CN106729987A - A kind of collagen/chitosan/Sodium Hyaluronate compound rest of load KGN - Google Patents

Abstract

The invention discloses a collagen/chitosan/sodium hyaluronate composite scaffold loaded with KGN. The scaffold is prepared from collagen, chitosan, sodium hyaluronate, KGN (kartogenin) and PLGA. The preparation method includes preparation of PLGA microspheres loaded with KGN small molecules, mixing of PLGA microspheres and collagen/chitosan/transparent Preparation steps of the sodium erythronate composite scaffold. The composite scaffold prepared by the invention has the characteristics of good biocompatibility, biodegradability, etc., and at the same time has the lubricating effect of sodium hyaluronate and the effect of KGN on promoting chondrogenic differentiation of bone marrow mesenchymal stem cells, which is beneficial to middle and early bone marrow. It is a good tissue regeneration material for osteoarthritis cartilage defect repair.

Description

A Collagen/Chitosan/Sodium Hyaluronate Composite Scaffold Loaded with KGN

technical field

The invention belongs to the field of biomedicine, and in particular relates to a collagen/chitosan/sodium hyaluronate composite bracket loaded with KGN.

Background technique

In recent years, with the acceleration of population aging, more and more people are troubled by articular cartilage damage, especially patients with osteoarthritis (Osteoarthritis, OA). OA is a multifactorial degenerative disease characterized by degeneration of articular cartilage, reactive hyperplasia of joint margins and subchondral bone, clinically manifested as slowly developing joint pain, tenderness, stiffness, joint swelling, limited mobility and joint deformity etc. The most effective method to treat OA clinically is joint replacement, but this method may cause many adverse reactions and is expensive, so it is generally only used in advanced OA patients. At present, there is no effective treatment for patients with early and middle stages of OA, and most methods can only temporarily relieve the symptoms of patients, but cannot delay or prevent the progress of OA. Therefore, it is imminent to develop an effective OA treatment method and technology.

In recent years, a large number of studies have shown that OA is related to the degeneration of articular cartilage. Therefore, effective repair of articular cartilage degeneration in OA patients may be an effective way for the clinical treatment of OA. In the repair of articular cartilage defects, the application of stem cell-directed differentiation to regenerate articular cartilage has become a research hotspot. Mesenchymal stem cells (Mesenchymal stem cells, MSCs) have multi-directional cell differentiation potential, and it is of great significance for the treatment of OA to reasonably promote the directional differentiation of MSCs in OA patients to chondrocytes.

In 2012, a new small molecule compound kartogenin (KGN) was discovered. It is a compound screened by Johnson et al. from more than 22,000 heterocyclic drug molecules with different structures. Mesenchymal stem cells differentiate into chondrocytes. Therefore, using KGN to promote cartilage-oriented differentiation of MSCs to develop a cartilage repair scaffold loaded with KGN may be effective in the repair of cartilage defects in OA.

Collagen (Collagen, COL) is an extracellular matrix component of many tissues and has been used in a variety of tissue engineering scaffold materials. However, due to its shortcomings such as fast degradation rate and poor mechanical properties when used alone, it is often combined with other materials.

Chitosan (CS) is a natural polymer biomaterial, mainly found in the shells of marine organisms and fungal cell walls. It is a degradable biomaterial with a wide range of sources, easy access and good biocompatibility. . It has excellent properties such as non-toxicity, non-irritation, biocompatibility, biodegradability, and easy modification. It has a good application prospect.

Sodium hyaluronate (Hyaluronic acid sodium salt, HAS) is a natural biomolecule widely present in skin and other tissues, and is the main component of connective tissues such as human interstitial cells and joint synovial fluid. It is almost in pure form in the joint cavity. exist. 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 OA non-surgical treatment, supplementing exogenous sodium hyaluronate is one of the common methods, which can increase the content of sodium hyaluronate in synovial fluid, improve the physiological function of synovial fluid under pathological conditions, relieve joint pain, protect cartilage and synovial fluid. Membrane from damage and so on.

The invention synthesizes the respective advantages of KGN, natural collagen, chitosan and sodium hyaluronate to prepare a composite bracket that can be used for repairing cartilage defects in osteoarthritis. It can promote the cartilage differentiation of autologous bone marrow-derived MSCs by releasing loaded KGN, and at the same time release sodium hyaluronate through scaffold degradation to enhance the repair effect, so as to relieve and treat OA.

Contents of the invention

The object of the present invention is to provide a collagen/chitosan/sodium hyaluronate composite scaffold loaded with KGN for repairing osteoarthritis cartilage defect and a preparation method thereof. The prepared composite material has the characteristics of good biocompatibility, degradability and non-immunogenicity. At the same time, the scaffold material can effectively promote the reconstruction and regeneration of articular cartilage. Medical material for osteoarthritis treatment.

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

A collagen/chitosan/sodium hyaluronate composite scaffold loaded with KGN is prepared from collagen solution, chitosan solution, sodium hyaluronate solution and PLGA microspheres loaded with KGN small molecules as main raw materials, the collagen Solution concentration is 0.6wt%, chitosan solution concentration is 1wt%, sodium hyaluronate solution concentration is 1wt%; the volume ratio of described collagen solution, chitosan solution and sodium hyaluronate solution is 1～10:10 : 1 ~ 10; the mass percent of the PLGA microspheres loaded with KGN small molecules in the composite scaffold is 0.03wt%.

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

The preparation method of the PLGA microspheres loaded with KGN small molecules is as follows:

(A) Accurately weigh 200 mg of PLGA and dissolve in 10 mL of dichloromethane, and ultrasonically oscillate to form an oil phase (O);

(B) Accurately weigh 10 mg of KGN and place it in 1 mL of a mixture of ultrapure water and methanol (the volume ratio of ultrapure water to methanol is 1:1), and dissolve it completely at 37°C to form an inner aqueous phase (W1);

(C) Slowly inject the inner water phase (W1) described in step (B) into the oil phase (O) described in step (A) and continue stirring and emulsifying for 10 min to make it uniformly dispersed to form a colostrum emulsion (W1/O);

(D) The colostrum emulsion (W1/O) described in step (C) was dropped into 50 mL of 1wt% PVA solution containing 5 mL of 0.1wt% Tween-20 at 60 drops/min to form an external water phase (W2), Continuous stirring to form double milk (W1/O/W2);

(E) Fully centrifuge the double emulsion (W1/O/W2) described in step (D), discard the supernatant, wash and centrifuge repeatedly with ultrapure water for 3 times, freeze-dry, and store in the dark at -20°C.

The preparation method of the above-mentioned collagen/chitosan/sodium hyaluronate composite scaffold loaded with KGN comprises the following steps: (1) dissolving collagen and chitosan in 0.1wt% glacial acetic acid to prepare a collagen solution and chitosan solution;

(2) Prepare hyaluronic acid solution by dissolving sodium hyaluronate in distilled water or deionized water;

(3) Mix the collagen solution obtained in step (1) with the chitosan solution, and vortex to mix evenly;

(4) Slowly add the sodium hyaluronate solution obtained in step (2) into the mixing and swirling solution in step (3), and mix well;

(5) Add the PLGA microspheres loaded with KGN small molecules into the mixed solution obtained in step (4), stir overnight with magnetic force, freeze-dry and add a cross-linking agent for cross-linking;

(6) After cross-linking, wash repeatedly with Na ₂ HPO ₃ , NaCl and distilled water until neutral, and freeze-dry for the second time to obtain the collagen/chitosan/sodium hyaluronate composite scaffold loaded with KGN.

The cross-linking agent described in step (5) is composed of 2-(N-morpholine) ethanesulfonic acid monohydrate, 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide and N- Hydroxysuccinimide is prepared by dissolving 40% ethanol solution in which 2-(N-morpholine)ethanesulfonic acid monohydrate, 1-ethyl-3(3-dimethylaminopropyl The molar ratio of carbodiimide to N-hydroxysuccinimide is 50:33:8.

The Na ₂ HPO ₃ and NaCl in step (6) are 0.1M Na ₂ HPO ₃ , 1M NaCl and 2M NaCl, respectively.

The beneficial effect of the present invention is that: the KGN-loaded collagen/chitosan/sodium hyaluronate composite scaffold prepared by the present invention has a three-dimensional network structure, has good hygroscopicity, and has a simple and mature preparation process. The obtained material can improve the shortcomings of insufficient mechanical properties and rapid degradation of collagen, and has good biocompatibility, non-toxicity, degradability and non-immunogenicity, among which hyaluronic acid can play an important role in lubricating and improving joint physiological processes At the same time, the release of KGN loaded in the material can be as long as 12 weeks, and it is a better medical material suitable for the treatment of middle and early osteoarthritis.

Description of drawings

Fig. 1 is the water absorption figure of the collagen/chitosan/sodium hyaluronate composite scaffold loaded with KGN;

Figure 2 (A) is a scanning electron micrograph of the collagen/chitosan/sodium hyaluronate composite scaffold loaded with KGN prepared in the present invention;

Figure 2(B) is the scanning electron micrograph of the collagen/chitosan/sodium hyaluronate composite scaffold without KGN loaded.

detailed description

The present invention will be described in further detail below in conjunction with the accompanying drawings and embodiments.

Embodiment one

(1) Accurately weigh 200 mg of PLGA and dissolve in 4 mL of dichloromethane, and shake to form an oil phase (O);

(2) Accurately weigh 10 mg KGN and place it in 1 mL of a mixture of ultrapure water and methanol, and dissolve it completely at 37°C to form an inner water phase (W1); the volume ratio of ultrapure water to methanol is 1 :1;

(3) Slowly inject the inner water phase (W1) in step (2) into the oil phase (O) in step (1) and continue stirring and emulsifying for 10 min, so that it can be evenly dispersed to form a colostrum emulsion (W1/O);

(4) Drop the colostrum emulsion (W1/O) described in step (3) into 50 mL of 1wt% PVA solution containing 5 mL of 0.1wt% Tween-20 at 60 drops/min to form an external water phase (W2), Continuous stirring to form double milk (W1/O/W2);

(5) Fully centrifuge the double emulsion (W1/O/W2) described in step (4), discard the supernatant, then repeatedly wash and centrifuge with ultrapure water for 3 times, freeze-dry, and store in the dark at -20°C. PLGA microspheres loaded with KGN small molecules were prepared;

(6) Weigh 45.1127 g of bovine tendon collagen and dissolve it in 100 mL of glacial acetic acid solution with a concentration of 0.1 wt %, place it on a stirrer and stir to dissolve, and prepare a collagen (COL) solution with a mass fraction of 0.6 %;

(7) Weigh 1.0 g of chitosan and dissolve it in 100 mL of 0.1% glacial acetic acid solution, place it on a stirrer and stir to dissolve, and prepare a chitosan (CS) solution with a mass fraction of 1%;

(8) Weigh 0.5 g of sodium hyaluronate and dissolve it in 50 mL of distilled water or deionized water solution, place it on a stirrer and stir to dissolve, and prepare a hyaluronic acid (HSA) solution with a mass fraction of 1%;

(9) Add the bovine tendon collagen solution into a 50 mL centrifuge tube, vortex on a vortex, then add chitosan solution to the vortex to mix, then slowly add sodium hyaluronate solution to make a mixed collagen/shell Polysaccharide/sodium hyaluronate solution;

(10) Add the PLGA microspheres loaded with KGN small molecules prepared in step (5) to the collagen/chitosan/sodium hyaluronate mixed solution at 500 μg/mL; put the mixed solution on a magnetic stirrer Stir overnight, wherein the volume ratio of 0.6% COL, 1% CS and 1% HAS solution is 1:1:1;

(11) Freeze-drying in a freeze dryer; use a cross-linking agent to cross-link the freeze-dried composite material, and repeatedly wash with 0.1M Na ₂ HPO ₃ , 1M NaCl, 2M NaCl distilled water until neutral, and the cross-linking agent consists of 2- (N-Morpholine)ethanesulfonic acid monohydrate (MES), 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide dissolved in The volume concentration is prepared in 40% ethanol solution, wherein the mass ratio of MES/EDC/NHS is 50:33:8; then freeze-dried twice to obtain the collagen/chitosan/sodium hyaluronate composite scaffold loaded with KGN.

Embodiment two

In step (6) of Example 1, change bovine tendon collagen to fish skin collagen; in step (10), change the volume ratio of 0.6% COL, 1% CS and 1% HAS solution to 5:10 : 10, the other steps are the same as in Example 1, preparing the collagen/chitosan/sodium hyaluronate composite scaffold loaded with KGN.

Embodiment Three

In step (6) of Example 1, change bovine tendon collagen to pig skin collagen; in step (10), change the volume ratio of 0.6% COL, 1% CS and 1% HAS solution to 1:10 : 10, the other steps are the same as in Example 1, preparing the collagen/chitosan/sodium hyaluronate composite scaffold loaded with KGN.

Embodiment four

In step (6) of Example 1, the bovine tendon collagen was changed to bovine skin collagen; in step (10), the volume ratio of 0.6% COL, 1% CS and 1% HAS solution was changed to 10:10: 5. The other steps were the same as in Example 1, and collagen/chitosan/sodium hyaluronate composite scaffolds loaded and unloaded with KGN were prepared respectively.

Embodiment five

In step (10) of Example 1, change the volume ratio of 0.6% COL, 1% CS and 1% HAS solution to 10:10:1, and the other steps are the same as in Example 1 to prepare KGN-loaded collagen /chitosan/sodium hyaluronate composite scaffold.

Water absorption test of collagen/chitosan/sodium hyaluronate composite scaffold loaded with KGN:

The dry-loaded KGN collagen/chitosan/sodium hyaluronate composite scaffold prepared in the above example was taken out with a hole puncher to take a circle with a diameter of 7 mm, weighed its mass W1, immersed in PBS (Ph=7.4), and swelled in a water bath at 37°C for 24 After h, take it out, blot the surface moisture, weigh its weight W0, calculate the water absorption rate according to the formula, perform the experiment 3 times in parallel, and take the average value.

Water swelling rate (%)=(W0-W1)/W1×100%

In the formula, W1 is the weight of the dry material (mg), and W0 is the weight of the material after swelling (mg).

The results are shown in Figure 1. It can be seen from Figure 1 that the collagen/chitosan/sodium hyaluronate composite scaffolds loaded with KGN in different proportions prepared in the above examples all showed good water absorption performance, and the ratio of 0.5:1:1 was the best, and other materials There is no difference in water absorption.

The composite scaffolds were characterized by SEM. The results showed that the prepared and unloaded collagen/chitosan/sodium hyaluronate composite scaffolds showed a three-dimensional network structure (Fig. The PLGA microspheres loaded with KGN were clearly visible in the composite scaffold (Fig. 2(B)).

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 (8)

1. A collagen/chitosan/sodium hyaluronate composite scaffold loaded with KGN, characterized in that: the PLGA microspheres of collagen solution, chitosan solution, sodium hyaluronate solution and loaded KGN small molecules are main raw materials Prepared, the collagen solution concentration is 0.6wt%, the chitosan solution concentration is 1wt%, the sodium hyaluronate solution concentration is 1wt%; the volume ratio of the collagen solution, chitosan solution and sodium hyaluronate solution 1~10:10:1~10. 2. The collagen/chitosan/sodium hyaluronate composite scaffold loaded with KGN according to claim 1, characterized in that: the mass percent of PLGA microspheres loaded with KGN small molecules in the composite scaffold is 0.03wt%. 3. The collagen/chitosan/sodium hyaluronate composite scaffold loaded with KGN according to claim 1, characterized in that: the collagen is derived from fish skin, pig skin, cowhide or bovine tendon. 4. the collagen/chitosan/sodium hyaluronate composite support of load KGN according to claim 1, is characterized in that: the PLGA microsphere preparation method of described load KGN small molecule is as follows: (A) Accurately weigh 200 mg of PLGA and dissolve in 10 mL of dichloromethane, and ultrasonically oscillate to form an oil phase; (B) Accurately weigh 10 mg of KGN and place it in a mixture of 1 mL of ultrapure water and methanol, and dissolve it completely at 37°C to form an inner water phase; (C) Slowly inject the inner water phase described in step (B) into the oil phase described in step (A) and continue to stir and emulsify for 10 minutes to make it evenly dispersed to form a colostrum emulsion; (D) drop the colostrum emulsion in step (C) into 50 mL 1wt% PVA solution containing 5 mL 0.1wt% Tween-20 at 60 drops/min to form an external aqueous phase, and continue stirring to form double emulsion; (E) Fully centrifuge the double emulsion described in step (D), discard the supernatant, wash and centrifuge repeatedly with ultrapure water for 3 times, freeze-dry, and store in the dark at -20°C. 5. The collagen/chitosan/sodium hyaluronate composite scaffold loaded with KGN according to claim 4, characterized in that: in the mixed solution of ultrapure water and methanol described in step (B), ultrapure water and The volume ratio of methanol is 1:1. 6. a preparation method of the collagen/chitosan/sodium hyaluronate composite support of load KGN as described in any one in claim 1～5, it is characterized in that: comprise the steps: (1) Collagen and chitosan were dissolved in 0.1wt% glacial acetic acid to prepare collagen solution and chitosan solution; (2) Prepare hyaluronic acid solution by dissolving sodium hyaluronate in distilled water or deionized water; (3) Mix the collagen solution obtained in step (1) with the chitosan solution, and vortex to mix evenly; (4) Slowly add the sodium hyaluronate solution obtained in step (2) into the mixing and swirling solution in step (3), and mix well; (5) Add the PLGA microspheres loaded with KGN small molecules into the mixed solution obtained in step (4), stir overnight with magnetic force, freeze-dry and add a cross-linking agent for cross-linking; (6) After cross-linking, wash repeatedly with Na ₂ HPO ₃ , NaCl and distilled water until neutral, and freeze-dry for the second time to obtain the collagen/chitosan/sodium hyaluronate composite scaffold loaded with KGN. 7. A method for preparing a KGN-loaded collagen/chitosan/sodium hyaluronate composite scaffold according to claim 5, wherein the cross-linking agent in step (5) is composed of 2-(N- Morpholine)ethanesulfonic acid monohydrate, 1-ethyl-3(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide dissolved in 40% ethanol solution Prepared, wherein moles of 2-(N-morpholine)ethanesulfonic acid monohydrate, 1-ethyl-3(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide The ratio is 50:33:8. 8. A method for preparing a KGN-loaded collagen/chitosan/sodium hyaluronate composite scaffold according to claim 5, characterized in that: the Na ₂ HPO ₃ and NaCl in step (6) are 0.1M respectively _Na2HPO3 , 1M _NaCl and 2M NaCl.