Circular Halbach array for fast magnetic separation of hyaluronan-expressing tissue progenitors.

Connective tissue progenitors (CTPs) are a promising therapeutic agent for bone repair. Hyaluronan, a high molecular mass glycosaminoglycan, has been shown by us to be a suitable biomarker for magnetic separation of CTPs from bone marrow aspirates in a canine model. For the therapy to be applicable in humans, the magnetic separation process requires scale-up without compromising the viability of the cells. The scaled-up device presented here utilizes a circular Halbach array of diametrically magnetized, cylindrical permanent magnets. This allows precise control of the magnetic field gradient driving the separation, with theoretical analysis favoring a hexapole field. The separation vessel has the external diameter of a 50 mL conical centrifuge tube and has an internal rod that excludes cells from around the central axis. The magnet and separation vessel (collectively dubbed the hexapole magnet separator or HMS) was tested on four human and four canine bone marrow aspirates. Each CTP-enriched cell product was tested using cell culture bioassays as surrogates for in vivo engraftment quality. The magnetically enriched cell fractions showed statistically significant, superior performance compared to the unenriched and depleted cell fractions for all parameters tested, including CTP prevalence (CTPs per 10(6) nucleated cells), proliferation by colony forming unit (CFU) counts, and differentiation by staining for the presence of osteogenic and chondrogenic cells. The simplicity and speed of the HMS operation could allow both CTP isolation and engraftment during a single surgical procedure, minimizing trauma to patients and lowering cost to health care providers.

Slowing the Onset of Hypoxia Increases Colony Forming Efficiency of Connective Tissue Progenitor Cells In Vitro.

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.

In vivo transplantation of autogenous marrow-derived cells following rapid intraoperative magnetic separation based on hyaluronan to augment bone regeneration.

INTRODUCTION: This project was designed to test the hypothesis that rapid intraoperative processing of bone marrow based on hyaluronan (HA) could be used to improve the outcome of local bone regeneration if the concentration and prevalence of marrow-derived connective tissue progenitors (CTPs) could be increased and nonprogenitors depleted before implantation. METHODS: HA was used as a marker for positive selection of marrow-derived CTPs using magnetic separation (MS) to obtain a population of HA-positive cells with an increased CTP prevalence. Mineralized cancellous allograft (MCA) was used as an osteoconductive carrier scaffold for loading of HA-positive cells. The canine femoral multidefect model was used and four cylindrical defects measuring 10 mm in diameter and 15 mm in length were grafted with MCA combined with unprocessed marrow or with MS processed marrow that was enriched in HA(+) CTPs and depleted in red blood cells and nonprogenitors. Outcome was assessed at 4 weeks using quantitative 3D microcomputed tomography (micro-CT) analysis of bone formation and histomorphological assessment. RESULTS: Histomorphological assessment showed a significant increase in new bone formation and in the vascular sinus area in the MS-processed defects. Robust bone formation was found throughout the defect area in both groups (defects grafted with unprocessed marrow or with MS processed marrow.) Percent bone volume in the defects, as assessed by micro-CT, was greater in defects engrafted with MS processed cells, but the difference was not statistically significant. CONCLUSION: Rapid intraoperative MS processing to enrich CTPs based on HA as a surface marker can be used to increase the concentration and prevalence of CTPs. MCA grafts supplemented with heparinized bone marrow or MS processed cells resulted in a robust and advanced stage of bone regeneration at 4 weeks. A greater new bone formation and vascular sinus area was found in defects grafted with MS processed cells. These data suggest that MS processing may be used to enhance the performance of marrow-derived CTPs in clinical bone regeneration procedures. Further assessment in a more stringent bone defect model is proposed.

Hyaluronan as a novel marker for rapid selection of connective tissue progenitors.

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.