利用干细胞衍生的生长因子实现新型无细胞骨再生。

Novel cell-free regeneration of bone using stem cell-derived growth factors.

机构信息

Department of Oral and Maxillofacial Surgery, Nagoya University Graduate School of Medicine, Nagoya, Japan.

出版信息

Int J Oral Maxillofac Implants. 2013 Jul-Aug;28(4):1009-16. doi: 10.11607/jomi.3036.

DOI:10.11607/jomi.3036

PMID:23869359

Abstract

PURPOSE

Tissue engineering and regenerative medicine may now be used for the treatment of maxillofacial defects, but the related procedures have several limitations, including high capital investment, expensive cell culture techniques, and complicated safety and quality management issues. Stem cells secrete many cytokines that can affect cell mobilization and differentiation that accumulate in conditioned media. This study investigated the effects of stem cell-conditioned media from human bone marrow-derived mesenchymal stem cells (MSCs) on bone regeneration and its ability to induce endogeneous stem cell mobilization and bone regeneration.

MATERIALS AND METHODS

Human MSCs that were 70% to 80% confluent were refed with serum-free Dulbecco's modified Eagle medium, and the cell-cultured conditioned media were collected after 48 hours of incubation. The collected media were defined as cultured conditioned media from MSCs (MSC-CM). Rat bone-marrow-derived stem cells (rMSCs) were cultured with MSC-CM for 48 hours; then, cell mobilization and the expression of osteogenic-related genes were investigated. The presence of cytokines in MSC-CM was determined by enzyme-linked immunosorbent assay. Collagen sponge was then soaked in MSC-CM and grafted into rat calvarial bone defects. At 2 or 4 weeks after implantation of graft materials, implanted sections were evaluated by microcomputed tomography and histologic analysis.

RESULTS

MSC-CM enhanced the migration, proliferation, and expression of osteogenic marker genes, such as alkaline phosphatase, osteocalcin, and Runx2, of rMSCs in vitro. Cytokines such as insulinlike growth factor-1, vascular endothelial growth factor, transforming growth factor-β1, and hepatocyte growth factor were present in the MSC-CM. MSC-CM significantly increased the migration and expression of osteogenic-related genes of rMSCs. Early bone regeneration in rat calvaria was also observed.

CONCLUSION

These results suggest that MSC-CM may have potential for novel cell-free regeneration of bone.

摘要

目的

组织工程和再生医学现在可用于治疗颌面缺损，但相关程序有几个限制，包括高资本投资、昂贵的细胞培养技术以及复杂的安全和质量管理问题。干细胞分泌许多细胞因子，可影响细胞动员和分化，这些细胞因子在条件培养基中积累。本研究调查了人骨髓间充质干细胞（MSCs）来源的干细胞条件培养基对骨再生的影响及其诱导内源性干细胞动员和骨再生的能力。

材料和方法

当人 MSCs 达到 70%-80%汇合时，用无血清 Dulbecco 修改的 Eagle 培养基重新喂养，孵育 48 小时后收集细胞培养的条件培养基。收集的培养基被定义为 MSC 培养的条件培养基（MSC-CM）。将大鼠骨髓来源的干细胞（rMSCs）与 MSC-CM 共培养 48 小时；然后，研究细胞动员和骨生成相关基因的表达。通过酶联免疫吸附试验确定 MSC-CM 中细胞因子的存在。然后将胶原海绵浸泡在 MSC-CM 中并移植到大鼠颅骨骨缺损中。在植入材料后 2 或 4 周，通过 microCT 和组织学分析评估植入物切片。

结果

MSC-CM 增强了 rMSCs 的迁移、增殖和碱性磷酸酶、骨钙素和 Runx2 等成骨标志物基因的表达。胰岛素样生长因子-1、血管内皮生长因子、转化生长因子-β1 和肝细胞生长因子等细胞因子存在于 MSC-CM 中。MSC-CM 显著增加了 rMSCs 的迁移和骨生成相关基因的表达。在大鼠颅骨中也观察到早期骨再生。