Search

CN-122005937-A - Injection bone collagen gel capable of resisting irradiation sterilization and preparation method thereof

CN122005937ACN 122005937 ACN122005937 ACN 122005937ACN-122005937-A

Abstract

The invention discloses an injection bone collagen gel capable of tolerating irradiation sterilization and a preparation method thereof. By combining the bone targeting factor TBMP-calcined bone scaffold with an injectable polyphosphazene-collagen-glycerol gel system, a novel injectable bone grafting composite material is constructed. Aiming at overcoming the multiple bottlenecks of insufficient osteogenesis capability, unsatisfactory repairing effect, large operation wound and the like in the current bone defect treatment, and providing an innovative treatment strategy for the clinical repair of large-volume maxillofacial bone defects. After the TBMP2@TBC-polyphosphazene-collagen@glycerol injectable gel is subjected to irradiation sterilization, the gel morphology is unaffected, and the proliferation and osteogenic differentiation of BMSCs can be obviously promoted.

Inventors

  • LI QIHONG
  • ZHAO YANTAO
  • LI CHEN
  • LIU YICHEN
  • RONG SHIPENG
  • WANG XIAONING
  • WANG WENYING

Assignees

  • 中国人民解放军总医院第五医学中心

Dates

Publication Date
20260512
Application Date
20260121

Claims (9)

  1. 1. An injectable bone collagen gel resistant to irradiation sterilization, characterized in that it is constructed by combining a bone targeting factor TBMP-calcined bone scaffold with a polyphosphazene-collagen-glycerol gel system.
  2. 2. The method for preparing radiation sterilization resistant injectable collagen gel according to claim 1, wherein the steps are as follows: (1) The preparation of calcined bone material includes taking fresh animal bone, eliminating soft tissue and outer cortical bone, cutting cancellous bone into small blocks, flushing with high pressure water gun to eliminate blood and bone marrow, degreasing, decellularizing, stoving, calcining, soaking the calcined bone in NaH 2 PO 4 solution for normal temperature incubation, regulating pH to neutrality, washing and freeze drying for preservation; (2) Preparing TBMP2@TBC, namely diluting TBMP < 2 > with PBS buffer solution, soaking the calcined bone prepared in the step (1), refrigerating for 18-30h, and then vacuum freeze-drying to obtain TBMP2@TBC; (3) The preparation of bone collagen gel comprises the steps of taking fresh animal bones, removing soft tissues, cutting cortical bones into small blocks, flushing by a high-pressure water gun to remove blood and bone marrow residues, degreasing and decellularizing sequentially, grinding into particles with the particle size of 80-120 meshes by adopting a grinding instrument, removing non-collagen impurities, decalcification, regulating the pH value of the decalcified bone meal particles to 1.5-2, adding 0.5-1.5 mol/L of acetic acid and 8-12% of the bone mass into pepsin, extracting collagen by a shaking table at room temperature for 5-9d, centrifuging an acid and an enzymolysis sample, taking supernatant, regulating the pH value to 6-6.5, salting out for 18-30h, dialyzing with acetic acid and deionized water sequentially overnight, and freeze-drying a precipitate freeze-dryer after the dialysis is finished to obtain bone collagen; (4) The preparation of polyphosphazene-collagen@glycerol gel comprises dissolving lyophilized bone collagen in 0.3-0.7mol/L acetic acid solution, dialyzing with deionized water to remove acid, adjusting pH to neutrality, mixing bone collagen with polyphosphazene and glycerol, stirring, removing upper bubble layer, taking out, and storing in refrigerator at 2-8deg.C; (5) And (3) mixing the TBMP2@TBC prepared in the step (2) and the polyphosphazene-collagen@glycerol gel prepared in the step (4), stirring at room temperature, sterilizing by using gamma-ray irradiation of 20-30kGy, and storing in a refrigerator at a temperature of 2-8 ℃.
  3. 3. The method for preparing an injectable collagen gel which can withstand irradiation sterilization according to claim 2, wherein the animal bone is one or more of bovine bone, porcine bone, sheep bone and equine bone.
  4. 4. The method for preparing the injected bone collagen gel capable of resisting irradiation sterilization according to claim 2, wherein the calcining process in the step (1) is that the dried cancellous bone blocks are placed in a muffle furnace, the temperature is raised to 700-1000 ℃, the calcining furnace is maintained for 3-5 hours, the calcining furnace is closed, the natural cooling is carried out to room temperature, the calcined bone is taken out, immersed in monoammonium phosphate solution, subjected to constant-temperature hydrothermal treatment for 18-30 hours, dried, the bone blocks are placed in the muffle furnace for second calcination, the calcining time is raised to 700-1000 ℃ for 1-3 hours, then the calcining furnace is closed, the natural cooling is carried out to room temperature, and the calcined bone is taken out.
  5. 5. The method of preparing an injectable collagen gel which is resistant to sterilization by irradiation according to claim 2, wherein the removal of non-collagenous impurities in the step (3) is performed by using 0.05-0.15 mol/L NaOH, and the decalcification is performed by using 0.4-0.8 mol/L hydrochloric acid.
  6. 6. The method for preparing an injectable collagen gel which can withstand irradiation sterilization according to claim 2, wherein the degreasing is performed by using Triton X-100 and the decellularization is performed by using H 2 O 2 .
  7. 7. The method of preparing a radiation-sterilizable injectable collagen gel according to claim 2, wherein the TBMP is diluted in step (2) to a final concentration of 50-200 ug/ml.
  8. 8. The method for preparing the radiation sterilization resistant injectable collagen gel according to claim 2, wherein the bone collagen protein in the step (4) accounts for 2-5% of the mass of the acetic acid solution, and the mass ratio of the bone collagen protein to the polyphosphazene to the glycerol is 1 (0.5-2).
  9. 9. The method for preparing an injectable collagen gel which can withstand irradiation sterilization according to claim 2, wherein the mass ratio of the TBMP2@TBC and the polyphosphazene+collagen@glycerin gel in the step (5) is (0.5-2): 1.

Description

Injection bone collagen gel capable of resisting irradiation sterilization and preparation method thereof Technical Field The invention belongs to the technical field of bone repair, and particularly relates to an injection bone collagen gel capable of tolerating irradiation sterilization and a preparation method thereof. Background Jaw bone plays an important role in maintaining facial morphology, playing chewing function and the like, and jaw bone defects caused by factors such as trauma, infection, tumor, congenital diseases and the like are clinically repaired by adopting autologous bone, allogeneic bone and xenogeneic bone. Autologous bone is regarded as a gold standard because of good biocompatibility and strong osteogenesis capability, but is limited by insufficient bone mass and a second operation area, and allogeneic bone and xenogeneic bone have the problems of low induction activity, poor bone fusion quality, high absorption rate, immune rejection and the like, so that ideal repairing effect is difficult to realize. The oral environment is extremely complex due to the inclusion of neurovascular, dental, saliva and a variety of pathogens, further increasing the difficulty of repair. The traditional bone grafting material is often required to be shaped in operation, so that the operation time is prolonged, the pain of a patient is increased, and the pollution risk exists. Along with popularization of the minimally invasive concept, the injectable bone grafting material is receiving more and more clinical attention because the injectable bone grafting material can be injected in situ and molded rapidly, resists deformation effectively, and realizes accurate filling in a minimally invasive mode. BMP2, which is the most important regulator of bones, has a strong osteoinductive capacity, has been approved by the FDA and is widely used in bone tissue engineering. However, BMP2 is easy to cause problems of soft tissue inflammation, ectopic ossification and the like in application, so that the realization of slow release and bone targeted delivery of BMP2 is important. Aspartic acid-serine tripeptide (ASPSERSER) 6 HAs been shown to be an effective bone-targeting drug carrier molecule that can bind to low crystallinity Hydroxyapatite (HA) and accurately direct drugs to the osteogenic region. The TBMP2 with the 6 (ASPSERSER) is directly expressed by the genetic engineering technology in the earlier stage of the subject group, so that the problems of high cost, long time consumption and the like caused by later-stage connection are avoided (ZL 201510317322.3). The real bone ceramic (TBC) prepared by calcining bovine spongy bone at high temperature retains the hydroxyapatite component and the micropore structure of natural bone, has a calcium-phosphorus ratio close to that of human bone, and has good biocompatibility and bone conductivity (ZL 201510317324.2). However, TBCs lack osteoinductive capacity. The subject group realizes the sustained release of BMP2 and acts on the bone defect area for a long time by loading TBMP with bone targeting function while enhancing the bone induction capability of the material, thereby effectively improving the bone repair effect. Conventional liquid collagen is difficult to withstand sterilization by 60Co irradiation. Researches show that the viscosity of the material is seriously reduced after irradiation sterilization, even the viscosity is basically lost, and the material no longer has the performance of bearing powder or particles. This is probably due to the high water content of the hydrogel, combined with the high energy radiation induced hydrolysis, which may lead to degradation of some molecules and damage of the gel structure. It is therefore desirable to find a protective agent to improve the radiation resistance of collagen. Glycerol can be used for cryopreserving cells and tissues, and cell proliferation assay results indicate that glycerol and its decomposition products are biocompatible. The use of glycerol as a carrier by Osteotech (Eatontown, NJ, USA) produced a plastic Decalcified Bone Matrix (DBM) product and was clinically used, which fully demonstrates the safety of glycerol as an excipient carrier component and has no damaging effect on the performance of bone grafting materials. However, glycerol itself does not have gel characteristics, is easily soluble in water, and is difficult to meet the molding and operating requirements of injectable gel excipients when used alone. Disclosure of Invention The invention aims to provide an injection bone collagen gel capable of resisting irradiation sterilization and a preparation method thereof. An injectable collagen gel resistant to irradiation sterilization is constructed by combining a bone targeting factor TBMP-calcined bone scaffold with a polyphosphazene-collagen-glycerol gel system. The preparation method of the irradiation sterilization resistant injectable collagen gel comprises the following steps: (