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Background Hip fracture patients on oral anticoagulants (OACs) experience increased time-to-surgery and higher mortality compared to non-anticoagulated patients. However, it is unclear whether pre-injury OAC status and its associated operative delay are associated with worsening of peri-operative hemostasis or an increased risk of postoperative thromboembolism. Methods We performed a systematic review to identify studies that directly compared hemostatic and thromboembolic outcomes among hip fracture patients on an OAC prior to admission with those not on anticoagulants. Random effects meta-analyses were used to pool all outcomes of interest (estimated blood loss, transfusion requirements, and postoperative thromboembolism). Results Twenty-one studies involving 21 417 patients were included. Estimated blood loss was higher among patients presenting with OACs compared to those not anticoagulated (mean difference 31.0 mL, 95% confidence interval [CI] 6.2-55.7). Anticoagulated patients also had a 1.3-fold higher risk of receiving red blood cell transfusions (odds ratio [OR] 1.34, 95% CI 1.20-1.51); however, rates of postoperative thromboembolism were similar regardless of anticoagulation status (OR 0.96, 95% CI 0.40-2.79 for venous thromboembolism; OR 0.58, 95% CI 0.25-1.36 for arterial thromboembolism). No subgroup effect was found based on anticoagulant type or degree of surgical delay. Conclusion Hip fracture patients on OACs experience increased surgical blood loss and higher risk of red blood cell transfusions. However, the degree of surgical delay did not mitigate this risk, and there was no difference in postoperative thromboembolism. The impact of appropriate, timely OAC reversal on blood conservation and expedited surgery in anticoagulated hip fracture patients warrants urgent evaluation.

Mast cells contain an abundance of tryptase, and preclinical models have shown elevated serum mast cell tryptase (SMCT) in the setting of posttraumatic joint contractures. Therefore, SMCT emerged as a potential biomarker to help recognize patients with more severe injuries and a higher likelihood of developing contractures. The objective of this study is to assess SMCT levels in participants with varying severity of elbow fractures and/or dislocations. A prospective cohort including 13 participants with more severe injuries that required an operation and 28 participants with less severe injuries managed nonoperatively were evaluated. A control group of eight individuals without elbow injuries was also evaluated. The SMCT levels were measured using an enzyme-linked immunosorbent assay kit specific for human mast cell tryptase. A one-way analysis of variance and Tukey's Honest Significance test was used to assess for statistical significance among and between the three groups. The average time from injury to the collection of the blood samples was 4 ± 2 days. Highly significant differences were identified between the operative, nonoperative, and control groups (P = .0005). In the operative group, SMCT levels were significantly higher than the nonoperative group (P = .0005) and the control group (P = .009), suggesting a correlation between SMCT levels and injury severity. There was no statistically significant difference in SMCT levels between the nonoperative and control groups. The SMCT levels were elevated in participants with acute elbow injuries requiring operative intervention, suggesting that SMCT levels were higher in injuries regarded as more severe.

Background Osteoarthritis (OA) is one of the leading causes of disability worldwide. Previous history of knee injury is a significant risk factor for OA. It has been established that low-level chronic inflammation plays a pivotal role in the onset and pathogenesis of OA. The primary aim of this research was to determine if a history of knee joint injury is associated with systemic inflammation. A secondary aim was to determine if systemic inflammation is related to knee pain and joint structure. Methods Differences in serum cytokine association networks, knee joint structural changes (MRI), and self-reported pain (i.e., Knee Injury and Osteoarthritis Outcome Score Pain subscale, KOOSPAIN and Intermittent and Constant Osteoarthritis Pain score, ICOAP) between individuals who had sustained a youth (aged 15–26 years) sport-related knee injury 3–10 years previously and age- and sex-matched controls were examined. Proteins of interest were also examined in an OA rat model. Results Cytokine association networks were found to differ significantly between study groups, yet no significant associations were found between networks and KOOSPAIN or MRI-defined OA. A group of cytokines (MCP1/CCL2, CCL22 and TNFα) were differentially associated with other cytokines between study groups. In a pre-clinical rat OA model, serum CCL22 levels were associated with pain (r = 0.255, p = 0.045) and structural changes to the cartilage. CCL22 expression was also observed in human OA cartilage and furthermore, CCL22 induced apoptosis of isolated human chondrocytes. Discussion These results suggest that CCL22 may be an early factor in the onset/pathogenic process of cartilage degeneration and/or related to pain OA.

Epidural fat is commonly discarded during spine surgery to increase the operational field. However, mesenchymal progenitor cells (MPCs) have now been identified in human epidural fat and within the murine dura mater. This led us to believe that epidural fat may regulate homeostasis and regeneration in the vertebral microenvironment. Using two MPC lineage tracing reporter mice (Prx1 and Hic1), not only have we found that epidural fat MPCs become incorporated in the dura mater over the course of normal skeletal maturation, but have also identified these cells as an endogenous source of repair and regeneration post-dural injury. Moreover, our results reveal a partial overlap between Prx1+ and Hic1+ populations, indicating a potential hierarchical relationship between the two MPC populations. This study effectively challenges the notion of epidural fat as an expendable tissue and mandates further research into its biological function and relevance.

Posttraumatic joint contracture is a debilitating complication following an acute fracture or intra-articular injury that can lead to loss of motion and an inability to complete activities of daily living. In prior studies using an established in vivo model, we found that ketotifen fumarate (KF), a mast cell stabilizer, was associated with a significant reduction in the severity of posttraumatic joint contracture. Our primary research question in the current study was to determine whether a dose-response relationship exists between KF and posttraumatic joint contracture reduction. Methods A standardized operative method to create posttraumatic joint contracture in a knee was performed on skeletally mature New Zealand White rabbits. The animals were randomly assigned to 1 of 5 groups (n = 10 per group): a nonoperative control group, an operative control group, or 1 of 3 experimental KF groups (0.01 mg/kg [the KF 0.01 group], 0.1 mg/kg [KF 0.1], or 5.0 mg/kg [KF 5.0]). Flexion contractures were measured following 8 weeks of knee immobilization using a hydraulic material-testing machine. The posterior knee joint capsules were then harvested for quantification of myofibroblast and mast cell numbers with immunohistochemistry analysis. Results Forty-five rabbits were used in the final analysis. Contracture severity was significantly reduced in the KF 0.1 group (p = 0.016) and the KF 5.0 group (p = 0.001) compared with the operative control group. When converted to a percent response, posttraumatic joint contracture reduction was 13%, 45%, and 63% for the KF 0.01, KF 0.1, and KF 5.0 groups, respectively. A half-maximal effective concentration (EC50) for KF of 0.22 mg/kg was established. There was also a decrease in myofibroblasts, mast cells, and substance P-containing nerve fiber counts with increasing doses of KF. Conclusions Using a preclinical, rabbit in vivo model of posttraumatic joint contracture, increasing doses of KF were associated with decreasing biomechanical estimates of knee posttraumatic joint contracture as well as decreasing numbers of myofibroblasts, mast cells, and substance P-containing nerve fibers. Clinical relevance KF has been used safely in humans for more than 40 years and, to our knowledge, is the first and only agent ready to be potentially translated into an effective treatment for posttraumatic joint contracture.

A new therapeutic option for treating arthritis pain., Chronic joint pain such as mechanical allodynia is the most debilitating symptom of arthritis, yet effective therapies are lacking. We identify the pannexin-1 (Panx1) channel as a therapeutic target for alleviating mechanical allodynia, a cardinal sign of arthritis. In rats, joint pain caused by intra-articular injection of monosodium iodoacetate (MIA) was associated with spinal adenosine 5′-triphosphate (ATP) release and a microglia-specific up-regulation of P2X7 receptors (P2X7Rs). Blockade of P2X7R or ablation of spinal microglia prevented and reversed mechanical allodynia. P2X7Rs drive Panx1 channel activation, and in rats with mechanical allodynia, Panx1 function was increased in spinal microglia. Specifically, microglial Panx1-mediated release of the proinflammatory cytokine interleukin-1β (IL-1β) induced mechanical allodynia in the MIA-injected hindlimb. Intrathecal administration of the Panx1-blocking peptide 10panx suppressed the aberrant discharge of spinal laminae I-II neurons evoked by innocuous mechanical hindpaw stimulation in arthritic rats. Furthermore, mice with a microglia-specific genetic deletion of Panx1 were protected from developing mechanical allodynia. Treatment with probenecid, a clinically used broad-spectrum Panx1 blocker, resulted in a striking attenuation of MIA-induced mechanical allodynia and normalized responses in the dynamic weight-bearing test, without affecting acute nociception. Probenecid reversal of mechanical allodynia was also observed in rats 13 weeks after anterior cruciate ligament transection, a model of posttraumatic osteoarthritis. Thus, Panx1-targeted therapy is a new mechanistic approach for alleviating joint pain.

Significance: The pathogenesis of fibrogenic wound and connective tissue healing is complex and incompletely understood. Common observations across a vast array of human and animal models of fibroproliferative conditions suggest neuroinflammatory mechanisms are important upstream fibrogenic events. Recent Advances: As detailed in this review, mast cell hyperplasia is a common observation in fibrotic tissue. Recent investigations in human and preclinical models of hypertrophic wound healing and post-traumatic joint fibrosis provides evidence that fibrogenesis is governed by a maladaptive neuropeptide-mast cell-myofibroblast signaling pathway. Critical Issues: The blockade and manipulation of these factors is providing promising evidence that if timed correctly, the fibrogenic process can be appropriately regulated. Clinically, abnormal fibrogenic healing responses are not ubiquitous to all patients and the identification of those at-risk remains an area of priority. Future Directions: Ultimately, an integrated appreciation of the common pathobiology shared by many fibrogenic connective tissue conditions may provide a scientific framework to facilitate the development of novel antifibrotic prevention and treatment strategies.

Previous studies have implicated a myofibroblast-mast cell-neuropeptide axis of fibrosis in pathologic joint capsules from post-traumatic contractures. The hypothesis to be tested is that joint capsule cells (JC) from human elbows with post-traumatic contractures and their interactions with mast cells (MC) and neuropeptides in the microenvironment underlie the pathogenesis of contractures. The hypothesis was tested using an in vitro collagen gel contraction model. The JC were isolated from human elbow capsules and mixed with neutralized PureCol collagen I. The gels were treated in various ways, including addition of MC (HMC-1), the neuropeptide substance P (SP), an NK1 receptor (SP receptor) antagonist RP67580 and the mast cell stabilizer ketotifen fumarate (KF). The collagen gels were released from the wells and gel size (contraction) was measured optically at multiple time points. The JC contracted collagen gels in a dose-dependent manner. This was enhanced in the presence of MC and increased further with SP. Increasing concentrations of the SP receptor antagonist, RP67580 or the mast cell stabilizer, KF decreased the magnitude of contraction. These observations identify putative mechanistic components of a myofibroblast-mast cell-neuropeptide axis of fibrosis in the joint capsules in post-traumatic contractures and potential prophylactic or therapeutic interventions.

To determine if decreased vascular responsiveness in the medial collateral ligament (MCL) of anterior cruciate ligament transected (ACL-t) rabbit knees is due to pericyte deficiency associated with angiogenesis. Vascular responses to potassium chloride (KCl), phenylephrine, acetylcholine, and sodium nitroprusside (SNP) were evaluated in ACL-t rabbit knees (n = 6) and control knees (n = 5) using laser speckle perfusion imaging. Ligament degeneration was determined by ultrasound imaging. Vascular and pericyte volume were measured using quantitative immunohistochemical volumetric analysis using CD31 and α-smooth muscle actin antibodies with co-localization analysis. Perfusion was increased in the ACL-t rabbits 2.5-fold. Responsiveness to phenylephrine, SNP, and acetylcholine was significantly decreased in the ACL knee while no change in KCl responses was seen. MCL ultrasound imaging revealed decreased collagen organization, increased ligament thickness, and increased water content in the ACL-t MCL. Vascular Volume was increased fourfold in ACL deficient knees, while pericyte volume to endothelial volume was not changed. No difference in CD31 and α-SMA co-localization was found. Blood vessels in the MCL of ACL-t knees do not lack smooth muscle. The MCL vasculature can undergo constrictive response to KCl, but have impaired receptor mediated responses and impaired nitric oxide signaling.

Proteoglycan 4 (PRG4/lubricin) is secreted by cells that reside in articular cartilage and line the synovial joint. Lubricin may play a role in modulating inflammatory responses through interaction with CD44. This led us to examine if lubricin could be playing a larger role in the modulation of inflammation/immunity through interaction with Toll-like receptors (TLRs). Human Embryonic Kidney (HEK) cells overexpressing TLRs 2, 4 or 5 and surface plasmon resonance were employed to determine if full length recombinant human lubricin was able to bind to and activate TLRs. Primary human synovial fibroblasts were also examined using flow cytometry and Luminex multiplex ELISA. A rat destabilization model of osteoarthritis (OA) was used to determine if lubricin injections were able to regulate pain and/or inflammation in vivo. Lubricin can bind to and regulate the activity of TLRs, leading to downstream changes in inflammatory signalling independent of HA. We confirmed these findings in vivo through intra-articular injections of lubricin in a rat OA model where the inhibition of systemic inflammatory signaling and reduction in pain were observed. Lubricin plays an important role in regulating the inflammatory environment under both homeostatic and tissue injury states.

Using a rabbit model of post-traumatic joint contractures, we investigated whether treatment with a mast cell stabilizer after joint injury would lessen the molecular manifestations of joint capsule fibrosis. Surgical joint injury was used to create stable post-traumatic contractures of the knee in skeletally mature New Zealand white rabbits. Four groups of animals were studied: a non-operated control group (n = 8), an operated contracture group (n = 13) and two operated groups treated with the mast cell stabilizer, ketotifen, at doses of 0.5 mg/kg (n = 9) and 1.0 mg/kg (n = 9) twice daily. Joint capsule fibrosis was assessed by quantifying the mRNA and protein levels of α-SMA, tryptase, TGF-β1, collagen I and collagen III. Significance was tested using an ANOVA analysis of variance. The protein and mRNA levels of α-SMA, TGF-β1, tryptase and collagen I and III were significantly elevated in the operated contracture group compared to control (p

Background: The propensity of joints to become stiff after trauma is widely appreciated, and the joint capsule is commonly recognized as the major motion-limiting anatomical structure. Affected joint capsules become fibrotic, characterized by myofibroblast and collagen hyperplasia. Mast cell hyperplasia is common within fibrotic tissue, and mast cells are known to synthesize many profibrotic mediators. We hypothesized that mast cell inhibition after skeletal injury would lessen contracture severity and reduce myofibroblast hyperplasia within the joint capsule. Methods: Posttraumatic contractures of the knee were created with use of a combination of intra-articular injury and internal immobilization in skeletally mature New Zealand White rabbits. Four groups of animals were studied: a nonoperative control group, a group with the operatively created contracture and no pharmacological treatment (the operative contracture group), and two groups with the operatively created contracture that were treated with a mast cell stabilizer, ketotifen fumarate, at a dose of either 0.5 or 1.0 mg/kg twice daily (the 0.5-mg/kg and 1.0-mg/kg ketotifen groups). After eight weeks of immobilization, flexion contractures were measured and the posterior aspect of the joint capsule was harvested for quantification of myofibroblast and mast cell numbers. Results: Flexion contractures developed in the operative contracture group (mean and standard deviation, 58° ± 14°), and the severity of the contractures was reduced in both the group treated with 0.5 mg/kg of ketotifen (42° ± 17°) and the group treated with 1.0 mg/kg of ketotifen (45° ± 10°) (p < 0.02). The joint capsule myofibroblast and mast cell numbers in the operative contracture group were significantly increased compared with the values in the control group (p < 0.001), and the myofibroblast and mast cell numbers in both ketotifen groups were significantly reduced compared with the values in the operative contracture group (p < 0.001). Conclusions: The use of a mast cell stabilizer, ketotifen, was effective in reducing the biomechanical and cellular manifestations of joint capsule fibrosis in a rabbit model of posttraumatic joint contracture. This finding suggests that an inflammatory pathway, mediated by mast cell activation, is involved in the induction of joint capsule fibrosis after traumatic injury. Clinical Relevance: These results suggest that mast cell activation is an important event in the genesis of posttraumatic joint contractures. Additional work is needed to determine if mast cell inhibition has a role in the prevention of posttraumatic joint contractures in humans.

BackgroundThe drastic difference in healing capacity between the anterior cruciate ligament and the medial collateral ligament is still largely unexplained. Few studies have compared the profiles of messenger ribonucleic acid expression for healing-associated molecules in ligaments during the course of healing.HypothesisInjury responses of the injured anterior cruciate ligament and medial collateral ligament are characterized by very different profiles of angiogenesis-promoting and repair-associated gene expression during the healing process.Study DesignControlled laboratory study.MethodsReverse-transcriptase polymerase chain reaction was used to assay expression of messenger ribonucleic acid for 11 healing- and angiogenesis-associated molecules at 3 days and 2, 6, and 16 weeks after anterior cruciate ligament or medial collateral ligament injury in adult female New Zealand White rabbits.ResultsMarked differences were found in the postinjury changes in messenger ribonucleic acid levels in the anterior cruciate ligament compared to the medial collateral ligament. Notably, messenger ribonucleic acid levels for the important repair-associated growth factor transforming growth factor-β1 did not increase in injured anterior cruciate ligament at any time point. Similarly, Unlike the injured medial collateral ligament, no statistically significant increases in messenger ribonucleic acid levels for the important scar matrix protein collagen III were detected in injured anterior cruciate ligament. In contrast, matrix metalloproteinase Messenger ribonucleic acid levels were markedly elevated in injured anterior cruciate ligament but only modestly increased in Medial collateral ligament.ConclusionThe results suggest that injury leads to an antifibrotic, catabolic response in the rabbit anterior cruciate ligament, Possibly to prevent fibrosis and diminish the risk for loss of joint motion.Clinical RelevanceThe development of effective biologically based treatments for anterior cruciate ligament injuries will need to incorporate strategies to deal with the significant differences in the molecular responses to injury of these tissues.

Loss of joint motion, or joint contracture, is a common complication following injuries.1 The joint capsule is a key contributor to the formation of contractures and surgical releases to improve joint motion invariably include its removal.2 Although the anatomic importance of the joint capsule in the pathogenesis of joint contractures has been known for many years, only recently has there been description of the cellular changes associated with posttraumatic contractures.3–5 It was determined that the myofibroblast, a specialized fibro-blast expressing the contractile smooth muscle protein α-smooth muscle actin (α-SMA) and associated with contracture and fibrosis in many organ systems, is increased in numbers in joint capsules from posttraumatic contractures when compared to similar tissues from normal joints.4–6 This is true for the chronic stages of posttraumatic contractures in human elbow joints and a rabbit knee model of posttraumatic contractures.3–5 Myofibroblast numbers are increased very early in the contracture process, as early as 4 weeks after injury, in this rabbit knee model.7 The question remains as to what is contributing to this early and sustained increase in myofibroblast numbers. One possibility contributing to the early and sustained increase in myofibroblast numbers is a mast cell–neuropeptide fibrosis axis as described in skin wound healing.8 Mast cells are widely distributed throughout connective tissues, including skin and joint capsules.8–14 Mast cell granules contain preformed mediators associated with fibrosis including platelet-derived growth factor-A, basic fibroblast growth factor and endothelin-1, while the profibrotic TGF-β1 is formed upon mast cell stimulation.9,12 It is generally believed that mast cells induce a fibrotic response via involvement of connective tissue fibroblasts and myofibroblasts.9,12,15,16 It has been well described that the neuropeptides Substance P and CGRP can cause mast cell degranulation.8,11,13,14,17,18 Peptidergic nerve terminalshavebeen detectedin close association with mast cells in skin, ligament, joint capsule, and the gut.10,11,13,14,19 It was recently reported that both the density of Substance P positive fibers and the number of mast cells were increased in the pathologic palmar fascia of patients with Dupuytren’s contracture when compared to nonpathologic palmar fascia of control individuals.19 Thus, the myofibroblast–mast cell–neuropeptide pathway may represent what is abnormal (myofibroblasts), how this abnormality comes about (mast cell degranulation liberating growth factors), and why this may occur (neuropeptide induced degranulation of mast cells). As a first step to evaluate the potential involvement of this pathway in joint contracture development, the hypothesis that joint capsule mast cell and neuropeptide containing nerve fiber numbers are increased in posttraumatic contractures is tested. The objective is to evaluate this hypothesis in the acute and chronic stages of the rabbit knee model of posttraumatic contractures, as well as the chronic stages of human posttraumatic elbow contractures.

Previous work has shown that innervation participates in normal ligament healing. The present study was performed to determine if exogenous nerve growth factor (NGF) would improve the healing of injured ligament by promoting reinnervation, blood flow, and angiogenesis. Two groups of 30 Sprague-Dawley rats underwent unilateral medial collateral ligament transection (MCL). One group was given 10 microg NGF and the other was given PBS via osmotic pump over 7 days after injury. After 7, 14, and 42 days, in vivo blood flow was measured using laser speckle perfusion imaging (LSPI). Morphologic assessments of nerve density, vascularity, and angiogenesis inhibitor production were done in three animals at each time point by immunohistochemical staining for the pan-neuronal marker PGP9.5, the endothelial marker vWF, and the angiogenesis inhibitor thrombospondin-2 (TSP-2). Ligament scar material and structural mechanical properties were assessed in seven rats at each time point. Increased nerve density was promoted by NGF at both 14 and 42 days. Exposure to NGF also led to increased ligament vascularity, as measured by histologic assessment of vWF immunohistochemistry, although LSPI-measured blood flow was not significantly different from controls. NGF treatment also led to decreased expression of TSP-2 at 14 days. Mechanical testing revealed that exposure to NGF increased failure load by 40%, ultimate tensile strength by 55%, and stiffness by 30% at 42 days. There were no detectable differences between groups in creep properties. The results suggest that local application of NGF can improve ligament healing by promoting both reinnervation and angiogenesis, and results in scars with enhanced mechanical properties.

Background Articular cartilage has been the focus of multiple strategies to improve its regenerative/ repair capacity. The Murphy Roths Large (MRL/MpJ) “super-healer” mouse demonstrates an unusual enhanced regenerative capacity in many tissues and provides an opportunity to further study endogenous cartilage repair. The objective of this study was to test whether the super-healer phenotype could be transferred from MRL/MpJ to non-healer C57Bl/6 mice by allogeneic bone marrow transplant. Methodology The healing of 2mm ear punches and full thickness cartilage defects was measured 4 and 8 weeks after injury in control C57Bl/6 and MRL/MpJ “super-healer” mice, and in radiation chimeras reconstituted with bone marrow from the other mouse strain. Healing was assessed using ear hole diameter measurement, a 14 point histological scoring scale for the cartilage defect and an adapted version of the Osteoarthritis Research Society International scale for assessment of osteoarthritis in mouse knee joints. Principal Findings Normal and chimeric MRL mice showed significantly better healing of articular cartilage and ear wounds along with less severe signs of osteoarthritis after cartilage injury than the control strain. Contrary to our hypothesis, however, bone marrow transplant from MRL mice did not confer improved healing on the C57Bl/6 chimeras, either in regards to ear wound healing or cartilage repair. Conclusion and Significance The elusive cellular basis for the MRL regenerative phenotype still requires additional study and may possibly be dependent on additional cell types external to the bone marrow.

Summary Objective Murine brachymorphism ( bm ) results from an autosomal recessive mutation of the Papss2 gene that encodes 3′-phosphoadenosine 5′-phosphosulfate synthetase 2, one of the principal enzymes required for the sulfation of extracellular matrix molecules in cartilage and other tissues. A spondyloepimetaphyseal dysplasia has been identified in Pakistani kindred having a mutation of PAPSS2 . In addition to skeletal malformations that include short stature evident at birth due to limb shortening, brachydactyly, and kyphoscoliosis [Ahmad M, Haque MF, Ahmad W, Abbas H, Haque S, Krakow D, et al. Distinct, autosomal recessive form of spondyloepimetaphyseal dysplasia segregating in an inbred Pakistani kindred. Am J Med Genet 1998;78:468–73], affected individuals demonstrate premature onset degenerative joint disease. We investigated whether loss of Papss2 activity would similarly lead to degenerative joint disease in mice. Methods Mice carrying the bm mutation on a C57BL/6 background were obtained from the Jackson Laboratory. Limbs were analyzed by micro-computed tomography (μCT) and histology. Results At 12 months of age both male and female bm mice exhibited severe degenerative knee joint disease, with cartilage damage being primarily evident in the patello-femoral and medial compartments. Control 12–14-month-old C57BL/6 mice, in contrast, only occasionally demonstrated minimal cartilage damage. μCT imaging of bm limbs revealed shortened diaphyses associated with flared metaphyses in the proximal elements of both fore and hind limbs. Additionally, the bm hind limbs demonstrated extensive structural alterations, characterized by distortion of the patello-femoral groove, and prominent bowing of both tibia and fibula. Conclusions The bm mutant, which develops severe articular cartilage lesions of the knee joint by approximately 12 months of age, represents a novel example of murine degenerative joint disease, possibly representing a model of human PAPSS2 deficiency-associated arthrosis.

Little is known about the contribution of innervation to ligament healing after traumatic disruption, although there is good evidence of an important role for the peripheral nervous system in the healing of fractures and skin injuries. Tissues such as ligament, with a low resting blood supply, are dependent on substantial increases in blood flow and vascular volume during the initial stages of repair. We hypothesized that this initial healing response would be strongly promoted by neurogenic inflammation. Since the saphenous nerve (a major sensory branch of the femoral nerve) supplies the medial half of the knee joint, we elected to use femoral nerve transection as a model to determine the role of sensory and autonomic innervation in the initial outcome of repair of the injured medial collateral ligament. Twelve adult, female NZW rabbits underwent right medial collateral ligament transection. Of these, six rabbits underwent right femoral nerve transection to disrupt the somatic sensory and autonomic nerve supply to the knee joint and six were kept neurologically intact (controls). At six weeks post-injury, the animals were assessed by laser Doppler perfusion imaging (LDI) to determine the local blood flow, at both the injury site and at the uninjured contralateral ligament. The animals were then killed, the knee joints were removed and the biomechanical characteristics of the healing bone-median collateral ligament (MCL)-bone complexes assessed. In a separate cohort of 16 rabbits, vascular volumes of the injured ligaments were measured by infusion of a carmine red/gelatin solution. At six weeks post-injury, in vivo measurement of perfusion with LDI revealed that normally innervated ligaments had an almost three-fold higher average blood flow. Carmine red/gelatin infusion revealed a 50% higher density of blood vessels as compared to denervated ligaments. The force required for ultimate failure was found to be 50% higher in normally innnervated MCL's as compared to denervated MCL's: 153.14 +/- 20.71 N versus 101.29 +/- 17.88 N (p < 0.05). Static creep was increased by 66% in denervated MCL's: 2.83 +/- 0.45% versus 1.70 +/- 0.12% (p < 0.05). Total creep was increased by 45% in denervated MCL's: 5.29 +/- 0.62% compared to 3.64 +/- 0.31% in innervated MCL's (p < 0.05). We conclude that intact innervation makes a critical contribution to the early healing responses of the MCL of adult rabbits.

Previous work has shown a progressive, age-related loss of knee joint innervation in the C57BL6Nia mouse. We did three experiments to describe further the loss and determine whether it might contribute to the development of knee osteoarthrosis in this model. Immunocytochemistry showed that the percentage of neurons expressing substance P and calcitonin gene-related peptide increased with age, indicating a relatively selective loss of mechanoreceptors. Histological examination of knee joints of mice at various ages showed that loss of joint innervation always preceded histological changes of cartilage degeneration. The mice usually developed a mild form of osteoarthrosis, but surgical ablation of joint innervation caused the development of severe patellofemoral osteoarthrosis. The findings are consistent with the hypothesis that an age-related loss of joint innervation may contribute to the development of osteoarthrosis.

Metabolic diseases such as diabetes mellitus type-II (DM-II) may increase the risk of suffering painful connective tissue disorders and tendon ruptures. The pathomechanisms, however, by which diabetes adversely affects connective tissue matrix metabolism and regeneration, still need better definition. Our aim was to study the effect of DM-II on expressional changes of neuro- and angiotrophic mediators and receptors in intact and healing Achilles tendon. The right Achilles tendon was transected in 5 male DM-II Goto-Kakizaki (GK) and 4 age-matched Wistar control rats. The left Achilles tendons were left intact. At week 2 post-injury, NGF, BDNF, TSP, and receptors TrkA, TrkB and Nk1 gene expression was studied by quantitative RT-PCR (qRT-PCR) and their protein distribution by immunohistochemistry in intact and injured tendons. The expression of tendon-related markers, Scleraxis (SCX) and Tenomodulin (TNMD), was evaluated by qRT-PCR in intact and injured tendons. Injured tendons of diabetic GK rats exhibited significantly down-regulated Ngf and Tsp1 mRNA and corresponding protein levels, and down-regulated Trka gene expression compared to injured Wistar controls. Intact tendons of DM-II GK rats displayed reduced mRNA levels for Ngf, Tsp1 and Trkb compared to corresponding intact non-diabetic tendons. Up-regulated Scx and Tnmd gene expression was observed in injured tendons of normal and diabetic GK rats compared to intact Wistar controls. However, these molecules were not up-regulated in injured DM-II GK rats compared to their corresponding controls. Our results suggest that DM-II has detrimental effects on neuro- and angiotrophic pathways, and such effects may reflect the compromised repair seen in diabetic Achilles tendon. Thus, novel approaches for regeneration of injured, including tendinopathic, and surgically repaired diabetic tendons may include therapeutic molecular modulation of neurotrophic pathways such as NGF and its receptors.

The regulatory mechanisms involved in tendon homeostasis and repair are not fully understood. Accumulating data, however, demonstrate that the nervous system, in addition to afferent (sensory) functions, through efferent pathways plays an active role in regulating pain, inflammation, and tissue repair. In normal-, healing- and tendinopathic tendons three neurosignalling pathways consisting of autonomic, sensory and glutamatergic neuromediators have been established. In healthy tendons, neuromediators are found in the paratenon, whereas the proper tendon is practically devoid of nerves, reflecting that normal tendon homeostasis is regulated by pro- and anti-inflammatory mediators from the tendon surroundings. During tendon repair, however, there is extensive nerve ingrowth into the tendon proper and subsequent time-dependent appearance of sensory, autonomic and glutamatergic mediators, which amplify and fine-tune inflammation and tendon regeneration. In tendinopathy, excessive and protracted sensory and glutamatergic signalling may be involved in inflammatory, painful and hypertrophic tissue reactions. As our understanding of these processes improves, neuronal mediators may prove to be useful in the development of targeted pharmacotherapy and tissue engineering approaches to painful, degenerative and traumatic tendon disorders.

Objective To derive a clinical decision guide (CDG) to identify patients best suited for cervical diagnostic facet joint blocks. Design Prospective cohort study. Setting Pain management center. Participants Consecutive patients with neck pain (N=125) referred to an interventional pain management center were approached to participate. Interventions Subjects underwent a standardized testing protocol, performed by a physiotherapist, prior to receiving diagnostic facet joint blocks. All subjects received the reference standard diagnostic facet joint block protocol, namely controlled medial branch blocks (MBBs). The physicians performing the MBBs were blinded to the local anesthetic used and findings of the clinical tests. Main Outcome Measures Multivariate regression analyses were performed in the derivation of the CDGs. Sensitivity, specificity, positive and negative likelihood ratios, and 95% confidence intervals (CIs) were calculated for the index tests and CDGs. Results A CDG involving the findings of the manual spinal examination (MSE), palpation for segmental tenderness (PST), and extension-rotation (ER) test demonstrated a specificity of 84% (95% CI, 77–90) and a positive likelihood ratio of 4.94 (95% CI, 2.8–8.2). Sensitivity of the PST and MSE were 94% (95% CI, 90–98) and 92% (95% CI, 88–97), respectively. Negative findings on the PST were associated with a negative likelihood ratio of .08 (95% CI, .03–.24). Conclusions MSE, PST, and ER may be useful tests in identifying patients suitable for diagnostic facet joint blocks. Further research is needed to validate the CDGs prior to their routine use in clinical practice.

Retrograde tracing with Fluoro-Gold was used to identify the complete population of knee joint afferents in the lumbar dorsal root ganglia of adult female Wistar rats. There was an average of 581 +/- 31 (mean +/- S.D.) afferents supplying each joint. These were found distributed from L1 to L5 with the great majority localised in the L3 and L4 ganglia. Electron microscopy of the posterior articular nerve of the knee revealed an average of 103 +/- 15 (mean +/- S.D.) myelinated and 513 +/- 39 unmyelinated axonal profiles. Since about 50-60% of the unmyelinated profiles would be expected to be sympathetic efferents, these numbers are consistent with the numbers of afferents found by Fluoro-Gold retrograde tracing and suggest that the posterior articular nerve contains about 50% of the total number of knee joint afferents in the rat. Immunohistochemistry revealed that an average of 10% of identified joint afferents expressed substance P-like immunoreactivity and that 33% expressed calcitonin gene-related peptide-like immunoreactivity.

Posttraumatic elbow stiffness remains a common and challenging clinical problem. In the setting of a congruent articular surface, the joint capsule is regarded as the major motion-limiting anatomic structure. The affected joint capsule is characterized by irreversible biomechanical and biochemical fibrogenic changes strikingly similar to those observed in many other fibroproliferative human conditions. Studies in humans and preclinical animal models are providing emergent evidence that neuroinflammatory mechanisms are critical upstream events in the pathogenesis of posttraumatic connective tissue fibrogenesis. Maladaptive recruitment and activation of mast cell infiltrates coupled with the aberrant expression of growth factors such as transforming growth factor-beta, nerve growth factor, and neuropeptides such as substance P are common observations in posttraumatic joint contractures and many other fibroproliferative disorders. Blockade of these factors is providing promising evidence that if treatment is timed correctly, the fibrogenic process can be interrupted or impeded. This review serves to highlight opportunities derived from these recent discoveries across many aberrant fibrogenic disorders as we strive to develop novel, targeted antifibrotic prevention and treatment strategies for posttraumatic elbow stiffness.

Type 2 diabetes adversely affects the properties of native connective tissue. The underlying mechanisms, however, by which diabetes alters connective tissue metabolism, especially tendon, are poorly defined. The aim of this study was to determine the effect of type 2 diabetes on the mechanical, histological, and molecular properties of the intact and healing Achilles tendon. The right Achilles tendon was transected in 11 male diabetic Goto-Kakizaki (GK) and 10 age- and sex-matched Wistar control rats, while the left Achilles tendon was left intact. At 2 wk postinjury the intact and injured tendons were assessed by biomechanical testing and histology. The gene expression of collagen I and III, biglycan, versican, MMP-13, and MMP-3 was measured by quantitative RT-PCR, and their protein distribution was studied by immunohistochemistry. Intact tendons exhibited only small differences between the groups. In injured tendons, however, a significantly smaller transverse area and lower stiffness was found in diabetic GK compared with Wistar control rats. This correlated with impaired structural organization of collagen fibers and a reduced expression of collagen I and III in the injured tendons of the diabetic GK compared with Wistar control. Moreover, MMP-3 gene expression was downregulated in the injured diabetic GK tendons compared with injured Wistar controls. Our results indicate that in a rat model of diabetes tendon healing is impaired mainly due to altered expression of collagen and MMPs reflecting decreased degradation of matrix proteins and impaired tissue remodeling. Further our data suggest that therapeutic modulation of collagens or MMPs might be targets for new regenerative approaches in operated, injured, or maybe also degenerative tendon diseases in diabetes.

Abiotrophy is hypothesized to explain the onset and time course of deficits in Parkinson’s disease (PD) Abiotrophy includes: 1) exposure to agent(s) causing the death of dopaminergic nigrostriatal neurons (DNSns), 2) gradual death of DNSns with age, 3) summation of 1) and 2) until DNSn numbers fall below a threshold for detectable neurological deficits. Murine DNSn death following methyl-phenyl-tetrahydropyridine (MPTP) exposure occurs according to an exponential relationship while age-related death of DNSns occurs according to a second exponential relationship. Summing the two exponential losses overestimates experimental DNSn death showing a simple abiotrophic model is not sufficient. Aged murine DNSns greatly increase their dopamine synthesis and the density of their striatal axon terminals which may explain the above threshold. Murine DNSns die gradually after MPTP exposure and L-deprenyl treatment rescues MPTP-damaged DNSns by a previously undiscovered action, altering the abiotrophic interactions and possibly explaining the slowed progression of PD found with deprenyl treatment.

Chronic inflammation associated with osteoarthritis (OA) alters normal responses and modifies the functionality of the articular vasculature. Altered responsiveness of the vasculature may be due to excessive neural activity associated with chronic pain and inflammation, or from the production of inflammatory mediators which induce vasodilation. Using laser speckle perfusion imaging (LSPI), blood flow to the medial collateral ligament (MCL) of adult rabbits was measured in denervated ACL transected knees (n = 6) and compared to unoperated control (n = 6) and 6-week anterial cruciate ligament (ACL)-transected knees (n = 6). Phenylephrine and neuropeptide Y were applied to the MCL vasculature in topical boluses of 100 microL (dose range 10(-14) to 10(-8) mol and 10(-14) to 10(-9) mol, respectively). Denervation diminished vasoconstrictive responsiveness to phenylephrine compared to both control and ACL-transected knees. Denervation minimally enhanced vascular responses to neuropeptide Y (NPY) compared to ACL deficiency alone, which nevertheless remained significantly diminished from control responses. To evaluate the potential role of inflammatory dilators in the diminished contractile responses, phenylephrine was coadministered with histamine, substance P, and prostaglandin E(2). High-dose histamine, and low-dose substance P and PGE(2) were able to inhibit contractile responses in the MCL of control knees. Excessive neural input does not mediate diminished vasoconstrictive responses in the ACL transected knee; inflammatory mediators may play a role in the deficient vascular responsiveness of the ACL transected knee.

Denervation substantially impairs healing of the medial collateral ligament (MCL). Because normal ligaments are sparsely innervated, we hypothesized that neuropeptide-containing neurons would sprout or proliferate after ligament transection, followed by later regression with healing, in a manner analogous to blood vessels.We transected the right MCL in 9 mature female New Zealand white rabbits and killed 3 rabbits at 2, 6 or 14 weeks. Alternate sets of 12-mm serial sections of healing MCL scars were examined by fluorescent immunohistochemistry for substance P (SP), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY) and pan-neuronal marker PGP9.5.Normal MCLs had few peptidergic fibres located in the epiligament in a perivascular pattern. At 2 weeks, PGP9.5-, SP-and CGRP-positive fibres had increased in the epiligament adjacent to the injury. By 6 weeks, there were increases in CGRP-and PGP9.5-positive fibres in epiligament and scar, with similar but less marked increases in SP-positive fibres. At 14 weeks, there was notable regression of immunostained peptidergic nerve fibres in the scar.This experiment shows evidence for a remarkable plasticity of ligament innervation after injury, supporting the idea that neuronal factors play a fundamental role in wound healing.

An organ culture model of four bovine anterior cruciate ligaments was used to compare synovial fluid with bovine serum, lactated Ringer's solution, and Basal Medium Eagle's (BME) to determine their relative abilities to support in vitro conversion of proline into hydroxyproline. There was no difference between serum and synovial fluid in their ability to support collagen synthesis. These results suggest that bovine synovial fluid is not inhibitory in that it is similar to bovine serum in supporting ligament collagen synthesis under the conditions described.

Chronic inflammation associated with osteoarthritis (OA) may alter normal vascular responses and contribute to joint degradation. Vascular responses to vasoactive mediators were evaluated in the medial collateral ligament (MCL) of the anterior cruciate ligament (ACL)-deficient knee. Chronic joint instability and progressive OA were induced in rabbit knees by surgical transection of the ACL. Under halothane anesthesia, laser speckle perfusion imaging (LSPI) was used to measure MCL blood flow in unoperated control ( n = 12) and 6-wk ACL-transected knees ( n = 12). ACh, bradykinin, histamine, substance P (SP), and prostaglandin E2 (PGE2) were applied to the MCL vasculature in topical boluses of 100 μl (dose range 10−14 to 10−8 mol). In normal joints, ACh, bradykinin, histamine, and PGE2 evoked a dilatory response. Substance P caused a biphasic response that was dilatory from 10−14 to 10−11 mol and constricting at higher doses. In ACL-deficient knees, ACh, bradykinin, histamine, and SP decreased perfusion, whereas PGE2 had a biphasic response that decreased perfusion at 10−14 to 10−11 mol and was dilatory at higher concentrations. Sodium nitroprusside increased perfusion in resting and phenylephrine-precontracted vessels with no significant differences between ACL-transected and control knees. Femoral artery occlusion and release increased perfusion by 74.3 ± 11.1% in control knees but only by 25.8 ± 4.4% in ACL-deficient knees. The altered responsiveness of the MCL vasculature to these inflammatory mediators may indicate endothelial dysfunction in the MCL, which may contribute to the progression and severity of OA and to the adaptation of the joint in an altered mechanical environment.

Previous experiments revealed that denervation impairs healing of the MCL. This suggested the hypothesis that denervation would decrease repair-associated mRNA levels in the injured MCL when compared with normally innervated injured MCL. Adult, skeletally mature female rabbits were assigned to one of four groups: unoperated control, femoral nerve transection alone (denervated controls), MCL partial tear or denervated MCL partial tear. At three days, two weeks, six weeks or sixteen weeks post-surgery, cohorts of 6 rabbits from each experimental group were killed. Ligaments were harvested, RNA extracted and RT-PCR was performed using rabbitspecific primers. In the denervated injury group, mRNA levels for the angiogenesis-associated gene MMP-13, matrix components Collagen I and III, growth factor TGF-β and angiogenesis inhibitors TIMP-3, and TSP-1 had all increased by two-weeks post-injury, in comparison to the non-denervated injury group (p ≤ 0.01). An increased level of TSP-1 mRNA was also detected in the denervated injured group at sixteen weeks post injury (p ≤ 0.01). Contrary to the initial hypothesis, denervation led to increased mRNA levels for many relevant molecules during the early stages of MCL healing. Thus, inappropriate timing of over-expression of some molecules may potentially contribute to the decreased quality of the scar tissue, particularly molecules such as TSP-1. Neuronal derived factors strongly influence the in vivo metabolic activity of ligament and scar fibroblasts in the initial phases of healing. © 2006 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res

Sympathetic-derived neuropeptide Y (NPY) helps regulate inflammatory responses in injury and disease, is a vasoconstrictor, and stimulates angiogenesis. Rupture of the anterior cruciate ligament (ACL) is a common clinical presentation that results in tissue inflammation, hyperemia, and angiogenesis in the intact medial collateral ligament (MCL). This study is the first to examine the vasoregulatory role of NPY in ACL-deficient knee joints by using the newly developed technique of laser speckle perfusion imaging (LSPI). MCL blood flow was measured in two groups of adult rabbits: unoperated control ( n = 6), and 6-wk ACL transected ( n = 5). Under anesthesia, the MCL was surgically exposed and tissue blood flow was imaged at high resolution using LSPI. NPY was applied to the MCL vasculature in topical boluses of 100 μl (dose range 10−14 to 10−9 mol), and the α-adrenoceptor agonist phenylephrine was applied in doses of 10−14, 10−10, and 10−7 mol. In control rabbits, topical administration of NPY or phenylephrine produced dose-dependent vasopressor responses (maximal effect at 10−9 mol NPY and 10−7 mol phenylephrine). In ACL-transected knees, there was little or no vasoconstrictive response to NPY at any dose. The response to phenylephrine was significantly reduced compared with control ligaments. Possible causes of the reduced vasoconstrictive response to NPY in the MCL after 6 wk of ACL deficiency include development of tolerance to the peptide due to a prolonged increase in sympathetic nerve activity or change in the distribution or functionality of the NPY Y1 receptors. Chronic ACL deficiency leads to profound and protracted hyperemia in associated articular tissues. Abrogation of a vasoconstrictor response to both NPY and phenylephrine in the MCL indicates that ACL deficiency induces major changes in the vascular physiological homeostasis.

Retrograde tracing with Fluoro-Gold (FG) was used to identify the complete population of knee joint sympathetic postganglionic efferents in the lumbar sympathetic chain of adult female Wistar rats. In 6 rats, the total number and distribution of FG-labelled neurons in the lumbar sympathetic chain was determined. The rat knee joint is supplied by an average of 187+/-57 sympathetic afferents with the majority at the L3 and L4 levels. Immunohistochemistry using antibodies specific for tyrosine hydroxylase (TH), somatostatin (SS) or vasoactive intestinal polypeptide (VIP) revealed that 33 % of knee joint sympathetic afferents contained TH, 42 % contained VIP, and none contained somatostatin. Retrograde tracing with FG provided accurate and reproducible labelling of the joint-innervating subpopulation of sympathetic efferent neurons. This model lends itself to the further study of the molecular responses of this neuronal population in the various disorders and conditions affecting joints.

A 57-year-old man was referred by his family physician to our spine clinic for assessment of left-sided leg pain of six months’ duration. His history was significant only for a spontaneous onset of lumbar back pain noted immediately after awkward bending six months prior, followed two to three

The regulatory mechanisms involved in tendon homeostasis and repair are not fully understood. Accumulating data, however, demonstrate that the nervous system, in addition to afferent (sensory) functions, through efferent neuronal pathways plays an active role in regulating pain, inflammation, and tissue repair processes. Thus, in normal-, healing- and tendinopathic tendons three major neuronal signalling pathways consisting of autonomic, sensory and glutamatergic neuromediators have been established. In healthy tendons, these neural elements are found in the paratenon, whereas the proper tendon is practically devoid of nerves, reflecting that normally tendon homeostasis is regulated by pro- and anti-inflammatory mediators from the tendon surroundings. During tendon repair, however, there is extensive nerve ingrowth into the tendon proper and subsequent time-dependent appearance of sensory, autonomic and glutamatergic mediators, which amplify and fine-tune inflammation and tendon regeneration. In tendinopathy excessive and protracted sensory and glutamatergic signalling may be involved in inflammatory, painful and hypertrophic tissue reactions. In a future perspective, neuronal mediators may prove to be useful in targeted pharmacotherapy and tissue engineering in painful, degenerative and traumatic tendon disorders.