MicroRNA-29a: a novel target for non-operative management of symptomatic lumbar spinal stenosis.

PURPOSE: Lumbar spinal stenosis (LSS) is the most common reason for spinal surgery in patients over the age of 65, and there are few effective non-surgical treatments. Therefore, the development of novel treatment or preventative modalities to decrease overall cost and morbidity associated with LSS is an urgent matter. The cause of LSS is multifactorial; however, a significant contributor is ligamentum flavum hypertrophy (LFH) which causes mechanical compression of the cauda equina or nerve roots. We assessed the role of a novel target, microRNA-29a (miR-29a), in LFH and investigated the potential for using miR-29a as a therapeutic means to combat LSS. METHODS: Ligamentum flavum (LF) tissue was collected from patients undergoing decompressive surgery for LSS and assessed for levels of miR-29a and pro-fibrotic protein expression. LF cell cultures were then transfected with either miR-29a over-expressor (agonist) or inhibitor (antagonist). The effects of over-expression and under-expression of miR-29a on expression of pro-fibrotic proteins was assessed. RESULTS: We demonstrated that LF at stenotic levels had a loss of miR-29a expression. This was associated with greater LF tissue thickness and higher mRNA levels of collagen I and III. We also demonstrated that miR29-a plays a direct role in the regulation of collagen gene expression in ligamentum flavum. Specifically, agents that increase miR-29a may attenuate LFH, while those that decrease miR-29a promote fibrosis and LFH. CONCLUSION: This study demonstrates that miR-29a may potentially be used to treat LFH and provides groundwork to initiate the development of a therapeutic product for LSS.

The Glasgow Microenvironment Score associates with prognosis and adjuvant chemotherapy response in colorectal cancer.

BACKGROUND: The Glasgow Microenvironment Score (GMS) combines peritumoural inflammation and tumour stroma percentage to assess interactions between tumour and microenvironment. This was previously demonstrated to associate with colorectal cancer (CRC) prognosis, and now requires validation and assessment of interactions with adjuvant therapy. METHODS: Two cohorts were utilised; 862 TNM I-III CRC validation cohort, and 2912 TNM II-III CRC adjuvant chemotherapy cohort (TransSCOT). Primary endpoints were disease-free survival (DFS) and relapse-free survival (RFS). Exploratory endpoint was adjuvant chemotherapy interaction. RESULTS: GMS independently associated with DFS (p = 0.001) and RFS (p < 0.001). GMS significantly stratified RFS for both low risk (GMS 0 v GMS 2: HR 3.24 95% CI 1.85-5.68, p < 0.001) and high-risk disease (GMS 0 v GMS 2: HR 2.18 95% CI 1.39-3.41, p = 0.001). In TransSCOT, chemotherapy type (pinteraction = 0.013), but not duration (p = 0.64) was dependent on GMS. Furthermore, GMS 0 significantly associated with improved DFS in patients receiving FOLFOX compared with CAPOX (HR 2.23 95% CI 1.19-4.16, p = 0.012). CONCLUSIONS: This study validates the GMS as a prognostic tool for patients with stage I-III colorectal cancer, independent of TNM, with the ability to stratify both low- and high-risk disease. Furthermore, GMS 0 could be employed to identify a subset of patients that benefit from FOLFOX over CAPOX.

Tissue Engineering for Musculoskeletal Regeneration and Disease Modeling.

Musculoskeletal injuries and associated conditions are the leading cause of physical disability worldwide. The concept of tissue engineering has opened up novel approaches to repair musculoskeletal defects in a fast and/or efficient manner. Biomaterials, cells, and signaling molecules constitute the tissue engineering triad. In the past 40 years, significant progress has been made in developing and optimizing all three components, but only a very limited number of technologies have been successfully translated into clinical applications. A major limiting factor of this barrier to translation is the insufficiency of two-dimensional cell cultures and traditional animal models in informing the safety and efficacy of in-human applications. In recent years, microphysiological systems, often referred to as organ or tissue chips, generated according to tissue engineering principles, have been proposed as the next-generation drug testing models. This chapter aims to first review the current tissue engineering-based approaches that are being applied to fabricate and develop the individual critical elements involved in musculoskeletal organ/tissue chips. We next highlight the general strategy of generating musculoskeletal tissue chips and their potential in future regenerative medicine research. Exemplary microphysiological systems mimicking musculoskeletal tissues are described. With sufficient physiological accuracy and relevance, the human cell-derived, three-dimensional, multi-tissue systems have been used to model a number of orthopedic disorders and to test new treatments. We anticipate that the novel emerging tissue chip technology will continually reshape and improve our understanding of human musculoskeletal pathophysiology, ultimately accelerating the development of advanced pharmaceutics and regenerative therapies.

The efficacy and safety of tranexamic acid for reducing blood loss following simultaneous bilateral total knee arthroplasty: a multicenter retrospective study.

BACKGROUND: The purpose of the study was to evaluate whether tranexamic acid (TXA) administration could reduce blood loss and transfusion risk after simultaneous bilateral total knee arthroplasty (SBTKA). METHODS: As a multicenter retrospective study, a total of 575 patients were assigned into three groups on the basis of TXA usage, including intravenous (IV) group (1 g IV TXA 5-10 min prior to the incision), combined group (1 g IV TXA combined with intra-articular injection of 1 g TXA prior to the closure every knee) and control group (no TXA use). The primary outcomes were total blood loss (TBL). The secondary outcomes were maximum hemoglobin (Hb) and hematocrit (Hct) drop, transfusion rate, drain volume, length of stay, hospitalization expenses and the incidence of complications. RESULTS: The mean TBL in control group (1685.0 ± 571.4 mL) were higher than that in IV group (1061.1 ± 689.6 mL, p = 0.006 and combined group (988.3 ± 559.3 mL, p = 0.003). The maximum Hb and Hct drop in combined group (28.5 ± 13.4 g/L, p = 0.016; 0.074 ± 0.053, p < 0.001) and IV group (28.8 ± 14.5 g/L, p = 0.025; 0.082 ± 0.056, p = 0.001) were lower than those in control group (33.4 ± 14.0; 0.131 ± 0.049). But the difference between IV and combined groups was not significant. The similar trend was detected on drain volume, length of stay and hospitalization expenses. The incidence of complications did not differ significantly among the three groups (p > 0.05). CONCLUSIONS: The study indicates that TXA could reduce blood loss with no apparent increase in the incidence of complications during SBTKA.

Prenatal exposure to environmental factors and congenital limb defects.

Limb congenital defects afflict approximately 0.6:1000 live births. In addition to genetic factors, prenatal exposure to drugs and environmental toxicants, represents a major contributing factor to limb defects. Examples of well-recognized limb teratogenic agents include thalidomide, warfarin, valproic acid, misoprostol, and phenytoin. While the mechanism by which these agents cause dymorphogenesis is increasingly clear, prediction of the limb teratogenicity of many thousands of as yet uncharacterized environmental factors (pollutants) remains inexact. This is limited by the insufficiencies of currently available models. Specifically, in vivo approaches using guideline animal models have inherently deficient predictive power due to genomic and anatomic differences that complicate mechanistic comparisons. On the other hand, in vitro two-dimensional (2D) cell cultures, while accessible for cellular and molecular experimentation, do not reflect the three-dimensional (3D) morphogenetic events in vivo nor systemic influences. More robust and accessible models based on human cells that accurately replicate specific processes of embryonic limb development are needed to enhance limb teratogenesis prediction and to permit mechanistic analysis of the adverse outcome pathways. Recent advances in elucidating mechanisms of normal development will aid in the development of process-specific 3D cell cultures within specialized bioreactors to support multicellular microtissues or organoid constructs that will lead to increased understanding of cell functions, cell-to-cell signaling, pathway networks, and mechanisms of toxicity. The promise is prompting researchers to look to such 3D microphysiological systems to help sort out complex and often subtle interactions relevant to developmental malformations that would not be evident by standard 2D cell culture testing. Birth Defects Research (Part C) 108:243-273, 2016. © 2016 Wiley Periodicals, Inc.

Platelet-Rich Plasma Inhibits Mechanically Induced Injury in Chondrocytes.

PURPOSE: To investigate the effect of platelet-rich plasma (PRP) on mechanically injured chondrocytes. METHODS: PRP from bovine whole blood was activated to prepare platelet-rich plasma releasate (PRPr). Bovine articular chondrocytes were subjected to 16%, 0.5-Hz biaxial cyclic tensile strain (CTS) for 48 hours and cultured for another 24 hours without cell stretching as an in vitro model of mechanically injured chondrocytes. Culture medium in the 3 PRP- and CTS-treated groups was supplemented with 10% PRPr at the start of CTS, after 24 hours of CTS, and after 48 hours of CTS, respectively. Gene expression levels of type II collagen, aggrecan, matrix metalloproteinase (MMP)-3, MMP-13, inducible nitric oxide synthase, and cyclooxygenase 2 were quantitatively evaluated. Changes in the content of nitric oxide (NO), prostaglandin E2 (PGE2), MMP-3, and tissue inhibitor of metalloproteinase 1 in the culture medium were also measured. RESULTS: PRPr increased type II collagen and aggrecan messenger RNA expression; diminished CTS-dependent up-regulation of MMP-3, inducible nitric oxide synthase, and cyclooxygenase 2 gene expression; and reduced CTS-induced overproduction of NO and PGE2 when PRPr was applied early at the start of CTS. The addition of PRPr after 24 hours of CTS only inhibited MMP-3 gene up-regulation and the increase of NO and PGE2 induced by CTS. These changes were not observed when PRPr was supplemented after 48 hours of CTS. PRPr mitigated the increased MMP-3 production and decreased tissue inhibitor of metalloproteinase 1 secretion resulting from CTS in a time-dependent manner. CONCLUSIONS: PRP treatment ameliorated multiple CTS-mediated catabolic and inflammatory responses in chondrocytes. More beneficial effects were observed with early PRP application. CLINICAL RELEVANCE: Intra-articular PRP injections at the beginning of strenuous exercises may be used to protect chondrocytes from mechanical injury, thus preventing joints from increased wear.

Stem cell-based microphysiological osteochondral system to model tissue response to interleukin-1ß.

Osteoarthritis (OA) is a chronic degenerative disease of the articular joint that involves both bone and cartilage degenerative changes. An engineered osteochondral tissue within physiological conditions will be of significant utility in understanding the pathogenesis of OA and testing the efficacy of potential disease-modifying OA drugs (DMOADs). In this study, a multichamber bioreactor was fabricated and fitted into a microfluidic base. When the osteochondral construct is inserted, two chambers are formed on either side of the construct (top, chondral; bottom, osseous) that is supplied by different medium streams. These medium conduits are critical to create tissue-specific microenvironments in which chondral and osseous tissues will develop and mature. Human bone marrow stem cell (hBMSCs)-derived constructs were fabricated in situ and cultured within the bioreactor and induced to undergo spatially defined chondrogenic and osteogenic differentiation for 4 weeks in tissue-specific media. We observed tissue specific gene expression and matrix production as well as a basophilic interface suggesting a developing tidemark. Introduction of interleukin-1ß (IL-1ß) to either the chondral or osseous medium stream induced stronger degradative responses locally as well as in the opposing tissue type. For example, IL-1ß treatment of the osseous compartment resulted in a strong catabolic response in the chondral layer as indicated by increased matrix metalloproteinase (MMP) expression and activity, and tissue-specific gene expression. This induction was greater than that seen with IL-1ß application to the chondral component directly, indicative of active biochemical communication between the two tissue layers and supporting the osteochondral nature of OA. The microtissue culture system developed here offers novel capabilities for investigating the physiology of osteochondral tissue and pathogenic mechanisms of OA and serving as a high-throughput platform to test potential DMOADS.

Enhanced osteochondral repair by leukocyte-depleted platelet-rich plasma in combination with adipose-derived mesenchymal stromal cells encapsulated in a three-dimensional photocrosslinked injectable hydrogel in a rabbit model.

INTRODUCTION: Intra-articular injection of adipose-derived mesenchymal stromal cells (ASCs) and/or platelet-rich plasma (PRP) have been reported to independently and synergistically improve healing of osteochondral lesions in animal models. However, their independent and combined effects when localized to an osteochondral lesion by encapsulation within a photocrosslinkable methacrylated gelatin hydrogel (GelMA) have not been explored. Herein we investigated a unique combination of allogeneic ASCs and PRP embedded in GelMA as a single-stage treatment for osteochondral regeneration in a rabbit model. METHODS: Thirty mature rabbits were divided into six experimental groups: (1) Sham; (2) Defect; (3) GelMA; (4) GelMA + ASCs; (5) GelMA + PRP; and (6) GelMA + ASCs + PRP.At 12 weeks following surgical repair, osteochondral regeneration was assessed on the basis of gross appearance, biomechanical properties, histological and immunohistochemical characteristics, and subchondral bone volume. RESULTS: In terms of mechanical property reflecting the ability of neotissue to bear stress, PRP only group were significantly lower than the Sham group (p = 0.0098). On the other hand, ASCs only and ASCs combined with PRP groups did not exhibit significantly difference, which suggesting that incorporation of ASCs assists in restoring the ability of the neotissue to bear stresses similarly to native tissue (p = 0.346, p = 0.40, respectively). Safranin O in ASCs combined with PRP group was significantly higher than the Defect and GelMA only groups (p = 0.0009, p = 0.0017, respectively). Additionally, ASCs only and ASCs combined with PRP groups presented especially strong staining for collagen type II. Surprisingly, PRP only and PRP + ASCs groups tended to exhibit higher collagen type I and collagen type X staining compared to ASCs only group, suggesting a potential PRP-mediated hypertrophic effect. CONCLUSION: Regeneration of a focal osteochondral defect in a rabbit model was improved by a single-stage treatment of a photocrosslinked hydrogel containing allogenic ASCs and autologous PRP, with the combination of ASCs and PRP producing superior benefit than either alone. No experimental construct fully restored all properties of the native, healthy osteochondral unit, which may require longer follow-up or further modification of PRP and/or ASCs characteristics.

Quantitative Real-Time Polymerase Chain Reaction May Serve as a Useful Adjunct to Conventional Culture in The Detection of Cutibacterium acnes in the Glenohumeral Joint: A Study of 100 Consecutive Patients.

Objectives: Synovial fluid or tissue culture is the current gold standard for diagnosis of infection, but Cutibacterium acnes (C. acnes) is a frequent cause of shoulder PJI and is a notoriously fastidious organism. The purpose of this study was to compare quantitative real-time polymerase chain reaction (qRT-PCR) to standard culture as a more rapid, sensitive means of identifying C. acnes from the glenohumeral joint. We hypothesized that qRT-PCR would be more effective than standard culture at identifying C. acnes and would have greater sensitivity and specificity for detecting infection. Methods: This was a prospective observational study with 100 consecutive patients undergoing arthroscopic or open shoulder surgery with known positive and negative controls. Intraoperatively, synovial fluid and tissue was obtained for C. acnes qRT-PCR and results were blinded to the gold standard microbiology cultures. Results: Clinical review demonstrated 3 patients (3%) with positive cultures, none of which were positive for C. acnes. Of the samples tested by the C. acnes qRT-PCR standard curve, 12.2% of tissue samples and 4.5% of fluid samples were positive. Culture sensitivity was 60.0%, specificity was 100.0%, PPV was 100.0%, and NPV was 97.9%. C. acnes qRT-PCR standard curve sensitivity, specificity, PPV, and NPV was 60.0%, 90.3%, 25.0%, and 97.7% respectively for tissue specimens and 0%, 95.2%, 0%, and 95.2% respectively, for fluid specimens. For combination of culture and tissue qRT-PCR, the sensitivity, specificity, PPV and NPV was 100%, 90.3%, 35.7%, and 100%, respectively. Conclusion: We report that qRT-PCR for C. acnes identified the organism more frequently than conventional culture. While these findings demonstrate the potential utility of qRT-PCR, the likelihood of false positive results of qRT-PCR should be considered. Thus, qRT-PCR may be useful as an adjuvant to current gold standard workup of synovial fluid or tissue culture for the diagnosis of infection.

JAK/STAT3 represents a therapeutic target for colorectal cancer patients with stromal-rich tumors.

Colorectal cancer (CRC) is a heterogenous malignancy underpinned by dysregulation of cellular signaling pathways. Previous literature has implicated aberrant JAK/STAT3 signal transduction in the development and progression of solid tumors. In this study we investigate the effectiveness of inhibiting JAK/STAT3 in diverse CRC models, establish in which contexts high pathway expression is prognostic and perform in depth analysis underlying phenotypes. In this study we investigated the use of JAK inhibitors for anti-cancer activity in CRC cell lines, mouse model organoids and patient-derived organoids. Immunohistochemical staining of the TransSCOT clinical trial cohort, and 2 independent large retrospective CRC patient cohorts was performed to assess the prognostic value of JAK/STAT3 expression. We performed mutational profiling, bulk RNASeq and NanoString GeoMx® spatial transcriptomics to unravel the underlying biology of aberrant signaling. Inhibition of signal transduction with JAK1/2 but not JAK2/3 inhibitors reduced cell viability in CRC cell lines, mouse, and patient derived organoids (PDOs). In PDOs, reduced Ki67 expression was observed post-treatment. A highly significant association between high JAK/STAT3 expression within tumor cells and reduced cancer-specific survival in patients with high stromal invasion (TSPhigh) was identified across 3 independent CRC patient cohorts, including the TrasnSCOT clinical trial cohort. Patients with high phosphorylated STAT3 (pSTAT3) within the TSPhigh group had higher influx of CD66b + cells and higher tumoral expression of PDL1. Bulk RNAseq of full section tumors showed enrichment of NFκB signaling and hypoxia in these cases. Spatial deconvolution through GeoMx® demonstrated higher expression of checkpoint and hypoxia-associated genes in the tumor (pan-cytokeratin positive) regions, and reduced lymphocyte receptor signaling in the TME (pan-cytokeratin- and αSMA-) and αSMA (pan-cytokeratin- and αSMA +) areas. Non-classical fibroblast signatures were detected across αSMA + regions in cases with high pSTAT3. Therefore, in this study we have shown that inhibition of JAK/STAT3 represents a promising therapeutic strategy for patients with stromal-rich CRC tumors. High expression of JAK/STAT3 proteins within both tumor and stromal cells predicts poor outcomes in CRC, and aberrant signaling is associated with distinct spatially-dependant differential gene expression.

Pathogenesis of acquired heterotopic ossification: Risk factors, cellular mechanisms, and therapeutic implications.

Heterotopic ossification (HO), including hereditary and acquired HO, is the formation of extraskeletal bone in skeletal muscle and surrounding soft tissues. Acquired HO is often caused by range of motion, explosion injury, nerve injury or burns. Severe HO can lead to pain and limited joint activity, affecting functional rehabilitation and quality of life. Increasing evidence shows that inflammatory processes and mesenchymal stem cells (MSCs) can drive HO. However, explicit knowledge about the specific mechanisms that result in HO and related cell precursors is still limited. Moreover, there are no effective methods to prevent or reduce HO formation. In this review, we provide an update of known risk factors and relevant cellular origins for HO. In particular, we focus on the underlying mechanisms of MSCs in acquired HO, which follow the osteogenic program. We also discuss the latest therapeutic value and implications for acquired HO. Our review highlights the current gaps in knowledge regarding the pathogenesis of acquired HO and identifies potential targets for the prevention and treatment of HO.

The development of a mouse model to investigate the formation of heterotopic ossification.

BACKGROUND: Muscle injury and concomitant bone injury are important drivers to induce heterotopic ossification (HO). However, the related roles of muscle and concomitant bone injury in HO formation are still unclear. This study aims to develop a mouse model through the combination of hindlimb amputation (Am) and cardiotoxin (CTX) injection to investigate the mechanism of HO formation. METHOD: The mice were randomly divided into Am group (Am of right hindlimb, n = 12), CTX group (CTX injection in the calf muscle of left hindlimb, n = 12) and Am + CTX group (the combination of Am of right hindlimb and CTX injection of left hindlimb, n = 18). MicroCT was used to evaluate the incidence of HO. Histology was used to investigate the progression of HO. RESULTS: The MicroCT showed that only Am or CTX injection failed to induce HO while the combination of Am and CTX injection successfully induced HO. The incidence of HO was significant in Am + CTX group on day 7 (0% vs 0% vs 83.3%, p = 0.001) and day 14 (0% vs 0% vs 83.3%, p = 0.048). HO was located on the left hindlimb where CTX was injected. Moreover, the bone volume and bone density on day 14 were higher than those on day 7 in Am + CTX group. Histology revealed the evidence of calcification and expression of osteogenic markers in calcification sites in Am + CTX group. CONCLUSION: In summary, the combination of Am and CTX injection could successfully induce dystrophic calcification/HO, which occurs in the location of muscle injury.

Bubble-PAPR: a phase 1 clinical evaluation of the comfort and perception of a prototype powered air-purifying respirator for use by healthcare workers in an acute hospital setting.

OBJECTIVES: We aimed to design and produce a low-cost, ergonomic, hood-integrated powered air-purifying respirator (Bubble-PAPR) for pandemic healthcare use, offering optimal and equitable protection to all staff. We hypothesised that participants would rate Bubble-PAPR more highly than current filtering face piece (FFP3) face mask respiratory protective equipment (RPE) in the domains of comfort, perceived safety and communication. DESIGN: Rapid design and evaluation cycles occurred based on the identified user needs. We conducted diary card and focus group exercises to identify relevant tasks requiring RPE. Lab-based safety standards established against British Standard BS-EN-12941 and EU2016/425 covering materials; inward particulate leakage; breathing resistance; clean air filtration and supply; carbon dioxide elimination; exhalation means and electrical safety. Questionnaire-based usability data from participating front-line healthcare staff before (usual RPE) and after using Bubble-PAPR. SETTING: Overseen by a trial safety committee, evaluation progressed sequentially through laboratory, simulated, low-risk, then high-risk clinical environments of a single tertiary National Health Service hospital. PARTICIPANTS: 15 staff completed diary cards and focus groups. 91 staff from a range of clinical and non-clinical roles completed the study, wearing Bubble-PAPR for a median of 45 min (IQR 30-80 (15-120)). Participants self-reported a range of heights (mean 1.7 m (SD 0.1, range 1.5-2.0)), weights (72.4 kg (16.0, 47-127)) and body mass indices (25.3 (4.7, 16.7-42.9)). OUTCOME MEASURES: Preuse particulometer 'fit testing' and evaluation against standards by an independent biomedical engineer.Primary:Perceived comfort (Likert scale).Secondary: Perceived safety, communication. RESULTS: Mean fit factor 16 961 (10 participants). Bubble-PAPR mean comfort score 5.64 (SD 1.55) vs usual FFP3 2.96 (1.44) (mean difference 2.68 (95% CI 2.23 to 3.14, p<0.001). Secondary outcomes, Bubble-PAPR mean (SD) versus FFP3 mean (SD), (mean difference (95% CI)) were: how safe do you feel? 6.2 (0.9) vs 5.4 (1.0), (0.73 (0.45 to 0.99)); speaking to other staff 7.5 (2.4) vs 5.1 (2.4), (2.38 (1.66 to 3.11)); heard by other staff 7.1 (2.3) vs 4.9 (2.3), (2.16 (1.45 to 2.88)); speaking to patients 7.8 (2.1) vs 4.8 (2.4), (2.99 (2.36 to 3.62)); heard by patients 7.4 (2.4) vs 4.7 (2.5), (2.7 (1.97 to 3.43)); all p<0.01. CONCLUSIONS: Bubble-PAPR achieved its primary purpose of keeping staff safe from airborne particulate material while improving comfort and the user experience when compared with usual FFP3 masks. The design and development of Bubble-PAPR were conducted using a careful evaluation strategy addressing key regulatory and safety steps. TRIAL REGISTRATION NUMBER: NCT04681365.

Creation of a Knee Joint-on-a-Chip for Modeling Joint Diseases and Testing Drugs.

The high prevalence of debilitating joint diseases like osteoarthritis (OA) poses a high socioeconomic burden. Currently, the available drugs that target joint disorders are mostly palliative. The unmet need for effective disease-modifying OA drugs (DMOADs) has been primarily caused by the absence of appropriate models for studying the disease mechanisms and testing potential DMOADs. Herein, we describe the establishment of a miniature synovial joint-mimicking microphysiological system (miniJoint) comprising adipose, fibrous, and osteochondral tissue components derived from human mesenchymal stem cells (MSCs). To obtain the three-dimensional (3D) microtissues, MSCs were encapsulated in photocrosslinkable methacrylated gelatin before or following differentiation. The cell-laden tissue constructs were then integrated into a 3D-printed bioreactor, forming the miniJoint. Separate flows of osteogenic, fibrogenic, and adipogenic media were introduced to maintain the respective tissue phenotypes. A commonly shared stream was perfused through the cartilage, synovial, and adipose tissues to enable tissue crosstalk. This flow pattern allows the induction of perturbations in one or more of the tissue components for mechanistic studies. Furthermore, potential DMOADs can be tested via either "systemic administration" through all the medium streams or "intraarticular administration" by adding the drugs to only the shared "synovial fluid"-simulating flow. Thus, the miniJoint can serve as a versatile in vitro platform for efficiently studying disease mechanisms and testing drugs in personalized medicine.

Protein expression of S100A2 reveals it association with patient prognosis and immune infiltration profile in colorectal cancer.

PURPOSE: Colorectal cancer (CRC) is the third most diagnosed cancer worldwide. Despite a well-established knowledge of tumour development, biomarkers to predict patient outcomes are still required. S100 calcium-binding protein A2 (S100A2) has been purposed as a potential marker in many types of cancer, however, the prognostic value of S100A2 in CRC is rarely reported. MATERIAL AND METHODS: In this study, immunohistochemistry (IHC) was performed to identify the prognostic role of S100A2 protein expression in the tumour core of the tissue microarrays (TMAs) in colorectal cancer patients (n=787). Bulk RNA transcriptomic data was used to identify significant genes compared between low and high cytoplasmic S100A2 groups. Multiplex immunofluorescence (mIF) was performed to further study and confirm the immune infiltration in tumours with low and high cytoplasmic S100A2. RESULTS: Low cytoplasmic protein expression of S100A2 in the tumour core was associated with poor survival (HR 0.539, 95%CI 0.394-0.737, P<0.001) and other adverse tumour phenotypes. RNA transcriptomic analysis showed a gene significantly associated with the low cytoplasmic S100A2 group (AKT3, TAGLN, MYLK, FGD6 and ETFDH), which correlated with tumour development and progression. GSEA analysis identifies the enriched anti-tumour and immune activity group of genes in high cytoplasmic S100A2. Additionally, mIF staining showed that high CD3+FOXP3+ and CD163+ inversely associated with low cytoplasmic S100A2 (P<0.001, P=0.009 respectively). CONCLUSION: Our finding demonstrates a prognostic value of S100A2 together with the correlation with immune infiltration in CRC.

Potential Methods of Targeting Cellular Aging Hallmarks to Reverse Osteoarthritic Phenotype of Chondrocytes.

Osteoarthritis (OA) is a chronic degenerative joint disease that causes pain, physical disability, and life quality impairment. The pathophysiology of OA remains largely unclear, and currently no FDA-approved disease-modifying OA drugs (DMOADs) are available. As has been acknowledged, aging is the primary independent risk factor for OA, but the mechanisms underlying such a connection are not fully understood. In this review, we first revisit the changes in OA chondrocytes from the perspective of cellular hallmarks of aging. It is concluded that OA chondrocytes share many alterations similar to cellular aging. Next, based on the findings from studies on other cell types and diseases, we propose methods that can potentially reverse osteoarthritic phenotype of chondrocytes back to a healthier state. Lastly, current challenges and future perspectives are summarized.

Generation of hyaline-like cartilage tissue from human mesenchymal stromal cells within the self-generated extracellular matrix.

Chondrocytic hypertrophy, a phenotype not observed in healthy hyaline cartilage, is often concomitant with the chondrogenesis of human mesenchymal stromal cells (hMSCs). This undesired feature represents one of the major obstacles in applying hMSCs for hyaline cartilage repair. Previously, we developed a method to induce hMSC chondrogenesis within self-generated extracellular matrix (mECM), which formed a cartilage tissue with a lower hypertrophy level than conventional hMSC pellets. In this study, we aimed to test the utility of hypoxia and insulin-like growth factor-1 (IGF1) on further reducing hypertrophy. MSC-mECM constructs were first subjected to chondrogenic culture in normoxic or hypoxic (5%) conditions. The results indicated that hMSC-derived cartilage formed in hypoxic culture displayed a significantly reduced hypertrophy level than normoxic culture. However, hMSC chondrogenesis was also suppressed under hypoxic culture, partially due to the reduced activity of the IGF1 pathway. IGF1 was then supplemented in the chondrogenic medium, which promoted remarkable hMSC chondrogenesis under hypoxic culture. Interestingly, the IGF1-enhanced hMSC chondrogenesis, under hypoxic culture, was not at the expense of promoting significantly increased hypertrophy. Lastly, the cartilage tissues created by hMSCs with different conditions were implanted into osteochondral defect in rats. The results indicated that the tissue formed under hypoxic condition and induced with IGF1-supplemented chondrogenic medium displayed the best reparative results with minimal hypertrophy level. Our results demonstrate a new method to generate hyaline cartilage-like tissue from hMSCs without using exogenous scaffolds, which further pave the road for the clinical application of hMSC-based cartilage tissue engineering. STATEMENT OF SIGNIFICANCE: In this study, hyaline cartilage-like tissues were generated from human mesenchymal stromal cells (hMSCs), which displayed robust capacity in repairing the osteochondral defect in rats. In particular, the extracellular matrix created by hMSCs was used, so no exogenous scaffold was needed. Through a series of optimization, we defined that hypoxic culture and supplementation of insulin-like growth factor-1 (IGF-1) in chondrogenic medium resulted in robust cartilage formation with minimal hypertrophy. We also demonstrated that hypoxic culture suppressed chondrogenesis and hypertrophy through modulating the Wnt/ß-catenin and IGF1 pathways, respectively. Our results demonstrate a new method to generate hyaline cartilage-like tissue from hMSCs without using exogenous scaffolds, which will further pave the road for the clinical application of hMSCs-based cartilage tissue engineering.

Role of Canonical Wnt/ß-Catenin Pathway in Regulating Chondrocytic Hypertrophy in Mesenchymal Stem Cell-Based Cartilage Tissue Engineering.

In the past 3 decades, the cartilage repair potential of mesenchymal stromal cells, or mesenchymal stem cells (MSCs), has been widely examined in animal studies. Unfortunately, the phenotype and physical properties of MSC-derived cartilage tissue are not comparable to native hyaline cartilage. In particular, chondrocytic hypertrophy, a phenotype that is not observed in healthy hyaline cartilage, is concomitant with MSC chondrogenesis. Given that hypertrophic chondrocytes potentially undergo apoptosis or convert into osteoblasts, this undesired phenotype needs to be prevented or minimized before MSCs can be used to repair cartilage injuries in the clinic. In this review, we first provide an overview of chondrocytic hypertrophy and briefly summarize current methods for suppressing hypertrophy in MSC-derived cartilage. We then highlight recent progress on modulating the canonical Wnt/ß-catenin pathway for inhibiting hypertrophy. Specially, we discuss the potential crosstalk between Wnt/ß-catenin with other pathways in regulating hypertrophy. Lastly, we explore future perspectives to further understand the role of Wnt/ß-catenin in chondrocytic hypertrophy.

Experimental models to study osteoarthritis pain and develop therapeutics.

Pain is the predominant symptom of osteoarthritis (OA) that drives patients to seek medical care. Currently, there are no pharmacological treatments that can reverse or halt the progression of OA. Safe and efficacious medications for long-term management of OA pain are also unavailable. Understanding the mechanisms behind OA pain generation at onset and over time is critical for developing effective treatments. In this narrative review, we first summarize our current knowledge on the innervation of the knee joint, and then discuss the molecular mechanism(s) currently thought to underlie OA pain. In particular, we focus on the contribution of each joint component to the generation of pain. Next, the current experimental models for studying OA pain are summarized, and the methods to assess pain in rodents are presented. The potential application of emerging microphysiological systems in OA pain research is especially highlighted. Lastly, we discuss the current challenge in standardizing models and the selection of appropriate systems to address specific questions.

Modeling early changes associated with cartilage trauma using human-cell-laden hydrogel cartilage models.

BACKGROUND: Traumatic impacts to the articular joint surface are known to lead to cartilage degeneration, as in post-traumatic osteoarthritis (PTOA). Limited progress in the development of disease-modifying OA drugs (DMOADs) may be due to insufficient mechanistic understanding of human disease onset/progression and insufficient in vitro models for disease and therapeutic modeling. In this study, biomimetic hydrogels laden with adult human mesenchymal stromal cells (MSC) are used to examine the effects of traumatic impacts as a model of PTOA. We hypothesize that MSC-based, engineered cartilage models will respond to traumatic impacts in a manner congruent with early PTOA pathogenesis observed in animal models. METHODS: Engineered cartilage constructs were fabricated by encapsulating adult human bone marrow-derived mesenchymal stem cells in a photocross-linkable, biomimetic hydrogel of 15% methacrylated gelatin and promoting chondrogenic differentiation for 28 days in a defined medium and TGF-ß3. Constructs were subjected to traumatic impacts with different strains or 10 ng/ml IL-1ß, as a common comparative method of modeling OA. Cell viability and metabolism, elastic modulus, gene expression, matrix protein production and activation of catabolic enzymes were assessed. RESULTS: Cell viability staining showed that traumatic impacts of 30% strain caused an appropriate level of cell death in engineered cartilage constructs. Gene expression and histo/immunohistochemical analyses revealed an acute decrease in anabolic activities, such as COL2 and ACAN expression, and a rapid increase in catabolic enzyme expression, e.g., MMP13, and inflammatory modulators, e.g., COX2. Safranin O staining and GAG assays together revealed a transient decrease in matrix production 24 h after trauma that recovered within 7 days. The decrease in elastic modulus of engineered cartilage constructs was coincident with GAG loss and mediated by the encapsulated cells. The acute and transient changes observed after traumatic impacts contrasted with progressive changes observed using continual IL-1ß treatment. CONCLUSIONS: Traumatic impacts delivered to engineered cartilage constructs induced PTOA-like changes in the encapsulated cells. While IL-1b may be appropriate in modeling OA pathogenesis, the results of this study indicate it may not be appropriate in understanding the etiology of PTOA. The development of a more physiological in vitro PTOA model may contribute to the more rapid development of DMOADs.