Cobalt Doped Nano-Hydroxyapatite Incorporated Gum Tragacanth-Alginate Polymeric Beads: An Angiogenic-Osteogenic Matrix for Bone Tissue Engineering

Abstract

The success of a tissue engineered bone graft depends on its ability to induce both angiogenesis and osteogenesis in vivo upon implantation. Angiogenesis is the formation of new blood vessels essential for the transportation of the nutrients and gases at the healing site whereas osteogenesis is the process that leads to the synthesis of new bone. The strategies that have been explored so far to ensure both osteogenesis and angiogenesis in bone tissue engineering include the application of growth factors, genetic manipulation of the cells, use of the micro-patterned scaffold, and exploitation of bio-physical stimuli. However, the aforesaid strategies are associated with high cost, process complexity and poor control over the therapeutic outcome. A comprehensive review of the pertinent literature revealed that there is a dearth of cost-effective material/system which can induce both angiogenesis and osteogenesis by its own without the assistance of externally applied growth factors or other cues. The aforementioned limitation can be surmounted by adopting a biomaterial-directed approach for engineering angiogenesis and osteogenesis. Keeping this perspective in mind, here we have developed an angiogenic-osteogenic 3D matrix by combining osteogenic and angiogenic biomaterials into a single platform. The present thesis reports three scientific contributions pertaining to the development of the aforesaid angiogenic-osteogenic 3D matrix.

Firstly, we synthesized an angiogenic hydroxyapatite by doping bivalent cobalt ions into synthetic hydroxyapatite. Cobalt induces angiogenesis through the activation of the hypoxia inducible factor 1 alpha (HIF-1α) pathway. Hydroxyapatites (HAp), doped with varying concentration of cobalt were prepared by ammoniacal precipitation method and the extent of doping was measured by inductively coupled plasma optical emission spectrometry (ICP-OES). Physicochemical characterization of the cobalt doped hydroxyapatites (Co-HAp) was done by X-ray diffraction (XRD) study, Fourier transformed infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). Analysis revealed that the doping of cobalt upto a threshold limit supports the viability, proliferation and differentiation of bone cells (MG-63, a human osteoblast cell line). Western blot analysis in conjugation with ELISA study showed that the Co-HAp samples significantly increased the expression of HIF-1α and vascular endothelial growth factor (VEGF) in MG-63 cells. The angiogenic potential of Co-HAp was further confirmed by the in vitro tube formation assay using human umbilical vein endothelial cells (HUVECs). The studies clearly suggested that a particular composition of Co-HAp (HAN2) is an angiogenic biomaterial suitable for bone tissue engineering.

Secondly, we prepared an osteogenic matrix by incorporating gum tragacanth (GT) into calcium alginate (CA) hydrogel beads. Here, we showed that the incorporation of GT in the bead-composition significantly improved the molecular transport, swelling and degradation properties of the CA bead. We showed that the presence of GT in the beadcomposition resulted in improved viability, proliferation, and differentiation of encapsulated bone cells (MG-63). We further demonstrated that the bone cell loaded CAGT beads are capable of inducing angiogenesis. In conclusion, we proved that a selective composition of CA-GT is osteogenic in nature.

Finally, we developed an angiogenic-osteogenic platform by incorporating the angiogenic Co-HAp into osteogenic CA-GT hydrogel beads. We showed that the incorporation of needle-shaped nano-particulate cobalt doped nano-HAp (Co-nHAp) into the CA-GT did not affect the physicochemical properties of the CA-GT beads (swelling, degradation and molecular transport). Co-nHAp incorporated CA-GT beads significantly enhanced the osteogenic differentiation of encapsulated human mesenchymal stem cells (hMSCs) and HUVECs mediated angiogenesis. These studies clearly implied that Co-nHAp incorporated CA-GT bead is an angiogenic-osteogenic system suitable for bone tissue engineering application.