Buried filet of limb flaps for the reconstruction of forequarter and hindquarter amputations.

BACKGROUND AND OBJECTIVES: Forequarter and hindquarter amputations have traditionally been closed with local tissues, but the technique is plagued by a high rate of complication such as marginal necrosis, seroma, infection, and dehiscence. Filet of limb flaps have been used when local tissues are insufficient for closure, and despite their use in more extensive and complex wounds, outcomes seem to be better in these cases. Recognizing that filet of limb flaps not only serve to cover the wound, but also eliminate dead space, supplement at-risk and/or radiated tissue, pad underlying hard structures, and facilitate neuroma prevention with target muscle reinnervation, we have change our practice to utilize buried filet of limb flaps even when local tissues are technically "sufficient" to close the wound. The purpose of this article to organize and describe the ways in which buried filet-of-limb flaps can be used to achieve important and discrete surgical objectives in forequarter and hindquarter amputation, and to facilitate increased recognition of collaborative interdisciplinary opportunities for spare-part reconstruction. METHODS: Retrospective data from the medical records of seven patients, collected between 2010 to 2023 at our single tertiary referral center, were reviewed. This included all patients for whom a buried (or partially buried) filet of limb flap was attempted for forequarter or hindquarter amputation reconstruction. RESULTS: Five males and two females ranging 55 to 75 years of age, met the inclusion criteria. Three cases of forequarter amputation and four cases of hindquarter amputation were included. Six flaps were successfully transferred without major flap-related complications. The mean follow-up period was eight and a half months. CONCLUSION: Even when local tissues are technically "sufficient" to close forequarter and hindquarter amputation wounds, we have found buried filet of limb flaps to be useful in several ways. These include occupying dead space, providing double-layer coverage, padding hard structures, preventing neuromas, and reconstructing sacro- and spino-pelvic continuity. Our approach emphasizes interdisciplinary collaboration and highlights the potential advantages of buried filet of limb flaps in optimizing patient outcomes for complex limb amputations.

Did the Physical and Mental Health of Orthopaedic Patients Change After the Onset of the COVID-19 Pandemic?

BACKGROUND: The 2019 novel coronavirus (COVID-19) pandemic has been associated with poor mental health outcomes and widened health disparities in the United States. Given the inter-relationship between psychosocial factors and functional outcomes in orthopaedic surgery, it is important that we understand whether patients presenting for musculoskeletal care during the pandemic were associated with worse physical and mental health than before the pandemic's onset. QUESTIONS/PURPOSES: (1) Did patients seen for an initial visit by an orthopaedic provider during the COVID-19 pandemic demonstrate worse physical function, pain interference, depression, and/or anxiety than patients seen before the pandemic, as measured by the Patient-Reported Outcomes Measurement Information System (PROMIS) instrument? (2) During the COVID-19 pandemic, did patients living in areas with high levels of social deprivation demonstrate worse patterns of physical function, pain interference, depression, or anxiety on initial presentation to an orthopaedic provider than patients living in areas with low levels of social deprivation, compared with prepandemic PROMIS scores? METHODS: This was a retrospective, comparative study of new patient evaluations that occurred in the orthopaedic department at a large, urban tertiary care academic medical center. During the study period, PROMIS computer adaptive tests were routinely administered to patients at clinical visits. Between January 1, 2019, and December 31, 2019, we identified 26,989 new patients; we excluded 4% (1038 of 26,989) for being duplicates, 4% (1034 of 26,989) for having incomplete demographic data, 44% (11,925 of 26,989) for not having a nine-digit home ZIP Code recorded, and 5% (1332 of 26,989) for not completing all four PROMIS computer adaptive tests of interest. This left us with 11,660 patients in the "before COVID-19" cohort. Between January 1, 2021 and December 31, 2021, we identified 30,414 new patients; we excluded 5% (1554 of 30,414) for being duplicates, 4% (1142 of 30,414) for having incomplete demographic data, 41% (12,347 of 30,414) for not having a nine-digit home ZIP Code recorded, and 7% (2219 of 30,414) for not completing all four PROMIS computer adaptive tests of interest. This left us with 13,152 patients in the "during COVID-19" cohort. Nine-digit home ZIP Codes were used to determine patients' Area Deprivation Indexes, a neighborhood-level composite measure of social deprivation. To ensure that patients included in the study represented our overall patient population, we performed univariate analyses on available demographic and PROMIS data between patients included in the study and those excluded from the study, which revealed no differences (results not shown). In the before COVID-19 cohort, the mean age was 57 ± 16 years, 60% (7046 of 11,660) were women, 86% (10,079 of 11,660) were White non-Hispanic, and the mean national Area Deprivation Index percentile was 47 ± 25. In the during COVID-19 cohort, the mean age was 57 ± 16 years, 61% (8051 of 13,152) were women, 86% (11,333 of 13,152) were White non-Hispanic, and the mean national Area Deprivation Index percentile was 46 ± 25. The main outcome measures in this study were the PROMIS Physical Function ([PF], version 2.0), Pain Interference ([PI], version 1.1), Depression (version 1.0), and Anxiety (version 1.0). PROMIS scores follow a normal distribution with a mean t-score of 50 and a standard deviation of 10. Higher PROMIS PF scores indicate better self-reported physical capability, whereas higher PROMIS PI, Depression, and Anxiety scores indicate more difficulty managing pain, depression, and anxiety symptoms, respectively. Clinically meaningful differences in PROMIS scores between the cohorts were based on a minimum clinically important difference (MCID) threshold of 4 points. Multivariable linear regression models were created to determine whether presentation to an orthopaedic provider during the pandemic was associated with worse PROMIS scores than for patients who presented before the pandemic. Regression coefficients (ß) represent the estimated difference in PROMIS scores that would be expected for patients who presented during the pandemic compared with patients who presented before the pandemic, after adjusting for confounding variables. Regression coefficients were evaluated in the context of clinical importance and statistical significance. Regression coefficients equal to or greater than the MCID of 4 points were considered clinically important, whereas p values < 0.05 were considered statistically significant. RESULTS: We found no clinically important differences in baseline physical and mental health PROMIS scores between new patients who presented to an orthopaedic provider before the COVID-19 pandemic and those who presented during the COVID-19 pandemic (PROMIS PF: ß -0.2 [95% confidence interval -0.43 to 0.03]; p = 0.09; PROMIS PI: ß 0.06 [95% CI -0.13 to 0.25]; p = 0.57; PROMIS Depression: ß 0.09 [95% CI -0.14 to 0.33]; p = 0.44; PROMIS Anxiety: ß 0.58 [95% CI 0.33 to 0.84]; p < 0.001). Although patients from areas with high levels of social deprivation had worse PROMIS scores than patients from areas with low levels of social deprivation, patients from areas with high levels of social deprivation demonstrated no clinically important differences in PROMIS scores when groups before and during the pandemic were compared (PROMIS PF: ß -0.23 [95% CI -0.80 to 0.33]; p = 0.42; PROMIS PI: ß 0.18 [95% CI -0.31 to 0.67]; p = 0.47; PROMIS Depression: ß 0.42 [95% CI -0.26 to 1.09]; p = 0.23; PROMIS Anxiety: ß 0.84 [95% CI 0.16 to 1.52]; p = 0.02). CONCLUSION: Contrary to studies describing worse physical and mental health since the onset of the COVID-19 pandemic, we found no changes in the health status of orthopaedic patients on initial presentation to their provider. Although large-scale action to mitigate the effects of worsening physical or mental health of orthopaedic patients may not be needed at this time, orthopaedic providers should remain aware of the psychosocial needs of their patients and advocate on behalf of those who may benefit from intervention. Our study is limited in part to patients who had the self-agency to access specialty orthopaedic care, and therefore may underestimate the true changes in the physical or mental health status of all patients with musculoskeletal conditions. Future longitudinal studies evaluating the impact of specific COVID-19-related factors (for example, delays in medical care, social isolation, or financial loss) on orthopaedic outcomes may be helpful to prepare for future pandemics or natural disasters. LEVEL OF EVIDENCE: Level II, prognostic study.

FoxO1 is a crucial mediator of TGF-ß/TAK1 signaling and protects against osteoarthritis by maintaining articular cartilage homeostasis.

Transforming growth factor-ß (TGF-ß) signaling is a critical regulator for articular cartilage tissue maintenance and chondrocyte homeostasis. Nonetheless, the regulatory networks and downstream signaling pathways that govern the chondroprotective function of TGF-ß in the context of osteoarthritis (OA) are not fully defined. Recent studies reveal that mice with postnatal deletion of triple forkhead box class Os (FoxOs) (1, 3, and 4) spontaneously develop OA-like pathologies. The OA phenotype largely recapitulates that observed in mice with loss of TGF-ßR2. In the present study, we investigated the role of FoxOs as downstream mediators of TGF-ß signaling and define their role in articular cartilage homeostasis. Among the three FoxOs (1, 3, and 4), TGF-ß signaling exclusively regulates FoxO1 in a TGF-ß activated kinase 1 (TAK1)-dependent manner. Furthermore, FoxO1 was genetically ablated in mice in a tissue-specific manner in articular cartilage or overexpressed in adult cartilage immediately followed by meniscal/ligament injury (MLI). Histological and microcomputed tomography (micro-CT) analyses demonstrated that loss of FoxO1 postnatally in articular cartilage leads to OA-like pathologies, and gain of FoxO1 in adult cartilage has both preventative and therapeutic effects on surgically induced OA. Mechanistically, FoxO1 was found to maintain articular chondrocyte homeostasis through induction of anabolic and autophagy-related gene expressions. Importantly, overexpression of FoxO1 markedly rescued the OA phenotypes caused by deficiency in TGF-ß signaling in chondrocytes. Our study identifies that TGF-ß/TAK1-FoxO1 is a key signaling cascade in regulation of articular cartilage autophagy and homeostasis and is a potentially important therapeutic target for OA-like joint diseases.

Otto Aufranc Award: Identification of Key Molecular Players in the Progression of Hip Osteoarthritis Through Transcriptomes and Epigenetics.

BACKGROUND: This study aimed: (1) to compare the transcriptome profile of articular cartilage in cam-FAI (early stage) to advanced OA secondary to cam-FAI (late stage) and (2) to investigate epigenetic changes through the expression of DNA methylation enzymes DNMT3B, DNMT1, and DNMT3A and peroxisome proliferator-activated receptor gamma (PPARγ) in human cartilage samples during the progression of hip OA. METHODS: Full-thickness cartilage samples were collected from the anterolateral head-neck junction (impingement zone) of 22 patients (9 early-FAI and 13 late-FAI). RNA sequencing and in vitro cartilage cultures with histological analysis and immunohistochemistry staining for PPARγ and DNMT3B were performed. Target gene validation was confirmed with RT-PCR. RESULTS: Fifty genes and 42 pathways were identified differentially between early and late-FAI (fold change <-1.5 or >1.5, P < .01). PPARγ and DNMT3B were gradually suppressed with disease progression. Contrarily, disease progression induced expression of DNMT1/3A. CONCLUSION: By comparing comprehensive gene expression in early and late stage hip degeneration at the whole-genome level, distinct transcriptome profiles for early and late stage disease were identified along with key molecular contributors to the progression of hip OA. Preservation of endogenous PPARγ may have therapeutic potential to delay or prevent hip OA.

Ablation of Dnmt3b in chondrocytes suppresses cell maturation during embryonic development.

DNA methylation is a major mode of epigenetic regulation in the mammalian genome and is essential for embryonic development. The three catalytic DNA methyltransferases (Dnmts), Dnmt1, Dnmt3a, and Dnmt3b, catalyze the methylation of cytosine. Dnmt3b is highly expressed in chondrocytes and global knockout of Dnmt3b led to skeletal deformations and embryonic lethality, suggesting an essential role of Dnmt3b in endochondral bone formation. To further define the role of Dnmt3b in skeletal development, Dnmt3b was deleted in Col2 positive chondrocyte lineage cells. Both axial and appendicular skeletal size were reduced and bone mineralization was delayed in Col2Cre+ ;Dnmt3bf/f (Dnmt3bCol2 ) mice at E14.5 and E18.5. While Alcian Blue Hematoxylin/Orange G (ABH/OG) staining showed normal chondrocyte columns in control growth plates, the length of hypertrophic chondrocyte zone and type X collagen expression were decreased in E18.5 growth plates from Dnmt3bCol2 mice. TUNEL and PCNA staining demonstrated that the delay in chondrocyte maturation observed in the Dnmt3bCol2 growth plates was not secondary to altered chondrocyte apoptosis or proliferation. Complementary in vitro experiments were performed on primary sternal chondrocytes isolated from control and Dnmt3bCol2 mice. Gene expression studies confirmed delayed terminal maturation as Mmp13 and Col10a1 expression was down-regulated in Dnmt3bCol2 chondrocytes. In addition, alkaline phosphatase (ALP) and Alizarin Red staining confirmed that Dnmt3b deletion in chondrocytes delays in vitro chondrocyte hypertrophic differentiation and matrix mineralization. Mechanistically, Dnmt3b gene deletion resulted in decreased BMP signaling through reduction of Smad1 phosphorylation. These findings show that epigenetic factor, Dnmt3b is necessary for normal chondrocyte hypertrophic maturation and limb development.

BMP-TAK1 (MAP3K7) Induces Adipocyte Differentiation Through PPARγ Signaling.

BMPs have been shown to promote adipocyte differentiation through SMAD-dependent signaling. However, the role of TGF-ß-activated kinase 1 (TAK1) in non-canonical BMP signaling in adipocyte differentiation remains unclear. Here, we show that TAK1 inhibition decreases lipid accumulation in C3H10T1/2 mesenchymal stem cells (MSCs) induced to differentiate into adipocytes. TAK1 knockdown by siRNA further confirms that TAK1 is required for adipocyte commitment of MSCs. Additionally, TAK1 knockdown inhibits adipogenesis of 3T3-L1 preadipocytes, indicating that TAK1 is not only needed for adipocyte commitment, but also required for adipocyte terminal differentiation. Furthermore, TAK1 ablation specifically in adipocytes reduced high fat diet-induced weight gain and improved glucose tolerance. Mechanistically, we demonstrate that TAK1 is required for PPARγ transactivation and promotes PPARγ transcriptional activity synergistically with TAK1 binding protein 1 (TAB1). Collectively, our results demonstrate that TAK1 plays a critical role in BMP-mediated adipocyte differentiation. J. Cell. Biochem. 118: 204-210, 2017. © 2016 Wiley Periodicals, Inc.

PGE2 Receptor Subtype 1 (EP1) Regulates Mesenchymal Stromal Cell Osteogenic Differentiation by Modulating Cellular Energy Metabolism.

Mesenchymal stromal cells (MSCs) are multipotent progenitors capable of differentiation into osteoblasts and can potentially serve as a source for cell-based therapies for bone repair. Many factors have been shown to regulate MSC differentiation into the osteogenic lineage such as the Cyclooxygenase-2 (COX2)/Prostaglandin E2 (PGE2) signaling pathway that is critical for bone repair. PGE2 binds four different receptors EP1-4. While most studies focus on the role PGE2 receptors EP2 and EP4 in MSC differentiation, our study focuses on the less studied, receptor subtype 1 (EP1) in MSC function. Recent work from our laboratory showed that EP1-/- mice have enhanced fracture healing, stronger cortical bones, higher trabecular bone volume and increased in vivo bone formation, suggesting that EP1 is a negative regulator of bone formation. In this study, the regulation of MSC osteogenic differentiation by EP1 receptor was investigated using EP1 genetic deletion in EP1-/- mice. The data suggest that EP1 receptor functions to maintain MSCs in an undifferentiated state. Loss of the EP1 receptor changes MSC characteristics and permits stem cells to undergo more rapid osteogenic differentiation. Notably, our studies suggest that EP1 receptor regulates MSC differentiation by modulating MSC bioenergetics, preventing the shift to mitochondrial oxidative phosphorylation by maintaining high Hif1α activity. Loss of EP1 results in inactivation of Hif1α, increased oxygen consumption rate and thus increased osteoblast differentiation. J. Cell. Biochem. 118: 4383-4393, 2017. © 2017 Wiley Periodicals, Inc.

Inflammation and epigenetic regulation in osteoarthritis.

Osteoarthritis (OA) was once defined as a non-inflammatory arthropathy, but it is now well-recognized that there is a major inflammatory component to this disease. In addition to synovial cells, articular chondrocytes and other cells of diarthrodial joints are also known to express inflammatory mediators. It has been proposed that targeting inflammation pathways could be a promising strategy to treat OA. There have been many reports of cross-talk between inflammation and epigenetic factors in cartilage. Specifically, inflammatory mediators have been shown to regulate levels of enzymes that catalyze changes in DNA methylation and histone structure, as well as alter levels of non-coding RNAs. In addition, expression levels of a number of these epigenetic factors have been shown to be altered in OA, thereby suggesting potential interplay between inflammation and epigenetics in this disease. This review provides information on inflammatory pathways in arthritis and summarizes published research on how epigenetic regulators are affected by inflammation in chondrocytes. Furthermore, we discuss data showing how altered expression of some of these epigenetic factors can induce either catabolic or anti-catabolic effects in response to inflammatory signals. A better understanding of how inflammation affects epigenetic factors in OA may provide us with novel therapeutic strategies to treat this condition.

Factors Associated With Early Improvement in Low Back Pain After Total Hip Arthroplasty: A Multi-Center Prospective Cohort Analyses.

This study identified factors associated with an improvement in low back pain (LBP) at six-month follow-up after total hip arthroplasty (THA). Data from a national registry of 3054 patients were analyzed. Factors under analysis included demographics, comorbid conditions, operative and nonoperative joint pain severity, physical function, and mental health. Differences in these factors between patients with and without improvement in LBP were examined. Among patients reporting severe or moderate LBP preoperatively, 56% improved 6 months after surgery. Patients without improvement were more likely to be on Medicare, have a high school education or less, have household income less than $45,000 and have one or more comorbid conditions. Patients with improvement in LBP experienced more resolution of pain in both the operative and nonoperative hip.

TGF-ß1 Suppresses Plasmin and MMP Activity in Flexor Tendon Cells via PAI-1: Implications for Scarless Flexor Tendon Repair.

Flexor tendon injuries caused by deep lacerations to the hands are a challenging problem as they often result in debilitating adhesions that prevent the movement of the afflicted fingers. Evidence exists that tendon adhesions as well as scarring throughout the body are largely precipitated by the pleiotropic growth factor, Transforming Growth Factor Beta 1(TGF-ß1), but the effects of TGF-ß1 are poorly understood in tendon healing. Using an in vitro model of tendon healing, we previously found that TGF-ß1 causes gene expression changes in tenocytes that are consistent with scar tissue and adhesion formation, including upregulation of the anti-fibrinolytic protein, PAI-1. Therefore, we hypothesized that TGF-ß1 contributes to scarring and adhesions by reducing the activity of proteases responsible for ECM degradation and remodeling, such as plasmin and MMPs, via upregulation of PAI-1. To test our hypothesis, we examined the effects of TGF-ß1 on the protease activity of tendon cells. We found that flexor tendon tenocytes treated with TGF-ß1 had significantly reduced levels of active MMP-2 and plasmin. Interestingly, the effects of TGF-ß1 on protease activity were completely abolished in tendon cells from homozygous plasminogen activator inhibitor 1 (PAI-1) knockout (KO) mice, which are unable to express PAI-1. Our findings support the hypothesis that TGF-ß1 induces PAI-1, which suppresses plasmin and plasmin-mediated MMP activity, and provide evidence that PAI-1 may be a novel therapeutic target for preventing adhesions and promoting a scarless, regenerative repair of flexor tendon injuries.

TAK1 regulates SOX9 expression in chondrocytes and is essential for postnatal development of the growth plate and articular cartilages.

TAK1 is a MAP3K that mediates non-canonical TGF-ß and BMP signaling. During the embryonic period, TAK1 is essential for cartilage and joint development as deletion of Tak1 in chondro-osteo progenitor cells leads to severe chondrodysplasia with defects in both chondrocyte proliferation and maturation. We have investigated the role of TAK1 in committed chondrocytes during early postnatal development. Using the Col2a1-CreER(T2); Tak1(f/f) mouse model, we induced deletion of Tak1 at postnatal day 7 and characterized the skeletal phenotypes of these mice at 1 and 3 months of age. Mice with chondrocyte-specific Tak1 deletion exhibited severe growth retardation and reduced proteoglycan and type II collagen content in the extracellular matrix of the articular cartilage. We found reduced Col2a1 and Acan expression, but increased Mmp13 and Adamts5 expression, in Tak1-deficient chondrocytes along with reduced expression of the SOX trio of transcription factors, SOX9, SOX5 and SOX6. In vitro, BMP2 stimulated Sox9 gene expression and Sox9 promoter activity. These effects were reduced; however, following Tak1 deletion or treatment with a TAK1 kinase inhibitor. TAK1 affects both canonical and non-canonical BMP signal transduction and we found that both of these pathways contribute to BMP2-mediated Sox9 promoter activation. Additionally, we found that ATF2 directly binds the Sox9 promoter in response to BMP signaling and that this effect is dependent upon TAK1 kinase activity. These novel findings establish that TAK1 contributes to BMP2-mediated Sox9 gene expression and is essential for the postnatal development of normal growth plate and articular cartilages.

Cartilage-specific RBPjκ-dependent and -independent Notch signals regulate cartilage and bone development.

The Notch signaling pathway has emerged as an important regulator of endochondral bone formation. Although recent studies have examined the role of Notch in mesenchymal and chondro-osteo progenitor cell populations, there has yet to be a true examination of Notch signaling specifically within developing and committed chondrocytes, or a determination of whether cartilage and bone formation are regulated via RBPjκ-dependent or -independent Notch signaling mechanisms. To develop a complete understanding of Notch signaling during cartilage and bone development we generated and compared general Notch gain-of-function (Rosa-NICD(f/+)), RBPjκ-deficient (Rbpjκ(f/f)), and RBPjκ-deficient Notch gain-of-function (Rosa-NICD(f/+);Rbpjκ(f/f)) conditional mutant mice, where activation or deletion of floxed alleles were specifically targeted to mesenchymal progenitors (Prx1Cre) or committed chondrocytes (inducible Col2Cre(ERT2)). These data demonstrate, for the first time, that Notch regulation of chondrocyte maturation is solely mediated via the RBPjκ-dependent pathway, and that the perichodrium or osteogenic lineage probably influences chondrocyte terminal maturation and turnover of the cartilage matrix. Our study further identifies the cartilage-specific RBPjκ-independent pathway as crucial for the proper regulation of chondrocyte proliferation, survival and columnar chondrocyte organization. Unexpectedly, the RBPjκ-independent Notch pathway was also identified as an important long-range cell non-autonomous regulator of perichondral bone formation and an important cartilage-derived signal required for coordinating chondrocyte and osteoblast differentiation during endochondral bone development. Finally, cartilage-specific RBPjκ-independent Notch signaling likely regulates Ihh responsiveness during cartilage and bone development.

Mitochondrial dysfunction and permeability transition in osteosarcoma cells showing the Warburg effect.

Metabolic reprogramming in cancer is manifested by persistent aerobic glycolysis and suppression of mitochondrial function and is known as the Warburg effect. The Warburg effect contributes to cancer progression and is considered to be a promising therapeutic target. Understanding the mechanisms used by cancer cells to suppress their mitochondria may lead to development of new approaches to reverse metabolic reprogramming. We have evaluated mitochondrial function and morphology in poorly respiring LM7 and 143B osteosarcoma (OS) cell lines showing the Warburg effect in comparison with actively respiring Saos2 and HOS OS cells and noncancerous osteoblastic hFOB cells. In LM7 and 143B cells, we detected markers of the mitochondrial permeability transition (MPT), such as mitochondrial swelling, depolarization, and membrane permeabilization. In addition, we detected mitochondrial swelling in human OS xenografts in mice and archival human OS specimens using electron microscopy. The MPT inhibitor sanglifehrin A reversed MPT markers and increased respiration in LM7 and 143B cells. Our data suggest that the MPT may play a role in suppression of mitochondrial function, contributing to the Warburg effect in cancer.

Deletion of the transforming growth factor ß receptor type II gene in articular chondrocytes leads to a progressive osteoarthritis-like phenotype in mice.

OBJECTIVE: While transforming growth factor ß (TGFß) signaling plays a critical role in chondrocyte metabolism, the TGFß signaling pathways and target genes involved in cartilage homeostasis and the development of osteoarthritis (OA) remain unclear. Using an in vitro cell culture method and an in vivo mouse genetic approach, we undertook this study to investigate TGFß signaling in chondrocytes and to determine whether Mmp13 and Adamts5 are critical downstream target genes of TGFß signaling. METHODS: TGFß receptor type II (TGFßRII)-conditional knockout (KO) (TGFßRII(Col2ER)) mice were generated by breeding TGFßRII(flox/flox) mice with Col2-CreER-transgenic mice. Histologic, histomorphometric, and gene expression analyses were performed. In vitro TGFß signaling studies were performed using chondrogenic rat chondrosarcoma cells. To determine whether Mmp13 and Adamts5 are critical downstream target genes of TGFß signaling, TGFßRII/matrix metalloproteinase 13 (MMP-13)- and TGFßRII/ADAMTS-5-double-KO mice were generated and analyzed. RESULTS: Inhibition of TGFß signaling (deletion of the Tgfbr2 gene in chondrocytes) resulted in up-regulation of Runx2, Mmp13, and Adamts5 expression in articular cartilage tissue and progressive OA development in TGFßRII(Col2ER) mice. Deletion of the Mmp13 or Adamts5 gene significantly ameliorated the OA-like phenotype induced by the loss of TGFß signaling. Treatment of TGFßRII(Col2ER) mice with an MMP-13 inhibitor also slowed OA progression. CONCLUSION: Mmp13 and Adamts5 are critical downstream target genes involved in the TGFß signaling pathway during the development of OA.

RBP-Jκ-dependent Notch signaling is required for murine articular cartilage and joint maintenance.

OBJECTIVE: Osteoarthritis (OA) is a degenerative disease resulting in severe joint cartilage destruction and disability. While the mechanisms underlying the development and progression of OA are poorly understood, gene mutations have been identified within cartilage-related signaling molecules, implicating impaired cell signaling in OA and joint disease. The Notch pathway has recently been identified as a crucial regulator of growth plate cartilage development, and components are expressed in joint tissue. This study was undertaken to investigate a novel role for Notch signaling in joint cartilage development, maintenance, and the pathogenesis of joint disease in a mouse model. METHODS: We performed the first mouse gene study in which the core Notch signaling component, RBP-Jκ, was tissue specifically deleted within joints. The Prx1Cre transgene removed Rbpjk loxP-flanked alleles in mesenchymal joint precursor cells, while the Col2Cre(ERT2) transgene specifically deleted Rbpjk in postnatal chondrocytes. Murine articular chondrocyte cultures were also used to examine Notch regulation of gene expression. RESULTS: Loss of Notch signaling in mesenchymal joint precursor cells did not affect embryonic joint development in mice, but rather, resulted in an early, progressive OA-like pathology. Additionally, partial loss of Notch signaling in murine postnatal cartilage resulted in progressive joint cartilage degeneration and an age-related OA-like pathology. Inhibition of Notch signaling altered the expression of the extracellular matrix (ECM)-related factors type II collagen (COL2A1), proteoglycan 4, COL10A1, matrix metalloproteinase 13, and ADAMTS. CONCLUSION: Our findings indicate that the RBP-Jκ-dependent Notch pathway is a novel pathway involved in joint maintenance and articular cartilage homeostasis, a critical regulator of articular cartilage ECM-related molecules, and a potentially important therapeutic target for OA-like joint disease.

High incidence of hemiarthroplasty for shoulder osteoarthritis among recently graduated orthopaedic surgeons.

BACKGROUND: Primary glenohumeral osteoarthritis is a common indication for shoulder arthroplasty. Historically, both total shoulder arthroplasty (TSA) and hemi-shoulder arthroplasty (HSA) have been used to treat primary glenohumeral osteoarthritis. The choice between procedures is a topic of debate, with HSA proponents arguing that it is less invasive, faster, less expensive, and technically less demanding, with quality of life outcomes equivalent to those of TSA. More recent evidence suggests TSA is superior in terms of pain relief, function, ROM, strength, and patient satisfaction. We therefore investigated the practice of recently graduated orthopaedic surgeons pertaining to the surgical treatment of this disease. QUESTIONS/PURPOSES: We hypothesized that (1) recently graduated, board eligible, orthopaedic surgeons with fellowship training in shoulder surgery are more likely to perform TSA than surgeons without this training; (2) younger patients are more likely to receive HSA than TSA; (3) patient sex affects the choice of surgery; (4) US geographic region affects practice patterns; and (5) complication rates for HSA and TSA are not different. METHODS: We queried the American Board of Orthopaedic Surgery's database to identify practice patterns of orthopaedic surgeons taking their board examination. We identified 771 patients with primary glenohumeral osteoarthritis treated with TSA or HSA from 2006 to 2011. The rates of TSA and HSA were compared based on the treating surgeon's fellowship training, patient age and sex, US geographic region, and reported surgical complications. RESULTS: Surgeons with fellowship training in shoulder surgery were more likely (86% versus 72%; OR 2.32; 95% CI, 1.56-3.45, p<0.001) than surgeons without this training to perform TSA rather than HSA. The mean age for patients receiving HSA was not different from that for patients receiving TSA (66 versus 68, years, p=0.057). Men were more likely to receive HSA than TSA when compared to women (RR 1.54; 95% CI, 1.19-2.00, p=0.0012). The proportions of TSA and HSA were similar regardless of US geographic region (Midwest HSA 21%, TSA 79%; Northeast HSA 25%, TSA 75%; Northwest HSA 16%, TSA 84%; South HSA 27%, TSA 73%; Southeast HSA 24%, TSA 76%; Southwest HSA 23%, TSA 77%; overall p=0.708). The overall complication rates were not different with the numbers available: 8.4% (15/179) for HSA and 8.1% (48/592) for TSA (p=0.7555). CONCLUSIONS: The findings of this study are at odds with the recommendations in the current clinical practice guidelines for the treatment of glenohumeral osteoarthritis published by the American Academy of Orthopaedic Surgeons. These guidelines favor using TSA over HSA in the treatment of shoulder arthritis. Further investigation is needed to clarify if these practice patterns are isolated to recently graduated board eligible orthopaedic surgeons or if the use of HSA continues with orthopaedic surgeons applying for recertification. LEVEL OF EVIDENCE: Level III, therapeutic study. See Instructions for Authors for a complete description of levels of evidence.

BMP2, but not BMP4, is crucial for chondrocyte proliferation and maturation during endochondral bone development.

The BMP signaling pathway has a crucial role in chondrocyte proliferation and maturation during endochondral bone development. To investigate the specific function of the Bmp2 and Bmp4 genes in growth plate chondrocytes during cartilage development, we generated chondrocyte-specific Bmp2 and Bmp4 conditional knockout (cKO) mice and Bmp2,Bmp4 double knockout (dKO) mice. We found that deletion of Bmp2 and Bmp4 genes or the Bmp2 gene alone results in a severe chondrodysplasia phenotype, whereas deletion of the Bmp4 gene alone produces a minor cartilage phenotype. Both dKO and Bmp2 cKO mice exhibit severe disorganization of chondrocytes within the growth plate region and display profound defects in chondrocyte proliferation, differentiation and apoptosis. To understand the mechanism by which BMP2 regulates these processes, we explored the specific relationship between BMP2 and Runx2, a key regulator of chondrocyte differentiation. We found that BMP2 induces Runx2 expression at both the transcriptional and post-transcriptional levels. BMP2 enhances Runx2 protein levels through inhibition of CDK4 and subsequent prevention of Runx2 ubiquitylation and proteasomal degradation. Our studies provide novel insights into the genetic control and molecular mechanism of BMP signaling during cartilage development.

Inhibition of the Wnt-ß-catenin and Notch signaling pathways sensitizes osteosarcoma cells to chemotherapy.

Osteosarcoma (OS) is one of the most common malignant bone tumors in early adolescence. Multi-drug chemotherapy has greatly increased the five year survival rate from 20% to 70%. However, the rate has been staggering for 30 years and the prognosis is particularly poor for patients with recurrence and metastasis. Our study aimed to investigate the role of Wnt-ß-catenin, Notch and Hedgehog pathway in OS development because all these pathways are involved in skeletal development, tumorigenesis and chemoresistance. Our results showed that the major components in Wnt-ß-catenin pathway, e.g. Wnt3a, ß-catenin and Lef1, were consistently upregulated in human osteosarcoma cell line Saos2 cells compared to human fetal osteoblasts (hFOB), whereas the changes in the expression levels of Notch and Hh signaling molecules were not consistent. Knocking down ß-catenin increased the Saos2 sensitivity to methotrexate (MTX) induced cell death. Consistently, the expression level of ß-catenin protein correlated with the invasiveness of OS, as evidenced by more intensive ß-catenin immunoreactivity in higher grade OS samples. Chemical inhibition of the Wnt-ß-catenin signaling enhanced MTX mediated death of Saos2 cells. A synergistic effect with MTX was observed when both inhibitors for Wnt-ß-catenin and Notch pathways were simultaneously used, while the addition of the Hh inhibitor did not further improve the efficacy. Our findings provide some novel insight to OS pathogenesis and lay a foundation for future application of Wnt-ß-catenin and Notch inhibitors together with the currently used chemotherapeutic drugs to improve the outcome of OS treatment.