Messenger RNA (mRNA) is a new class of drug that can be used to express a therapeutic protein and, in contrast to DNA, is safer and inexpensive. Among its advantages, mRNA will immediately begin to express its encoded protein in the cell cytoplasm. The protein will be expressed for a period of time, after which the mRNA is degraded. There is no risk of genetic damage, one of the concerns with plasmid DNA (pDNA) used in traditional gene therapy approaches. Nevertheless, mRNA application in tissue regeneration and regenerative medicine remains limited. In this case, mRNA must overcome its main hurdles: immunogenicity, lack of stability, and intracellular delivery. Research has been done to overcome these limitations, and the future of mRNA seems promising for tissue repair. 1,2. This keynote talk will address questions including: What are the opportunities for mRNA to improve outcomes in musculoskeletal tissue repair, in particular bone and cartilage? What are the key factors and challenges to expediting this technology to patient treatment (beyond COVID-19 vaccination)?. Acknowledgements: E.R.B thanks the cmRNAbone project funded by the European Union's Horizon 2020 research and innovation program under the grant agreement no. 874790 and the NIH R01 AR074395 from NIAMS for funding her mRNA work

Joint tissues release extracellular vesicles (EVs) that potentially sustain joint homeostasis and contribute to osteoarthritis (OA) pathogenesis. EVs are putative novel therapeutics for OA, and transport biologically active molecules (including small non-coding RNAs (SNCRNAs)) between cells. This study identified altering SNCRNA cargo in EVs in OA which may act as early diagnostic markers and treatment targets. OA was surgically induced in four skeletally mature Standardbred horses using an osteochondral fragment model in the left middle carpal joint. The right joint underwent sham surgery. Synovial fluid (SF) and plasma were obtained weekly throughout the 70-day study. EVs were isolated using size exclusion chromatography and characterised using nanoparticle tracking (Nanosight), and exosome fluorescence detection and tetraspanin phenotyping (Exoview). RNA was extracted from EVs derived from SF (sham and OA joints) and plasma collected at days 10, 35, 42, 49, 56, 63, and subjected to small RNA sequencing on a NovaSeq SP100 flow cell (Illumina). Nanosight-derived EV characteristics of size and concentration were not significantly different following disease induction. The diameter of the temporal population of plasma and SF-derived exosomes changed significantly for CD9 and CD81 following OA induction with significant temporal, and disease-related changes in CD63 and CD81 protein expressin in plasma and SF. In SF and plasma-derived EVs snoRNAs, snRNAs, tRNAs, lncRNA, y-RNA, piRNAs and scRNA were found. Following pairwise analysis of all-time points we identified 27 miRs DE in plasma and 45 DE miRs in SF. Seven were DE in plasma and SF; miR-451, miR-25, miR-215, miR-92a, miR-let-7c, miR-486-5p, miR-23a. In plasma and SF 35 and 21 snoRNAs were DE with four DE in plasma and SF; U3, snord15, snord46, snord58. This work has identified alterations to OA EV sncRNAs in plasma and SF providing a greater understanding of the role of EVs in early OA

Objectives. Studies which consider the molecular mechanisms of degeneration and regeneration of cartilaginous tissues are seriously hampered by problematic ribonucleic acid (RNA) isolations due to low cell density and the dense, proteoglycan-rich extracellular matrix of cartilage. Proteoglycans tend to co-purify with RNA, they can absorb the full spectrum of UV light and they are potent inhibitors of polymerase chain reaction (PCR). Therefore, the objective of the present study is to compare and optimise different homogenisation methods and RNA isolation kits for an array of cartilaginous tissues. Materials and Methods. Tissue samples such as the nucleus pulposus (NP), annulus fibrosus (AF), articular cartilage (AC) and meniscus, were collected from goats and homogenised by either the MagNA Lyser or Freezer Mill. RNA of duplicate samples was subsequently isolated by either TRIzol (benchmark), or the RNeasy Lipid Tissue, RNeasy Fibrous Tissue, or Aurum Total RNA Fatty and Fibrous Tissue kits. RNA yield, purity, and integrity were determined and gene expression levels of type II collagen and aggrecan were measured by real-time PCR. Results. No differences between the two homogenisation methods were found. RNA isolation using the RNeasy Fibrous and Lipid kits resulted in the purest RNA (A260/A280 ratio), whereas TRIzol isolations resulted in RNA that is not as pure, and show a larger difference in gene expression of duplicate samples compared with both RNeasy kits. The Aurum kit showed low reproducibility. Conclusion. For the extraction of high-quality RNA from cartilaginous structures, we suggest homogenisation of the samples by the MagNA Lyser. For AC, NP and AF we recommend the RNeasy Fibrous kit, whereas for the meniscus the RNeasy Lipid kit is advised. Cite this article: M. Peeters, C. L. Huang, L. A. Vonk, Z. F. Lu, R. A. Bank, M. N. Helder, B. Zandieh Doulabi. Optimisation of high-quality total ribonucleic acid isolation from cartilaginous tissues for real-time polymerase chain reaction analysis. Bone Joint Res 2016;5:560–568. DOI: 10.1302/2046-3758.511.BJR-2016-0033.R3

When joints sustain injury, the release of inflammation cytokines can cleavage matrix proteins and result in cartilage degradation and the subsequent osteoarthritis. RNA therapeutics emerging recently is a very promising approach to efficiently and specifically inhibit disease gene expression. However, the major challenge is how to deliver therapeutic RNA into joint and cartilage. Janus base nanotubes are self-assembled from synthetic Janus bases inspired from DNA base pairs. Based on the charge interaction, we are able to “sandwich” small RNAs among Janus base nanotubes to form tiny, nano-rod shaped delivery vehicles. Such vehicles can be engineered into different sizes and shapes. We have found that short and slim morphologies can greatly increase their penetration to extracellular matrix and delivery into “difficult-to-reach” tissues, such as cartilage and brain. Moreover, by delivering therapeutic siRNA, we have demonstrated its high-efficacy in inhibiting expression of an inflammatory regulator, Interleukin-1 receptor (IL-1R) in articular cartilage. Moreover, the inhibition effect is long-lasting so that joint inflammation and cartilage degradation caused by meniscus injury are greatly inhibited in a mouse model. Therefore, the Janus base nanotubes present a great potential in engineering into nano-structures for RNA delivery. Such approach may become an effective therapeutic against joint inflammation and arthritis

RNA-Seq or whole transcriptome shotgun sequencing has been adopted in the last years as a reference technique to determine the presence and the quantity of different species of RNA in determined biological samples, thanks to it allows the identification every single RNA species transcribed from a reference genome. Meta-profiling takes advantage of the public availability of an increasing set of RNA-Seq data produced by different laboratories to summarize the expression levels of the different RNA species of many samples according to their biological context, giving the opportunity to perform comparisons on the gene expression profiles of different tissues by integrating data derived from a high number of studies. By using Genevestigator™; a platform which integrates RNA-Seq data into meta-profiles, we have performed a comparison between the gene expression profiles of bone, cartilage, muscle tendon and skin by means of interrogating its database with different gene sets and families with relevance to the function of the tissues of the musculoskeletal system. The collagen gene family and genes coding for proteoglycans, matrix metalloproteinases and tissue inhibitors of metalloproteinases, mechanotransduction-related proteins and signalling pathways involved in tissue development and differentiation have been analysed. Hierarchical clustering for every gene set was performed for the understanding the differences and similarities between the different tissues included in the analyses. The results of this study will help to improve our understanding of the musculoskeletal system, and will help to identify new biomarkers and signalling pathways of specific relevance for the bone, cartilage, muscle and tendon

Abstract. Objectives. The term heterotopic ossification (HO) describes lamellar bone formation within soft tissues following injury. A genome-wide scan of patients after hip arthroplasty has identified that variation within the lncRNA CASC20 is associated with HO susceptibility. Previous findings in our lab have demonstrated upregulation of CASC20 during BMP2-induced osteodifferentiation of adipose-derived stem cells (hMAD) alongside osteodifferentiation markers, RUNX2 and OSX. We hypothesize that CASC20 is a novel regulator of bone formation and aim to investigate CASC20 function in bone formation. Methods. 1) We used miRanda prediction algorithm and the ENCORI database to respectively predict which miRNAs CASC20 interacts with and to select for experimentally validated miRNAs. 2) We characterized the expression and functional role of CASC20-interacting miRNAs by respectively analyzing publicly available datasets (GSE107279 and pubmed.ncbi.nlm.nih.gov/26175215/) and by using Gene Ontology (GO) analysis. 3) We overexpressed CASC20 in hMAD using a lentiviral system and tested the effect of CASC20 overexpression in osteodifferentiation and expression of putative CASC20-interacting miRNAs. Results. 1) We identified 64 experimentally validated miRNAs that are predicted to interact with CASC20. 2) GO analysis revealed that the most frequently targeted molecular functions included SMADs, MAPKK and other kinase activities known to play a central role in osteo and chondrogenesis. We found 10 miRNAs including hsa-miR-485-3p that demonstrated down-regulation in both osteo- and chondrogenesis. 3) We found that CASC20-overexpression augmented the osteodifferentiation of hMAD measured in mineralization using Alizarin Red S. CASC20 overexpression increased the expression of osteogenic marker ALP and decreased the expression of hsa-miR-485-3p. Conclusion. Here we show how CASC20 may regulate bone formation by acting as a competitive endogenous RNA (ceRNA). We are currently using CASC20 overexpression model in osteo- and chondrogenesis, and testing CASC20-miRNA interaction to establish the underlying mechanism for the observed associations

Heterotopic ossification (HO) is lamellar bone formation that occurs within tissues that do not normally have properties of ossification. The pathoaetiology of HO is poorly understood. We conducted a genome wide association study to better understand the genetic architecture of HO. 891 patients of European descent (410 HO cases) following THA for primary osteoarthritis were recruited from the UK. HO was assessed from plain AP radiographs of the pelvis. Genomic DNA was extracted, genotyped using the Illumina 610 beadchip and referenced using the 1000 Genome Project panel. HO susceptibility case-control analysis and an evaluation of disease severity in those with HO was undertaken using SNPTESTv2.3.0 on>10 million variants. We tested variants most strongly associated with HO in an independent UK THA replication cohort comprising 209 cases and 211 controls. The datasets were meta-analysed using PLINK. In the discovery cohort 70 signals with an index variant at p<9×10–5 were suggestively associated with HO susceptibility. The strongest signal lay just downstream of the gene ARHGAP18 (rs59084763, effect allele frequency (EAF) 0.19, OR1.87 [1.48–2.38], p=2.48×10–8), the second strongest signal lay within the long non-coding (LNC) RNA gene CASC20 (rs11699612, EAF 0.25, OR1.73 [1.1.40–2.16, p=9.3×10–8). In the discovery cohort 73 signals with an index variant at p<9×10–5 were associated with HO severity. At replication, 12 of the leading 14 susceptibility signals showed a concordant direction of allelic effect and 5 replicated at nominal significance. Following meta-analysis, the lead replicating susceptibility signal was the CASC20 variant rs11699612 (p=2.71×10–11). We identify consistent replicating association of variation within the LNC RNA CASC20 with HO susceptibility after THA. Although the function of CASC20 is currently unknown, possible mechanisms include transcriptional, post-transcriptional and epigenetic regulation of downstream target genes. The work presented here provides new avenues for the development of novel predictive and therapeutic approaches towards HO

Summary. RNAi targeting TNF-alpha inhibits particle-induced inflammation and osteolysis. Introduction. Over 1000,000 joint prostheses are implanted every year in the world. Aseptic joint loosening is a key factor that reduces the longevity of joint prosthesis. Prosthetic wear particles are thought to play a central role in the initiation and development of periprosthetic osteolysis, leading to aseptic loosening of prostheses. This study aims to investigate the effect of RNA interference (RNAi) targeting tumor necrosis factor-alpha (TNF-α) gene on particle-induced inflammation and osteolysis in macrophages in vitro and in vivo. Methods. An in vitro-transcribed small interfering (siRNA) sequences targeting mouse TNF-alpha gene from four candidates was screened and identified and then a lentivirus vector expressing short hairpin RNA (shRNA) was constructed to allow an efficient expression of TNF-alpha-siRNA. Lentivirus-mediated shRNA was transduced into mouse macrophages cells line RAW 264.7. Ceramic particles and titanium particles were added to stimulate cells 24 h after lentivirus transduction. TNF-alpha expression at both mRNA and protein levels were detected quantitatively by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) at different time intervals. Lentivirus-mediated shRNA suspension was locally administered into the murine calvarial model following local particle injection. Multi-slice spiral CT scan was used to evaluate the osteolysis of calvaria by detecting the width of the cranial sutures. Results. Lentivirus-mediated shRNA was effectively transfected, and inhibited the expression of TNF-alpha both in mRNA and protein level in RAW 264.7. Multi-slice spiral CT scan showed that lentivirus-mediated shRNA significantly suppressed osteolysis of mouse calvaria. Conclusion. Our investigation demonstrated that lentivirus-mediated shRNA targeting TNF-alpha gene could inhibit particle-induced inflammation and osteolysis in vitro and in vivo. Therefore, lentivirus-mediated gene therapy has the potential to be developed into a novel therapeutic strategy in the treatment of aseptic joint loosening

Summary. RNAi targeting p110β reduces TNF-alpha production and osteolysis in response to wear particles. Introduction. Aseptic joint loosening is a key factor that reduces the life span of joint prosthesis. Prosthetic wear particles are thought to play a central role in the initiation and development of periprosthetic osteolysis, leading to aseptic loosening of prostheses. This study aims to explore the effect of p110β-targeted small interfering RNA (siRNA) and lentivirus on particle-induced inflammatory cytokine expression in murine macrophage. Methods. siRNA and lentivirus targeting p110β were transfected and infected prior to particle stimulation, respectively. Ceramic and titanium particles of different sizes were prepared to stimulate macrophages. Fluorescence microscopy showed that the siRNA transfection and lentivirus infection efficiency were 74.2 ± 4.2% and 92.3 ± 2.6%, respectively. Results. Real-time polymerase chain reaction (PCR) showed that the levels of tumor necrosis factor-alpha (TNF-alpha) mRNA in the particle stimulation plus RNA interference (RNAi) groups were significantly lower compared with the particle stimulation-only groups (P<0.05), respectively. Similarly, enzyme-linked immunosorbent assay (ELISA) showed that protein levels of TNF-alpha in RNAi-treated groups were significantly decreased after transfection or infection (P<0.05), respectively. Western Blot showed that Phospho-Akt activation was significantly reduced by RNAi. As assessed by CT and micro-CT, particle implantation induced a significant osteolysis effect in mice calvaria, which was limited by p110β-lentivirus addition. Conclusions. p110β subtype of PI3K, followed by activation of phosphor-AKT (Ser473), may possibly participate in the regulation of activating macrophages by wear particles, ultimately resulting in the secretion of TNF-α and osteolysis

Introduction

During osteoarthritis (OA) progression the articular chondrocyte undergoes a phenotypic switch in which the chondrocyte acquires a catabolic and hypertrophy-like state. Bone morphogenetic protein (BMP)-7 is known for its anti-catabolic and pro-anabolic properties in cartilage repair and in OA chondrocytes. In its anabolic state the chondrocyte”s metabolism and protein synthesis are up-regulated. In order to meet a higher demand of protein synthesis, it is expected that the translational capacity of the chondrocyte is increased after exposure to BMP-7. The cellular availability of maturated ribosomal RNAs (rRNA) is rate-limiting in the assembly of ribosomes and previously it has been shown that BMP-7 treatment resulted in increased expression levels of bagpipe homeobox homolog 1 (BAPX-1/NKX3.2). We therefore hypothesize that BMP-7 enhances the translational capacity of articular chondrocytes via BAPX-1/NKX3.2-dependent synthesis of rRNAs.

We found that adipose stem cells are poorly differentiated into bone and that their ability to differentiate into bone varies from cell line to cell line. The osteogenic differentiation ability of the adipose stem cell lines was distinguished through Alzarin Red Staining, and the cell lines that performed well and those that did not were subjected to RNA-seq analysis. The selected gene GSTT1 (glutathione S-transferase theta-1) gene is a member of a protein superfamily that catalyzes the conjugation of reduced glutathione to a variety of hydrophilic and hydrophobic compounds. The purpose of this study is to treat avascular necrosis and bone defect by improving bone regeneration with adipose stem cells introduced with a new GSTT1 gene related to osteogenic differentiation of adipose stem cells. In addition, the GSTT1 gene has the potential as a genetic marker that can select a specific cell line in the development of an adipose stem cell bone regeneration drug. Total RNA was extracted from each sample using the TRIzol reagent. Its concentration and purity were determined based on A260 and A260/A280, respectively, using a spectrophotometer. RNA sequencing library of each sample was prepared using a TruSeq RNA Library Prep Kit. RNA-seq experiments were performed for hADSCs. Cells were transfected with either GSTT1 at 100 nM or siControl (scramble control) by electroporation using a 1050 pulse voltage for 30 ms with 2 pulses using a 10 μl pipette tip. The purpose of this study is to discover genetic markers that can promote osteogenic differentiation of adipose stem cells (hADSCs) through mRNA-seq gene analysis. The selected GSTT1 gene was found to be associated with the enhancement of osteogenic differentiation of adipose stem cells. siRNA against GSTT1 reduced osteogenic differentiation of hADSCs, whereas GSTT1 overexpression enhanced osteogenic differentiation of hADSCs under osteogenic conditions. In this study, GSTT1 transgenic adipose stem cells could be used in regenerative medicine to improve bone differentiation. In addition, the GSTT1 gene has important significance as a marker for selecting adipose stem cells with potential for bone differentiation in the development of a therapeutic agent for bone regeneration cells

Osteoarthritis (OA) is a common cause of chronic pain. Subchondral bone is highly innervated, and bone structural changes directly correlate with pain in OA. Mechanisms underlying skeletal–neural interactions are under-investigated. Bone derived axon guidance molecules are known to regulate bone remodelling. Such signals in the nervous system regulate neural plasticity, branching and neural inflammation. Perturbation of these signals during OA disease progression may disrupt sensory afferents activity, affecting tissue integrity, nociception, and proprioception. Osteocyte mechanical loading and IL-6 stimulation alters axon guidance signalling influencing innervation, proprioception, and nociception. Human Y201 MSC cells, embedded in 3D type I collagen gels (0.05 × 106 cell/gel) in 48 well plastic or silicone (load) plates, were differentiated to osteocytes for 7 days before stimulation with IL-6 (5ng/ml) with soluble IL-6 receptor (sIL-6r (40ng/ml) or unstimulated (n=5/group), or mechanically loaded (5000 μstrain, 10Hz, 3000 cycles) or not loaded (n=5/group). RNA extracted 1hr and 24hrs post load was quantified by RNAseq whole transcriptome analysis (NovaSeq S1 flow cell 2 × 100bp PE reads and differentially expressed neurotransmitters identified (>2-fold change in DEseq2 analysis on normalised count data with FDR p<0.05). After 24 hours, extracted IL-6 stimulated RNA was quantified by RT-qPCR for neurotrophic factors using 2–∆∆Ct method (efficiency=94-106%) normalised to reference gene GAPDH (stability = 1.12 REfinder). Normally distributed data with homogenous variances was analysed by two-tailed t test. All detected axonal guidance genes were regulated by mechanical load. Axonal guidance genes were both down-regulated (Netrin1 0.16-fold, p=0.001; Sema3A 0.4-fold, p<0.001; SEMA3C (0.4-fold, p<0.001), and up-regulated (SLIT2 2.3-fold, p<0.001; CXCL12 5-fold, p<0.001; SEMA3B 13-fold, p<0.001; SEMA4F 2-fold, p<0.001) by mechanical load. IL6 and IL6sR stimulation upregulated SEMA3A (7-fold, p=0.01), its receptor Plexin1 (3-fold, p=0.03). Neutrophins analysed in IL6 stimulated RNA did not show regulation. Here we show osteocytes regulate multiple factors which may influence innervation, nociception, and proprioception upon inflammatory or mechanical insult. Future studies will establish how these factors may combine and affect nerve activity during OA disease progression

To address the current challenge of anterior cruciate ligament (ACL) reconstruction, this study is the first to fabricate a braided collagen rope (BCR) which mimics native hamstring for ACL reconstruction. The study aims to evaluate the biological and biomechanical properties of BCR both in vivo and vitro. Rabbit ACL reconstruction model using collagen rope and autograft (hamstring tendon) was conducted. The histological and biomechanical evaluations were conducted at 6-, 12-, 18, 26-week post-operation. In vitro study included cell morphology analysis, cell function evaluation and RNA sequencing of the tenocytes cultured on BCR. A cadaver study was also conducted to verify the feasibility of BCR for ACL reconstruction. BCR displays satisfactory mechanical strength similar to hamstring graft for ACL reconstruction in rabbit. Histological assessment showed BCR restore ACL morphology at 26 weeks similar to native ACL. The superior dynamic ligamentization in BCR over autograft group was evidenced by assessment of cell and collagen morphology and orientation. The in vitro study showed that the natural collagen fibres within BCR enables to signal the morphology adaptation and orientation of human tenocytes in bioreactor. BCR enables to enhance cell proliferation and tenogenic expression of tenocytes as compared to hydrolysed collagen. We performed an RNA-Sequencing (RNA-seq) experiment where RNA was extracted from tenocyte seeded with BCR. Analysis of enriched pathways of the up-regulated genes revealed that the most enriched pathways were the Hypoxia-inducible factor 1-alpha (HIF1A) regulated networks, implicating the possible mechanism BCR induced ACL regeneration. The subsequent cadaver study was conducted to proof the feasibility of BCR for ACL reconstruction. This study demonstrated the proof-of-concept of bio-textile braided collagen rope for ACL reconstruction, and the mechanism by which BCR induces natural collagen fibres that positively regulate morphology and function of tenocytes

Stimulation of the mechanosensitive ion channel, Piezo1 promotes bone anabolism and SNPs in the Piezo1 locus are associated with changes in fracture risk. Osteocytes function as critical regulators of bone homeostasis by sensing mechanical signals. The current study used a human, cell-based physiological, 3D in vitro model of bone to determine whether loading of osteocytes in vitro results in upregulation of the Piezo1 pathway. Human Y201 MSCs, embedded in type I collagen gels and differentiated to osteocytes for 7-days, were subjected to pathophysiological load (5000 µstrain, 10Hz, 5 mins; n=6) with unloaded cells as controls (n=4). RNA was extracted 1-hr post load and assessed by RNAseq analysis. To mimic mechanical load and activate Piezo1, cells were differentiated to osteocytes for 13 days and treated ± Yoda1 (5µM, 2- and 24-hs, n=4); vehicle treated cells served as controls (n=4). RNA was subjected to RT-qPCR and data normalised to the housekeeping gene, YWHAZ. Media was analysed for IL6 release by ELISA. Mechanical load upregulated Piezo1 gene expression (16.5-fold, p<0.001) and expression of the transcription factor NFATc1, and matricellular protein CYR61, known regulators of Piezo1 mechanotransduction (3-fold; p= 5.0E-5 and 6.8-fold; p= 6.0E-5, respectively). After 2-hrs, Yoda1 increased the expression of the early mechanical response gene, cFOS (11-fold; p=0.021), mean Piezo1 expression (2.3-fold) and IL-6 expression (103-fold, p<0.001). Yoda1 increased the release of IL6 protein after 24 hours (7.5-fold, p=0.001). This study confirms Piezo1 as an important mechanosensor in osteocytes. Piezo1 activation mediated an increase in IL6, a cytokine that drives inflammation and bone resorption providing a direct link between mechanical activation of Piezo1, bone remodeling and inflammation, which may contribute to mechanically induced joint degeneration in diseases such as osteoarthritis. Mechanistically, we hypothesize this may occur through promoting Ca2+ influx and activation of the NFATc1 signaling pathway

Varus malalignment increases the susceptibility of cartilage to mechanical overloading, which stimulates catabolic metabolism to break down the extracellular matrix and lead to osteoarthritis (OA). The altered mechanical axis from the hip, knee to ankle leads to knee joint pain and ensuing cartilage wear and deterioration, which impact millions of the aged population. Stabilization of the remaining damaged cartilage, and prevention of further deterioration, could provide immense clinical utility and prolong joint function. Our previous work showed that high tibial osteotomy (HTO) could shift the mechanical stress from an imbalanced status to a neutral alignment. However, the underlying mechanisms of endogenous cartilage stabilization after HTO remain unclear. We hypothesize that cartilage-resident mesenchymal stem cells (MSCs) dampen damaged cartilage injury and promote endogenous repair in a varus malaligned knee. The goal of this study is to further examine whether HTO-mediated off-loading would affect human cartilage-resident MSCs' anabolic and catabolic metabolism. This study was approved by IACUC at Xi'an Jiaotong University. Patients with medial compartment OA (52.75±6.85 yrs, left knee 18, right knee 20) underwent open-wedge HTO by the same surgeons at one single academic sports medicine center. Clinical data was documented by the Epic HIS between the dates of April 2019 and April 2022 and radiographic images were collected with a minimum of 12 months of follow-up. Medial compartment OA with/without medial meniscus injury patients with unilateral Kellgren /Lawrence grade 3–4 was confirmed by X-ray. All incisions of the lower extremity healed well after the HTO operation without incision infection. Joint space width (JSW) was measured by uploading to ImageJ software. The Knee injury and Osteoarthritis Outcome Score (KOOS) toolkit was applied to assess the pain level. Outerbridge scores were obtained from a second-look arthroscopic examination. RNA was extracted to quantify catabolic targets and pro-inflammatory genes (QiaGen). Student's t test for two group comparisons and ANOVA analysis for differences between more than 2 groups were utilized. To understand the role of mechanical loading-induced cartilage repair, we measured the serial changes of joint space width (JSW) after HTO for assessing the state of the cartilage stabilization. Our data showed that HTO increased the JSW, decreased the VAS score and improved the KOOS score significantly. We further scored cartilage lesion severity using the Outerbridge classification under a second-look arthroscopic examination while removing the HTO plate. It showed the cartilage lesion area decreased significantly, the full thickness of cartilage increased and mechanical strength was better compared to the pre-HTO baseline. HTO dampened medial tibiofemoral cartilage degeneration and accelerate cartilage repair from Outerbridge grade 2 to 3 to Outerbridge 0 to 1 compared to untreated varus OA. It suggested that physical loading was involved in HTO-induced cartilage regeneration. Given that HTO surgery increases joint space width and creates a physical loading environment, we hypothesize that HTO could increase cartilage composition and collagen accumulation. Consistent with our observation, a group of cartilage-resident MSCs was identified. Our data further showed decreased expression of RUNX2, COL10 and increased SOX9 in MSCs at the RNA level, indicating that catabolic activities were halted during mechanical off-loading. To understand the role of cartilage-resident MSCs in cartilage repair in a biophysical environment, we investigated the differentiation potential of MSCs under 3-dimensional mechanical loading conditions. The physical loading inhibited catabolic markers (IL-1 and IL-6) and increased anabolic markers (SOX9, COL2). Knee-preserved HTO intervention alleviates varus malalignment-related knee joint pain, improves daily and recreation function, and repairs degenerated cartilage of medial compartment OA. The off-loading effect of HTO may allow the mechanoregulation of cartilage repair through the differentiation of endogenous cartilage-derived MSCs

Abstract. Objectives. Osteocytes function as critical regulators of bone homeostasis by sensing mechanical signals. Stimulation of the mechanosensitive ion channel, Piezo1 promotes bone anabolism and deletion of Piezo1 in osteoblasts and osteocytes decreases bone mass and bone strength in mice. This study determined whether loading of osteocytes in vitro results in upregulation of the Piezo1 pathway. Methods. Human MSC cells (Y201), embedded in type I collagen gels and differentiated to osteocytes in osteogenic media for 7-days, were subjected to pathophysiological load (5000 µstrain, 10Hz, 5 mins; n=6) with unloaded cells as controls (n=4). RNA was extracted 1-hr post load and Piezo1 activation assessed by RNAseq analysis (NovaSeq S1 flow cell 2 × 100bp PE reads). To mimic mechanical load and activate Piezo1, Y201s were differentiated to osteocytes in 3D gels for 13 days and treated, with Yoda1 (5µM, 2 hours, n=4); vehicle treated cells served as controls (n=4). Extracted RNA was subjected to RT-qPCR and data analysed by Minitab. Results. Low mRNA expression of PIEZO1 in unloaded cells was upregulated 5-fold following 1-hr of mechanical load (p=0.003). In addition, the transcription factor NFATc1, a known regulator of Piezo1 mechanotransduction, was also upregulated by load (2.4-fold; p=0.03). Y201 cells differentiated in gels expressed the osteocyte marker, SOST. Yoda1 upregulated PIEZO1 (1.7-fold; p=0.057), the early mechanical response gene, cFOS (4-fold; p=0.006), COL1A1 (3.9-fold; p=0.052), and IL-6 expression (7.7-fold; p=0.001). Discussion. This study reveals PIEZO1 as an important mechanosenser in osteocytes. Piezo 1 mediated increases in the bone matrix protein, type I collagen, and IL-6, a cytokine that drives inflammation and bone resorption. This provides a direct link between mechanical activation of Piezo 1, bone remodelling and inflammation, which may contribute to mechanically-induced joint degeneration in osteoarthritis. Mechanistically, we hypothesise this may occur through promoting Ca2+ influx and activation of the NFAT1 signalling pathway

Tendon healing is a complex process that often results in compromised healing of the tendon tissue. It has recently been shown that temporal changes in the expression profile and the histological tissue quality of the tendons occur during the early healing process after acute Achilles tendon rupture. Whether these changes are accompanied by an altered healing process, is not yet known and was the aim of the present study. Tendon biopsies were obtained from 24 patients with acute Achilles tendon rupture at the time of surgery (2–9 days after rupture) and examined histologically as well as on RNA level. Histologically, the tendon architecture, the amount of aligned collagen, glycosaminoglycan and fat as well as the cellularity, vascularity and immune cell infiltration were determined. On RNA level the expression of markers for the modeling/remodeling (MMPs and TIMPs), collagens (1, 3, 5), tendon markers (scleraxis, tenomodulin), pro- and anti-inflammatory markers (IL-1beta, IL6, IL10, IL33, TNFa, TGF-beta1, COX2) and immune cell markers (CD3, CD68, CD80, CD206) were analyzed by Real-Time PCR. To determine the clinical outcome, the patients were followed up 12 months after the operation and the following scores were recorded: Subjective score, Tegner score, Visual Analog Scale (VAS) pain, VAS function, Matles Test, Achilles tendon total rupture score (ATRS), Therman 100-points score, Heel rise test. Statistics: Spearman correlation analysis. Correlation analysis shows that early post-rupture surgery is associated with better clinical outcome (ATRS Score: p=0.022). Histologically, a good functional healing outcome shows a positive correlation to the amount of aligned collagen (Heel Rise Test: p = 0.009) and glycosaminoglycans in the tendon (Heel Rise Test: p = 0.026, Matles difference: p = 0.029), as well as a negative correlation to the fat content (Thermann score: p = 0.018, subjective score: p = 0.027, VAS function: p = 0.031). On RNA level, a good healing outcome correlates with increased expression of MMP13, collagen 1, 3, 5 (Heel Rise Test: p = 0.019, p = 0.048, p = 0.030), and TIMP2 (Tegner Score: p = 0.040), TGF-beta1 (Thermann Score: p = 0.032) and CD80 (ATRS: p = 0.025, Thermann score:, p = 0.032). Whereas a limited healing outcome is associated with an increased expression of MMP2 (Heel Rise Test: p = 0.033), MMP3 (Matles Test: p=0.001, Heal Rise test p = 0.017), and IL33 (Tegner Score: p = 0.047). The results of the study show a clear relationship between the tendon biology at the time of the surgery and the clinical and functional healing outcome 12 months after the operation. Especially matrix formation and remodeling play a crucial role, while the examined immunological factors seem to influence the tendon healing to a lesser extent. The modulation of matrix formation could potentially lead to improved treatment options in the future

To date, few studies have investigated the feasibility of the loop-mediated isothermal amplification (LAMP) assay for identifying pathogens in tissue samples. This study aimed to investigate the feasibility of LAMP for the rapid detection of methicillin-susceptible or methicillin-resistant Staphylococcus aureus (MSSA or MRSA) in tissue samples, using a bead-beating DNA extraction method. Twenty tissue samples infected with either MSSA (n = 10) or MRSA (n = 10) were obtained from patients who underwent orthopedic surgery for suspected musculoskeletal infection between December 2019 and September 2020. DNA was extracted from the infected tissue samples using the bead-beating method. A multiplex LAMP assay was conducted to identify MSSA and MRSA infections. To recognize the Staphylococcus genus, S. aureus, and methicillin resistance, 3 sets of 6 primers for the 16S ribosomal ribonucleic acid (rRNA) and the femA and mecA genes were used, respectively. The limit of detection and sensitivity (detection rate) of the LAMP assay for diagnosing MSSA and MRSA infection were analyzed. The results of this study suggest that the LAMP assay performed with tissue DNA samples can be a useful diagnostic method for the rapid detection of musculoskeletal infections caused by MSSA and MRSA

More and more evidences showed that cartilage harbored local progenitor cells that could differentiate toward osteoblast, chondrocyte, and adipocyte. However, our previous results showed that osteoarthritis derived chondroprogenitor cells (OA-CPC) exhibited strong osteogenic potential even in chondrogenic condition. How to promote their chondrogenic potential is the key for cartilage repair and regeneration in osteoarthritis. Recently, lipid availability was proved to determine skeletal progenitor fate. Therefore, we aim to determine whether lipid inhibition under 3D culture condition could enhance OA-CPC chondrogenesis. Moreover, glucose concentration was also evaluated for chondrogenic capacity. Although there are many researches showed that lower glucose promotes chondrogenesis, in our results, we found that OA-CPC in high concentration of glucose (4.5g/L) with lipid inhibitor (GW1100) showed strongest chondrogenic potential, which could form largest cell pellet with strong proteoglycan staining, COL II expression and no COL I expression. Besides, COL2A1 was increased and COL10A1 was decreased significantly by GW1100 under high glucose condition in 2D culture. Interestingly, although the expression level of MMP13 was not changed by GW1100 at RNA and protein level, less MMP13 protein secreted out of cell nuclear. In summary, we estimated that higher glucose and lower lipid supplies benefit OA-CPC chondrogenesis and cartilage repair

Heterotopic ossification (HO) is defined as aberrant bone formation in extraskeletal locations. In this process, local stromal cells of mesenchymal origin abnormally differentiate, resulting in pathologic cartilage and bone matrix deposition. However, the specific cell type and mechanisms beyond this process are not well understood, in part due to the heterogeneity of progenitor cells involved. Here, a combination of single cell RNA sequencing (scRNA-Seq) and lineage tracing, defined the extent to which synovial / tendon sheath progenitor cells contribute to HO. For this purpose, a Tppp3 (tubulin polymerization-promoting protein family member 3) inducible reporter model was used, in combination with either Scx (Scleraxis) or Pdgfra (Platelet derived growth factor receptor alpha) reporter animals. Both arthroplasty-induced and tendon injury-mouse experimental HO models were utilized. ScRNA-Seq of tendon-induced traumatic HO suggested that Tppp3 is a progenitor cell marker for either osteochondral or tendon or cells. After HO induction, Tppp3 reporter+ cell population expanded in number and contributed to cartilage and bone formation in tendon and joint-associated HO. Using double reporter animals, we found that both Pdgfra+Tppp3+ and Pdgfra+Tppp3- progenitor cells produced HO-associated cartilage. Finally, the examination of human samples showed a significant population of TPPP3+ cells overlapping with osteogenic markers in areas of HO. Overall, these results provide novel observations that peritenon and synovial progenitor cells undergo abnormal osteochondral differentiation and contribute to heterotopic bone formation after trauma