Parathyroid hormone (PTH) analogs are the main anabolic pharmacological agent for osteoporosis. PTH is an endogenous hormone, of which amino acids 1-34 bind the parathyroid hormone receptor (PTH1r), a G-coupled protein receptor expressed in kidney, fat, and bone. PTH increases trabecular bone mass by promoting the differentiation of the mesenchymal stem cell (MSC) into the osteogenic lineage. The Zinc Finger Protein 467 (Zfp467) is a potential downstream target of PTH1r and an important mediator of the MSC into the adipogenic lineage. Taken together, we ask whether Zfp467 knockout cells will show greater osteogenic potential and increased sensitivity to PTH treatment. We also seek to investigate a mechanistic signaling pathway of PTH1r involving Zfp467. Calvarial osteoblast (COB) and bone marrow stromal cells (BMSCs) from Zfp467 wild type (WT) and knockout (KO) mice were osteogenically differentiated and treated with either continuous (48h) or intermittent (6h/42h) PTH for 7-14 days. At 7 and 14 days, alkaline phosphatase (ALP) and von Kossa staining were conducted, respectively. At 7 days after differentiation, qPCR was used to analyze genes involved in osteogenesis, adipogenesis, WNT signaling, and mitochondrial respiration. ELISA was used to measure cAMP levels. Seahorse XF96 assays were used to measure oxygen consumption rates (OCRs) and extracellular acidification rates (ECARs). Western blot was used to measure PTH1r. Additionally, adipogenic differentiation and Oil Red O staining were performed on BMSCs. ALP and von Kossa results showed that Zfp467 KO cells exhibited increased osteogenesis and an increased response to PTH treatment (continuous and intermittent) as compared to WT controls. qPCR analysis of Alp, Rankl, and Sp7 further supported an increased osteogenic potential of the KO. Also, Oil Red O staining revealed suppressed adipogenesis in KO BMSCs and qPCR analysis showed suppressed Adiponectin and Ppary in KO COBs. Additionally, Pth1r and PTH1r expressions were significantly higher in KO and short PTH treatments (~10m) induced a remarkable reduction in Zfp467 of WT cells. Furthermore, the KO showed suppressed Pgc1a, similar OCR, and increased ECAR as compared to WT. The KO also exhibited higher cAMP levels and was more responsive to PTH-induced increases of cAMP at 10 minutes of PTH exposure. However, qPCR analysis of Lef1 and Sost showed no difference regarding the WNT pathway. Our data support an anti-osteogenic and pro-adipogenic role for Zfp467. The KO displays less adipogenesis, more osteogenesis, and is consistently more sensitive to the osteogenic effects of PTH. The upregulation of Pth1r and PTH1r in KO cells offers an explanation for this increased sensitivity. We propose a mechanism where the suppression of Zfp467 upregulates Pth1r and PTH1r activation suppresses Zfp467, resulting in a constitutively active positive feedback loop. Further still, the KO shows potentially suppressed mitochondrial biogenesis (through Pgc1a analysis), similar oxidative phosphorylation (through OCRs), increased glycolysis (through ECARs), and increased PKA signaling (through cAMP assays), yet their exact connections to the PTH1r-Zfp467 signaling pathway have yet to be investigated.

The objective of the present study was to synthesize porous, biodegradable poly (D, l- lactide-co-glycolide) PLGA-B-HA (Bovine hydroxyapatite) composite and evaluate the effect of ceramic content on bone marrow cell differentiation in vitro. A macroporous biodegradable PLGA-B-HA composite with the pore size varying from 0.1 to 1000?? and a highly interconnected structure was fabricated using the freeze-drying/lyophilization technique. A pilot study was done to determine the effects of B-HA on to the osteoblast function. The main study was done to determine the effect of the increase in B-HA concentration on to the mesenchymal stem cell differentiation. Morphological characteristics of the composites were analyzed using FTIR and SEM/EDX analysis. The composites were seeded with neonatal rat calvarial osteoblasts (NRCO). The polymer: ceramic ratio in this study was 35%:65%. For comparison parallel experiments involving pure HA-200 discs were performed. SEM results indicated a higher proliferation and mineralization on PLGA-B-HA composites than pure HA discs. In addition, we evaluated the in vitro characteristics of PLGA-B-HA composites with varying ratios, i.e., 1:1, 1:2 and 1:3, seeded with rat marrow cells. FTIR indicated an increase in the area under the ceramic peak as ceramic concentration was increased. In addition, the average roughness values increased in the order of 1:3 andgt; 1:2 andgt; 1:1. Both compressive strength and modulus of 1:1 were significantly higher than 1:2 and 1:3 PLGA-B-HA composites. No significant difference in compressive modulli and strengths could be observed for 1:2 and 1:3 PLGA-B-HA composites. Cellular activity was determined by measuring AP activity, total protein analysis and osteocalcin concentration. Evaluation of alkaline phosphatase activity showed bone cells attached to 1:3 (PLGA-B-HA) expressed significantly higher alkaline phosphatase as compared to 1:1 and 1:2 PLGA-B-HA composites. In addition, cells seeded on to 1:3 composites secreted significantly higher osteocalcin and at a relatively short time period as compared to the other samples. Corrosion studies (ICP) and pH values indicate minimal difference in the concentration of Ca and P and pH in tissue culture media for all the samples at the end of all time periods. Hence we conclude that an increase in the ceramic concentration stimulated mesenchymal stem cell differentiation thereby promoting osteogenesis.