The regulation of osteogenesis : the role of abaloparatide and other osteogenic factors in vitro and abaloparatide-loaded hydrogel in vivo

Abstract

With the advance of tissue engineering, synthetic bone substitutes constitute an attractive alternative to the traditional treatment of bone defects by autografts and allografts. Together with the modulation of the scaffold physical characteristics, the incorporation of therapeutic drugs, growth factors, or ions into bone substitutes has proven to be an efficient strategy to improve bone regeneration. However, developing materials that are safe and efficient in various pathological contexts remain challenging and requires a better knowledge of material- and osteogenic factor-driven osteogenesis.

Abaloparatide, as an analogue of human recombinant parathyroid hormone protein (PTHrp), has been considered as a drug for treating postmenopausal osteoporosis since 2017. Several in vivo studies of delivering teriparatide with polymeric scaffolds in treating bone defects have been reported. Besides, a clinical study demonstrates that less bone resorption effect and fewer side-effects (such as hypercalcemia) were induced by subcutaneous injected abaloparatide in patients, compared to the teriparatide. These findings suggest abaloparatide could be an alternative to teriparatide in the bone defect treatments. However, its potential to favor bone formation when associated with polymeric scaffolds remains to be fully demonstrated. In the current work, we investigated the bone regeneration potential of abaloparatide-loaded GelMA hydrogel. As a first step, abaloparatide treatment of MC3T3-E1 pre-osteoblast cells was shown to significantly promote cell viability and osteogenic differentiation in vitro. Abaloparatide was then added to a methacrylated gelatin (GelMA) hydrogel as a drug delivery system. The 3D porous GelMA was found effective in prolonging the release of abaloparatide (more than 10 days) in vitro. Therefore, injectable photo crosslinked GelMA hydrogel was used in this study to prolong the release of abaloparatide. Overall, my study reveals that abaloparatide alone enhanced pre-osteoblast proliferation and osteogenic differentiation and implies that abaloparatide-loaded GelMA hydrogel is effective in stimulating bone regeneration compared to the control (no treatment) group and GelMA control (GelMA without drug) group.

In the second part of the thesis I evaluated the proliferation and osteogenic differentiation of human mesenchymal stem cells (hMSCs) from five different donors in response to osteogenic medium, bone-morphogenetic protein-2 (BMP-2), lysophosphatidic acid (LPA), epidermal growth factor (EGF), interleukin-3 (IL-3) and silicate (Si) ions. BMP-2 was identified as the most promising agent, increasing alkaline phosphatase (ALP) activity, expression of osteogenic marker genes as well as increasing mineralization of hMSCs from all the donors. LPA and Si ions could also be regarded as potential agents as these two factors increased the osteogenic differentiation of hMSCs from two donors. IL-3 did not seem to affect cell proliferation and osteogenic differentiation, whereas EGF showed enhanced cell proliferation but different influences in osteogenic differentiation of hMSCs from different donors. This study paves the way for further in vitro studies of osteogenic differentiation by proteomics in order to elucidate the molecular level osteogenic differentiation mechanisms in more detail.

Taken together, these two studies identify the osteogenic effects of abaloparatide as well as multiple growth factors and Si ions in pre-osteoblasts and hMSCs, respectively, and provides a potential strategy to locally treat bone defects with abaloparatide-loaded GelMA hydrogel.