Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.
Proc Natl Acad Sci U S A. 2010 Feb 23;107(8):3311-6. doi: 10.1073/pnas.0905445107. Epub 2010 Feb 2.
Clinical protocols utilize bone marrow to seed synthetic and decellularized allogeneic bone grafts for enhancement of scaffold remodeling and fusion. Marrow-derived cytokines induce host neovascularization at the graft surface, but hypoxic conditions cause cell death at the core. Addition of cellular components that generate an extensive primitive plexus-like vascular network that would perfuse the entire scaffold upon anastomosis could potentially yield significantly higher-quality grafts. We used a mouse model to develop a two-stage protocol for generating vascularized bone grafts using mesenchymal stem cells (hMSCs) from human bone marrow and umbilical cord-derived endothelial cells. The endothelial cells formed tube-like structures and subsequently networks throughout the bone scaffold 4-7 days after implantation. hMSCs were essential for stable vasculature both in vitro and in vivo; however, contrary to expectations, vasculature derived from hMSCs briefly cultured in medium designed to maintain a proliferative, nondifferentiated state was more extensive and stable than that with hMSCs with a TGF-beta-induced smooth muscle cell phenotype. Anastomosis occurred by day 11, with most hMSCs associating closely with the network. Although initially immature and highly permeable, at 4 weeks the network was mature. Initiation of scaffold mineralization had also occurred by this period. Some human-derived vessels were still present at 5 months, but the majority of the graft vasculature had been functionally remodeled with host cells. In conclusion, clinically relevant progenitor sources for pericytes and endothelial cells can serve to generate highly functional microvascular networks for tissue engineered bone grafts.
临床方案利用骨髓为合成和去细胞同种异体骨移植物接种种子，以增强支架重塑和融合。骨髓衍生的细胞因子在移植物表面诱导宿主新生血管形成，但缺氧条件会导致核心细胞死亡。添加能够产生广泛原始丛状血管网络的细胞成分，这些网络在吻合后可以灌注整个支架，可能会产生质量更高的移植物。我们使用小鼠模型开发了一种两阶段方案，使用来自人骨髓和脐带衍生内皮细胞的间充质干细胞 (hMSC) 生成血管化骨移植物。内皮细胞在植入后 4-7 天形成管状结构，随后在骨支架中形成网络。hMSC 对于体外和体内稳定的血管都是必不可少的；然而，与预期相反，在设计用于维持增殖、非分化状态的培养基中短暂培养的 hMSC 衍生的血管比具有 TGF-β诱导的平滑肌细胞表型的 hMSC 衍生的血管更广泛和稳定。吻合发生在第 11 天，大多数 hMSC 与网络密切相关。尽管最初不成熟且高度渗透，但在 4 周时网络已经成熟。支架矿化也已经开始。在 5 个月时仍存在一些人源性血管，但大多数移植物血管已经被宿主细胞进行了功能重塑。总之，用于周细胞和内皮细胞的临床相关祖细胞来源可用于为组织工程骨移植物生成高度功能性的微血管网络。
