Cell transplantation of stem cells has tremendous potential for craniofacial regenerative applications; yet, identification of the appropriate cell types and cell processing protocols are two of the most critical determinants in producing successful outcomes. In the present study, our aim was to assess the capacity of a cell production process, which utilizes an automated closed-system bioreactor, to produce clinical-scale numbers of autologous cells (BRCs) capable of regenerating clinically viable bone. Through cell surface marker characterization of BRCs, it was determined that the SPP ex vivo cell expansion processing of bone marrow aspirate was capable of producing cell populations highly enriched for mesenchymal and vascular progenitor cells. This was confirmed through cell characterization of BRCs, where cells within this heterogeneous population demonstrated the capacity to be induced to differentiate down chondrogenic, adipogenic, osteogenic, and angiogenic lineages. Finally, the test of the clinical regenerative capacity of BRCs demonstrated their ability to regenerate highly vascular bone in a human jawbone defect.
The cell-processing system employed to produce these cells utilizes an SPP protocol.20,22,25,35
Gastens et al
examined this system and its ability to expand bone-marrow-derived cells to produce clinical-scale cell numbers. These initial studies compared cell phenotypes of cells produced with this closed SPP system to phenotypes of cells cultured with an open system utilizing conventional tissue culture parameters. Although mesenchymal cell numbers were observed to be higher in cells cultured with the SPP closed system, the limitation of the study was that the initial BMMNC fractions tested in the two different systems came from different donors; thus, differences observed between the final cell products could have been, at least in part, attributed to donor–donor variability. In the present study, although we did not compare cell populations from closed and open culture systems, we did compare BRCs directly to the phenotypes of the BMMNCs from which they were produced. The final BRC population showed marked enrichment for mesenchymal and vascular cell phenotypes, suggesting that for therapeutic regenerative strategies, the SPP process supports the production of a more favorable cell population for transplantation than protocols using transplants comprised of unfractioned, whole bone marrow. Although there is often a wide degree of variability in the phenotypic expression of these markers from donor to donor, the relative differences in cell phenotype before and after cell processing is consistent in that the final product is enriched in expression of mesenchymal and angiogenic phenotypes after cell processing with the SPP system. Additional studies need to be performed comparing cell phenotypes and differentiation potential of cells derived from the same donor, when processed with either this closed system or a traditional tissue culture open system technique. It is also important to note that although the objectives of the differentiation assays were to examine the osteogenic and angiogenic potential of the BRCs, it is not possible to regrow or resume culture of cells in the Replicell system once they have been removed from the system. As such, further culture of BRCs required conventional tissue culturing techniques. However, it is recognized that this additional culture step could have potentially resulted in a cell population not identical to the population produced from the Replicell system; yet, because the Replicell system is a closed system, there is not a viable alternative to performing or adapting the aforementioned assays to cells cultured while in the Replicell system.
The ability of the SPP process to form bone-forming cells has also been recently studied in vitro
and in ectopic animal models.26
In these studies, the levels of bone formation seen in vivo
followed the same trends of the osteogenic differentiation observed in vitro
. Additionally, cell surface markers, including CD90+ (Thy1) and CD105+ (endoglin), were positively correlated with ectopic bone formation in mice. CD105, originally identified as a marker of mesenchymal stem cells,36
has more recently been associated with vascular endothelium in angiogenic tissues37
and expression of CD90 has been linked to bone marrow subpopulations of colony-forming mesenchymal stem cells (CFU-F, colony-forming unit–fibroblasts).38
In our study, cell surface marker expression of BRCs from the subject who underwent cell transplantation in the bone defect showed 65-fold and 5-fold increases in CD90+ and CD105+, respectively (data not shown). This served as an indication that the BRC product was highly enriched with cells possessing mesenchymal and angiogenic potential and is in accordance with previous reports demonstrating enrichment of theses cell types with this cell-processing protocol.26,35
Although the cell product is highly enriched for vascular and mesenchymal cells as indicated by cell surface marker expression and in vitro
differentiation capacity, it is clear that in vitro
osteogenic differentiation of cell populations does not guarantee bone-forming capacity in vivo
. Meijer et al
. performed a clinical study evaluating the repair of jawbone defects in six subjects treated with autologous cells expanded using an open system,39
similar to the protocol used in traditional tissue culture of mesencyhmal stem cells.40,41
In this study, cells were cultured anywhere from 12 to 25 days before implantation and the last 7 days in culture cells were grown on a mineral substrate, hydroxyapatite (HA) particles, in the presence of the osteoinductive agent dexamethasone. When cells grown under these osteogenic conditions were analyzed for their osteogenic capacity, all six bone marrow specimens produced cells capable of osteogenic differentiation in vitro
(as determined by ALP expression) and bone formation in vivo
(subcutaneous implants of HA/cell constructs in athymic mice). However, after implantation of these HA/cell constructs into various human jawbone defects of the six patients, biopsy specimens taken at 4 months showed that bone formation by implanted cells was able to regenerate bone in only one of the six patients treated. The authors made the important observation that in vitro
osteogenic assays and bone formation in preclinical mouse models may not necessarily correlate to successful bone regeneration in the more challenging clinical applications. They concluded further that inadequate vascularity could have resulted in the reported disappointing outcome of the study.39
In accordance with the authors' conclusion from this study, it is our belief that not only is vascularization from the host environment essential to clinical bone regeneration, but even further, that the angiogenic potential of the transplanted cells themselves should play an active role in this vascularization process.
The BRCs used to treat the human jawbone defect in our study were not produced in the presence of any osteogenic factors, and a gelatin sponge (with no known osteoinductive or osteoconductive properties) was used as a carrier matrix to transplant the cells, as opposed to a mineralized matrix such as HA. Additionally, the biopsy specimen harvested at 6 weeks showed significant new bone formation containing abundant blood vessels. Although no direct evidence (i.e., labeling) is provided relative to the source of cells that produced the regenerated tissue, we make the assumption that the transplanted cells at least partly contributed to the regeneration because the bone core specimen analyzed was harvested from the central region of the defect and graft site. We were able to identify this exact region through the use of surgical measurement templates/guides. Yet, even still, despite these promising clinical results, the fact that they were obtained in a single patient can be viewed as a study limitation and minimizes the general conclusions that can be drawn. An additional note is that this case presentation is part of a larger, U.S. FDA-regulated, randomized, controlled Phase I/II trial where a larger number of patients are to be treated with BRCs. This larger study is still ongoing as it includes a 1-year patient follow-up; however, upon study completion, all the clinical data will be analyzed and the results outlined in a future report. While it is realized that the feasibility of this process for routine tooth extraction surgeries is most likely not practical, this study was conducted as an FDA Phase I/II study to examine safety and efficacy of this therapy for regeneration of craniofacial bone. If results are favorable, this type of therapy may certainly be feasible for larger, more challenging craniofacial reconstructions.