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Mechanical loading is an important aspect of post-surgical fracture care. The timing of load application relative to the injury event may differentially regulate repair depending on the stage of healing. Here, we used a novel mechanobiological model of cortical defect repair that offers several advantages including its technical simplicity and spatially confined repair program, making effects of both physical and biological interventions more easily assessed. Using this model, we showed that daily loading (5N peak load, 2Hz, 60 cycles, 4 consecutive days) during hematoma consolidation and inflammation disrupted the injury site and activated cartilage formation on the periosteal surface adjacent to the defect. We also showed that daily loading during the matrix deposition phase enhanced both bone and cartilage formation at the defect site, while loading during the remodeling phase resulted in an enlarged woven bone regenerate. All loading regimens resulted in abundant cellular proliferation throughout the regenerate and fibrous tissue formation directly above the defect demonstrating that all phases of cortical defect healing are sensitive to physical stimulation. Stress was concentrated at the edges of the defect during exogenous loading, and finite element (FE)-modeled longitudinal strain (εzz) values along the anterior and posterior borders of the defect (~2200με) was an order of magnitude larger than strain values on the proximal and distal borders (~50-100με). It is concluded that loading during the early stages of repair may impede stabilization of the injury site important for early bone matrix deposition, whereas loading while matrix deposition and remodeling are ongoing may enhance stabilization through the formation of additional cartilage and bone.

Adult skeletal tissue responds to increasing mechanical stimulation by forming new bone to resist damage; however, aging can diminish mechanoresponsiveness and lead to bone loss and increased fracture risk. In pursuit of therapies to maintain healthy bone, we assessed age-associated changes in the periosteal osteoprogenitor cell population, and accompanying load-induced periosteal bone formation, cortical geometry, and trabecular microarchitecture. We hypothesized that aging mice have reduced osteogenic capacity in response to mechanical loading due to decreased osteoprogenitor cell numbers and reduced cellular proliferation. Right tibiae of 16-wk-old adult and 52-wk-old aged female, n=6, C57BL/6 mice were subjected to axial compression (5N, 2Hz, 60 cycles, 3 days/week) for two weeks, and cortical and trabecular bone assessed with micro-computed tomography and dynamic histomorphometry. Baseline periosteal cell number and nuclear morphology at tibial midshaft were quantified using two-photon imaging of intact DAPI-stained tibiae in adult and aged mice. In addition, acute periosteal responses were assessed in a separate group of adult and aged mice subjected to four consecutive days of strain-matched axial compression (1400mE, 120 cycles, 2Hz). In this group, cell proliferation and osteoprogenitor number were quantified in the periosteum by immunohistochemical staining of proliferating cell nuclear antigen and paired-related homeobox1 (Prrx1, osteoprogenitor marker) on transverse midshaft sections from loaded and non-loaded tibiae. A Student's t-test determined significance at p<0.05. Two-weeks of loading resulted in increasing trends in Ct.Ar, Imax, Imin, pMOI, and measures of trabecular microarchitecture, though not significant in either age group. Age-related differences were detected in load-induced periosteal bone formation (p<0.05), which increased significantly in adults but not in aged. At baseline, adult mice exhibit greater periosteal cell number, nuclear area, and circularity (all p<0.01) than aged mice (Fig 1a). In the acutely loaded groups, both adult and aged tibiae responded with similar increases in periosteal cell proliferation, 140% and 111%, respectively (Fig 1b); however, Prrx1-positive cells are reduced by a third in aged midshaft tibiae (Fig 1c). We conclude that the aged-related attenuation of load-induced periosteal bone formationmay be explained, in part, by decreased Prrx1+ osteoprogenitors residing in periosteal stem cell niche. (Figure Presented)

Mesenchymal stem cell (MSC) differentiation can be manipulated by nanotopographic interface providing a unique strategy to engineering stem cell therapy and circumventing complex cellular reprogramming. However, our understanding of the nanotopographic-mechanosensitive properties of MSCs and the underlying biophysical linkage of the nanotopography-engineered stem cell to directed commitment remains elusive. Here, we show that osteogenic differentiation of human MSCs (hMSCs) can be largely promoted using our nanoengineered topographic glass substrates in the absence of dexamethasone, a key exogenous factor for osteogenesis induction. We demonstrate that hMSCs sense and respond to surface nanotopography, through modulation of adhesion, cytoskeleton tension, and nuclear activation of TAZ (transcriptional coactivator with PDZ-binding motif), a transcriptional modulator of hMSCs. Our findings demonstrate the potential of nanotopographic surfaces as noninvasive tools to advance cell-based therapies for bone engineering and highlight the origin of biophysical response of hMSC to nanotopography.

The Wnt pathway is a new target in bone therapeutic space. WNT proteins are potent stem cell activators and pro-osteogenic agents. Here, we gained insights into the molecular and cellular mechanisms responsible for liposome-reconstituted recombinant human WNT3A protein (L-WNT3A) efficacy to treat osteonecrotic defects. Skeletal injuries were coupled with cryoablation to create non-healing osteonecrotic defects in the diaphysis of the murine long bones. To replicate clinical therapy, osteonecrotic defects were treated with autologous bone graft, which were simulated by using bone graft material from syngeneic ACTB-eGFP-expressing mice. Control osteonecrotic defects received autografts alone; test sites received autografts treated ex vivo with L-WNT3A. In vivo microCT monitored healing over time and immunohistochemistry were used to track the fate of donor cells and assess their capacity to repair osteonecrotic defects according to age and WNT activation status. Collectively, analyses demonstrated that cells from the autograft directly contributed to repair of an osteonecrotic lesion, but this contribution diminished as the age of the donor increased. Pre-treating autografts from aged animals with L-WNT3A restored osteogenic capacity to autografts back to levels observed in autografts from young animals. A WNT therapeutic approach may therefore have utility in the treatment of osteonecrosis, especially in aged patients.

INTRODUCTION: Bone fractures reduce quality of life, increase mortality, and are associated with an economic burden of $19 billion/year in the US [1]. A common complication of fractures is non-union resulting from poor vascularization [2]. Angiogenesis, the process by which new vessels emerge from existing vessels, is critical for normal skeletal development, homeostasis, and repair. At the fracture site, there is close proximity between cells of the osteoblastic lineage (OBs) and vessel-forming endothelial cells (ECs) [3], suggesting co-regulation of these cell types. Recent studies have shown that osterix (Osx)-positive osteoblast precursors migrate to the site of injury at the same time as vascular infiltration [4]. Adding to the complexity, progenitors from the periosteum (Ps) and endosteum (Es) have shown differences in their differentiation capacity during bone repair [5]. It is still unclear from which envelopes vessels originate, and which signals control their directional infiltration and remodeling during the bone repair process. At the cellular level, it is unclear which cell types and signals orchestrate OB and EC progenitor function. In this study, we used an intramembranous bone repair model and a threedimensional (3D), high-resolution imaging platform to quantify the spatiotemporal profile of vascular and osteogenic progenitor distribution during healing. This experimental setup allowed quantification of 3D vessel geometry, the distribution of osteoprogenitors in relation to vessels, and OB/EC progenitor proliferation at the site of repair. We hypothesize that osteogenic and angiogenic cells co-localize during the repair process and that angiogenic and osteogenic progenitor cells at the periosteal and endosteal surfaces show differential proliferative activity. METHODS: Bone repair model: Bilateral tibial monocortical defects (1.0 mm, circular) were created in female adult C57BL/6 mice, which were sacrificed at post-surgical day (PSD) 2, 3, 5, 7, 10 (N = 4 at each time point). This model produces a consistent repair process [6]. The NYU School of Medicine Institutional Animal Care and Use Committee approved all procedures. Preparation and immunostaining of thick sections: Freshly dissected tibiae were fixed in 4% paraformaldehyde at 4 C for 4 hours and decalcified in 0.5 M EDTA, pH 7.5 at 4 C for 36 hours. Samples were cryoembedded, thick-sectioned, and stained with primary antibodies against osteoprogenitor marker Osx (sc-22536-R, Santa Cruz, 1:100), proliferation marker Ki-67 (M-19, Santa Cruz, 1:100), and endothelial marker EMCN (V.7C7, Santa Cruz, 1:100). After three washes with PBS, the samples were stained with Alexa Fluor secondary antibodies (1:400). Nuclei were stained with DAPI (0.5 mug/ml). Confocal and two-photon imaging: 3D fluorescent images were taken with a Zeiss LSM710 microscope with both confocal and two-photon imaging capabilities. A 20x water-immersion objective (1.7 mm working distance) was used for image capture. Z-stacks of 100 mum in height were captured at 512x512 pixel resolution with a 2 mum z-step. The two-photon mode was used to image collagen (420-465 nm filter), DAPI (465-500 nm filter), Osterix (Osx) (520-555 nm filter), and Ki-67 (575-645 nm filter), all using 840 nm excitation from a Mai Tai Sapphire laser. The confocal mode was used to image endothelial cell marker endomucin (EMCN) (650-735 nm filter) with 633 nm HeNe laser excitation. 3D image analysis: Z-stacks obtained from two-photon microscopy were rendered in 3D using Bitplane Imaris v7.4.1 and Fiji. To assess the spatial distribution of cellular expression of angiogenic, osteogenic, and proliferation markers, four volumes of interest (VOI) of 260x230x52 mum were selected at the: (1) rim of the defect near the intact cortical bone, (2) center of the defect, (3) periosteal surface, and (3) endosteal surface bordering the intact cortical shell. Vessel surface area and volume, cellular localization, and OB and EC cell numbers were quantified at the defect site in 3D space. RESULTS: At the defect rim and center, vessel surface area and volume (data not shown) followed a similar increasing trend from PSD 2 to 5, reaching a plateau from PSD 5 to 7 (Fig. 1B, C), with surface area greater at the center vs. rim of the defect (Fig. 1C). Early during repair (PSD 2-3), the number of Osx+ cells was higher in the Es vs. Ps (Fig. 1D), and higher at the center vs. the rim of the defect (Fig. 1E). On PSD 2, the Es vs. Ps exhibited more Osx+ cells that were Ki-67+ (65+/-18% and 5+/-5%, respectively) (Fig. 1F) and that were co-localized with vessels (60+/-20% and 11+/-11%, respectively) (Fig. 1G). DISCUSSION: Blood vessel formation and skeletal development are coupled during growth [7]; and recent evidence suggests that the vasculature network acts as a template for bone deposition [8]. Our data showing that approximately 60% of Osx+ cells at the Es co-localize with vessels during the early phases of healing, suggesting that angio-osteo coupling is recapitulated during bone repair. In addition, that greater numbers of Osx+ cells were observed at the Es vs. Ps, and that this difference was maintained throughout the healing process, which suggests that the Es cellular niche is a primary contributor of osteogenic cells to repair. Previous studies show that both the Ps and Es are important sources of osteoblasts during healing [5], and our data further suggest that there are differences in the relative contribution of cells from these disparate niches. In intact bone, Osx+ cells preferentially colocalize with vessels that (1) highly express both EMCN and CD31 (Type H vessels), and (2) are primarily situated along the endosteal surface in mouse long bone [3]. Our data showing that a greater percentage of Osx+ cells at the Es vs. Ps co-localize with EMCN+ during the early repair process supports the idea that this Osx+ cell-rich Es perivascular niche is an important contributor to bone repair. Interestingly, we found that over the course of healing, the center of the defect is more highly vascularized than the rim of the defect. Since we expect vessels originating from the Es and Ps to pass through this region to vascularize the regenerate, these data suggest that vessels from either the marrow or soft tissues above the defect are the first to infiltrate the defect. Vessel origin and mechanisms underlying cross-talk and co-regulation between osteoprogenitors and endothelial cells during regenerative angiogenesis and defect repair are currently under investigation

INTRODUCTION: According to a recent study from the Center of Disease Control and Prevention, 1 in every 10 Americans aged 12 and older reported chronic use of antidepressants. Chronic use of serotonin re-uptake inhibitors (SSRI) has been linked to impaired bone mineral accrual during skeletal development and osteoporosis [1,2]. We investigated the effect of fluoxetine, the most commonly prescribed SSRI in the U.S., on the complex program of bone regeneration in two disparate models of fracture repair in mice, followed by a thorough assessment of the in vitro mineralization capacity of primary osteoprogenitor cells (OPCs). We hypothesized that fluoxetine exerts a negative effect on osteoblast proliferation and differentiation during the process of fracture repair, resulting in a less mineralized and weaker bony callus. METHODS: Twelve week-old C57BL/6J mice were used following the IACUC guidelines at our institution. Fluoxetine was delivered in the drinking water at 10 mg/kg/day dose during the 3 weeks before surgeries to simulate chronic SSRI use [3]. Bone fracture repair through endochondral ossification was analyzed using a well-established femur fracture model stabilized with an intramedullary rod. Fracture callus was examined at 14 and 28 days. Intramembranous ossification was analyzed using a 1-mm monocortical tibial defect model. Here, injuries were allowed to heal for 7 or 14 days. Samples were subjected to microCT analysis, histomorphometry, TRAP and ALP histochemistry and immunolabeling for osteocalcin and runx2. A set of fractured femurs at d28 was subjected to 4-point biomechanical bending tests. All mice were continuously treated with fluoxetine during the repair period, except for a group of mice in which we aimed at understanding how discontinuation of the SSRI at the time of fracture would affect fracture healing (Fig. 1A). For the in vitro studies, bone marrow stromal cells were cultured in growth media alone or in presence of 5, 10 or 20 microM fluoxetine, with and without serotonin. Cell proliferation was measured using a BrdU colorimetric assay and apoptotic cells were detected by TUNEL labeling. bMSCs were also cultured in osteogenic differentiation media alone or with the aforementioned fluoxetine concentrations. Mineralization activity was analyzed by alizarin red staining and ALP activity and the expression of osteogenic markers was evaluated by qRT-PCR. An additional set of in vitro experiments was carried out with serotonin supplementation at 50mM in growth media or osteogenic media. Cell proliferation and osteogenic differentiation were examined. Student's t test with Holm-Sidak correction were used to quantify differences described in this study. Error bars represent standard deviation. An asterisk symbol (*) denotes a p value of less than 0.05. RESULTS: Fluoxetine-treated mice developed a normal cartilaginous callus at 14 days after fracture. At 28 days, the fluoxetine-treated animals demonstrated a significantly smaller and biomechanically weaker bony callus (Fig. 1B). In order to further dissect the mechanism that resulted in a smaller osseous regenerate, we studied the healing process of monocortical tibial defects as an intramembranous model of bone healing, which confirmed a direct effect of fluoxetine on osteoblast differentiation and mineralization. In vitro studies established that fluoxetine treatment decreases osteogenic differentiation and mineralization and that this effect is serotonin-independent. Finally, in a translational approach, we tested whether cessation of the medication would result in restoration of the regenerative potential. Interestingly, histologic and microCT analysis revealed non-union formation in these animals with fibrous tissue interposition within the callus (Fig. 1). DISCUSSION: In summary, our current study shows that chronic fluoxetine treatment negatively affects bone healing by inhibiting proliferation, osteoblast differentiation and mineralization. Data from this study and others provide strong evidence that chronic SSRI use leads to osteoporosis, which is associated with an increased fracture risk. In a translation arm of our study, we aimed at studying the effect of fluoxetine cessation at the time of fracture. In this group, we surprisingly encountered the consistent formation of non-unions with persistent fibrous tissue interposition. Further studies are now focusing at understanding this intriguing finding. (Figure Presented)

Chronic use of selective serotonin re-uptake inhibitors (SSRI) for the treatment of depression has been linked to osteoporosis. In this study, we investigated the effect of chronic SSRI use on fracture healing in two murine models of bone regeneration. First, we performed a comprehensive analysis of endochondral bone healing in a femur fracture model. C57/BL6 mice treated with fluoxetine, the most commonly prescribed SSRI, developed a normal cartilaginous soft-callus at 14 days after fracture and demonstrated a significantly smaller and biomechanically weaker bony hard-callus at 28 days. In order to further dissect the mechanism that resulted in a smaller bony regenerate, we used an intramembranous model of bone healing and revealed that fluoxetine treatment resulted in a significantly smaller bony callus at 7 and 14 days post-injury. In order to test whether the smaller bony regenerate following fluoxetine treatment was caused by an inhibition of osteogenic differentiation and/or mineralization, we employed in vitro experiments, which established that fluoxetine treatment decreases osteogenic differentiation and mineralization and that this effect is serotonin-independent. Finally, in a translational approach, we tested whether cessation of the medication would result in restoration of the regenerative potential. However, histologic and microCT analysis revealed non-union formation in these animals with fibrous tissue interposition within the callus. In conclusion, fluoxetine exerts a direct, inhibitory effect on osteoblast differentiation and mineralization, shown in two disparate murine models of bone repair. Discontinuation of the drug did not result in restoration of the healing potential, but rather led to complete arrest of the repair process. Besides the well-established effect of SSRIs on bone homeostasis, our study provides strong evidence that fluoxetine use negatively impacts fracture healing

Angiogenesis is a process by which new blood vessels emerge from existing vessels through endothelial cell sprouting, migration, proliferation, and tubule formation. Angiogenesis during skeletal growth, homeostasis and repair is a complex and incompletely understood process. As the skeleton adapts to mechanical loading, we hypothesized that mechanical stimulation regulates "osteo-angio" crosstalk in the context of angiogenesis. We showed that conditioned media (CM) from osteoblasts exposed to fluid shear stress enhanced endothelial cell proliferation and migration, but not tubule formation, relative to CM from static cultures. Endothelial cell sprouting was studied using a dual-channel collagen gel-based microfluidic device that mimics vessel geometry. Static CM enhanced endothelial cell sprouting frequency, whereas loaded CM significantly enhanced both frequency and length. Both sprouting frequency and length were significantly enhanced in response to factors released from osteoblasts exposed to fluid shear stress in an adjacent channel. Osteoblasts released angiogenic factors, of which osteopontin, PDGF-AA, IGBP-2, MCP-1, and Pentraxin-3 were upregulated in response to mechanical loading. These data suggest that in vivo mechanical forces regulate angiogenesis in bone by modulating "osteo-angio" crosstalk through release of paracrine factors, which we term "osteokines".