CN112203702A - 富含脱细胞化骨细胞外基质水凝胶的脱细胞化骨生物材料 - Google Patents
富含脱细胞化骨细胞外基质水凝胶的脱细胞化骨生物材料 Download PDFInfo
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- CN112203702A CN112203702A CN201980028528.0A CN201980028528A CN112203702A CN 112203702 A CN112203702 A CN 112203702A CN 201980028528 A CN201980028528 A CN 201980028528A CN 112203702 A CN112203702 A CN 112203702A
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Abstract
本发明涉及生物材料，该生物材料从脱细胞化的动物骨骼组织形成并且用凝胶形式的骨细胞外基质包被，其能够提供有效的机械和生物支持，此外在其它生物材料的处理、研究和开发中用作骨移植物、生物反应器或载体时允许富含细胞、纳米复合物或药物；也就是说，它已经从脱细胞化的、冻干的、多孔的且刚性的、可操作的、安全的且非免疫原性的骨材料形成，包被有并且富含特异刺激骨组织的物质，以颗粒或块形式呈现/使用，因此它具有当用作生物反应器时体外促进成熟细胞系或祖细胞系形成的能力，并且当用作体内移植物时具有得到证明的整合以及加快骨折愈合和骨缺损的填充的能力；该生物材料还允许基于骨组织有机细胞外基质的完整性得到维持的实情促进细胞形成，并且它能够改善愈合时间，降低成本并为基础研究做出科学贡献，证明了脱细胞化有机基质的生物技术重要性、研究性需要和对生物材料的适用性。
Description
本专利涉及富含骨细胞外基质水凝胶的脱细胞化骨生物材料，更具体地涉及天然生物材料，其仅由脱细胞化的、冻干的、多孔的且刚性的、可操作的、安全的和非免疫原性的骨生物材料形成，包被有并且富含对骨组织特异性的刺激性物质，以颗粒或块形成呈现/使用。因此，当用作生物反应器时，它具有体外促进成熟细胞系或祖细胞系形成的能力，并且当体内用作骨移植物时，其显示出高的整合能力以及更快的骨折愈合和骨缺损的填充的速率。
到目前为止，对于骨组织再生，自体骨移植物(自体移植物)，接着是使用尸体人骨(同种异体移植物)或动物骨(异种移植物)，是最常用的备选，在治疗和矫正骨衰竭(bonefailure)中效果最佳。寻找克服同种异体移植物和异种移植物临床疗效的新方法仍然是组织生物工程学的一个挑战(SHRIVATS；MCDERMOTT；HOLLINGER,2014)。
为了产生理想的骨组织框架，最重要的标准之一是该产品由高度互连的多孔网络组成，该多孔网络具有足够大的尺寸以用于细胞迁移、流体交换以及最终组织生长和血管形成。然而，仅促进细胞发育是不够的，骨组织的生理作用要求植入缺损部位的生物材料除了不产生免疫响应外还能够承受与骨骼功能性压迫性刺激相关的机械负荷(GRUSKIN等人，2012)。
脱蛋白化(deproteinized)和/或冻干的生物材料是由天然的骨头和珊瑚制成的，并且已被大规模使用。它们具有继承原始材料作为孔结构的特性的优点，但是，由于所有有机材料都通过逐步退火(高达300℃)并随后冻干而去除，因此许多重要的物质和组织再生的刺激因素丧失。这种类型的无机多孔材料已在多种牙科和整形外科应用中获得了广泛的接受，尽管它们是纯矿物和骨传导性骨架，但由于它不呈现相关的骨诱导特性，因此无法改善骨再生。在这种情况下，脱矿物化(demineralized)的骨修复材料，例如
通过“匐行置换(creeping substitution)”细胞过程表现出相对较弱的临床效果，这限制了其在大骨缺损中的应用(LEI等人，2015)。然而，仅在美国，每年集中在这些常规骨移植产品使用的市场交易额就超过10亿美元(GRUSKIN等，2012)。
当前，已经开发了多种生物材料用作骨替代物，并且更广泛地分类为无机和有机材料，其包括天然来源的或合成的成分。诸如β-磷酸三钙(0-TCP)、羟磷灰石(HA)和生物活性玻璃陶瓷
的无机材料由于它们在结构和成分方面与骨本身的无机元素的相似性而已用于骨组织工程目的。这些无机生物材料甚至具有如下的优势：通常等于或大于骨组织的骨传导性和压缩能力的潜力，但是，由于它们具有天然脆弱的结构，因此对于必须承受高负荷的生物应用总是引起极大担忧(FERNANDEZ-YAGUE等，2015)。
无机材料的替代物是有机天然聚合物或化学合成的。这些替代材料具有鼓励其在组织工程中应用的特性。从天然来源，例如胶原、透明质酸、纤维素、丝、藻酸盐和壳聚糖衍生的生物材料通常以生物相容性，允许细胞在其结构内粘附和迁移为特征。特别地，胶原海绵已被用于提供生长因子并促进骨再生。尽管种类繁多，但天然聚合物的主要局限性包括加工和纯化困难以及对免疫原性的担忧。另外，产品和材料批次变化性的可能性降低了临床结果的可预测性。最后，没有天然来源的有机生物材料能够结合包含有机和无机成分的骨骼组织的机械特性(SHRIVATS；MCDERMOTT；HOLLINGER,2014)。
旨在用于组织工程的有机聚合物的合成领域已经显著增长，主要涉及框架的聚合技术以最小化批次变化性。具有特定的微观和宏观特征的合成生物材料正在开发中。微观特征包括组成、结构和连接基团，而宏观特征包括孔隙率、刚度和弹性。关于组成，经常合成用于再生骨组织生物材料的聚合物包括聚乳酸(PLA)、聚乙醇酸(PGA)、PLGA、聚己内酯(PCL)、聚乙烯(PE)、聚乙二醇(PEG)和聚甲基丙烯酸甲酯(PMMA)等等。尽管受到生物学启发并且用途广泛，但合成聚合物作为组织工程模型也存在缺陷。生物活性的缺乏限制了生物材料与宿主之间的正向相互作用，其方式与在天然具有用于组织细胞外基质(ECM)的结合域的天然来源聚合物中观察到的方式相反。此外，合成聚合物的降解产物通常包括酸副产物，例如PLA或PGA，它们可阻碍再生过程(SHRIVATS；MCDERMOTT；HOLLINGER,2014)。
最近，基于骨再生框架的开发的一些研究的临床成功似乎与通过开发无机和有机生物材料的协同组合来克服单相生物材料所呈现的局限性有关。在此意义上，在寻找杂合产品方面已经取得了令人感兴趣的进展。通过将有机和无机材料结合实现有希望的支架的生产，从而创造生物相容性模型，其为先前仅具有骨传导性的材料赋予一定的骨诱导能力，并在骨缺损区域中具有所需要的压缩强度。例如，在不损害胶原的粘合特性或聚己内酯微纤维(PCL)的机械抗性的情况下，实现胶原纳米纤维和PCL的组合。生物成型剂(biomolders)中壳聚糖和羟磷灰石的混合物产生的材料具有机械性能、孔隙率和生物活性以支持细胞生长和新的骨形成，如
产品(生物材料和生长因子的成功结合)中见到。已经有富含胶原、生长因子或骨形态发生蛋白(BMP)，尤其是促进成骨分化的BMP-2的矿物质。然而，尽管已证明BMP的疗效，但由于其生物半衰期低、系统性副作用以及在损伤部位的快速清除，其临床应用仍然是复杂的。最近，研究集中在递送系统，该递送系统使BMP远离其治疗目标的扩散最小化，不仅改善骨形成，而且还限制不必要的反应。具有组合产品的生物材料的其他示例包括胶原和HA、PGA和βTCP，以及PEG、PCL、胶原和纳米HA的特别令人感兴趣的关联(SHRIVATS；MCDERMOTT；HOLLINGER,2014)。
生产用于组织再生的生物材料的主要目的之一是支持和促进损伤部位所需要的生理功能。通常，这包括为再生细胞群体的迁移和特化以及隔离细胞外基质成分(ECM)和局部生长因子提供理想的框架。这种多维支持的行为有利于固定、锚定、分化、增殖和细胞功能性的能力。已知哺乳动物组织的细胞外基质可以被分离，脱细胞化并用作支架，它们已被显示为促进不同组织的功能恢复。从ECM的构建性重塑机制包括募集祖细胞、促进细胞迁移和增殖、区域性血管生成以及在宿主组织界面和生物支架处促进有利的M2巨噬细胞表型。尽管ECM已成功用于非同源位点，但最近的研究证明了特异性，即当从特定组织中衍生生物基质时出现其他功能并复杂的组织形成(Sawkins等，2013)。也充分描述了除了制备、加工和采购地点的差异外，与其他组织相似，供体的年龄也对ECM的性质及其临床表现产生重大影响(BENDERS等,2013；SAWKINS等,2013；WILLIAMS等,2014)。
在这种情况下，去矿物质化骨基质(DBM)被开发为骨替代品，以克服常规移植物的局限性，并在植入部位处提供更大的组织特异性。传导性小骨DBM是通过酸提取同种异体或异种骨的矿物质内容物产生的，并含有生长因子、非胶原蛋白和I型胶原(SAWKINS等，2013)。尽管具有可变性，DBM的骨诱导作用已经在动物研究中得到充分的描述，但是在人类的临床研究中却缺乏类似的信息。脱矿物质化过程的最终产品是通常与粘性载体结合的DBM粉末，其意图促进处理、配制和最小限度的临床使用，因为它不能有效地提供连续性和必要的物理支持以校正缺陷评论。粘性载体通常是水溶性聚合物，例如透明质酸钠或羧甲基纤维素，或无水可混溶的溶剂，例如甘油，它们可能具有肾毒性作用。设计为对DBM有效性测试媒介物使用的研究是有限的。迄今为止已知的是，成骨活性似乎存在差异，其可能与使用不同的媒介物，悬浮的DBM的量以及媒介物将DBM颗粒递送到骨骼缺损部位处足够的时间以促进骨骼再生的能力有关。最近的一项研究表征了对四种市售骨移植物替代品的炎症响应，并且发现这三种DBM材料比合成的羟磷灰石化合物产生更多的炎症。然而，尚不确定DBM材料或媒介物是否引起炎症响应(GRUSKIN等，2012；CHENG；SOLORIO；ALSBERG，2014)。
正由于越来越特异性的支架的开发似乎对组织工程的治疗成功至关重要，似乎也明显的是，具有时间、空间和生物刺激给药参数的多种生长因子的可用性对于成功的再生疗法的发展至关重要。实现以适当的剂量进行药物递送和生长因子的序贯递送的越来越富集的生物材料的改善可能是在胚胎发生和骨折愈合过程中重建自然发生的骨再生过程的关键(SHRIVATS；MCDERMOTT；HOLLINGER，2014)。
迄今为止，组织生物工程尚未能够为骨组织再生产生令人信服的疗法，并且还没有将细胞用途与生物材料整合在一起的产品(SHRIVATS；MCDERMOTT；HOLLINGER，2014)。临床上寻找骨再生的新的有效替代已经产生了几种可在临床上产生影响的可能性，尽管它们都没有优先考虑使用保存完好的脱细胞化骨细胞外基质和/或细胞富集作为治疗的可能性和组织工程的潜力副产物(LI等，2015)。下一代用于骨再生的生物材料不仅应在物理上支持骨缺损，而且还应在化学和生物学上维持存在的生长因子和细胞(PAUL等，2016)。
在受控环境中操作的添加三体一组(triad)：干细胞、生长因子和支架的诸如生物反应器等技术的开发使离体组织的创建成为实际可能性。3D培养物中提供的刺激物可以能够指导细胞分化和行为，产生用于体内植入的特化组织。尽管在技术和产品质量的标准化方面仍面临挑战，但期望生物反应器的临床应用将会取得进展，因为该技术提供的益处，尤其是在移植物血管化知识方面的益处可能是创建更有希望的产品和疗法的关键(BARTNIKOWSKI等，2014)。
超越生物反应器技术，下一代离体组织生产可以通过计算机制作的织物技术的发展来推进，该技术可以将天然和合成聚合物以及无机材料结合起来以生产生物材料，包括热塑性塑料、水凝胶和更复杂的复合材料框架，这将允许对仅受分布技术分辨率限制的其结构和组成进行精确控制。这可为生产具有每个患者特异性的几何参数的支架和组织打开大门，这是当前方法无法实现的壮举。对于制作尺寸和组织工程三体一组要素的功能融合，必须不断推进基础研究(FERNANDEZ-YAGUE等，2015)。
最后，在坚持不懈寻找促进骨生长的生物材料的开发中，尽管有需求且即使有值得注意的产品在研究上取得了一定的临床成功，但迄今为止，它们无一能够克服自体、同源或异种移植物在其治疗临界尺寸缺损的能力方面的功效。已开发出几种具有一定生物相容性的生物材料，因为它们被设计为尽可能模拟天然骨细胞外基质的多孔网络，但是，就结构和功能而言，它们仍无法与天然骨相比较(SHRIVATS；MCDERMOTT；HOLLINGER，2014)。
鉴于上述当时使用的技术，有可能鉴定它们的主要局限性和缺点，例如：
-自体移植物对供体的有限的可用性和高发病率；
-骨库材料的可用性和质量低；
-冷冻/冻干同种异体移植物和异种移植物产生免疫原性排斥的可能性和疾病传播的潜在风险；
-传统疗法中不令人满意且不利的临床结果的可能性；
-无机生物材料相对较弱的临床效果和较低的骨诱导能力；
-对某些材料的机械压缩和脆性的支撑能力低；
-缺乏制备技术和产品的批次质量的标准化；
-缺乏确保生长因子的足够质量和可用性的产品；
-缺乏具有允许包含细胞的技术的集成产品；
-缺乏具有多种生长因子的复合产品；
-缺乏骨组织特定来源的安全产品；
-缺乏从骨组织特定区域产生的安全产品；
-缺乏由特定年龄的骨组织生产的安全产品；
-缺乏脱细胞化骨基质的安全产品；
-缺乏针对每种类型的患者、损伤或生物学目的的定制产品；
-测试的现有生物反应器模型的效率低；
-缺乏用于体外研究的产品；
-杂合产品的成本很高，尤其是与生长因子相关时。
在此意义上讲，为了解决甚至克服已鉴定的缺点，开发了富含骨细胞外基质水凝胶的脱细胞化骨生物材料。它代表了生物材料，该生物材料从脱细胞化的动物骨骼组织开发，并包被有凝胶形式的骨细胞外基质，其能够赋予有效的机械和生物支持，并且在其他生物材料的处理、研究和开发中用作骨移植物、生物反应器或媒介物时进一步富集细胞系、纳米复合物或药物。本专利的产品与其他技术不同自开始以来就一直开发为通过维持骨骼组织有机细胞外基质的完整性促进细胞形成，能够改善愈合时间，降低成本并为基础科学地做出贡献，证明生物材料中脱细胞化的有机基质的生物技术重要性、研究需要和适用性。
通过根据以下附图的详细描述，可以更好地理解富含骨细胞外基质水凝胶的脱细胞化骨生物材料，其中：
图01显示了在富含骨细胞外基质水凝胶的脱细胞化骨生物材料的脱细胞化过程中浸入溶液之前(左)和浸入溶液后(右)的骨组织碎片的图片。
图02显示了富含骨细胞外基质水凝胶的脱细胞化骨生物材料的脱骨化骨基质的冻干颗粒(左)和水凝胶(右)的图片。
图03显示了富含骨细胞外基质水凝胶的脱细胞化骨生物材料的放大倍数8倍(a)、12.5倍(b)和20倍(c)下的脱细胞化骨生物材料的立体显微术(steromicroscopy)图像。
图04显示了富含骨细胞外基质水凝胶的脱细胞化骨生物材料的放大倍数50倍(A)、100倍(b)、200倍(c)和350倍(d)下的脱细胞化骨生物材料的扫描显微术图像。
根据以上附图，可以看出富含脱细胞化骨细胞外基质水凝胶的脱细胞化骨生物材料对应于天然生物材料，其仅由脱细胞化、冻干、多孔且刚性、可操作、安全且非免疫原性物质开发，包被有并且富含对骨组织骨特异性的刺激物质，以颗粒或块形式呈现/使用，具有被健康科学(医学，兽医和牙科)和生物技术中的产业生产和基础科学利用的潜力。当用作生物反应器时，它具有促进体外成熟细胞系或祖细胞系的形成的能力，并且当用作体内移植物时表现较高的整合能力和较大的骨折愈合速度和骨缺损的填充速度。
因此，从屠宰场收集并送往实验室的收集的动物骨骼具有经监管的来源/经过认证(Federal Inspection Service of the Ministry of Agriculture,Livestock andSupply-SIF/MAPA/巴西)。在清洁和解剖收集的骨之后，将选择并处理骨组织的特定区域，以最大程度地保留存在的有机细胞外基质和组织的物理、生物学和形态功能特征。通过在200-500rpm搅拌下在去污剂溶液(Triton 1-3％，十二烷基硫酸钠-SDS 0.1-2.5％或其他)中浸泡24-96小时，直到材料呈现出一定数量的小于50ng的样本DNA来处理选定的组织。脱细胞化后，将所得固体基质用缓冲溶液(PBS pH 7.4-7.8或其他溶液)反复洗涤，在25-50℃的受控温度下烘箱干燥至少12-48小时，然后冻干，在环氧乙烷中灭菌并保留用于凝胶包被。
脱细胞化的骨细胞外基质凝胶由收集的相同材料生成。在选择骨组织之后，将液氮中冷冻的材料磨成小碎片，或者直到它变成粉末。然后在室温下在以200-500rpm搅拌下于酸性溶液(0.1-2.5N HCl或其他溶液)中进行脱矿物质化24-96小时，并用蒸馏水彻底洗涤。干燥后，将材料在氯仿/甲醇溶液中，在200-500rpm的搅拌下，在室温下脱脂1-3小时，并坚持用蒸馏水洗涤。干燥后，通过在37℃在200-500rpm搅拌下浸入酶溶液(0.01％-0.5％胰蛋白酶和0.01-0.2％EDTA或其他溶液)将材料进行脱细胞化12-48小时，直至材料具有小于50ng样本DNA。在那之后，在4℃下200-500rpm搅拌下添加1％的抗生素和抗真菌溶液(链霉素/青霉素，庆大霉素或其他)达12-48小时。
在此时段之后，对培养物中的内容物进行污染检测，冻干并保存在-80℃的冰箱中。从无菌冻干的内容物中，在室温下在磁力搅拌下用胃蛋白酶0.5-2.5mg/mL的酸性溶液(HCl 0.01-0.1N)进行酶消化48小时-120小时。此后，将称为消化基质的材料保存在-80℃的冰箱中。从消化的基质中，在4℃下用0.05-0.5N NaOH溶液和缓冲溶液(PBS pH 7.4-7.8或其他溶液)进行中和。为了形成水凝胶，将材料在37℃下放置至少1-6小时。
凭借产生的脱细胞化材料、固体基质和凝胶，生物材料如下产生：将固体基质浸入由脱细胞化基质本身产生的凝胶中，从而使水凝胶填充其中存在的孔并能够包被整个材料。然后，可以将生物材料冻干并且在-80℃冰箱中保存直至使用。
鉴于上述技术的描述，以及专利申请之后的优选实施方案和可能的实施方式，其遵循的方式不应限制本发明，并且可能存在等同的构造变体，但是它们没有脱离本发明的保护范围。
Claims (11)
1.富含脱细胞化骨细胞外基质水凝胶的脱细胞化骨生物材料，其特征在于它是从成年、青年、新生儿或胎儿的天然动物或人骨骼获得的，在清洗、分选、研磨、浸泡、洗涤、干燥、冷冻干燥和冷冻几个阶段后，进行化学和酶促脱细胞化过程。
2.根据权利要求1所述的富含脱细胞化骨细胞外基质水凝胶的脱细胞化骨生物材料，其特征在于，它是使用从特定年龄的供体收集的材料从脱细胞化过程中获得的。
3.根据权利要求1和2所述的富含脱细胞化骨细胞外基质水凝胶的脱细胞化骨生物材料，其特征在于，它是使用从骨组织，例如骨膜、骨内膜、生长区、不同骨化区域、关节表面，骨密质，骨松质，骨髓腔或其他区域的特定区域收集的材料从脱细胞化过程获得的。
4.根据权利要求1、2和3所述的富含脱细胞化骨细胞外基质水凝胶的脱细胞化骨生物材料，其特征在于，它是以不同浓度，单独或组合使用去污剂如Triton、十二烷基硫酸钠-SDS、乙酰氨基甲基-二乙基乙酸铵(Abietamidomethyl-diethylammonium Acetate)或类似物以保留组织的细胞外基质从脱细胞化过程中获得的。
5.根据权利要求1、2、3和4所述的富含脱细胞化骨细胞外基质水凝胶的脱细胞化骨生物材料，其特征在于，它是从凝胶化或干燥的脱细胞化细胞外基质水凝胶涂层中获得的。
6.根据权利要求1、2、3、4和5所述的富含脱细胞化骨细胞外基质水凝胶的脱细胞化骨生物材料，用作以粉末、不同粒度测量的颗粒、块或凝胶形式呈现的骨移植物。
7.根据权利要求1、2、3、4、5和6所述的富含脱细胞化骨细胞外基质水凝胶的脱细胞化骨生物材料，其特征在于其用于动物和/或人类牙程序、拔牙术、神经根切断术(rhizectomy)、三壁骨缺损、骨坑、深而狭窄的半腐败物、牙根间缺损以及与其他提取物、骨质增大或植入术的技术结合使用。
8.根据权利要求1、2、3、4、5、6和7所述的富含脱细胞化骨细胞外基质水凝胶的脱细胞化骨生物材料，其用于动物和/或人类的整形外科程序，用于骨质增大，骨折的矫正和/或骨骼不同区域的骨缺损。
9.根据权利要求1、2、3、4、5、6、7和8所述的富含脱细胞化骨细胞外基质水凝胶的脱细胞化骨生物材料，其特征在于它与适合用于指定适应症的纳米复合材料、合成药物和/或其他赋形剂结合。
10.根据权利要求1、2、3、4、5、6、7、8和9所述的富含脱细胞化骨细胞外基质水凝胶的脱细胞化骨生物材料，其特征在于其用作生物反应器或框架，用于在间充质细胞、祖细胞或成熟细胞的培养物或谱系的形成/建立中的组织工程应用。
11.根据权利要求1、2、3、4、5、6、7、8、9和10所述的富含脱细胞化骨细胞外基质水凝胶的脱细胞化骨生物材料，其用作3D打印中的原料。
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| US20200405912A1 (en) | 2020-12-31 |
| KR20210005562A (ko) | 2021-01-14 |
| BR102018003726B1 (pt) | 2023-04-11 |
| BR102018003726A2 (pt) | 2019-09-10 |
| JP2021514820A (ja) | 2021-06-17 |
| EP3760240A1 (en) | 2021-01-06 |
| MX2020008912A (es) | 2021-01-08 |
| IL276956A (en) | 2020-10-29 |
| WO2019161465A1 (pt) | 2019-08-29 |
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