CN116492510A - 一种基于脱细胞基质微球的组织填充修复材料及其制备方法 - Google Patents
一种基于脱细胞基质微球的组织填充修复材料及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种基于脱细胞基质微球的组织填充修复材料及其制备方法，所述材料是由脱细胞基质微球及凝胶介质混合制备而成。所述脱细胞基质微球既具有良好的生物相容性和降解时间可控性，还通过脱细胞基质的三维组成和结构，为组织的修复提供天然细胞外基质再生微环境，促进组织细胞增殖，效果持久且自然无痕。所述的凝胶介质用于脱细胞基质微球的均匀分散与体内递送。本发明操作工艺简单、实施条件温和，制备的组织填充修复材料，在医学美容、组织损伤修复等领域中具有广阔的应用前景。
Description
技术领域
本发明涉及生物医用材料技术与医学美容领域，具体涉及一种基于脱细胞基质微球的组织填充修复材料及其制备方法。
背景技术
目前的组织填充材料主要有生物性材料和人工合成材料。生物性可注射填充材料主要包括自体、同种异体及异种材料，如自体脂肪、动物源胶原蛋白、以及透明质酸钠凝胶等。上述生物性可注射填充材料均不可避免的面临持久性不佳等缺点。人工合成的可注射填充材料如聚左旋乳酸PLLA、聚己内酯PCL等可降解塑料制备的微球（不可降解微球已逐渐被市场摒弃），基于异物刺激和炎症修复原理，诱导胶原无序增生，具有较为长效的软组织填充效果，是目前医学美容领域正处于增长态势的注射材料。但其生物相容性差，基于病理学原理而非生理学再生，所以注射后易产生炎症反应，形成包膜、硬结、肉芽肿等问题。因此，亟需开发理想的可注射组织充填材料，使其具备生物相容性好、无毒、无致癌性、无免疫原性、效果持久且效果完美无痕等特点。
细胞外基质（extracellular matrix，ECM）是存在于细胞之间的结构蛋白、多糖、活性因子组成的复杂网络，既可为细胞提供机械性物理支撑，还通过细胞粘附域序列与细胞表面整合素家族等相结合，发挥连接、牵引等力学作用。此外，ECM中存在大量生长因子及酶类的稳定结合位点，是细胞外活性物质的储存池。因此，ECM是具备物理、生物属性的最为重要的细胞外微环境。
脱细胞细胞外基质（decellularized extracellular matrix，dECM）也称为脱细胞基质，是从组织中去除细胞成分制备的天然材料，最大程度保留天然ECM原有的生化活性成分和三维结构，有利于细胞粘附、增殖、分化及其他生物学功能的发挥。不仅如此，由于ECM成分在不同物种间的相对保守性，脱细胞基质材料几乎没有或仅有很小的免疫原性，在组织修复与再生中具有其他生物材料无法比拟的优势。
由dECM制备的水凝胶可以同时满足可注射性及可贴敷性，然而，dECM水凝胶也存在其自身的局限性。首先，脱细胞基质材料的来源是影响其再生修复活性的重要方面之一，对于惰性组织器官如真皮、肌腱、心脏等来源的dECM制备的水凝胶来说，其本身具有较差的生物活性；其次，dECM水凝胶在成胶后的支撑性及组织牵引活性等生物力学性能相对较差；另外，dECM水凝胶的体内降解时间相对较短。因此只有少部分活性细胞或组织来源的（干细胞、肝脏等）dECM水凝胶可通过一定的诱导再生能力发挥较为长效的组织修复作用。
而本发明制备的dECM微球作用于生物组织后，具有更高的局部dECM含量及更高的与自体ECM的整合程度、更高的生物力学支撑及牵引性能、更长且可控的降解时间，因此具有更强的生物刺激活性，从而使得不同来源的dECM材料均可发挥较好的诱导再生效果，是脱细胞基质应用的一大突破。
因此，dECM制备的微球具有明显优于dECM水凝胶的体内维持时间和刺激再生活性，使得材料来源不受组织器官限制。利用天然dECM微球替代可降解塑料微球作为注射填充材料，既具有良好的生物相容性，还通过脱细胞基质的生物学功能，为组织提供天然活性微环境，并促进组织再生，发挥不同于传统微球基于炎症修复和胶原增生的组织填充效果，效果持久且自然无痕。目前还未见利用脱细胞基质微球混合凝胶介质的组织填充修复材料。
发明内容
本发明的目的在于提供一种基于脱细胞基质微球的组织填充修复材料，该材料由脱细胞基质微球及凝胶介质混合制备而成。该材料可首先通过基质微球及凝胶介质的物理占位发挥即刻填充效果；其次通过其生物活性成分，活化损伤/衰老组织，促进细胞再生；还通过其天然的生物力学牵引促进活化的细胞向损伤/衰老处迁移，从而发挥长效填充及修复效果。
根据本发明的一个方面，提供一种基于脱细胞基质微球的组织填充修复材料，所述材料是由脱细胞基质微球及凝胶介质混合制备而成。优选的，所述脱细胞基质微球的含量不超过50%（w/v）。优选的，所述脱细胞基质微球的粒径为1~1000 µm；更优选的，所述脱细胞基质微球为多种粒径的混合物。
进一步的，所述脱细胞基质微球的制备方法包括以下步骤：
(1)制备脱细胞基质；优选的，所述脱细胞基质来源为培养或分离的细胞、组织或器官，经脱细胞工艺制得；优选的，使用组织或器官；更优选的，组织或器官的来源选选自肝脏、真皮、肌肉、肌腱、心脏、胰腺、脾脏、睾丸、肺、肾脏、大网膜、脂肪、小肠、脐带、胎盘、血管、神经、骨、软骨中的一种或多种；更优选的，利用浓度为0.5~2.0 mg/mL的胃蛋白酶，在稀盐酸或醋酸配制的酸性溶液中搅拌消化去除端肽；
（2）将步骤（1）制得的脱细胞基质冻干制备脱细胞基质粗制微球，依次过梯度筛网；优选的，经切割、粉碎、研磨或震荡中的一种或多种方式处理得到脱细胞基质粗制微球。优选的，所述脱细胞基质进行冻干前、后可做匀浆或研磨处理；
（3）将步骤（2）制得的脱细胞基质粗制微球用缓冲液充分溶解作为脱细胞基质溶液水相，按水相:油相介于1:1~1:20的比例高速混合得到分散均匀的脱细胞基质乳液；优选的，所述缓冲液为磷酸盐缓冲液；更优选的，所述缓冲液的pH值为2.0-6.5；
（4）将脱细胞基质乳液恒温孵育，使脱细胞基质天然成分在水相液滴中交联固化，制成脱细胞基质微球；优选的所述孵育在恒温箱中进行；
（5）依次过梯度筛网，分别收集过滤产物，得到不同粒径范围的脱细胞基质微球；
进一步的，所述步骤（2）中脱细胞基质粗制微球的直径为不大于1.0 mm，梯度筛网孔径范围为1~1000 µm。进一步的，所述步骤（3）中油相选自甲苯、液体石蜡、正己烷、二氯甲烷、三氯甲烷、乙酸乙酯、植物油中的一种或几种。进一步的，所述步骤（4）中孵育温度为不高于70℃；优选的，孵育温度为不高于40℃；更优选的，孵育过程中适当旋转或搅拌。
所述步骤（4）中交联方式为自主装交联或添加交联剂；优选的，制备脱细胞基质微球的交联剂选自二醛、二乙烯基砜、缩水甘油醚、三羟甲基丙烷-三（3-吖丙啶基丙酸酯）、多聚环氧化合物、京尼平、二胺、多胺中的一种或多种；更优选的，交联方式为不添加任何交联剂的自组装交联。进一步的，所述步骤（5）中梯度筛网孔径范围为1~1000 µm。
进一步的，所述凝胶介质为交联和/或未交联的液体状、凝胶悬液状或膏状，以满足可注射性或可涂抹性或可贴附性；优选的，制备凝胶介质的溶质为透明质酸钠、羧甲基纤维素、羧甲基纤维素钠、羟丙基甲基纤维素、胶原蛋白、壳聚糖、海藻酸钠、凡士林、淀粉、无机盐、聚谷氨酸、脱细胞基质、聚丙烯N,N-二甲基二烯丙基胺盐共聚物、聚丙烯酰胺中的一种或多种；优选的，多种分子量梯度的介质溶液混合使用；优选的，制备交联型凝胶介质的交联剂为二醛、二乙烯基砜、缩水甘油醚、三羟甲基丙烷-三（3-吖丙啶基丙酸酯）、多聚环氧化合物、京尼平、二胺、多胺中的一种或多种。
进一步的，所述的基于脱细胞基质微球的组织填充修复材料的制备方法包括以下步骤：
（i）将脱细胞基质微球缓缓倒入凝胶介质溶液中；优选的，所述脱细胞基质微球为粗制微球或乳化法制得的微球；更优选的，所述脱细胞基质微球为乳化法制得的微球；更优选的，所述脱细胞基质微球的使用方式为一种或多种粒径混合使用；更优选的，不同粒径的脱细胞基质微球的使用方式为多种粒径混合使用。
（ii）使脱细胞基质微球与凝胶介质溶液充分接触并搅拌，直到凝胶介质中没有结块为止；
（iii）除气泡处理后制备出基于脱细胞基质微球的组织填充修复材料。优选的，所述除气泡处理采用真空搅拌脱泡机。
进一步的，所述步骤（i）中脱细胞基质微球为乳化法制得的微球或脱细胞基质粗制微球。进一步的，所述步骤（i）中脱细胞基质微球为一种或多种粒径混合使用；优选的，不同粒径的脱细胞基质微球混合使用。
根据本发明的另一个方面，提供一种基于脱细胞基质微球的组织填充修复材料的制备方法，包括如下步骤：
（1）制备脱细胞基质；
（2）将步骤（1）制得的脱细胞基质冻干制备脱细胞基质粗制微球，依次过梯度筛网；
（3）将步骤（2）制得的脱细胞基质粗制微球用缓冲液充分溶解作为脱细胞基质溶液水相，按水相:油相介于1:1~1:20的比例高速混合得到分散均匀的脱细胞基质乳液；
（4）将脱细胞基质乳液恒温孵育，使脱细胞基质天然成分在水相液滴中交联固化，制成脱细胞基质微球；优选的，所述恒温孵育在恒温箱中进行；
（5）依次过梯度筛网，分别收集过滤产物，得到不同粒径范围的脱细胞基质微球；优选的，所述梯度筛网孔径范围为1~1000 µm；
（6）将脱细胞基质微球缓缓倒入凝胶介质中；
（7）使脱细胞基质微球与凝胶介质溶液充分接触并搅拌，直到凝胶介质中没有结块为止；
（8）除气泡处理后制备出基于脱细胞基质微球的组织填充修复材料。
进一步的，所述凝胶介质为交联和/或未交联的液体状、凝胶悬液状或膏状，以满足可注射性或可涂抹性或可贴附性；优选的，制备凝胶介质的溶质为透明质酸钠、羧甲基纤维素、羧甲基纤维素钠、羟丙基甲基纤维素、胶原蛋白、壳聚糖、海藻酸钠、凡士林、淀粉、无机盐、聚谷氨酸、脱细胞基质、聚丙烯N,N-二甲基二烯丙基胺盐共聚物、聚丙烯酰胺中的一种或多种；优选的，为满足可注射性，多种分子量梯度的介质溶液混合使用，以防止凝胶介质在同一时间降解，从而维持脱细胞基质微球长期稳定悬浮；优选的，制备交联型凝胶介质的交联剂为二醛、二乙烯基砜、缩水甘油醚、三羟甲基丙烷-三（3-吖丙啶基丙酸酯）、多聚环氧化合物、京尼平、二胺、多胺中的一种或多种。
根据本发明的另一方面，提供一种所述的基于脱细胞基质微球的组织填充修复材料或所述制备方法获得的基于脱细胞基质微球的组织填充修复材料的用途，用于制备医学美容或组织损伤修复试剂。
本发明的方法建立的基于脱细胞基质微球的组织填充修复材料，与现有微球产品相比，既具有良好的生物相容性，还通过脱细胞基质的生物学功能，为组织提供天然活性微环境，促进多种自身细胞外基质成分的有序再生，发挥不同于炎症修复和胶原增生的组织填充和修复效果，效果持久且自然无痕。
附图说明
图1 脱细胞基质微球促进自体成纤维细胞增殖试验
图2 脱细胞基质微球的显微图片。
图3 基于脱细胞基质微球的组织填充修复材料促进自体组织bFGF、VEGF分泌的RT-PCR检测。
图4 基于脱细胞基质微球的组织填充修复材料促进自体组织细胞外基质中胶原蛋白、弹性蛋白、纤连蛋白高表达的RT-PCR检测。
图5 基于脱细胞基质微球的组织填充修复材料不诱发自体组织IL-1、TNF-α等炎症因子高表达的RT-PCR检测。
具体实施方式
以下通过实施例来进一步说明本发明，但并不限于此。
实施例1脱细胞基质微球的制备
将冻干的脱细胞基质在胃蛋白酶酸性溶液中搅拌消化10 h以去除端肽；收集消化液作为水相，按水相:油相为1:5的比例与二氯甲烷高速混合得到分散均匀的脱细胞基质乳液。将乳液在37℃恒温孵箱中孵育5 h，使脱细胞基质天然成分在水相液滴中自组装交联固化，制成脱细胞基质微球，依次过25 µm、50 µm、100 µm、250 µm筛网，分别收集得到平均粒径为0~25 µm、25~50 µm、50~100 µm、100~250 µm的脱细胞基质微球。
实施例2脱细胞基质微球在动物皮下注射后的降解实验
实验动物（SD大鼠）用3％的戊巴比妥腹腔注射（40.0 mg/kg）麻醉后，背部皮肤碘酒、乙醇消毒。用PBS溶液混合不同规格（平均粒径为0~25 µm、25~50 µm、50~100 µm、100~250µm的脱细胞基质微球）的脱细胞基质制备的微球，缓慢注射到大鼠背部皮下组织中，注射剂量25 mg，对照组为PBS溶解相同剂量脱细胞基质制备的水凝胶。在注射后第1、2、3、4、5、6、7、8、9个月对各组动物随机在无菌条件下取注射位点皮下组织及其周边组织，固定于4%多聚甲醛中。1周后，经梯度乙醇脱水，二甲苯透明，石蜡包埋，切片。然后脱蜡复水，用Harris苏木素染色，0.5%盐酸乙醇分色，95%乙醇伊红染色，梯度乙醇脱水，二甲苯透明，中性树胶封固，显微镜下观察观察注射部位植入剂的吸收降解情况。
结果表明，脱细胞基质水凝胶及微球在注射后均可完全降解，其中水凝胶体内维持时间相对较短，而微球具有更长的体内降解时间，因而具有更好的针对缺损组织的填充效果，且降解时间随着粒径的增大而延长，说明微球具有良好降解时间可控性（表1）。
实施例3脱细胞基质微球促进自体成纤维细胞增殖试验
实验动物（SD大鼠）用3％的戊巴比妥腹腔注射（40.0 mg/kg）麻醉后，背部皮肤碘酒、乙醇消毒。用PBS溶液分别混合真皮组织来源（惰性组织器官的典型代表）、肝脏组织来源（活性组织器官的典型代表）的平均粒径为25~50 µm脱细胞基质微球，缓慢注射到大鼠背部皮下组织中，注射剂量25 mg。另外，相同剂量的真皮组织来源（惰性组织器官的典型代表）、肝脏组织来源（活性组织器官的典型代表）的脱细胞基质制备的水凝胶分别按相同方法注射。对照组为PBS缓冲液。分别在注射后第2、4、6周及半年对相应组动物在无菌条件下取注射位点皮下组织及其周边组织，固定于4%多聚甲醛中。1周后，经梯度乙醇脱水，二甲苯透明，石蜡包埋，切片。
增殖细胞核抗原（Proliferative cell nuclear antigen，PCNA）是一种DNA聚合酶辅助因子，直接参与DNA合成，是增殖细胞的特异性标记。对切片进行PCNA免疫组化染色并计数阳性细胞比例步骤如下：对切片进行脱蜡、水化，用磷酸盐缓冲液充分冲洗，用3%的过氧化氢封闭内源性过氧化物酶，用磷酸盐缓冲液充分冲洗，抗原修复，用羊血清封闭非特异性蛋白，加入已稀释的兔抗大鼠PCNA一抗，放入湿盒中4℃过夜后常温复温30 min，用磷酸盐缓冲液充分冲洗，加入已稀释的羊抗兔二抗后放入湿盒中常温1 h，用磷酸盐缓冲液充分冲洗，加入SP后放入常温30 min，用磷酸盐缓冲液充分冲洗，加入DAB显色时间控制在3-10 min由镜下观察颜色控制时间，复染、脱水、透明、封片，在显微镜下计数PCNA阳性细胞占比。
结果显示：只有活性组织来源的脱细胞基质水凝胶注射后可显著促进自身成纤维细胞增殖，而惰性组织未表现出任何影响；本发明利用惰性组织及活性组织制备的脱细胞基质微球注射后均可显著提高自身成纤维细胞PCNA的阳性率，具有明显优于脱细胞基质水凝胶的生物活性和体内维持时间，且在微球完全降解一段时间后，不再继续促进机体自身成纤维细胞增殖，具有良好的生物安全性（图1；***p＜0.001 vs. PBS缓冲液）。
实施例4凝胶介质的制备
凝胶介质的主要作用是使脱细胞基质微球分散，方便涂抹及注射，且可能发挥一定的物理支撑性，而发挥修复活性的主要为脱细胞基质微球，所以凝胶介质溶液有多种选择，本实施例使用羧甲基纤维素钠溶液，但不应被限定于此。在超净台中向烧杯中倒入一定量的磷酸盐缓冲液；缓缓倒入一定量的市售羧甲基纤维素钠粉末（终产品中的占比为3%（w/v）），使粉末与缓冲液充分接触并涡旋搅拌2 h，保证凝胶中没有粉末结块；向凝胶中加入终产品中占比为1 %（w/v）的甘油，充分搅拌5 min，制备出羧甲基纤维素钠凝胶介质溶液。
实施例5基于脱细胞基质微球的组织填充修复材料的制备
在超净台中向烧杯中倒入一定量的羧甲基纤维素钠介质溶液；分别缓缓倒入一定量的不同粒径范围的脱细胞基质微球，终产品中的占比为5%，使微球与介质溶液充分接触并搅拌2 h，保证凝胶中没有微球结块；使用真空搅拌脱泡机进行搅拌除气泡处理，制备出基于脱细胞基质微球的组织填充修复材料，于相差显微镜下拍照（图2）。
实施例6基于脱细胞基质微球的组织填充修复材料活化自体组织微环境的bFGF、VEGF表达试验
实验动物（SD大鼠）用3％的戊巴比妥腹腔注射（40.0 mg/kg）麻醉后，背部皮肤碘酒、乙醇消毒。用1 mL注射器分别将介质溶液、平均粒径为25~50 µm的基于脱细胞基质微球的组织填充修复材料、某市售PCL微球产品分3点缓慢注射到同一只大鼠背部皮下组织中，每个位点注射500 μL。将注射后的动物按取材时间点随机分为3组，每组不小于5只，分别在注射后第2、4、6个月对相应组动物在无菌条件下取注射位点皮下组织及其周边组织，进行RNA提取、反转录cDNA后各组的碱性成纤维细胞生长因子（bFGF）、血管内皮细胞生长因子（VEGF）的基因表达水平进行RT-qPCR检测。
结果显示：本发明制备的组织填充修复材料皮下注射后可促进自体bFGF、VEGF的基因表达，且在微球完全降解一段时间后，不再继续促进机体bFGF、VEGF的基因表达，具有良好的生物安全性；而市售PCL微球产品与介质对照无显著差异（图3. A. bFGF的相对表达；图3. B. VEGF的相对表达；**p＜0.01， ***p＜0.001 vs.介质溶液）
实施例7基于脱细胞基质微球的组织填充修复材料促进自体皮下组织细胞外基质中胶原蛋白、弹性蛋白、纤连蛋白表达试验
实验动物（SD大鼠）用3％的戊巴比妥腹腔注射（40.0 mg/kg）麻醉后，背部皮肤碘酒、乙醇消毒。用1 mL注射器分别将介质溶液、平均粒径为25~50 µm的基于脱细胞基质微球的组织填充修复材料、某市售PCL微球产品分3点缓慢注射到同一只大鼠背部皮下组织中，每个位点注射500 μL。将注射后的动物按取材时间点随机分为3组，每组不小于5只，分别在注射后第2、4、6个月对相应组动物在无菌条件下取注射位点皮下组织及其周边组织，进行RNA提取、反转录cDNA后各组的胶原蛋白、弹性蛋白、纤连蛋白等细胞外基质主要蛋白的基因表达水平进行RT-qPCR检测。
结果显示：本发明制备的组织填充修复材料皮下注射后可促进自身胶原蛋白、弹性蛋白等细胞外基质主要蛋白基因的表达，且在微球完全降解一段时间后，本材料不再继续促进机体自身胶原蛋白、弹性蛋白、纤连蛋白等细胞外基质主要蛋白的基因表达，具有良好的生物安全性；而市售PCL微球产品仅促进胶原蛋白增生（图4 .A. 胶原蛋白的相对表达；图4 .B. 弹性蛋白的相对表达；图4 .C. 纤连蛋白的相对表达；***p＜0.001 vs.介质溶液）。
实施例8基于脱细胞基质微球的组织填充修复材料的长效填充不诱发局部病理性炎症增生
实验动物（SD大鼠）用3％的戊巴比妥腹腔注射（40.0 mg/kg）麻醉后，背部皮肤碘酒、乙醇消毒。用1 mL注射器分别将介质溶液、平均粒径为25~50 µm的基于脱细胞基质微球的组织填充修复材料、某市售PCL微球产品分3点缓慢注射到同一只大鼠背部皮下组织中，每个位点注射500 μL。将注射后的动物按取材时间点随机分为3组，每组不小于5只，分别在注射后第2、4、6个月对相应组动物在无菌条件下取注射位点皮下组织及其周边组织，进行RNA提取、反转录cDNA后各组的IL-1、TNF-α等炎症因子的基因表达水平进行RT-qPCR检测。
结果显示：本发明制备的组织填充修复材料皮下注射后的修复机理并非通过促发炎症反应实现，具有良好的生物安全性；而市售PCL微球产品相较于介质对照，炎症因子IL-1、TNF-α的表达明显增高（图5. A. IL-1的相对表达；图5. B.TNF-α的相对表达；***p＜0.001 vs. 介质溶液）。
以上实施例表明，本材料可活化组织微环境，促进组织细胞的分裂及细胞外基质复合成分的有序再生，其修复机理不同于可降解塑料微球产品基于异物刺激的炎症修复原理造成的胶原无序增生。
所述仅为本发明的较佳实施例而已，并非用以限定本发明的实质技术内容范围，本发明的实质技术内容是广义地定义于申请的权利要求范围中，任何他人完成的技术实体或方法，若是与申请的权利要求范围所定义的完全相同，也或是一种等效的变更，均将被视为涵盖于该权利要求范围之中。
Claims (10)
1.一种基于脱细胞基质微球的组织填充修复材料，其特征在于：所述材料是由脱细胞基质微球及凝胶介质混合制备而成。
2.根据权利要求1所述的基于脱细胞基质微球的组织填充修复材料，其特征在于：所述脱细胞基质微球的制备方法包括以下步骤：
(1) 制备脱细胞基质；
（2）将步骤（1）制得的脱细胞基质冻干制备脱细胞基质粗制微球，依次过梯度筛网；
（3）将步骤（2）制得的脱细胞基质粗制微球用缓冲液在酸性条件下充分溶解作为脱细胞基质溶液水相，按水相:油相介于1:1~1:20的比例高速混合得到分散均匀的脱细胞基质乳液；
（4）将脱细胞基质乳液恒温孵育，使脱细胞基质天然成分在水相液滴中交联固化，制成脱细胞基质微球；
（5）依次过梯度筛网，分别收集过滤产物，得到不同粒径范围的脱细胞基质微球。
3.根据权利要求2所述的组织填充修复材料，其特征在于：所述步骤（2）中脱细胞基质粗制微球的直径为不大于1.0 mm，梯度筛网孔径范围为1~1000 µm；和/或所述步骤（3）中油相选自甲苯、液体石蜡、正己烷、二氯甲烷、三氯甲烷、乙酸乙酯、植物油中的一种或几种。
4.根据权利要求2所述的组织填充修复材料，其特征在于：所述步骤（4）中交联方式为自主装交联或添加交联剂，和/或所述步骤（5）中梯度筛网孔径范围为1~1000 µm。
5.根据权利要求1所述的基于脱细胞基质微球的组织填充修复材料，其特征在于：所述凝胶介质为交联和/或未交联的液体状、凝胶悬液状或膏状，以满足可注射性或可涂抹性或可贴附性。
6.根据权利要求1所述的基于脱细胞基质微球的组织填充修复材料，其特征在于：所述的基于脱细胞基质微球的组织填充修复材料的制备方法包括以下步骤：
（i）将脱细胞基质微球缓缓倒入凝胶介质溶液中；
（ii）使脱细胞基质微球与凝胶介质溶液充分接触并搅拌，直到凝胶介质中没有结块为止；
（iii）除气泡处理后制备出基于脱细胞基质微球的组织填充修复材料。
7.根据权利要求6所述的组织填充修复材料，其特征在于：所述步骤（i）中脱细胞基质微球为乳化法制得的微球或脱细胞基质粗制微球，和/或所述步骤（i）中脱细胞基质微球为一种或多种粒径混合使用。
8.一种基于脱细胞基质微球的组织填充修复材料的制备方法，其特征在于包括如下步骤：
（1）制备脱细胞基质；
（2）将步骤（1）制得的脱细胞基质冻干制备脱细胞基质粗制微球，依次过梯度筛网；
（3）将步骤（2）制得的脱细胞基质粗制微球用缓冲液在酸性条件下充分溶解作为脱细胞基质溶液水相，按水相:油相介于1:1~1:20的比例高速混合得到分散均匀的脱细胞基质乳液；
（4）将脱细胞基质乳液恒温孵育，使脱细胞基质天然成分在水相液滴中交联固化，制成脱细胞基质微球；
（5）依次过梯度筛网，分别收集过滤产物，得到不同粒径范围的脱细胞基质微球；
（6）将脱细胞基质微球缓缓倒入凝胶介质中；
（7）使脱细胞基质微球与凝胶介质溶液充分接触并搅拌，直到凝胶介质中没有结块为止；
（8）除气泡处理后制备出基于脱细胞基质微球的组织填充修复材料。
9.如权利要求8所述的一种基于脱细胞基质微球的组织填充修复材料的制备方法，其特征在于：所述凝胶介质为交联和/或未交联的液体状、凝胶悬液状或膏状，以满足可注射性或可涂抹性或可贴附性。
10.一种如权利要求1-7中任一项所述的基于脱细胞基质微球的组织填充修复材料或权利要求8-9中任一项所述制备方法获得的基于脱细胞基质微球的组织填充修复材料的用途，其特征在于，用于制备医学美容或组织损伤修复制剂。
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