Heylman Christopher M, Caralla Tonya N, Boehm Cynthia A, Patterson Thomas E, Muschler George F
Department of Biomedical Engineering, Cleveland Clinic, 9500 Euclid Ave ND20, Cleveland, OH 44195.
Department of Biomedical Engineering, Cleveland Clinic 9500, Euclid Ave ND20, Cleveland, OH 44195.
J Regen Med Tissue Eng. 2013 Sep 26;2. doi: 10.7243/2050-1218-2-7.
Survival and colony formation by transplanted tissue derived connective tissue progenitor cells (CTPs) are thought to be important factors in the success of clinical tissue engineering strategies for bone regeneration. Transplantation of cells into defects larger than a few millimeters expose cells to a profoundly hypoxic environment. This study tested the hypothesis that delaying the onset of hypoxia will improve the survival and performance of CTPs .
To mimic declines seen in an avascular bone defect, colony forming efficiency by marrow derived nucleated cells was assessed under osteogenic conditions. Variation in the rate of oxygen decline from an oxygen tension of 21% to 0.1% oxygen was explored using an incubator with programmable active control of gas concentrations. The effect of doping cultures with defined concentrations of RBCs was also used to evaluate the potential for RBCs to serve as a natural buffer in the setting of declining oxygen levels.
A delay in onset of hypoxia over 96 hours resulted in a 3-fold increase in the relative colony forming efficiency (rCFE) of CTPs as compared to an immediate onset of hypoxia. The presence of RBCs inhibited the rCFE of CTPs. Given the negative effects of RBCs, methods of RBC removal were evaluated and compared for their effectiveness of RBC removal and retention of colony forming efficiency.
These data suggest that conditions of hypoxia compromise colony forming efficiency in marrow derived CTPs. However, slowing the rate of decline of oxygen preserved colony forming efficiency at levels achieved in a stable normoxic (3% O) environment. These data also suggest that RBCs are detrimental to the rCFE of CTPs and that buffy coat is an effective and preferred method for removing RBCs from marrow aspirates while preserving CTPs. These findings may inform clinical strategies for CTP transplantation.
移植的组织来源的结缔组织祖细胞（CTP）的存活和集落形成被认为是骨再生临床组织工程策略成功的重要因素。将细胞移植到大于几毫米的缺损中会使细胞暴露于严重缺氧的环境中。本研究检验了延迟缺氧发作将改善CTP存活和性能的假设。
为模拟无血管骨缺损中出现的氧含量下降情况，在成骨条件下评估骨髓来源的有核细胞的集落形成效率。使用具有可编程气体浓度主动控制功能的培养箱，探索从21%氧张力降至0.1%氧的氧气下降速率的变化。还用特定浓度的红细胞对培养物进行掺杂，以评估红细胞在氧水平下降时作为天然缓冲剂的潜力。
与立即出现缺氧相比，缺氧发作延迟超过96小时导致CTP的相对集落形成效率（rCFE）增加了3倍。红细胞的存在抑制了CTP的rCFE。鉴于红细胞的负面影响，评估并比较了去除红细胞的方法在去除红细胞和保留集落形成效率方面的有效性。
这些数据表明，缺氧条件会损害骨髓来源的CTP的集落形成效率。然而，减缓氧气下降速率可在稳定的常氧（3%O）环境中达到的水平下保留集落形成效率。这些数据还表明，红细胞对CTP的rCFE有害，而血沉棕黄层是从骨髓抽吸物中去除红细胞同时保留CTP的有效且首选的方法。这些发现可能为CTP移植的临床策略提供参考。
