Interactions between bone marrow stromal stem cells and nano-hydroxyapatite

doi:10.3969/j.issn.2095-4344.0693

Abstract

Abstract:

BACKGROUND: As a kind of bioactive material, nano-hydroxyapatite has received extensive attention for its excellent biological performance. Its effect on the proliferation, differentiation, mineralization and cytotoxicity of bone marrow stromal stem cells is an important factor affecting its osteogenesis performance, and it is also a current hot topic in the field of bone tissue engineering.
OBJECTIVE: To explore and analyze the effect of nano-hydroxyapatite on bone marrow stromal stem cells and to provide guidance for further study of tissue engineered bone.
METHODS: Relevant literature published from January 1, 2006 to July 26, 2018 was retrieved by the first author in PubMed, CNKI, VIP, WanFang and Google Scholar. The keywords were “hydroxyapatite, nano-hydroxyapatite, biological matrix material, bone marrow stromal stem cells, osteogenic activity, biocompatibility, cytotoxicity, bone defect, bone tissue engineering, tissue engineered bone” in English and Chinese, respectively. Sixty-six eligible articles were finally reviewed.
RESULTS AND CONCLUSION: A large number of studies have shown that the nano-hydroxyapatite and its composite materials with bone marrow stromal stem cells have good affinity, adhesion and biocompatibility, and can induce the proliferation and differentiation of bone marrow stromal stem cells. Nano-hydroxyapatite is an ideal material for bone defect repair, which can be widely used in bone defect repair and surface modification of implants, thereby promoting early new bone formation and strengthening the binding between implants and bone tissues. Nano-hydroxyapatite composite materials are also used to develop biological artificial ligaments and guided bone tissue regeneration membrane materials as well as to treat femoral head necrosis. Further investigations on the interaction between nano-hydroxyapatite and bone marrow stromal stem cells will certainly promote the research progress and clinical application of tissue-engineered bone.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Hydroxyapatites, Bone Marrow, Mesenchymal Stem Cells, Tissue Engineering

CLC Number:

Li Lu, Shu Jingyuan, Zheng Lixia, Cui Yingying, Niu Yueyue, Wang Qian, Wang Qingshan. Interactions between bone marrow stromal stem cells and nano-hydroxyapatite[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(1): 132-138.

Figures/Tables 3

尹美林等[18]采用化学沉淀方法制备nHA，并对其进行表征，扫描电镜下见羟基磷灰石为针状晶体，结晶程度好，粒径分布均匀。 2.2 BMSCs生物学特性 20世纪60年代Friedenstein等在骨髓细胞培养中发现一群成纤维样、贴壁生长与造血干细胞不同的细胞，其主要来源于中胚层，在骨膜、肌肉、外周血以及结缔组织中均有存在，以骨髓和脐血中含量最为丰富。研究发现其具有较强的自我更新、多向分化潜能，可在不同的诱导条件下分化为成骨细胞、软骨细胞、脂肪细胞、肌细胞等多种细胞。徐俊昌等[19]在体外分离培养BMSCs并对其表面标志物、多向分化特性进行鉴定，结果显示BMSCs表面表达CD105、CD166、CD29，成骨诱导分化后，胞体变宽，钙结节茜素红染色、碱性磷酸酶染色为阳性，成软骨分化3周后阿利辛染色阳性，成脂肪诱导后细胞胞浆内出现脂肪小体。 2.3 nHA及其复合材料对BMSCs的影响 2.3.1 nHA微粒浓度对BMSCs的影响 Liu等[20]研究了不同浓度nHA微粒对BMSCs增殖分化的影响，研究结果显示，当nHA微粒浓度<20 μg/104个细胞时，nHA颗粒可促进BMSCs的增殖，但差异无显著性意义，nHA微粒浓度为20 μg/104个细胞时，可显著促进BMSCs的增殖，nHA微粒浓度大于20 μg/104个细胞时，可显著抑制BMSCs的增殖；在无血清培养条件下，nHA浓度为20 μg/104个细胞时，没有促进BMSCs增殖的作用，而浓度大于20 μg/104个细胞时，nHA微粒显著抑制BMSCs的增殖；在成骨诱导培养液中，nHA微粒浓度< 20 μg/104个细胞时，对BMSCs矿化无显著促进作用，随着微粒浓度的增加，其对BMSCs成骨分化的促进作用逐渐增强，呈浓度依从性关系，但当浓度大于20 μg/104个细胞时，其对BMSCs矿化促进作用随微粒浓度的增加而逐渐减弱。研究发现，羟基磷灰石生物毒性受颗粒直径、接触剂量和接触方式的影响[21]。Remya等[22]将不同质量浓度nHA(10，50，100，500，1 000 mg/L)与BMSCs相互作用，结果显示，暴露在高质量浓度(1 000 mg/L)nHA溶液中悬浮细胞出现纺锤形改变和颗粒状细胞质的变化，随着nHA质量浓度的增加，细胞形态出现极其不规则状，细胞扩散模式受限制；通过检测线粒体活动对细胞生存能力进行评估，结果表明，低质量浓度中的细胞生存能力与对照组无显著差异，高质量浓度中的细胞存活率约60%；对细胞氧化应激进行检测发现，高质量浓度nHA溶液中细胞皱褶变化高，而早期细胞凋亡则出现在nHA较低的质量浓度中。nHA微粒甚至更高的质量浓度只改变了细胞形态，并不诱导细胞凋亡。 nHA在复合材料中所占比例同样对BMSCs有明显影响。Zhu等[23]以含体积分数为10%胎牛血清的DMEM完全培养基为阴性实验对照组，以0.64%质量浓度苯酚为阳性实验对照组，将BMSCs与材料浸提液共同培养为实验组，观察第3，5，7天培养的BMSCs相对生长情况。结果显示，实验组细胞吸光度值显著增加，BMSCs的相对生长高于对照组和阴性组。在纳米羟基磷灰石/聚乙交酯-丙交酯(nanograde hydroxyapatite/poly lactic-co-glycolic acid，nHA/PLGA)复合支架材料中，当nHA/PLGA复合体积比为50/50时，BMSCs在黏附性、增殖率、成骨分化等方面均优于其他比例，表明合适比例的nHA在生物活性及骨传导方面扮演者重要的角色[24]。谭羽莹等[25]观察改性PLGA/HA复合材料与BMSCs体外培养后的细胞活性及生物相容性，MTT比色法检测在不同浓度PLGA/HA浸提液(10%，30%，50%，80%)中BMSCs的增殖情况，观察材料表面细胞的黏附性及细胞形态，结果显示，高浓度材料浸提液(50%，80%)对BMSCs的体外增殖呈负影响，但CTS均在1级，符合安全评价标准；实验还表明，BMSCs能够黏附并伸展形成伪足固定于支架材料表面，部分细胞已融合成细胞层，可见细胞外基质。 2.3.2 nHA支架材料表面微形貌对BMSCs的影响生物材料表面状态及物理结构对生物性能有较大的影响，天然骨组织中的羟基磷灰石以数十纳米的晶体形式存在，而正是这些数十纳米晶体的有序排列构成了骨组织的力学属性。nHA表面与细胞相互作用，其化学成分和表面形貌强烈影响着细胞的生存、附着、迁移分化和增殖[26]。周琰春等[27]采用十六烷基三甲基溴化铵(cetyltrimethylammonium bromide，CTAB)为添加剂，在水相体系中制备了形貌均匀的nHA粉末，以无CTAB添加剂的普通羟基磷灰石粉末为对照，分别制备成羟基磷灰石膜并对其物相进行表征，nHA粉末外貌呈球形，为多晶状态，分散好，大小均匀，具有较好的浸润性及亲水性。普通羟基磷灰石粉末外貌呈条棒状，单晶态，浸润性及亲水性不及nHA。将分离培养后的BMSCs在两种基质上复合，观察BMSCs增殖、分化状况，结果证明，nHA基质表面细胞数量显著高于普通羟基磷灰石基质，而且能获得更高的碱性磷酸酶、骨钙素表达，说明羟基磷灰石支架材料表面微形貌的构建对BMSCs增殖、分化具有明显影响，见图1，2。"

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