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Articular cartilage injuries have a limited healing capacity and, due to inflammatory and catabolic activities, often experience progressive degeneration towards osteoarthritis. Current repair techniques generally provide short-term symptomatic relief; however, the regeneration of hyaline cartilage remains elusive, leaving both the repair tissue and surrounding healthy tissue susceptible to long-term wear. Therefore, methods to preserve cartilage following injury, especially from matrix loss and catabolism, are needed to delay, or even prevent, the deteriorative process. The goal of this study was to develop and evaluate a cartiage-penetrating hyaluronic-acid (HA) hydrogel to improve damaged cartilage biomechanics and prevent tissue degeneration. At time zero, the HA-based hydrogel provided a 46.5% increase in compressive modulus and a decrease in permeability after simulated degeneration of explants (collagenase application). Next, in a degenerative culture model (interleukin-1 β [IL-1β] for 2 weeks), hydrogel application prior to or midway through the culture mitigated detrimental changes to compressive modulus and permeability observed in non-treated explants. Furthermore, localized loss of proteoglycan was observed in degenerative culture conditions alone (non-treated), but hydrogel administration significantly improved the retention of matrix elements. Finally, NITEGE staining and gene expression analysis showed the ability of the HA gel to decrease chondrocyte catabolic activity. These results highlight the importance of reinforcing damaged cartilage with a biomaterial system to both preserve tissue content and reduce catabolism associated with injury and inflammation.

BackgroundThe healing environment within the posterolateral lumbar spine is one of the most clinically challenging bone healing environments in all of orthopaedics due to a lack of a contained space and the need to formde novobone in a non-bony environment. Our group has previously published data that suggests that sclerostin in expressed locally at high levels throughout the process of a spinal fusion mass maturing.MethodsWe computationally identified multiple FDA-approved drugs, as well as a novel drug, for their ability to disrupt the interaction between sclerostin and its receptor, LRP5/6. The drugs were tested in several in vitro biochemical assays using murine MC3T3 and MSCs, assessing their ability to (1) enhance canonical Wnt signaling, (2) promote the accumulation of the active (non-phosphorylated) form of β-catenin, and (3) enhance the intensity and signaling duration of BMP signaling. These drugs were then tested subcutaneously in rats as standalone osteoinductive agents on plain collagen sponges. Finally, the top drug candidates (called VA1 and C07) were tested in a rabbit posterolateral spine fusion model for their ability to achieve a successful fusion.ResultsWe show that by controlling GSK3b phosphorylation, these SMIs simultaneously enhance canonical Wnt signaling and potentiate canonical BMP signaling intensity and duration. We also demonstrate that the SMIs produce dose-dependent ectopic mineralizationin vivoin rats as well as significantly increase posterolateral spine fusion rates in rabbitsin vivo, both as standalone osteogenic drugs and in combination with autologous iliac crest bone graft.ConclusionsFew if any osteogenic small molecules have been described that possess the osteoinductive potency of BMP itself – that is, the ability to formde novoectopic bone as a standalone agent, particularly in stringentin vivoenvironments. Herein, we describe two such SMIs that have this unique ability and thus may have potential application as novel cost-effective biologic bone graft substitutes for achieving consistent spinal fusion or even or critical-sized fracture defects.FundingThis work was supported by a Veteran Affairs Career Development Award (IK2-BX003845).

The bone healing environment in the posterolateral spine following arthrodesis surgery is one of the most challenging in all of orthopedics and our understanding of the molecular signaling pathways mediating osteogenesis during spinal fusion is limited. In this study, the spatial and temporal expression pattern of Wnt signaling factors and inhibitors during spinal fusion was assessed for the first time. Bilateral posterolateral spine arthrodesis with autologous iliac crest bone graft was performed on 21 New Zealand White rabbits. At 1‐, 2‐, 3‐, 4‐, and 6‐weeks, the expression of sclerostin and a variety of canonical and noncanonical Wnts signaling factors was measured by qRT‐PCR from tissue separately collected from the transverse processes, the Outer and Inner Zones of the fusion mass, and the adjancent paraspinal muscle. Immunohistochemistry for sclerostin protein was also performed. Sclerostin and many Wnt factors, especially Wnt3a and Wnt5a, were found to have distinct spatial and temporal expression patterns. For example, harvesting ICBG caused a significant increase in sclerostin expression. Furthermore, the paraspinal muscle immediately adjacent to the transplanted ICBG also had significant increases in sclerostin expression at 3 weeks, suggesting new potential mechanisms for pseudarthroses following spinal arthrodesis. The presented work is the first description of the spatial and temporal expression of sclerostin and Wnt signaling factors in the developing spine fusion, filling an important knowledge gap in the basic biology of spinal fusion and potentially aiding in the development of novel biologics to increase spinal fusion rates., In this study, the temporal and spatial expression of sclerostin and Wnt signaling factors during a developing posterolateral spine fusion was determined for the first time. Most of the canonical and noncanonical Wnt factors and inhibitors that were assessed were found to have distinct spatial and temporal expression patterns and those patterns were found to differ between successful fusions and nonunions, suggesting new molecular mechanisms for the development of pseudarthroses following spinal arthrodesis. This fills an important knowledge gap in the basic biology of spinal fusion and may aid in the development of novel biologics to increase spinal fusion rates.

Context Progesterone (P) resistance is a hallmark of endometriosis, but the underlying mechanism(s) for loss of P sensitivity leading to lesion establishment remains poorly understood. Objective To evaluate the association between Notch-1 signaling activation and P resistance in the progression of endometriosis. Design Case control study; archived formalin-fixed, paraffin-embedded tissues. Setting University hospitals (United States, Taiwan). Patients Women with endometriosis; human endometrial stromal cell line (HESC). Intervention Eutopic endometria (EU) and ectopic lesions (ECs) were collected from surgically diagnosed patients. Archived tissue sections of EU and ECs were identified. HESCs were treated with N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) and valproic acid (VPA) to, respectively, suppress and induce Notch-1 activation. Outcome Measures Tissues were analyzed for Notch Intra-Cellular Domain 1 (NICD1) and progesterone receptor (PGR) protein expression by immunohistochemistry and for transcript levels of NICD1 target genes HES1, PGR, and PGR-B by quantitative reverse transcription polymerase chain reaction. DAPT- or VPA-treated HESCs with and without P cotreatment were evaluated for cell numbers and for PGR, HES1, and PGR target gene DKK1 transcript levels. Results Nuclear-localized stromal NICD1 protein levels were inversely associated with those of total PGR in EU and ECs. Stromal ECs displayed higher HES1 and lower total PGR and PGR-B transcript levels than EU. In HESCs, DAPT reduction of NICD1 decreased cell numbers and increased PGR transcript and nuclear PGR protein levels and, with P cotreatment, maintained P sensitivity. Conversely, VPA induction of NICD1 decreased PGR transcript levels and, with P cotreatment, abrogated P-induced DKK1 and maintained HES1 transcript levels. Conclusions Aberrant Notch-1 activation is associated with decreased PGR that contributes to P resistance in endometriosis., Notch-1 activation is associated with loss of progesterone receptor expression in ectopic lesions of endometriotic patients and with loss of progesterone sensitivity in endometrial stromal cells.

OBJECTIVES: Fracture is a traumatic event that can initiate systemic bone loss and lead to increased future risk of fracture, especially in the elderly. We sought to determine whether modulation of the gut microbiome using a daily probiotic supplement would dampen systemic inflammatory responses to fracture to accelerate bone repair and preserve distant bone sites. METHODS: Twelve-week-old male C57BL/6 J mice were randomly assigned to receive the probiotic Bifidobacterium adolescentis (1 × 10(8) CFU) via a daily oral gavage or PBS vehicle control. After two weeks, experimental mid-diaphyseal femoral fractures were created. Bones were analyzed using micro-CT at 10 and 18-days post-fracture. Midsagittal histological sections of fractured femora were stained with TRAP to quantify osteoclast number within the callus. RNA isolated from the small intestines was analyzed for gene expression of tight junction proteins. Serum levels of 20 cytokines were measured and analyzed using principal components analysis (PCA). RESULTS: Micro-CT of fractured limbs revealed a significant increase in callus bone volume (+28%) at day 18 post-fracture in mice receiving the B. adolescentis supplement. The total number of osteoclasts within the fracture callus at day 18 was also significantly increased (+158%) in the probiotic group suggesting more advanced remodeling of the callus. Micro-CT analyses of the L3 vertebral body, a distant bone site, revealed significantly higher trabecular bone volume at day 10 (+ 22%) and 18 (+11%) post-fracture in the probiotic group. PCA of serum cytokines revealed distinct clustering of the B. adolescentis and PBS groups indicating an anti-inflammatory effect by B. adolescentis. In the small intestines, probiotic supplementation significantly increased gene expression of tight junction proteins occludin, Jam3, ZO-1, and claudin-15 at both day 10 and day 18 post-fracture. CONCLUSIONS: Daily B. adolescentis supplementation not only accelerated fracture healing but also protected against vertebral body bone loss following fracture. These effects are likely a consequence of tightened intestinal barrier integrity, thus preventing systemic inflammation stemming from fracture-induced gut permeability. FUNDING SOURCES: Funding provided by Emory University and the Burroughs Wellcome Fund.

Cytosolic Malic Enzyme (ME1) provides reduced NADP for anabolism and maintenance of redox status. To examine the role of ME1 in tumor genesis of the gastrointestinal tract, we crossed mice having augmented intestinal epithelial expression of ME1 (ME1-Tg mice) with ApcMin/+ mice to obtain male ApcMin/+/ME1-Tg mice. ME1 protein levels were significantly greater within gut epithelium and adenomas of male ApcMin/+/ME1-Tg than ApcMin/+ mice. Male ApcMin/+/ME1-Tg mice had larger and greater numbers of adenomas in the small intestine (jejunum and ileum) than male ApcMin/+ mice. Male ApcMin/+/ME1-Tg mice exhibited greater small intestine crypt depth and villus length in non-adenoma regions, correspondent with increased KLF9 protein abundance in crypts and lamina propria. Small intestines of male ApcMin/+/ME1-Tg mice also had enhanced levels of Sp5 mRNA, suggesting Wnt/β-catenin pathway activation. A small molecule inhibitor of ME1 suppressed growth of human CRC cells in vitro, but had little effect on normal rat intestinal epithelial cells. Targeting of ME1 may add to the armentarium of therapies for cancers of the gastrointestinal tract.

Trichloroethylene (TCE) is a widespread environmental pollutant associated with immunotoxicity and autoimmune disease. Previous studies showed that mice exposed from gestation through early life demonstrated CD4(+) T cell alterations and autoimmune hepatitis. Determining the role of one environmental risk factor for any disease is complicated by the presence of other stressors. Based on its known effects, we hypothesized that developmental overnutrition in the form of a moderately high-fat diet (HFD) consisting of 40% kcal fat would exacerbate the immunotoxicity and autoimmune-promoting effects of low-level (

Metabolic stress in the early-life environment as a consequence of maternal overnutrition and obesity leads to an increased risk of adult metabolic syndrome in offspring. Given the greater risk for cancer development at a number of tissue sites for obese individuals, exposure of the highly developmentally “plastic” fetus and neonate to a dysregulated maternal endocrine milieu may similarly result in increased cancer susceptibility as adults. In rodent models, from which this concept has gained the most direct experimental support, the feedforward circuitry for cancer propensity appears to be generationally transmitted, in part, via epigenetic biochemical marks. Here, we review the current state of this nascent field with attention given to tissues that are likely impacted by the recent epidemic of maternal obesity. We highlight current thinking on underlying molecular mechanisms and discuss how such knowledge may be used to design interventional strategies for obese pregnant women to counter increased risk for malignancy in their offspring.