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Large animal testing and clinical trials using bioengineered bladder for augmentation have revealed that large grafts fail due to insufficient blood supply. To address this critical issue, an in vivo staged implant strategy was developed and evaluated to create autologous, vascularized bioengineered bladder tissue with potential for clinical translation. Pig bladders were used to create acellular urinary bladder matrices (UBMs), which were implanted on the rectus abdominus muscles of rats and pigs to generate cellular and vascular grafts. Rectus-regenerated bladder grafts (rrBGs) were highly cellularized and contained an abundance of CD31-positive blood vessels, which were shown to be functional by perfusion studies. Muscle patterns within grafts showed increased smooth muscle formation over time and specifically within the detrusor compartment, with no evidence of striated muscle. Large, autologous rrBGs were transplanted to the pig bladder after partial cystectomy and compared to transplantation of control UBMs at 2 weeks and 3 months post-transplant. Functional, ink-perfused blood vessels were found in the central portion of all rrBGs at 2 weeks, while UBM grafts were significantly deteriorated, contracted and lacked central cellularization and vascularization. By 3 months, rrBGs had mature smooth muscle bundles and were morphologically similar to native bladder. This staged implantation technique allows for regeneration and harvest of large bladder grafts that are morphologically similar to native tissue with functional vessels capable of inosculating with host bladder vessels to provide quick perfusion to the central area of the large graft, thereby preventing early ischemia and contraction.

ObjectivePost-traumatic osteoarthritis (PTOA) is a degenerative joint disease initiated by injury. Early phase (0-7 days) treatments often include rest (unloading) and anti-inflammatory medications, but how those early interventions impact PTOA progression is unknown. We hypothesized that early unloading and anti-inflammatory treatment would diminish joint inflammation and slow PTOA progression.DesignMice were injured with non-invasive ACL rupture followed by hindlimb unloading (HLU) or normal cage activity (ground control: GC) for 7 days, after which all mice were allowed normal cage activity. HLU and GC mice were treated with daily celecoxib (CXB; 10mg/kg IP) or vehicle. Protease activity was evaluated using in vivo fluorescence imaging, osteophyte formation and epiphyseal trabecular bone were quantified using micro-computed tomography, and synovitis and articular cartilage were evaluated using whole-joint histology at 7, 14, 21, and 28 days post-injury.ResultsHLU significantly reduced protease activity (-22-30% compared to GC) and synovitis (-24-50% relative to GC) at day 7 post-injury (during unloading), but these differences were not maintained at later timepoints. Similarly, trabecular bone volume was partially preserved in HLU mice at during unloading (-14-15% BV/TV for HLU mice, -21-22% for GC mice relative to uninjured), but these differences were not maintained during reloading. Osteophyte volume was reduced by both HLU and CXB, but there was not an additive effect of these treatments (HLU:-46%, CXB:-30%, HLU+CXB:-35% relative to vehicle GC at day 28).ConclusionsThese data suggest that early unloading following joint injury can reduce inflammation and potentially slow PTOA progression.

The utility of nonsurgical, mechanical compression-based joint injury models to study osteoarthritis pathogenesis and treatments is increasing. Joint injury may be induced via cyclic compression loading or acute overloading to induce anterior cruciate ligament rupture. Models utilizing mechanical testing systems are highly repeatable, require little expertise, and result in a predictable onset of osteoarthritis-like pathology on a rapidly progressing timeline. In this chapter, we describe the procedures and equipment needed to perform mechanical compression-induced initiation of osteoarthritis in mice and rats.

Fracture induces systemic bone loss in mice and humans, and a first (index) fracture increases the risk of future fracture at any skeletal site more in men than women. The etiology of this sex difference is unknown, but fracture may induces a greater systemic bone loss response in males. Also sex differences in systemic muscle loss after fracture have not been examined. We investigated sex differences in systemic bone and muscle loss after transverse femur fracture in 3-month-old male and female C57BL/6J mice. Whole-body and regional bone mineral content and density (BMC and BMD), trabecular and cortical bone microstructure, muscle contractile force, muscle mass, and muscle fiber size were quantified at multiple time points post-fracture. Serum concentrations of inflammatory cytokines (IL-1s, IL-6, and TNF-α) were measured 1-day post-fracture. One day post-fracture, IL-6 and Il-1B were elevated in fracture mice of both sexes, but TNF-α was only elevated in male fracture mice. Fracture reduced BMC, BMD, and trabecular bone microstructural properties in both sexes 2 weeks post-fracture, but declines were greater in males. Muscle contractile force, mass, and fiber size decreased primarily in the fractured limb at 2 weeks post-fracture and females showed a trend towards greater muscle loss. Bone and muscle properties recovered by 6 weeks post-fracture. Overall, post-fracture systemic bone loss is greater in males, which may contribute to sex differences in subsequent fracture risk. In both sexes, muscle loss is primarily confined to the injured limb and fracture may induce greater inflammation in males. This article is protected by copyright. All rights reserved.

In Duchenne muscular dystrophy (DMD), a lack of functional dystrophin leads to myofiber instability and progressive muscle damage that results in fibrosis. While fibrosis is primarily characterized by an accumulation of extracellular matrix (ECM) components, there are changes in ECM architecture during fibrosis that relate more closely to functional muscle stiffness. One of these architectural changes in dystrophic muscle is collagen cross-linking, which has been shown to increase the passive muscle stiffness in models of fibrosis including the mdx mouse, a model of DMD. We tested whether the intraperitoneal injections of beta-aminopropionitrile (BAPN), an inhibitor of the cross-linking enzyme lysyl oxidase, would reduce collagen cross- linking and passive stiffness in young and adult mdx mice compared to saline-injected controls. We found no significant differences between BAPN treated and saline treated mice in collagen cross-linking and stiffness parameters. However, we observed that while collagen cross-linking and passive stiffness scaled positively in dystrophic muscles, collagen fiber alignment scaled with passive stiffness distinctly between muscles. We also observed that the dystrophic diaphragm showed the most dramatic fibrosis in terms of collagen content, cross-linking, and stiffness. Overall, we show that while BAPN was not effective at reducing collagen cross- linking, the positive association between collagen cross-linking and stiffness in dystrophic muscles still show cross-linking as a viable target for reducing passive muscle stiffness in DMD or other fibrotic muscle conditions.Key PointsBAPN did not reduce collagen cross-linking or passive stiffness in any muscleCollagen cross-links scaled with passive stiffness in dystrophic musclesThe mdx diaphragm showed the most dramatic fibrosis related to collagen content, cross- linking, and passive stiffnessCollagen fiber alignment scales with passive stiffness differently between muscles

Mechanical unloading causes rapid loss of bone structure and strength, which gradually recovers after resuming normal loading. However, it is not well established how this adaptation to unloading and reloading changes with age. Clinically, elderly patients are more prone to musculoskeletal injury and longer periods of bedrest, therefore it is important to understand how periods of disuse will affect overall skeletal health of aged subjects. Bone also undergoes an age-related decrease in osteocyte density, which may impair mechanoresponsiveness. In this study, we examined bone adaptation during unloading and subsequent reloading in mice. Specifically, we examined the differences in bone adaptation between young mice (3-month-old), old mice (18-month-old), and transgenic mice that exhibit diminished osteocyte density at a young age (3-month-old BCL-2 transgenic mice). Mice underwent 14days of hindlimb unloading followed by up to 14days of reloading. We analyzed trabecular and cortical bone structure in the femur, mechanical properties of the femoral cortical diaphysis, osteocyte density and cell death in cortical bone, and serum levels of inflammatory cytokines. We found that young mice lost ~10% cortical bone volume and 27-42% trabecular bone volume during unloading and early reloading, with modest recovery of metaphyseal trabecular bone and near total recovery of epiphyseal trabecular bone, but no recovery of cortical bone after 14days of reloading. Old mice lost 12-14% cortical bone volume and 35-50% trabecular bone volume during unloading and early reloading but had diminished recovery of trabecular bone during reloading and no recovery of cortical bone. In BCL-2 transgenic mice, no cortical bone loss was observed during unloading or reloading, but 28-31% trabecular bone loss occurred during unloading and early reloading, with little to no recovery during reloading. No significant differences in circulating inflammatory cytokine levels were observed due to unloading and reloading in any of the experimental groups. These results illustrate important differences in bone adaptation in older and osteocyte deficient mice, suggesting a possible period of vulnerability in skeletal health in older subjects during and following a period of disuse that may affect skeletal health in elderly patients.

Myocardial infarction (MI) and osteoporotic fracture are leading causes of morbidity and mortality, and epidemiological evidence linking their incidence suggests possible crosstalk. MI can exacerbate atherosclerosis through the sympathetic nervous system (SNS) activation and β3 adrenoreceptor-mediated release of hematopoietic stem cells, leading to monocytosis. We hypothesized that this same pathway initiates systemic bone loss following MI,since osteoclasts differentiate from monocytes. In this study, MI was created with left anterior descendingartery ligation in 12-week-old male mice (n = 24) randomized to β3 -adrenergic receptor (AR) antagonist (SR 59230A) treatment or no treatment for 10 days postoperatively. Additional mice (n = 21, treated and untreated) served as unoperated controls. Bone mineral density (BMD), bone mineral content (BMC), and body composition were quantified at baseline and 10 days post-MI using dual-energy x-ray absorptiometry; circulating monocyte levels were quantified and the L5 vertebral body and femur were analyzed with microcomputed tomography 10 days post-MI. We found that MI led to circulating monocyte levels increases, BMD and BMC decreases at the femur and lumbar spine in MI mice (-6.9% femur BMD, -3.5% lumbar BMD), and trabecular bone volume decreases in MI mice compared with control mice. β3 -AR antagonist treatment appeared to diminish the bone loss response (-5.3% femur BMD, -1.2% lumbar BMD), though these results were somewhat inconsistent. Clinical significance:These results suggest that MI leads to systemic bone loss, but that the SNS may not be a primary modulator of this response; bone loss and increased fracture risk may be important clinical comorbidities following MI or other ischemic injuries.

Joint trauma results in the production of inflammatory cytokines that stimulate the secretion of catabolic enzymes, which degrade articular cartilage. Molecular fragments of the degraded articular cartilage further stimulate inflammatory cytokine production, with this process eventually resulting in post-traumatic osteoarthritis (PTOA). The loss of matrix component aggrecan occurs early in the progression of PTOA and results in the loss of compressive stiffness in articular cartilage. Aggrecan is highly sulfated, associates with hyaluronic acid (HA), and supports the compressive stiffness in cartilage. Presented here, we conjugated the HA-binding peptide GAHWQFNALTVRGSG (GAH) to anionic nanoparticles (hNPs). Nanoparticles conjugated with roughly 19 GAH peptides, termed 19 GAH-hNP, bound to HA in solution and increased the dynamic viscosity by 94.1% compared to an HA solution treated with unconjugated hNPs. Moreover, treating aggrecan-depleted (AD) cartilage explants with 0.10 mg of 19 GAH-hNP restored the cartilage compressive stiffness to healthy levels six days after a single nanoparticle treatment. Treatment of AD cartilage with 0.10 mg of 19 GAH-hNP inhibited the degradation of articular cartilage. Treated AD cartilage had 409% more collagen type II and 598% more GAG content than untreated-AD explants. The 19 GAH-hNP therapeutic slowed ECM degradation in AD cartilage explants, restored the compressive stiffness of damaged cartilage, and showed promise as a localized treatment for PTOA.

The single best predictor of future fracture risk is a prior fracture at any skeletal site. However, the mechanisms underlying this relationship remain incompletely understood. Here we review three potential mechanisms underlying increased risk. An index fracture may reflect preexisting deficits in bone quality or impairments in physical function. Second, fracture may cause biomechanical changes that increase future fracture risk. Third, fracture induces a period of systemic bone loss that is never fully recovered. Further investigation into these mechanisms can inform treatments that prevent future fractures for individuals with a prior history of fracture.

In Duchenne muscular dystrophy (DMD), a lack of functional dystrophin leads to myofiber instability and progressive muscle damage that results in fibrosis. While fibrosis is primarily characterized by an accumulation of extracellular matrix (ECM) components, there are changes in ECM architecture during fibrosis that relate more closely to functional muscle stiffness. One of these architectural changes in dystrophic muscle is collagen cross-linking, which has been shown to increase the passive muscle stiffness in models of fibrosis including the mdx mouse, a model of DMD. We tested whether the intraperitoneal injections of beta-aminopropionitrile (BAPN), an inhibitor of the cross-linking enzyme lysyl oxidase, would reduce collagen cross-linking and passive stiffness in young and adult mdx mice compared to saline-injected controls. We found no significant differences between BAPN treated and saline treated mice in collagen cross-linking and stiffness parameters. However, we observed that while collagen cross-linking and passive stiffness scaled positively in dystrophic muscles, collagen fiber alignment scaled with passive stiffness distinctly between muscles. We also observed that the dystrophic diaphragm showed the most dramatic fibrosis in terms of collagen content, cross-linking, and stiffness. Overall, we show that while BAPN was not effective at reducing collagen cross-linking, the positive association between collagen cross-linking and stiffness in dystrophic muscles still show cross-linking as a viable target for reducing passive muscle stiffness in DMD or other fibrotic muscle conditions.

ObjectiveTo compare the early responses to joint injury in conventional and germ-free mice.DesignPost-traumatic osteoarthritis (PTOA) was induced using a non-invasive anterior cruciate ligament rupture model in 20-week old germ-free (GF) and conventional C57BL/6 mice. Injury was induced in the left knees of n=8 GF and n=10 conventional mice. To examine the effects of injury, n=5 GF and n=9 conventional naïve control mice were used. Mice were euthanized 7 days post-injury, followed by synovial fluid recovery for global metabolomic profiling and analysis of epiphyseal trabecular bone by micro-computed tomography (μCT). Global metabolomic profiling assessed metabolic differences in the joint response to injury between GF and conventional mice. Magnitude of trabecular bone volume loss measured using μCT assessed early OA progression in GF and conventional mice.ResultsμCT found that GF mice had significantly less trabecular bone loss compared to conventional mice, indicating that the GF status was protective against early OA changes in bone structure. Global metabolomic profiling showed that conventional mice had greater variability in their metabolic response to injury, and a more distinct joint metabolome compared to their corresponding controls. Furthermore, differences in the response to injury in GF compared to conventional mice were linked to mouse metabolic pathways that regulate inflammation associated with the innate immune system.ConclusionsThese results suggest that the gut microbiota promote the development of PTOA during the acute phase following joint trauma possibly through the regulation of the innate immune system.

Osteoarthritis (OA) is a painful and debilitating disease characterized by the chronic and progressive degradation of articular cartilage. Post-traumatic OA (PTOA) is a secondary form of OA that develops in ~50% of cases of severe articular injury. Inflammation and re-occurring injury have been implicated as contributing to the progression of PTOA after the initial injury. However, there is very little known about external factors prior to injury that could affect the risk of PTOA development. To examine how the gut microbiome affects PTOA development we used a chronic antibiotic treatment regimen starting at weaning for six weeks prior to ACL rupture, in mice. A six-weeks post-injury histological examination showed more robust cartilage staining on the antibiotic (AB)-treated mice than the untreated controls (VEH), suggesting slower disease progression in AB cohorts. Injured joints also showed an increase in the presence of anti-inflammatory M2 macrophages in the AB group. Molecularly, the phenotype correlated with a significantly lower expression of inflammatory genes Tlr5, Ccl8, Cxcl13, and Foxo6 in the injured joints of AB-treated animals. Our results indicate that a reduced state of inflammation at the time of injury and a lower expression of Wnt signaling modulatory protein, Rspo1, caused by AB treatment can slow down or improve PTOA outcomes.

Inflammation following joint trauma contributes to cartilage degradation and progression of post traumatic osteoarthritis (PTOA). Therefore, drug delivery vehicles that deliver effective anti-inflammatory treatments have the potential to prevent PTOA. We have developed solid and hollow, thermoresponsive nanoparticles for the controlled release of our anti-inflammatory MK2-inhibiting (MK2i) peptide for intra-articular injection to halt inflammation that contributes to the advancement of PTOA. This system exploits the thermosensitive characteristic of N-isopropyl acrylamide (NIPAm) to transition phases when passing through its lower critical solution temperature (LCST). The nanoparticles (NPs) swell below the LCST and constrict above it. Non-crosslinked poly(NIPAm) (pNIPAm), held above its LCST, formed hydrophobic cores around which shells composed of NIPAm, degradable crosslinker N, N′-bis (acryloyl) cystamine (BAC), sulfated 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS), and acrylic acid (AAc) were polymerized. Removal of the non-crosslinked pNIPAm cores via diffusion produced thermosensitive, degradable nanoparticles with low density, or hollow, cores. The data presented here revealed low-density, termed hollow, nanoparticles (hNPs) load and release significantly more MK2i than solid nanoparticles (sNPs). Furthermore, drug loading below the LCST of NIPAm results in roughly 2.5 times more therapeutic encapsulation compared to loading particles in their constricted state. Hollow nanoparticles increase drug loading compared to solid nanoparticles, are taken up into chondrocytes within 24 h, cleared from the cells within 6 days, significantly decrease the secretion of the proinflammatory cytokine IL-6, and, via intra-articular injection, are successfully delivered into the joint space of rats. The peptide loaded nanoparticles provide a reproducible platform for intra-articular delivery of therapeutics.

Myocardial infarction (MI) and osteoporotic fracture (Fx) are leading causes of morbidity and mortality worldwide, and there is epidemiological evidence linking their incidence, suggesting possible crosstalk. MI can exacerbate underlying atherosclerosis through sympathetic nervous system activation and β3 adrenoreceptor-mediated release of hematopoietic stem cells (HSCs), leading to monocytosis. We hypothesized that this same pathway may initiate systemic bone loss following MI, since osteoclasts differentiate from monocytes. In this study, MI was performed in 12-week-old male mice (n=24), and mice were randomized to treatment with a β3-adrenergic receptor antagonist (SR 59230A) or no treatment for 10 days post-operatively; additional mice (n=21, treated and untreated) served as un-operated controls. Bone mineral density (BMD), bone mineral content (BMC), and body composition were quantified at baseline and 10 days post-MI using DXA; at 10 days post-MI circulating monocyte and neutrophil levels were quantified, and the L5 vertebral body and femur were analyzed with micro-computed tomography. We found that MI led to increases in circulating monocyte and neutrophil levels, but contrary to our initial hypothesis, β3-antagonist treatment further increased circulating monocytes compared to untreated mice. BMD and BMC of MI mice decreased at the femur and lumbar spine (−6.9% femur BMD, -3.5% lumbar BMD); β3-antagonist treatment diminished this bone loss response (−5.3% femur BMD, -1.2% lumbar BMD). Similarly, trabecular bone volume was decreased in MI mice compared to control mice, and this bone loss was partially attenuated by β3-antagonist treatment. These results suggest that MI leads to bone loss at both the axial and appendicular skeleton, and that the sympathetic nervous system may be a modulator of this response. This study is the first to mechanistically show bone loss following an ischemic injury; these findings suggest that bone loss and increased fracture risk may be important clinical co-morbidities following MI or other ischemic injuries.

Aging and injury are two major risk factors for osteoarthritis (OA). Yet, very little is known about how aging and injury interact and contribute to OA pathogenesis. In the present study, we examined age- and injury-related molecular changes in mouse knee joints that could contribute to OA. Using RNA-seq, first we profiled the knee joint transcriptome of 10-week-old, 62-week-old, and 95-week-old mice and found that the expression of several inflammatory-response related genes increased as a result of aging, whereas the expression of several genes involved in cartilage metabolism decreased with age. To determine how aging impacts post-traumatic arthritis (PTOA) development, the right knee joints of 10-week-old and 62-week-old mice were injured using a non-invasive tibial compression injury model and injury-induced structural and molecular changes were assessed. At six-week post-injury, 62-week-old mice displayed significantly more cartilage degeneration and osteophyte formation compared with young mice. Although both age groups elicited similar transcriptional responses to injury, 62-week-old mice had higher activation of inflammatory cytokines than 10-week-old mice, whereas cartilage/bone metabolism genes had higher expression in 10-week-old mice, suggesting that the differential expression of these genes might contribute to the differences in PTOA severity observed between these age groups.

Osteoarthritis (OA) is a debilitating and painful disease characterized by the progressive loss of articular cartilage. Post‐traumatic osteoarthritis (PTOA) is an injury‐induced type of OA that persists in an asymptomatic phase for years before it becomes diagnosed in ~50% of injured individuals. Although PTOA is not classified as an inflammatory disease, it has been suggested that inflammation could be a major driver of PTOA development. Here we examined whether a state of systemic inflammation induced by lipopolysaccharide (LPS) administration 5‐days before injury would modulate PTOA outcomes. RNA‐seq analysis at 1‐day post‐injury followed by micro‐computed tomography (μCT) and histology characterization at 6 weeks post‐injury revealed that LPS administration causes more severe PTOA phenotypes. These phenotypes included significantly higher loss of cartilage and subchondral bone volume. Gene expression analysis showed that LPS alone induced a large cohort of inflammatory genes previously shown to be elevated in synovial M1 macrophages of rheumatoid arthritis (RA) patients, suggesting that systemic LPS produces synovitis. This synovitis was sufficient to promote PTOA in MRL/MpJ mice, a strain previously shown to be resistant to PTOA. The synovium of LPS‐treated injured joints displayed an increase in cellularity, and immunohistological examination confirmed that this increase was in part attributable to an elevation in type 1 macrophages. LPS induced the expression of Tlr7 and Tlr8 in both injured and uninjured joints, genes known to be elevated in RA. We conclude that inflammation before injury is an important risk factor for the development of PTOA and that correlating patient serum endotoxin levels or their state of systemic inflammation with PTOA progression may help develop new, effective treatments to lower the rate of PTOA in injured individuals. © 2020 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.

Patients with anterior cruciate ligament (ACL) rupture are two times as likely to develop posttraumatic osteoarthritis (PTOA). Annually, there are ∼900,000 knee injuries in the United States, which account for ∼12% of all osteoarthritis (OA) cases. PTOA leads to reduced physical activity, deconditioning of the musculoskeletal system, and in severe cases requires joint replacement to restore function. Therefore, treatments that would prevent cartilage degradation post-injury would provide attractive alternatives to surgery. Sclerostin (Sost), a Wnt antagonist and a potent negative regulator of bone formation, has recently been implicated in regulating chondrocyte function in OA. To determine whether elevated levels of Sost play a protective role in PTOA, we examined the progression of OA using a noninvasive tibial compression overload model in SOST transgenic (SOSTTG ) and knockout (Sost-/- ) mice. Here we report that SOSTTG mice develop moderate OA and display significantly less advanced PTOA phenotype at 16 weeks post-injury compared with wild-type (WT) controls and Sost-/- . In addition, SOSTTG built ∼50% and ∼65% less osteophyte volume than WT and Sost-/- , respectively. Quantification of metalloproteinase (MMP) activity showed that SOSTTG had ∼2-fold less MMP activation than WT or Sost-/- , and this was supported by a significant reduction in MMP2/3 protein levels, suggesting that elevated levels of SOST inhibit the activity of proteolytic enzymes known to degrade articular cartilage matrix. Furthermore, intra-articular administration of recombinant Sost protein, immediately post-injury, also significantly decreased MMP activity levels relative to PBS-treated controls, and Sost activation in response to injury was TNFα and NF-κB dependent. These results provide in vivo evidence that sclerostin functions as a protective molecule immediately after joint injury to prevent cartilage degradation. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.

Background Context The basivertebral foramen (BF), located in the middle posterior wall of the vertebral body, may induce local weakness and contribute to the formation of a retropulsed bone fragment (RBF) in thoracolumbar burst fracture (TLBF). We hypothesize that the mechanism of TLBF is related to the BF. Purpose This study aimed to clarify the relationship between RBFs and the BF in TLBFs, and to explain the results using biomechanical experiments and micro-computed tomography (micro-CT). Study Design A comprehensive research involving clinical radiology, micro-CT, and biomechanical experiments on cadaveric spines was carried out. Patient Sample A total of 162 consecutive patients diagnosed with TLBF with RBFs, drawn from 256 patients who had reported accidents or injuries to their thoracolumbar spine, comprised the patient sample. Outcome Measures Dimensions and location of the RBFs in relation to the BF were the outcome measures. Materials and Methods Computed tomography reconstruction imaging was used to measure the dimensions and location of RBFs in 162 patients (length, height, width of RBF and vertebral body). Furthermore, micro-CT scans were obtained of 10 cadaveric spines. Each vertebral body was divided into three layers (superior, middle, and inferior), and each layer was divided further into nine regions (R1–R9). Microarchitecture parameters were calculated from micro-CT scans, including bone volume fraction (BV/TV), connectivity (Conn.D), trabecular number (Tb.N), trabecular thickness (Tb.Th), and bone mineral density (BMD). Differences were analyzed between regions and layers. Burst fractures were simulated on cadaveric spines to explore the fracture line location and test the relationship between RBFs and BF. Results Retropulsed bone fragment width was usually one-third of the width of the vertebral body, whereas RBF length and height were approximately half of the corresponding vertebral body dimensions. Measures of trabecular bone quality were generally lowest in those central and superior regions of the vertebral body which are adjacent to the BF and which are most affected by burst fracture. In simulated TLBFs, the fracture line went across the vertex or upper surface of the BF. Conclusions The most vulnerable regions in the vertebral body lie within or just superior to the BF. The central MR2 region in particular is at risk of fracture and RBF formation.

Category: Midfoot/Forefoot, Trauma Introduction/Purpose: Lisfranc injuries occurring between the medial cuneiform and base of the 2nd metatarsal require anatomic fixation. Suture button and screws are standard techniques for fixation, but the screw may decrease physiologic motion, whereas suture buttons may cause increased soft tissue irritation and iatrogenic cartilage damage. Potential benefits of the InternalBrace include physiologic motion, decreased iatrogenic damage, collagen ingrowth, limited bony erosion and decreased soft tissue irritation. In light of these potential benefits, no studies have investigated the biomechanical properties of the InternalBrace in a Lisfranc injury model. However, it is unknown whether there is significant difference in the biomechanical properties of the IB compared to the screw, or SB during load to failure, and cyclical loading. Methods: Three groups of sawbones were fixed together with either a 3.5 mm screw, SB, or IB, composed of a curved button, fibertape, and 4.75 mm biotenodesis screw. Sawbone constructs were held in a mechanical testing system (Model 809, MTS Systems Corp, Minneapolis MN). The first three groups of 10 were loaded in axial tension at 0.5mm/sec until failure to determine load-displacement data. Yield, stiffness, ultimate strength (US), yield energy, post-yield energy and ultimate strength energy were calculated. Three more groups of 8 constructs were loaded in-vitro at cyclical physiologic loads until displacement of 1.5 mm occurred. Constructs were first loaded for 10,000 cycles at 69 N (estimate for 50% body weight or assisted walking). Surviving specimens were loaded at 138 N (normal walk) for an additional 10,000 cycles and then 207 N (jog) for an additional 10,000 cycles. Displacement was recorded. The biomechanical properties were then compared between groups. Results: When loaded in axial tension at 0.5mm/sec until failure, the screw was found to be the stiffest construct (2,240 N/mm), while the InternalBrace (200 N/mm) was stiffer than the suture button (133 N/mm). Qualitatively, the InternalBrace was also found to hold load more consistently and for larger displacement prior to failure when compared to the suture button. Cyclic loading was performed with 10,000 cycles of 69 N, 138 N, and 207 N. The screw had the greatest resistance to fatigue. The InternalBrace maintained stiffness as well or better than the suture button, but the fatigue life was shorter than that of the suture button. Conclusion: To our knowledge, the biomechanical properties of the IB have not been compared to screw and SB for ligamentous lisfranc injuries. This study gives valuable information about the mechanical integrity of InternalBrace and supports continued use. However, further studies are warranted before making conclusions regarding early weight bearing.

A history of prior fracture is the most reliable indicator of prospective fracture risk. Increased fracture risk is not confined to the region of the prior fracture, but is operant at all skeletal sites, providing strong evidence of systemic bone loss after fracture. Animal and human studies suggest that systemic bone loss begins shortly after fracture and persists for several years in humans. In fact, bone quantity and bone quality may never fully return to their pre-fracture levels, especially in older subjects, demonstrating a need for improved understanding of the mechanisms leading to systemic bone loss after fracture in order to reduce subsequent fracture risk. Although the process remains incompletely understood, mechanical unloading (disuse), systemic inflammation, and hormones that control calcium homeostasis may all contribute to systemic bone loss. Additionally, individual factors can potentially affect the magnitude and time course of systemic bone loss and recovery. The magnitude of systemic bone loss correlates positively with injury severity and age. Men may also experience greater bone loss or less recovery than women after fracture. This review details the current understanding of systemic bone loss following fracture, including possible underlying mechanisms and individual factors that may affect this injury response.

Knee injuries cause structural damage and acute inflammation that initiates the development of post-traumatic osteoarthritis (PTOA). NADPH oxidase 4 (Nox4), a member of a family of enzymes that generates reactive oxygen species (ROS), plays a pivotal role in normal development of the musculoskeletal system, but may increase ROS production to harmful levels after joint injury. The role of ROS in both normal joint homeostasis and injury is poorly understood, but inhibition of excessive ROS production by Nox4 after joint injury could be protective to the joint, decreasing oxidative stress, and initiation of PTOA. Knee injuries were simulated using inflammatory cytokines in cultured primary human chondrocytes and a non-invasive mouse model of PTOA in C57BL/6N and Nox4 knockout mice. There is an acute decrease in Nox4 activity within 24 h after injury in both systems, followed by a subsequent sustained low-level increase, a novel finding not seen in any other system. Inhibition of Nox4 activity by GKT137831 was protective against early structural changes after non-invasive knee injury in a mouse model. Nox4 knockout mice had significant differences in structural and mechanical properties of bone, providing further evidence for the role of Nox4 in development of joint tissues and biochemical response after joint injury. Nox4 plays a significant role in the acute phase after joint injury, and targeted inhibition of inflammation caused by Nox4 may be protective against early joint changes in the pathogenesis of PTOA. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2429-2436, 2019.

IMPACT: Our research would be the first therapeutic to both prevent and treat osteoarthritis - helping 27 millions U.S. citizens alone immediately. OBJECTIVES/GOALS: Our objective is to conjugate hyaluronic acid binding peptides (HABP) to anionic hollow nanoparticle (hNP), and allowing the HABP-hNP complex to penetrate into osteoarthritic cartilage, bind to exposed HA, prevent further degradation, and restore the compressive strength of articular cartilage. METHODS/STUDY POPULATION: N-isopropyl acrylamide, 2-acrylamindo-2-methyl-1-propanesulfonic acid, N,N’-bis(acryoyl)cystamine, and Acrylic Acid, in fluorescent batches rhodamine b isothiocyanate (RBITC), were polymerized via precipitation reaction. HA binding peptide, GAHWQFNALTVRGSG-Hydrazide (GAH-Hyd), was covalently bonded to the hNP using DMTMM chemistry. The reaction was halted by diluting the solution 10:1 with milliQ water and purified using tangential flow filtration. The dynamic viscosity of the six treatments were analyzed in a 70 kDa HA. Using a rheometer (Discovery HR-3) with a 20 mm parallel plate geometry, TA Instruments, New Castle, DE), a frequency sweep (0.01 -1000 Hz, 2.512 Pa) was conducted to measure the storage modulus of each solution. RESULTS/ANTICIPATED RESULTS: GAH-Hyd was successfully conjugated to the surface of the hNP and zeta-potential shows a significant increase in surface charge from -21.41 mV for unconjugated hNP to -8.94 mV for 65 GAH conjugated hNP, confirming conjugation. The hNPs need 65 ±10 GAH per nanoparticle to significantly bind to HA, shown by increasing the dynamic viscosity of the solution. The minimum concentration of 65 GAH-hNP required to significantly bind to HA is 313 µM. These data from our study display the ability to functionalized the surface of polymeric hNPs with site specific peptides and their ability to bind to diseased tissue. We expect the GAH-hNP system will restore the compressive strength of OA cartilage and prevent further HA degradation in ex vivo aggrecan depleted cartilage plugs. DISCUSSION/SIGNIFICANCE OF FINDINGS: Binding to exposed HA within the ECM of cartilage protects the HA from further degradation, halting the progression of OA. 65 GAH-hNP binds to HA at a 313 µM. Our system can be translated and used to treat a multitude of conditions by conjugating tissue specific peptides to the surface of our hNPs and delivery site specific therapeutics to diseases tissue.

We previously showed that after femur fracture, mice lose bone at distant skeletal sites, including the lumbar vertebrae. This bone loss may increase the risk of subsequent vertebral fractures, particularly if bone is lost from high-strain bone regions, which are most commonly found adjacent to the superior and inferior endplates of the vertebral body. To determine regional bone loss from the lumbar spine following femur fracture, we evaluated the cranial, center, and caudal portions of the L5 vertebral bodies of Young (3 month-old) and Middle-Aged (12 month-old) female C57BL/6 mice two weeks after a transverse femur fractures compared to Young and Middle-Aged uninjured control mice. We hypothesized that greater bone loss would be observed in the cranial and caudal regions than in the center region in both Young and Middle-Aged mice. Trabecular and cortical bone microstructure were evaluated using micro-computed tomography, and osteoclast number and eroded surface were evaluated histologically. In Young Mice, fracture led to decreased trabecular and cortical bone microstructure primarily in the cranial and caudal regions, but not the center region, while Middle-Aged mice demonstrated decreases in trabecular bone in all regions, but did not exhibit any changes in cortical bone microstructure after fracture. No significant differences in osteoclast number or eroded surface were observed at this time point. These data suggest that bone loss following fracture in Young Mice is concentrated in areas that contain a large amount of high-strain tissue, whereas bone loss in Middle-Aged mice is less region-dependent and is restricted to the trabecular bone compartment. These results illustrate how systemic bone loss after fracture could lead to decreases in vertebral strength, and how distinct regional patterns and age-dependent differences in bone loss may differentially affect vertebral fracture risk.

Recently, autophagy-related proteins were shown to regulate osteoclast mediated bone resorption, a critical process in autoimmune diseases such as rheumatoid arthritis. However, the role of autophagy-linked FYVE containing protein, WDFY3, in osteoclast biology remains elusive. WDFY3 is a master regulator in selective autophagy for clearing ubiquitinated protein aggregates and has been linked with rheumatoid arthritis. Herein, we used a series of WDFY3 transgenic mice (Wdfy3(lacZ) and Wdfy3(loxP)) to investigate the function of WDFY3 in osteoclast development and function. Our data demonstrate that WDFY3 is highly expressed at the growth plate of neonatal mice and is expressed in osteoclasts invitro cultures. Osteoclasts derived from WDFY3 conditional knockout mice (Wdfy3(loxP/loxP)-LysM-Cre(+)) demonstrated increased osteoclast differentiation as evidenced by higher number and enlarged size of TRAP(+) multinucleated cells. Western blot analysis also revealed up-regulation of TRAF6 and an increase in RANKL-induced NF-κB signaling in WDFY3-deficient bone marrow-derived macrophages compared to wild type cultures. Consistent with these observations WDFY3-deficient cells also demonstrated an increase in osteoclast-related genes Ctsk, Acp5, Mmp9 and an increase of dentine resorption in invitro assays. Importantly, invivo RANKL gene transfer exacerbated bone loss in WDFY3 conditional knockout mice, as evidenced by elevated serum TRAP, CTX-I and micro-CT analysis of distal femurs compared to wild type littermates. Taken together, our data highlight a novel role for WDFY3 in osteoclast development and function, which can be exploited for the treatment of musculoskeletal diseases.

Osteophytes are a typical radiographic finding during osteoarthritis (OA). Osteophytes are thought to form in response to joint instability; however, the time course of osteophyte formation and joint stabilization following joint injury is not well understood. In this study, we investigated the time course of osteophyte formation and joint function following non-invasive knee injury in mice. We hypothesized that initial joint instability following knee injury would initiate osteophyte formation, which would in turn restabilize the joint and reduce range of motion (ROM). Mice were subjected to non-invasive anterior cruciate ligament (ACL) rupture. Anterior-posterior (AP) joint laxity, ROM, and chondro/osteophyte formation were measured immediately after injury, and 2, 4, 6, and 8 weeks post-injury. Chondrophyte areas at each time point were measured with histology, while mineralized osteophyte volume was determined using micro-computed tomography. Immediately after ACL rupture, AP joint laxity was increased twofold, while ROM was increased 11.7%. Chondrophytes appeared by 2 weeks post-injury, corresponding with a decrease in AP joint laxity and ROM. By 8 weeks post-injury, considerable osteophyte formation was observed around the joint, AP joint laxity returned to control levels, and joint ROM decreased to 61% of control values. These data support a role for chondro/osteophytes in joint restabilization after injury, and provide crucial insight into the time course and pathology of joint degeneration during OA development in the mouse. Statement of Clinical Significance: Results from this study increase understanding of conditions leading to osteophyte formation.© 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:466-473, 2017.

Development of osteoarthritis commonly involves degeneration of epiphyseal trabecular bone. In previous studies, we observed 30-44% loss of epiphyseal trabecular bone (BV/TV) from the distal femur within 1 week following non-invasive knee injury in mice. Mechanical unloading (disuse) may contribute to this bone loss; however, it is unclear to what extent the injured limb is unloaded following injury, and whether disuse can fully account for the observed magnitude of bone loss. In this study, we investigated the contribution of mechanical unloading to trabecular bone changes observed following non-invasive knee injury in mice (female C57BL/6N). We investigated changes in gait during treadmill walking, and changes in voluntary activity level using Open Field analysis at 4, 14, 28, and 42 days post-injury. We also quantified epiphyseal trabecular bone using μCT and weighed lower-limb muscles to quantify atrophy following knee injury in both ground control and hindlimb unloaded (HLU) mice. Gait analysis revealed a slightly altered stride pattern in the injured limb, with a decreased stance phase and increased swing phase. However, Open Field analysis revealed no differences in voluntary movement between injured and sham mice at any time point. Both knee injury and HLU resulted in comparable magnitudes of trabecular bone loss; however, HLU resulted in considerably more muscle loss than knee injury, suggesting another mechanism contributing to bone loss following injury. Altogether, these data suggest that mechanical unloading likely contributes to trabecular bone loss following non-invasive knee injury, but the magnitude of this bone loss cannot be fully explained by disuse. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1680-1687, 2016.