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

Objectives. Given the function of adiponectin (ADIPOQ) on the inflammatory condition of obesity and osteoarthritis (OA), we hypothesized that the ADIPOQ gene might be a candidate gene for a marker of susceptibility to OA. Methods. We systematically screened three tagging polymorphisms (rs182052, rs2082940 and rs6773957) in the ADIPOQ gene, and evaluated the association between the genetic variants and OA risk in a case-controlled study that included 196 OA patients and 442 controls in a northern Chinese population. Genotyping was performed using the Sequenom MassARRAY iPLEX platform. Results. The single nucleotide polymorphism (SNP) rs182052 was found to be potentially associated with knee OA risk (additive model: odds ratio = 1.38; 95% confidence interval 1.07 to 1.76; p = 0.012). Furthermore, a non-significant association was observed for rs182052 and body mass index with regard to OA risk in interaction analyses (p = 0.063). Similarly, no significant interaction was detected for rs182052 and age with regard to OA risk (p = 0.614). Conclusion. These findings suggest that the SNP rs182052 in the ADIPOQ gene may potentially modify individual susceptibility to knee OA in the Chinese population. Further studies are warranted to investigate our findings in more depth. Cite this article: L. Jiang, X. Zhu, J. Rong, B. Xing, S. Wang, A. Liu, M. Chu, G. Huang. Obesity, osteoarthritis and genetic risk: The rs182052 polymorphism in the ADIPOQ gene is potentially associated with risk of knee osteoarthritis. Bone Joint Res 2018;7:494–500. DOI: 10.1302/2046-3758.77.BJR-2017-0274.R1

Staphylococcus aureus (SA), the predominant pathogen in human osteomyelitis, is known to persist by forming intracellular reservoirs, including in bone cells (Schwarz et al., 2019, Yang et al., 2018, Krauss et al., 2019, Gao et al., 2020, Bosse et al., 2005), promoting decreased antibiotic susceptibility. However, there are no evidence-based treatment guidelines for intracellular SA infections in osteomyelitis. We sought to address this by systematically reviewing the literature and, testing a selection of antibiotic treatments in a clinically relevant in vitro assay. We conducted a systematic review of the literature to determine the current evidence for the efficacy of antibiotics against intracellular SA infections relevant to osteomyelitis. For the antibiotics identified as potentially useful, we determined their minimal inhibitory concentration (MIC) against 11 clinical osteomyelitis SA- isolates. We selected those for further testing reported able to reach a higher concentration in the bone than the identified MIC against the majority of strains. Thus, rifampicin, oxacillin, linezolid, levofloxacin, oritavancin and doxycycline were tested in human SaOS-2-osteocyte infection models (Gunn et al., 2021) of acute (1d) or chronic (14d) infection to clear intracellular SA. Antibiotics were tested at 1x/4x/10x the MIC for the duration of 1d or 7d in each model. A systematic review found that osteoblasts and macrophages have mostly been used to test immediate short-term activity against intracellular SA, with a high variability in methodology. However, some extant evidence supports that rifampicin, oritravancin, linezolid, moxifloxacin and oxacillin may be effective intracellular treatments. While studies are ongoing, in vitro testing in a clinically relevant model suggests that rifampicin, oxacillin and doxycycline could be effectively used to treat osteomyelitic intracellular SA infections. Importantly, these have lower MICs against multiple clinical isolates than their respective clinically-achievable bone concentrations. The combined approach of a systematic review and disease-relevant in vitro screening will potentially inform as to the best approach for treating osteomyelitis where intracellular SA infection is confirmed or suspected

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

Objectives. Several genome-wide association studies (GWAS) of bone mineral density (BMD) have successfully identified multiple susceptibility genes, yet isolated susceptibility genes are often difficult to interpret biologically. The aim of this study was to unravel the genetic background of BMD at pathway level, by integrating BMD GWAS data with genome-wide expression quantitative trait loci (eQTLs) and methylation quantitative trait loci (meQTLs) data. Method. We employed the GWAS datasets of BMD from the Genetic Factors for Osteoporosis Consortium (GEFOS), analysing patients’ BMD. The areas studied included 32 735 femoral necks, 28 498 lumbar spines, and 8143 forearms. Genome-wide eQTLs (containing 923 021 eQTLs) and meQTLs (containing 683 152 unique methylation sites with local meQTLs) data sets were collected from recently published studies. Gene scores were first calculated by summary data-based Mendelian randomisation (SMR) software and meQTL-aligned GWAS results. Gene set enrichment analysis (GSEA) was then applied to identify BMD-associated gene sets with a predefined significance level of 0.05. Results. We identified multiple gene sets associated with BMD in one or more regions, including relevant known biological gene sets such as the Reactome Circadian Clock (GSEA p-value = 1.0 × 10. -4. for LS and 2.7 × 10. -2. for femoral necks BMD in eQTLs-based GSEA) and insulin-like growth factor receptor binding (GSEA p-value = 5.0 × 10. -4. for femoral necks and 2.6 × 10. -2. for lumbar spines BMD in meQTLs-based GSEA). Conclusion. Our results provided novel clues for subsequent functional analysis of bone metabolism, and illustrated the benefit of integrating eQTLs and meQTLs data into pathway association analysis for genetic studies of complex human diseases. Cite this article: W. Wang, S. Huang, W. Hou, Y. Liu, Q. Fan, A. He, Y. Wen, J. Hao, X. Guo, F. Zhang. Integrative analysis of GWAS, eQTLs and meQTLs data suggests that multiple gene sets are associated with bone mineral density. Bone Joint Res 2017;6:572–576

Abstract. Objectives. A damaged vertebral body can exhibit accelerated ‘creep’ under constant load, leading to progressive vertebral deformity. However, the risk of this happening is not easy to predict in clinical practice. The present cadaveric study aimed to identify morphometric measurements in a damaged vertebral body that can predict a susceptibility to accelerated creep. Methods. Mechanical testing of 28 human spinal motion segments (three vertebrae and intervening soft tissues) showed how the rate of creep of a damaged vertebral body increases with increasing “damage intensity” in its trabecular bone. Damage intensity was calculated from vertebral body residual strain following initial compressive overload. The calculations used additional data from 27 small samples of vertebral trabecular bone, which examined the relationship between trabecular bone damage intensity and residual strain. Results. Calculations from trabecular bone samples showed a strong non-linear relationship between residual strain and trabecular bone damage intensity (R. 2. = 0.78, P < 0.001). In damaged vertebral bodies, damage intensity as calculated from residual strain was then related to vertebral creep rate (R. 2. = 0.39, P = 0.001). This procedure enabled accelerated vertebral body creep to be predicted from morphological changes (residual strains) in the damaged vertebral body. Conclusion. These findings suggest that morphometric measurements obtained from fractured vertebrae can be used to quantify vertebral damage intensity and hence to predict progressive vertebral deformity

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

Background. Despite the known multifactorial nature of scaphoid wrist fracture non-union, a possible genetic predisposition for the development of this complication remains unknown. This pilot study aimed to address this issue by performing Single Nucleotide Polymorphisms (SNPs) analysis of specific genes known to regulate fracture healing. Materials and Methods. We reviewed 120 patients in a retrospective case-control study from the Hand Surgery Department of Asepeyo Hospital. The case group comprised 60 patients with confirmed scaphoid wrist non-union, diagnosed by Magnetic Resonance Imaging (MRI) and Computed Tomography (CT). The control group comprised 60 patients with scaphoid fracture and complete bone consolidation. Sampling was carried out with a puncture of a finger pad using a sterile, single-use lancet. SNPs were determined by real-time polymerase chain reaction (PCR) using specific, unique probes with the analysis of the melting temperature of hybrids. The X2 test compared genotypes between groups. Multivariate logistic regression analysed the significance of many covariates and the incidence of scaphoid wrist non-union. Results. We found significant differences in subjects who had a smoking habit (p=0.001), high blood pressure (p<0.001), and surgical treatment (p=0.002) in patients with scaphoid non-union. There were more Caucasians (p=0.04) and males (p=0.001) in the case group. Falls were the main mechanism of fracture. The CC genotype in GDF5 (rs143383) was more frequent in patients with scaphoid non-union compared to the controls (p=0.02). CT was prevalent in the controls (p=0.02). T allele in GDF5 was more frequent in patients without non-union (p=0.001). Conclusions. Individuals who were carriers of the CC genotype in GDF5 showed higher susceptibility to suffering scaphoid wrist non-union. Furthermore, being a carrier of CT and T allele suggests that this could be behave as a protection factor against non-union. This is the first clinical study to investigate the potential existence of genetic susceptibility to scaphoid wrist fracture non-union. Level of evidence. Level III, Cross Sectional Study, Epidemiology Study

Summary Statement. Combination of antibiotics with N-acetylcisteine and sub-MIC concentration of erythromycin was evaluated in two collection and 16 clinical strains of staphylococci isolated from PJI. The results were strain-dependent, so it evidences the necessity of perform individual studies of biofilm susceptibility. Objectives. Staphylococci are the most common cause of prosthetic joint infections (PJI) (1), making the treatment of this disease difficult due to the increased resistance to antibiotics of biofilms. Combination between antibiotics and other compounds could be a good alternative. The aim of this study was to evaluate the effect of the combination of two compounds with nine antibiotics in biofilms formed by staphylococcal strains isolated from PJI. Methods. 16 clinical strains (8 S. aureus and 8 S. epidermidis) isolated from patients with PJI as well as 2 collection strains (S. aureus 15981 and S. epidermidis ATCC 35984) were tested against 9 antibiotics (rifampin, vancomycin, tigecycline, clindamycin, cotrimoxazole, ciprofloxacin, cloxacillin, daptomycin and fosfomycin) in combination with NAC 1024 μg/mL and erythromycin at subinhibitory concentration (0.12 μg/mL), which was established after the determination of MIC according to EUCAST recommendations. The Calgary Biofilm Device (CBD) was used to determine the susceptibility of the biofilms to these combinations. The Minimal Biofilm Eradication Concentration (MBEC) for the all the antibiotics alone was determined in a previous study. All the experiments were performed by triplicate. Results. All the S. aureus strains showed homogeneous results, and the addition of NAC or erythromycin at the tested concentrations has not a clear effect in the antibiotic susceptibility of the biofilm, although combination of tigecycline with NAC seems even to increase the MBEC in most cases. Almost all clinical strains were MRSA. Regarding S. epidermidis strains, the results were strain-dependant. The combination with NAC seems to increase the MBEC for rifampin and tigecycline in some strains. However, there was a slight MBEC decrease with cotrimoxazole, ciprofloxacin, cloxacillin, daptomycin and fosfomycin. Erythromycin combinations resulted to be successful only in a few cases, and there is no an apparent relationship between erythromycin resistance and combination results. It is especially remarkable that one strain (P-23.2) showed a slight decrease of the MBEC combining both substances with most of the antibiotics tested. Conclusion. None of the combinations tested was clearly effective against biofilms for all strains of both species. This heterogeneity showed the necessity of making an individual study of each strain. The search of new strategies to fight against PJI is mandatory and further investigation is needed

Osteoporosis is a disease when bone mass and tissue is lost, with a consequent increase in bone fragility and increase susceptibility to develop fracture. The osteoporosis prevalence increases markedly with age, from 2% at 50 years to more than 25% at 80 years. 1. in women. The vast majority of distal radius fractures (DRFs) can be considered fragility fractures. The DRF is usually the first medical presentation of these fractures. With an aging population, all fracture clinics should have embedded screening for bone health and falls risk. DRF is the commonest type of fracture in perimenopausal women and is associated with an increased risk of later non-wrist fracture of up to one in five in the subsequent decade. 2. . According to the national guidelines in managing the fragility fractures of distal radius with regards the bone health review, we, as orthopedic surgeons, are responsible to detect the risky patients, refer them to the responsible team to perform the required investigations and offer the treatment. We reviewed our local database (E-trauma) all cases of fracture distal radius retrospectively during the period from 01/08/2019 to 29/09/2019. We included total of 45 patients who have been managed conservatively and followed up in fracture clinic. Our inclusion criteria was: women aged 65 years and over, men aged 75 years and over with risk factors, patients who are more than 50 years old and sustained low energy trauma whatever the sex is or any patient who has major risk factor (current or frequent recent use of oral or systemic glucocorticoids, untreated premature menopause or previous fragility fracture). We found that 96% of patients were 50 years old or more and 84% of the patients were females. 71% of patients were not referred to Osteoporosis clinic and 11% were already under the orthogeriatric care and 18% only were referred. Out of the 8 referred patients, 3 were referred on 1st appointment, 1 on the 3rd appointment, 1 on discharge from fracture clinic to GP again and 3 were without clear documentation of the time of referral. We concluded that we as trust are not compliant to the national guidelines with regards the osteoporosis review for the DRF as one of the first common presentations of fragility fractures. We also found that the reason for that is that there is no definitive clear pathway for the referral in our local guidelines. We recommended that the Osteoporosis clinic referral form needs to be available in the fracture clinic in an accessible place and needs to be filled by the doctor reviewing the patient in the fracture clinic in the 1st appointment. A liaison nurse also needs to ensure these forms have been filled and sent to the orthogeriatric team. Alternatively, we added a portal on our online database (e-trauma), therefore the patient who fulfils the criteria for bone health review should be referred to the orthogeriatric team to review

Lumbar disc herniation represents by far the most prevalent pathology, causing pain and sciatica and constitutes an important cause of disability and one of the most cost-intensive health problems. The aetiology is very complex. In recent years, it has been suggested in twin and family studies that genetic risk factors contribute to the development of LDH. Our purpose is to analyse genetic susceptibility to symptomatic LDH in Spanish surgical patients treated with different surgical techniques. Single-nucleotide polymorphisms (SNPs) in VDR, GDF5, Col1A1, THBS2 and CHST were genotyped in a case-control study with 50 symptomatic LDH in Spanish surgical patients and 50 Spanish health controls. All patients provided signed informed consent. Sampling was carried out with a puncture of the pad of a finger using a sterile, single-use lancet. SNPs were determined by real-time polymerase chain reaction (PCR) using specific, unique probes with the analysis of the melting temperature of hybrids. The X2 test compared genotypes between groups. Multivariate logistic regression analysed the significance of many covariates and the incidence of LDH. We found significant differences in age, gender and smoking status between the two groups. There were significant differences in the CC (rs2228570) genotype in VDR in patients with LDH (p<0.05). There were significant differences in the GT (rs1800012) genotype in Col1A1 in patients with LDH (p=0.001). In Col1A1, T allele was more frequent in the case group than in the control group (p<0.001). Regarding surgical techniques, of the 50 patients included in the cases group, 25 were treated with open microdiscectomy and 25 received endoscopic discectomy. Outcomes were assessed at 12 months using VAS, and NASS instrument. Postoperative pain and pain medication were significantly reduced in the endoscopic group. Patient satisfaction is greater in the endoscopic group, with shorter hospital stays and earlier return to normal activity. GT genotype in Col1A1 was more frecuent in the endoscopic group compared to the microdiscectomy group (p=0.002). CC genotype in VDR and GT genotype in Col1A1 are associated with symptomatic LDH susceptibility in Spanish surgical patients. GT genotype in Col1A1 is associated with symptomatic LDH treated with full-endoscopic discectomy

Osteoarthritis is the most prevalent joint disease, causing severe pain, deformity and a loss of mobility. Low back pain (LBP), frequently associated with degeneration of the intervertebral disc (IVD), is the No.1 cause of Years Lived with Disability. Age is a major risk factor for both conditions. However, the reasons why susceptibility to these conditions increases with age are poorly understood. The circadian (24 hourly) clocks in the brain and periphery direct key aspects of physiology through rhythmic control of tissue-specific sets of downstream genes. Work from our group focuses on the roles of circadian clocks in the articular cartilage and IVD. We show that the daily rhythm in these tissues becomes dampened and out-of-phase during ageing. Further, our data identify circadian clock disruption in cartilage and IVD as a new target of inflammation. Moreover, we show that mice with targeted knockout of an essential clock gene (BMAL1) in chondrocytes and disc cells have profound, yet tissue-specific degeneration in the articular cartilage and IVD. These findings implicate the local skeletal clock as a key regulatory mechanism for tissue homeostasis. This new avenue of research holds potential to better understand, and eventually treat these debilitating conditions

Modular hip prostheses were introduced to optimize the intra-surgical adaptation of the implant design to the native anatomy und biomechanics of the hip. The downside of a modular implant design with an additional modular interface is the potential susceptibility to fretting, crevice corrosion and wear. For testing hip implants with proximal femoral modularity according to ISO & ASTM, sodium chloride solutions are frequently used to determine the fatigue strength and durability of the stem-neck connection. The present study illustrate that the expansion of standard requirements of biomechanical testing is necessary to simulate metal ion release as well as fretting and crevice corrosion by using alternative test fluids. To assess the primary stability of tibial plateaus in vitro, different approaches had been undergone: cement penetration depth analysis, static tension or compression loading until interface failure. However, these test conditions do not reflect the in vivo physiologic loading modes, where the tibial plateau is predominantly subjected to combined compression and shear forces. The objectives were to evaluate the impact of the tibial keel & stem length on the primary stability of a posterior-stabilised tibial plateau under dynamic compression-shear loading conditions in human tibiae

Familial osteochondritis dissecans (FOCD) is an inherited defect of cartilage and bone characterized by development of large cartilage lesions in multiple joints, short stature and early onset osteoarthritis. We have studied a family from Northern Sweden with FOCD over five generations. All affected family members have a heterozygous missense mutation on exon 17 of the aggrecan gene, resulting in a Val-Met amino acid replacement in the G3 aggrecan C-type lectin domain (CLD). Aggrecan, a major proteoglycan of articular cartilage produced by chondrocytes, has a large protein core richly substituted with sulfated glycosaminoglycan chains. The unique structure, its high concentration within the cartilage extracellular matrix and its ability to form a supermolecular complex with hyaluronan and bind to other matrix proteins all profoundly influence the biomechanical properties of the tissue. Deletion of CLD in a chick aggrecan construct was found to influence its secretion from chondrocytes and human aggrecan constructs carrying the V2303M mutation showed diminished interactions with the ECM proteins tenascin-R, fibulin-1 and fibulin-2. To investigate the pathogenesis of FOCD, we studied chondrogenic differentiation of patient bone marrow mesenchymal stem cells and induced pluripotent stem cells. We demonstrated that the mutation results in accumulation of unfolded or misfolded aggrecan within the lumen of the chondrocyte endoplasmic reticulum. Associated with this is the failure to assemble a normal extracellular matrix. This explains the susceptibility of these patients to cartilage injury and the degenerative changes that lead to early onset osteoarthritis

Tendon and ligament injuries represent highly prevalent and unmet clinical challenge that may significantly benefit from tissue engineering therapeutic strategies, once optimal cell source and biomolecules regulating tendon homeostasis are properly defined. Herein, we aimed to evaluate the expression of tendon/ligament markers in two novel cell populations, namely human dental pulp stem cells (DPSCs) and periodontal ligament cells (PDLCs), in response to supplementation with TGF-β ligands relevant for tendon development and healing, as well as under standard tri-lineage differentiation conditions. DPSCs and PDLCs were isolated from sound human permanent molars removed for orthodontic reasons. Pulp tissue and periodontal ligament were minced and digested with collagenase (3mg/mL) and cells were expanded in α-MEM supplemented with 10% fetal bovine serum (basal medium). To evaluate the susceptibility of DPSCs and PDLCs to tenogenic induction, cells were seeded at density of 1000 cells/cm. 2. and cultured up to 21 days in basal medium or media supplemented with TGF-β3 (10ng/ml), or GDF-5 (50 ng/ml). Cell response was evaluated weakly by analysis of expression of tendon, bone and cartilage markers, employing real time RT-PCR and immunocytochemistry. A significant increase in collagen I and collagen III expression was observed with the culture progression in all conditions, with abundant matrix being deposited by day 14. A significant upregulation of scleraxis expression was demonstrated in response to supplementation with TGF-β3 in both cell populations, when compared to basal medium and medium with GDF-5. It was concluded that TGF-β3 may represent an effective inducer of stem cell tenogenic differentiation

Background. Rotator cuff disease (RCD) is the most common cause of shoulder pain and limitation of activities in sports and in repetitive work. The aetiology of RCD is not well established. A number of gene pathways are altered in RCD. Polymorphisms in Col1A1, Col5A1 (encoding collagen) and GDF5 (TGF-beta superfamily) can be associated with RCD susceptibility. Materials and Methods. Single-nucleotide polymorphisms (SNPs) in Col1A1, GDF5 and Col5A1 were genotyped in a case-control study with 103 RCD patients and 104 controls in Caucasian and African populations who suffered from injuries in any other anatomical location. All patients provided signed informed consent. Sampling was carried out with a puncture of the pad of a finger using a sterile, single-use lancet. NSPs were determined by real-time polymerase chain reaction (PCR) using specific, unique probes with the analysis of the melting temperature of hybrids. The X2 test compared genotypes between groups. Multivariate logistic regression analysed the significance of many covariates and the incidence of RCD. Results. We found significant differences in subjects who were exposed to heavy physical exertion involving the upper limbs (p=0.002). There was a significant difference in the distribution of the three polymorphisms in the GDF5 gene between the two races that were studied, with a higher frequency of TC heterozygotes in Caucasians and TT homozygotes in Africans (p=0.05). There were significant differences in the CC (rs143383) polymorphism in the GDF5 gene in patients with RCD (p<0.04)

Dupuytren Disease (DD), the most common connective tissue disease in man, presents as a benign fibromatosis of the hands and fingers resulting in the formation of nodules and cords and often leading to flexion contractures in association with keloids or Peyronie disease. Surgical resection of the fibrotic nodules, and more recently intra-lesional collagenase injection are the main therapeutic options for these patients. While the exact cause of DD is still unknown, linkage and Genome Wide Association Studies (GWAS) showed molecular heterogeneity with at least 10 different susceptibility loci 6 of which are close to genes encoding proteins in the Wnt-signaling pathway. We aim to identify the molecular basis of Dupuytren Disease (DD). Twenty patients with Dupuytren disease (including 3 patients with autosomal dominant inheritance, 1 with keloids and congenital torticollis, 2 with Peronie disease), were included in this study. Chromosome Microarray Analysis (CMA), Whole Exome Sequencing (WES) of gDNA and proteomic analysis by LC-Tandem Mass Spectrometry (LC-MSMS) studies were performed. Expression and Network analysis of LCMSMS results was performed using Principal Component Analysis (PCA), ANOVA and Ingenuity Pathway Analysis (IPA). No pathogenic copy number variants (CNVs) were found in CMA (n = 3). WES showed potentially pathogenic variants in POSTN, WNT11, MMP1 and COL3A1. PCA showed three differentially expressed clusters and network-IPA identified ACTB, BAX, COL3A1, FBN1, FN1, MMP1 as potential biomarkers. Comprehensive multi-OMIC analysis of gDNA and tissue proteins in patients with DD identified several connective tissue biomarkers potentially important in the pathogenesis of DD

Platelet-rich plasma is a new inductive therapy which is being increasingly used for the treatment of the complications of bone healing, such as infection and nonunion. The activator for platelet-rich plasma is a mixture of thrombin and calcium chloride which produces a platelet-rich gel. We analysed the antibacterial effect of platelet-rich gel in vitro by using the platelet-rich plasma samples of 20 volunteers. In vitro laboratory susceptibility to platelet-rich gel was determined by the Kirby-Bauer disc-diffusion method. Baseline antimicrobial activity was assessed by measuring the zones of inhibition on agar plates coated with selected bacterial strains. Zones of inhibition produced by platelet-rich gel ranged between 6 mm and 24 mm (mean 9.83 mm) in diameter. Platelet-rich gel inhibited the growth of Staphylococcus aureus and was also active against Escherichia coli. There was no activity against Klebsiella pneumoniae, Enterococcus faecalis, and Pseudomonas aeruginosa. Moreover, platelet-rich gel seemed to induce the in vitro growth of Ps. aeruginosa, suggesting that it may cause an exacerbation of infections with this organism. We believe that a combination of the inductive and antimicrobial properties of platelet-rich gel can improve the treatment of infected delayed healing and nonunion

The most common reasons for total joint arthroplasty (TJA) failure are aseptic loosening (AL) and prosthetic joint infection (PJI). There is a big clinical challenge to identify the patients with high risk of AL/PJI before the TJA surgery. Although there is evidence that genetic factors contribute to the individual susceptibility to AL/PJI, a predictive model for identification of patients with a high genetic risk of TJA failure has not been developed yet. We aimed to develop a risk evaluation tool utilising the AL/PJI-associated polymorphisms for identification of patients with high genetic risk of TJA failure based on inflammation-gene polymorphism panel. Based on allele and genotype frequencies of twenty-five single nucleotide polymorphisms (SNPs) in TNF, IL2, IL6, IL10, IL1b, IL-1Ra, MBL2, MMP1, FTO genes and those influencing the serum levels of biomarkers of TJA outcomes (IL6, CCL2/MCP-1, CRP, ESR) in peripheral blood obtained from patients with TJA (AL, n=110; PJI, n=93; no complications, n=123), we calculated a hazard ratio and a relative entropy of alleles and genotypes associated with AL and PJI and their combinations in patient subgroups. We conducted a risk evaluation tool based on the presence of risk alleles and genotypes in TNF (rs361525, rs1800629), DARC (rs12075), MBL2 (rs11003125) and FTO (rs9939609, rs9930506) genes associated with implant failure (AL/PJI). Of these, FTO gene variations (rs9939609, rs9930506) were associated mainly with PJI (P=0.001, OR=2.04, 95%CI=1.132–2.603; P=0.011, OR=1.72, 95%CI=1.338–3.096) and DARC (rs12075) with AL (P=0.005, OR=1.79, 95%CI=1.193–2.696). This tool calculates a hazard ratio of a combination of SNPs associated with AL and PJI for identification of patients with high and low risk of AL/PJI TJA failure. We proposed a risk evaluation tool for stratification of patients before the TJA surgery based on the genetic risk of AL/PJI development. The effect size for each genotype combination described in the study is small. Further multiparametric data analysis and studies on an extended patient cohort and other non-genetic and genetic parameters are ongoing. Grant support: AZV MZ CR VES16-131852A, VES15-27726A, IGA LF UP_2016_011

TRIM32 is a candidate gene at the 9q33.1 genetic susceptibility locus for hip osteoarthritis (OA). Increased cartilage degradation typical of OA has previously been demonstrated in Trim32 knockout mice. Our aim is to investigate the role of TRIM32 in human and murine articular tissue. TRIM32 expression in human articular cartilage was examined by immunostaining. TRIM32 expression was compared in femoral head chondrocytes from patients with and without primary hip OA (n=6/group) and examined by Western blotting. Aggrecanolysis by femoral head explants from Trim32 knockout (T32KO) and wild-type (WT) mice was compared following stimulation with IL1α or retinoic acid (RA) and was assessed by DMMB assay (n=4/group). Expression of chondrocyte phenotype markers was measured by qPCR and compared between articular chondrocytes from WT and T32KO mice following catabolic (IL1α/TNFα) or anabolic (Oncostatin-M (OSM)/IGF1) stimulation. TRIM32 expression was demonstrated in human articular cartilage; TRIM32 expression by chondrocytes was reduced in patients with hip OA (p=0.03). Greater aggrecanolysis occurred in cartilage explants from T32KO mice after treatment with no stimulation (p=0.03), IL1α (p=0.02), and RA (p=0.001). Unstimulated T32KO chondrocytes expressed reduced Col2a1 (p=8.53×10. −5. ), and Sox9 (p=2.35×10. −6. ). Upon IL1α treatment, T32KO chondrocytes expressed increased Col10a1 (p=0.0003). Upon anabolic stimulation, T32KO chondrocytes expressed increased Col2a1 (OSM: p=0.001; IGF: p=0.001), and reduced Sox9 (OSM: p=0.0002; IGF: p=0.0006). These results indicate that altered TRIM32 expression in human articular tissue is associated with OA, and that Trim32 knockout results in increased cartilage degradation in murine femoral head explants. Predisposition to cartilage degeneration with reduced Trim32 expression may involve increased chondrocyte hypertrophy upon catabolic cytokine stimulation and dysregulation of Col2a1 and Sox9 expression upon anabolic stimulation