Slowing the onset of hypoxia increases colony forming efficiency of connective tissue progenitor cells in vitro

Abstract

Background 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 in vitro. Methods To mimic declines seen in an avascular in vivo 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. Results 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 in vitro 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. Conclusions 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% O2) 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.

Assessment of Methods for Rapid Intraoperative Concentration and Selection of Marrow-Derived Connective Tissue Progenitors for Bone Regeneration Using the Canine Femoral Multidefect Model

“…This study also highlights the important effect of CFE when making estimates of true CTP prevalence ( t P CTP-O ) based on measurement of observed CTP-O prevalence ( o P CTP-O ) since o P CTP-O = t P CTP-O CFE. Consistent with our previous report, 19 we found evidence that the high concentration of RBCs in raw marrow aspirates appears to compromise CFE, resulting in an underestimation of t P CTP-O unless RBC concentration is reduced by centrifugation. This finding highlights a limitation in our ability to precisely characterize the effect of DS processing.…”

“…Therefore, RBCs could account for the entire suspected effect on CFE. 19 However, in this case, there was also the possibility that the ACD used as an anticoagulant may have an adverse effect on CFE. To assess the possibility of an adverse effect of ACD on CFE, an additional follow-up experiment was performed.…”

“…However, roughly twice as many CTP-Os were measured in the final concentrate than in the initial aspirate. This raised the possibility that the prevalence of CTP-Os in the starting samples of DS processed marrow may have been systematically underestimated due to a reduction of colony-forming efficiency (CFE), either resulting from the abundance of RBCs (previously noted to compromise CFE) 19 or a potential effect of the ACD anticoagulant.…”

“… 5 An increased prevalence of CTP-Os means that the implanted CTP-Os will have fewer cells to compete with for limited supply of oxygen in the defect site. 14 , 19–21 Removal of RBCs limits the debris that is placed into the defect site and the associated inflammatory response needed to clear the debris from the site where the bone is desired. 22–24 This study provides the first attempt to objectively evaluate and compare these methods for processing marrow-derived cells, using a biologically relevant large animal model.…”

Tethering of Epidermal Growth Factor (EGF) to Beta Tricalcium Phosphate (βTCP) via Fusion to a High Affinity, Multimeric βTCP-Binding Peptide: Effects on Human Multipotent Stromal Cells/Connective Tissue Progenitors

“…Optimization of CTP isolation [ 4 ] and transplantation strategies [ 8 – 10 ] has led to improved bone healing in animal models [ 9 , 11 – 13 ]. However, the hypoxic, nutrient-limited, and inflammatory microenvironment of the bone wound can cause death of a substantial fraction of transplanted cells within the first few days [ 14 – 17 ], reducing the effective number of osteoprogenitors that contribute to the proliferative and remodeling stage of wound healing [ 18 ]. We thus hypothesize that providing bioactive cues that stimulate survival and proliferation of connective tissue progenitors within grafts, without interrupting terminal osteogenic differentiation, will improve the outcome of bone healing [ 1 , 3 , 18 , 19 ].…”