Genetic and pharmacologic suppression of PPARγ enhances NELL-1-stimulated bone regeneration.

Recent investigations into mechanisms behind the development of osteoporosis suggest that suppressing PPARγ-mediated adipogenesis can improve bone formation and bone mineral density. In this study, we investigated a co-treatment strategy to enhance bone formation by combining NELL-1, an osteogenic molecule that has been extensively studied for its potential use as a therapeutic for osteoporosis, with two methods of PPARγ suppression. First, we suppressed PPARγ genetically using lentiviral PPARγ-shRNA in immunocompromised mice for a proof of concept. Second, we used a PPARγ antagonist to suppress PPARγ pharmacologically in immunocompetent senile osteopenic mice for clinical transability. We found that the co-treatment strategy significantly increased bone formation, increased the proliferation stage cell population, decreased late apoptosis of primary mouse BMSCs, and increased osteogenic marker mRNA levels in comparison to the single agent treatment groups. The addition of PPARγ suppression to NELL-1 therapy enhanced NELL-1's effects on bone formation by upregulating anabolic processes without altering NELL-1's inhibitory effects on osteoclastic and adipogenic activities. Our findings suggest that combining PPARγ suppression with therapeutic NELL-1 may be a viable method that can be further developed as a novel strategy to reverse bone loss and decrease marrow adiposity in age-related osteoporosis.

Peroxisome Proliferator-Activated Receptor-γ Knockdown Impairs Bone Morphogenetic Protein-2-Induced Critical-Size Bone Defect Repair.

The Food and Drug Administration-approved clinical dose (1.5 mg/mL) of bone morphogenetic protein-2 (BMP2) has been reported to induce significant adverse effects, including cyst-like adipose-infiltrated abnormal bone formation. These undesirable complications occur because of increased adipogenesis, at the expense of osteogenesis, through BMP2-mediated increases in the master regulatory gene for adipogenesis, peroxisome proliferator-activated receptor-γ (PPARγ). Inhibiting PPARγ during osteogenesis has been suggested to drive the differentiation of bone marrow stromal/stem cells toward an osteogenic, rather than an adipogenic, lineage. We demonstrate that knocking down PPARγ while concurrently administering BMP2 can reduce adipogenesis, but we found that it also impairs BMP2-induced osteogenesis and leads to bone nonunion in a mouse femoral segmental defect model. In addition, in vitro studies using the mouse bone marrow stromal cell line M2-10B4 and mouse primary bone marrow stromal cells confirmed that PPARγ knockdown inhibits BMP2-induced adipogenesis; attenuates BMP2-induced cell proliferation, migration, invasion, and osteogenesis; and escalates BMP2-induced cell apoptosis. More important, BMP receptor 2 and 1B expression was also significantly inhibited by the combined BMP2 and PPARγ knockdown treatment. These findings indicate that PPARγ is critical for BMP2-mediated osteogenesis during bone repair. Thus, uncoupling BMP2-mediated osteogenesis and adipogenesis using PPARγ inhibition to combat BMP2's adverse effects may not be feasible.

The Effects of Systemic Therapy of PEGylated NEL-Like Protein 1 (NELL-1) on Fracture Healing in Mice.

Fractures are common, with an incidence of 13.7 per 1000 adults annually. Systemic agents have been widely used for enhancing bone regeneration; however, the efficacy of these therapeutics for the management and prevention of fracture remains unclear. NEL-like protein 1 (NELL-1) is a potent pro-osteogenic cytokine that has been modified with polyethylene glycol (PEG)ylation [PEGylated NELL-1 (NELL-PEG)] to enhance its pharmacokinetics for systemic therapy. Our aim was to investigate the effects of systemic administration of NELL-PEG on fracture healing in mice and on overall bone properties in uninjured bones. Ten-week-old CD-1 mice were subjected to an open osteotomy of bilateral radii and treated with weekly injections of NELL-PEG or PEG phosphate-buffered saline as control. Systemic injection of NELL-PEG resulted in improved bone mineral density of the fracture site and accelerated callus union. After 4 weeks of treatment, mice treated with NELL-PEG exhibited substantially enhanced callus volume, callus mineralization, and biomechanical properties. NELL-PEG injection significantly augmented bone regeneration, as confirmed by high expression of bone turnover rate, bone formation rate, and mineral apposition rate. Consistently, the immunohistochemistry results also confirmed a high bone remodeling activity in the NELL-PEG-treated group. Our findings suggest that weekly injection of NELL-PEG may have the clinical potential to accelerate fracture union and enhance overall bone properties, which may help prevent subsequent fractures.

Efficacy of Intraperitoneal Administration of PEGylated NELL-1 for Bone Formation.

Systemically delivered NEL-like molecule-1 (NELL-1), a potent pro-osteogenic protein, promotes bone formation in healthy and osteoporotic mouse models. PEGylation of NELL-1 (NELL-PEG) increases the half-life of the protein in a mouse model without compromising its osteogenic potential, thereby improving its pharmacokinetics upon systemic delivery. This study consists of a twofold approach: a biodistribution test and an in vivo osteogenic potential test. The biodistribution test compared two commonly used administration methods for drug delivery other than intravenous-intraperitoneal (IP) and subcutaneous (SC)-to examine NELL-PEG biodistribution in mice. Compared to a single-dose SC injection (1.25 mg/kg), a single-dose IP administration yielded a higher protein uptake in the targeted bone sites. When the IP injection dose was doubled to 2.5 mg/kg, the protein remained in the femurs, tibias, and vertebrae for up to 72 h. Next, based on the results of the biodistribution study, IP administration was selected to further investigate the in vivo osteogenic effects of weekly NELL-PEG injection (q7d). In vivo, the IP administered NELL-PEG group showed significantly greater bone mineral density, bone volume fraction, and trabecular bone formation in the targeted bone sites compared to the phosphate-buffered saline control. In summary, weekly NELL-PEG injection via IP administration successfully enhanced the overall bone quality. These findings demonstrate that systemic delivery of NELL-PEG via IP administration may serve as an effective osteogenic therapy for preventing and treating osteoporosis.

Guidelines for Dual Energy X-Ray Absorptiometry Analysis of Trabecular Bone-Rich Regions in Mice: Improved Precision, Accuracy, and Sensitivity for Assessing Longitudinal Bone Changes.

Trabecular bone is frequently studied in osteoporosis research because changes in trabecular bone are the most common cause of osteoporotic fractures. Dual energy X-ray absorptiometry (DXA) analysis specific to trabecular bone-rich regions is crucial to longitudinal osteoporosis research. The purpose of this study is to define a novel method for accurately analyzing trabecular bone-rich regions in mice via DXA. This method will be utilized to analyze scans obtained from the International Space Station in an upcoming study of microgravity-induced bone loss. Thirty 12-week-old BALB/c mice were studied. The novel method was developed by preanalyzing trabecular bone-rich sites in the distal femur, proximal tibia, and lumbar vertebrae via high-resolution X-ray imaging followed by DXA and micro-computed tomography (micro-CT) analyses. The key DXA steps described by the novel method were (1) proper mouse positioning, (2) region of interest (ROI) sizing, and (3) ROI positioning. The precision of the new method was assessed by reliability tests and a 14-week longitudinal study. The bone mineral content (BMC) data from DXA was then compared to the BMC data from micro-CT to assess accuracy. Bone mineral density (BMD) intra-class correlation coefficients of the new method ranging from 0.743 to 0.945 and Levene's test showing that there was significantly lower variances of data generated by new method both verified its consistency. By new method, a Bland-Altman plot displayed good agreement between DXA BMC and micro-CT BMC for all sites and they were strongly correlated at the distal femur and proximal tibia (r=0.846, p<0.01; r=0.879, p<0.01, respectively). The results suggest that the novel method for site-specific analysis of trabecular bone-rich regions in mice via DXA yields more precise, accurate, and repeatable BMD measurements than the conventional method.

Pharmacokinetics and osteogenic potential of PEGylated NELL-1 in vivo after systemic administration.

Osteoporosis is a skeletal disorder attributable to an imbalance in osteoblast and osteoclast activity. NELL-1, a secretory protein that promotes osteogenesis while suppressing osteoclastic activity, holds potential as an osteoporosis therapy. Recently, we demonstrated that PEGylation of NELL-1 significantly improves its thermostability while preserving its bioactivity in vitro. However, the effect of PEGylation on the pharmacokinetics and osteogenic potential of NELL-1 in vivo have yet to be investigated. The present study demonstrated that PEGylation of NELL-1 significantly increases the elimination half-life time of the protein from 5.5 h to 15.5 h while distributing more than 2-3 times the amount of protein to bone tissues (femur, tibia, vertebrae, calvaria) in vivo when compared to naked NELL-1. In addition, microCT and DXA analyses demonstrated that systemic NELL-PEG therapy administered every 4 or 7 days significantly increases not only femoral and lumbar BMD and percent bone volume, but also new bone formation throughout the overall skeleton after four weeks of treatment. Furthermore, immunohistochemistry revealed increased osteocalcin expression, while TRAP staining showed reduced osteoclast numbers in NELL-PEG groups. Our findings suggest that the PEGylation technique presents a viable and promising approach to further develop NELL-1 into an effective systemic therapeutic for the treatment of osteoporosis.