Altered Sox9 and FGF signaling gene expression in Aga2 OI mice negatively affects linear growth.

Osteogenesis imperfecta (OI), or brittle bone disease, is a disorder characterized by bone fragility and increased fracture incidence. All forms of OI also feature short stature, implying an effect on endochondral ossification. Using the Aga2+/- mouse, which has a mutation in type I collagen, we show an affected growth plate primarily due to a shortened proliferative zone. We used single-cell RNA-Seq analysis of tibial and femoral growth plate tissues to understand transcriptional consequences on growth plate cell types. We show that perichondrial cells, which express abundant type I procollagen, and growth plate chondrocytes, which were found to express low amounts of type I procollagen, had ER stress and dysregulation of the same unfolded protein response pathway as previously demonstrated in osteoblasts. Aga2+/- proliferating chondrocytes showed increased FGF and MAPK signaling, findings consistent with accelerated differentiation. There was also increased Sox9 expression throughout the growth plate, which is expected to accelerate early chondrocyte differentiation but reduce late hypertrophic differentiation. These data reveal that mutant type I collagen expression in OI has an impact on the cartilage growth plate. These effects on endochondral ossification indicate that OI is a biologically complex phenotype going beyond its known impacts on bone to negatively affect linear growth.

Identification of osteogenic progenitor cell-targeted peptides that augment bone formation.

Activation and migration of endogenous mesenchymal stromal cells (MSCs) are critical for bone regeneration. Here, we report a combinational peptide screening strategy for rapid discovery of ligands that not only bind strongly to osteogenic progenitor cells (OPCs) but also stimulate osteogenic cell Akt signaling in those OPCs. Two lead compounds are discovered, YLL3 and YLL8, both of which increase osteoprogenitor osteogenic differentiation in vitro. When given to normal or osteopenic mice, the compounds increase mineral apposition rate, bone formation, bone mass, and bone strength, as well as expedite fracture repair through stimulated endogenous osteogenesis. When covalently conjugated to alendronate, YLLs acquire an additional function resulting in a "tri-functional" compound that: (i) binds to OPCs, (ii) targets bone, and (iii) induces "pro-survival" signal. These bone-targeted, osteogenic peptides are well suited for current tissue-specific therapeutic paradigms to augment the endogenous osteogenic cells for bone regeneration and the treatment of bone loss.

Selective inhibition of progesterone receptor in osteochondral progenitor cells, but not in mature chondrocytes, modulated subchondral bone structures.

OBJECTIVE: The presence or relative proportion of progesterone nuclear receptors (PR) in different tissues may contribute to sexual dimorphism in these tissues. PR is expressed in chondrocytes, but its function is mostly unknown. We hypothesized that the PR may regulate chondrocyte metabolism and affect subchondral bone structure. METHODS: We utilized genetic fate mapping and immunohistochemistry to elucidate PR expression in and effect on cartilage. To define sex-dependent and chondrocyte-specific effects of the PR on subchondral bone, we selectively deleted PR in osteochondrogenic progenitor cells marked by Prx1 (Prx1; PRcKO) and Collagen 2 (Col2; PRcKO), or in matured chondrocytes marked by aggrecan (Acan; PRcKO) and evaluated subchondral bone structure at 4 months of age. Chondrocyte aging was monitored by anti-senescence marker p16INK4a, and MMP13, one of the Senescence-Associated Secretary Phenotype (SASP) components. RESULTS: Compared to wild-type (WT) mice, the female Prx1; PRcKO and the Col2; PRcKO mice had greater total subchondral bone volume and greater subchondral cortical bone thickness, with increased estimated subchondral bone stiffness and failure load in both female and male Col2; PRcKO mice. Moreover, Col2; PRcKO mice from both sexes had greater bone formation and bone strength at the femurs. In contrast, we did not observe any subchondral bone changes in Acan; PRcKO mice other than higher work-to-failure observed in the male Acan; PRcKO mice. Despite no detected difference in articular cartilage between the WT and the PR; chondrocyte conditional deletion mice, there were greater numbers of senescent chondrocytes and increased MMP13 expression, especially in the male mutant mice. CONCLUSION: These findings suggest that selective inhibition of PR in osteoprogenitor cells, but not in terminally differentiated chondrocytes, induced an increased subchondral bone phenotype and high estimated subchondral bone strength, which might be associated with the development of osteoarthritis in older age.

Sex dimorphic regulation of osteoprogenitor progesterone in bone stromal cells.

Increasing peak bone mass is a promising strategy to prevent osteoporosis. A mouse model of global progesterone receptor (PR) ablation showed increased bone mass through a sex-dependent mechanism. Cre-Lox recombination was used to generate a mouse model of osteoprogenitor-specific PR inactivation, which recapitulated the high bone mass phenotype seen in the PR global knockout mouse mode. In this work, we employed RNA sequencing analysis to evaluate sex-independent and sex-dependent differences in gene transcription of osteoprogenitors of wild-type and PR conditional knockout mice. PR deletion caused marked sex hormone-dependent changes in gene transcription in male mice as compared to wild-type controls. These transcriptional differences revealed dysregulation in pathways involving immunomodulation, osteoclasts, bone anabolism, extracellular matrix interaction and matrix interaction. These results identified many potential mechanisms that may explain our observed high bone mass phenotype with sex differences when PR was selectively deleted in the MSCs.

Acceleration of Fracture Healing by Overexpression of Basic Fibroblast Growth Factor in the Mesenchymal Stromal Cells.

In this study, we engineered mesenchymal stem cells (MSCs) to over-express basic fibroblast growth factor (bFGF) and evaluated its effects on fracture healing. Adipose-derived mouse MSCs were transduced to express bFGF and green fluorescence protein (ADSCbFGF -GFP). Closed-femoral fractures were performed with osterix-mCherry reporter mice of both sexes. The mice received 3 × 105 ADSCs transfected with control vector or bFGF via intramuscular injection within or around the fracture sites. Mice were euthanized at days 7, 14, and 35 to monitor MSC engraftment, osteogenic differentiation, callus formation, and bone strength. Compared to ADSC culture alone, ADSCbFGF increased bFGF expression and higher levels of bFGF and vascular endothelial growth factor (VEGF) in the culture supernatant for up to 14 days. ADSCbFGF treatment increased GFP-labeled MSCs at the fracture gaps and these cells were incorporated into the newly formed callus. quantitative reverse transcription polymerase chain reaction (qRT-PCR) from the callus revealed a 2- to 12-fold increase in the expression of genes associated with nervous system regeneration, angiogenesis, and matrix formation. Compared to the control, ADSCbFGF treatment increased VEGF expression at the periosteal region of the callus, remodeling of collagen into mineralized callus and bone strength. In summary, MSCbFGF accelerated fracture healing by increasing the production of growth factors that stimulated angiogenesis and differentiation of MSCs to osteoblasts that formed new bone and accelerated fracture repair. This novel treatment may reduce the time required for fracture healing. Stem Cells Translational Medicine 2017;6:1880-1893.

Sex-Dependent, Osteoblast Stage-Specific Effects of Progesterone Receptor on Bone Acquisition.

The role of the progesterone receptor (PR) in the regulation of sexual dimorphism in bone has yet to be determined. Here we utilized genetic fate mapping and Western blotting to demonstrate age-dependent PR expression in the mouse femoral metaphysis and diaphysis. To define sex-dependent and osteoblast stage-specific effects of PR on bone acquisition, we selectively deleted PR at different stages of osteoblast differentiation. We found that when Prx1-Cre mice were crossed with PR floxed mice to generate a mesenchymal stem cell (MSC) conditional KO model (Prx1; PRcKO), the mutant mice developed greater trabecular bone volume with higher mineral apposition rate and bone formation. This may be explained by increased number of MSCs and greater osteogenic potential, particularly in males. Age-related trabecular bone loss was similar between the Prx1; PRcKO mice and their WT littermates in both sexes. Hormone deficiency during the period of rapid bone growth induced rapid trabecular bone loss in both the WT and the Prx1; PRcKO mice in both sexes. No differences in trabecular bone mass was observed when PR was deleted in mature osteoblasts using osteocalcin-Cre (Bglap-Cre). Also, there were no differences in cortical bone mass in all three PRcKO mice. In conclusion, PR inactivation in early osteoprogenitor cells but not in mature osteoblasts influenced trabecular bone accrual in a sex-dependent manner. PR deletion in osteoblast lineage cells did not affect cortical bone mass. © 2017 American Society for Bone and Mineral Research.

A Novel Hybrid Compound LLP2A-Ale Both Prevented and Rescued the Osteoporotic Phenotype in a Mouse Model of Glucocorticoid-Induced Osteoporosis.

Prolonged glucocorticoid (GC) administration causes secondary osteoporosis (GIOP) and non-traumatic osteonecrosis. LLP2A-Ale is a novel bone-seeking compound that recruits mesenchymal stem cells to the bone surface, stimulates bone formation, and increases bone mass. The purpose of this study was to determine if treatment with LLP2A-Ale alone or in combination with parathyroid hormone (PTH) could prevent or treat GIOP in a mouse model. Four-month-old male Swiss-Webster mice were randomized to a prevention study with placebo, GC (day 1-28), and GC + LLP2A-Ale (IV, day 1) or a treatment study with placebo, GC (days 1-56), GC + LLP2A-Ale (IV, day 28), GC + PTH, and GC + LLP2A-Ale + PTH (days 28-56). Mice were killed on day 28 (prevention study) or on day 56 (treatment study). The study endpoints included bone mass, bone strength, serum markers of bone turnover (P1NP and CTX-I) and angiogenesis (VEGF-A), surface-based bone turnover, and blood vessel density. LLP2A-Ale prevented GC-induced bone loss and increased mechanical strength in the vertebral body (days 28 and 56) and femur (day 56). LLP2A-Ale, PTH, and LLP2A-Ale + PTH treatment significantly increased the mineralizing surface, bone formation rate, mineral apposition rate, double-labeled surface, and serum P1NP level on day 56. LLP2A-Ale and PTH treatment increased femoral blood vessel density and LLP2A-Ale increased serum VEGF-A on day 28. Therefore, LLP2A-Ale monotherapy could be a potential option to both prevent and treat GC-induced osteoporosis and bone fragility.

Improved Mobilization of Exogenous Mesenchymal Stem Cells to Bone for Fracture Healing and Sex Difference.

Mesenchymal stem cell (MSC) transplantation has been tested in animal and clinical fracture studies. We have developed a bone-seeking compound, LLP2A-Alendronate (LLP2A-Ale) that augments MSC homing to bone. The purpose of this study was to determine whether treatment with LLP2A-Ale or a combination of LLP2A-Ale and MSCs would accelerate bone healing in a mouse closed fracture model and if the effects are sex dependent. A right mid-femur fracture was induced in two-month-old osterix-mCherry (Osx-mCherry) male and female reporter mice. The mice were subsequently treated with placebo, LLP2A-Ale (500 µg/kg, IV), MSCs derived from wild-type female Osx-mCherry adipose tissue (ADSC, 3 x 105 , IV) or ADSC + LLP2A-Ale. In phosphate buffered saline-treated mice, females had higher systemic and surface-based bone formation than males. However, male mice formed a larger callus and had higher volumetric bone mineral density and bone strength than females. LLP2A-Ale treatment increased exogenous MSC homing to the fracture gaps, enhanced incorporation of these cells into callus formation, and stimulated endochondral bone formation. Additionally, higher engraftment of exogenous MSCs in fracture gaps seemed to contribute to overall fracture healing and improved bone strength. These effects were sex-independent. There was a sex-difference in the rate of fracture healing. ADSC and LLP2A-Ale combination treatment was superior to on callus formation, which was independent of sex. Increased mobilization of exogenous MSCs to fracture sites accelerated endochondral bone formation and enhanced bone tissue regeneration. Stem Cells 2016;34:2587-2600.

ß-Ecdysone Augments Peak Bone Mass in Mice of Both Sexes.

BACKGROUND: One of the strongest predictors for osteoporosis is peak bone mass. Interventions to augment peak bone mass have yet to be developed. ß-Ecdysone (ßEcd), a natural steroid-like compound produced by arthropods to initiate metamorphosis, is believed to have androgenic effects and so may be used to augment bone mass. QUESTIONS/PURPOSES: The purpose of this study was to use both male and female (1) gonadal-sufficient; and (2) -insufficient mice to investigate sex differences in terms of bone development and structure after ßEcd administration. METHODS: Two-month-old male and female Swiss-Webster mice were randomized to receive either vehicle or ßEcd (0.5 mg/kg) for 3 weeks. In a separate experiment to evaluate the effects of ßEcd on sex hormone-deficient mice, gonadectomy was performed in male (orchiectomy [ORX]) and female mice (ovariectomy [OVX]). Sham-operated and the ORX/OVX mice were then treated for 3 weeks with ßEcd. Primary endpoints for the study were trabecular bone structure and bone strength. RESULTS: In male mice, the trabecular bone volume was 0.18±0.02 in the placebo-treated (PL) and 0.23±0.02 in the ßEcd-treated group (p<0.05 versus PL); and 0.09±0.01 in the ORX group (p<0.05 versus PL) and 0.12±0.01 in the ORX+ßEcd group. Vertebral bone strength (maximum load) was 43±2 in PL and 51±1 in the ßEcd-treated group (p<0.05 versus PL); and 30±4 in the ORX group (p<0.05 versus PL) and 37±3 in the ORX+ßEcd group. In female mice, trabecular bone volume was 0.23±0.02 in PL and 0.26±0.02 in the ßEcd-treated group (p<0.05 versus PL); and 0.15±0.01 in the OVX group (p<0.05 versus PL) and 0.14±0.01 in the OVX+ßEcd group. Maximum load of the vertebrae was 45±2 in PL and 48±4 in the ßEcd-treated group; and 39±4 in the OVX group (p<0.05 versus PL) and 44±4 in the OVX+ßEcd group. CONCLUSIONS: These findings suggest the potential use of ßEcd in the augmentation of bone mass in growing male and female mice. It may also partially prevent the detrimental effects of gonadectomy on trabecular bone. CLINICAL RELEVANCE: Our results support the potential use of ßEcd or nature products that are rich in ßEcd to augment peak bone mass. ßEcd may differ from the other anabolic hormone treatments that may have severe side effects such as serious cardiac complications. However, its effects on humans remain to be determined.

Prevention of glucocorticoid induced bone changes with beta-ecdysone.

Beta-ecdysone (ßEcd) is a phytoecdysteroid found in the dry roots and seeds of the asteraceae and achyranthes plants, and is reported to increase osteogenesis in vitro. Since glucocorticoid (GC) excess is associated with a decrease in bone formation, the purpose of this study was to determine if treatment with ßEcd could prevent GC-induced osteoporosis. Two-month-old male Swiss-Webster mice (n=8-10/group) were randomized to either placebo or slow release prednisolone pellets (3.3mg/kg/day) and treated with vehicle control or ßEcd (0.5mg/kg/day) for 21days. GC treatment inhibited age-dependent trabecular gain and cortical bone expansion and this was accompanied by a 30-50% lower bone formation rate (BFR) at both the endosteal and periosteal surfaces. Mice treated with only ßEcd significantly increased bone formation on the endosteal and periosteal bone surfaces, and increased cortical bone mass were their controls to compare to GC alone. Concurrent treatment of ßEcd and GC completely prevented the GC-induced reduction in BFR, trabecular bone volume and partially prevented cortical bone loss. In vitro studies determined that ßEcd prevented the GC increase in autophagy of the bone marrow stromal cells as well as in whole bone. In summary, ßEcd prevented GC induced changes in bone formation, bone cell viability and bone mass. Additional studies are warranted of ßEcd for the treatment of GC induced bone loss.