CN116271232A - 一种基于原位矿化类骨羟基磷灰石的复合凝胶的合成方法及其产品和应用 - Google Patents
一种基于原位矿化类骨羟基磷灰石的复合凝胶的合成方法及其产品和应用 Download PDFInfo
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- CN116271232A CN116271232A CN202211095858.1A CN202211095858A CN116271232A CN 116271232 A CN116271232 A CN 116271232A CN 202211095858 A CN202211095858 A CN 202211095858A CN 116271232 A CN116271232 A CN 116271232A
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Abstract
本发明公开了一种基于原位矿化类骨羟基磷灰石的复合凝胶的合成方法：取动物骨膜，依次用免疫染色通透液和裂解液处理；将处理后的骨膜组织灭菌、脱毒、洗涤后冷冻干燥；将骨膜组织研磨成粉末后在溶液中消化，得到细胞外基质；将氯化钙溶解于乙醇中，搅拌下加入三乙胺，随后滴加磷酸的乙醇溶液搅拌，离心弃上清，并用乙醇洗涤重悬，得到磷酸钙矿化前驱体；将磷酸钙矿化前驱体与细胞外基质混合均匀得到凝胶，并在凝胶中原位仿生矿化得到类骨羟基磷灰石，作为复合凝胶。本发明还提供了上述合成方法制备得到的复合凝胶及其在制备骨缺损修复产品中的应用。该复合凝胶在体内外具有优异的生物相容性、骨传导性、骨诱导性、成血管能力以及机械性能。
Description
技术领域
本发明属于生物医用材料合成技术领域，尤其涉及一种基于原位矿化类骨羟基磷灰石的复合凝胶的合成方法及其产品和应用。
背景技术
骨骼作为支撑人体的主要硬组织，由纳米羟基磷灰石(长度30-50nm，宽度20-25nm，厚度1.5-4nm)与有机基质分级组装形成。由于骨组织成分与结构的复杂性，其缺损的愈合需要经历多重生理过程，包括早期炎症免疫调节、血管生成、成骨分化和生物矿化。而大面积骨缺损(例如颌面创伤、肿瘤消融、椎间盘损伤或退变等)往往难以通过自身愈合，是骨科最常见且具挑战性的临床问题。目前往往需要移植手术(包括自体移植、同种异体移植或外源性生物材料的移植)的介入，为骨组织的有效愈合提供适当的环境。但这些移植材料存在一些局限性：(1)自体移植物可能涉及组织发病率和供体的可用有限性；(2)同种异体移植物具有严重感染及免疫原性排斥的风险；(3)金属或陶瓷植入物等固态生物材料的大小和形状不易适应缺损组织。尽管三维打印技术的最新进展为材料的可塑提供可能性，但在植入手术中适应受损部位的形态也不易应用。因此，需构建一种在骨缺损处可完全适应的新型生物材料，从而引导细胞生长和分化并促进新骨组织的沉积。
近年来，多功能水凝胶材料具备良好的细胞相容性、细胞粘附性和对细胞的增殖、迁移及分化的促进作用，现已广泛应用于生物医用材料领域。在骨缺损修复中，仿生复合水凝胶为理想型材料，其粘弹性接近天然组织，可以很好地模拟成骨微环境，从而促进骨组织的修复与再生。且可充分适应不同形状和深度的骨缺损，并应用于微创植入手术，与宿主组织建立紧密接触限制纤维化并有利于骨传导性。该类复合水凝胶材料选用生物相容性良好的有机高分子(如海藻酸盐、壳聚糖、透明质酸、聚乙二醇、明胶等)形成有机基质网络。但其缺少成骨相关的生物活性分子，在骨组织修复中应用具有局限性。为了解决该缺陷，由组织脱细胞产生的细胞外基质(ECM)被研发应用，可较为完整保留其原始组织的生物活性及功能复杂性。然而这种基于ECM的水凝胶材料的机械性能较弱，往往需要掺杂无机颗粒增加其机械支持力。纳米羟基磷灰石作为天然骨组织的无机成分，经常被添加入有机基质网络中，可增强材料的骨传导性、骨诱导性以及成血管能力。但该人工合成的纳米羟基磷灰石与天然骨组织的形貌、结构并不相似，对骨缺损修复的生理过程缺乏全面的精确调控。且由于其与有机网络没有较强的相互作用，导致羟基磷灰石团聚、分散不均匀，在临床应用中具有局限性。而骨组织中的羟基磷灰石生成往往需要经过复杂的生物矿化过程，由无机离子或类液体前驱体在相关有机基质的调控下形成片板状的纳米羟基磷灰石与胶原进行组装。由于水凝胶中水含量较高，导致无机物含量很低，且无机物与有机基质间的相互作用较弱，导致两相易分离，因此在水凝胶中合成类骨羟基磷灰石一直为材料合成中的技术难题。
综上，需要一种在水凝胶中合成类骨羟基磷灰石的方法，从而制备出具有生物相容性、骨传导性、骨诱导性、成血管能力以及机械支持力的仿生复合凝胶，在骨缺损修复中促进炎症免疫调节、血管生成、成骨分化及生物矿化。
发明内容
本发明提供的目的在于提供一种基于原位矿化类骨羟基磷灰石的复合凝胶的合成方法及其产品和应用，合成的复合凝胶在体内外具有优异的生物相容性、骨传导性、骨诱导性、成血管能力以及机械性能，为骨缺损修复提供合适的应用材料。
本发明提供如下技术方案：
一种基于原位矿化类骨羟基磷灰石的复合凝胶的合成方法，所述合成方法包括以下步骤：
(1)取动物骨膜组织，依次用免疫染色通透液和裂解液处理，破坏骨膜组织内部的细胞；
(2)将步骤(1)处理后的骨膜组织灭菌、脱毒、洗涤后冷冻干燥；
(3)将步骤(2)的骨膜组织研磨成粉末后在溶液中消化，离心弃沉淀，调节pH至中性，得到骨膜组织脱细胞的细胞外基质；
(4)将氯化钙溶解于乙醇中，搅拌下加入三乙胺，随后滴加磷酸的乙醇溶液搅拌，离心弃上清，并用乙醇洗涤重悬，得到磷酸钙矿化前驱体；
(5)将步骤(4)制备的磷酸钙矿化前驱体与步骤(3)制备的骨膜组织脱细胞的细胞外基质混合均匀得到凝胶，磷酸钙矿化前驱体在凝胶中原位仿生矿化得到类骨羟基磷灰石，作为复合凝胶。
在本发明中，对于步骤(1)和步骤(2)，为了保证骨膜组织脱细胞后的细胞外基质具有良好的功能完整性和生物相容性，免疫染色通透液和裂解液的浓度和作用时间需要调整至合适范围，同时过氧乙酸、Tris-HCl缓冲液、冰乙酸及胃蛋白酶的浓度与作用时间也需进行调整。
在步骤(1)中，所述免疫染色通透液为聚乙二醇辛基苯基醚，所述裂解液为SDS裂解液、NP-40裂解液或RIPA裂解液。
优选的，在步骤(1)中，所述免疫染色通透液为聚乙二醇辛基苯基醚，其浓度为0.3-1％，处理时间为12-24h；所述裂解液选为SDS裂解液，其浓度为0.5-1％，处理时间为3-6h。
进一步优选的，在步骤(1)中，所述免疫染色通透液的浓度为1％，处理时间为12h。所述裂解液选为SDS裂解液，其浓度为1％，处理时间为3h。
免疫染色通透液选择为聚乙二醇辛基苯基醚，浓度为1％，处理时间为12h，这种非离子型表面活性剂可以助于原骨膜组织中的细胞细胞膜通透。裂解液选为SDS裂解液，浓度为1％，处理时间为3h，作为阴离子去垢剂，从而促进细胞膜的裂解，特别适用于骨膜组织中的细胞骨架蛋白的溶解。以上两个步骤，可以进一步保证骨膜组织的细胞完全脱干净，同时维持良好的功能完整性。
优选的，在步骤(2)中，采用过氧乙酸灭菌和Tris-HCl缓冲液脱毒；所述过氧乙酸的浓度为1-3％，处理时间为1-4h，所述Tris-HCl缓冲液脱毒时间为24-48h，所述冷冻干燥时间为24-48h。
进一步优选的，在步骤(2)中，所述过氧乙酸的浓度为3％，处理时间为2h，所述Tris-HCl缓冲液脱毒时间为36h，所述冷冻干燥时间为24h。
灭菌过程中应用的过氧乙酸的浓度为3％，处理时间为2h，Tris-HCl缓冲液脱毒时间为36h，该浓度的过氧乙酸可完全杀死骨膜组织脱细胞的细胞外基质中的细菌，利用长时间缓冲液浸泡脱毒，期间多次定期换液，保证该细胞外基质的生物相容性。
优选的，在步骤(3)中，采用冰乙酸和胃蛋白酶溶液消化，所述冰乙酸的浓度为0.1-1M，胃蛋白酶浓度为0.5-2mg/mL，消化时间为36-72h。所述离心速度为500-800g，离心时间为10-15min，所述pH调整为7.0-7.5。
进一步优选的，在步骤(3)中，所述冰乙酸的浓度为0.5M，胃蛋白酶浓度为0.8mg/mL，消化时间为48h。所述离心速度为500g，离心时间为10min。所述pH调整为7.4。
在冰乙酸与胃蛋白酶的消化过程中，骨膜组织脱细胞的细胞外基质中基本单元会发生凝胶化的交联，而后调整pH为7.4，维持良好的生物相容性。
为了调控磷酸钙矿化前驱体的尺寸及结构，确保在凝胶中的仿生矿化，所述合成方法中磷酸钙、三乙胺和磷酸的反应浓度和反应时间均需进行优化。
其中，在步骤(3)中，骨膜组织脱细胞的细胞外基质在4℃保存。
优选的，在步骤(4)中，所述氯化钙的浓度为20-40mM，三乙胺的浓度为0.4-2.0M，磷酸的浓度为10-30mM，搅拌速度为800-1200rpm，搅拌时间为4-12h。
进一步优选的，在步骤(4)中，所述氯化钙的浓度为30mM，三乙胺的浓度为0.8M，磷酸的浓度为20mM，搅拌速度为800rpm，搅拌时间为12h。
在合成磷酸钙矿化前驱体的过程中，优化的氯化钙与磷酸的浓度可将产物调控为无定形磷酸钙前驱体，且三乙胺的浓度略微过量，一直处于搅拌的状态，可将磷酸钙矿化前驱体的尺寸维持在纳米级别。
为了保证仿生复合凝胶中类骨羟基磷灰石的原位矿化，有机相和无机相间具有合适的相互作用力，提供良好的骨传导性、骨诱导性和机械强度，为骨缺损愈合提供良好的微环境，所述合成方法中，骨膜组织脱细胞后的细胞外基质与磷酸钙矿化前驱体的反应浓度与反应时间均需要进行优化。
优选的，在步骤(5)中，所述的磷酸钙矿化前驱体浓度为10-20mg/mL，离心速度为6000-8000rpm，离心时间为5-10min，骨膜组织脱细胞的细胞外基质浓度为4-8mg/mL，磷酸钙矿化前驱体在凝胶中的质量百分比控制为60-70％，原位矿化反应时间为12-48h。
进一步优选的，在步骤(5)中，所述的磷酸钙矿化前驱体浓度为10mg/mL，离心速度为6000rpm，离心时间为5min，骨膜组织脱细胞的细胞外基质为8mg/mL，磷酸钙矿化前驱体在凝胶中的质量百分比控制为70％，原位矿化反应时间为24h。
在合成类骨羟基磷灰石的过程中，利用前驱体的钙离子与细胞外基质的羧基形成较强的相互作用力形成有机-无机双重凝胶网络，并将前驱体在仿生复合凝胶中的质量百分数控制为70％，使无机前驱体能够在细胞外基质的调控下充分发生仿生矿化，在仿生凝胶中构建类骨羟基磷灰石。
其中，在步骤(5)中，在4℃混合均匀，在37℃原位矿化。
本发明还提供了一种根据上述合成方法制备得到的基于原位矿化类骨羟基磷灰石的复合凝胶。
本发明还提供了一种基于原位矿化类骨羟基磷灰石的复合凝胶在制备骨缺损修复产品中的应用。
本发明的技术构思在于选用骨膜组织脱细胞的细胞外基质作为原位仿生矿化的有机基质，可通过免疫染色通透液与裂解液将组织中细胞脱除完全，再灭菌、脱毒、消化得到功能完整且生物相容性良好的骨膜组织脱细胞的细胞外基质；选用无定形磷酸钙前驱体作为类骨羟基磷灰石的矿化前驱体，在三乙胺的封端作用下，可得到纳米尺寸的无定形磷酸钙矿化前驱体。利用无定形磷酸钙前驱体与天然的有机基质之间形成强相互作用形成复合凝胶，并在有机基质的调控下原位仿生矿化构建类骨羟基磷灰石，为骨缺损的愈合提供良好的成骨修复微环境。
本发明通过骨膜组织脱细胞的细胞外基质调控磷酸钙矿化前驱体原位仿生矿化得到类骨羟基磷灰石，并在分子尺度形成有机与无机的强相互作用，形成仿生复合凝胶，通过有机基质优异的生物功能性与生物相容性以及类骨羟基磷灰石良好的骨传导性、骨诱导性和机械强度，促进骨修复过程中炎症免疫调节、血管生成、成骨分化及生物矿化，为骨缺损愈合提供了安全有效且应用灵活的生物医用材料；并成功应用至临界尺寸的骨缺损修复，大幅度提升不可自修复的骨组织修复，为骨组织修复提供一种有效的新型生物医用复合材料。
附图说明
图1中A为骨膜组织脱细胞前后的组织切片苏木精-伊红染色(HE)、马松染色(Masson)、I型胶原免疫组化染色(Col1)、DAPI细胞核荧光染色图；B为骨膜组织脱细胞前后内部的DNA含量统计；C和D分别为磷酸钙矿化前驱体的透射电子显微镜图和XRD图谱。
图2中A为仿生复合凝胶的透射电子显微镜图；B为类骨羟基磷灰石长度、宽度与厚度统计；C、D和E分别为仿生复合凝胶的XRD图谱、扫描电子显微镜图与X射线能谱元素像分析图；F为仿生复合凝胶(BOH)、骨膜组织脱细胞后的细胞外基质(PECM)与在PECM中加入人工合成的羟基磷灰石(PECMH)的压缩模量。
图3中A和B分别为骨髓间充质干细胞(BMSC)在空白对照、PECM、PECMH与BOH组孵育7天后的碱性磷酸酶(ALP)染色图和含量定量统计；C和D分别为BMSC在空白对照、PECM、PECMH与BOH组孵育14天后的茜素红(ARS)染色图和定量统计；E和F为人脐静脉内皮细胞(HUVEC)在空白对照、PECM、PECMH与BOH组孵育6小时后的显微镜图和分支定量统计。
图4为在大鼠临界性颅骨缺损模型中，在空白对照、PECM、PECMH与BOH组缺损处4周与8周的(A)Micro CT成像、(B)骨体积分数及(C)骨密度的定量统计。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例对本发明进行进一步的详细说明。应当理解，此处所描述的具体实施方式仅仅用以解释本发明，并不限定本发明的保护范围。实施例1利用无定形磷酸钙矿化前驱体在仿生复合凝胶中合成类骨羟基磷灰石
剥取大白猪股骨上的骨膜，用去离子水冲洗三次至无血水。用免疫染色通透液(1％聚乙二醇辛基苯基醚)处理12h后，去离子水漂洗净。再用1％SDS裂解液处理3h，去离子水冲洗净。随后将样品用3％过氧乙酸灭菌2h后，转入Tris-HCl缓冲液进行脱毒36h，用PBS缓冲液洗涤后，冷冻干燥24h。将冷冻干燥后的样品研磨成粉末后，在0.5M冰乙酸与0.8mg/mL胃蛋白酶的溶液中进行消化48h，500g离心10min后弃沉淀，用10M氢氧化钠溶液调节pH至7.4，置于4℃保存，得到骨膜组织脱细胞的细胞外基质(PECM)备用。11.76g二水合氯化钙溶解于1.60L乙醇中，在800rpm磁力搅拌下，将221.79mL三乙胺加入到上述溶液中，随后滴加磷酸的乙醇溶液(4.18mL磷酸溶于80mL乙醇中)搅拌，离心弃上清，并用乙醇洗涤重悬，得到磷酸钙矿化前驱体，用去离子水洗涤三次，用去离子水分散成浓度为10mg/mL后取20mL与PECM(8mg/mL)10mL在4℃混合搅拌均匀，在37℃孵育24h，得到BOH。同样在取相同浓度人工合成的纳米羟基磷灰石20mL，在4℃与PECM(8mg/mL)10mL混合，在37℃孵育24h，得到PECMH。
如图1所示，骨膜组织脱细胞前后的组织切片通过苏木精-伊红染色(HE)、马松染色(Masson)、I型胶原免疫组化染色(Col1)、DAPI细胞核荧光染色以及其内部DNA含量的统计对比，骨膜组织在经过脱细胞处理后，细胞已经完全脱离组织，内部DNA含量显著清零，且组织原本的胶原纤维结构没有发生改变。通过透射电子显微镜观察磷酸钙矿化前驱体的尺寸，使用该方法合成的磷酸钙矿化前驱体可均匀分散且粒径大约在2-5nm。通过XRD观察，该磷酸钙矿化前驱体为无定形，可被用于后续的仿生矿化。
如图2所示，仿生复合凝胶(BOH)通过透射电子显微镜观察，其内部的无定形磷酸钙矿化前驱体已被骨膜组织脱细胞后的细胞外基质调控为结晶形态。经过统计该晶体长度为46.29±6.31nm，宽度为20.52±3.33nm，厚度为4.00±1.16nm，与天然骨羟基磷灰石的形貌与尺寸相似。通过XRD观察仿生复合凝胶在(002)，(211)，(310)，(222)，(213)与(004)均有特征峰出现，与天然骨羟基磷灰石相似。通过扫描电子显微镜观察到，BOH具有微米级别的孔，有利于细胞的生长。且通过X射线能谱元素像分析可得其内部的有C、O、Ca、P元素组成，也证实了仿生复合凝胶有机与无机相的组成。同时可观察到，该仿生复合凝胶的压缩模量为51.71±6.90kPa，比PECM(15.37±1.97kPa)与PECMH(26.27±2.42kPa)均有显著提升，也证实了单纯混合人工合成的纳米羟基磷灰石与有机基质的相互作用有限，纳米尺寸的磷酸钙矿化前驱体与有机基质间存在较强相互作用。
实施例2具有类骨羟基磷灰石的仿生复合凝胶在体内外对骨缺损愈合的应用
将骨膜组织脱细胞后的细胞外基质凝胶(PECM)、在PECM中加入人工合成的羟基磷灰石(PECMH)、仿生复合凝胶(BOH)分别加入96孔板，每孔加入75μL凝胶，以不加入材料组的孔作为空白对照。在每个孔中接种骨髓间充质干细胞(BMSC)，密度为2×103个/孔。用添加了10％胎牛血清、100U/mL青霉素和100μg/mL链霉素的高糖DMEM作为培养基，在5％CO2的37℃细胞培养箱条件下进行培养。培养7d后，吸去培养基，用PBS缓冲液冲洗2次，每孔加入400μL碱性磷酸酶(ALP)染液，避光，置于摇床上1h，吸去染色液，用PBS缓冲液冲洗2次。培养14d后，吸去培养基，用PBS缓冲液冲洗2次，每孔加入4％多聚甲醛溶液，室温固定30min，吸去固定液，用PBS缓冲液冲洗2次，每孔加入茜素红染色工作液，染色10min，吸去染色液，用PBS缓冲液冲洗2次。用光学显微镜观察BMSC在不同条件下的染色情况。将PECM、PECMH与BOH分别加入96孔板，以不加入材料组的孔作为空白对照。在每孔中加入100μL人脐静脉内皮细胞(HUVEC)，接种密度为1×105个/mL，孵育6h。用光学显微镜观察HUVEC在不同条件下的生长状态。
48只8周龄的Sprague Dawley雄性大鼠(体重为250±50g)随机平均分为PECM组、PECMH组和BOH组，以不加入材料作为空白对照。麻醉后，对大鼠颅骨皮肤进行表面消毒，用手术刀切口，在大量无菌盐水冲洗下用手术钻平转双侧全层临界尺寸的颅骨缺损(直径为5mm)。用PECM、PECMH或BOH水凝胶注射至颅骨缺损处后，用2-0尼龙线其缝合骨膜和头皮，以不注射任何材料的组作为空白对照。将大鼠单独饲养在笼子中，并保持在23±3℃的恒温。在移植后的第3天、第4周和第8周(每个时间点，每组取4只)，通过CO2窒息和颈椎脱位对动物实施安乐死。获取头骨样品并固定在4％多聚甲醛中用于随后的免疫学检查。
如图3所示，BMSC在BOH水凝胶中培养7天后碱性磷酸酶的含量比在PECM与PECMH中均有显著提升。培养14天后，经茜素红染色，在BOH水凝胶中培养的BMSC细胞外沉积的钙结节比在PECM与PECMH中显著增多。证明具有类骨羟基磷灰石的仿生复合凝胶在体外具有显著提升BMSC成骨分化及其生物矿化的功能，且比单纯混合人工合成的羟基磷灰石更有促进作用。HUVEC在BOH凝胶中培育后，较在PECM与PECMH中形成小管的形态显著完整，同时形成分支也显著增多。证明BOH在体外具有良好的促成血管功能。
如图4所示，在大鼠的临界尺寸颅骨缺损模型中，不注射任何材料的空白对照组，几乎不能进行自我修复，4周后骨体积分数仅有4.59±0.38％，骨密度为0.039±0.007g/cm3，8周骨体积分数为5.77±1.52％，骨密度为0.047±0.006g/cm3。注射PECM后，缺损处4周生长缓慢，骨体积分数为8.56±0.51％，骨密度为0.066±0.018g/cm3，直至8周骨体积分数略增长为11.06±1.36％，骨密度略增长为0.106±0.012g/cm3。在注射PECMH后，缺损处4周生长略提升，骨体积分数为13.20±1.23％，骨密度为0.143±0.016g/cm3，直至8周骨体积分数增长为20.91±2.26％，骨密度略增长为0.218±0.016g/cm3。而在注射BOH后，缺损处的修复速度与效果显著提升，4周时骨体积分数已增长为20.85±4.97％，骨密度为0.199±0.013g/cm3，8周时骨体积分数显著增长为36.01±2.06％，骨密度增长为0.398±0.024g/cm3。证明具有类骨羟基磷灰石的仿生复合水凝胶在体内对临界尺寸的骨缺损修复具有显著提升的效果。
以上所述的具体实施方式对本发明的技术方案和有益效果进行了详细说明，应理解的是以上所述仅为本发明的最优选实施例，并不用于限制本发明，凡在本发明的原则范围内所做的任何修改、补充和等同替换等，均应包含在本发明的保护范围之内。
Claims (9)
1.一种基于原位矿化类骨羟基磷灰石的复合凝胶的合成方法，其特征在于，所述合成方法包括以下步骤：
(1)取动物骨膜组织，依次用免疫染色通透液和裂解液处理，破坏骨膜组织内部的细胞；
(2)将步骤(1)处理后的骨膜组织灭菌、脱毒、洗涤后冷冻干燥；
(3)将步骤(2)的骨膜组织研磨成粉末后在溶液中消化，离心弃沉淀，调节pH至中性，得到骨膜组织脱细胞的细胞外基质；
(4)将氯化钙溶解于乙醇中，搅拌下加入三乙胺，随后滴加磷酸的乙醇溶液搅拌，离心弃上清，并用乙醇洗涤重悬，得到磷酸钙矿化前驱体；
(5)将步骤(4)制备的磷酸钙矿化前驱体与步骤(3)制备的骨膜组织脱细胞的细胞外基质混合均匀得到凝胶，磷酸钙矿化前驱体在凝胶中原位仿生矿化得到类骨羟基磷灰石，作为复合凝胶。
2.根据权利要求1所述的基于原位矿化类骨羟基磷灰石的复合凝胶的合成方法，其特征在于，在步骤(1)中，所述免疫染色通透液为聚乙二醇辛基苯基醚，所述裂解液为SDS裂解液、NP-40裂解液或RIPA裂解液。
3.根据权利要求2所述的基于原位矿化类骨羟基磷灰石的复合凝胶的合成方法，其特征在于，在步骤(1)中，所述免疫染色通透液为聚乙二醇辛基苯基醚，其浓度为0.3-1％，处理时间为12-24h；所述裂解液选为SDS裂解液，其浓度为0.5-1％，处理时间为3-6h。
4.根据权利要求1所述的基于原位矿化类骨羟基磷灰石的复合凝胶的合成方法，其特征在于，在步骤(2)中，采用过氧乙酸灭菌和Tris-HCl缓冲液脱毒；所述过氧乙酸的浓度为1-3％，处理时间为1-4h，所述Tris-HCl缓冲液脱毒时间为24-48h，所述冷冻干燥时间为24-48h。
5.根据权利要求1所述的基于原位矿化类骨羟基磷灰石的复合凝胶的合成方法，其特征在于，在步骤(3)中，采用冰乙酸与胃蛋白酶溶液消化；所述冰乙酸的浓度为0.1-1M，胃蛋白酶浓度为0.5-2mg/mL，消化时间为36-72h；所述离心速度为500-800g，离心时间为10-15min；所述pH调整为7.0-7.5。
6.根据权利要求1所述的基于原位矿化类骨羟基磷灰石的复合凝胶的合成方法，其特征在于，在步骤(4)中，所述氯化钙的浓度为20-40mM，三乙胺的浓度为0.4-2.0M，磷酸的浓度为10-30mM，搅拌速度为800-1200rpm，搅拌时间为4-12h。
7.根据权利要求1所述的基于原位矿化类骨羟基磷灰石的复合凝胶的合成方法，其特征在于，在步骤(5)中，所述的磷酸钙矿化前驱体浓度为10-20mg/mL，离心速度为6000-8000rpm，离心时间为5-10min，骨膜组织脱细胞的细胞外基质浓度为4-8mg/mL，磷酸钙矿化前驱体在凝胶中的质量百分比控制为60-70％，原位矿化反应时间为12-48h。
8.一种权利要求1-7任一所述的合成方法制备得到的基于原位矿化类骨羟基磷灰石的复合凝胶。
9.一种权利要求8所述的基于原位矿化类骨羟基磷灰石的复合凝胶在制备骨缺损修复产品中的应用。
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