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Background Mirikizumab (miri), a p19-directed IL-23 antibody, demonstrated efficacy and was well-tolerated in a phase 2 randomized clinical trial (NCT02891226) in patients with Crohn’s disease (CD). The histologic results at Week (W)12 induction and W52 maintenance are presented here. Methods Patients were randomized 2:1:1:2 across 4 treatment arms (PBO, 200, 600, 1000mg miri) administered intravenously (IV) every four weeks (Q4W) at Weeks 0, 4, and 8. Patients who received miri and achieved ≥1 point improvement from baseline (BL) at W12 in Simple Endoscopic Score for Crohn’s Disease (SES-CD) were re-randomized 1:1 into double-blind maintenance to continue their IV dose assignment Q4W (IV/IV) or to 300mg miri SC Q4W (IV/SC); maintenance IV arms were pooled for analysis. W12 non-improvers and all induction PBO patients received 1000mg miri Q4W from W12 through W52. Biopsies from terminal ileum and 4 colonic segments (ascending, transverse, descending, rectum) were obtained during endoscopy at W0, 12 and 52 and scored blind to treatment and response status. Ileum and colon scores were reported separately. Colon scores were derived from the sum of the 4 colonic segments, and total intestine scores from the 4 colonic segments plus ileum. Endpoints were defined post-hoc but prior to performing the analyses and only included patients with active histologic disease at BL (see Table). Histologic response was defined as: a) absence of neutrophils in lamina epithelialis, and absence of epithelial damage, erosions and ulceration or b) decrease of either RHI or GHAS ≥50% from BL. Histologic remission was defined as absence of mucosal neutrophils, epithelial damage, erosions, and ulceration. Deep histologic remission was defined as histologic remission combined with absence of chronic inflammatory cell infiltration of the lamina propria. For assessment of total intestine inflammation, the criteria for response, remission or deep remission must have been met in all 5 bowel segments. Results At W12, histologic response and remission were significantly higher in all segments of the 1000mg group versus PBO (response: ileum p Conclusion Patients treated with miri achieved and sustained histologic response and remission over 52 weeks of treatment. Among patients with W52 histologic remission, all but 2 patients achieved deep remission.

BACKGROUND Mirikizumab (miri), an anti-IL-23p19 inhibitor, has demonstrated efficacy in patients with ulcerative colitis and moderately to severely active Crohn’s disease (CD) in a Phase 2, randomised, double-blind, placebo-controlled study (NCT02891226). This analysis evaluated the effect of miri on patient perception of symptom severity as well as disease activity over 12-week (W) induction and 40W maintenance periods. METHODS During induction, patients were randomised 2:1:1:2 to placebo (PBO), 200mg, 600mg, or 1000mg miri administered intravenously (IV) every four weeks (Q4W). Patients who received miri during induction and achieved ≥1 point improvement in Simple Endoscopic Score for Crohn’s Disease (SES-CD) at W12 were randomised 1:1 to continue induction treatment (IV 200mg, 600mg, 1000mg miri Q4W) or receive 300mg miri Q4W administered subcutaneously (SC) for 40W (maintenance). Because of small patient numbers, the randomised maintenance groups results were combined for those who continued IV induction treatment (All IV-C) and those who received SC treatment (All SC). SES-CD non-improvers and patients who received PBO during induction received 1000mg miri IV Q4W during maintenance. The 6-point Patient’s Global Rating of Severity (PGRS) scale (1=none to 6=very severe) was recorded daily on an electronic diary, and the 7-point Patient’s Global Rating of Change (PGRC) scale (1=very much better to 7=very much worse) was recorded at W4, 12 and 52. Weekly average score for PGRS was calculated and the change from baseline in miri compared to PBO using a mixed model for repeated measures (MMRM) for induction period. Observed PGRC values at W4, 12 and 52 are presented. RESULTS Significant reduction in PGRS compared to PBO was observed as early as W4 in patients treated with 600mg miri (least square mean (LSM) difference ± standard error [95% confidence internal]: -0.44 ± 0.2 [-0.84, -0.04], p=0.031), and in all miri treated patients by W12: 200mg (-0.58 ± 0.2 [-1.05, -0.12], p=0.014), 600mg (-0.86 ± 0.2 [-1.31, -0.40], p CONCLUSION Mirikizumab treatment improved both patients’ self-assessment of disease severity and disease activity as early as week 4 and these improvements were sustained through 52 weeks in patients with moderately to severely active Crohn’s disease.

Objectives: To test the utility of clinical and circulating biomarkers to predict abdominal aortic aneurysm (AAA) growth rate and response to doxycycline therapy. Methods: Plasma samples were obtained in the Pharmaceutical Aneurysm Stabilization Trial that tested the effect of doxycycline (n = 44) vs. placebo (n = 49) in patients with a 35–50 mm AAA. Approximately 200 biomarkers were evaluated in a candidate approach that included markers of matrix turnover and cathepsin S activity and a broad-based approach of predominantly inflammation-related and clinical biomarkers. Results: In a recursive partitioning based analysis, total cholesterol, baseline AAA size, and apolipoprotein B were prognostic of AAA growth in the placebo group whereas elastin and biglycan degradation products were predictive of AAA growth with doxycycline treatment. Univariate analysis of these biomarkers showed that baseline total cholesterol (r = 0.38, unadjusted P = 0.011), apolipoprotein B (r = 0.41, unadjusted P = 0.005), and baseline AAA size (r = 0.35, unadjusted P = 0.013) correlated with AAA growth in the placebo but not the doxycycline group. Elastin fragments were associated with 18 month AAA growth (r = 0.33, unadjusted P = 0.031) in the doxycycline group. Limitations: Limitations of this study include small sample size, a retrospective growth analysis, and translatability of the method used to measure the analytes. Conclusions: This study implies that total cholesterol, baseline AAA size, and apolipoprotein B are predictors of AAA growth. Levels of elastin and biglycan fragments are predictive of doxycycline effects on AAA growth and provide a clue towards this unexpected negative effect.

Chondrocytes phenotype/markers were expressed in clinical samples of tendinopathy and calcifying tendinopathy. This study examined the spatial-temporal expression of chondro-osteogenic Bone Morphogenetic Proteins (BMPs), which might contribute to ectopic chondro-osteogenesis and failed healing process in tendinopathy. Collagenase was injected into patellar tendon of rats to induce ossified failed tendon healing. At week 2, 4, 8, 12, and 16, the patella tendon was harvested for immunohistochemical staining and analysis of BMP-2/4/7. BMP-4/7 showed similar expression patterns, which was different from BMP-2. The expression of BMP-2 in the tendon matrix increased at week 2 and was reduced to nearly undetectable level afterwards except at the chondro-ossification sites. However, the expression of BMP-4/7 in the healing tendon fibroblast-like cells and matrix increased at week 2, reduced at week 4 and 8 and increased again at week 12 and 16, consistent with transient healing at week 8 in this animal model. There was increasing strong expression of BMP-4/7 in the chondrocyte-like cells in the un-ossified and ossified areas from week 8-16. BMP-4/7, besides BMP-2, might also contribute to ectopic chondro-osteogenesis and failed healing in tendon injuries. BMP-4/7, but not BMP-2, might be involved in regulating late events in ossified failed tendon healing.

This study aimed to investigate the effect of repetitive tensile loading on the expression of BMP-2 and the effect of BMP-2 on the osteogenic differentiation of tendon-derived stem cells (TDSCs) in vitro. Repetitive stretching was applied to TDSCs isolated from rat patellar tendon at 0%, 4%, and 8%, 0.5 Hz. The expression of BMP-2 was detected by Western blotting and qPCR. To study the osteogenic effects of BMP-2 on TDSCs, BMP-2 was added to the TDSC monolayer for the detection of ALP activity and calcium nodule formation in a separate experiment. TDSCs adhered, proliferated, and aligned along the direction of externally applied tensile force while they were randomly oriented in the control group. Western blotting showed increased expression of BMP-2 in 4% and 8% stretching groups but not in the control group. Up-regulation of BMP-2 mRNA was also observed in the 4% stretching group. BMP-2 increased the osteogenic differentiation of TDSCs as indicated by higher ALP cytochemical staining, ALP activity, and calcium nodule formation. Repetitive tensile loading increased the expression of BMP-2 and addition of BMP-2 enhanced osteogenic differentiation of TDSCs. Activation of BMP-2 expression in TDSCs during tendon overuse might provide a possible explanation of ectopic calcification in calcifying tendinopathy. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:390–396, 2011

Objective: To explore whether Radix Dipsaci (RD) exhibits beneficial effects on tendon healing. Methods: An attempt was made to explore the in vitro effects of a hot water extract of RD on gene expression of procollagen Type I (COL1A1), procollagen Type III (COL3A1) and decorin in cultured tendon fibroblasts, and its in vivo effects in a well-established rat model of patellar tendon donor site injury. Results: It was found that gene expression of COL3A1 and decorin in cultured tendon fibroblasts was significantly increased by RD, but that COL1A1 was not affected. In vivo studies showed that RD increased blood vessels in the wound but did not significantly affect the expression of COL1A1, COL3A1 and decorin at day 14 post-injury. The ultimate tensile stress of the healing tendon was not significantly improved by either local injection or oral administration of hot water extract of RD (P > 0.05). Conclusion: The present findings imply that RD per se does not significantly improve tendon healing. Further investigation of RD in a herbal formula may be necessary to test its efficacy in tendon injuries.

Background Increase in expression of substance P (SP) and calcitonin gene-related peptide (CGRP) has been reported in clinical samples of tendinopathy. Purpose To examine the spatial-temporal expression of these neuropeptides as well as their association with activity-related tendon pain, matrix degeneration, failed healing, and pathologic calcification in an established collagenase-induced tendon injury rat model. Study Design Controlled laboratory study. Methods Collagenase or saline was injected into the patellar tendon of rats. At weeks 2, 4, 8, 12, and 16, just before the rats were sacrificed, the double-stance duration of rats was examined by gait analysis method. After sacrifice, the patellar tendons were harvested for histologic analysis and immunohistochemical staining of SP and CGRP. Results There was an increase of SP and CGRP immunopositivity in tendon fibroblasts at week 2. The immunopositive signals decreased at weeks 4 and 8 and were observed in chondrocyte-like cells. At weeks 12 and 16, the immunopositive staining increased again and was observed in cells embedded in calcific deposits in addition to tendon fibroblasts and chondrocyte-like cells. The expression pattern was consistent with matrix degeneration, calcification, and failed healing in the animal model. There were significant positive correlations of immunopositivity of SP (rho = .502, P = .002) and CGRP (rho = .483, P = .003) with double-stance duration after collagenase injection. Conclusion There was increased expression of SP and CGRP after collagenase-induced tendon injury, and their expression was positively associated with double-stance duration. Clinical Relevance Substance P and CGRP might be involved in the pathogenesis and origin of pain of tendinopathy and could be the targets for future intervention.

Chronic tendinopathy is characterized with longstanding activity-related pain with degenerative tendon injuries. An objective tool to measure painful responses in animal models is essential for the development of effective treatment for tendinopathy. Gait analysis has been developed to monitor the inflammatory pain in small animals. We reported the use of motion analysis to monitor gait changes in a rat model of degenerative tendon injury. Intratendinous injection of collagenase into the left patellar tendon of Sprague Dawley rat was used to induce degenerative tendon injury, while an equal volume of saline was injected in the control groups. Motion analyses with a high speed video camera were performed on all rats at pre-injury, 2, 4, 8, 12 or 16 weeks post injection. In the end-point study, the rats were sacrificed to obtain tendon samples for histological examination after motion analyses. In the follow-up study, repeated motion analyses were performed on another group of collagenase-treated and saline-treated rats. The results showed that rats with injured patellar tendon exhibited altered walking gait as compared to the controls. The change in double stance duration in the collagenase-treated rats was reversible by administration of buprenorphrine (p=0.029), it suggested that the detected gait changes were associated with pain. Comparisons of end-point and follow-up studies revealed the confounding effects of training, which led to higher gait velocities and probably a different adaptive response to tendon pain in the trained rats. The results showed that motion analysis could be used to measure activity-related chronic tendon pain.

We report chondrocyte phenotype and ectopic ossification in a collagenase-induced patellar tendon injury model. Collagenase or saline was injected intratendinously in one limb. The patella tendon was harvested for assessment at different times. There was an increase in cellularity, vascularity, and loss of matrix organization with time after collagenase injection. The tendon did not heal histologically until week 32. Ectopic mineralization as indicated by von Kossa staining started from week 8. Tendon calcification was mediated by endochondral ossification, as shown by expression of type X collagen. viva CT imaging and polarization microscopy showed characteristic bony porous structures and collagen fiber arrangement, respectively, in the calcific regions. Marrow-like cells and blood vessels were observed inside calcific deposits. Chondrocyte-like cells as indicated by morphology, expression of type II collagen, and sox 9 were seen around and embedded inside the calcific deposits. Fibroblast-like cells expressed type II collagen and sox 9 at earlier times, suggesting that erroneous differentiation of healing tendon fibroblasts may account for failed healing and ossification in collagenase-induced tendon degeneration. Because this animal model replicates key histopathological changes in calcific tendinopathy, it can be used as a model for the study of its pathogenesis at the patellar tendon. (J Histochem Cytochem 57:91–100, 2009)

Calcifying tendinopathy is a tendon disorder with calcium deposits in the mid-substance presented with chronic activity-related pain, tenderness, local edema and various degrees of incapacitation. Most of current treatments are neither effective nor evidence-based because its underlying pathogenesis is poorly understood and treatment is usually symptomatic. Understanding the pathogenesis of calcifying tendinopathy is essential for its effective evidence-based management. One of the key histopathological features of calcifying tendinopathy is the presence of chondrocyte phenotype which surrounds the calcific deposits, suggesting that the formation of calcific deposits was cell-mediated. Although the origin of cells participating in the formation of chondrocyte phenotype and ossification is still unknown, many evidences have suggested that erroneous tendon cell differentiation is involved in the process. Recent studies have shown the presence of stem cells with self-renewal and multi-differentiation potential in human, horse, mouse and rat tendon tissues. We hypothesized that the erroneous differentiation of tendon-derived stem cells (TDSCs) to chondrocytes or osteoblasts leads to chondrometaplasia and ossification and hence weaker tendon, failed healing and pain, in calcifying tendinopathy. We present a hypothetical model on the pathogenesis and evidences to support this hypothesis. Understanding the key role of TDSCs in the pathogenesis of calcifying tendinopathy and the mechanisms contributing to their erroneous differentiation would provide new opportunities for the management of calcifying tendinopathy. The re-direction of the differentiation of resident TDSCs to tenogenic or supplementation of MSCs programmed for tenogenic differentiation may be enticing targets for the management of calcifying tendinopathy in the future.

Chondrocytes phenotype/markers were expressed in clinical samples of tendinopathy and calcifying tendinopathy. This study examined the spatial-temporal expression of chondro-osteogenic Bone Morphogenetic Proteins (BMPs), which might contribute to ectopic chondro-osteogenesis and failed healing process in tendinopathy. Collagenase was injected into patellar tendon of rats to induce ossified failed tendon healing. At week 2, 4, 8, 12, and 16, the patella tendon was harvested for immunohistochemical staining and analysis of BMP-2/4/7. BMP-4/7 showed similar expression patterns, which was different from BMP-2. The expression of BMP-2 in the tendon matrix increased at week 2 and was reduced to nearly undetectable level afterwards except at the chondro-ossification sites. However, the expression of BMP-4/7 in the healing tendon fibroblast-like cells and matrix increased at week 2, reduced at week 4 and 8 and increased again at week 12 and 16, consistent with transient healing at week 8 in this animal model. There was increasing strong expression of BMP-4/7 in the chondrocyte-like cells in the un-ossified and ossified areas from week 8-16. BMP-4/7, besides BMP-2, might also contribute to ectopic chondro-osteogenesis and failed healing in tendon injuries. BMP-4/7, but not BMP-2, might be involved in regulating late events in ossified failed tendon healing.

Bench research, cross-sectional.To determine if the effects of low-intensity pulsed ultrasound (LIPUS) on matrix synthesis change at different stages of tendon healing.LIPUS is effective in promoting tendon healing by stimulation of matrix synthesis. The timing of initiation and duration of LIPUS treatment have been shown to affect its effectiveness to promote tendon healing, suggesting a change of tissue responses to LIPUS stimulation. Understanding how the cellular responses to LIPUS stimulation change during tendon healing is thus important.In a rat model of patellar tendon donor site injury, a single sonication of LIPUS or mock sonication was delivered to the injured knee of the rats on the fourth, 14th or 28th day postinjury. Tendon samples were harvested at 4 hours and 24 hours after sonication and the mRNA expression of COL1A1, COL3A1, decorin, biglycan, and TGF-beta 1 was analyzed.The results showed that a single sonication of LIPUS increased COL1A1 and COL3A1 mRNA in healing patellar tendons when administered on the fourth or 14th day postinjury, but not when administered on the 28th day postinjury. Both decorin and biglycan mRNA were decreased by treatment with LIPUS on the 28th day postinjury. Our results showed that LIPUS enhanced collagen synthesis in vivo only during the granulation phase. Matrix remodeling may be affected by LIPUS with the suppressed expression of decorin and biglycan.Our findings suggest that LIPUS should be applied during the granulation phase but not during the remodeling phase, to promote tendon healing.

Background Ectopic chondrogenesis and ossification were observed in a degenerative collagenase-induced calcific tendinopathy model and to a lesser extent, in a patellar tendon traumatic injury model. We hypothesized that expression of bone morphogenetic protein-2 (BMP-2) contributed to ectopic chondrogenesis and ossification. This study aimed to study the spatial and temporal expression of BMP-2 in our animal models. Methods Seventy-two rats were used, with 36 rats each subjected to central one-third patellar tendon window injury (C1/3 group) and collagenase-induced tendon injury (CI group), respectively. The contralateral limb served as controls. At week 2, 4 and 12, 12 rats in each group were sacrificed for immunohistochemistry and RT-PCR of BMP-2. Results For CI group, weak signal was observed at the tendon matrix at week 2. At week 4, matrix around chondrocyte-like cells was also stained in some samples. In one sample, calcification was observed and the BMP-2 signal was observed both in the calcific matrix and the embedded chondrocyte-like cells. At week 12, the staining was observed mainly in the calcific matrix. Similar result was observed in C1/3 group though the immunopositive staining of BMP-2 was generally weaker. There was significant increase in BMP-2 mRNA compared to that in the contralateral side at week 2 and the level became insignificantly different at week 12 in CI group. No significant increase in BMP-2 mRNA was observed in C1/3 group at all time points. Conclusion Ectopic expression of BMP-2 might induce tissue transformation into ectopic bone/cartilage and promoted structural degeneration in calcific tendinopathy.