Anabolic actions of PTH (1-34): Temporal and mechanistic impact on cell proliferation and differentiation during engineered bone regeneration.

Abstract

The use of pharmacologic agents, such as parathyroid hormone (PTH), capable of promoting bone formation offers exciting new approaches to address the critical need for skeletal regeneration. Although PTH is in clinical use for osteoporosis treatment and is being investigated for use in tissue engineering, fracture healing, and implant integration applications, its mechanisms of action remain unclear. The goals of this study were to determine the temporal and mechanistic impact of PTH on cell proliferation and differentiation using a novel bone regeneration model. Implanted bone marrow stromal cells (BMSCs) were used to generate ectopic bone nodules (i.e., ossicles). To determine the temporal dependence of PTH for anabolic actions in bone, mice with BMSC implants were administered PTH (40mug/kg/d) or vehicle for 1, 3, or 7wks, with treatment initiated 1 wk after cell implantation. Additionally, mice were treated with PTH for 3wks, initiated 12wks after BMSC implantation. Data indicated that PTH increased bone volume but the amount of bone growth was temporally dependent on the implanted BMSCs. PTH effects on cells at various stages of differentiation were determined by evaluating the temporal impact of 3wks of PTH on a synchronized cell population with treatment initiated 1d, 1, 2, or 3wks after implanting BMSCs. PTH increased bone in all groups with the greatest bone growth observed with PTH initiated 1wk after cell implantation. In addition, the effects of PTH on increasing BMSC numbers was evaluated in ossicles generated from BMSCs isolated from mice that constitutively expressed a light-producing enzyme, luciferase. Weekly bioluminescence imaging and enumeration of cells stained positive for the BrdU cell proliferation marker, revealed significant increases in BMSC numbers in the marrow of PTH-treated implants. PTH treatment was also combined with a potent bisphosphonate, zoledronic acid (ZA) (3mug/mouse/d), since bisphosphonates have been reported to blunt the anabolic actions of PTH in humans. ZA reduced PTH-mediated increases in proliferation but did not reduce bone area. These data suggest that PTH increases the number of bone-forming cells (i.e., osteoblasts) and not osteoblast recruitment <italic> in vivo</italic>, and that ZA hinders PTH-mediated increases in proliferation, but does not blunt increases in total bone formed. Hence, although PTH and ZA act via different mechanisms, simultaneous treatment with these two agents may exert additive effects on bone regeneration. Further, this thesis includes a comprehensive list of PTH-regulated genes generated from microarray experiments compiled to enhance knowledge of key genes involved in PTH-mediated bone formation. In conclusion, these studies provided a greater understanding of the temporal effects of PTH on bone and evidence that cell proliferation and differentiation play critical roles in PTH-facilitated bone regneneration. This investigation will contribute to optimizing the therapeutic potential of PTH and will be valuable in the development of new uses of PTH in bone regeneration applications.