Biomedical fields such as materials, drug controlled and sustained release systems, gene delivery carriers, cosmetic surgery, etc.

Hyaluronic acid is a natural biomolecule widely present in skin and other tissues. It is the main component of connective tissues such as human intercellular matrix and joint synovial fluid. It fills the space between cells and collagen fibers and covers some epidermal tissues. On the human body, it is widely distributed in the soft connective tissue of the human body, and exists in almost pure form in the joint cavity. It can promote cell proliferation and differentiation, scavenge oxygen free radicals, promote wound healing, regulate osmotic pressure, lubricate, promote cell repair and improve joints. In clinical KOA non-surgical treatment, supplementing exogenous hyaluronic acid is one of the commonly used methods, which can increase the content of hyaluronic acid in synovial fluid, improve the physiological function of synovial fluid under pathological conditions, relieve joint pain, protect cartilage and synovial membrane damaged etc.

The invention combines the advantages of natural collagen, silk fibroin and hyaluronic acid to prepare a composite material that can be used for knee osteoarthritis cartilage repair. It can be used as a scaffold for the adhesion, proliferation and differentiation of endogenous bone marrow-derived stem cells, and at the same time, the hyaluronic acid produced by material degradation can enhance the repair effect and achieve the purpose of alleviating and treating KOA.

Contents of the invention

The object of the present invention is to provide a composite material for knee osteoarthritis cartilage repair and a preparation method thereof. The obtained composite material has the characteristics of good biocompatibility, degradability and non-immunogenicity, and can effectively promote the reconstruction and regeneration of cartilage. Repairing medical materials.

To achieve the above object, the present invention adopts the following technical solutions:

A composite material for knee osteoarthritis cartilage repair is made from silk fibroin solution, collagen, and hyaluronic acid as raw materials, wherein the mass ratio of silk fibroin:collagen:hyaluronic acid is 30:60~70: 2.5~10.

The collagen is derived from fish skin, pig skin, cow hide or beef tendon.

The preparation method of the composite material for knee osteoarthritis cartilage repair comprises the following steps:

(1) Prepare silk fibroin solution;

(2) Dissolving collagen and hyaluronic acid in 1 mol/L malonic acid to prepare a certain concentration of collagen solution and hyaluronic acid solution; the mass ratio of collagen to malonic acid is 0.4:100; The mass ratio of described hyaluronic acid and malonic acid is 0.5:100;

(3) Mix the silk fibroin solution obtained in step (1) with the collagen solution and hyaluronic acid solution obtained in step (2), stir evenly, freeze-dry and add a cross-linking agent for cross-linking for 10 hours; the cross-linking agent consists of 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinyl (NHS) and 90% ethanol solution, wherein the mass ratio of EDC/NHS is 5:2.

(4) After cross-linking, rinse with distilled water or deionized water repeatedly to remove excess cross-linking agent, and freeze-dry the second time to obtain a composite material for knee osteoarthritis cartilage repair.

Wherein the preparation method of silk fibroin solution is as follows:

1) Put the shredded silk cocoons into Na ₂ CO ₃ solution, place them in a water bath for 30 minutes, remove the gum on the cocoons, repeat the operation 3 times, and then dry them to obtain silk fibroin; the mass fraction of the Na ₂ CO ₃ solution is 0.5%, the water bath temperature is 98±2℃.

2) Weigh the silk fibroin described in step 1), dissolve it in the mixed solution of CaCl ₂ /H ₂ O/C ₂ H ₅ OH at 78°C, and filter it through gauze to obtain the coarse filtrate; where CaCl ₂ /H ₂ O The molar ratio of CaCl ₂ /H ₂ O/C ₂ H ₅ OH in /C ₂ H ₅ OH mixed solution is 1:8:2; the mass ratio of silk fibroin to CaCl ₂ /H ₂ O/C ₂ H ₅ OH mixed solution for 1:25.

3) Pour the crude filtrate obtained in step 2) into a dialysis bag for dialysis for 3 days to remove calcium chloride and ethanol; then filter to obtain a silk fibroin solution; the molecular weight cut-off of the dialysis bag used is 8000-14000.

The composite material for knee osteoarthritis cartilage repair prepared by the invention has a three-dimensional network structure, good hygroscopicity, simple preparation process, low price and easy availability. The obtained material can improve the shortcomings of insufficient mechanical properties and rapid degradation of collagen, and at the same time, hyaluronic acid can play a lubricating role, promote cell proliferation, differentiation and improve important physiological processes of joints. The composite material has the characteristics of good biocompatibility, degradability and non-immunogenicity, and can effectively promote the reconstruction and regeneration of cartilage. of medical materials.

Description of drawings

Figure 1 is the infrared spectrum of the composite material used for knee osteoarthritis cartilage repair, a: before cross-linking, b: after cross-linking.

Fig. 2 is a scanning electron micrograph of the composite material used for knee osteoarthritis cartilage repair, and A-E respectively represent the composite materials obtained in the first group to the fifth group in the embodiment.

detailed description

Preparation of Composite Materials for Cartilage Repair in Osteoarthritis of the Knee

Example 1

1) Put the shredded silkworm cocoons into 0.5% Na ₂ CO ₃ solution, place them in a water bath at 98±2°C for 30 minutes, remove the gum on the silkworm cocoons, and repeat the operation 3 times. After drying in an oven at 60°C, silk fibroin is obtained. Weigh the above degummed silk fibroin, dissolve it in 78°C ternary solution CaCl ₂ /H ₂ O/C ₂ H ₅ OH solution (molar ratio 1:8:2), bath ratio 1:25, and filter through gauze. Pour the solution into a dialysis bag with a molecular weight cut-off of 8000-14000 and dialyze for 3 days to remove calcium chloride and ethanol; filter to obtain a silk fibroin solution.

2) Weigh 1.3 g of bovine tendon collagen and dissolve it in 323.7 g of 1 mol/L malonic acid solution, place it on a stirrer and stir to dissolve, and prepare a collagen solution with a mass fraction of 0.4%.

3) Weigh 0.089 g of hyaluronic acid and dissolve it in 19.9 g of malonic acid solution with a concentration of 1 mol/L, place it on a stirrer and stir to dissolve, and prepare a collagen solution with a mass fraction of 0.5%.

4) Mix the silk fibroin solution with the beef tendon collagen solution, put it on a magnetic stirrer and stir evenly, then slowly add the hyaluronic acid solution to make a uniformly mixed silk fibroin/collagen/hyaluronic acid solution, the silk fibroin The mass ratio of protein (SF)/collagen (Col)/hyaluronic acid (HA) is 30:67.5:2.5. Freeze-drying; use a cross-linking agent to cross-link the freeze-dried composite material once, and repeatedly rinse with deionized water to remove the excess cross-linking agent. The cross-linking agent is carbonized by 1-ethyl-3 (3-dimethylaminopropyl) Diimine, N-hydroxysuccinyl and 90% ethanol solution, in which the mass ratio of EDC/NHS is 5:2. Then the composite material used for knee osteoarthritis cartilage repair was obtained by secondary freeze-drying, which was recorded as the first group. The infrared images of the composite material before and after crosslinking are shown in Figure 1. It can be seen from Figure 1 that after cross-linking, the ratio of the absorption rate of the peaks at 1234 cm ^-1 and 1450 cm ^-1 of the composite material is close to 1, indicating that the collagen in the material still maintains a complete tertiary structure, and the biological activity is well preserved; Compared with before linking (Fig. 1a), the positions and intensities of the absorption peaks at 1630 cm ^-1 (the C=O stretching vibration peak of the amide I band) and 1234 cm ^-1 (the amide III band) of the composite material changed after cross linking. Effective crosslinking occurs, which is beneficial to the improvement of the degradation resistance and mechanical properties of the composite material.

Example 2

The mass ratio of silk fibroin/collagen/hyaluronic acid in step 4) was changed to 30:65:5, and other steps were the same as in Example 1 to prepare composite material C.

Example 3

The mass ratio of silk fibroin/collagen/hyaluronic acid in step 4) was changed to 30:62.5:7.5, and other steps were the same as in Example 1 to prepare composite material D.

Example 4

Composite material E was prepared by changing the mass ratio of silk fibroin/collagen/hyaluronic acid in step 4) to 30:60:10, and other steps were the same as in Example 1.

Comparative example:

The mass ratio of silk fibroin/collagen/hyaluronic acid in step 4) was changed to 30:70:0, and other steps were the same as in Example 1 to prepare composite material A.

The water absorption test of the above-mentioned composite material used for knee osteoarthritis cartilage repair:

Weigh 0.01 g of the composite material (W ₁ ) used for knee osteoarthritis cartilage repair, put it in a petri dish, add 1800 mL of PBS buffer solution to each well, place it at 37°C for 24 hours, and weigh its weight (W 1 ) respectively. ₂ ), water absorption (%)=(W ₂ -W ₁ )/W ₁ ×100%, each experiment was done 3 times in parallel, and the average value was taken, the results are as follows:

Table 1. The water absorption of the composite material obtained in the above examples.

It can be seen from the above table that the water absorption rate of the composite materials used for knee osteoarthritis cartilage repair obtained in the above examples and comparative examples decreases with the increase of hyaluronic acid content, but the overall difference is not significant.

The composite material obtained in the above examples for knee osteoarthritis cartilage repair has a three-dimensional network structure (as shown in Figure 2), and has the characteristics of good hygroscopicity, biocompatibility, degradability and non-immunogenicity .

The above descriptions are only preferred embodiments of the present invention, and all equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope of the present invention.

Claims (7)

1. the composite for knee osteoarthritis repair of cartilage, it is characterised in that: with fibroin albumen, collagen, transparent Matter acid is that raw material prepares, described fibroin albumen: collagen: the mass ratio of hyaluronic acid is 30:60 ~ 70:2.5 ~ 10.

The most according to claim 1 for the composite of knee osteoarthritis repair of cartilage, it is characterised in that: described collagen For coming from fish skin, Corii Sus domestica, Corii Bovis seu Bubali or beef tendon.

3. a preparation method for the composite for knee osteoarthritis repair of cartilage as claimed in claim 1, it is special Levy and be: comprise the steps:

(1) silk fibroin protein solution is prepared；

(2) respectively collagen, hyaluronic acid are dissolved in malonic acid and are prepared as certain density collagen solution and hyaluronic acid solution；

(3) step (1) gained silk fibroin protein solution is mixed with collagen solution and the hyaluronic acid solution of step (2) gained, stir Mix uniformly, add cross-linking agent after lyophilization and cross-link；

(4) repeatedly rinsing with water after crosslinking, secondary freeze drying i.e. obtains the composite for knee osteoarthritis repair of cartilage.

Composite material and preparation method thereof for knee osteoarthritis repair of cartilage the most according to claim 3, its feature exists In: collagen described in step (2) is 0.4:100 with the mass ratio of malonic acid；Described hyaluronic acid with the mass ratio of malonic acid is 0.5:100。

Composite material and preparation method thereof for knee osteoarthritis repair of cartilage the most according to claim 3, its feature exists In: cross-linking agent described in step (3) is by 1-ethyl-3(3-dimethyl aminopropyl) carbodiimides, N-maloyl be dissolved in Volumetric concentration be 90% ethanol solution in prepare, wherein 1-ethyl-3(3-dimethyl aminopropyl) carbodiimides and N-hydroxyl The mol ratio of succinyl is 5:2.

The preparation method of the composite for knee osteoarthritis repair of cartilage the most according to claim 3, its feature It is: the preparation method of described silk fibroin protein solution is as follows:

(1) Bombyx bombycis shredded being put into mass fraction is 0.5%Na₂CO₃Solution, places 30min in 98 ± 2 DEG C of water-baths, removes Colloid on Bombyx bombycis, 3 post-dryings of repeatable operation, obtain fibroin；

(2) fibroin described in step (1) is weighed, at 78 DEG C, at CaCl₂/H₂O/C₂H₅OH mixed solution dissolves, gauze mistake Filter, obtains coarse filtration liquid；Described CaCl₂/H₂O/C₂H₅The middle CaCl of OH mixed solution₂/H₂O/C₂H₅The mol ratio of OH is 1:8:2；

(3) step (2) gained coarse filtration liquid is poured 3 d that dialyse in bag filter into, remove calcium chloride and ethanol；Refilter, obtain silk Fibroin solution；The molecular cut off of described bag filter is 8000-14000.

Composite material and preparation method thereof for knee osteoarthritis repair of cartilage the most according to claim 4, its feature exists In: described fibroin and CaCl₂/H₂O/C₂H₅The mass ratio of OH mixed solution is 1:25.