阅读说明：本技术 一种耐溶蚀的骨诱导性丝素蛋白/羟基磷灰石/氧化镁凝胶海绵及制备方法 (Corrosion-resistant osteoinductive silk fibroin/hydroxyapatite/magnesium oxide gel sponge and preparation method thereof ) 是由 徐荷林 赵应征 袁健东 姚情 杨外庚 武子荃 于 2019-09-06 设计创作，主要内容包括：本发明公开了一种耐溶蚀的丝素蛋白/羟基磷灰石/氧化镁复合骨诱导性凝胶海绵及制备方法。本发明利用可溶性丝素蛋白为稳定剂高度分散羟基磷灰石与骨诱导性的氧化镁制备纳米混悬液,之后嵌入不溶性丝素蛋白纤维形成三维网络凝胶,利用冷冻干燥技术,制备丝素蛋白/羟基磷灰石/氧化镁凝胶海绵。该凝胶海绵体内移植后能吸收体液塑形滞留于损伤部位,能较长时间保持凝胶状态,不被溶解流失；在体液的作用下,该凝胶海绵中氧化镁缓慢降解产生镁离子和产生局部碱性微环境,促进骨髓间充质干细胞成骨分化,具有骨诱导作用；该凝胶海绵临床上可以直接填充于骨缺损部位,不存在炎症反应、异位骨化；该凝胶海绵制备方法简单。(The invention discloses a corrosion-resistant silk fibroin/hydroxyapatite/magnesium oxide composite osteoinductive gel sponge and a preparation method thereof. The invention utilizes soluble silk fibroin as a stabilizing agent to highly disperse hydroxyapatite and osteoinductive magnesium oxide to prepare nano suspension, then insoluble silk fibroin fibers are embedded to form three-dimensional network gel, and silk fibroin/hydroxyapatite/magnesium oxide gel sponge is prepared by utilizing a freeze drying technology. The gel sponge body can absorb body fluid to be shaped and retained at the injured part after being transplanted, can keep the gel state for a long time and cannot be dissolved and lost; under the action of body fluid, magnesium oxide in the gel sponge is slowly degraded to generate magnesium ions and a local alkaline microenvironment, thereby promoting osteogenic differentiation of mesenchymal stem cells and having osteoinductive effect; the gel sponge can be directly filled in bone defect parts clinically, and inflammatory reaction and ectopic ossification do not exist; the preparation method of the gel sponge is simple.)

1. An osteoinductive silk fibroin/hydroxyapatite/magnesium oxide gel sponge resistant to erosion, characterized in that: the silk fibroin porous sponge comprises silk fibroin, hydroxyapatite and magnesium oxide, wherein the silk fibroin comprises soluble silk fibroin and insoluble silk fibroin fibers.

2. The erosion-resistant osteoinductive silk fibroin/hydroxyapatite/magnesium oxide gel sponge according to claim 1, wherein: the nanometer hydroxyapatite and the magnesium oxide adsorb soluble silk fibroin, are uniformly embedded and grafted on the surface of insoluble silk fibroin fiber, and are freeze-dried to form the porous sponge.

3. The erosion-resistant osteoinductive silk fibroin/hydroxyapatite/magnesium oxide gel sponge according to claim 2, wherein: the mass ratio of the silk fibroin, the hydroxyapatite and the magnesium oxide is 100: (1-50): (1-50).

4. An erosion resistant osteoinductive silk fibroin/hydroxyapatite/magnesium oxide gel sponge according to claims 1-3, characterized in that: the silk fibroin is composed of soluble silk fibroin and insoluble silk fibroin fibers, and the mass ratio of the soluble silk fibroin to the insoluble silk fibroin is 100 (5-30).

5. An erosion resistant osteoinductive silk fibroin/hydroxyapatite/magnesium oxide gel sponge according to claims 1-3, characterized in that: after the gel cavernous body is filled with the bone defect part, the bone defect part cannot be quickly eroded by blood or tissue fluid, and can slowly absorb water and degrade to generate magnesium ions and a local weak alkaline microenvironment.

6. A method of preparing an erosion resistant osteoinductive silk fibroin/hydroxyapatite/magnesium oxide gel sponge as claimed in any of claims 1-3, characterized by comprising the steps of:

(1) adding hydroxyapatite and magnesium oxide into a soluble silk fibroin solution, and grinding by a wet method to form a nano suspension;

(2) adding insoluble silk fibroin fibers into the nano suspension obtained in the step (1), and stirring overnight to form silk fibroin/hydroxyapatite/magnesium oxide nano gel;

(3) and (3) freeze-drying the nano-gel prepared in the step (2) to obtain the silk fibroin/hydroxyapatite/magnesium oxide composite osteoinductive gel sponge.

7. The method of claim 6, wherein: the control conditions of the freeze drying in the step (3) are as follows: pre-freezing the medicinal micelle solution at minus 60 to minus 30 ℃ for 2 to 8 hours, then carrying out primary drying at minus 30 to 40 ℃ for 12 to 56 hours, and then carrying out secondary drying at 20 to 40 ℃ for 2 to 8 hours, wherein the time of the whole freeze-drying curve is 20 to 60 hours.

8. Use of an osteoinductive silk fibroin/hydroxyapatite/magnesium oxide gel sponge according to any one of claims 1 to 3 as bone graft repair material.

Technical Field

The invention belongs to the field of biological composite materials, particularly relates to the technical field of preparation of medical materials for bone defect repair, and particularly relates to a corrosion-resistant osteoinductive silk fibroin/hydroxyapatite/magnesium oxide gel sponge and a preparation method thereof.

Background

The bone grafting and repairing material is the key to realize the treatment of bone wound and mainly comprises autogenous bone, heteroplastic bone and artificial biological material. Autologous bone has good bone conduction, bone induction and osseointegration, and the axiom is the gold standard for bone wound repair. However, the limited availability of the bone-harvesting area of the body and the secondary trauma associated with the bone-harvesting area limit the clinical utility of autologous bone graft therapy. Although the origin of the xenogeneic bone from animals or corpses is far more than that of the autogenic bone, the xenogeneic bone has defects of transplant immune rejection, disease transmission and the like. In contrast, the artificial biomaterial has the advantages of abundant sources, good plasticity, close fit with the bone defect part, no risk of disease transmission, low immunological rejection reaction and the like. At present, the research on bone wound repair artificial biomaterials for complicating diabetes mainly utilizes bone morphogenetic growth factor (BMP-2) to compound natural or synthetic artificial materials, increases osteoblast-oriented bone formation and/or reduces osteoclast-oriented bone resorption, and has obvious in-situ or ectopic bone regeneration effects in numerous bone injury models. For example, BMP 2-collagen sponge induces osteoblast differentiation to repair diabetic bone defects to some extent (Int J Oral Maxillofac implanters 30(3) (2015) 707-14); the BMP 2-hyaluronic acid composite material can enhance osteoblast activity and inhibit osteoclast activity by inhibiting sclerostin, and promote fracture healing of diabetic rats. However, in clinical studies, BMP 2-collagen sponge has problems of fast in vivo erosion, inflammation, ectopic ossification, bone resorption and fat deposition, which limits its clinical therapeutic effect (Biomed Mater 11(5) (2016) 055011; Tissue Eng Part B Rev 22(4) (2016) 284-97).

Research reports that inorganic magnesium ions with proper concentration induce an osteogenic microenvironment and increase bone injury repair by regulating osteoblast adhesion and differentiation. The hard artificial metal material represented by metal magnesium or magnesium alloy has excellent bone induction capability and is widely applied to the fields of bone defect and bone fixation. The magnesium or magnesium alloy is slowly degraded in vivo after being transplanted to generate Mg^{2 +}Regulating osteoblast adhesion and differentiation, regulating callus matrix mineralization and playing an important role in bone formation and absorption processes. Yang et al found that magnesium metal still had osteoinductive effects on STZ-induced osteoporosis in diabetic rats (J biomedMater Res A99 (3) (2011) 386-94). Lin and the like use PLGA microspheres to wrap magnesium oxide as Mg²⁺Slowly releasing the storage, coating alginic acid on the microsphere, preparing spongy gel biomaterial, and accurately controlling Mg²⁺At a concentration of 50ppm, it was effective in enhancing osteoblast activity and stimulating the regeneration of femoral defects in rats (Biomaterials 174(2018)1-16), but the sponge gel had the drawback of rapid water absorption and erosion, in which the dispersed microspheres were easily lost at the site of injury. Chinese patent (publication No. CN 108939163A) discloses a magnesium-doped hydroxyapatite fibroin composite membrane with bone-promoting functionAccording to the invention, inorganic calcium ions, phosphate ions and magnesium ions are deposited on the surface of the silk fibroin film by taking the silk fibroin solution as a film forming material through an electrolytic method, and the prepared composite film has the defects of poor water absorption performance, slow degradation and incapability of generating a local alkaline microenvironment in vivo application.

Hydroxyapatite (HA) is a biological material with good bone conduction and osseointegration, and HAs been used for clinical bone repair and replacement. The HA hydration reaction product HAs the composition similar to bone mineral, can be combined with bone formation in a good chemical bond on an interface after being implanted into bone tissue, and degraded calcium ions and phosphate ions can be deposited into the bone tissue again to promote new bone formation. And HA HAs good in-situ self-curing performance, and can improve the plasticity of the organic artificial bone material and the fitting degree of the material and the bone defect part according to the bone defect area and shape. However, the hydroxyapatite is difficult to be completely replaced and utilized by organisms due to high crystallinity, large brittleness and slow in-vivo degradation, so that the clinical application of the hydroxyapatite is limited. Chinese patent (publication No. CN103432629A) prepares a silk fibroin/hydroxyapatite composite material by a chemical sedimentation method from inorganic calcium ions, phosphate ions and the like, and the material can improve the mechanical strength and porosity of HA, but HAs the defects of poor water absorption capacity, weak in-vivo osteoblast differentiation induction capacity and the like.

Disclosure of Invention

In order to solve the problems and the defects in the prior art, the invention aims to provide an osteoinductive silk fibroin/hydroxyapatite/magnesium oxide gel sponge with corrosion resistance and a preparation method thereof. The gel sponge can solve the problems of dissolution loss, inflammatory reaction, ectopic ossification, bone absorption, fat deposition and the like in the application of the existing BMP-2 gelatin sponge, and provides a safe and effective gel sponge substitute product with osteoinductive performance for clinical bone defect repair.

In order to achieve the above object, the first aspect of the present invention provides an erosion-resistant osteoinductive silk fibroin/hydroxyapatite/magnesium oxide gel sponge, which comprises a porous gel sponge composed of silk fibroin, hydroxyapatite and magnesium oxide, wherein the silk fibroin comprises soluble silk fibroin and insoluble silk fibroin fibers.

The gel sponge has a special microstructure, namely, soluble silk fibroin is adsorbed by nano-hydroxyapatite and magnesium oxide, and is uniformly embedded and grafted on the surface of insoluble silk fibroin fiber to form a porous gel sponge structure.

Further setting the mass ratio of the silk fibroin, the hydroxyapatite and the magnesium oxide as 100: (1-50): (1-50).

The silk fibroin is further arranged to be composed of soluble silk fibroin and insoluble silk fibroin fibers, and the mass ratio of the soluble silk fibroin to the insoluble silk fibroin is 100 (5-30).

The gel sponge is further provided with a structure that after the bone defect part in the gel sponge is filled, the gel sponge cannot be quickly corroded by blood or tissue fluid and can slowly absorb water and degrade to generate magnesium ions and a local weak alkaline microenvironment.

The second aspect of the invention provides a preparation method of the corrosion-resistant osteoinductive silk fibroin/hydroxyapatite/magnesium oxide gel sponge, which comprises the following steps

(1) Adding hydroxyapatite and magnesium oxide into a soluble silk fibroin solution, and grinding by a wet method to form a nano suspension;

(2) adding insoluble silk fibroin fibers into the nano suspension obtained in the step (1), and stirring overnight to form silk fibroin/hydroxyapatite/magnesium oxide nano gel;

(3) and (3) freeze-drying the nano-gel prepared in the step (2) to obtain the silk fibroin/hydroxyapatite/magnesium oxide composite osteoinductive gel sponge.

Further setting the control conditions of the freeze drying in the step (3) as follows: pre-freezing the medicinal micelle solution at minus 60 to minus 30 ℃ for 2 to 8 hours, then carrying out primary drying at minus 30 to 40 ℃ for 12 to 56 hours, and then carrying out secondary drying at 20 to 40 ℃ for 2 to 8 hours, wherein the time of the whole freeze-drying curve is 20 to 60 hours.

The invention also provides application of the osteoinductive silk fibroin/hydroxyapatite/magnesium oxide gel sponge as a bone transplantation repair material.

The invention has the advantages that: the bone-conductive hydroxyapatite and the bone-inductive magnesium oxide inorganic material are combined with the biocompatible silk fibroin gel to prepare the erosion-resistant gel sponge with bone-conductive and bone-inductive performances, which is used for repairing bone injury. Compared with the prior art, the method has the following advantages: (1) the gel sponge has the advantages that the soluble silk fibroin is adsorbed by the nano-hydroxyapatite and the magnesium oxide, and is uniformly embedded and grafted on the surface of the insoluble silk fibroin fiber to form a microstructure of a porous network; (2) the gel sponge body can absorb body fluid to be shaped and retained at the injured part after being transplanted, can keep the gel state for a long time and cannot be dissolved and lost; (3) magnesium oxide in the gel sponge is degraded under the action of body fluid to generate magnesium ions and a local alkaline microenvironment, so that osteogenic differentiation of stem cells is promoted, and the gel sponge has an osteoinductive effect; (4) the gel sponge is free of protein components such as BMP and the like, and the problems of inflammatory reaction, heterotopic ossification, bone absorption, fat deposition and the like do not exist in vivo application; (5) the invention directly utilizes the existing clinical materials to prepare the medicament by grinding/freeze-drying steps without adopting a complex chemical deposition method or an electrolysis method, has simple preparation method and is easy to realize large-scale production.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is within the scope of the present invention for those skilled in the art to obtain other drawings based on the drawings without inventive exercise.

FIG. 1 shows the results of the application examples of the gel sponges prepared in group 1, group 6, group 14 and comparative example 2 for promoting the adhesion and proliferation of MSCs;

FIG. 2 evaluation of biological Properties 1 the gel sponges prepared in group 1, group 6, group 14 and comparative example 3 induced osteogenic differentiation of MSCs in the experiment;

FIG. 3 biological Performance evaluation 2 the effect of the gel sponges prepared in groups 1, 6, 14, comparative example 1 and comparative example 3 on the repair of rat femoral defects (micro-CT image) in experiment 2.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings.

Preparing raw materials:

1. preparation of insoluble silk fibroin fiber

Cutting silkworm cocoon into pieces, adding 0.02M Na₂CO₃The solution was boiled at 100 ℃ for 1.5 hours, and washed with distilled water to remove sericin, thereby preparing insoluble silk fibroin fibers.

2. Preparation of soluble silk fibroin

Drying the prepared insoluble silk fibroin fibers, putting the dried insoluble silk fibroin fibers into a 9.3M LiBr solution with the constant temperature of 60 ℃ for deconstruction for 3 hours to prepare a 5.0% (w/v) silk fibroin solution, centrifuging and filtering the solution, putting the solution into a dialysis bag for dialysis for 3 days, and removing residual salt solution to obtain a soluble silk fibroin solution.