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Biology

使用间充质干细胞通过 体内 软骨内骨化进行整合骨形成

Published: July 14, 2023
doi:
10.3791/65573
, , ,
1Department of Molecular Craniofacial Embryology, Graduate School of Medical and Dental Sciences,Tokyo Medical and Dental University, 2Department of Maxillofacial Surgery, Graduate School of Medical and Dental Sciences,Tokyo Medical and Dental University, 3Department of Regenerative and Reconstructive Dental Medicine, Graduate School of Medical and Dental Sciences,Tokyo Medical and Dental University

Summary

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。

Abstract

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。

Introduction

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）。

Protocol

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 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?…

Representative Results

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…

Discussion

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…

Disclosures

The authors have nothing to disclose.

Acknowledgements

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）。

Materials

0.25w/v% Trypsin-1mmol/L EDTA.4Na Solution FUJIFILM Wako Pure Chemical  209-16941
Antisedan Nippon Zenyaku Kogyo
ascorbate-2-phosphate Nacalai Tesque 13571-14
Bambanker GC Lymphotec CS-02-001
basic fibroblastic growth factor Reprocell RCHEOT002 
bovine serum albumin FUJIFILM Wako Pure Chemical  012-23881 7.5 w/v%
Countess Automated Cell Counter with cell counting chamber slides and Trypan Blue stain 0.4% Invitrogen C10283
dexamethasone Merck D8893
Domitor Nippon Zenyaku Kogyo
Dormicum Astellas Pharma
Dulbecco's Modified Eagle Medium Merck D6429 high glucose
Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 Ham Merck D6421
Fetal bovine serum Hyclone SH30396.03
Gentamicin sulfate FUJIFILM Wako Pure Chemical  1676045  10 mg/mL
Haccpper Generator TechnoMax CH-400-5QB 50 ppm hypochlorous acid water
Human Mesenchymal Stem Cells Lonza PT-2501
HyStem Cell Culture Scaffold Kit Merck HYS020
IL-1ß PeproTech AF-200-01B
ITS-G supplement FUJIFILM Wako Pure Chemical  090-06741 ×100
L-Alanyl-L-Glutamine FUJIFILM Wako Pure Chemical  016-21841 200mmol/L (×100)
L-proline Nacalai Tesque 29001-42
L-Thyroxine Merck T1775
MSCGM Mesenchymal Stem Cell Growth Medium
BulletKit		 Lonza PT-3001
paraffin FUJIFILM Wako Pure Chemical  165-13375
PBS / pH7.4 100ml Medicago 09-2051-100
TGF-β3  Proteintech HZ-1090
Vetorphale Meiji Seika Kaisha
Visiocare Ointment SAVAVET/SAVA Healthcare
β-glycerophosphate FUJIFILM Wako Pure Chemical  048-34332
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Tags

Mesenchymal Stem CellsBone FormationIn VivoEndochondral OssificationArtificial CartilageBone RegenerationAngiogenesisScaffoldCartilage DifferentiationCritical Size Bone DefectECO-mediated Approach
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Yamazaki, S., Lin, Y., Marukawa, E., Ikeda, M. Integrated Bone Formation Through In Vivo Endochondral Ossification Using Mesenchymal Stem Cells. J. Vis. Exp. (197), e65573, doi:10.3791/65573 (2023).
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