Glenda J. Pettway

The release profiles and bioactivity of parathyroid hormone from poly(lactic-co-glycolic acid) microspheres

Poly(lactic-co-glycolic acid) (PLGA) microspheres containing bovine serum albumin (BSA) or human parathyroid hormone (PTH)(1-34) were prepared using a double emulsion method with high encapsulation efficiency and controlled particle sizes. The microspheres were characterized with regard to their surface morphology, size, protein loading, degradation and release kinetics, and in vitro and in vivo assessments of biological activity of released PTH. PLGA5050 microspheres degraded rapidly after a 3-week lag time and were degraded completely within 4 months. In vitro BSA release kinetics from PLGA5050 microspheres were characterized by a burst effect followed by a slow release phase within 1-7 weeks and a second burst release at 8 weeks, which was consistent with the degradation study. The PTH incorporated PLGA5050 microspheres released detectable PTH in the initial 24h, and the released PTH was biologically active as evidenced by the stimulated release of cAMP from ROS 17/2.8 osteosarcoma cells as well as increased serum calcium levels when injected subcutaneously into mice. Both in vitro and in vivo assays demonstrated that the bioactivity of PTH was maintained largely during the fabrication of PLGA microspheres and upon release. These studies illustrate the feasibility of achieving local delivery of PTH to induce a biologically active response in bone by a microsphere encapsulation technique.

Parathyroid Hormone Mediates Bone Growth through the Regulation of Osteoblast Proliferation and Differentiation

PTH (1-34) is the only FDA-approved anabolic agent for osteoporosis treatment in the U.S., but its mechanisms are not completely understood. This study investigated PTH effects on osteogenic cells at various stages of differentiation and proliferation using an engineered bone growth model in vivo. Ossicles were generated from bone marrow stromal cells (BMSCs) implanted in immunocompromised mice. Three weeks of PTH (40 microg/kg/day) or vehicle treatment initiated 1 day, 1, 2, or 3 weeks after BMSC implantation resulted in an anabolic response in PTH-treated implants (via histomorphometry and muCT) in all treatment groups. A novel in vivo tracking strategy with luciferase tagged BMSCs and weekly bioluminescent imaging of ossicles revealed increased donor cell proliferation in PTH-treated ossicles. The greatest increase occurred during the first week, and the activity remained elevated in PTH-treated implants over time. Zoledronic acid (ZA) was combined with PTH to delineate interactive mechanisms of these bone active agents. Combining ZA with PTH treatment reduced the PTH-mediated increase in luciferase BMSC activity, serum osteocalcin, and serum tartrate resistant acid phosphotase-5b (TRAP-5b) but ZA did not reduce the PTH-induced increase in total bone. Since zoledronic acid reduced PTH-induced proliferation without reducing bone volume, these data suggest that combining PTH and bisphosphonate therapy warrants further investigation in the treatment of bone disorders.

Cyclin D1 as a Target for the Proliferative Effects of PTH and PTHrP in Early Osteoblastic Cells

PTHrP induced a proliferative cyclin D1 activation in low‐density osteoblastic cells. The process was PKA and MAPK dependent and involved both AP‐1 and CRE sites. In ectopic ossicles generated from implanted bone marrow stromal cells, PTH upregulated cyclin D1 after acute or intermittent anabolic treatment. These data suggest a positive role of PTH and PTHrP in the cell cycle of early osteoblasts.

Proteoglycan 4: A Dynamic Regulator of Skeletogenesis and Parathyroid Hormone Skeletal Anabolism

Proteoglycan 4 (Prg4), known for its lubricating and protective actions in joints, is a strong candidate regulator of skeletal homeostasis and parathyroid hormone (PTH) anabolism. Prg4 is a PTH‐responsive gene in bone and liver. Prg4 null mutant mice were used to investigate the impact of proteoglycan 4 on skeletal development, remodeling, and PTH anabolic actions. Young Prg4 mutant and wild‐type mice were administered intermittent PTH(1–34) or vehicle daily from 4 to 21 days. Young Prg4 mutant mice had decreased growth plate hypertrophic zones, trabecular bone, and serum bone formation markers versus wild‐type mice, but responded with a similar anabolic response to PTH. Adult Prg4 mutant and wild‐type mice were administered intermittent PTH(1–34) or vehicle daily from 16 to 22 weeks. Adult Prg4 mutant mice had decreased trabecular and cortical bone, and blunted PTH‐mediated increases in bone mass. Joint range of motion and animal mobility were lower in adult Prg4 mutant versus wild‐type mice. Adult Prg4 mutant mice had decreased marrow and liver fibroblast growth factor 2 (FGF‐2) mRNA and reduced serum FGF‐2, which were normalized by PTH. A single dose of PTH decreased the PTH/PTHrP receptor (PPR), and increased Prg4 and FGF‐2 to a similar extent in liver and bone. Proteoglycan 4 supports endochondral bone formation and the attainment of peak trabecular bone mass, and appears to support skeletal homeostasis indirectly by protecting joint function. Bone‐ and liver‐derived FGF‐2 likely regulate proteoglycan 4 actions supporting trabeculae formation. Blunted PTH anabolic responses in adult Prg4 mutant mice are associated with altered biomechanical impact secondary to joint failure.

JunB as a downstream mediator of PTHrP actions in cementoblasts

The role of AP‐1 family members in the action of PTHrP was examined in cementoblasts. PTHrP increased mRNA and protein levels of all Fos members, but only one Jun member (JunB) was increased. Overexpression of JunB in cementoblasts mimicked actions of PTHrP to support osteoclastogenesis and inhibit cementoblast differentiation, suggesting that the actions of PTHrP on mesenchymal cells operate through JunB.

Ossicle and vossicle implant model systems

Bone regeneration and repair is a goal of many skeletal therapies and numerous agents positively or negatively impact these processes. New therapeutic agents and effective model systems are continually sought to identify agents and characterize their mechanisms of action are in constant demand. In addition, investigations of tumor cell-bone interaction in the skeletal metastatic microenvironment require well-defined and readily orchestrated models. This chapter describes a novel ectopic ossicle model and a vossicle modification that can be used to provide focused and rapid feedback of bone growth and bone-cellular interactions. The ossicle model is a bone marrow stromal cell (BMSC)-based model and the vossicle model is a neonatal vertebral bone transplant model. These models offer opportunities to mix and compare mesenchymal (donor derived) and hematopoietic elements (host derived). Multiple implants can be placed in one mouse to facilitate various outcome analyses, such as histomorphometry, micro-CT, gene expression studies, and cell tracking using markers such as luciferase, in response to pharma cological or genetic manipulation. Implants can also be combined with other cell types, such as cancer cells to evaluate the bone-tumor microenvironment.

Osteogenic and Adipogenic Cell Fractions Isolated from Postnatal Mouse Calvaria

The use of stem/progenitor cells represents a promising approach to treat craniofacial bone defects, but successful treatments will rely on the availability of cells that can be expanded in vitroand which will differentiate appropriately in vivo. The calvaria may represent a source of autologous cells for such purposes. We demonstrate expression of stem cell antigen-1 (Sca-1) in mouse calvaria. We isolated Sca-1+ and Sca-1– cells at high purity and tested the ability of these cells to differentiate into adipose and bone. We show that the Sca-1+ cell fraction has adipogenic differentiation potential and that the cell Sca-1– fraction has osteogenic differentiation potential. The Sca-1+ cell fraction partially retains its adipogenic differentiation potential and the Sca-1– cell fraction partially retains its osteogenic differentiation potential after in vitroexpansion. These data suggest that the calvaria may be used as a source of stem/progenitor cells that can be expanded in vitroand transplanted in vivofor craniofacial tissue regeneration.

JunB as a Potential Mediator of PTHrP Actions: New Gene Targets Ephrin B1 and VCAM-1

Parathyroid hormone-related protein (PTHrP) is an integral mediator of physiologic and pathologic processes and has demonstrated actions in the periodontium. PTHrP functions via AP-1, and specifically through JunB. This study identified JunB-dependent downstream mediators of PTHrP using OCCM cementoblastic transfectants with JunB over- or reduced expression. Over-expressing cells showed an increase in proliferation, while the opposite was seen in siRNA transfected cells. Microarray analysis of over-expressing cells revealed more than 1000 regulated genes. Three genes were investigated in more detail. The PTH/PTHrP receptor (PTHR1) and ephrin B1 (EfnB1) were down-regulated, and vascular cell adhesion molecule-1 (VCAM-1) was up-regulated with JunB over-expression. JunB siRNA transfectants had increased PTHR1, but reduced ephrin B1 and unaltered VCAM-1 in vitro. To validate these targets, parental OCCM cells and primary osteoblasts were treated with PTHrP, resulting in reduced PTHR1 and ephrin B1, and increased VCAM-1. Cell transfectants were implanted subcutaneously in vivo, and microarray analysis and RT-PCR performed. Over-expression of JunB down-regulated PTHR1 and ephrin B1, and increased VCAM-1. JunB siRNA transfectant implants had increased PTHR1 and ephrin B1, but no altered VCAM-1. These data highlight new gene targets for PTHrP and indicate JunB is a critical mediator of PTHrP actions.