Bone morphogenetic protein-2 (BMP-2) is a potent osteoinductive factor, yet its clinical use is limited by a short biological half-life, rapid local clearance and propensity for side effects. Heparin (HP), a highly sulfated glycosaminoglycan (GAG) that avidly binds BMP-2, has inherent biological properties that may circumvent these limitations. Here, we compared hyaluronan-based hydrogels formulated to include heparin (Heprasil™) with similar gels without heparin (Glycosil™) for their ability to deliver bioactive BMP-2 in vitro and in vivo. The osteogenic activity of BMP-2 released from the hydrogels was evaluated by monitoring alkaline phosphatase (ALP) activity and SMAD 1/5/8 phosphorylation in mesenchymal precursor cells. The osteoinductive ability of these hydrogels was determined in a rat ectopic bone model by 2D radiography, 3D µ-CT and histological analyses at 8 weeks post-implantation. Both hydrogels sustain the release of BMP-2. Importantly, the inclusion of a small amount of heparin (0.3% w/w) attenuated release of BMP-2 and sustained its osteogenic activity for up to 28 days. In contrast, hydrogels lacking heparin released more BMP-2 initially but were unable to maintain BMP-2 activity at later time points. Ectopic bone-forming assays using transplanted hydrogels emphasized the therapeutic importance of the initial burst of BMP-2 rather than its long-term osteogenic activity. Thus, tuning the burst release phase of BMP-2 from hydrogels may be advantageous for optimal bone formation.

Lowering the efficacious dose of bone morphogenetic protein-2 (BMP-2) for the repair of critical-sized bone defects is highly desirable, as supra-physiological amounts of BMP-2 have an increased risk of side effects and a greater economic burden for the healthcare system. To address this need, we explored the use of heparan sulfate (HS), a structural analog of heparin, to enhance BMP-2 activity. We demonstrate that HS isolated from a bone marrow stromal cell line (HS5) and heparin each enhances BMP-2-induced osteogenesis in C2C12 myoblasts, through increased ALP activity and osteocalcin mRNA expression. Commercially available HS variants from porcine kidney and bovine lung failed to generate similar effects. Heparin and HS5 influence BMP-2 activity by (i) prolonging BMP-2 half-life, (ii) reducing interactions between BMP-2 with its antagonist noggin, and (iii) modulating BMP2 distribution on the cell surface. Importantly, long-term supplementation of HS5 but not heparin greatly enhances BMP-2-induced bone formation in vitro and in vivo. These results show that bone marrow-derived HS effectively support bone formation, and suggests its applicability in bone repair by selectively facilitating the delivery and bioavailability of BMP-2.

The aim of the current study is to elucidate the mechanism of proline-rich tyrosine kinase 2 (Pyk2)-mediated cell proliferation and invasiveness in hepatocellular carcinoma (HCC) cells. Human HCC cell lines PLC and MHCC97L were stably transfected with either full-length Pyk2 or C-terminal non-kinase region of Pyk2 (PRNK). Functional studies on cell proliferation and invasion were conducted in vitro by colony formation assay, adhesion assay, migration assay and wound-healing assay. For the in vivo study, an orthotopic nude mice liver tumor model was applied to investigate the effects of Pyk2 overexpression on tumor growth and metastasis. Overexpression of Pyk2 in PLC cells resulted in an upregulation of colony formation (P = 0.021) and adhesion toward laminin (P = 0.018). Pyk2 promoted wound recovery by stimulation of actin stress fiber polymerization. In the in vivo study, transfection of PRNK in MHCC97L cells significantly decreased tumor volume (P = 0.001) and the incidence of lung metastasis (P = 0.014). Overexpression of Pyk2 promoted the activation of c-Src, formation of Pyk2/c-Src complex and activated the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK)-signaling pathway. Pyk2 upregulated the activation of ERK1/2 that is insensitive to MAPK/ERK kinase (MEK)1/2 inhibition. On the contrary, PRNK overexpression downregulated the activation of c-Src and ERK/MAPK-signaling pathways. Immunofluorescence staining showed that the focal adhesion localization of Pyk2 is a major determinant for c-Src and ERK/MAPK activation. In conclusion, our results showed that Pyk2 promoted cell proliferation and invasiveness by upregulation of the c-Src and ERK/MAPK-signaling pathways.