冻存骨髓基质细胞复合材料体内成骨基质的合成能力

背景:课题组以往研究显示：体外培养条件下，冻存骨髓基质细胞复苏后仍保持较高的细胞存活率、细胞增殖及成骨分化能力。上述结果仍然需要进一步在体内环境下证实。目的：观察经超低温冻存后的骨髓基质细胞和支架材料胶原膜BME-10X复合体植入裸鼠体内后Ⅰ型胶原的合成情况。方法：体外分离培养Beagle犬骨髓基质细胞，冻存12个月后复苏，体外构建骨髓基质细胞和胶原膜材料复合体。分别经矿化诱导培养液、基础培养液培养5 d后，植入裸鼠体内，于术后第4周取出标本，进行大体观察、组织病理学和免疫组化分析，并应用图像分析系统对各组标本中的Ⅰ型胶原进行定量分析。以矿化诱导培养液培养的单纯胶原膜材料为对照组。结果与结论：对照组在植入胶原膜后，胶原膜边界清晰，膜边缘及内部基本没有细胞生长，Ⅰ型胶原分布很少；在未诱导矿化组，术后第4周可见，胶原膜内有细胞长入，并有细小的条索状新生胶原形成，Ⅰ型胶原分布明显增多；在诱导矿化组，植入后也可见支架材料的分解降解和更多的细胞生长，大量新生的胶原形成类骨质样组织，与前两组对比，Ⅰ型胶原分布增多有显著性意义。结果表明冻存骨髓基质细胞复苏后进行体外培养扩增与诱导分化，并在体内环境下复合胶原支架材料，仍然具有较强成骨能力。

Osteogenic ability of cryopreserved bone marrow stromal cells complex in vivo

BACKGROUND:Our previous studies have demonstrated that cryopreserved bone marrow stromal cells (BMSCs) still maintain high survival rate, cell proliferation and osteogenic differentiation potentials after thawing. However, this result needs confirmed in vivo environment.  OBJECTIVE:To explore the effects of cryopreserved BMSCs and collagenic membrane BME-10X complex on type Ⅰ collagen synthesis in vivo. METHODS:Beagle dog BMSCs were cultured in vitro and cryopreserved for 12 months, which were thawed and prepared complexes with collagenic membrane. The complexes were cultured with mineralization induction medium or normal medium for 5 days, followed by implanting into nude mice. The specimens were harvested and analyzed by gross observation, histopathological and immunohistochemistry at 4 weeks after implantation. The collagenic membrane cultured with mineralization induction medium served as controls. RESULTS AND CONCLUSION:In the control group, the boundary of collagenic membrane was distinctly, without cell growth around boundary or intra collagenic membrane, additionally, there was little type Ⅰ collagen. In the non-induction group, cells grew into collagenic membrane, trabes-like collagen formed, and type Ⅰ collagen distribution increased at 4 weeks. In the induction group, scaffold degraded, more cells grew, and plenty of collagen formed osteoid-like tissues. The distribution of typeⅠcollagen was obviously increased than that of other groups. The findings demonstrated that cryopreserved BMSCs possess strong osteogenic differentiation potentials after proliferation and induction combined with collagenic membranes in vitro.