WO2023040853A1 - Periosteum-bone complex for reconstructing soft tissue-bone immune repair environment and preparation method therefor - Google Patents
Periosteum-bone complex for reconstructing soft tissue-bone immune repair environment and preparation method therefor Download PDFInfo
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- WO2023040853A1 WO2023040853A1 PCT/CN2022/118561 CN2022118561W WO2023040853A1 WO 2023040853 A1 WO2023040853 A1 WO 2023040853A1 CN 2022118561 W CN2022118561 W CN 2022118561W WO 2023040853 A1 WO2023040853 A1 WO 2023040853A1
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- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
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- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
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Definitions
- the present invention mainly relates to the biological field for bone tissue regeneration and repair, specifically a periosteum-bone complex and its preparation method aimed at rebuilding the soft tissue-bone immune repair environment.
- the impact of bone repair rational use of the advantages of spatial composite mode scaffolds to promote bone regeneration and repair.
- Bone defects caused by trauma, tumors and infections are common clinical diseases that affect human health, especially multiple complex bone defects, which often require repeated treatment, causing heavy psychological pressure and economic burden on individuals, and affecting social stability.
- the potential impact is an urgent problem to be solved in clinical orthopedics.
- Natural bone is a highly vascularized hard tissue covered by soft periosteum.
- the periosteum can serve as a cellular barrier against excessive penetration of inflammatory cells in a traumatic environment, and it is also a physical barrier that blocks muscle and fibrous tissue from filling the defect.
- the periosteum provides sufficient blood supply and nutrition for the cortical bone, and also provides a supporting microenvironment for the differentiation and maturation of mesenchymal stem cells.
- hydrogels derived from periosteal extracellular matrix can enhance the M2 polarization of macrophages in the early stage of bone injury and play an immunomodulatory role.
- the present invention obtains a space-patterned periosteum-bone complex through an innovative extracellular matrix preparation technology, proving that it has Regulate the performance of the immune microenvironment in the early stage of soft tissue-bone injury, coordinate subsequent angiogenesis and osteogenesis events, and promote bone healing.
- periosteum-bone complex there is no report on the preparation of periosteum-bone complex.
- the purpose of the present invention is to fill in the prior art, single-phase bone repair biological scaffolds are difficult to effectively avoid soft tissue-derived inflammatory infiltration, which aggravates the lack of immune regulation in the early stage of bone defect repair, and therefore provides a space pattern periosteum for rebuilding the soft tissue-bone immune repair environment - Bone complex and method for its preparation.
- the preparation method of the space pattern periosteum-bone complex of natural animal origin comprises the following steps:
- steps (5)-(9) rinse with double distilled water for 3-6 hours after each step is completed.
- the mass concentration of EDTA-Na 2 is 5%-20%;
- PBS buffer containing protease inhibitors the concentration of protease inhibitors is 10-50K IU/ml;
- the PBS buffer containing Triton X is Triton X-100 PBS buffer with a mass concentration of 0.01%-5%;
- the PBS buffer solution containing SLES is the PBS buffer solution of SLES with a mass concentration of 0.01%-5%;
- the PBS buffer solution containing DNase I is the PBS buffer solution with a concentration of 1-2mg/mL;
- Tris-HCl buffer solution Containing Tris-HCl buffer solution, the concentration of Tris-HCl is 5-20mM;
- the concentrations of penicillin and streptomycin in the mixed antibacterial solution were 20 U/ml and 20 ⁇ g/ml respectively; the ratio of penicillin and streptomycin was 1:1, and the volume ratio of the added mixed antibacterial solution to the original solution was 5:1.
- the present invention aims at the problems existing in clinical substitutes for soft tissue combined with bone injury, and establishes a preparation method of a periosteum-bone complex scaffold derived from natural allogeneic tissue. , PBS buffer containing protease inhibitors, PBS buffer containing Triton X-100, PBS buffer containing SLES, PBS buffer containing DNase I and Tris-HCl buffer after detoxification treatment.
- PBS buffer containing protease inhibitors PBS buffer containing Triton X-100
- PBS buffer containing SLES PBS buffer containing DNase I
- Tris-HCl buffer after detoxification treatment.
- the periosteum-bone scaffold used in the present invention is a space-patterned periosteum-bone complex obtained from an allogeneic natural source using an innovative method combining physical freeze-thaw, ultrasonic decalcification, and combined decellularization techniques.
- the material of the present invention is detoxified by repeated rinsing with Tris-HCl buffer and sterile saline, the scaffold has low cytotoxicity, has good biocompatibility and main bioactive components, and has a natural complex structure that is difficult to replicate .
- the material prepared by the present invention can be used as an immune barrier to participate in the transformation of macrophage phenotype in the early stage of bone repair.
- the material prepared by the present invention has the ability to coordinate early immune regulation and subsequent osteogenesis-angiogenesis events, and has the ability to promote bone repair.
- Fig. 1 is material general figure and CT scanning figure of the present invention
- Fig. 2 is material H&E staining of the present invention, DAPI staining figure and DNA quantitative detection figure;
- Fig. 3 is material Sirius red staining of the present invention, polarized light observation, collagen and GAGs quantitative detection figure;
- Fig. 4 is scanning electron micrograph of each part microstructure of material of the present invention.
- Fig. 5 is the atomic force microscope diagram, pressure-strain curve diagram and stiffness calculation value of the material of the present invention
- Figure 6 is a diagram showing that the material of the present invention inhibits the polarization of macrophage M1 and inhibits the expression of inflammation-related genes;
- Fig. 7 is a graph showing that the material of the present invention promotes the polarization of macrophage M2 and promotes the expression of anti-inflammatory related genes;
- Figure 8 is the material of the present invention promotes the expression of osteogenesis-related markers
- Fig. 9 is a diagram showing that the material of the present invention promotes blood vessel migration
- Fig. 10 is a CT scan and histological evaluation of bone repair promoted by the material of the present invention.
- Embodiment 1 The periosteum-bone complex and its preparation method aimed at rebuilding soft tissue-bone immune repair environment
- the above pretreated flaky bone was placed in 20% (w/v) EDTA-2Na solution for ultrasonic decalcification for 12 days, the ultrasonic parameter was 40kHZ, and the temperature was 22°C;
- Example 2 After each step in Example 1, a periosteum-bone complex with no cytotoxicity, low immunogenicity, good tissue active components and a spatial complex structure is finally obtained.
- the collagen arrangement, bone crystal structure and periosteum-bone interface connection structure of the present invention were observed by a scanning electron microscope, as shown in FIG. 3 .
- the atomic force microscope observes the microscopic surface topography of the present invention, as shown in Fig. 4 .
- Mechanomechanical test evaluation records the pressure-strain curve, and calculates the stiffness of the periosteal phase and bone phase of the periosteal bone scaffold in the space mode, as shown in Figure 5.
- the periosteum-bone complex scaffold affects the phenotype of macrophages.
- the periosteum phase inhibits the polarization of macrophages to the M1 direction and inhibits the expression of inflammation-related genes; the bone phase inevitably induces the polarization of macrophages to the M1 direction, as shown in Figure 6;
- the periosteum phase of the scaffold of the periosteum-bone complex induces the polarization of macrophages in the M2 direction and promotes the expression of anti-inflammatory related genes, as shown in Figure 7.
- Example 1 the periosteum-bone complex scaffold promotes osteogenesis performance verification
- the periosteum-bone complex scaffold promoted the expression of osteogenesis-related genes (Runx2, ALP, Col 1a1, OPN, OCN), as shown in Figure 8.
- Example 7 the periosteum-bone complex scaffold promotes angiogenesis performance verification
- the periosteum-bone complex scaffold promotes blood vessel formation and migration, as shown in Figure 9.
- Rats with bone defects were divided into four groups, namely the simple defect group, the softened acellular bone treatment group, and the periosteum-bone complex treatment group.
- the corresponding scaffolds were filled in the bone defects respectively. After 4 weeks and 8 weeks of treatment, it can be found that the effect of the periosteum-bone complex treatment group is significantly better than that of the other three groups.
- Figure 10 The effect of the periosteum-bone complex treatment group is significantly better than that of the other three groups.
- Embodiment 2 The periosteum-bone complex and its preparation method aimed at rebuilding soft tissue-bone immune repair environment
- the above pretreated flaky bone was placed in 20% (w/v) EDTA-2Na solution for ultrasonic decalcification for 12 days, the ultrasonic parameter was 40kHZ, and the temperature was 22°C;
- Embodiment 3 The periosteum-bone complex and its preparation method aimed at rebuilding soft tissue-bone immune repair environment
- the above pretreated flaky bone was placed in 20% (w/v) EDTA-2Na solution for ultrasonic decalcification for 12 days, the ultrasonic parameter was 40kHZ, and the temperature was 22°C;
- Embodiment 4 The periosteum-bone complex and its preparation method aimed at rebuilding soft tissue-bone immune repair environment
- the above pretreated flaky bone was placed in 20% (w/v) EDTA-2Na solution for ultrasonic decalcification for 12 days, the ultrasonic parameter was 40kHZ, and the temperature was 22°C;
- the above pretreated flaky bone was placed in 20% (w/v) EDTA-2Na solution for ultrasonic decalcification for 12 days, the ultrasonic parameter was 40kHZ, and the temperature was 22°C;
- the above pretreated flaky bone was placed in 20% (w/v) EDTA-2Na solution for ultrasonic decalcification for 12 days, the ultrasonic parameter was 40kHZ, and the temperature was 22°C;
- Example 7 The periosteum-bone complex and its preparation method aimed at rebuilding the soft tissue-bone immune repair environment
- the above pretreated flaky bone was placed in 20% (w/v) EDTA-2Na solution for ultrasonic decalcification for 12 days, the ultrasonic parameter was 40kHZ, and the temperature was 22°C;
- Examples 2-7 all can prepare periosteum-bone complex decellularized materials with complete decellularization, well-retained active ingredients and complete three-dimensional structure, and the corresponding results are basically consistent with those of Example 1.
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Abstract
Disclosed are a periosteum-bone complex for reconstructing a soft tissue-bone immune repair environment and a preparation method therefor. The preparation method comprises: cutting and proofing, repeatedly rinsing with deionized water, freezing and thawing with liquid nitrogen, performing ultrasonic decalcification, and treating with a PBS buffer solution containing a protease inhibitor, a PBS buffer solution containing Triton X-100, a PBS buffer solution containing SLES, a PBS buffer solution containing DNase I and a Tris-HCl buffer solution to obtain a cell-free periosteum-bone complex material. The complex retains a good periosteum-bone space structure and extracellular active ingredients, provides a "perichondrium-sclerotin" biphasic interface, effectively blocks excessive inflammation infiltration of a soft tissue source, participates in early immune remodeling of soft tissue-bone injury, and forms a benign immune-repair microenvironment by combining its own osteogenesis and vascularization advantages of the complex. A new way of thought is provided for promoting bone tissue regeneration and repair by using a biological material.
Description
本发明主要涉及用于骨组织再生修复的生物领域，具体是一种旨在重建软组织-骨免疫修复环境的骨膜-骨复合体及其制备方法，该材料关注骨缺损伴随的软组织损伤及其对骨修复的影响，合理运用空间复合模式支架的优势促进骨再生修复。The present invention mainly relates to the biological field for bone tissue regeneration and repair, specifically a periosteum-bone complex and its preparation method aimed at rebuilding the soft tissue-bone immune repair environment. The impact of bone repair, rational use of the advantages of spatial composite mode scaffolds to promote bone regeneration and repair.
由创伤、肿瘤和感染等原因引起的骨缺损是临床常见病，影响人类的健康，特别是多发的复杂性骨缺损，往往需要反复治疗，对个人造成沉重的心理压力及经济负担，对社会稳定产生潜在的影响，是临床骨科亟待解决的难题。Bone defects caused by trauma, tumors and infections are common clinical diseases that affect human health, especially multiple complex bone defects, which often require repeated treatment, causing heavy psychological pressure and economic burden on individuals, and affecting social stability. The potential impact is an urgent problem to be solved in clinical orthopedics.
近年，生物材料的替代治疗逐渐受到人们重视，其中，高刚度的单相骨替代品(如生物陶瓷、生物玻璃)由于与骨的矿物组成相似而得到广泛应用。骨愈合作为一个复杂的生理过程，包括了早期炎症调节、血管生成、成骨分化和生物矿化，这些替代材料无疑有助于成骨，但在免疫调节、血管生成方面的优势有限。通过加入化学分子、增加表面涂层等方式可以改善对局部免疫微环境的调节，但这些人工合成或局部添加的方式与生理条件仍有很大差异，而且在化学分子的用量、释放过程等多环节中存在控制难点。另外，严重的骨缺损通常也会伴随相邻软组织的损害，严重的肌肉创伤会加剧巨噬细胞向损伤部位的募集，从而损害骨愈合。因此，寻求更加合理的骨修复替代品仍然有重要意义。In recent years, the substitution therapy of biomaterials has gradually attracted people's attention. Among them, high-rigidity single-phase bone substitutes (such as bioceramics and bioglass) have been widely used because of their similar mineral composition to bone. Bone healing is a complex physiological process, including early inflammation regulation, angiogenesis, osteogenic differentiation, and biomineralization. These alternative materials undoubtedly contribute to osteogenesis, but have limited advantages in immune regulation and angiogenesis. The regulation of the local immune microenvironment can be improved by adding chemical molecules and adding surface coatings, but these methods of artificial synthesis or local addition are still very different from physiological conditions, and there are many factors such as the amount of chemical molecules and the release process. There are control difficulties in the link. In addition, severe bone defects are often accompanied by damage to adjacent soft tissues, and severe muscle trauma can exacerbate the recruitment of macrophages to the injury site, thereby impairing bone healing. Therefore, it is still of great significance to seek more reasonable bone repair substitutes.
天然骨是由软质的骨膜覆盖的高度血管化的硬质组织，骨膜可以作为抵抗创伤环境中炎性细胞过度渗透的细胞屏障，也是阻隔肌肉、纤维组织等填塞缺损部位的物理屏障。而且，骨膜为皮质骨提供了充足的血供和营养，也为间充质干细胞分化成熟提供了支持性的微环境。另外，报道显示，骨膜细胞外基质来源的水凝胶可在骨损伤早期增强巨噬细胞的M2极化，发挥免疫调节功能。Natural bone is a highly vascularized hard tissue covered by soft periosteum. The periosteum can serve as a cellular barrier against excessive penetration of inflammatory cells in a traumatic environment, and it is also a physical barrier that blocks muscle and fibrous tissue from filling the defect. Moreover, the periosteum provides sufficient blood supply and nutrition for the cortical bone, and also provides a supporting microenvironment for the differentiation and maturation of mesenchymal stem cells. In addition, reports have shown that hydrogels derived from periosteal extracellular matrix can enhance the M2 polarization of macrophages in the early stage of bone injury and play an immunomodulatory role.
鉴于骨膜在物理阻隔、免疫调节和血管生成等方面的天然优势，结合皮质骨本身的成骨性能，本发明通过创新的细胞外基质制备技术来获得空间模式的骨膜-骨复合体，证明其具备调节软组织-骨损伤早期免疫微环境的性能，并协调后续成血管、成骨事件，促进骨愈合。目前，尚无有关骨膜-骨复合体制备的报道。In view of the natural advantages of periosteum in terms of physical barrier, immune regulation and angiogenesis, combined with the osteogenic properties of cortical bone itself, the present invention obtains a space-patterned periosteum-bone complex through an innovative extracellular matrix preparation technology, proving that it has Regulate the performance of the immune microenvironment in the early stage of soft tissue-bone injury, coordinate subsequent angiogenesis and osteogenesis events, and promote bone healing. At present, there is no report on the preparation of periosteum-bone complex.
发明内容Contents of the invention
本发明目的在于填补现有技术中，单相骨修复生物支架难以有效避免软组织来源炎症浸润，加重骨缺损修复早期免疫调节不足，因此提供一种旨在重建软组织-骨免疫修复环境的空间模式骨膜-骨复合体及其制备方法。The purpose of the present invention is to fill in the prior art, single-phase bone repair biological scaffolds are difficult to effectively avoid soft tissue-derived inflammatory infiltration, which aggravates the lack of immune regulation in the early stage of bone defect repair, and therefore provides a space pattern periosteum for rebuilding the soft tissue-bone immune repair environment - Bone complex and method for its preparation.
为了实现上述目的，本发明采用如下技术方案，天然动物来源的空间模式骨膜-骨复合体的制备方法，包括以下步骤：In order to achieve the above object, the present invention adopts the following technical scheme, the preparation method of the space pattern periosteum-bone complex of natural animal origin, comprises the following steps:
(1)取宰杀12h以内的成年大白猪的股骨，分离干骺端，收集股骨干部分，去除内部骨髓腔，将股骨切割打样，分割成10mm*8mm片状；(1) Take the femur of an adult large white pig that was slaughtered within 12 hours, separate the metaphysis, collect the femoral shaft, remove the internal bone marrow cavity, cut the femur into samples, and divide it into 10mm*8mm slices;
(2)取一定量的片状骨，置于低温(4℃)摇床100rpm震荡，用含蛋白酶抑制剂的PBS缓冲液漂洗3次，每次10min，去除血液、贴附的脂肪碎片和其他杂质；(2) Take a certain amount of flaky bone, place it on a low-temperature (4°C) shaker at 100 rpm, rinse with PBS buffer containing protease inhibitors for 3 times, 10 min each time, to remove blood, attached fat fragments and other impurities;
(3)将上述预处理的片状骨置于乙二胺四乙基二钠(EDTA-Na2)溶液中超声脱钙12天；(3) Place the above-mentioned pretreated flake bone in EDTA-Na2 solution for ultrasonic decalcification for 12 days;
(4)将脱钙处理后的片状骨用液氮冻融3个循环(-80℃/22℃)；(4) Freezing and thawing the decalcified flake bone with liquid nitrogen for 3 cycles (-80°C/22°C);
(5)在含Triton X的PBS缓冲液中，低温(4℃)摇床100rpm震荡12-36小时；(5) In the PBS buffer solution containing Triton X, shake at 100 rpm on a low-temperature (4°C) shaker for 12-36 hours;
(6)在含十二烷基醚硫酸钠磺酸(SLES)的PBS缓冲液中，低温(4℃)摇床100rpm震荡1-4小时；(6) Shake in a PBS buffer solution containing sodium lauryl ether sulfate (SLES) at 100 rpm on a low-temperature (4°C) shaker for 1-4 hours;
(7)用封口膜具包裹骨膜-骨复合体的骨膜部分，使其尽可能不对外暴露；(7) Wrap the periosteum part of the periosteum-bone complex with a sealing film so that it is not exposed to the outside as much as possible;
(8)再次在含SLES的PBS缓冲液中，低温(4℃)摇床100rpm震荡12-36小时；(8) In the PBS buffer containing SLES again, shake at 100 rpm on a low-temperature (4°C) shaker for 12-36 hours;
(9)在含DNase I酶的PBS缓冲液中，37℃摇床100rpm震荡12-24小时；(9) In PBS buffer solution containing DNase I enzyme, shake at 100 rpm for 12-24 hours on a shaker at 37°C;
(10)在三羟甲基氨基甲烷盐酸盐(Tris-HCl)缓冲液中，低温(4℃)摇床100rpm震荡6-24小时；(10) In Tris-HCl buffer solution, shake at 100 rpm on a low-temperature (4°C) shaker for 6-24 hours;
(11)在无菌生理盐水中，加入混合抗菌溶液，低温(4℃)摇床100rpm震荡6-24小时；(11) Add the mixed antibacterial solution to the sterile physiological saline, shake at 100rpm on a low-temperature (4°C) shaker for 6-24 hours;
(12)上述(10)、(11)步骤重复3次，得到天然组织来源的骨膜-骨复合体材料。(12) The above steps (10) and (11) were repeated three times to obtain the periosteum-bone composite material derived from natural tissue.
步骤(5)-(9)中，每个步骤完成后均用双蒸水冲洗3-6小时。In steps (5)-(9), rinse with double distilled water for 3-6 hours after each step is completed.
另外，含EDTA-Na
2的脱钙液，EDTA-Na
2质量浓度为5％-20％；
In addition, for the decalcification solution containing EDTA-Na 2 , the mass concentration of EDTA-Na 2 is 5%-20%;
含蛋白酶抑制剂的PBS缓冲液，蛋白酶抑制剂浓度为10-50K IU/ml；PBS buffer containing protease inhibitors, the concentration of protease inhibitors is 10-50K IU/ml;
含Triton X的PBS缓冲液为质量浓度0.01％-5％的Triton X-100 PBS缓冲液；The PBS buffer containing Triton X is Triton X-100 PBS buffer with a mass concentration of 0.01%-5%;
含SLES的PBS缓冲液为质量浓度0.01％-5％的SLES的PBS缓冲液；The PBS buffer solution containing SLES is the PBS buffer solution of SLES with a mass concentration of 0.01%-5%;
含DNase Ⅰ的PBS缓冲液为浓度为1-2mg/mL的PBS缓冲液；The PBS buffer solution containing DNase Ⅰ is the PBS buffer solution with a concentration of 1-2mg/mL;
含Tris-HCl缓冲液,Tris-HCl浓度为5-20mM；Containing Tris-HCl buffer solution, the concentration of Tris-HCl is 5-20mM;
混合抗菌液中青霉素和链霉素的浓度分别为20U/ml，20μg/ml；青霉素和链霉素的比例为1:1，加入的混合抗菌液与原溶液体积比为5:1。The concentrations of penicillin and streptomycin in the mixed antibacterial solution were 20 U/ml and 20 μg/ml respectively; the ratio of penicillin and streptomycin was 1:1, and the volume ratio of the added mixed antibacterial solution to the original solution was 5:1.
本发明针对目前用于临床软组织合并骨损伤替代品存在的问题，建立了一种天然异体组织来源的骨膜-骨复合体支架的制备方法，该支架由成年大白猪股骨干切割打样、反复漂洗冻融、含蛋白酶抑制剂的PBS缓冲液、含Triton X-100的PBS缓冲液、含SLES的PBS缓冲液、含DNaseⅠ的PBS缓冲液和Tris-HCl缓冲液脱毒处理后获得。经由上述的制备方案可知，与现有技术相比，本发明公开具有以下主要优点：The present invention aims at the problems existing in clinical substitutes for soft tissue combined with bone injury, and establishes a preparation method of a periosteum-bone complex scaffold derived from natural allogeneic tissue. , PBS buffer containing protease inhibitors, PBS buffer containing Triton X-100, PBS buffer containing SLES, PBS buffer containing DNase I and Tris-HCl buffer after detoxification treatment. Via the above-mentioned preparation scheme, it can be seen that compared with the prior art, the disclosure of the present invention has the following main advantages:
(1)本发明所采用的骨膜-骨支架是异体天然来源的，运用物理冻融、超声脱钙、联合脱细胞技术相结合的创新方法获得的空间模式的骨膜-骨复合体。(1) The periosteum-bone scaffold used in the present invention is a space-patterned periosteum-bone complex obtained from an allogeneic natural source using an innovative method combining physical freeze-thaw, ultrasonic decalcification, and combined decellularization techniques.
(2)本发明材料通过Tris-HCl缓冲液及无菌生理盐水反复漂洗进行脱毒，支架细胞毒性低，拥有良好的生物相容性和主要的生物活性成分，并具有难以复制的天然复杂结构。(2) The material of the present invention is detoxified by repeated rinsing with Tris-HCl buffer and sterile saline, the scaffold has low cytotoxicity, has good biocompatibility and main bioactive components, and has a natural complex structure that is difficult to replicate .
(3)本发明所制备的材料在骨修复早期可作为免疫屏障参与巨噬细胞表型转化。(3) The material prepared by the present invention can be used as an immune barrier to participate in the transformation of macrophage phenotype in the early stage of bone repair.
(4)本发明所制备的材料具有协调早期免疫调节和后续成骨-血管生成事件，具备促进骨修复的性能。(4) The material prepared by the present invention has the ability to coordinate early immune regulation and subsequent osteogenesis-angiogenesis events, and has the ability to promote bone repair.
图1是本发明材料大体图及CT扫描图；Fig. 1 is material general figure and CT scanning figure of the present invention;
图2是本发明材料H&E染色、DAPI染色图和DNA定量检测图；Fig. 2 is material H&E staining of the present invention, DAPI staining figure and DNA quantitative detection figure;
图3是本发明材料天狼星红染色、偏正光观察，胶原和GAGs定量检测图；Fig. 3 is material Sirius red staining of the present invention, polarized light observation, collagen and GAGs quantitative detection figure;
图4是本发明材料各个部分微观结构扫描电子显微镜图；Fig. 4 is scanning electron micrograph of each part microstructure of material of the present invention;
图5是本发明材料原子力显微镜图、压力-应变曲线图和刚度计算值；Fig. 5 is the atomic force microscope diagram, pressure-strain curve diagram and stiffness calculation value of the material of the present invention;
图6是本发明材料抑制巨噬细胞M1极化、抑制炎性相关基因表达图；Figure 6 is a diagram showing that the material of the present invention inhibits the polarization of macrophage M1 and inhibits the expression of inflammation-related genes;
图7是本发明材料促进巨噬细胞M2极化、促进抗炎性相关基因表达图；Fig. 7 is a graph showing that the material of the present invention promotes the polarization of macrophage M2 and promotes the expression of anti-inflammatory related genes;
图8是本发明材料促进成骨相关标志物的表达；Figure 8 is the material of the present invention promotes the expression of osteogenesis-related markers;
图9是本发明材料促进血管迁入图；Fig. 9 is a diagram showing that the material of the present invention promotes blood vessel migration;
图10是本发明材料促进骨修复的CT扫描图和组织学评估图。Fig. 10 is a CT scan and histological evaluation of bone repair promoted by the material of the present invention.
下面结合附图及实施示例对本发明进行完整详细描述，但本发明的实施不仅限于此。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。The present invention will be fully described below in conjunction with the accompanying drawings and implementation examples, but the implementation of the present invention is not limited thereto. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.
实施例1：旨在重建软组织-骨免疫修复环境的骨膜-骨复合体及其制备方法Embodiment 1: The periosteum-bone complex and its preparation method aimed at rebuilding soft tissue-bone immune repair environment
(1)取材(1) Take materials
取宰杀12h以内健康成年大白猪的股骨，分离干骺端，收集股骨干部分，去除内部骨髓腔，将股骨干分割成10mm*8mm片状；Take the femur of a healthy adult Large White pig within 12 hours of slaughter, separate the metaphysis, collect the femoral shaft part, remove the internal bone marrow cavity, and divide the femoral shaft into 10mm*8mm pieces;
(2)预处理(2) Pretreatment
取一定量的片状骨，置于低温(4℃)摇床100rpm震荡，用含蛋白酶抑制剂的PBS缓冲液漂洗3次，每次10min，去除血液、贴附的脂肪碎片和其他杂质；Take a certain amount of flaky bone, place it on a low-temperature (4°C) shaker at 100 rpm, rinse with PBS buffer containing protease inhibitors for 3 times, 10 minutes each time, to remove blood, attached fat fragments and other impurities;
(3)脱钙(3) Decalcification
将上述预处理的片状骨置于20％(w/v)EDTA-2Na溶液中超声脱钙12天，超声参数为40kHZ，温度22℃；The above pretreated flaky bone was placed in 20% (w/v) EDTA-2Na solution for ultrasonic decalcification for 12 days, the ultrasonic parameter was 40kHZ, and the temperature was 22°C;
(4)骨膜-骨复合体获取(4) Acquisition of periosteum-bone complex
①液氮冻融3个循环(-80℃/37℃)；① Three cycles of liquid nitrogen freeze-thaw (-80°C/37°C);
②在浓度为1％的Triton X-100的PBS缓冲液中，1：1加入青霉素和链霉素(20U/ml，20μg/ml)混合抗菌液，低温(4℃)摇床100rpm震荡12小时；② Add penicillin and streptomycin (20U/ml, 20μg/ml) mixed antibacterial solution in 1% Triton X-100 PBS buffer solution at a ratio of 1:1, shake at 100rpm on a low temperature (4°C) shaker for 12 hours ;
③在浓度为1％的含SLES的去离子水中，1：1加入青霉素和链霉素(20U/ml，20μg/ml)混合抗菌液，低温(4℃)摇床100rpm震荡4小时；③ Add penicillin and streptomycin (20 U/ml, 20 μg/ml) mixed antibacterial solution in 1% deionized water containing SLES at a concentration of 1:1, shake at 100 rpm on a low temperature (4°C) shaker for 4 hours;
④用封口膜具包裹骨膜-骨复合体的骨膜部分，使其尽可能不对外暴露；④ Wrap the periosteum part of the periosteum-bone complex with a parafilm so that it is not exposed to the outside as much as possible;
⑤在浓度为1％的含SLES的去离子水中，1：1加入青霉素和链霉素(20U/ml，20μg/ml)混合抗菌液，低温(4℃)摇床100rpm震荡12小时；⑤ Add penicillin and streptomycin (20U/ml, 20μg/ml) mixed antibacterial solution 1:1 in deionized water containing SLES at a concentration of 1%, shake at 100rpm on a low-temperature (4°C) shaker for 12 hours;
⑥在浓度为1mg/ml的含DNase I的PBS缓冲液中，1：1加入青霉素和链霉素(20U/ml，20μg/ml)混合抗菌液，37℃摇床100rpm震荡12小时，即可得到骨膜骨支架。⑥Add penicillin and streptomycin (20U/ml, 20μg/ml) mixed antibacterial solution 1:1 to 1mg/ml PBS buffer solution containing DNase I, and shake at 100rpm for 12 hours on a shaker at 37°C. Obtain the periosteal bone scaffold.
(5)骨膜-骨复合体脱毒(5) Detoxification of the periosteum-bone complex
①在三羟甲基氨基甲烷盐酸盐(Tris-HCl)缓冲液中，低温(4℃)摇床100rpm震荡24小时；① In Tris-HCl buffer solution, shake at 100rpm on a low-temperature (4°C) shaker for 24 hours;
②在无菌生理盐水中，加入混合抗菌溶液，低温(4℃)摇床100rpm震荡24小时；②Add the mixed antibacterial solution to sterile normal saline, shake at 100rpm on a low-temperature (4°C) shaker for 24 hours;
③上述①、②两个步骤重复三次。③Repeat the above steps ① and ② three times.
经过实例1中各个步骤处理，最终获得无细胞毒性、低免疫原性、良好组织活性成分和空间复合结构的骨膜-骨复合体。After each step in Example 1, a periosteum-bone complex with no cytotoxicity, low immunogenicity, good tissue active components and a spatial complex structure is finally obtained.
1.实例1中，骨膜-骨复合体支架的大体观及脱细胞评估1. Gross view and decellularization evaluation of periosteum-bone complex scaffold in Example 1
骨膜-骨复合体支架的大体外观图及CT扫描图，如图1。The general appearance and CT scan of the periosteum-bone complex scaffold are shown in Figure 1.
2.实例1中，骨膜-骨复合体支架的脱细胞评估2. Decellularization Evaluation of the Periosteum-bone Complex Scaffold in Example 1
H&E染色显示无细胞及无细胞核成分残留，DNA定量检测几乎不含有DNA成分，如图2。H&E staining showed that there were no cells and no nuclear components remained, and DNA quantitative detection almost contained no DNA components, as shown in Figure 2.
3.实例1中，骨膜-骨复合体支架的微观结构评估3. Microstructural evaluation of the periosteum-bone complex scaffold in Example 1
扫描电子显微镜观察本发明的胶原排布、骨结晶结构以及骨膜-骨交界面连接结构，如图3。The collagen arrangement, bone crystal structure and periosteum-bone interface connection structure of the present invention were observed by a scanning electron microscope, as shown in FIG. 3 .
4.实例1中，骨膜-骨复合体支架的表面形貌及机械评估4. Surface topography and mechanical evaluation of the periosteum-bone complex scaffold in Example 1
原子力显微镜观察本发明微观表面形貌，如图4。The atomic force microscope observes the microscopic surface topography of the present invention, as shown in Fig. 4 .
机械力学测试评估记录压力-应变曲线，计算空间模式骨膜骨支架骨膜相和骨相的刚度，如图5。Mechanomechanical test evaluation records the pressure-strain curve, and calculates the stiffness of the periosteal phase and bone phase of the periosteal bone scaffold in the space mode, as shown in Figure 5.
5.实例1中，骨膜-骨复合体支架的免疫调节功能验证5. In example 1, the immunomodulatory function verification of the periosteum-bone complex scaffold
骨膜-骨复合体支架影响巨噬细胞表型，骨膜相抑制巨噬细胞向M1方向极化，抑制炎症相关基因表达；骨相无法避免地诱导巨噬细胞向M1方向极化，如图6；The periosteum-bone complex scaffold affects the phenotype of macrophages. The periosteum phase inhibits the polarization of macrophages to the M1 direction and inhibits the expression of inflammation-related genes; the bone phase inevitably induces the polarization of macrophages to the M1 direction, as shown in Figure 6;
骨膜-骨复合体支架骨膜相诱导巨噬细胞向M2方向极化，促进抗炎性相关基因表达，如图7。The periosteum phase of the scaffold of the periosteum-bone complex induces the polarization of macrophages in the M2 direction and promotes the expression of anti-inflammatory related genes, as shown in Figure 7.
6.实例1中，骨膜-骨复合体支架促进成骨性能验证6. In Example 1, the periosteum-bone complex scaffold promotes osteogenesis performance verification
骨膜-骨复合体支架促进成骨相关基因(Runx2,ALP,Col 1a1,OPN,OCN)表达，如图8。The periosteum-bone complex scaffold promoted the expression of osteogenesis-related genes (Runx2, ALP, Col 1a1, OPN, OCN), as shown in Figure 8.
7.实例1中，骨膜-骨复合体支架促进成血管性能验证7. In Example 1, the periosteum-bone complex scaffold promotes angiogenesis performance verification
骨膜-骨复合体支架促进血管形成及迁入，如图9。The periosteum-bone complex scaffold promotes blood vessel formation and migration, as shown in Figure 9.
8.实例1中，骨膜-骨复合体支架促进骨修复的评估8. Evaluation of periosteum-bone complex scaffolds for bone repair in Example 1
将骨缺损大鼠分为四组，分别为单纯缺损组、软化的脱细胞骨治疗组、骨膜-骨复合体治疗组。分别于骨缺损处填充相应支架。治疗4周及8周后可发现，骨膜-骨复合体治疗组效果显著优于其余三组。如图10。Rats with bone defects were divided into four groups, namely the simple defect group, the softened acellular bone treatment group, and the periosteum-bone complex treatment group. The corresponding scaffolds were filled in the bone defects respectively. After 4 weeks and 8 weeks of treatment, it can be found that the effect of the periosteum-bone complex treatment group is significantly better than that of the other three groups. Figure 10.
实施例2：旨在重建软组织-骨免疫修复环境的骨膜-骨复合体及其制备方法Embodiment 2: The periosteum-bone complex and its preparation method aimed at rebuilding soft tissue-bone immune repair environment
(1)取材(1) Take materials
取宰杀12h以内健康成年大白猪的股骨，分离干骺端，收集股骨干部分，去除内部骨髓腔，将股骨干分割成10mm*8mm片状；Take the femur of a healthy adult Large White pig within 12 hours of slaughter, separate the metaphysis, collect the femoral shaft part, remove the internal bone marrow cavity, and divide the femoral shaft into 10mm*8mm pieces;
(2)预处理(2) Pretreatment
取一定量的片状骨，置于低温(4℃)摇床100rpm震荡，用含蛋白酶抑制剂的PBS缓冲液漂洗3次，每次10min，去除血液、贴附的脂肪碎片和其他杂质；Take a certain amount of flaky bone, place it on a low-temperature (4°C) shaker at 100 rpm, rinse with PBS buffer containing protease inhibitors for 3 times, 10 minutes each time, to remove blood, attached fat fragments and other impurities;
(3)脱钙(3) Decalcification
将上述预处理的片状骨置于20％(w/v)EDTA-2Na溶液中超声脱钙12天，超声参数为40kHZ，温度22℃；The above pretreated flaky bone was placed in 20% (w/v) EDTA-2Na solution for ultrasonic decalcification for 12 days, the ultrasonic parameter was 40kHZ, and the temperature was 22°C;
(4)骨膜-骨复合体获取(4) Acquisition of periosteum-bone complex
①液氮冻融3个循环(-80℃/37℃)；① Three cycles of liquid nitrogen freeze-thaw (-80°C/37°C);
②在浓度为2％的Triton X-100的PBS缓冲液中，1：1加入青霉素和链霉素(20U/ml，20μg/ml)混合抗菌液，低温(4℃)摇床100rpm震荡24小时；②Add penicillin and streptomycin (20U/ml, 20μg/ml) mixed antibacterial solution 1:1 to 2% Triton X-100 in PBS buffer, shake at 100rpm for 24 hours on a low temperature (4°C) shaker ;
③在浓度为2％的含SLES的去离子水中，1：1加入青霉素和链霉素(20U/ml，20μg/ml)混合抗菌液，低温(4℃)摇床100rpm震荡4小时；③ Add penicillin and streptomycin (20U/ml, 20μg/ml) mixed antibacterial solution in 2% deionized water containing SLES at a concentration of 1:1, shake at 100rpm on a low temperature (4°C) shaker for 4 hours;
④用封口膜具包裹骨膜-骨复合体的骨膜部分，使其尽可能不对外暴露；④ Wrap the periosteum part of the periosteum-bone complex with a parafilm so that it is not exposed to the outside as much as possible;
⑤在浓度为2％的含SLES的去离子水中，1：1加入青霉素和链霉素(20U/ml，20μg/ml)混合抗菌液，低温(4℃)摇床100rpm震荡12小时；⑤ Add penicillin and streptomycin (20U/ml, 20μg/ml) mixed antibacterial solution 1:1 in deionized water containing SLES at a concentration of 2%, and shake at 100rpm on a low-temperature (4°C) shaker for 12 hours;
⑥在浓度为1mg/ml的含DNase I的PBS缓冲液中，1：1加入青霉素和链霉素(20U/ml，20μg/ml)混合抗菌液，37℃摇床100rpm震荡12小时，即可得到骨膜骨支架。⑥Add penicillin and streptomycin (20U/ml, 20μg/ml) mixed antibacterial solution 1:1 to 1mg/ml PBS buffer solution containing DNase I, and shake at 100rpm for 12 hours on a shaker at 37°C. Obtain the periosteal bone scaffold.
(5)骨膜-骨复合体脱毒(5) Detoxification of the periosteum-bone complex
①在三羟甲基氨基甲烷盐酸盐(Tris-HCl)缓冲液中，低温(4℃)摇床100rpm震荡24小时；① In Tris-HCl buffer solution, shake at 100rpm on a low-temperature (4°C) shaker for 24 hours;
②在无菌生理盐水中，加入混合抗菌溶液，低温(4℃)摇床100rpm震荡24小时；②Add the mixed antibacterial solution to sterile normal saline, shake at 100rpm on a low-temperature (4°C) shaker for 24 hours;
③上述①、②两个步骤重复三次。③Repeat the above steps ① and ② three times.
最终获得无细胞毒性、低免疫原性、良好组织活性成分和空间复合结构的骨膜-骨复合体。Finally, the periosteum-bone complex with no cytotoxicity, low immunogenicity, good tissue active components and spatial complex structure was obtained.
实施例3：旨在重建软组织-骨免疫修复环境的骨膜-骨复合体及其制备方法Embodiment 3: The periosteum-bone complex and its preparation method aimed at rebuilding soft tissue-bone immune repair environment
(1)取材(1) Take materials
取宰杀12h以内健康成年大白猪的股骨，分离干骺端，收集股骨干部分，去除内部骨髓腔，将股骨干分割成10mm*8mm片状；Take the femur of a healthy adult Large White pig within 12 hours of slaughter, separate the metaphysis, collect the femoral shaft part, remove the internal bone marrow cavity, and divide the femoral shaft into 10mm*8mm pieces;
(2)预处理(2) Pretreatment
取一定量的片状骨，置于低温(4℃)摇床100rpm震荡，用含蛋白酶抑制剂的PBS缓冲液漂洗3次，每次10min，去除血液、贴附的脂肪碎片和其他杂质；Take a certain amount of flaky bone, place it on a low-temperature (4°C) shaker at 100 rpm, rinse with PBS buffer containing protease inhibitors for 3 times, 10 minutes each time, to remove blood, attached fat fragments and other impurities;
(3)脱钙(3) Decalcification
将上述预处理的片状骨置于20％(w/v)EDTA-2Na溶液中超声脱钙12天，超声参数为40kHZ，温度22℃；The above pretreated flaky bone was placed in 20% (w/v) EDTA-2Na solution for ultrasonic decalcification for 12 days, the ultrasonic parameter was 40kHZ, and the temperature was 22°C;
(4)骨膜-骨复合体获取(4) Acquisition of periosteum-bone complex
①液氮冻融3个循环(-80℃/37℃)；① Three cycles of liquid nitrogen freeze-thaw (-80°C/37°C);
②在浓度为1％的Triton X-100的PBS缓冲液中，1：1加入青霉素和链霉素(20U/ml，20μg/ml)混合抗菌液，低温(4℃)摇床100rpm震荡12小时；② Add penicillin and streptomycin (20U/ml, 20μg/ml) mixed antibacterial solution in 1% Triton X-100 PBS buffer solution at a ratio of 1:1, shake at 100rpm on a low temperature (4°C) shaker for 12 hours ;
③在浓度为0.5％的含SLES的去离子水中，1：1加入青霉素和链霉素(20U/ml，20μg/ml)混合抗菌液，低温(4℃)摇床100rpm震荡4小时；③ Add penicillin and streptomycin (20 U/ml, 20 μg/ml) mixed antibacterial solution in 0.5% deionized water containing SLES at a ratio of 1:1, shake at 100 rpm on a low temperature (4°C) shaker for 4 hours;
④用封口膜具包裹骨膜-骨复合体的骨膜部分，使其尽可能不对外暴露；④ Wrap the periosteum part of the periosteum-bone complex with a parafilm so that it is not exposed to the outside as much as possible;
⑤在浓度为0.5％的含SLES的去离子水中，1：1加入青霉素和链霉素(20U/ml，20μg/ml)混合抗菌液，低温(4℃)摇床100rpm震荡24小时；⑤ Add penicillin and streptomycin (20 U/ml, 20 μg/ml) mixed antibacterial solution in 0.5% deionized water containing SLES at a ratio of 1:1, shake at 100 rpm on a low temperature (4°C) shaker for 24 hours;
⑥在浓度为1mg/ml的含DNase I的PBS缓冲液中，1：1加入青霉素和链霉素(20U/ml，20μg/ml)混合抗菌液，37℃摇床100rpm震荡12小时，即可得到骨膜骨支架。⑥Add penicillin and streptomycin (20U/ml, 20μg/ml) mixed antibacterial solution 1:1 to 1mg/ml PBS buffer solution containing DNase I, and shake at 100rpm for 12 hours on a shaker at 37°C. Obtain the periosteal bone scaffold.
(5)骨膜-骨复合体脱毒(5) Detoxification of the periosteum-bone complex
①在三羟甲基氨基甲烷盐酸盐(Tris-HCl)缓冲液中，低温(4℃)摇床100rpm震荡24小时；① In Tris-HCl buffer solution, shake at 100rpm on a low-temperature (4°C) shaker for 24 hours;
②在无菌生理盐水中，加入混合抗菌溶液，低温(4℃)摇床100rpm震荡24小时；②Add the mixed antibacterial solution to sterile normal saline, shake at 100rpm on a low-temperature (4°C) shaker for 24 hours;
③上述①、②两个步骤重复三次。③Repeat the above steps ① and ② three times.
最终获得无细胞毒性、低免疫原性、良好组织活性成分和空间复合结构的骨膜-骨复合体。Finally, the periosteum-bone complex with no cytotoxicity, low immunogenicity, good tissue active components and spatial complex structure was obtained.
实施例4：旨在重建软组织-骨免疫修复环境的骨膜-骨复合体及其制备方法Embodiment 4: The periosteum-bone complex and its preparation method aimed at rebuilding soft tissue-bone immune repair environment
(1)取材(1) Take materials
取宰杀12h以内健康成年大白猪的股骨，分离干骺端，收集股骨干部分，去除内部骨髓腔，将股骨干分割成10mm*8mm片状；Take the femur of a healthy adult Large White pig within 12 hours of slaughter, separate the metaphysis, collect the femoral shaft part, remove the internal bone marrow cavity, and divide the femoral shaft into 10mm*8mm pieces;
(2)预处理(2) Pretreatment
取一定量的片状骨，置于低温(4℃)摇床100rpm震荡，用含蛋白酶抑制剂的PBS缓冲液漂洗3次，每次10min，去除血液、贴附的脂肪碎片和其他杂质；Take a certain amount of flaky bone, place it on a low-temperature (4°C) shaker at 100 rpm, rinse with PBS buffer containing protease inhibitors for 3 times, 10 minutes each time, to remove blood, attached fat fragments and other impurities;
(3)脱钙(3) Decalcification
将上述预处理的片状骨置于20％(w/v)EDTA-2Na溶液中超声脱钙12天，超声参数为40kHZ，温度22℃；The above pretreated flaky bone was placed in 20% (w/v) EDTA-2Na solution for ultrasonic decalcification for 12 days, the ultrasonic parameter was 40kHZ, and the temperature was 22°C;
(4)骨膜-骨复合体获取(4) Acquisition of periosteum-bone complex
①液氮冻融3个循环(-80℃/37℃)；① Three cycles of liquid nitrogen freeze-thaw (-80°C/37°C);
②在浓度为2％的Triton X-100的PBS缓冲液中，1：1加入青霉素和链霉素(20U/ml，20μg/ml)混合抗菌液，低温(4℃)摇床100rpm震荡12小时；② Add penicillin and streptomycin (20U/ml, 20μg/ml) mixed antibacterial solution in 2% Triton X-100 PBS buffer solution at a ratio of 1:1, shake at 100rpm on a low temperature (4°C) shaker for 12 hours ;
③在浓度为0.5％的含SLES的去离子水中，1：1加入青霉素和链霉素(20U/ml，20μg/ml)混合抗菌液，低温(4℃)摇床100rpm震荡4小时；③ Add penicillin and streptomycin (20 U/ml, 20 μg/ml) mixed antibacterial solution in 0.5% deionized water containing SLES at a ratio of 1:1, shake at 100 rpm on a low temperature (4°C) shaker for 4 hours;
④用封口膜具包裹骨膜-骨复合体的骨膜部分，使其尽可能不对外暴露；④ Wrap the periosteum part of the periosteum-bone complex with a parafilm so that it is not exposed to the outside as much as possible;
⑤在浓度为0.5％的含SLES的去离子水中，1：1加入青霉素和链霉素(20U/ml，20μg/ml)混合抗菌液，低温(4℃)摇床100rpm震荡24小时；⑤ Add penicillin and streptomycin (20 U/ml, 20 μg/ml) mixed antibacterial solution in 0.5% deionized water containing SLES at a ratio of 1:1, shake at 100 rpm on a low temperature (4°C) shaker for 24 hours;
⑥在浓度为1mg/ml的含DNase I的PBS缓冲液中，1：1加入青霉素和链霉素(20U/ml，20μg/ml)混合抗菌液，37℃摇床100rpm震荡12小时，即可得到骨膜骨支架。⑥Add penicillin and streptomycin (20U/ml, 20μg/ml) mixed antibacterial solution 1:1 to 1mg/ml PBS buffer solution containing DNase I, and shake at 100rpm for 12 hours on a shaker at 37°C. Obtain the periosteal bone scaffold.
(5)骨膜-骨复合体脱毒(5) Detoxification of the periosteum-bone complex
①在三羟甲基氨基甲烷盐酸盐(Tris-HCl)缓冲液中，低温(4℃)摇床100rpm震荡24小时；① In Tris-HCl buffer solution, shake at 100rpm on a low-temperature (4°C) shaker for 24 hours;
②在无菌生理盐水中，加入混合抗菌溶液，低温(4℃)摇床100rpm震荡12小时；②Add the mixed antibacterial solution to sterile normal saline, and shake on a low temperature (4°C) shaker at 100rpm for 12 hours;
③上述①、②两个步骤重复三次。③Repeat the above steps ① and ② three times.
最终获得无细胞毒性、低免疫原性、良好组织活性成分和空间复合结构的骨膜-骨复合体。Finally, the periosteum-bone complex with no cytotoxicity, low immunogenicity, good tissue active components and spatial complex structure was obtained.
实施例5：旨在重建软组织-骨免疫修复环境的骨膜-骨复合体及其制备方法Example 5: Periosteum-Bone Complex Aiming at Reconstructing Soft Tissue-Bone Immunological Repair Environment and Its Preparation Method
(1)取材(1) Take materials
取宰杀12h以内健康成年大白猪的股骨，分离干骺端，收集股骨干部分，去除内部骨髓腔，将股骨干分割成10mm*8mm片状；Take the femur of a healthy adult Large White pig within 12 hours of slaughter, separate the metaphysis, collect the femoral shaft part, remove the internal bone marrow cavity, and divide the femoral shaft into 10mm*8mm pieces;
(2)预处理(2) Pretreatment
取一定量的片状骨，置于低温(4℃)摇床100rpm震荡，用含蛋白酶抑制剂的PBS缓冲液漂洗3次，每次10min，去除血液、贴附的脂肪碎片和其他杂质；Take a certain amount of flaky bone, place it on a low-temperature (4°C) shaker at 100 rpm, rinse with PBS buffer containing protease inhibitors for 3 times, 10 min each time, to remove blood, attached fat fragments and other impurities;
(3)脱钙(3) Decalcification
将上述预处理的片状骨置于20％(w/v)EDTA-2Na溶液中超声脱钙12天，超声参数为40kHZ，温度22℃；The above pretreated flaky bone was placed in 20% (w/v) EDTA-2Na solution for ultrasonic decalcification for 12 days, the ultrasonic parameter was 40kHZ, and the temperature was 22°C;
(4)骨膜-骨复合体获取(4) Acquisition of periosteum-bone complex
①液氮冻融3个循环(-80℃/37℃)；① Three cycles of liquid nitrogen freeze-thaw (-80°C/37°C);
②在浓度为0.5％的Triton X-100的PBS缓冲液中，1：1加入青霉素和链霉素(20U/ml，20μg/ml)混合抗菌液，低温(4℃)摇床100rpm震荡24小时；② Add penicillin and streptomycin (20U/ml, 20μg/ml) mixed antibacterial solution in 0.5% Triton X-100 PBS buffer solution at a ratio of 1:1, shake at 100rpm on a low temperature (4°C) shaker for 24 hours ;
③在浓度为0.5％的含SLES的去离子水中，1：1加入青霉素和链霉素(20U/ml，20μg/ml)混合抗菌液，低温(4℃)摇床100rpm震荡4小时；③ Add penicillin and streptomycin (20 U/ml, 20 μg/ml) mixed antibacterial solution in 0.5% deionized water containing SLES at a ratio of 1:1, shake at 100 rpm on a low temperature (4°C) shaker for 4 hours;
④用封口膜具包裹骨膜-骨复合体的骨膜部分，使其尽可能不对外暴露；④ Wrap the periosteum part of the periosteum-bone complex with a parafilm so that it is not exposed to the outside as much as possible;
⑤在浓度为0.5％的含SLES的去离子水中，1：1加入青霉素和链霉素(20U/ml，20μg/ml)混合抗菌液，低温(4℃)摇床100rpm震荡24小时；⑤ Add penicillin and streptomycin (20 U/ml, 20 μg/ml) mixed antibacterial solution in 0.5% deionized water containing SLES at a ratio of 1:1, shake at 100 rpm on a low temperature (4°C) shaker for 24 hours;
⑥在浓度为1mg/ml的含DNase I的PBS缓冲液中，1：1加入青霉素和链霉素(20U/ml，20μg/ml)混合抗菌液，37℃摇床100rpm震荡12小时，即可得到骨膜骨支架。⑥Add penicillin and streptomycin (20U/ml, 20μg/ml) mixed antibacterial solution 1:1 to 1mg/ml PBS buffer solution containing DNase I, and shake at 100rpm for 12 hours on a shaker at 37°C. Obtain the periosteal bone scaffold.
(5)骨膜-骨复合体脱毒(5) Detoxification of the periosteum-bone complex
①在三羟甲基氨基甲烷盐酸盐(Tris-HCl)缓冲液中，低温(4℃)摇床100rpm震荡24小时；① In Tris-HCl buffer solution, shake at 100rpm on a low-temperature (4°C) shaker for 24 hours;
②在无菌生理盐水中，加入混合抗菌溶液，低温(4℃)摇床100rpm震荡24小时；②Add the mixed antibacterial solution to sterile normal saline, shake at 100rpm on a low-temperature (4°C) shaker for 24 hours;
③上述①、②两个步骤重复三次。③Repeat the above steps ① and ② three times.
最终获得无细胞毒性、低免疫原性、良好组织活性成分和空间复合结构的骨膜-骨复合体。Finally, the periosteum-bone complex with no cytotoxicity, low immunogenicity, good tissue active components and spatial complex structure was obtained.
实施例6：旨在重建软组织-骨免疫修复环境的骨膜-骨复合体及其制备方法Example 6: Periosteum-Bone Complex Aiming at Reconstructing Soft Tissue-Bone Immunological Repair Environment and Its Preparation Method
(1)取材(1) Take materials
取宰杀12h以内健康成年大白猪的股骨，分离干骺端，收集股骨干部分，去除内部骨髓腔，将股骨干分割成10mm*8mm片状；Take the femur of a healthy adult Large White pig within 12 hours of slaughter, separate the metaphysis, collect the femoral shaft part, remove the internal bone marrow cavity, and divide the femoral shaft into 10mm*8mm pieces;
(2)预处理(2) Pretreatment
取一定量的片状骨，置于低温(4℃)摇床100rpm震荡，用含蛋白酶抑制剂的PBS缓冲液漂洗3次，每次10min，去除血液、贴附的脂肪碎片和其他杂质；Take a certain amount of flaky bone, place it on a low-temperature (4°C) shaker at 100 rpm, rinse with PBS buffer containing protease inhibitors for 3 times, 10 minutes each time, to remove blood, attached fat fragments and other impurities;
(3)脱钙(3) Decalcification
将上述预处理的片状骨置于20％(w/v)EDTA-2Na溶液中超声脱钙12天，超声参数为40kHZ，温度22℃；The above pretreated flaky bone was placed in 20% (w/v) EDTA-2Na solution for ultrasonic decalcification for 12 days, the ultrasonic parameter was 40kHZ, and the temperature was 22°C;
(4)骨膜-骨复合体获取(4) Acquisition of periosteum-bone complex
①液氮冻融3个循环(-80℃/37℃)；① Three cycles of liquid nitrogen freeze-thaw (-80°C/37°C);
②在浓度为1％的Triton X-100的PBS缓冲液中，1：1加入青霉素和链霉素(20U/ml，20μg/ml)混合抗菌液，低温(4℃)摇床100rpm震荡12小时；② Add penicillin and streptomycin (20U/ml, 20μg/ml) mixed antibacterial solution in 1% Triton X-100 PBS buffer solution at a ratio of 1:1, shake at 100rpm on a low temperature (4°C) shaker for 12 hours ;
③在浓度为1％的含SLES的去离子水中，1：1加入青霉素和链霉素(20U/ml，20μg/ml)混合抗菌液，低温(4℃)摇床100rpm震荡4小时；③ Add penicillin and streptomycin (20 U/ml, 20 μg/ml) mixed antibacterial solution in 1% deionized water containing SLES at a concentration of 1:1, shake at 100 rpm on a low temperature (4°C) shaker for 4 hours;
④用封口膜具包裹骨膜-骨复合体的骨膜部分，使其尽可能不对外暴露；④ Wrap the periosteum part of the periosteum-bone complex with a parafilm so that it is not exposed to the outside as much as possible;
⑤在浓度为1％的含SLES的去离子水中，1：1加入青霉素和链霉素(20U/ml，20μg/ml)混合抗菌液，低温(4℃)摇床100rpm震荡24小时；⑤ Add penicillin and streptomycin (20U/ml, 20μg/ml) mixed antibacterial solution 1:1 in deionized water containing SLES at a concentration of 1%, shake at 100rpm on a low temperature (4°C) shaker for 24 hours;
⑥在浓度为2mg/ml的含DNase I的PBS缓冲液中，1：1加入青霉素和链霉素(20U/ml，20μg/ml)混合抗菌液，37℃摇床100rpm震荡12小时，即可得到骨膜骨支架。⑥Add penicillin and streptomycin (20U/ml, 20μg/ml) mixed antibacterial solution 1:1 to 2mg/ml PBS buffer solution containing DNase I, and shake at 100rpm for 12 hours on a shaker at 37°C. Obtain the periosteal bone scaffold.
(5)骨膜-骨复合体脱毒(5) Detoxification of the periosteum-bone complex
①在三羟甲基氨基甲烷盐酸盐(Tris-HCl)缓冲液中，低温(4℃)摇床100rpm震荡24小时；① In Tris-HCl buffer solution, shake at 100rpm on a low-temperature (4°C) shaker for 24 hours;
②在无菌生理盐水中，加入混合抗菌溶液，低温(4℃)摇床100rpm震荡12小时；②Add the mixed antibacterial solution to sterile normal saline, and shake on a low temperature (4°C) shaker at 100rpm for 12 hours;
③上述①、②两个步骤重复三次。③Repeat the above steps ① and ② three times.
最终获得无细胞毒性、低免疫原性、良好组织活性成分和空间复合结构的骨膜-骨复合体。Finally, the periosteum-bone complex with no cytotoxicity, low immunogenicity, good tissue active components and spatial complex structure was obtained.
实施例7：旨在重建软组织-骨免疫修复环境的骨膜-骨复合体及其制备方法Example 7: The periosteum-bone complex and its preparation method aimed at rebuilding the soft tissue-bone immune repair environment
(1)取材(1) Take materials
取宰杀12h以内健康成年大白猪的股骨，分离干骺端，收集股骨干部分，去除内部骨髓腔，将股骨干分割成10mm*8mm片状；Take the femur of a healthy adult Large White pig within 12 hours of slaughter, separate the metaphysis, collect the femoral shaft part, remove the internal bone marrow cavity, and divide the femoral shaft into 10mm*8mm pieces;
(2)预处理(2) Pretreatment
取一定量的片状骨，置于低温(4℃)摇床100rpm震荡，用含蛋白酶抑制剂的PBS缓冲液漂洗3次，每次10min，去除血液、贴附的脂肪碎片和其他杂质；Take a certain amount of flaky bone, place it on a low-temperature (4°C) shaker at 100 rpm, rinse with PBS buffer containing protease inhibitors for 3 times, 10 minutes each time, to remove blood, attached fat fragments and other impurities;
(3)脱钙(3) Decalcification
将上述预处理的片状骨置于20％(w/v)EDTA-2Na溶液中超声脱钙12天，超声参数为40kHZ，温度22℃；The above pretreated flaky bone was placed in 20% (w/v) EDTA-2Na solution for ultrasonic decalcification for 12 days, the ultrasonic parameter was 40kHZ, and the temperature was 22°C;
(4)骨膜-骨复合体获取(4) Acquisition of periosteum-bone complex
①液氮冻融3个循环(-80℃/37℃)；① Three cycles of liquid nitrogen freeze-thaw (-80°C/37°C);
②在浓度为1％的Triton X-100的PBS缓冲液中，1：1加入青霉素和链霉素(20U/ml，20μg/ml)混合抗菌液，低温(4℃)摇床100rpm震荡12小时；② Add penicillin and streptomycin (20U/ml, 20μg/ml) mixed antibacterial solution in 1% Triton X-100 PBS buffer solution at a ratio of 1:1, shake at 100rpm on a low temperature (4°C) shaker for 12 hours ;
③在浓度为1％的含SLES的去离子水中，1：1加入青霉素和链霉素(20U/ml，20μg/ml)混合抗菌液，低温(4℃)摇床100rpm震荡4小时；③ Add penicillin and streptomycin (20 U/ml, 20 μg/ml) mixed antibacterial solution in 1% deionized water containing SLES at a concentration of 1:1, shake at 100 rpm on a low temperature (4°C) shaker for 4 hours;
④用封口膜具包裹骨膜-骨复合体的骨膜部分，使其尽可能不对外暴露；④ Wrap the periosteum part of the periosteum-bone complex with a parafilm so that it is not exposed to the outside as much as possible;
⑤在浓度为1％的含SLES的去离子水中，1：1加入青霉素和链霉素(20U/ml，20μg/ml)混合抗菌液，低温(4℃)摇床100rpm震荡24小时；⑤ Add penicillin and streptomycin (20U/ml, 20μg/ml) mixed antibacterial solution 1:1 in deionized water containing SLES at a concentration of 1%, shake at 100rpm on a low temperature (4°C) shaker for 24 hours;
⑥在浓度为2mg/ml的含DNase I的PBS缓冲液中，1：1加入青霉素和链霉素(20U/ml，20μg/ml)混合抗菌液，37℃摇床100rpm震荡12小时，即可得到骨膜骨支架。⑥Add penicillin and streptomycin (20U/ml, 20μg/ml) mixed antibacterial solution 1:1 to 2mg/ml PBS buffer solution containing DNase I, and shake at 100rpm for 12 hours on a shaker at 37°C. Obtain the periosteal bone scaffold.
(5)骨膜-骨复合体脱毒(5) Detoxification of the periosteum-bone complex
①在三羟甲基氨基甲烷盐酸盐(Tris-HCl)缓冲液中，低温(4℃)摇床100rpm震荡24小时；① In Tris-HCl buffer solution, shake at 100rpm on a low-temperature (4°C) shaker for 24 hours;
②在无菌生理盐水中，加入混合抗菌溶液，低温(4℃)摇床100rpm震荡24小时；②Add the mixed antibacterial solution to sterile normal saline, shake at 100rpm on a low-temperature (4°C) shaker for 24 hours;
③上述①、②两个步骤重复三次。③Repeat the above steps ① and ② three times.
最终获得无细胞毒性、低免疫原性、良好组织活性成分和空间复合结构的骨膜-骨复合体。Finally, the periosteum-bone complex with no cytotoxicity, low immunogenicity, good tissue active components and spatial complex structure was obtained.
实施例2-7均能制备出脱细胞完全、活性成分保留完好和空间三维结构完整的骨膜-骨复合体脱细胞材料，相应结果与实施例1基本一致。Examples 2-7 all can prepare periosteum-bone complex decellularized materials with complete decellularization, well-retained active ingredients and complete three-dimensional structure, and the corresponding results are basically consistent with those of Example 1.
Claims (9)
- 重建软组织-骨免疫修复环境骨膜-骨复合体及制备方法，其特征在于，该方法具体包括以下步骤：Reconstruction of soft tissue-bone immune repair environment periosteum-bone complex and its preparation method, characterized in that the method specifically includes the following steps:(1)取宰杀12h以内的猪的股骨，分离干骺端，收集股骨干部分，去除内部骨髓腔，将股骨切割打样，分割成10mm*8mm片状；(1) Take the femur of pigs slaughtered within 12 hours, separate the metaphysis, collect the femoral shaft, remove the internal bone marrow cavity, cut and sample the femur, and divide it into 10mm*8mm slices;(2)取一定量的片状骨，置于4℃摇床100rpm震荡，用含蛋白酶抑制剂的PBS缓冲液漂洗3次，每次10min，去除血液、贴附的脂肪碎片和其他杂质；(2) Take a certain amount of flaky bone, place it on a shaker at 4°C at 100 rpm, rinse with PBS buffer containing protease inhibitors for 3 times, each time for 10 minutes, to remove blood, attached fat fragments and other impurities;(3)将上述预处理的片状骨置于乙二胺四乙基二钠溶液中超声脱钙12天；(3) Place the above-mentioned pretreated flake bone in an edetyl disodium solution for ultrasonic decalcification for 12 days;(4)将脱钙处理后的片状骨用液氮冻融3个循环，温度范围为-80℃/22℃；(4) Freezing and thawing the decalcified flake bone with liquid nitrogen for 3 cycles, the temperature range is -80°C/22°C;(5)在含Triton X的PBS缓冲液中，置于4℃摇床100rpm震荡12-36小时；(5) In the PBS buffer solution containing Triton X, shake at 100 rpm at 4°C for 12-36 hours;(6)在含十二烷基醚硫酸钠磺酸的PBS缓冲液中，置于4℃摇床100rpm震荡1-4小时；(6) In the PBS buffer solution containing sodium lauryl ether sulfate sulfonic acid, shake at 100 rpm for 1-4 hours on a shaker at 4°C;(7)用封口膜具包裹骨膜-骨复合体的骨膜部分；(7) Wrap the periosteum part of the periosteum-bone complex with a parafilm;(8)再次在含SLES的PBS缓冲液中，置于4℃摇床100rpm震荡12-36小时；(8) In the PBS buffer containing SLES again, shake at 100 rpm for 12-36 hours on a shaker at 4°C;(9)在含DNase I酶的PBS缓冲液中，37℃摇床100rpm震荡12-24小时；(9) In PBS buffer solution containing DNase I enzyme, shake at 100 rpm for 12-24 hours on a shaker at 37°C;(10)在三羟甲基氨基甲烷盐酸盐缓冲液中，置于4℃摇床100rpm震荡6-24小时；(10) In the tris hydrochloride buffer solution, shake at 100 rpm on a shaker at 4°C for 6-24 hours;(11)在无菌生理盐水中，加入混合抗菌溶液，置于4℃摇床100rpm震荡6-24小时；(11) Add the mixed antibacterial solution to sterile physiological saline, and place it on a shaker at 4°C at 100 rpm for 6-24 hours;(12)上述步骤(10)、(11)重复3次，得到天然组织来源的骨膜-骨复合体材料。(12) The above steps (10) and (11) were repeated three times to obtain the periosteum-bone composite material derived from natural tissue.
- 根据权利要求1所述的重建软组织-骨免疫修复环境骨膜-骨复合体及制备方法，其特征在于：步骤(5)-(9)中，每个步骤完成后均用双蒸水冲洗3-6小时。The reconstructed soft tissue-bone immune repair environment periosteum-bone complex and its preparation method according to claim 1, characterized in that: in steps (5)-(9), each step is rinsed with double distilled water for 3- 6 hours.
- 根据权利要求1所述的重建软组织-骨免疫修复环境骨膜-骨复合体及制备方法，其特征在于：所述的乙二胺四乙基二钠溶液中EDTA-Na 2质量浓度为5％-20％。 According to claim 1, the reconstruction soft tissue-bone immune repair environment periosteum-bone complex and its preparation method are characterized in that: the mass concentration of EDTA-Na in the described ethylenediaminetetraethyl disodium solution is 5%- 20%.
- 根据权利要求1所述的重建软组织-骨免疫修复环境骨膜-骨复合体及制备方法，其特征在于：所述的含蛋白酶抑制剂的PBS缓冲液中蛋白酶抑制剂浓度为10-50K IU/ml。The reconstruction soft tissue-bone immune repair environment periosteum-bone complex and its preparation method according to claim 1, characterized in that: the protease inhibitor concentration in the PBS buffer containing protease inhibitor is 10-50K IU/ml .
- 根据权利要求1所述的重建软组织-骨免疫修复环境骨膜-骨复合体及制备方 法，其特征在于：所述的含Triton X的PBS缓冲液为质量浓度0.01％-5％的Triton X-100 PBS缓冲液。The reconstructed soft tissue-bone immune repair environment periosteum-bone complex and its preparation method according to claim 1, characterized in that: the PBS buffer containing Triton X is Triton X-100 with a mass concentration of 0.01%-5%. PBS buffer.
- 根据权利要求1所述的重建软组织-骨免疫修复环境骨膜-骨复合体及制备方法，其特征在于：含SLES的PBS缓冲液为质量浓度0.01％-5％的SLES的PBS缓冲液。The reconstructed soft tissue-bone immune repair environment periosteum-bone complex and its preparation method according to claim 1, characterized in that: the PBS buffer containing SLES is the PBS buffer of SLES with a mass concentration of 0.01%-5%.
- 根据权利要求1所述的重建软组织-骨免疫修复环境骨膜-骨复合体及制备方法，其特征在于：所述的含DNase Ⅰ的PBS缓冲液为质量浓度为1-2mg/mL的PBS缓冲液。The reconstruction soft tissue-bone immune repair environment periosteum-bone complex and its preparation method according to claim 1, characterized in that: the PBS buffer containing DNase I is a PBS buffer with a mass concentration of 1-2 mg/mL .
- 根据权利要求1所述的重建软组织-骨免疫修复环境骨膜-骨复合体及制备方法，其特征在于：所述的含Tris-HCl缓冲液,Tris-HCl浓度为5-20mM。The reconstructed soft tissue-bone immune repair environment periosteum-bone complex and its preparation method according to claim 1, characterized in that: the Tris-HCl-containing buffer solution has a Tris-HCl concentration of 5-20 mM.
- 根据权利要求1所述的重建软组织-骨免疫修复环境骨膜-骨复合体及制备方法，其特征在于：混合抗菌液中青霉素和链霉素的浓度分别为20U/ml，20μg/ml；青霉素和链霉素的比例为1:1，加入的混合抗菌液与原溶液体积比为5:1。According to claim 1, the reconstruction soft tissue-bone immune repair environment periosteum-bone complex and its preparation method are characterized in that: the concentrations of penicillin and streptomycin in the mixed antibacterial solution are respectively 20 U/ml and 20 μg/ml; penicillin and The ratio of streptomycin is 1:1, and the volume ratio of the added mixed antibacterial solution to the original solution is 5:1.
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