No abstract

Connective Tissue Progenitors (CTPs) in bone and marrow tissue are an important therapeutic target for bone regeneration. Concentration and selection of CTPs from the heterogenous population of cells in bone marrow remains a challenge due to their low prevalence. This study identifies surface-bound hyaluronan (HA), a component of the in vivo niche for CTPs, and evaluates HA as a useful surface marker for positive selection of CTPs. Mononuclear cells from bone marrow were labeled and magnetically separated on the basis of hyaluronan binding. HA(+++), HA(+) and HA(-) fractions were cultured and assayed for colony formation using a quantitative image-processing system. A mean of 2.7% of cells were retained in the HA(+++) fraction and were enriched by 3.4-fold (range of 95% CI: 2.3-4.8) in CTP prevalence when compared to the unselected buffy-coated bone marrow aspirate (BCM). In addition, colonies formed by HA(+++) CTPs demonstrated greater proliferation (more cells per colony) and greater alkaline phosphatase activity than CTP colonies derived from unselected BCM. These data demonstrate that one or more subsets of human marrow-derived CTPs retain a HA rich matrix on their surface at the time of harvest. HA(+++) CTPs may offer a useful cell population for regenerative therapies.

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.