这项工作得到了日本科学振兴会（JSPS）的科学研究补助金（KAKENHI）的支持（资助号）。JP19K10259 和 22K10032 到 MAI）。

该方案使用4周龄的雄性裸鼠。在22-24°C和50%-70%相对湿度下，在12小时光/暗循环下将四只小鼠饲养在笼子中。所有动物实验均按照东京医科齿科大学机构动物护理和使用委员会批准的指南（批准编号：A2019-204C、A2020-116A 和 A2021-121A）进行。
1. 缓冲液和试剂的制备
<ol><li>通过将MSC生长培养基的补充试剂盒添加到MSC基础培养基中来制备间充质干细胞生长培养基（MSC生长培养基），并将其储存在4°C。</li>
	<li>通过向培养基中加入10%胎牛血清（FBS），50μg/ mL庆大霉素，200μML-丙氨酰-L-谷氨酰胺和1ng / mL碱性成纤维细胞生长因子来制备Dulbecco改良的Eagle培养基和Ham's F12（DMEM / F-12）培养基，并将其储存在4°C。</li>
	<li>使用前向DMEM中加入1%ITS-G添加剂，0.12%牛血清白蛋白，50μg/ mL庆大霉素，0.35mM L-脯氨酸，100nM地塞米松和10ng / mL TGF-β3来制备软骨培养基。</li>
	<li>使用前向DMEM中加入10mM β-甘油磷酸盐，0.12%牛血清白蛋白，10nM地塞米松，200μM抗坏血酸-2-磷酸盐，50nM L-甲状腺素，50μg/ mL庆大霉素和50pg / mL IL-1β来制备肥厚培养基。</li>
	<li>用200μL在60°C预热的熔化石蜡涂覆24孔培养皿。 在室温下静置，直到石蜡凝固。</li>
</ol>2. 人类间充质干细胞的扩增
注：在开始实验之前，应在微量培养物中选择具有高MSC软骨形成潜力的MSC，如17所述。
<ol><li>根据制造商的说明，在37°C下，在5%CO2和95%加湿空气培养箱中，用MSC生长培养基在10cm培养皿中以5×103个细胞/ cm 2的密度培养原代人骨髓来源的MSC（传代2）。</li>
	<li>每 2-3 天更换一次培养基。一旦细胞达到 80%-90% 汇合度，使用真空泵从细胞培养皿中吸出培养基，用 5 mL PBS 洗涤，然后用真空泵吸出溶液。</li>
	<li>向培养皿中加入 1 mL 0.05% 胰蛋白酶/0.02% EDTA 溶液。将培养皿在37°C孵育5-10分钟。</li>
	<li>加入 1 mL MSC 生长培养基以终止酶促反应。将细胞悬液转移到 50 mL 试管中。</li>
	<li>将来自其他培养皿的细胞悬浮液混合在 50 mL 试管中，并将其保持在冰上。</li>
	<li>使用细胞计数器通过将 10 μL 细胞悬液与 10 μL 0.4% 台盼蓝染色剂混合来计数细胞。</li>
	<li>在室温下以220× g 离心剩余的细胞悬浮液3分钟。除去上清液，加入浓度为1.0×10 6个细胞/ mL的冻存溶液。</li>
	<li>将1mL细胞悬液分配到每个冷冻管中，并在-80°C下储存12小时，然后转移到液氮中。由此产生的冷冻库存（第3代）用于该协议。</li>
	<li>对于实验，将储存的MSC以5×103 个细胞/cm 2的密度接种在10cm培养皿中。在 DMEM/F-12 培养基中培养细胞直至 80%-90% 汇合（第 4 代）。</li>
</ol>3. MSC包封水凝胶的制备
<ol><li>胰蛋白酶消化细胞并制备细胞悬液，如步骤2.3-2.6所述。</li>
	<li>要制备 10 个构建体（每个构建体 2.5 x 10 5 个细胞），将含有 2.5 x 106 个细胞的细胞悬液等分到1.5 mL 微管中。</li>
	<li>将悬浮液以220× g 离心3分钟，用真空泵除去上清液，并将其保持在冰上。</li>
	<li>将巯基改性透明质酸 （HA）、巯基反应联剂、聚乙二醇二丙烯酸酯和脱气水瓶置于室温。</li>
	<li>在无菌条件下，使用带针头的注射器将 1.0 mL 脱气水添加到含有 HA 的瓶子中（参见制造商的说明）。</li>
	<li>偶尔涡旋升温至37°C，直到溶液变清，然后置于冰上。固体完全溶解需要不到 30 分钟的时间。</li>
	<li>在无菌条件下，使用带针的注射器向交联剂瓶中加入 0.5 mL 脱气水。倒置数次溶解，然后放在冰上。</li>
	<li>将 120 μL 溶解的 HA 溶液加入等分的细胞沉淀（2.5 x 106 个细胞）中。通过来回移液重悬细胞沉淀。</li>
	<li>将30μL溶解的交联剂溶液加入含有HA和细胞的试管中（步骤3.8），并通过敲击试管合并，然后短暂旋转。将 HA 溶液和交联剂溶液以 4：1 的比例混合。</li>
	<li>将15μL含有MSCs（2.5×10,5个细胞）的组合溶液滴到石蜡包被的24孔板上，并使其在37°C下固化30分钟（图2）。由于粘度高，因此准备至少 10% 的额外量的细胞/水凝胶混合物。 注：水凝胶的特性在前面已经描述过39。</li>
</ol>4. 体外 分化条件
<ol><li>将 0.5 mL 软骨分化培养基加入 MSC 接种的 HA 水凝胶构建体中。每 2-3 天更换一次培养基。</li>
	<li>3 周后，改用肥厚分化培养基（每孔 0.5 mL）并将构建体再培养 2 周。</li>
</ol>5 . 间 充质干细胞体内植入
<ol><li>在总共5周的体外培养后，将构建体皮下植入裸鼠的背部，如前所述38,40。</li>
	<li>称量小鼠并通过腹膜内注射给予用0.75mg / kg体重（b.w.）美托咪定，4.0mg / kg b.w.咪达唑仑和5.0mg / kg b.w.布托啡诺制备的组合麻醉剂，如所述38。</li>
	<li>通过对手术刺激的不动来确认充分的麻醉，并在手术期间通过缓慢、有规律的胸部运动和定期通过粉红色粘膜的颜色适当供氧来监测呼吸深度。在眼睛上使用兽药膏，以防止麻醉期间干燥。</li>
	<li>将小鼠放在俯卧位的无菌手术单上，用50ppm次氯酸水（pH 5.0;HAW），并在小鼠上放置穿孔手术单。 注意： 在高压灭菌器、环氧乙烷气体或 HAW 中对所有手术材料进行灭菌。外科医生应清洗手指并穿上手术衣和手套。</li>
	<li>用剪刀在肩部和臀部区域沿脊柱中心线做两个相距 2 厘米的 5 毫米长的皮肤切口。</li>
	<li>将刮刀皮下插入切口，形成皮下口袋。</li>
	<li>用镊子将两到三个构建体（每个~15μL，步骤5.1）插入每个口袋中。植入同一口袋的 HA 构建体非常容易发生融合。为防止融合，请在每个口袋中插入一个 HA 构建体。</li>
	<li>用 4-0 编织丝线缝合切口。向小鼠注射拮抗剂，以0.75mg / kg b.w.阿替美唑制备麻醉剂，如所述38。</li>
	<li>当小鼠从麻醉中恢复时，将小鼠置于无菌恢复笼中，该笼含有无菌地板垫料，没有食物和水瓶，并持续监测体温，脉搏和呼吸。</li>
	<li>不要让小鼠无人看管，直到它恢复足够的意识以保持胸骨卧位。完全康复后，将动物与其他动物一起放回饲养室。</li>
	<li>在愈合期间，每隔几天监测一次动物是否有任何可能的并发症。通过吸入二氧化碳对小鼠实施安乐死，在植入后4周和8周几乎没有痛苦。</li>
	<li>切开移植部位的皮肤，并用镊子取出植入的结构。将构建体固定在4%多聚甲醛中16小时，然后对其进行分析。</li>
</ol>6. 统计分析
<ol><li>以平均值±标准差 （SD） 的形式报告定量数据。使用商业软件进行统计分析。</li>
	<li>使用学生 t 检验或单因素方差分析，然后使用 Tukey 的多重比较检验来确定组间的显着差异。p<0.05和p<0.01表示两组间差异有统计学意义。</li>
</ol>

人工软骨植入物通过软骨内骨化进行骨再生

作者宣称不存在任何利益冲突。

使用适当的支架材料来促进从肥厚软骨到骨骼的过渡，是扩大基于 MSC 的工程肥厚性软骨移植物和治疗具有临床意义的骨缺损的有前途的方法。在这里，我们表明 HA 是一种极好的支架材料，可在体外支持基于 MSC 的肥厚软骨组织的分化并促进体内软骨内骨形成 38。此外，在体内，HA构建体被证明可以促进植入同一口袋中的多个构建体的融合，以形成单个整合的...

自体骨仍然是修复因创伤、先天性缺损和手术切除引起的骨缺损的金标准。然而，自体骨移植具有显着的局限性，包括供体疼痛、感染风险和可从患者中分离出的骨量有限 1,2,3,4。许多生物材料已被开发为骨替代品，将天然或合成聚合物与矿化材料（如磷酸钙或羟基磷灰石）相结合 5,6。这些工程材料中的骨形成通常使用矿化材料作为启动材料来实现，以使干细胞通过膜内骨化 （IMO） 过程直接分化成成骨细胞7。该过程缺乏血管生成步骤，导致植入后移植物的体内血管化不足8,9,10，因此，使用这种过程的方法可能不是治疗大骨缺损的最佳方法11。
用于概括软骨内骨化 （ECO） 过程的策略已被证明可以克服与传统的基于 IMO 的方法相关的重大问题。在 ECO 中，软骨模板中的软骨细胞释放血管内皮生长因子 （VEGF），促进血管浸润和软骨模板重塑为骨12。ECO 介导的通过软骨重塑和血管生成进行成骨的方法，在骨折修复过程中也被激活，使用人工创建的源自 MSC 的软骨组织作为启动材料。软骨细胞可以耐受骨缺损的缺氧，诱导血管生成，并将游离软骨移植物转化为血管生成组织。许多研究报告说，基于 MSC 的软骨移植物通过实施这样的 ECO 程序在体内产生骨 13,14,15,16,17,18,19,20,21。
这种 ECO 介导的方法的临床应用的一个基本要求是如何在临床环境中制备所需数量的软骨移植物。制备适合实际骨缺损尺寸的临床软骨是不切实际的。因此，移植软骨在植入多个碎片时必须整体形成骨22.水凝胶可能是一种有吸引力的工具，用于扩大用于软骨内骨化的组织工程移植物。许多天然来源的水凝胶在体外支持MSC软骨形成，在体内支持ECO 23,24,25,26,27,28,29,30,31,32;然而，满足临床应用要求的最佳支撑材料仍未确定。透明质酸 （HA） 是一种可生物降解且具有生物相容性的多糖，存在于软骨33 的细胞外基质中。HA 通过 CD44 等表面受体与 MSC 相互作用，以支持软骨生成分化 25,26,28,30,31,32,34。此外，HA支架促进IMO介导的人牙髓干细胞成骨分化35，支架与胶原蛋白结合促进ECO介导的成骨36,37。
在这里，我们提出了一种使用骨髓来源的成人人间充质干细胞制备 HA 水凝胶的方法，以及它们在体外用于肥厚软骨生成和随后的体内软骨内骨化 38。我们将 HA 的特性与胶原蛋白的特性进行了比较，胶原蛋白是一种广泛用于骨组织工程的材料，具有 MSC，并且是用于扩大人工移植物用于软骨内骨化的有用材料17。在免疫功能低下的小鼠模型中，通过皮下植入评估接种了人间充质干细胞的 HA 和胶原构建体的体内 ECO 潜力。结果表明，HA水凝胶非常适合作为MSCs的支架，以产生人工软骨移植物，从而通过ECO形成骨骼。
该协议分为两个步骤。首先，制备接种在透明质酸水凝胶上的人间充质干细胞的构建体，并在 体外分化为肥厚软骨。接下来，将分化的构建体皮下植入裸模型中以诱导 体内 软骨内骨化（图1）。

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			<td>0.25w/v% Trypsin-1mmol/L EDTA.4Na Solution</td>
			<td>FUJIFILM Wako Pure Chemical&#160;</td>
			<td>209-16941</td>
			<td></td>
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			<td>Antisedan</td>
			<td>Nippon Zenyaku Kogyo</td>
			<td></td>
			<td></td>
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			<td>ascorbate-2-phosphate</td>
			<td>Nacalai Tesque</td>
			<td>13571-14</td>
			<td></td>
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			<td>Bambanker</td>
			<td>GC Lymphotec</td>
			<td>CS-02-001</td>
			<td></td>
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			<td>basic fibroblastic growth factor</td>
			<td>Reprocell</td>
			<td>RCHEOT002&#160;</td>
			<td></td>
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			<td>bovine serum albumin</td>
			<td>FUJIFILM Wako Pure Chemical&#160;</td>
			<td>012-23881</td>
			<td>7.5 w/v%</td>
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			<td>Countess Automated Cell Counter with cell counting chamber slides and Trypan Blue stain 0.4%</td>
			<td>Invitrogen</td>
			<td>C10283</td>
			<td></td>
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			<td>Merck</td>
			<td>D8893</td>
			<td></td>
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			<td>Domitor</td>
			<td>Nippon Zenyaku Kogyo</td>
			<td></td>
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			<td>Dormicum</td>
			<td>Astellas Pharma</td>
			<td></td>
			<td></td>
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			<td>Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Merck</td>
			<td>D6429</td>
			<td>high glucose</td>
		</tr>
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			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium/Nutrient Mixture F-12 Ham</td>
			<td>Merck</td>
			<td>D6421</td>
			<td></td>
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			<td>Fetal bovine serum</td>
			<td>Hyclone</td>
			<td>SH30396.03</td>
			<td></td>
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			<td>Gentamicin sulfate</td>
			<td>FUJIFILM Wako Pure Chemical&#160;</td>
			<td>1676045</td>
			<td>&#160;10 mg/mL</td>
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			<td>Haccpper Generator</td>
			<td>TechnoMax</td>
			<td>CH-400-5QB</td>
			<td>50 ppm hypochlorous acid water</td>
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			<td>Human Mesenchymal Stem Cells</td>
			<td>Lonza</td>
			<td>PT-2501</td>
			<td></td>
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			<td>HyStem Cell Culture Scaffold Kit</td>
			<td>Merck</td>
			<td>HYS020</td>
			<td></td>
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			<td>IL-1&#223;</td>
			<td>PeproTech</td>
			<td>AF-200-01B</td>
			<td></td>
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			<td>ITS-G supplement</td>
			<td>FUJIFILM Wako Pure Chemical&#160;</td>
			<td>090-06741</td>
			<td>&#215;100</td>
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			<td>L-Alanyl-L-Glutamine</td>
			<td>FUJIFILM Wako Pure Chemical&#160;</td>
			<td>016-21841</td>
			<td>200mmol/L (&#215;100)</td>
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			<td>L-proline</td>
			<td>Nacalai Tesque</td>
			<td>29001-42</td>
			<td></td>
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			<td>L-Thyroxine</td>
			<td>Merck</td>
			<td>T1775</td>
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			<td>Lonza</td>
			<td>PT-3001</td>
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			<td>paraffin</td>
			<td>FUJIFILM Wako Pure Chemical&#160;</td>
			<td>165-13375</td>
			<td></td>
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			<td>PBS / pH7.4 100ml</td>
			<td>Medicago</td>
			<td>09-2051-100</td>
			<td></td>
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			<td>TGF-&#946;3&#160;</td>
			<td>Proteintech</td>
			<td>HZ-1090</td>
			<td></td>
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			<td>Meiji Seika Kaisha</td>
			<td></td>
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			<td>Visiocare Ointment</td>
			<td>SAVAVET/SAVA Healthcare</td>
			<td></td>
			<td></td>
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			<td>&#946;-glycerophosphate</td>
			<td>FUJIFILM Wako Pure Chemical&#160;</td>
			<td>048-34332</td>
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</table>

integrated bone formation through in vivo endochondral ossification using mesenchymal stem cells

使用间充质干细胞 （MSC） 的常规骨再生疗法很难应用于大于临界尺寸的骨缺损，因为它没有诱导血管生成的机制。植入由间充质干细胞制成的人造软骨组织通过软骨内骨化 （ECO） 在 体内 诱导血管生成和骨形成。因此，这种ECO介导的方法可能是未来一种很有前途的骨再生疗法。这种 ECO 介导的方法临床应用的一个重要方面是建立一个方案，用于准备足够的软骨以植入以修复骨缺损。设计大小符合实际骨缺损形状的单个移植软骨块尤其不切实际。因此，被移植的软骨必须具有多块植入时整体形成骨骼的特性。水凝胶可能是一种有吸引力的工具，用于扩大用于软骨内骨化的组织工程移植物以满足临床要求。尽管许多天然衍生的水凝胶在 体外 支持MSC软骨形成，在 体内支持ECO，但满足临床应用需求的最佳支架材料尚未确定。透明质酸（HA）是软骨细胞外基质的重要组成部分，是一种可生物降解和生物相容的多糖。在这里，我们表明HA水凝胶具有优异的特性，可以支持基于MSC的软骨组织的 体外 分化，并促进 体内软骨内骨的形成。

Autologous bone is still the gold standard for repairing bone defects due to trauma, congenital defects, and surgical resection. However, autogenous bone grafting has significant limitations, including donor pain, risk of infection, and limited bone volume that can be isolated from the patients1,2,3,4. Numerous biomaterials have been developed as bone substitutes, combining natural or synthetic polymers with mineralized materials such as calcium phosphate or hydroxyapatite5,6. Bone formation in these engineered materials is usually achieved using the mineralized material as a priming material to allow stem cells to differentiate directly into osteoblasts through the intramembrane ossification (IMO) process7. This process lacks the angiogenic step, resulting in insufficient in vivo vascularization of the graft after implantation8,9,10, and therefore, approaches using such a process may not be optimal for treating large bone defects11.
Strategies applied to recapitulate the endochondral ossification (ECO) process, an innate mechanism in skeletogenesis during development, have been shown to overcome significant problems associated with traditional IMO-based approaches. In ECO, chondrocytes in the cartilage template release vascular endothelial growth factor (VEGF), which promotes vascular infiltration and remodeling of the cartilage template into bone12. The ECO-mediated approach to osteogenesis via cartilage remodeling and angiogenesis, which is also activated during fracture repair, uses artificially created cartilage tissue derived from MSCs as a priming material. Chondrocytes can tolerate hypoxia in bone defects, induce angiogenesis, and convert a vascular-free cartilage graft into angiogenic tissue. Numerous studies have reported that MSC-based cartilage grafts generate bone in vivo by implementing such an ECO program13,14,15,16,17,18,19,20,21.
An essential requirement for the clinical application of this ECO-mediated approach is how to prepare the desired amount of cartilage graft in a clinical setting. Preparing clinical cartilage of a size that fits the actual bone defect is not practical. Therefore, graft cartilage must form bone integrally when multiple fragments are implanted22. Hydrogels may be an attractive tool for scaling up tissue-engineered grafts for endochondral ossification. Many naturally derived hydrogels support MSC cartilage formation in vitro and ECO in vivo23,24,25,26,27,28,29,30,31,32; however, the optimal support material to meet the clinical application requirements has remained undetermined. Hyaluronic acid (HA) is a biodegradable and biocompatible polysaccharide present in the extracellular matrix of cartilage33. HA interacts with MSCs via surface receptors such as CD44 to support chondrogenic differentiation25,26,28,30,31,32,34. In addition, HA scaffolds promote IMO-mediated osteogenic differentiation of human dental pulp stem cells35, and scaffolds combined with collagen promote ECO-mediated osteogenesis36,37.
Here, we present a method for preparing HA hydrogels using bone marrow-derived adult human MSCs and their use for hypertrophic chondrogenesis in vitro and subsequent endochondral ossification in vivo38. We compared the characteristics of HA with those of collagen, a material widely applied in bone tissue engineering with MSCs and a useful material for scaling up artificial grafts for endochondral ossification17. In an immunocompromised mouse model, HA and collagen constructs seeded with human MSCs were evaluated for in vivo ECO potential by subcutaneous implantation. The results show that HA hydrogels are excellent as a scaffold for MSCs to create artificial cartilage grafts that allow bone formation through ECO.
The protocol is divided into two steps. First, constructs of human MSCs seeded on hyaluronan hydrogel are prepared and differentiated into hypertrophic cartilage in vitro. Next, the differentiated constructs are implanted subcutaneously in a nude model to induce endochondral ossification in vivo (Figure 1).

作者聚焦：通过血管化人工软骨整合增强骨再生 

使用间充质干细胞通过 体内 软骨内骨化进行整合骨形成

Our research intends to develop a method that improves the lack of blood flow and can be applied to large bone defects. The protocol presented here is one with the approaches we have taken to this end. The approach to bone formation using artificial cartilage the structure that mimics the natural process of skeletal development during the embryogenesis.This approach is relatively new in bone regeneration research. The advantage we inducing cartilage neogenesis, which is not an existent method currently used in clinical practice exists for vascularized bone grafts. Traditionally, bone regeneration research has used the techniques that combine stem cells, biomaterials, and growth factors alone or in combination.The method of bone formation by implanting artificial cartilage uses a techniques that makes artificial cartilage by mixing stem cells with biomaterials that serve the scaffolding material, and inducing cartilage differentiation. Healing our patients'bone defect clinically requires a large quantity of high quality artificial cartilage. Our protocol addresses the challenge of producing high quality artificial cartilage, and how to make multiple cartilage fuse with each other to form a unified bone.Our protocol helps generate larger, higher quality cartilage with fewer cells than protocols that use or do not use other scaffold materials to create artificial cartilage. Furthermore, the protocol can generate one integrated piece of bone, even with multiple pieces implanted in vivo. This is important because preparing clinical cartilage of a shape and size that fits the bone defect is impractical.

将MSC封装的HA水凝胶在补充有TGFβ3（软骨生成诱导剂41 ）的软骨形成培养基中培养（步骤4.1）。我们将 HA 的特性与胶原蛋白的特性进行了比较，胶原蛋白已被证明可有效创建用于软骨内骨化的基于 MSC 的人工软骨移植物，如前所述38。本研究中未将未分化的 MSC 列为阴性对照，因为已经证明未分化的 MSC 需要矿化表面作为启动基质，才能通过成骨分化（即膜内骨<s...

Watch this Scientific Journal Video about Enhancing Bone Regeneration with Vascularized Artificial Cartilage Integration at JoVE.com

通过植入由间充质干细胞产生的人工软骨组织，通过软骨内骨化进行骨治疗有可能规避传统疗法的缺点。透明质酸水凝胶可有效扩大均匀分化的软骨移植物，并在 体内融合移植物之间形成具有血管化的整合骨。

Conventional bone regeneration therapy using mesenchymal stem cells (MSCs) is difficult to apply to bone defects larger than the critical size because it ...

我们的研究旨在开发一种改善血流不足并可应用于大骨缺损的方法。这里介绍的协议是我们为此目的所采取的方法之一。使用人造软骨进行骨骼形成的方法，这种结构模仿胚胎发生过程中骨骼发育的自然过程。这种方法在骨再生研究中相对较新。我们诱导软骨新生的优势是目前临床实践中不存在的用于血管化骨移植物的方法。传统上，骨再生研究使用单独或组合使用干细胞、生物材料和生长因子相结合的技术。通过植入人造软骨形成骨的方法使用一种技术，通过将干细胞与用作支架材料的生物材料混合并诱导软骨分化来制造人造软骨。临床上治愈患者的骨缺损需要大量高质量的人造软骨。我们的方案解决了生产高质量人造软骨的挑战，以及如何使多个软骨相互融合以形成统一的骨骼。与使用或不使用其他支架材料来制造人造软骨的方案相比，我们的方案有助于用更少的细胞产生更大、更高质量的软骨。此外，该方案可以生成一块完整的骨骼，即使体内植入了多块骨骼。这很重要，因为准备适合骨缺损的形状和大小的临床软骨是不切实际的。

integrated-bone-formation-through-vivo-endochondral-ossification

Research

JoVE Journal

Biology

Integrated Bone Formation Through In Vivo Endochondral Ossification Using Mesenchymal Stem Cells

781 次观看.  Tokyo Medical and Dental University. 我们的研究旨在开发一种改善血流不足并可应用于大骨缺损的方法。这里介绍的协议是我们为此目的所采取的方法之一。使用人造软骨进行骨骼形成的方法，这种结构模仿胚胎发生过程中骨骼发育的自然过程。这种方法在骨再生研究中相对较新。我们诱导软骨新生的优势是目前临床实践中不存在的用于血管化骨移植物的方法。传统上，骨再生研究使用单独或组合使用干?...

视频: 作者聚焦：通过血管化人工软骨整合增强骨再生 

Conventional bone regeneration therapy using mesenchymal stem cells (MSCs) is difficult to apply to bone defects larger than the critical size because it does not have a mechanism to induce angiogenesis. Implanting artificial cartilage tissue fabricated from MSCs induces angiogenesis and bone formation in vivo via endochondral ossification (ECO). Therefore, this ECO-mediated approach may be a promising bone regeneration therapy in the future. An important aspect of the clinical application of this ECO-mediated approach is establishing a protocol for preparing enough cartilage to be implanted to repair the bone defect. It is especially not practical to design a single mass of grafted cartilage of a size that conforms to the shape of the actual bone defect. Therefore, the cartilage to be transplanted must have the property of forming bone integrally when multiple pieces are implanted. Hydrogels may be an attractive tool for scaling up tissue-engineered grafts for endochondral ossification to meet clinical requirements. Although many naturally derived hydrogels support MSC cartilage formation in vitro and ECO in vivo, the optimal scaffold material to meet the needs of clinical applications has yet to be determined. Hyaluronic acid (HA) is a crucial component of the cartilage extracellular matrix and is a biodegradable and biocompatible polysaccharide. Here, we show that HA hydrogels have excellent properties to support in vitro differentiation of MSC-based cartilage tissue and promote endochondral bone formation in vivo.

Bone therapy via endochondral ossification by implanting artificial cartilage tissue produced from mesenchymal stem cells has the potential to circumvent the drawbacks of conventional therapies. Hyaluronic acid hydrogels are effective in scaling up uniformly differentiated cartilage grafts as well as creating integrated bone with vascularization between fused grafts in vivo.

科研

生物学

Preparation and In Vitro Differentiation of Mesenchymal Stem Cell-Encapsulated Hydrogels for Bone Formation

Begin by trypsinizing the human mesenchymal stem cells to create a cell suspension. Next, aliquot the cell suspension into a 1.5-milliliter tube to create 10 constructs. After centrifuging the suspension at 220g for three minutes, use a vacuum pump to remove the supernatant.Now, place the cell pellet on ice. Next, bring the thiol-modified hyaluronic acid, thiol-reactive crosslinker, polyethylene glycol diacrylate, and degassed water bottles to room temperature. Under sterile conditions, use a needle equipped syringe to add one milliliter of degassed water to the bottle containing hyaluronic acid.Vortex the solution, occasionally heating it to 37 degrees Celsius until it becomes clear. Then, place the solution on ice. Under sterile conditions, add 5 milliliters of degassed water to the crosslinker bottle.Dissolve by inverting repeatedly before placing it on ice. Now, add 120 microliters of the dissolved hyaluronic acid solution to the aliquoted cell pellet and resuspend the cell pellet by pipetting the solution back and forth. Add 30 microliters of the dissolved crosslinker solution into the tube with the resuspended cell pellet.Ensure proper mixing by tapping on the tube. After mixing, briefly spin down the solution. Drop 15 microliters of the combined solution onto a paraffin-coated 24-well plate.Allow it to solidify at 37 degrees Celsius for 30 minutes. For the in vitro differentiation, add 0.5 milliliters of chondrogenic differentiation medium to the cell seated hydrogels. After three weeks, switch to the hypertrophic differentiation medium, adding 0.5 milliliters per well.Chondrogenic differentiation resulted in sulfated glycosaminoglycans in type 2 collagen positive extracellular matrix in both the hyaluronic acid or, HA, and collagen constructs. However, the distribution was more homogenous in the hyaluronic acid constructs. The collagen constructs demonstrated peripheral cells with heterogeneous morphology.The average roundness of cells in the HA constructs was higher than in the collagen constructs. Calcium deposition was detected at the outer edges of both constructs at five weeks. In the in vivo HA constructs, all implants were attached to each other in subcutaneous pockets and formed integrated bone tissue.40%of the in vivo collagen constructs were independent. Osteoid tissue with lamellar morphology was formed in the outer regions of both in vivo constructs at eight weeks post-implantation. Loss of cartilage was also observed in the HA construct.Multiple in vivo HA constructs appeared to form integrated bone tissue as indicated by the connection to the bone tissues and the presence of joint fibrous tissue. While 40%of the collagen constructs were united, the remaining constructs existed apart or were only attached without bony tissue connections. Both constructs showed positively labeled vessels in the bone marrow, and the inner cartilage was surrounded by multinucleated osteoclast cells.The mineral was deposited in the outer osteoid region of both constructs with higher mineral volume and HA constructs due to their larger size.