Summary Posterior Hox genes (Hox9-13) are critical for patterning the limb skeleton along the proximodistal axis during embryonic development. Here we show that Hox11 paralogous genes, which developmentally pattern the zeugopod (radius/ulna and tibia/fibula), remain regionally expressed in the adult skeleton. Using Hoxa11eGFP reporter mice, we demonstrate expression exclusively in multi-potent mesenchymal stromal cells (MSCs) in the bone marrow of the adult zeugopod. Hox-positive cells express PDGFRα and CD51, are marked by LepR-Cre, exhibit CFU-F activity and tri-lineage differentiate in vitro. Loss of Hox11 function leads to fracture repair defects, including reduced cartilage formation and delayed ossification. Hox mutant cells are defective in osteoblastic and chondrogenic differentiation in tri-lineage differentiation experiments and these defects are zeugopod-specific. In the stylopod (humerus and femur) and sternum, BM-MSCs express other regionally restricted Hox genes and femur fractures heal normally in Hox11 mutants. Together, our data supports regional Hox expression and function in skeletal MSCs.

Intermittent parathyroid hormone (PTH) administration increases systemic and craniofacial bone mass. However, the effect of PTH therapy on healing of tooth extraction sites is unknown. The aims of this study were to determine the effect of PTH therapy on tooth extraction socket healing and to examine whether PTH intra-oral injection promotes healing. The mandibular first molars were extracted in rats, and subcutaneous PTH was administered intermittently for 7, 14, and 28 days. In a second study, maxillary second molars were extracted, and PTH was administered by either subcutaneous or intra-oral injection to determine the efficacy of intra-oral PTH administration. Healing was assessed by micro-computed tomography and histomorphometric analyses. PTH therapy accelerated the entire healing process and promoted both hard-and softtissue healing by increasing bone fill and connective tissue maturation. PTH therapy by intra-oral injection was as effective as subcutaneous injection in promoting tooth extraction socket healing. The findings suggest that PTH therapy promotes tooth extraction socket healing and that intra-oral injections can be used to administer PTH. A supplemental appendix to this article is published electronically only at http://jdr.sagepub.com/supplemental.

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.

Background Therapeutic augmentation of fracture site angiogenesis with Deferoxamine (DFO) has proven to increase vascularity, callus size and mineralization in long-bone fracture models. We posit that the addition of DFO would enhance pathological fracture healing in the setting of radiotherapy in a model where non-unions are the most common outcome. Methods Sprague-Dawley rats (n = 35) were divided into 3 groups. Fracture (Fx), radiated fracture (XFx) and radiated fracture + DFO (XFxDFO). Groups XFx and XFxDFO received a human equivalent dose of radiotherapy (7 Gy/day × 5 days = 35 Gy) 2 weeks prior to mandibular osteotomy and external fixation. The XFxDFO group received injections of DFO into the fracture callus after surgery. Following a 40-day healing period, mandibles were dissected, clinically assessed for bony-union, imaged with Micro-CT, and tension tested to failure. Results Compared to radiated fractures, metrics of callus size, mineralization and strength in DFO treated mandibles were significantly increased. These metrics were restored to a level demonstrating no statistical difference from control fractures. In addition we observed an increased rate of achieving bony unions in the XFxDFO treated group when compared to XFx (67% vs. 20% respectively). Conclusions Our data demonstrate near total restoration of callus size, mineralization, and biomechanical strength, as well as a 3-fold increase in the rate of union with the use of DFO. Our results suggest that the administration of DFO may have the potential for clinical translation as a new treatment paradigm for radiation induced pathologic fractures. Level of Evidence Animal study, not gradable for level of evidence.

Background: IL-6 signaling plays a role in immune and skeletal systems. Results: sIL-6R mediated PTH-dependent hematopoietic cell expansions and blocking sIL-6R reduced PTH anabolic actions in mice. Conclusion: sIL-6R is a mediator of PTH hematopoietic actions in marrow and anabolic actions in bone. Significance: Novel orphan sIL-6R functions support PTH actions in bone and bone marrow.

The processes that govern fracture repair rely on many mechanisms that recapitulate embryonic skeletal development. Hox genes are transcription factors that perform critical patterning functions in regional domains along the axial and limb skeleton during development. Much less is known about roles for these genes in the adult skeleton. We recently reported that Hox11 genes, which function in zeugopod development (radius/ulna and tibia/fibula), are also expressed in the adult zeugopod skeleton exclusively in PDGFRα+/CD51+/LepR+ mesenchymal stem/stromal cells (MSCs). In this study, we use a Hoxa11eGFP reporter allele and loss-of-function Hox11 alleles, and we show that Hox11 expression expands after zeugopod fracture injury, and that loss of Hox11 function results in defects in endochondral ossification and in the bone remodeling phase of repair. In Hox11 compound mutant fractures, early chondrocytes are specified but show defects in differentiation, leading to an overall deficit in the cartilage production. In the later stages of the repair process, the hard callus remains incompletely remodeled in mutants due, at least in part, to abnormal bone matrix organization. Overall, our data supports multiple roles for Hox11 genes following fracture injury in the adult skeleton.

The role of exercise in promoting bone health is typically attributed to increased mechanical loading, which induces functional adaptation. Recent evidence suggests that habitual aerobic exercise has influence at the cellular level as well. The effect of aerobic capacity on osteoblast-lineage cell differentiation and function as well as skeletal phenotype is unknown. Using a rat model of high-capacity and low-capacity runners (HCRs and LCRs, respectively), in which an intrinsic functional genomic difference in aerobic capacity exists between nontrained animals, this study evaluated the effects of aerobic capacity on measures of bone mass and strength as well as osteoblast activity following ovariectomy. The ovariectomized rat emulates the clinical features of the estrogen-depleted human skeleton and represents a valuable model for studying short-term upregulation of osteoblast activity. We hypothesized that intrinsically high aerobic capacity would augment osteoblast response, which would mitigate the deleterious effects of hormone withdrawal. Femora and tibiae were assessed by micro-computed tomography, mechanical testing, and dynamic histomorphometry. HCRs had enhanced femoral tissue mineral density and estimated elastic modulus relative to LCRs. At 4 weeks postovariectomy, HCRs demonstrated a more robust osteoblast response. Markers of bone formation were upregulated to a greater extent in HCRs than LCRs, suggesting a role for aerobic capacity in governing osteoblast activity. Results from this and future studies will help to identify the influence of cellular aerobic metabolism on bone health, which may lead to new strategies for targeting diseases of the skeleton.

ErbB2 expression in early breast cancer can predict tumor aggressiveness and clinical outcomes in large patient populations. Accurate assessment with physical biopsy and conventional pathology can be limited by tumor heterogeneity. We aim to demonstrate real-time optical sectioning using a near-infrared labeled ErbB2 peptide that generates tumor-specific contrast in human xenograft breast tumors in vivo. We used IRDye800CW as the fluorophore, validated performance characteristics for specific peptide binding to cells in vitro, and investigated peak peptide uptake in tumors using photoacoustic tomography. We performed real-time optical imaging using a handheld dual-axes confocal fluorescence endomicroscope that collects light off-axis to reduce tissue scattering for greater imaging depths. Optical sections in either the vertical or horizontal plane were collected with sub-cellular resolution. Also, we found significantly greater peptide binding to pre-clinical xenograft breast cancer in vivo and to human specimens of invasive ductal carcinoma that express ErbB2 ex vivo. We used a scrambled peptide for control. Peptide biodistribution showed high tumor uptake by comparison with other organs to support safety. This novel integrated imaging strategy is promising for visualizing ErbB2 expression in breast tumors and serve as an adjunct during surgery to improve diagnostic accuracy, identify tumor margins, and stage early cancers.