Intervertebral disc (IVD) degeneration is a major cause of low back pain (LBP). Degenerate discs are associated with accelerated cellular senescence. Cell senescence is associated with a secretory phenotype characterised by increased production of catabolic enzymes and cytokines. However, to date, the mechanism of cell senescence within disc degeneration is unclear. Senescence can be induced by increased replication or induced by stress such as reactive oxygen species or cytokines. This study investigated the association of cellular senescence with markers of DNA damage and presence of cytoplasmic DNA (which in cancer cells has been shown to be a key regulator of the secretory phenotype), to determine mechanisms of senescence in disc degeneration. Immunohistochemistry for the senescence marker: p16INK4A was firstly utilised to screen human intervertebral discs for discs displaying at least 30% immunopostivity. These discs were then subsequently analysed for immunopostivity for DNA damage markers γH2AX and cGAS and the presence of cytoplasmic DNA. The number of immunopositive cells for p16 INK4A positively correlated with the expression of γH2AX and cGAS. Senescent cells were also associated with the presence of cytoplasmic DNA. These new findings elucidated a role of cGAS and γH2AX as a link from genotoxic stress to cytokine expression which is associated with senescent cells. The findings indicate that cellular senescence in vivo is associated with DNA damage and presence of cytoplasmic DNA. Whether this DNA damage is a result of replicative senescence or stress induced is currently being investigated in vitro

There is an evolving body of evidence that demonstrates the role of epigenetic mechanisms, such as DNA-methylation in the pathogenesis of OA. This systematic review aims to summarize the current evidence of DNA methylation and its influence on the pathogenesis of OA. A pre-defined protocol in alignment with the PRISMA guidelines was employed to systematically review eight bibliographic databases, to identify associations between DNA-methylation of articular chondrocytes and osteoarthritis. A search of Medline (Ovid), Embase, Web-of-Science, Scopus, PubMed, Cinahl (EBSCOhost), Cochrane Central and Google Scholar was performed between 1st January 2015 to 31st January 2021. Data extraction was performed by two independent reviewers. During the observation period, we identified 15 gene specific studies and 24 genome wide methylation analyses. The gene specific studies mostly focused on the expression of pro-inflammatory markers, such as IL8 and MMP13 which are overexpressed in OA chondrocytes. DNA hypomethylation in the promoter region resulted in overexpression, whereas hypermethylation was seen in non-OA chondrocytes. Others reported on the association between OA risk genes and the DNA methylation pattern close to RUNX2, which is an important OA signal. The genome wide methylation studies reported mostly on differentially methylated regions comparing OA chondrocytes and non-OA chondrocytes. Clustering of the regions identified genes that are involved in skeletal morphogenesis and development. Differentially methylated regions were seen in hip OA and knee OA chondrocytes, and even within different regions of an OA affected knee joint, differentially methylated regions were identified depending on the disease stage. This systematic review demonstrates the growing evidence of epigenetic mechanisms, such as DNA methylation, in the pathogenesis of OA. In recent years, there has been a focus on the interplay between OA risk genes and DNA methylation changes which revealed a reactivation of genes responsible for endochondral ossification during development. These are important findings and may help to identify eventual future therapeutic targets. However, the current body of literature is mostly showing the differences in DNA methylation of OA chondrocytes and non-OA chondrocytes, but a true longitudinal analysis demonstrating the DNA methylation changes actually happening is still not available

Introduction and Objective. Senescent bone cell overburden accelerates osteoporosis. Epigenetic alteration, including microRNA signalling and DND methylation, is one of prominent features of cellular senescence. This study aimed to investigate what role microRNA-29a signalling may play in the development of senile osteoporosis. Materials and Methods. Bone biopsy and serum were harvested from 13 young patients and 15 senior patients who required spine surgery. Bone mass, microstructure, and biomechanics of miR-29a knockout mice (miR-29aKO) and miR-29a transgenic mice (miR-29aTg) were probed using mCT imaging and three-point bending material test. Senescent cells were probed using senescence-associated b-galactosidase (SA-b-gal) staining. Transcriptomic landscapes of osteoblasts were characterized using whole genome microarray and KEGG bioinformatics. miR-29a and senescence markers p16. INK4a. , p21. Waf/cipl. and inflammatory cytokines were quantified using RT-PCR. DNA methylome was probed using methylation-specific PCR and 5-methylcytosine immunoblotting. Results. Senescent osteoblast overburden, DNA hypermethylation and oxidative damage together with significant decreases in serum miR-29a levels were present in bone specimens of aged patients. miR-29aKO mice showed a phenotype of skeletal underdevelopment, low bone mineral density and weak biomechanics. miR-29a knockout worsened age-induced bone mass and microstructure deterioration. Of note, aged miR-29aTg mice showed less bone loss and fatty marrow than aged wild-type mice. Transgenic overexpression of miR-29s compromised age-dysregulated osteogenic differentiation capacity of bone-marrow mesenchymal cells. In vitro, miR-29a promoted transcriptomic landscapes of antioxidant proteins in osteoblasts. The microRNA interrupted DNA methyltransferase (Dnmt3b)-mediated DNA methylation, inhibiting reactive oxygen radicals burst, IL-6 and RANKL production, and a plethora of senescent activity, including increased p16. INK4a. , p21. Waf/cipl. signalling and SA-b-gal activity. Conclusions. miR-29a loss is correlated with human age-mediated osteoporosis. miR-29a signalling is indispensable in bone mase homeostasis and microstructure integrity. Gain of miR-29a function is advantageous to delay age-induced bone loss through promoting antioxidant proteins to inhibit DNA hypermethylation-mediated osteoblast senescence. Collective investigations shine light onto the anabolic effects miR-29a signalling to bone integrity and highlight a new epigenetic protection strategy through controlling microRNA signalling to delay osteoblast senescence and senile osteoporosis development

Previous research has shown an increase in chromosomal aberrations in patients with worn implants. The type of aberration depended on the type of metal alloy in the prosthesis. We have investigated the metal-specific difference in the level of DNA damage (DNA stand breaks and alkali labile sites) induced by culturing human fibroblasts in synovial fluid retrieved at revision arthroplasty. All six samples from revision cobalt-chromium metal-on-metal and four of six samples from cobalt-chromium metal-on-polyethylene prostheses caused DNA damage. By contrast, none of six samples from revision stainless-steel metal-on-polyethylene prostheses caused significant damage. Samples of cobalt-chromium alloy left to corrode in phosphate-buffered saline also caused DNA damage and this depended on a synergistic effect between the cobalt and chromium ions. Our results further emphasise that epidemiological studies of orthopaedic implants should take account of the type of metal alloy used

Fatty marrow and bone loss are prominent pathologic features of osteoporosis. DNA hypermethylation shifts mesenchymal stem cells towards adipocytes impairing bone formation. Brown adipocytes produce growth factors advantageous to osteogenesis, whereas white adipocytes secrete pro-inflammatory cytokines deleterious to bone homeostasis. We assess DNA methylation inhibitor action to brown and white adipocyte formation in marrow fat of osteoporotic skeletons. Osteoporotic skeletons in mice were induced by glucocorticoid, ovariectomy or ageing. Marrow adipose volume and bone structure were quantified using OsO4 contrast-μCT imaging. Brown and white adipocytes were probed using immunostaining, RT-PCR and primary bone-marrow mesenchymal stem cell cultures. Abundant marrow fat and spare trabecular bone existed in osteoporotic skeletons. Osteoporosis increased expressions of general adipogenic markers PPARγ2 and FABP4 and white adipocyte markers TCF21 and HOXc9, whereas expressions of brown adipocyte markers PGC-1α and UCP-1 and osteogenic markers Runx2 and osteocalcin were significantly decreased. Number of UCP-1 immunostaining-positive brown adipocytes also reduced in osteoporotic bone. In vitro, DNA methylation inhibitor 5'-aza-deoxycystidine significantly increased brown adipocyte formation and osteogenic differentiation and mitigated dexamethasone-induced white adipocyte formation in mesenchymal stem cells. 5'-aza-deoxycystidine control of brown adipogenesis and white fat formation appeared to be regulated by increasing Wnt3a/β-catenin and reducing Dkk1. Disintegrated brown adipocyte and white fat cell differentiation contribute to osteoporosis pathogenesis. Maintaining DNA hypomethylation promotes Wnt signalling and brown adipocyte differentiation facilitating osteogenic differentiation. This study shed a new light to the contribution of brown adipocytic cells to bone metabolism during osteoporosis

INTRODUCTION. Many patients suffering from osteoarthritis (OA) take daily glucosamine (GlcN) in the hope of slowing down disease progression and ameliorating pain. However, the physiological basis of this effect is not known. We previously presented preliminary data suggesting that GlcN prevented the increase in interleukin-1beta (IL-1b) expression caused by addition of inflammatory cytokines to cultures of healthy human articular chondrocytes. Previous studies had also shown that, in OA, epigenetic DNA methylation loss at specific CpG sites in relevant promoters ‘unsilences’ the genes and that this DNA de-methylation underlies the aberrant gene expression of proteases (Arthritis Rheum 52;3110-24). Furthermore, exogenous inflammatory cytokines have the capacity to cause DNA de-methylation in the IL-1b promoter (Arthritis Rheum. 2009, 60, 3303-3313). The aims of the present study were to investigate whether GlcN not only prevents the increased IL-1b expression, but also inhibits epigenetic unsilencing by preventing the cytokine-induced loss of DNA methylation. METHODS. Healthy chondrocytes were isolated from the articular cartilage of four femoral heads, after operations following femoral neck fracture (ethic permission was obtained). The chondrocytes were cultured for 5 weeks in four treatment groups: no treatment (control); with IL-1b and oncostatin M (IL1b+OSM); with 2.0mM GlcN; and with IL1b+OSM+GlcN. Total RNA and genomic DNA were extracted. The % DNA methylation at the CpG site at -299bp (previously identifies as the crucial CpG site) was determined after bisulphite modification with a pyrosequencer. Gene expression of IL-1B was quantified by SybrGreen-based qRT-PCR. RESULTS. The chondrocytes from one patient grew too slowly to obtain results. In two patients (aged 80 and 85), exogenous IL-1b increased expression of IL-1b several 100-fold and reduced the % DNA methylation from 60% to 40%. GlcN alone showed no significant difference to the control group. When GlcN was present together with IL1b, the increase in expression of IL1B was only 1/3 of that cause by IL-1b and the loss of DNA methylation was prevented. The final sample (patient aged 94), showed a low % DNA methylation (46%) even in the control culture and glucosamine had no effect. This may be an anomaly due to the great age of the patient. DISCUSSION. The results suggest that GlcN is capable of ameliorating the cytokine-induced loss of DNA methylation. However, patient numbers are too low to have statistical significance. Further work is clearly needed confirm the hypothesis that DNA de-methylation of the IL-1b promoter caused by exogenous IL-1b is reversed by GlcN

In osteoarthritis (OA), articular chondrocytes undergo a phenotypic change and acquire a gene expression repertoire that is characterized by the aberrant expression of numerous catabolic genes including matrix metalloproteinases 3, 9 and 13, ADAMTS-4 and interleukin-1beta (IL1B = gene, IL-1b=protein). Previous studies (Arthritis Rheum 52;3110-24) have shown that epigenetic DNA demethylation at specific CpG sites in the relevant promoters accounts for the aberrant expression and that inflammatory cytokines (TNF-alpha, oncostatin M, IL-1b) can cause both aberrant expression and loss of DNA methylation, at least in vitro (Arthritis Rheum. 2009, 60,3303-3313). If the mechanisms of DNA de-methylation were understood, they might provide a new molecular target for therapeutic intervention. We hypothesize that nuclear translocation of the transcription factor NF-kB is involved in de-methylation because 1) we and others have demonstrated that cytokine-induced expression of IL1B in healthy chondrocytes requires NF-kB and 2) DNA de-methylation during B cell maturation was crucially dependent on the rel/NF-kB family (Nat Genet. 1996, 13,435-441). The aims of the study were to determine whether the NF-kB inhibitor BAY 11-7082 (BAY) could prevent the cytokine-induced loss of DNA de-methylation and thereby show that NF-kB is required for DNA de-methylation. METHODS. Healthy chondrocytes were isolated from the articular cartilage of six femoral heads, obtained with ethical permission after operations following neck of femur fractures. Chondrocytes were cultured for 5 weeks in 4 separate groups: without treatment (control culture); with 2.5ng/ml IL-1b and 2.5ng/ml oncostatin M (IL-1b+OSM); with 1.0mM BAY alone; and IL-1b+OSM+BAY. Total RNA and genomic DNA were extracted from each sample. Gene expression of IL1B was determined by SybrGreen-based qRT-PCR. The % DNA methylation at a specific CpG site in the IL1B promoter (which had previously been identified as a crucial CpG site) was quantified after bisulfite modification with a pyrosequencer (Biotage). The data for IL1B expression and % DNA methylation were analyzed in Microsoft Excel using Wilcoxon's signed rank test. P values < 0.05 were considered significant. RESULTS. Although there was considerable variation between samples, expression of IL1B was increased by > 1000 fold in the IL-1b+OSM group compared with control culture, confirming previous results. When BAY was present together with IL-1b+OSM, the increase in IL1B expression was reduced from ∼1000-fold to ∼200-300-fold (P< 0.01). In addition, the % DNA methylation had changed. At the -299 CpG site of IL1B promoter the % methylation was 57% in control culture and 60% in the BAY alone group. IL-1b+OSM caused a decrease to 37% (P<0.01 compared with all other groups), whereas presence of BAY prevented this loss, since the % methylation was 58% in IL-1b+OSM+BAY group. DISCUSSION. The novel findings of this study are that when nuclear translocation of NF-kB is inhibited by BAY, the IL-1b induced increase of IL1B expression was ameliorated and the loss of DNA methylation in the IL1B promoter was prevented. The data confirm our hypothesis that NF-kB is required for the DNA de-methylation initiated by IL-1b+OSM

Mitochondrial dysfunction has been demonstrated in aging and osteoarthritic tissues. We investigated knee joints of prematurely aging mitochondrial DNA mutator mice (PolgD275A) to evaluate a relationship between mitochondrial dysfunction and osteoarthritis. Cartilage damage was evaluated using OARSI histopathology grading and osteoclast numbers were quantified by tartrate-resistant acid phosphatase staining in wild type, heterozygous and homozygous PolgD275A mice. Subchondral cortical plate and epiphyseal trabecular bone structures were determined by micro-computed tomography. Apoptosis in cartilage and subchondral bone tissues was studied using an indirect TUNEL method. Homozygous mutants displayed osteopenia of the epiphyseal trabecular bone and subchondral cortical plate in comparison to wild type and heterozygous mutants. Subchondral osteopenia was associated with a strong increase of osteoclast numbers (0.88±0.30/mm bone perimeter) compared to heterozygous (0.25±0.03/mm) and wild type mice (0.12±0.04/mm). Wild type mice as well as hetero- and homozygous mutants displayed low-grade cartilage degeneration due to loss of cartilage proteoglycans. In contrast, chondrocyte hypertrophy was more abundant in the homozygous mice. There were no differences in chondrocyte apoptosis rates between groups. Prematurely ageing mtDNA mutator mice with or without further mechanic or metabolic stimuli might serve as a valuable model for further experimental studies on aging-induced osteoporotic OA phenotype

Systemic concentrations of metal ions (cobalt and chromium) are persistently elevated in patients with metal-on-metal hip resurfacing (MOMHR) compared to conventional total hip arthroplasty (THA). Several studies by us and others have described the detrimental effects of metal exposure on survival and function of various cell types in-vitro, but the mechanisms for these effects remain unclear. Epigenetic modifications following chronic metal exposure is a possible mechanism that could mediate these effects. Here we test the methylation status in genomic DNA from MOMHR (“cases”) and THA (“controls”) patient-groups, and its correlation with circulating metal levels. The cohort consisted of 34 patients with a well-functioning MOMHR at a median follow-up of 9.75 years. These were individually matched for gender, age and time-since-surgery to a non-exposure group consisting of patients with THA. Genomic DNA was isolated from blood samples and cell composition estimated using the ‘estimateCellCounts’ function in ‘minfi R-package’. Methylation was assessed using the Illumina 450k BeadChip array analysing 426,225 probes. Logit model was fitted at each probe with case/control status as independent variable and covariates of gender, age, time-since-surgery, smoking, non-arthroplasty metal exposure, and cell composition. DNA methylation age was assessed using an online calculator (. https://dnamage.genetics.ucla.edu/. ) and comparisons made between cases and controls, and correlated with circulating metal levels. Cell distributions did not differ between the cases and controls (Wilcoxon test p<0.17) with no probe having an association at 5% FDR. Circulating metal levels and LVEDD also had no association with any probe at 5% FDR. There was no preferential age acceleration between cases and controls (Wilcox p<0.7), and it had no correlation with plasma-chromium or blood-cobalt levels (p<0.9). In summary, large methylation changes following MOMHR seem to be absent, compared to THA. Future research with larger samples will be needed to clarify the presence and extent of small methylation changes

INTRODUCTION. Bone marrow derived mesenchymal stem cells are a potential source of cells for the repair of articular cartilage defects. Hypoxia has been shown to improve chondrogenesis in adult stem cells. In this study we characterised bone marrow derived stem cells and investigated the effects of hypoxia on gene expression changes and chondrogenesis. MATERIALS AND METHODS. Adherent colony forming cells were isolated and cultured from the stromal component of bone marrow. The cells at passage 2 were characterised for stem cell surface epitopes, and then cultured as cell aggregates in chondrogenic medium under normoxic (20% oxygen) or hypoxic (5% oxygen) conditions for 14 days. Gene expression analysis, glycosoaminoglycan and DNA assays, and immunohistochemical staining were determined to assess chondrogenesis. RESULTS. Bone marrow derived adherent colony forming cells stained strongly for markers of adult mesenchymal stem cells including CD44, CD90 and CD105, and they were negative for the haematopoietic cell marker CD34 and for the neural and myogenic cell marker CD56. Interestingly, a high number of cells were also positive for the pericyte marker 3G5. Cell aggregates showed a chondrogenic response and in lowered oxygen there was increased matrix accumulation of proteoglycan, but less cell proliferation, which resulted in 3.2-fold more glycosoaminoglycan per DNA after 14 days of culture. In hypoxia there was increased expression of key transcription factor SOX6, and the expression of collagens II and XI, and aggrecan was also increased. DISCUSSION. Pericytes are a candidate stem cell in many tissue and our results show that bone marrow derived mesenchymal stem cells express the pericyte marker 3G5. The response to chondrogenic culture in these cells was enhanced by lowered oxygen tension, which up-regulated SOX6 and increased the synthesis and assembly of matrix during chondrogenesis. This has important implications for tissue engineering applications of bone marrow derived stem cells

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

Decellularization techniques have advanced to reduce the risk of immune rejection in transplantation. Validation of these protocols typically relies on Crapo's criteria. 1. , which include the absence of visible nuclei and low DNA content. In our study, five decellularization protocols were compared to determine the optimal approach for human fascia lata (HFL) samples. However, our findings raised questions as to why recipients can still develop immunity despite meeting validation criteria. HFL samples were decellularized using four protocols with SDS-Triton X100-DNase (D1 to D4-HFL) and one protocol using solvent-detergent-based baths (D5-HFL). The decellularized samples (D-HFL) were compared to native samples (N-HFL) using histology, and DNA content was measured. The human leukocyte antigen (HLA) content within the matrix was assessed using western blot analysis. Both D-HFL and N-HFL samples, along with negative control patches, were implanted in the backs of 28 Wistar rats. Anti-human IgG serum levels were evaluated after one month. H&E and Hoechst staining revealed the absence of residual cells in all decellularization protocols. DNA content was consistently below the critical threshold (p<0.05). All implanted D-HFL samples resulted in significantly lower anti-human IgG levels compared to N-HFL (p<0.01). However, 2.5 out of 4 rats developed immunity after being implanted with D1 to D4-HFL, with varying levels of anti-human IgG. Only rats implanted with D5-HFL showed undetectable levels of IgG and were considered non-immunized. Western blot analysis indicated that only D5-HFL had a residual HLA content below 1%. The literature on decellularization has primarily relied on Crapo's criteria, which do not consider the role of HLA mismatch in acute immune rejection. Our results suggest that a residual HLA content below 1% should also be considered to prevent immunization, even if other validation criteria are met. Further research is needed to evaluate the impact of residual HLA levels on human allotransplantation outcomes

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

Intervertebral disc (IVD) degeneration occurs with aging, leading to low back pain (LBP), which is one of the leading conditions of disability worldwide. With the lack of effective treatment, decellularized extracellular matrix (dECM) – based biomaterials have been proposed for IVD regeneration. However, the impact of donor ages on tissue repair had never been explored before in the disc field. Therefore, we aimed to address this question. For that, a decellularization protocol for bovine nucleus pulposus (NP) of different aged donors (fetus, young and old) was optimized by testing several detergents (SDS and Triton). The process efficiency was evaluated in terms of DNA and cell removal, as well as ECM preservation. Afterwards, dECMs were repopulated with bovine NP cells and cultured ex vivo. At day 7, cell behavior, ECM de novo synthesis and remodeling were evaluated [1]. Moreover, dECMs’ inflammatory response was assessed after in vivo CAM assay. Finally, inflammatory and angiogenic cytokines were analyzed in the conditioned media-derived from dECMs by using a cytokine array. As results, an optimal decellularization protocol (SDS 0.1%, 1h), efficient at removing cells and DNA from bovine NPs, while preserving ECM cues of native tissues, was developed. After repopulation, aggrecan increased in younger NPs, while collagen 2 decreased which may be indicative of matrix remodeling [1]. After in vivo CAM assay, fetal dECMs showed the highest inflammatory response. Finally, no statistically significant changes of cytokines were detected in the matrices, despite for a trend of higher IFN-α, IFN-γ and LIF in fetal dECMs, IL-1β in young dECMs and Decorin in old dECMs. Overall, this work uncovered the importance of tissue donor ages for tissue regenerative purpose, opening new avenues for the development of appropriate therapeutic strategies for IVD degeneration. Acknowledgments: FCT, EUROSPINE, ON Foundation

The purpose of this study is to enhance massive bone allografts osseointegration used to reconstruct large bone defects. These allografts show >50% complication rate requiring surgical revision in 20% cases. A new protocol for total bone decellularisation exploiting the vasculature can offer a reduction of postoperative complication by annihilating immune response and improving cellular colonization/ osseointegration. The nutrient artery of 18 porcine bones - humerus/femur/radius/ulna - was cannulated. The decellularization process involved immersion and sequential perfusion with specific solvents over a course of one week. Perfusion was realized by a peristaltic pump (mean flow rate: 6ml/min). The benefit of arterial perfusion was compared to a control group kept in immersion baths without perfusion. Bone samples were processed for histology (HE, Masson's trichrome and DAPI for cell detection), immunohistochemistry (IHC : Collagen IV/elastin for intraosseous vascular system evaluation, Swine Leukocyte Antigen – SLA for immunogenicity in addition to cellular clearance) and DNA quantification. Sterility and solvent residues in the graft were also evaluated with thioglycolate test and pH test respectively. Compared to native bones, no cells could be detected and residual DNA was <50ng/mg dry weight. Intramedullary spaces were completely cleaned. IHC showed the preservation of intracortical vasculature with channels bounded by Collagen IV and elastin within Haversian systems. IHC also showed a significant decrease in SLA signaling. All grafts were sterile at the last decellularization step and showed no solvent residue. The control group kept in immersion baths, paired with 6 perfused radii/ulnae, showed that the perfusion is mandatory to ensure complete decellularisation. Our results prove the effectiveness of a new concept of total bone decellularisation by perfusion. These promising results could lead to a new technique of Vascularized Composite Allograft transposable to pre-clinical and clinical models

We have developed a novel technique to analyse bone, using imaging mass cytometry (IMC) without the constraints of using immunofluorescent histochemistry. IMC can measure the expression of over 40 proteins simultaneously, without autofluorescence. We analysed mitochondrial respiratory chain (RC) protein deficiencies in human bone which are thought to contribute to osteoporosis with increasing age. Osteoporosis is characterised by reduced bone mineral density (BMD) and fragility fractures. Humans accumulate mitochondrial mutations and RC deficiency with age and this has been linked to the changing phenotype in advancing age and age-related disease. Mitochondrial mutations are detectable from the age of 30 onwards, coincidently the age BMD begins to decline. Mitochondria contain their own genome which accumulates somatic variants at around 10 times the rate of nuclear DNA. Once these mutations exceed a threshold, RC deficiency and cellular dysfunction occur. The PolgD257A/D257A mouse model expresses a proof-reading deficient version of PolgA, a mtDNA polymerase. These mice accumulate mutations 3-5 times higher than wild-type mice showing enhanced levels of age-related osteoporosis and RC deficiency in osteoblasts. Bone samples were analysed from young and old patients, developing a protocol and analysis framework for IMC in bone tissue sections to analyse osteoblasts in-situ for RC deficiency. Samples from the femoral neck of 10 older healthy volunteers aged 40 – 85 were compared with samples from young patients aged 1-19. We have identified RC complex I defect in osteoblasts from 6 of the older volunteers, complex II defects in 2 of the older volunteers, complex IV defect in just 1 older volunteer, and complex V defect in 4 of the older volunteers. These observations are consistent with the PolgD257A/D257A mouse-model and suggest that RC deficiency, due to age-related pathogenic mitochondrial DNA mutations, may play a significant role in the pathogenesis of human age-related osteoporosis

Successful anterior cruciate ligament (ACL) reconstructions strive a firm ligament-bone integration. Therefore, the aim of this study was to address in more detail the enthesis as the thriphasic bone attachment of the ACL using a tissue engineering approach. To establish a tissue-engineered enthesis-like construct, triphasic scaffolds embroidered from poly(L-lactide-co-caprolactone) and polylactic acid functionalized with collagen foam were colonized with osteogenically differentiated human mesenchymal stromal cells (hMSCs) and lapine (L) ACL fibroblasts. These triphasic scaffolds with a bone-, a fibrocartilage transition- and a ligament phase were seeded directly after spheroid assembly or with 14 days precultured LACL fibroblast spheroids and 14 days osteogenically differentiated hMSCs spheroids (=longer preculture) and cultured for further 14 days. Cell survival was tested. Collagen type I and vimentin were immunolabeled and the content of DNA and sulfated glycosaminoglycan (sGAG) was quantified. The relative gene expression of tenascin C, type I and X collagens, Mohawk and Runx2 was analyzed. Compared to the LACL spheroids the hMSC spheroids adhered better to the scaffold surface with faster cell outgrowth on the fibers. Collagen type I and vimentin were mainly detected in the hMSCs colonizing the bone zone. The DNA content was generally higher in the bone (hMSCs) than in the ligament zones and after short spheroid preculture higher than after longer preculture whereas the sGAG content was greater after longer preculture for both cell types. The longer precultivated hMSCs expressed more type I collagen in comparison to those only shortly precultured before scaffold seeding. Type I collagen and tenascin C were higher expressed in scaffolds directly colonized with LACL compared to those seeded after longer spheroid preculture. The gene expression of ECM components and transcription factors depended on cell type and preculturing condition. Zonal colonization of triphasic scaffolds using the spheroid method is possible offering a novel approach for enthesis tissue engineering

Abstract. Objectives. The objective of this study is to investigate if genomic sequencing is a useful method to diagnose orthopaedic infections. Current methods used to identify the species of bacteria causing orthopaedic infections take considerable time and the results are frequently insufficient for guiding antibiotic treatment. The aim here is to investigate if genomic sequencing is a faster and more reliable method to identify the species of bacteria causing infections. Current methods include a combination of biochemical markers and microbiological cultures which frequently produce false positive results and false negative results. Methods. Samples of prosthetic fluid were obtained from surgical interventions to treat orthopaedic infections. DNA is extracted from these samples lab and nanopore genomic sequencing is performed. Initial investigations informed that a sequencing time of 15 minutes was sufficient. The resulting genomic sequence data was classified using Basic Local Alignment Tool (BLAST) against the NCBI bacterial database and filtered by only including reads with an identity score of 90 and E-value of 1e-50. An E-value of 1e-50 suggests a high-quality result and is commonly used when analysing genomic data. This data was then filtered in R Studio to identify if any species were associated with orthopaedic infections. The results from genomic sequencing were compared to microbiology results from the hospital to see if the same species had been identified. The whole process from DNA extraction to output took approximately 2 hours, which was faster than parallel microbiological cultures. Results. In these preliminary analyses, 15 samples have been collected from patients with confirmed/suspected orthopaedic infections. To date, 11 samples from confirmed infected patients have been sequenced and a summary of the findings are presented in the table attached. As well as finding bacteria species to match microbiological cultures, genomic sequencing has also identified bacteria when culture results have been negative, but the patient is known to have an infection due to clinical indication and previous culture results. This example suggests genomic sequencing may have higher sensitivity than microbiological cultures at detecting bacteria causing orthopaedic infections. Results in table indicate the identification of bacteria from genomic sequencing that match microbiological cultures are high quality. Conclusions. Preliminary data presented using genomic sequencing suggests that the technique may be useful to identify bacterial species causing orthopaedic infections and can do so in a shorter time frame than current microbial methods. The results from genomic sequencing all produced a number of false positive results which hopefully can be reduced by improving the bioinformatic techniques used and increasing the sample number to include individuals without an infection. Further analysis will also look at identifying antibiotic resistance genes in the sequencing data and seeing if this ca be used to predict which patients will and will not respond to antibiotic treatment. The aim at the end of this project is to demonstrate if genomic sequencing is a more sensitive method to identify bacteria causing orthopaedic infections that current methods and if it can be used to guide antibiotic treatment. Include limitations, next steps and bigger picture. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Aim. Osteoarthritis (OA) is caused by complex interactions between genetic and environmental factors. Epigenetic mechanisms control the expression of genes and are likely to regulate the OA transcriptome. We performed integrative genomic analyses to define methylation-gene expression relationships in osteoarthritic cartilage. Patients and Methods. Genome-wide DNA methylation profiling of articular cartilage from five patients with OA of the knee and five healthy controls was conducted using the Illumina Infinium HumanMethylation450 BeadChip (Illumina, San Diego, California). Other independent genome-wide mRNA expression profiles of articular cartilage from three patients with OA and three healthy controls were obtained from the Gene Expression Omnibus (GEO) database. Integrative pathway enrichment analysis of DNA methylation and mRNA expression profiles was performed using integrated analysis of cross-platform microarray and pathway software. Gene ontology (GO) analysis was conducted using the Database for Annotation, Visualization and Integrated Discovery (DAVID). Results. We identified 1265 differentially methylated genes, of which 145 are associated with significant changes in gene expression, such as DLX5, NCOR2 and AXIN2 (all p-values of both DNA methylation and mRNA expression < 0.05). Pathway enrichment analysis identified 26 OA-associated pathways, such as mitogen-activated protein kinase (MAPK) signalling pathway (p = 6.25 × 10-4), phosphatidylinositol (PI) signalling system (p = 4.38 × 10-3), hypoxia-inducible factor 1 (HIF-1) signalling pathway (p = 8.63 × 10-3 pantothenate and coenzyme A (CoA) biosynthesis (p = 0.017), ErbB signalling pathway (p = 0.024), inositol phosphate (IP) metabolism (p = 0.025), and calcium signalling pathway (p = 0.032). Conclusion. We identified a group of genes and biological pathwayswhich were significantly different in both DNA methylation and mRNA expression profiles between patients with OA and controls. These results may provide new clues for clarifying the mechanisms involved in the development of OA. Cite this article: A. He, Y. Ning, Y. Wen, Y. Cai, K. Xu, Y. Cai, J. Han, L. Liu, Y. Du, X. Liang, P. Li, Q. Fan, J. Hao, X. Wang, X. Guo, T. Ma, F. Zhang. Use of integrative epigenetic and mRNA expression analyses to identify significantly changed genes and functional pathways in osteoarthritic cartilage. Bone Joint Res 2018;7:343–350. DOI: 10.1302/2046-3758.75.BJR-2017-0284.R1

Abstract. Introduction. Bone grafts are utilised in a range of surgical procedures, from joint replacements to treatment of bone loss resulting from cancer. Decellularised allograft bone is a regenerative, biocompatible and immunologically safe potential source of transplant bone. Objectives. To compare the structural and biomechanical parameters of decellularised and unprocessed (cellular) trabecular bone from the human femoral head (FH) and tibial plateau (TP). Methods. Bone pins were harvested from 10 FHs and 11 TPs (27, 34 respectively). Pins were decellularised (0.1% w/v sodium dodecyl sulphate) or retained as cellular controls. QA testing was carried out to assess protocol efficacy (total DNA and histological analysis). Cellular and decellularised FH (n=7) and TP (n=10) were uCT scanned. Material density (MD); apparent density (BV/TV); trabecular connectivity; trabecular number; trabecular thickness (Tb-t) and trabecular spacing were measured. Pins were then compression tested to determine ultimate compressive stress (UCS), Young's modulus and 0.2% proof stress. Results. Total DNA levels of decellularised bone were below 50 ng.mg. −1. dry weight. Cell nuclei and marrow were largely removed. No significant differences in properties were found between decellularised and cellular bone from either anatomical region (p>0.05, Mann-Whitney). No significant differences in biomechanical properties were found between cellular FH and cellular TP (p>0.05) though significant differences in structural properties were found (MD: TP>FH, p=0.001; BV/TV: FH>TP, p=0.001; and Tb-t: FH>TP, p=0.005). Significant differences were found between decellularised FH and decellularised TP (UCS: FH>TP, p=0.001; Young's modulus: FH>TP, p=0.002; proof stress; FH>TP, p=0.001; MD: TP>FH, p<0.001; BV/TV: FH>TP, p<0.001 and Tb-t: FHT>P p<0.001. Conclusion. Decellularisation did not affect the properties of human trabecular bone. Differences were found between the mechanical and structural properties of decellularised FH and TP which could facilitate stratified bone grafts for different applications. Declaration of Interest. (a) fully declare any financial or other potential conflict of interest