CN117618660A

CN117618660A - Injectable photo-curing double-layer integrated hydrogel composite material and preparation method and application thereof

Info

Publication number: CN117618660A
Application number: CN202410112604.9A
Authority: CN
Inventors: 张学慧; 邓旭亮; 卢妍惠; 宋佳
Original assignee: Peking University School of Stomatology
Current assignee: Peking University School of Stomatology
Priority date: 2024-01-26
Filing date: 2024-01-26
Publication date: 2024-03-01

Abstract

The invention discloses an injectable photo-curing double-layer integrated hydrogel composite material and a preparation method and application thereof. The preparation method of the invention comprises the following steps: (1) Preparing a loose inner layer comprising a modified chondroitin sulfate hydrogel; (2) Forming a dense outer layer containing modified silk fibroin hydrogel on the basis of the loose inner layer. The double-layer integrated hydrogel prepared by the invention has the advantages of injectability, shape plasticity and the like, has low technical sensitivity and convenient clinical operation, and simultaneously improves the tight combination degree of the internal bone filling material and the external barrier layer. In addition, the degradation speed of the double-layer integrated hydrogel is matched with the bone regeneration speed, and the double-layer integrated hydrogel has good biocompatibility. The inner hydrogel can effectively guide bone tissue regeneration, and the outer hydrogel can be well attached to the tissue after being implanted, so that the effects of sealing a bone defect space and preventing gingival fibrous tissue from climbing and growing in are achieved, the fusion degree of bone repair is remarkably improved, and the method has wide application prospects in filling and repairing irregular bone defects.

Description

Injectable photo-curing double-layer integrated hydrogel composite material and preparation method and application thereof

Technical Field

The invention belongs to the field of biomedical materials, and particularly relates to an injectable photo-curing double-layer integrated hydrogel composite material, and a preparation method and application thereof.

Background

In oral clinics, the combined use of bone grafting materials, including bone filler materials (filling the bone defect area to induce osteogenesis) and bone regeneration membrane materials (preventing epithelial cell ingrowth to exert barrier function) is a traditional clinical approach to treating jaw defects. For example, bone filler materials, which are widely used in oral clinics in China, mainly comprise hydroxyapatite, are mainly obtained from bovine bone sources, and bone regeneration guiding membrane materials are mainly derived from pigskin or bovine hide collagen. The clinical treatment effects of the two materials have certain limitations, and are mainly reflected in that the bone filling material is still in a loose sand shape after being implanted into a bone defect area, and the bone fusion effect is poor. After the bone regeneration membrane material is implanted into the defect, the mechanical support is insufficient due to the too fast degradation of collagen components, and the defect bone formation deficiency is easily caused to collapse. In addition, there is still a lack of effective repair materials in the filling repair of irregular bone defects.

The hydrogel has bionic properties, researches show that the hydrogel has good biocompatibility and biodegradability, and can be used as a bone tissue scaffold to promote cell adhesion, cell migration and cell growth, so as to guide bone regeneration. Various physical and chemical methods such as photo-thermal reaction, supermolecule assembly strategy, micro-flow control and the like are tried by scholars at home and abroad to prepare a novel hydrogel system, including improving the drug loading capacity of a hydrogel material and adjusting the release rate of bioactive molecules to prepare the nanoparticle composite hydrogel.

While nanocomposite hydrogels can release growth factors to promote bone regeneration, barrier function is not ideal. The ideal oral bone filling material provides a good bone induction environment for the formation of new bones, simultaneously provides a barrier function for preventing the growth of gingival fibroblasts, and improves the effects of new bone formation and bone fusion on the basis of ensuring the three-dimensional space of the bone formation. Nowadays, hydrogel materials are widely focused by researchers, and the materials can realize bionic activity through design and processing, so as to provide a proper microenvironment for cell function differentiation and new bone formation. Therefore, research on an integrated hydrogel with both osteoinductive activity and barrier function and high stability becomes a problem to be solved and explored in the field of oral medical implant materials.

Although some injectable hydrogels based on metal peroxide compounds are developed at present, metal ions exist after the metal oxide reaction, and the long-term existence in human bodies is unfavorable for health. Some researches and developments use traditional Chinese medicine molecules as doping agents, the traditional Chinese medicine molecules and conductive polypyrrole are synthesized into hydrogel, and the hydrogel is coated and embedded into a titanium alloy network, but the efficacy of the traditional Chinese medicine molecules is difficult to maintain for a long time, and the problem of taking out a titanium alloy stent is also faced after operation, so that secondary wounds are easy to cause. In addition, some researches focus on the preparation of nano-hydroxyapatite-PLA-PEG-PLA block copolymer composite materials by nano-hydroxyapatite and block copolymers, but the hydroxyapatite is gradually degraded after being implanted into a human body for a long time, the chimerism with the block copolymers is weakened, and the mechanical properties are gradually reduced.

Therefore, there is still a need for a hydrogel bone grafting material with high biological safety, good mechanical properties, and simultaneously having osteoinductive activity and barrier function.

Disclosure of Invention

The invention provides a partially injectable photo-cured double-layer integrated hydrogel integrating bone and barrier support and a preparation method thereof, which aims to solve the technical problems of poor bone fusion and lack of integration of osteogenesis and barrier function of the existing oral clinical bone filling material. Specifically, the present invention includes the following.

In a first aspect of the present invention, there is provided a method for preparing a partially injectable photocurable bilayer integrated osteogenic hydrogel composite material comprising the steps of:

(1) Preparing a loose inner layer comprising a modified chondroitin sulfate hydrogel;

(2) Forming a dense outer layer comprising a modified silk fibroin hydrogel on the basis of the porous inner layer.

In certain embodiments, the methods of preparation according to the present invention, wherein the modified silk fibroin and modified chondroitin sulfate each contain a photosensitive group.

In certain embodiments, the preparation method according to the present invention comprises the steps of:

(1) Respectively carrying out acylation reaction on the silk fibroin and the chondroitin sulfate to obtain the silk fibroin containing photosensitive groups and the chondroitin sulfate containing photosensitive groups;

(2) Preparing silk fibroin sol and chondroitin sulfate sol from the silk fibroin with the photosensitive group and the chondroitin sulfate with the photosensitive group respectively;

(3) And (3) respectively photo-curing the chondroitin sulfate sol and the silk fibroin sol to form a double-layer integrated hydrogel with the loose inner layer and the compact outer layer.

In certain embodiments, the methods of preparation according to the present invention, wherein the concentration of silk fibroin in the silk fibroin sol is 100-500 mg/mL and the concentration of chondroitin sulfate in the chondroitin sulfate sol is 10-300 mg/mL.

In certain embodiments, the method of making according to the present invention, wherein the loose inner layer and the dense outer layer are joined by chemical bonds or physical chimerism into a double layer integral structure.

In certain embodiments, the method of making according to the present invention, wherein the time of photocuring is from 10 to 60 s.

In certain embodiments, the method of making according to the present invention, wherein the bulk inner layer comprises 70-90% by volume of the bilayer integrated hydrogel.

In certain embodiments, the method of making according to the present invention, wherein the dense outer layer comprises 10-30% by volume of the bilayer integrated hydrogel.

In a second aspect of the invention, there is provided a partially injectable photocurable two-layer integrated osteogenic hydrogel composite obtained by the method of the first aspect and comprising a loose inner layer of modified chondroitin sulfate hydrogel and a dense outer layer of modified silk fibroin hydrogel, said loose inner layer and dense outer layer being joined by chemical bonding or physical interlocking into a two-layer integrated structure.

In a third aspect of the present invention, there is provided a method of using the locally injectable photocurable bi-layer integrated osteogenic hydrogel composite of the present invention comprising the step of bringing the loose inner layer into proximity with and in contact with at least a portion of bone tissue.

In a fourth aspect of the invention, a partially injectable photocurable two-layer integrated osteogenic hydrogel composite is provided for use in the preparation of bone grafting materials or bone repair materials or irregular bone defect filling repair materials.

The beneficial effects of the invention include, but are not limited to:

(1) The preparation method comprises the steps of introducing photosensitive groups on silk fibroin and chondroitin sulfate through an acylation reaction, mixing the photosensitive groups with PBS and LAP to obtain sol-state hydrogel, and finally carrying out photo-curing treatment to enable the material to undergo sol-gel transformation to obtain the hydrogel with a double-layer integrated structure.

(2) The double-layer integrated hydrogel prepared by the invention can reside in the bone defect after being injected and cured by light, fully contacts with each wall of the bone defect, and is uniform and completely fills the bone defect.

(3) The double-layer integrated hydrogel prepared by the invention realizes firm combination due to the inherent covalent bond connection, thereby preventing gingival fibroblast from growing in during osseointegration.

(4) The photo-curing double-layer integrated hydrogel capable of being locally injected, which is prepared by the invention, has excellent effects of repairing bones and inducing bone regeneration, and particularly has wide application prospects in filling and repairing irregular bone defects.

(5) The preparation process is simple, and the preparation method can be industrially produced in batches and popularized and applied.

Drawings

Figure 1 shows the double layer integrated structure, injectability and shape plasticity of the hydrogel.

Fig. 2 shows a scanning electron microscope image of a double-layer integrated structure of a hydrogel, in which SF layer is an outer silk fibroin layer, LCS layer is low concentration chondroitin sulfate of example 2, and HCS layer is high concentration chondroitin sulfate of example 1.

Figure 3 shows the structural stability of the bilayer integrated hydrogel at 25 hours to reach swelling equilibrium.

Figure 4 shows the function of the upper hydrogel to block gingival fibroblast ingrowth.

FIG. 5 shows the depth of cell ingrowth over time of culture.

Fig. 6 shows that the upper hydrogel layer has good tissue adhesion properties.

Fig. 7 shows that the bilayer integrated hydrogel has good synergistic osteogenic function.

Fig. 8 shows the degradability of the bilayer integrated hydrogel, with the left column degraded and the right column osteogenesis in each group.

Figure 9 shows bone neogenesis in mice treated with the examples and comparative materials for 4 weeks.

Fig. 10 shows the bone mineralization density of mice treated with the examples and the comparative materials for 4 weeks.

Figure 11 shows bone neogenesis in mice treated with the examples and comparative materials for 8 weeks.

Fig. 12 shows the bone mineralization density of mice treated with the examples and the comparative materials for 8 weeks.

Figure 13 shows bone neogenesis in mice treated with the examples and comparative materials for 12 weeks.

Fig. 14 shows the bone mineralization density of mice treated with the examples and the comparative materials for 12 weeks.

Detailed Description

Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in the present invention, it is understood that the upper and lower limits of the ranges and each intermediate value therebetween are specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

Herein, the terms "bilayer integrated osteogenic hydrogel", "bilayer hydrogel", "photocurable bilayer integrated osteogenic hydrogel", "bilayer integrated hydrogel" have the same meaning, interchangeably used to refer to bilayer hydrogel structures having different pore sizes connected by chemical bonds or physical chimerism.

Preparation method

In one aspect of the invention, a method is provided for preparing a partially injectable photocurable bilayer integrated osteogenic hydrogel composite comprising (1) preparing a loose inner layer comprising a modified chondroitin sulfate hydrogel; (2) Forming a dense outer layer comprising a modified silk fibroin hydrogel on the basis of the porous inner layer.

In a preferred embodiment, the method comprises the steps of:

(1) Respectively carrying out acylation reaction on the silk fibroin and the chondroitin sulfate to obtain the silk fibroin containing the photosensitive group and the chondroitin sulfate containing the photosensitive group;

(2) Preparing the silk fibroin containing the photosensitive group and the chondroitin sulfate containing the photosensitive group into silk fibroin sol and chondroitin sulfate sol respectively;

(3) And (3) respectively photo-curing the chondroitin sulfate sol and the silk fibroin sol to form a double-layer integrated hydrogel with a loose inner layer and a compact outer layer.

In step (1), the acylation reaction is intended to modify the silk fibroin and chondroitin sulfate so that they contain a photosensitive group. The terms "photosensitive group", "photocuring group", "double bond group" and "photo-energetic group" are used interchangeably herein to refer to a class of chemical groups that are sensitive to light and that can chemically react or change their physical properties upon absorption of light quanta, e.g., during photocuring, the photosensitive group, upon absorption of light energy, initiates a polymerization reaction that converts the resin from a liquid state to a solid state.

As used herein, the term "acylation reaction" refers to a reaction in which an acyl group is introduced at an atom such as carbon, nitrogen, oxygen or sulfur in the molecular structure in the presence of an acylating agent. "acylating agent" refers to an agent that provides an acyl group in an acylation reaction. In the present invention, the acylating agent is not particularly limited as long as it can achieve a sufficient degree of acylation of silk fibroin and chondroitin sulfate, and examples thereof include, but are not limited to, amides (e.g., formamide, acetamide, methacrylamide, etc.), carboxylic acids (e.g., formic acid, acetic acid, acrylic acid, malonic acid, etc.), carboxylic acid esters (e.g., monoethyl malonate, dimethyl malonate, etc.), acid anhydrides (e.g., acetic anhydride, propionic anhydride, benzoic anhydride, methacrylic anhydride, etc.), and/or acid halides (e.g., acid chloride, acid bromide, acid iodide, etc.). In a preferred embodiment, the acylating agent is glycerol methacrylate. In another preferred embodiment, the acylating agent is methacrylic anhydride.

Herein, "degree of acylation" refers to the degree of substitution of an amino group. The inventor discovers that the acylation degree influences the photocuring performance and the structural stability of the double-layer integrated hydrogel. In order to realize the long-term stability of the double-layer integrated hydrogel structure and avoid unstable structure caused by insufficient photocuring, the invention optimizes the acylation degree, thereby optimizing the introduction of photosensitive groups, and further ensuring the strength of the cured hydrogel and simultaneously facilitating the cell culture in the gel. Preferably, the degree of acylation is from 30 to 90%, more preferably from 30 to 70%, even more preferably from 30 to 60%, for example from 30%, 35%, 40%, 45%, 50%, 55%, 60%. In a preferred embodiment, the degree of acylation is 30%.

In the present invention, the preparation of the modified silk fibroin and modified chondroitin sulfate is not particularly limited, and can be carried out by methods known in the art. In some embodiments, silk is boiled with an alkali solution, such as sodium carbonate, degummed, dissolved in lithium bromide, and subsequently reacted with glycerol methacrylate to produce a modified silk fibroin bearing photo-energetic groups. In a preferred embodiment, sliced cocoons are placed in a range of 0.01 to 0.1. 0.1M, preferably 0.02M Na ₂ CO ₃ In the solution, sericin is removed by boiling at a high temperature, preferably 90 to 120 ℃, and still preferably 100 ℃, and then washing with distilled water and stirring several times. The degummed silk is then dried at room temperature and then dissolved in a solution of lithium bromide in the range of 0.1-2 g/mL, preferably 0.8 g/mL, at 40-80 ℃, preferably 60 ℃. After solubilization, 0.1-5 mL, preferably 2-4 mL, still preferably 3 mL of 200-600 mM, preferably 300-500 mM, still preferably 424 mM of glycerol methacrylate solution is added, and 1-8 h, still preferably 2-4 h, most preferably 3 h, is stirred at 40-80 ℃, preferably 60 ℃, to obtain silk fibroin with a photo-energetic group.

In some embodiments, a bicine buffer (0.5 mol/L, pH=9.0) was prepared by first preparing 0.1-1 mol/L, preferably 0.5 mol/L, bicine buffer, dissolving N, N-bis (2-hydroxyethyl) glycine in 4 ml deionized water, slowly dropwise adding NaOH solution with stirring to give a pH acidometer reading of 9.0, and adding deionized water to a constant volume of 50 ml. Dissolving chondroitin sulfate in bicine buffer solution at 20-60 ℃, preferably 30-50 ℃ and still preferably 40 ℃, cooling to room temperature to prepare chondroitin sulfate solution, preserving heat in a constant-temperature water bath (such as 37 ℃), removing air from the solution by using nitrogen, and then dropwise adding lithium bromide for acylation reaction to obtain chondroitin sulfate stock solution with a light energy group. In a further embodiment, chondroitin sulfate is dissolved in deionized water and methacrylic anhydride is added in a molar ratio of 10-40:1, preferably 15-30:1, still preferably 15-25:1, still preferably 18-22:1, methacrylic anhydride to chondroitin sulfate hydroxyl groups. The pH is then adjusted to alkaline, preferably to pH 8, and the reaction solution is then stirred in an ice bath for 12-48, h, preferably 20-24 h.

The present inventors have further found that, in step (2), the concentration of chondroitin sulfate in the inner layer is in a suitable range to provide a bilayer integrated hydrogel having good osteogenic properties. If the concentration of the inner layer chondroitin sulfate is too low, the requirement of critical bone defect repair cannot be met, and the osteogenic performance of the photo-curable double-layer integrated hydrogel is insufficient and the bone defect repair performance is poor. Therefore, the invention adopts chondroitin sulfate with higher concentration to prepare hydrogel, and achieves the stability of the bionic bone repair structure by optimizing the photo-curing time.

In some embodiments, the concentration of chondroitin sulfate in the chondroitin sulfate sol is 10-300 mg/mL, preferably 20-250 mg/mL, still preferably 30-200 mg/mL, more preferably 50-200 mg/mL, such as 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 mg/mL.

In some embodiments, the inner layer of chondroitin sulfate comprises 50% to 99%, preferably 60% to 95%, and more preferably 70% to 90%, such as 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90%, etc., of the volume fraction of the bilayer integrated hydrogel.

In addition, the invention also discovers that too high or too low a concentration of silk fibroin in the silk fibroin sol can affect the mechanical support and barrier properties of the dense outer layer. In some embodiments, the concentration of silk fibroin in the silk fibroin sol is 100-500 mg/mL, preferably 150-450 mg/mL, still preferably 200-400 mg/mL, more preferably 250-350 mg/mL, such as 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350 mg/mL.

In step (3), the photo-curing is a reaction performed in the presence of a photoinitiator and visible light. In the present invention, the photoinitiator is not particularly limited as long as it can achieve a desired photocuring effect, and examples thereof include, but are not limited to, radical photoinitiators (e.g., 2,4, 6-trimethylbenzoyl-diphenylphosphine oxide (TPO), 2-hydroxy-2-methyl-1- [4- (2-hydroxyethoxy) phenyl ] -1-propanone (MBF), etc.), cationic photoinitiators (e.g., diaryliodonium salts, triaryliodonium salts, alkyl iodonium salts, isopropylferrocenium hexafluorophosphate, etc.). In a preferred embodiment, the photoinitiator is lithium phenyl-2, 4, 6-trimethylbenzoyl phosphinate (LAP).

In some embodiments, the silk fibroin outer layer comprises 1% -50%, preferably 5-40%, still preferably 10-30%, such as 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30%, etc., by volume of the bilayer integrated hydrogel.

In the present invention, the concentration of photoinitiator used to prepare the photocurable silk fibroin sol is 10-30 mg/mL, preferably 15-25 mg/mL, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 mg/mL. The concentration of photoinitiator used to prepare the photocurable chondroitin sulfate sol is in the range of 0.1-10 mg/mL, preferably 0.5-5 mg/mL, and also preferably 1-5 mg/mL, e.g., 1, 2, 3, 4, 5 mg/mL.

The solvent used in the process of preparing the silk fibroin sol or the chondroitin sulfate sol is not particularly limited, and solvents that can be used in the present invention include, but are not limited to, PBS, physiological saline, deionized water, purified water, or the like.

The present inventors have also found through studies that stability of the double-layer integrated hydrogel can be achieved only for a suitable photo-curing time. For example, if the photo-curing time is too short, the two-layer integrated hydrogel structure is weak. In the present invention, the photo-curing time is 10 to 60 s, preferably 15 to 55 s, further preferably 20 to 50 s, more preferably 25 to 45 s, more preferably 25 to 35 s, such as 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 s.

In the present invention, the photo-curing is performed in the presence of visible light, wherein the wavelength of the visible light is preferably 400-480 nm, more preferably 400-460 nm, still more preferably 400-440 nm, more preferably 400-420 nm, such as 405 nm, 410 nm, 415 nm, 420 nm. In a preferred embodiment, the visible light is blue light of 405 nm wavelength.

Double-layer integrated hydrogel

In one aspect of the invention, a partially injectable photocurable two-layer integrated osteogenic hydrogel composite is provided that includes a loose inner layer of modified chondroitin sulfate hydrogel and a dense outer layer of modified silk fibroin hydrogel that are chemically bonded or physically chimeric into a two-layer integrated structure. It will be appreciated by those skilled in the art that chemical bonding includes bonding of the two-layer integral structure by means such as covalent bonding, and physical interlocking includes bonding of the two-layer integral structure by means such as friction or adsorption.

In the present invention, the bulk inner layer comprises 70-90%, preferably 75-85%, such as 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85% by volume of the bilayer integrated hydrogel. The dense outer layer comprises 10-30%, preferably 15-25%, for example 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25% by volume of the bilayer integrated hydrogel.

In the invention, the compact outer layer has high mechanical support strength and barrier function. The loose inner layer has osteoinductive activity, integrates bones and barriers, has synergistic effect, and has osteogenic performance superior to that of traditional bone powder. In addition, the bilayer integrated hydrogels prepared according to the present invention do not contain any growth factors, particularly for bone formation-related growth factors (including, but not limited to, insulin-like growth factor (IGF-1), epidermal Growth Factor (EGF), fibroblast Growth Factor (FGF), platelet-derived proliferation factor (PDGF), and growth hormone release-inhibiting factor (SRIH), and are not required to be used as a scaffold.

The double-layer integrated hydrogel prepared by the invention has low technical sensitivity and convenient clinical operation, improves the tight combination degree of an internal bone filling material and an external barrier membrane, can effectively guide bone tissue regeneration, can well attach tissues after the outer-layer hydrogel is implanted, achieves the effects of sealing a bone defect space, preventing climbing and growing in of gum fibrous tissues, and improves the fusion degree of bone repair.

The double-layer integrated hydrogel prepared by the invention has injectability, and can reach the position needing bone grafting or bone repair by injection. Meanwhile, the double-layer integrated hydrogel prepared by the method has good shape plasticity and can adapt to any bone defect shape.

The double-layer integrated hydrogel prepared by the invention belongs to a degradable material, can be slowly degraded after implantation, and has a degradation speed matched with an osteogenesis speed, and can avoid the problem of chemical toxicity caused by material degradation because the double-layer integrated hydrogel is a natural polymer.

Application method

The present invention also provides a method of using a locally injectable photocurable bi-layer integrated osteogenic hydrogel composite comprising the step of bringing the loose inner layer into proximity with bone tissue and into contact with at least a portion of the bone tissue. In a specific embodiment, a porous inner layer of modified chondroitin sulfate is obtained by photocuring at the bone defect site, and then a dense outer layer is formed by photocuring on the basis of the inner layer. It will be appreciated by those skilled in the art that the irregular bone defect filling and repair can also be achieved by prefabricating a double-layer integral structure of suitable dimensions (matching the contours of the bone defect or the site to be repaired) in vitro and further bringing the loose inner layer into close proximity to and in contact with at least part of the bone tissue. In certain embodiments, the method is an in vivo method. In further embodiments, the method is an in vitro method.

Application of

In one aspect, the invention provides an application of a partially injectable photo-cured double-layer integrated osteogenic hydrogel composite material in bone grafting material or bone repair material or irregular bone defect filling repair.

In the present invention, the bone grafting material or bone repairing material is not particularly limited, and examples thereof include materials for use in applications such as tibial bone grafting material, femoral bone grafting material, humeral bone grafting material, ulna bone grafting material, radius bone grafting material, skull bone grafting material, oral bone grafting, and the like.

Example 1

The embodiment shows a preparation method of a partially injectable photo-curing double-layer integrated osteogenic hydrogel composite material, which comprises the following steps:

(1) Firstly, boiling silk for degumming, naturally drying in the air after cleaning, dissolving by using lithium bromide LiBr, then carrying out acylation reaction to obtain a silk fibroin stock solution with a light energy group, dialyzing the silk fibroin stock solution with the light energy group by using deionized water, and then carrying out freeze drying to obtain the silk fibroin with a photosensitive group. Specifically, 5 g slice cocoons were placed in 1L of 0.02M Na ₂ CO ₃ Boiling at 100deg.C for 30 min, removing sericin, washing with distilled water, and stirring for 20 min. Subsequently, the degummed silk was dried at room temperature and then dissolved 1 h in a 5 mL solution containing 4.03 g lithium bromide (LiBr) at 60 ℃. Immediately after dissolution of LiBr, 0.3 g mL (424 g mM) of Glycerol Methacrylate (GMA) solution was added and stirred at 300 rpm at 60 c for 3 g h to give a high yield reaction between GMA and SF. The resulting solution was then centrifuged and dialyzed against distilled water using Slide-A-Lyzer dialysis cartridges for 3 days. Finally, 12 h was frozen at-20℃and 48 h was freeze-dried. Storing the freeze-dried SF and Sil-MA powder at the temperature of-20 ℃ to obtain the silk fibroin with the light energy groups;

(2) Chondroitin sulfate powder (CS) was dissolved in deionized water. After sufficient dissolution, methacrylic Anhydride (MA) was dropped into CS. The molar ratio of MA to CS hydroxyl groups was 20 times. Then, naOH solution was carefully added to adjust the pH to 8. The reaction solution was stirred in an ice bath for 24 hours. After the reaction period is finished, dialyzing the reaction mixture with deionized water to remove residual unreacted MA and any byproducts, and then freeze-drying CS-MA to obtain chondroitin sulfate particles with a light energy group;

(3) Dissolving the silk fibroin obtained in the step (1) of 300 mg and 20 mg photoinitiator LAP in 1 mL PBS to obtain a photo-curable silk fibroin sol;

(4) And (3) dissolving the chondroitin sulfate particles obtained in the step (2) of 100mg and a photoinitiator LAP 2 mg in 1 mL of PBS to obtain the photo-curable chondroitin sulfate sol.

(5) Injecting the obtained photo-curable chondroitin sulfate sol and silk fibroin sol into the defect of the skull of the mouse respectively, curing for 30 seconds by using photo-curing lamplight, taking blank treatment as a control group, taking materials for Micro-CT, hard tissue section dyeing and immunofluorescence dyeing respectively 4 weeks, 8 weeks and 12 weeks after operation, focusing on observation of the new bone formation condition and material degradation condition, and analyzing the mineralization density of the new bone. As shown in the drawing, the detection results of the embodiment show that the animal experiment results show that the new bone growth amount is more prominent than that of the control group in 4 weeks after operation, the material is stable, the new bone growth condition is poor in the blank control group due to no bone induction active material filling, the new bone growth amount of the hydrogel group is obviously increased in 8 weeks after operation, and compared with the hydrogel group and the control group, the difference is more obvious. The new bone maturity of the hydrogel group is increased after 12 weeks of operation, the bone pit is obvious, the material is basically degraded, and the new bone formation condition and the new bone maturity of the control group are weaker than those of the hydrogel group.

Example 2

(1) Firstly, boiling silk for degumming, naturally air-drying after cleaning, dissolving by using lithium bromide LiBr, and then carrying out acylation reaction to obtain silk fibroin stock solution with a light energy group;

(2) Dialyzing the silk fibroin stock solution with the light energy groups obtained in the step (1) by deionized water, and freeze-drying to obtain silk fibroin with photosensitive groups;

(3) N, N-bis (2-hydroxyethyl) glycine was dissolved in 4 mL deionized water, 1 mol/L NaOH solution was slowly added dropwise with stirring to give a pH acidometer reading of 9.0, and deionized water was added to a constant volume of 50 mL, which buffer was referred to as bicine buffer (0.5 mol/L, pH=9.0). Dissolving CS in bicine buffer solution at 40 ℃, cooling to room temperature to prepare CS solution, then preserving heat in water bath at 37 ℃, removing air from the solution by using nitrogen, then dropwise adding lithium bromide LiBr, and performing acylation reaction (an acylating reagent is methacrylamide) to obtain chondroitin sulfate stock solution with a light energy group;

(4) Dialyzing the chondroitin sulfate stock solution with the light energy groups obtained in the step (3) by using deionized water, and freeze-drying to obtain chondroitin sulfate particles with photosensitive groups;

(5) Dissolving the silk fibroin obtained in the step (2) of 300 mg and 20 mg photoinitiator LAP in 1 mL PBS to obtain a photo-curable silk fibroin sol;

(6) Dissolving 50 mg chondroitin sulfate particles obtained in the step (4) and a photoinitiator LAP 2 mg in 1 mL PBS to obtain a photocurable chondroitin sulfate sol;

the double-layer integrated hydrogel in a sol state is obtained through the steps, and the main components of the double-layer integrated hydrogel comprise silk fibroin sol with the concentration of 300 mg/mL and chondroitin sulfate sol with the concentration of 50 mg/mL;

(7) Injecting the obtained photo-curable chondroitin sulfate sol and silk fibroin sol into the defect of the skull of a mouse, curing for 30 seconds by using photo-curing lamplight, taking blank treatment as a control group, taking materials for 4 weeks, 8 weeks and 12 weeks after operation, respectively carrying out Micro-CT, hard tissue section dyeing and immunofluorescence dyeing, focusing on observation of the formation of new bones and degradation of materials, and analyzing the mineralization density of the new bones.

Comparative example 1

The following shows a method for preparing a locally injectable hydrogel composite material, comprising the steps of:

(1) Weighing GelMA, dissolving by using lithium bromide LiBr, and then carrying out acylation reaction to obtain GelMA stock solution with a light energy group;

(2) Dialyzing the GelMA stock solution with the light energy groups obtained in the step (1) by using deionized water, and freeze-drying to obtain GelMA particles with photosensitive groups;

(3) Dissolving 100mg GelMA particles obtained in the step (2) and 20 mg photoinitiator LAP in 1 mL PBS to obtain photo-curable GelMA sol;

the GelMA hydrogel in a sol state is obtained through the steps, and the main component is GelMA sol with the concentration of 100 mg/mL.

(4) Injecting the obtained photo-curable GelMA sol into the defect of the skull of the mouse, curing for 30 seconds by using photo-curing light, taking blank treatment as a control group, taking materials for 4 weeks, 8 weeks and 12 weeks after operation, respectively carrying out Micro-CT, hard tissue section dyeing and immunofluorescence dyeing, mainly observing the formation condition of new bones and the degradation condition of materials, and analyzing the mineralization density of the new bones.

In contrast to example 1, it has a hydrogel structure but no bilayer and osteoinductive activity.

Comparative example 2

The use of a repair membrane comprising commercial Bio-OSS bone powder in bone defects is shown below.

The commercial Bio-OSS bone powder is selected to be fully filled in the small tertiary skull defect, then a commercial Bio-Gide@membrane slightly larger than the bone defect area is cut to cover the bone powder, the skin incision is closed, micro-CT, hard tissue section staining and immunofluorescence staining are respectively carried out on the materials obtained 4 weeks, 8 weeks and 12 weeks after the operation, the new bone formation condition and the material degradation condition are observed in an important way, and the mineralization density of the new bone is analyzed.

As shown in the middle part (Bio) of fig. 7, the defect site is filled with only a large amount of bone powder. In addition, fig. 9 shows bone neogenesis in mice treated with the examples and comparative materials for 4 weeks. Fig. 10 shows the bone mineralization density of mice treated with the examples and the comparative materials for 4 weeks. Figure 11 shows bone neogenesis in mice treated with the examples and comparative materials for 8 weeks. Figure 12 shows bone neogenesis in mice treated with the examples and comparative materials for 12 weeks. Fig. 13 shows the bone mineralization density of mice treated with the examples and the comparative materials for 12 weeks. The result shows that the partially injectable photo-curing double-layer integrated hydrogel prepared by the invention has excellent bone repair and bone regeneration induction effects.

Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art will understand that: the technical scheme described in the foregoing embodiments can be modified or some of the technical features thereof can be replaced by equivalents. Such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The preparation method of the injectable photo-curing double-layer integrated hydrogel composite material is characterized by comprising the following steps of:

2. The method of preparing an injectable photocurable bilayer integrated hydrogel composite material according to claim 1, wherein the modified silk fibroin and modified chondroitin sulfate each contain a photosensitive group.

3. The method of preparing an injectable photocurable bilayer integrated hydrogel composite material according to claim 1 or 2, comprising the steps of:

4. The method for preparing an injectable photocurable bilayer integrated hydrogel composite according to claim 3, wherein the concentration of silk fibroin in the silk fibroin sol is 100-500 mg/mL and the concentration of chondroitin sulfate in the chondroitin sulfate sol is 10-300 mg/mL.

5. The method of preparing an injectable photocurable bilayer integrated hydrogel composite according to any one of claims 1-4, wherein the loose inner layer and the dense outer layer are combined into a bilayer integrated structure by chemical bonding and/or physical embedding.

6. The method of preparing an injectable photocurable bilayer integrated hydrogel composite material as recited in claim 5, wherein the time of photocuring is from 10 to 60 s.

7. The method of preparing an injectable photocurable bilayer integrated hydrogel composite material according to claim 6, wherein the loose inner layer comprises 70-90% by volume of the bilayer integrated hydrogel and the dense outer layer comprises 10-30% by volume of the bilayer integrated hydrogel.

8. An injectable photocurable bilayer integrated hydrogel composite material obtained according to the preparation method of any one of claims 1-7.

9. The method of using an injectable photocurable bilayer integrated hydrogel composite material as recited in claim 8, comprising the step of bringing the loose inner layer into proximity with and in contact with at least a portion of bone tissue.

10. Use of the injectable photocurable bilayer integrated hydrogel composite material according to claim 8 for the preparation of bone grafting material, bone repair material or irregular bone defect filling repair material.