Bone regeneration through tissue engineering is emerging as a means of replacing and treating malfunctioning or depleted bones. Bone graphs are second to only blood transfusion as the most implanted material; however current grafts are less than ideal. Approximately 850,000 procedures each year require bone grafts with additional applications in both dental prostheses and middle inner ear implants available. Currently, synthetic materials are responsible for only 10% of the market while autografts (52%) and allografts (38%) dominate. Autografts are considered the current gold standard yet have high costs and require traumatic surgery while allografts generate concerns due to the possibility of virus and contaminating agent transmittance. Due to these limitations a tissue engineered bone graft which could improve on today's current standard is desired.

Currently the most commonly used synthetic material for such procedures is hydroxyapatite (HAP). Hydroxyapatite is a bioactive ceramic with a unique crystal structure (P6₃/m) and is both crystallographically and chemical (Ca₁₀(PO₄)₆(OH)₂) similar to native bone. It has generated great interest in the field of tissue engineering because it has shown favorable biological response and bonds with surrounding tissue. However due to the difficulty densifying HAP there are significant concerns using the material for bone loading applications and therefore it is typically limited to coatings on metallic implants and as defect fillers. In order to overcome these concerns it is believed that synthesis and processing must be carried out at the nanoscale.

The goal of this research is to develop a clinically and commercially viable tissue engineered bone construct using nanocrystalline hydroxyapatite (nanoHAP, Angstrom Medica, Woburn, MA) combined with an available and renewable cell source, such as human mesenchymal stem cells (hMSC). A routinely applicable procedure for the ex vivo expansion and differentiation of stems cells on advanced materials such as nanoHAP has not yet been developed and optimized. To this end, we have systematically examined the impacts of culture conditions and medium components on the proliferation, differentiation and metabolism of hMSCs on nanoHAP.

hMSC's were cultured on nanoHAP and treated culture plastic (TCP) and the culture substrates were analyzed on the basis of their efficacy in promoting cell adhesion and adhesion dependent functions, in particular proliferation rate. Cell proliferation was assessed by quantifying culture DNA contents using a florescence based assay at various times following cell seeding and differentiation.

Osteogenic differentiation was induced using known mineralization factors i.e. dexamethasone, b-glycerolphosphate, ascorbic acid and Vitamin D₃. Effective factor concentrations for media components such as amino acids and dexamethasone, were explored using single-factor dose response experiments. Extent of osteogenic differentiation was assessed using confocal microscopy to analyze cell morphology and real-time RT-PCR to quantitatively measure both early and late gene expression markers (e.g. bone sialoprotein II and osteocalcin) with respect to a housekeeping gene (18S). Metabolic assessments were performed by time series measurements on an array of primary metabolites in the extracellular medium using High Performance Liquid Chromatography (HPLC) as well as a series of colorimetric assays.

In on-going work, we are correlating the above effects to the metabolic changes underlying cellular expansion and differentiation using metabolic flux analysis (MFA). A flux based stoichiometric model containing 127 reactions and 97 metabolites was designed based upon central carbon metabolism for collagen I production. Steady-state material balance equations were written for each reaction. The equations were expressed in matrix notation, S * v = b, where S is the stoichiometric matrix whose entries S_ij are the stoichiometric coefficients of metabolite i in reaction j. The vector v has entries v_j which is the flux through reaction j. Alternatively, b was determined experimentally by taking measurements of extracelluar metabolite concentrations as a function of time The model was initially solved to determine theoretical optimal collagen and biomass using linear programming methods. Results were then compared with actual data to determine which pathways were active and which amino acids were critical to collagen synthesis, a characteristic of osteogenesis.

Our results indicate that cell adhesion is similar for both nHAP and TCP substrates, and although the initial proliferation rate is greater on TCP, nHAP shows long term stability. Furthermore, our results show that supplementation of the culture medium with amino acids significantly increases hMSC proliferation on both nanoHAP and standard tissue culture substrate without loss of osteogenic differentiation potential. On the other hand, an increase in cell density through delayed induction of osteogenesis negatively impacted the differentiation potential. Our data also shows that the initial period of hMSC differentiation corresponds to a sharp increase in glycolytic activity and significant changes in amino acid fluxes with osteogenesis.

Taken together, our results to date indicate that the use of nanoHAP accelerates osteogenic differentiation compared to conventional tissue culture substrates. Moreover, hMSC differentiation is accompanied by significant metabolic shifts, suggesting that metabolic approaches may be available to enhance and/or accelerate in vitro osteoblast formation.