COMPARISON OF MESENCHYMAL STEM CELLS OSTEOGENESIS IN BIOCOMPOSITE HYDROGELS FOR REPAIR OF CRANIOFACIAL DEFECTS: AN IN VITRO/IN VIVO STUDY

*Yang F; #Haerian HD; *Pow-Anpongkul N; +*Elisseeff, JH +*Johns Hopkins University, Baltimore, MD # University of Maryland, College Park, MD INTRODUCTION: Craniofacial reconstruction is an area of medicine where tissue engineering has a potential to significantly improve surgical options by providing a source for tissue augmentation. Mesenchymal stem cells (MSC) are progenitor cells which can differentiate down multiple lineages including bone. We hypothesized that incorporating bone matrix components (such as hydroxyapatite, type I collagen etc) into the scaffolds may promote osteogenesis. The aim of this study was to examine the potential of various biological-synthetic composites for osteogenesis of human mesenchymal stem cells both in vitro and in vivo. METHODS: Human MSCs (Cambrex) were predifferentiated in standard osteogenic medium for 12 days in monolayer before photoencapsulation. Five experimental groups of biocomposite materials were examined: PODA (poly (ethylene oxide) diacrylate) +HA, Gelrin (fibrinogen-PEG cogel containing 2% of the protein), Gelrin+HA, Collagen I +HA, Cymentra+HA (human alloderm extract, primarily composed of type I collagen). For all the groups supplemented with HA (hydroxyapatite powder, 10um-40um in size), 1% w/v of HA was added into the hydrogel solution. All experimental groups were mixed with PODA to make a composite material of a total 15% w/v to ensure the same pore size. Cells were also encapsulated in PODA alone as a control. Cells (15 million/ml) were then suspended in polymer solution in all 6 groups and exposed to light (365nm, 5mins) to solidify. For in vitro study, all the constructs were cultured in standard osteogenic medium and harvested at a 3-week time point (n=10/group). For in vivo study, two symmetrical defects (d=3.5mm) were made in athymic mouse cranium and constructs with desired shape and size were implanted (Fig. 7). Samples were harvested at 2, 4, and 7 weeks. Biochemical, histological, gene expression and x-ray radiography analyses were performed to evaluate the differentiation of MSCs into an osteogenic phenotype and the accumulation of bone-specific products in the hydrogels. Statistical significance was determined by analysis of variance (ANOVA single factor) and set as P<0.05 (*). RESULTS & DISCUSSION: Human MSCs underwent osteogenesis pathway in all hydrogels, as indicated by the opaque appearance, (Fig 1), positive expression of various bone marker genes (Fig 5) and detection of bone matrix proteins (Fig 3, 4). Gels implanted in vivo became opaque at harvest and exhibited radioopacity close to natural bone by xray imaging. HA increased the production of bone-specific proteins such as alkaline phosphatase and may increase the collagen production (Fig 3). However, effects of HA also depend on other components of the scaffold. Upregulation of Col I was only seen when HA was combined with Gelrin (Fig 6) while no similar effects were observed with other HA groups. Our results suggest that HA-containing hydrogel composite may provide a better environment for MSC based craniofacial repair. 6mm osteogenic medium. Gelrin group accumulated less amount of calcium in the scaffold but more in the pericellular regions. Groups supplemented with HA showed a stronger staining of mineralization.

100 m PODA Gelrin Col+HA

PODA+HA Gelrin+HA Cym+HA Figure 3. Picro Red staining (collagen) exhibited collagen production in the pericellular regions of all the groups, with a stronger staining in PODA-HA group compared to PODA alone. While such effects was not observed with Gelrin-HA group compared to Gelrin alone. Figure 4. Quantitative ALP assay demonstrated hydroxyapatite (HA) significantly increased alkaline phosphatase production in PODA+HA and Gelrin+HA groups compared to PODA and Gelrin alone.

Figure 5. Expression of early bone markers (cbfa-1, ON) and late bone marker (col I), suggesting cells have undergone osteogenesis pathway in all the groups.

Figure 6. Quantitative realtime PCR showed strongest col I expression in GelrinHA group.

PODA PODA-HA Gelrin Gelrin-HA Col-HA Cym-HA Figure 1. Constructs after 21 days cultivation in osteogenic medium.

100m Figure 7. In implantation. vivo

Figure 8. X-ray images morphology at harvest. PODA Gelrin:Gelrin 2 weeks -HA 4 weeks

and

gross

Cym-HA 7 weeks

Acknowledgement: The authors would like to acknowledge NIH for funding. Figure 2. Von Kossa staining of constructs after 21 days cultivation in

52nd Annual Meeting of the Orthopaedic Research Society

Paper No: 0897

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