Thrombin has many biological properties similar to those of growth factors. In a previous study, we showed that thrombin improves healing of the rat tendo Achillis. Low molecular weight heparin (LMWH) inhibits the activity and the generation of thrombin. We therefore considered that LMWH at a thromboprophylactic dose might inhibit tendon repair. Transection of the tendo Achillis was carried out in 86 rats and the healing tested mechanically. Low molecular weight heparin (dalateparin) was either injected a few minutes before the operation and then given continuously with an osmotic mini pump for seven days, or given as one injection before the operation. In another experiment ,we gave LMWH or a placebo by injection twice daily. The anti-factor Xa activity was analysed. Continuous treatment with LMWH impaired tendon healing. After seven days, this treatment caused a 33% reduction in force at failure, a 20% reduction in stiffness and a 67% reduction in energy uptake. However, if injected twice daily, LMWH had no effect on tendon healing. Anti-factor Xa activity was increased by LMWH treatment, but was normal between intermittent injections. Low molecular weight heparin delays tendon repair if given continuously, but not if injected intermittently, probably because the anti-factor Xa activity between injections returns to normal, allowing sufficient thrombin stimulation for repair. These findings indicate the need for caution in the assessment of long-acting thrombin and factor Xa inhibitors

Nanometre-sized particles of ultra-high molecular weight polyethylene have been identified in the lubricants retrieved from hip simulators. Tissue samples were taken from seven failed Charnley total hip replacements, digested using strong alkali and analysed using high-resolution field emission gun-scanning electron microscopy to determine whether nanometre-sized particles of polyethylene debris were generated in vivo. A randomised method of analysis was used to quantify and characterise all the polyethylene particles isolated. We isolated nanometre-sized particles from the retrieved tissue samples. The smallest identified was 30 nm and the majority were in the 0.1 μm to 0.99 μm size range. Particles in the 1.0 μm to 9.99 μm size range represented the highest proportion of the wear volume of the tissue samples, with 35% to 98% of the total wear volume comprised of particles of this size. The number of nanometre-sized particles isolated from the tissues accounted for only a small proportion of the total wear volume. Further work is required to assess the biological response to nanometre-sized polyethylene particles

Objectives

Ligaments which heal spontaneously have a healing process that is similar to skin wound healing. Menopause impairs skin wound healing and may likewise impair ligament healing. Our purpose in this study was to investigate the effect of surgical menopause on ligament healing in a rabbit medial collateral ligament model.

Objectives. Adult mice lacking the transcription factor NFAT1 exhibit osteoarthritis (OA). The precise molecular mechanism for NFAT1 deficiency-induced osteoarthritic cartilage degradation remains to be clarified. This study aimed to investigate if NFAT1 protects articular cartilage (AC) against OA by directly regulating the transcription of specific catabolic and anabolic genes in articular chondrocytes. Methods. Through a combined approach of gene expression analysis and web-based searching of NFAT1 binding sequences, 25 candidate target genes that displayed aberrant expression in Nfat1. -/-. AC at the initiation stage of OA, and possessed at least four NFAT1 binding sites in the promoter of each gene, were selected and tested for NFAT1 transcriptional activities by chromatin immunoprecipitation (ChIP) and promoter luciferase reporter assays using chondrocytes isolated from the AC of three- to four-month-old wild-type mice or Nfat1. -/-. mice with early OA phenotype. Results. Chromatin immunoprecipitation assays revealed that NFAT1 bound directly to the promoter of 21 of the 25 tested genes encoding cartilage-matrix proteins, growth factors, inflammatory cytokines, matrix-degrading proteinases, and specific transcription factors. Promoter luciferase reporter assays of representative anabolic and catabolic genes demonstrated that NFAT1-DNA binding functionally regulated the luciferase activity of specific target genes in wild-type chondrocytes, but not in Nfat1. -/-. chondrocytes or in wild-type chondrocytes transfected with plasmids containing mutated NFAT1 binding sequences. Conclusion. NFAT1 protects AC against degradation by directly regulating the transcription of target genes in articular chondrocytes. NFAT1 deficiency causes defective transcription of specific anabolic and catabolic genes in articular chondrocytes, leading to increased matrix catabolism and osteoarthritic cartilage degradation. Cite this article: M. Zhang, Q. Lu, T. Budden, J. Wang. NFAT1 protects articular cartilage against osteoarthritic degradation by directly regulating transcription of specific anabolic and catabolic genes. Bone Joint Res 2019;8:90–100. DOI: 10.1302/2046-3758.82.BJR-2018-0114.R1

Objectives. The aim of this study was to investigate the role of miR-126 in the development of osteoarthritis, as well as the potential molecular mechanisms involved, in order to provide a theoretical basis for osteoarthritis treatment and a novel perspective for clinical therapy. Methods. Human chondrocyte cell line CHON-001 was administrated by different doses of interleukin (IL)-1β to simulate inflammation. Cell viability, migration, apoptosis, IL-6, IL-8, and tumour necrosis factor (TNF)-α expression, as well as expression of apoptosis-related factors, were measured to assess inflammation. miR-126 expression was measured by quantitative polymerase chain reaction (qPCR). Cells were then transfected with miR-126 inhibitor to assess the effect of miR-126 on IL-1β-injured CHON-001 cells. Expression of B-cell lymphoma 2 (Bcl-2) and the activity of mitogen-activated protein kinase (MAPK) / Jun N-terminal kinase (JNK) signaling pathway were measured by Western blot to explore the underlying mechanism through which miR-126 affects IL-1β-induced inflammation. Results. After IL-1β administration, cell viability and migration were suppressed while apoptosis was enhanced. Expression of IL-6, IL-8, and TNF-α were all increased, and miR-126 was upregulated. In IL-1β-administrated CHON-001 cells, miR-126 inhibitor suppressed the effect of IL-1β on cell viability, migration, apoptosis, and inflammatory response. Bcl-2 expression was negatively regulated with miR-126 in IL-1β-administrated cells, and thus affected expressions of phosphorylated MAPK and JNK. Conclusion. IL-1β-induced inflammatory markers and miR-126 was upregulated. Inhibition of miR-126 decreased IL-1β-induced inflammation and cell apoptosis, and upregulated Bcl-2 expression via inactivating the MAKP/JNK signalling pathway. Cite this article: C. D. Yu, W. H. Miao, Y. Y. Zhang, M. J. Zou, X. F. Yan. Inhibition of miR-126 protects chondrocytes from IL-1β induced inflammation via upregulation of Bcl-2. Bone Joint Res 2018;7:414–421. DOI: 10.1302/2046-3758.76.BJR-2017-0138.R1

Objectives. The role of mechanical stress and transforming growth factor beta 1 (TGF-β1) is important in the initiation and progression of osteoarthritis (OA). However, the underlying molecular mechanisms are not clearly known. Methods. In this study, TGF-β1 from osteoclasts and knee joints were analyzed using a co-cultured cell model and an OA rat model, respectively. Five patients with a femoral neck fracture (four female and one male, mean 73.4 years (68 to 79)) were recruited between January 2015 and December 2015. Results showed that TGF-β1 was significantly upregulated in osteoclasts by cyclic loading in a time- and dose-dependent mode. The osteoclasts were subjected to cyclic loading before being co-cultured with chondrocytes for 24 hours. Results. A significant decrease in the survival rate of co-cultured chondrocytes was found. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL) assay demonstrated that mechanical stress-induced apoptosis occurred significantly in co-cultured chondrocytes but administration of the TGF-β1 receptor inhibitor, SB-505124, can significantly reverse these effects. Abdominal administration of SB-505124 can attenuate markedly articular cartilage degradation in OA rats. Conclusion. Mechanical stress-induced overexpression of TGF-β1 from osteoclasts is responsible for chondrocyte apoptosis and cartilage degeneration in OA. Administration of a TGF-β1 inhibitor can inhibit articular cartilage degradation. Cite this article: R-K. Zhang, G-W. Li, C. Zeng, C-X. Lin, L-S. Huang, G-X. Huang, C. Zhao, S-Y. Feng, H. Fang. Mechanical stress contributes to osteoarthritis development through the activation of transforming growth factor beta 1 (TGF-β1). Bone Joint Res 2018;7:587–594. DOI: 10.1302/2046-3758.711.BJR-2018-0057.R1

Objectives. In order to screen the altered gene expression profile in peripheral blood mononuclear cells of patients with osteoporosis, we performed an integrated analysis of the online microarray studies of osteoporosis. Methods. We searched the Gene Expression Omnibus (GEO) database for microarray studies of peripheral blood mononuclear cells in patients with osteoporosis. Subsequently, we integrated gene expression data sets from multiple microarray studies to obtain differentially expressed genes (DEGs) between patients with osteoporosis and normal controls. Gene function analysis was performed to uncover the functions of identified DEGs. Results. A total of three microarray studies were selected for integrated analysis. In all, 1125 genes were found to be significantly differentially expressed between osteoporosis patients and normal controls, with 373 upregulated and 752 downregulated genes. Positive regulation of the cellular amino metabolic process (gene ontology (GO): 0033240, false discovery rate (FDR) = 1.00E + 00) was significantly enriched under the GO category for biological processes, while for molecular functions, flavin adenine dinucleotide binding (GO: 0050660, FDR = 3.66E-01) and androgen receptor binding (GO: 0050681, FDR = 6.35E-01) were significantly enriched. DEGs were enriched in many osteoporosis-related signalling pathways, including those of mitogen-activated protein kinase (MAPK) and calcium. Protein-protein interaction (PPI) network analysis showed that the significant hub proteins contained ubiquitin specific peptidase 9, X-linked (Degree = 99), ubiquitin specific peptidase 19 (Degree = 57) and ubiquitin conjugating enzyme E2 B (Degree = 57). Conclusion. Analysis of gene function of identified differentially expressed genes may expand our understanding of fundamental mechanisms leading to osteoporosis. Moreover, significantly enriched pathways, such as MAPK and calcium, may involve in osteoporosis through osteoblastic differentiation and bone formation. Cite this article: J. J. Li, B. Q. Wang, Q. Fei, Y. Yang, D. Li. Identification of candidate genes in osteoporosis by integrated microarray analysis. Bone Joint Res 2016;5:594–601. DOI: 10.1302/2046-3758.512.BJR-2016-0073.R1

Objectives. The molecular mechanism of rheumatoid arthritis (RA) remains elusive. We conducted a protein-protein interaction network-based integrative analysis of genome-wide association studies (GWAS) and gene expression profiles of RA. Methods. We first performed a dense search of RA-associated gene modules by integrating a large GWAS meta-analysis dataset (containing 5539 RA patients and 20 169 healthy controls), protein interaction network and gene expression profiles of RA synovium and peripheral blood mononuclear cells (PBMCs). Gene ontology (GO) enrichment analysis was conducted by DAVID. The protein association networks of gene modules were generated by STRING. Results. For RA synovium, the top-ranked gene module is HLA-A, containing TAP2, HLA-A, HLA-C, TAPBP and LILRB1 genes. For RA PBMCs, the top-ranked gene module is GRB7, consisting of HLA-DRB5, HLA-DRA, GRB7, CD63 and KIT genes. Functional enrichment analysis identified three significant GO terms for RA synovium, including antigen processing and presentation of peptide antigen via major histocompatibility complex class I (false discovery rate (FDR) = 4.86 × 10 – 4), antigen processing and presentation of peptide antigen (FDR = 2.33 × 10 – 3) and eukaryotic translation initiation factor 4F complex (FDR = 2.52 × 10 – 2). Conclusion. This study reported several RA-associated gene modules and their functional association networks. Cite this article: X. Xiao, J. Hao, Y. Wen, W. Wang, X. Guo, F. Zhang. Genome-wide association studies and gene expression profiles of rheumatoid arthritis: an analysis. Bone Joint Res 2016;5:314–319. DOI: 10.1302/2046-3758.57.2000502

Objectives. Ubiquitin E3 ligase-mediated protein degradation regulates osteoblast function. Itch, an E3 ligase, affects numerous cell functions by regulating ubiquitination and proteasomal degradation of related proteins. However, the Itch-related cellular and molecular mechanisms by which osteoblast differentiation and function are elevated during bone fracture repair are as yet unknown. Methods. We examined the expression levels of E3 ligases and NF-κB members in callus samples during bone fracture repair by quantitative polymerase chain reaction (qPCR) and the total amount of ubiquitinated proteins by Western blot analysis in wild-type (WT) mice. The expression levels of osteoblast-associated genes in fracture callus from Itch knockout (KO) mice and their WT littermates were examined by qPCR. The effect of NF-κB on Itch expression in C2C12 osteoblast cells was determined by a chromatin immunoprecipitation (ChIP) assay. Results. The expression levels of WW Domain Containing E3 Ubiquitin Protein Ligase 1 (Wwp1), SMAD Specific E3 Ubiquitin Protein Ligase 1 (Smurf1), SMAD Specific E3 Ubiquitin Protein Ligase 2 (Smurf2) and Itch were all significantly increased in the fracture callus of WT mice, which was associated with elevated expression of NF-κB members and total ubiquitinated proteins. Callus tissue isolated from Itch KO mice expressed higher levels of osteoblast-associated genes, including Runx2, a positive regulator of osteoblast differentiation, but osteoclast-associated genes were not increased. Both NF-κB RelA and RelB proteins were found to bind to the NF-κB binding site in the mouse Itch promoter. Conclusions. Our findings indicate that Itch depletion may have a strong positive effect on osteoblast differentiation in fracture callus. Thus, ubiquitin E3 ligase Itch could be a potential target for enhancing bone fracture healing. Cite this article: J. Liu, X. Li, H. Zhang, R. Gu, Z. Wang, Z. Gao, L. Xing. Ubiquitin E3 ligase Itch negatively regulates osteoblast function by promoting proteasome degradation of osteogenic proteins. Bone Joint Res 2017;6:154–161. DOI: 10.1302/2046-3758.63.BJR-2016-0237.R1

Objective. Excessive mechanical stress on synovial joints causes osteoarthritis (OA) and results in the production of prostaglandin E2 (PGE2), a key molecule in arthritis, by synovial fibroblasts. However, the relationship between arthritis-related molecules and mechanical stress is still unclear. The purpose of this study was to examine the synovial fibroblast response to cyclic mechanical stress using an in vitro osteoarthritis model. Method. Human synovial fibroblasts were cultured on collagen scaffolds to produce three-dimensional constructs. A cyclic compressive loading of 40 kPa at 0.5 Hz was applied to the constructs, with or without the administration of a cyclooxygenase-2 (COX-2) selective inhibitor or dexamethasone, and then the concentrations of PGE2, interleukin-1β (IL-1β), tumour necrosis factor-α (TNF-α), IL-6, IL-8 and COX-2 were measured. Results. The concentrations of PGE2, IL-6 and IL-8 in the loaded samples were significantly higher than those of unloaded samples; however, the concentrations of IL-1β and TNF-α were the same as the unloaded samples. After the administration of a COX-2 selective inhibitor, the increased concentration of PGE2 by cyclic compressive loading was impeded, but the concentrations of IL-6 and IL-8 remained high. With dexamethasone, upregulation of PGE2, IL-6 and IL-8 was suppressed. Conclusion. These results could be useful in revealing the molecular mechanism of mechanical stress in vivo for a better understanding of the pathology and therapy of OA. Cite this article: Bone Joint Res 2014;3:280–8

Malrotation of the femoral component is a cause of patellofemoral maltracking after total knee arthroplasty. Its precise effect on the patellofemoral mechanics has not been well quantified. We have developed an in vitro method to measure the influence of patellar maltracking on contact. Maltracking was induced by progressively rotating the femoral component either internally or externally. The contact mechanics were analysed using Tekscan. The results showed that excessive malrotation of the femoral component, both internally and externally, had a significant influence on the mechanics of contact. The contact area decreased with progressive maltracking, with a concomitant increase in contact pressure. The amount of contact area that carries more than the yield stress of ultra-high molecular weight polyethylene significantly increases with progressive maltracking. It is likely that the elevated pressures noted in malrotation could cause accelerated and excessive wear of the patellar button

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

The formation of restrictive adhesions around the musculotendinous unit after injury is one of the most vexing processes faced by the surgeon. In flexor tendons it has been shown that the synovial tissue is the source of aggressive fibroblasts which contribute to this process. Using a rabbit model, we have examined the effects of treating the synovial sheath with the antimetabolite 5-fluorouracil (5-FU) for five minutes. Inflammatory, proliferative and molecular markers were compared in the response of the treated and control tendons to injury. Compared with a control group we found that the proliferative and inflammatory responses were significantly reduced (p < 0.001) in the treated tendons. Not only was there a reduction in the cellular and cytokine response, but there also was a significant (p < 0.001) reduction in the level of activity of the known pro-scarring agent, transforming growth factor beta 1 (TGF-β1). These pilot studies indicate that the formation of restrictive adhesions may be modulated using a simple single-touch technique in the hope of producing a better return of function

Bone morphogenetic protein (BMP) has a crucial role in osteochondrogenesis of bone formation as well as in the repair of fractures. The interaction between hedgehog protein and BMPs is inferred from recent molecular studies. Hedgehog genes encode secreted proteins which mediate patterning and growth during skeletal development. We have shown that Indian hedgehog gene (Ihh) is expressed in cartilage anlage and later in mature and hypertrophic chondrocytes. This finding suggests that Ihh may regulate the development of chondrocytes. Our results in this study have shown that Ihh transcripts were expressed in hypertrophic chondrocytes in mice at three days but not at three weeks, although a similar expression pattern of α1 (X) collagen could be observed in both types of cartilage. To investigate the possibility that there are direct and age-dependent functions of Ihh in chondrocytes, cultured chondrocytes were treated with the amino-terminal fragment of Sonic hedgehog protein (Shh-N) which can functionally substitute for Ihh protein. Shh-N did not affect the proliferation and differentiation of chondrocytes from three-week-old mice but had a significant effect on three-day-old mice. It enhanced proliferation up to 128% of the control culture in a dose-dependent manner. Although there was no effect in Shh-N-treated cultures, Shh-N enhanced the stimulatory effect of parathyroid hormone (PTH) on the synthesis of proteoglycans. Because the effects of Shh-N on chondrocyte differentiation in this culture system differed from those of bone morphogenetic protein-2 (BMP2) and PTH, in terms of proteoglycan synthesis and ALPase activity, it is unlikely that BMP2 or PTH/PTH-related protein mediates the direct effects of Ihh in chondrocytes. Our study shows that Ihh can function in chondrocytes in a direct and age-dependent fashion

We developed a rat model of limb lengthening to study the basic mechanism of distraction osteogenesis, using a small monolateral external fixator. In 11-week-old male rats we performed a subperiosteal osteotomy in the midshaft of the femur with distraction at 0.25 mm every 12 hours from seven days after operation. Radiological and histological examinations showed a growth zone of constant thickness in the middle of the lengthened segment, with formation of new bone at its proximal and distal ends. Osteogenic cells were arranged longitudinally along the tension vector showing the origin and the fate of individual cells in a single section. Typical endochondral bone formation was prominent in the early stage of distraction, but intramembraneous bone formation became the predominant mechanism of ossification at later stages. We also showed a third mechanism of ossification, ‘transchondroid bone formation’. Chondroid bone, a tissue intermediate between bone and cartilage, was formed directly by chondrocyte-like cells, with transition from fibrous tissue to bone occurring gradually and consecutively without capillary invasion. In situ hybridisation using digoxigenin-11-UTP-labelled complementary RNAs showed that the chondroid bone cells temporarily expressed type-II collagen mRNA. They did not show the classical morphological characteristics of chondrocytes, but were assumed to be young chondrocytes undergoing further differentiation into bone-forming cells. We found at least three different modes of ossification during bone lengthening by distraction osteogenesis. We believe that this is the first report of such a rat model, and have shown the validity of in situ hybridisation techniques for the study of the cellular and molecular mechanisms involved in distraction osteogenesis

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.

Objectives

The aim of this study was to investigate the effect of hyperglycaemia on oxidative stress markers and inflammatory and matrix gene expression within tendons of normal and diabetic rats and to give insights into the processes involved in tendinopathy.

Objectives

Osteoporosis is a chronic disease. The aim of this study was to identify key genes in osteoporosis.

Objectives

The objectives of this study were: 1) to examine osteophyte formation, subchondral bone advance, and bone marrow lesions (BMLs) in osteoarthritis (OA)-prone Hartley guinea pigs; and 2) to assess the disease-modifying activity of an orally administered phosphocitrate ‘analogue’, Carolinas Molecule-01 (CM-01).

Objectives

Degenerative disc disease (DDD) and osteoarthritis (OA) are relatively frequent causes of disability amongst the elderly; they constitute serious socioeconomic costs and significantly impair quality of life. Previous studies to date have found that aggrecan variable number of tandem repeats (VNTR) contributes both to DDD and OA. However, current data are not consistent across studies. The purpose of this study was to evaluate systematically the relationship between aggrecan VNTR, and DDD and/or OA.

Objectives

In this study, we compared the pain behaviour and osteoarthritis (OA) progression between anterior cruciate ligament transection (ACLT) and osteochondral injury in surgically-induced OA rat models.

Objectives

To determine the pattern of mutations of the WISP3 gene in clinically identified progressive pseudorheumatoid dysplasia (PPD) in an Indian population.

Objectives

Triamcinolone acetonide (TA) is widely used for the treatment of rotator cuff injury because of its anti-inflammatory properties. However, TA can also produce deleterious effects such as tendon degeneration or rupture. These harmful effects could be prevented by the addition of platelet-rich plasma (PRP), however, the anti-inflammatory and anti-degenerative effects of the combined use of TA and PRP have not yet been made clear. The objective of this study was to determine how the combination of TA and PRP might influence the inflammation and degeneration of the rotator cuff by examining rotator cuff-derived cells induced by interleukin (IL)-1ß.

Objectives

The objective of this study was to develop a test for the rapid (within 25 minutes) intraoperative detection of bacteria from synovial fluid to diagnose periprosthetic joint infection (PJI).

Objectives

Recently, high failure rates of metal-on-metal (MOM) hip implants have raised concerns of cobalt toxicity. Adverse reactions occur to cobalt nanoparticles (CoNPs) and cobalt ions (Co²⁺) during wear of MOM hip implants, but the toxic mechanism is not clear.

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

Objectives

Sustained intra-articular delivery of pharmacological agents is an attractive modality but requires use of a safe carrier that would not induce cartilage damage or fibrosis. Collagen scaffolds are widely available and could be used intra-articularly, but no investigation has looked at the safety of collagen scaffolds within synovial joints. The aim of this study was to determine the safety of collagen scaffold implantation in a validated in vivo animal model of knee arthrofibrosis.

Objectives

This study looked to analyse the expression levels of microRNA-140-3p and microRNA-140-5p in synovial fluid, and their correlations to the severity of disease regarding knee osteoarthritis (OA).

Objectives

Osteoarthritis (OA) is characterised by articular cartilage degradation. MicroRNAs (miRNAs) have been identified in the development of OA. The purpose of our study was to explore the functional role and underlying mechanism of miR-138-5p in interleukin-1 beta (IL-1β)-induced extracellular matrix (ECM) degradation of OA cartilage.

Objectives

Vancomycin and fosfomycin are antibiotics commonly used to treat methicillin-resistant Staphylococcus aureus (MRSA) infection. This study compares the in vitro inhibitory effects against MRSA of articulating cement spacers impregnated with either vancomycin or fosfomycin.

Objectives

The present study describes a novel technique for revitalising allogenic intrasynovial tendons by combining cell-based therapy and mechanical stimulation in an ex vivo canine model.

Objectives

Osteophytes are products of active endochondral and intramembranous ossification, and therefore could theoretically provide significant efficacy as bone grafts. In this study, we compared the bone mineralisation effectiveness of osteophytes and cancellous bone, including their effects on secretion of growth factors and anabolic effects on osteoblasts.

Objectives

All-suture anchors are increasingly used in rotator cuff repair procedures. Potential benefits include decreased bone damage. However, there is limited published evidence for the relative strength of fixation for all-suture anchors compared with traditional anchors.

Objectives

Rotator cuff tears are among the most frequent upper extremity injuries. Current treatment strategies do not address the poor quality of the muscle and tendon following chronic rotator cuff tears. Hypoxia-inducible factor-1 alpha (HIF-1α) is a transcription factor that activates many genes that are important in skeletal muscle regeneration. HIF-1α is inhibited under normal physiological conditions by the HIF prolyl 4-hydroxylases (PHDs). In this study, we used a pharmacological PHD inhibitor, GSK1120360A, to enhance the activity of HIF-1α following the repair of a chronic cuff tear, and measured muscle fibre contractility, fibrosis, gene expression, and enthesis mechanics.

In vivo animal experimentation has been one of the cornerstones of biological and biomedical research, particularly in the field of clinical medicine and pharmaceuticals. The conventional in vivo model system is invariably associated with high production costs and strict ethical considerations. These limitations led to the evolution of an ex vivo model system which partially or completely surmounted some of the constraints faced in an in vivo model system. The ex vivo rodent bone culture system has been used to elucidate the understanding of skeletal physiology and pathophysiology for more than 90 years. This review attempts to provide a brief summary of the historical evolution of the rodent bone culture system with emphasis on the strengths and limitations of the model. It encompasses the frequency of use of rats and mice for ex vivo bone studies, nutritional requirements in ex vivo bone growth and emerging developments and technologies. This compilation of information could assist researchers in the field of regenerative medicine and bone tissue engineering towards a better understanding of skeletal growth and development for application in general clinical medicine.

Cite this article: A. A. Abubakar, M. M. Noordin, T. I. Azmi, U. Kaka, M. Y. Loqman. The use of rats and mice as animal models in ex vivo bone growth and development studies. Bone Joint Res 2016;5:610–618. DOI: 10.1302/2046-3758.512.BJR-2016-0102.R2.

This review briefly summarises some of the definitive studies of articular cartilage by microscopic MRI (µMRI) that were conducted with the highest spatial resolutions. The article has four major sections. The first section introduces the cartilage tissue, MRI and µMRI, and the concept of image contrast in MRI. The second section describes the characteristic profiles of three relaxation times (T₁, T₂ and T_1ρ) and self-diffusion in healthy articular cartilage. The third section discusses several factors that can influence the visualisation of articular cartilage and the detection of cartilage lesion by MRI and µMRI. These factors include image resolution, image analysis strategies, visualisation of the total tissue, topographical variations of the tissue properties, surface fibril ambiguity, deformation of the articular cartilage, and cartilage lesion. The final section justifies the values of multidisciplinary imaging that correlates MRI with other technical modalities, such as optical imaging. Rather than an exhaustive review to capture all activities in the literature, the studies cited in this review are merely illustrative.

Objectives

We have observed clinical cases where bone is formed in the overlaying muscle covering surgically created bone defects treated with a hydroxyapatite/calcium sulphate biomaterial. Our objective was to investigate the osteoinductive potential of the biomaterial and to determine if growth factors secreted from local bone cells induce osteoblastic differentiation of muscle cells.

Objectives

Recent studies have shown that systemic injection of rapamycin can prevent the development of osteoarthritis (OA)-like changes in human chondrocytes and reduce the severity of experimental OA. However, the systemic injection of rapamycin leads to many side effects. The purpose of this study was to determine the effects of intra-articular injection of Torin 1, which as a specific inhibitor of mTOR which can cause induction of autophagy, is similar to rapamycin, on articular cartilage degeneration in a rabbit osteoarthritis model and to investigate the mechanism of Torin 1’s effects on experimental OA.

Background

Resveratrol is a polyphenolic compound commonly found in the skins of red grapes. Sirtuin 1 (SIRT1) is a human gene that is activated by resveratrol and has been shown to promote longevity and boost mitochondrial metabolism. We examined the effect of resveratrol on normal and osteoarthritic (OA) human chondrocytes.

Objectives

The aim of this study was to examine whether asymmetric loading influences macrophage elastase (MMP12) expression in different parts of a rat tail intervertebral disc and growth plate and if MMP12 expression is correlated with the severity of the deformity.

Objectives

Rotator cuff tears are among the most common and debilitating upper extremity injuries. Chronic cuff tears result in atrophy and an infiltration of fat into the muscle, a condition commonly referred to as ‘fatty degeneration’. While stem cell therapies hold promise for the treatment of cuff tears, a suitable immunodeficient animal model that could be used to study human or other xenograft-based therapies for the treatment of rotator cuff injuries had not previously been identified.

Objectives

The major problem with repair of an articular cartilage injury is the extensive difference in the structure and function of regenerated, compared with normal cartilage. Our work investigates the feasibility of repairing articular osteochondral defects in the canine knee joint using a composite lamellar scaffold of nano-ß-tricalcium phosphate (ß-TCP)/collagen (col) I and II with bone marrow stromal stem cells (BMSCs) and assesses its biological compatibility.

We have designed a prospective study to evaluate the usefulness of prolonged incubation of cultures from sonicated orthopaedic implants. During the study period 124 implants from 113 patients were processed (22 osteosynthetic implants, 46 hip prostheses, 54 knee prostheses, and two shoulder prostheses). Of these, 70 patients had clinical infection; 32 had received antibiotics at least seven days before removal of the implant. A total of 54 patients had sonicated samples that produced positive cultures (including four patients without infection). All of them were positive in the first seven days of incubation. No differences were found regarding previous antibiotic treatment when analysing colony counts or days of incubation in the case of a positive result. In our experience, extending incubation of the samples to 14 days does not add more positive results for sonicated orthopaedic implants (hip and knee prosthesis and osteosynthesis implants) compared with a conventional seven-day incubation period.

Cite this article: Bone Joint J 2013;95-B:1001–6.

Trauma and orthopaedics is the largest of the surgical specialties and yet attracts a disproportionately small fraction of available national and international funding for health research. With the burden of musculoskeletal disease increasing, high-quality research is required to improve the evidence base for orthopaedic practice. Using the current research landscape in the United Kingdom as an example, but also addressing the international perspective, we highlight the issues surrounding poor levels of research funding in trauma and orthopaedics and indicate avenues for improving the impact and success of surgical musculoskeletal research.

Cite this article: Bone Joint J 2014; 96-B:1578–85.

The treatment of chronic osteomyelitis often includes surgical debridement and filling the resultant void with antibiotic-loaded polymethylmethacrylate cement, bone grafts or bone substitutes. Recently, the use of bioactive glass to treat bone defects in infections has been reported in a limited series of patients. However, no direct comparison between this biomaterial and antibiotic-loaded bone substitute has been performed.

In this retrospective study, we compared the safety and efficacy of surgical debridement and local application of the bioactive glass S53P4 in a series of 27 patients affected by chronic osteomyelitis of the long bones (Group A) with two other series, treated respectively with an antibiotic-loaded hydroxyapatite and calcium sulphate compound (Group B; n = 27) or a mixture of tricalcium phosphate and an antibiotic-loaded demineralised bone matrix (Group C; n = 22). Systemic antibiotics were also used in all groups.

After comparable periods of follow-up, the control of infection was similar in the three groups. In particular, 25 out of 27 (92.6%) patients of Group A, 24 out of 27 (88.9%) in Group B and 19 out of 22 (86.3%) in Group C showed no infection recurrence at means of 21.8 (12 to 36), 22.1 (12 to 36) and 21.5 (12 to 36) months follow-up, respectively, while Group A showed a reduced wound complication rate.

Our results show that patients treated with a bioactive glass without local antibiotics achieved similar eradication of infection and less drainage than those treated with two different antibiotic-loaded calcium-based bone substitutes.

Cite this article: Bone Joint J 2014; 96-B:845–50.

Osteoarthritis (OA) is an important cause of pain, disability and economic loss in humans, and is similarly important in the horse. Recent knowledge on post-traumatic OA has suggested opportunities for early intervention, but it is difficult to identify the appropriate time of these interventions. The horse provides two useful mechanisms to answer these questions: 1) extensive experience with clinical OA in horses; and 2) use of a consistently predictable model of OA that can help study early pathobiological events, define targets for therapeutic intervention and then test these putative therapies. This paper summarises the syndromes of clinical OA in horses including pathogenesis, diagnosis and treatment, and details controlled studies of various treatment options using an equine model of clinical OA.

Introduction

Osteoarthritis (OA) is a progressively debilitating disease that affects mostly cartilage, with associated changes in the bone. The increasing incidence of OA and an ageing population, coupled with insufficient therapeutic choices, has led to focus on the potential of stem cells as a novel strategy for cartilage repair.

Objective

To study the effect of hyaluronic acid (HA) on local anaesthetic chondrotoxicity in vitro.

The role of inflammatory cells and their products in tendinopathy is not completely understood. Pro-inflammatory cytokines are upregulated after oxidative and other forms of stress. Based on observations that increased cytokine expression has been demonstrated in cyclically-loaded tendon cells we hypothesised that because of their role in oxidative stress and apoptosis, pro-inflammatory cytokines may be present in rodent and human models of tendinopathy. A rat supraspinatus tendinopathy model produced by running overuse was investigated at the genetic level by custom micro-arrays. Additionally, samples of torn supraspinatus tendon and matched intact subscapularis tendon were collected from patients undergoing arthroscopic shoulder surgery for rotator-cuff tears and control samples of subscapularis tendon from ten patients with normal rotator cuffs undergoing arthroscopic stabilisation of the shoulder were also obtained. These were all evaluated using semiquantitative reverse transcription polymerase chain-reaction and immunohistochemistry.

We identified significant upregulation of pro-inflammatory cytokines and apoptotic genes in the rodent model (p = 0.005). We further confirmed significantly increased levels of cytokine and apoptotic genes in human supraspinatus and subscapularis tendon harvested from patients with rotator cuff tears (p = 0.0008).

These findings suggest that pro-inflammatory cytokines may play a role in tendinopathy and may provide a target for preventing tendinopathies.

Post-natal vasculogenesis, the process by which vascular committed bone marrow stem cells or endothelial precursor cells migrate, differentiate and incorporate into the nacent endothelium and thereby contribute to physiological and pathological neurovascularisation, has stimulated much interest. Its contribution to neovascularisation of tumours, wound healing and revascularisation associated with ischaemia of skeletal and cardiac muscles is well established. We evaluated the responses of endothelial precursor cells in bone marrow to musculoskeletal trauma in mice.

Bone marrow from six C57 Black 6 mice subjected to a standardised, closed fracture of the femur, was analysed for the combined expression of cell-surface markers stem cell antigen 1 (sca-1⁺) and stem cell factor receptor, CD117 (c-kit⁺) in order to identify the endothelial precursor cell population. Immunomagnetically-enriched sca-1⁺ mononuclear cell (MNC^sca-1+) populations were then cultured and examined for functional vascular endothelial differentiation. Bone marrow MNC^{sca-1+,c-kit+} counts increased almost twofold within 48 hours of the event, compared with baseline levels, before decreasing by 72 hours.

Sca-1⁺ mononuclear cell populations in culture from samples of bone marrow at 48 hours bound together Ulex Europus-1, and incorporated fluorescent 1,1′-dioctadecyl- 3,3,3,’3′-tetramethylindocarbocyanine perchlorate-labelled acetylated low-density lipoprotein intracellularily, both characteristics of mature endothelium.

Our findings suggest that a systemic provascular response of bone marrow is initiated by musculoskeletal trauma. Its therapeutic manipulation may have implications for the potential enhancement of neovascularisation and the healing of fractures.

We studied bone-tendon healing using immunohistochemical methods in a rabbit model.

Reconstruction of the anterior cruciate ligament was undertaken using semitendinosus tendon in 20 rabbits. Immunohistochemical evaluations were performed at one, two, four and eight weeks after the operation. The expression of CD31, RAM-11, VEGF, b-FGF, S-100 protein and collagen I, II and III in the bone-tendon interface was very similar to that in the endochondral ossification. Some of the type-III collagen in the outer layer of the graft, which was deposited at a very early phase after the operation, was believed to have matured into Sharpey-like fibres. However, remodelling of the tendon grafted into the bone tunnel was significantly delayed when compared with this ossification process. To promote healing, we believe that it is necessary to accelerate remodelling of the tendon, simultaneously with the augmentation of the ossification.

We used a biodegradable mesh to convert an acetabular defect into a contained defect in six patients at total hip replacement. Their mean age was 61 years (46 to 69). The mean follow-up was 32 months (19 to 50). Before clinical use, the strength retention and hydrolytic in vitro degradation properties of the implants were studied in the laboratory over a two-year period. A successful clinical outcome was determined by the radiological findings and the Harris hip score.

All the patients had a satisfactory outcome and no mechanical failures or other complications were observed. No protrusion of any of the impacted grafts was observed beyond the mesh. According to our preliminary laboratory and clinical results the biodegradable mesh is suitable for augmenting uncontained acetabular defects in which the primary stability of the implanted acetabular component is provided by the host bone. In the case of defects of the acetabular floor this new application provides a safe method of preventing graft material from protruding excessively into the pelvis and the mesh seems to tolerate bone-impaction grafting in selected patients with primary and revision total hip replacement.

This study was designed to test the hypothesis that the sensory innervation of bone might play an important role in sensing and responding to low-intensity pulsed ultrasound and explain its effect in promoting fracture healing. In 112 rats a standardised mid-shaft tibial fracture was created, supported with an intramedullary needle and divided into four groups of 28. These either had a sciatic neurectomy or a patellar tendon resection as control, and received the ultrasound or not as a sham treatment. Fracture union, callus mineralisation and remodelling were assessed using plain radiography, peripheral quantitative computed tomography and histomorphology.

Daily ultrasound treatment significantly increased the rate of union and the volumetric bone mineral density in the fracture callus in the neurally intact rats (p = 0.025), but this stimulating effect was absent in the rats with sciatic neurectomy. Histomorphology demonstrated faster maturation of the callus in the group treated with ultrasound when compared with the control group. The results supported the hypothesis that intact innervation plays an important role in allowing low-intensity pulsed ultrasound to promote fracture healing.

Despite worldwide clinical use of bio-absorbable devices for internal fixation in orthopaedic surgery, the degradation behaviour and tissue replacement of these implants are not fully understood.

In a long-term experimental study, we have determined the patterns of tissue restoration 36 and 54 months after implantation of polyglycolic acid and poly-laevo-lactic acid screws in the distal femur of the rabbit.

After 36 months in the polyglycolic acid group the specimens showed no remaining polymer and loose connective tissue occupied 80% of the screw track. Tissue restoration remained poor at 54 months, the amounts of trabecular bone and haematopoietic elements being significantly lower than those in the intact control group. The amount of trabecular bone within the screw track at 54 months in the polyglycolic acid group was less than in the empty drill holes (p = 0.04). In the poly-laevo-lactic acid group, polymeric material was present in abundance after 54 months, occupying 60% of the cross-section of the core area of the screw track.

When using absorbable internal fixation implants we should recognise that the degradation of the devices will probably not be accompanied by the restoration of normal trabecular bone.

Although success has been achieved with implantation of bone marrow mesenchymal stem cells (bMSCs) in degenerative discs, its full potential may not be achieved if the harsh environment of the degenerative disc remains. Axial distraction has been shown to increase hydration and nutrition. Combining both therapies may have a synergistic effect in reversing degenerative disc disease. In order to evaluate the effect of bMSC implantation, axial distraction and combination therapy in stimulating regeneration and retarding degeneration in degenerative discs, we first induced disc degeneration by axial loading in a rabbit model.

The rabbits in the intervention groups performed better with respect to disc height, morphological grading, histological scoring and average dead cell count. The groups with distraction performed better than those without on all criteria except the average dead cell count.

Our findings suggest that bMSC implantation and distraction stimulate regenerative changes in degenerative discs in a rabbit model.

Allograft bone is widely used in orthopaedic surgery, but peri-operative infection of the graft remains a common and disastrous complication. The efficacy of systemic prophylactic antibiotics is unproven, and since the graft is avascular it is likely that levels of antibiotic in the graft are low.

Using an electrical potential to accelerate diffusion of antibiotics into allograft bone, high levels were achieved in specimens of both sheep and human allograft. In human bone these ranged from 187.1 mg/kg in endosteal (sd 15.7) to 124.6 (sd 46.2) in periosteal bone for gentamicin and 31.9 (sd 8.9) in endosteal and 2.9 (sd 1.1) in periosteal bone for flucloxacillin. The antibiotics remained active against bacteria in vitro after iontophoresis and continued to elute from the allograft for up to two weeks.

Structural allograft can be supplemented directly with antibiotics using iontophoresis. The technique is simple and inexpensive and offers a potential means of reducing the rate of peri-operative infection in allograft surgery. Iontophoresis into allograft bone may also be applicable to other therapeutic compounds.

Objectives

There is increasing application of bone morphogenetic proteins (BMPs) owing to their role in promoting fracture healing and bone fusion. However, an optimal delivery system has yet to be identified. The aims of this study were to synthesise bioactive BMP-2, combine it with a novel α-tricalcium phosphate/poly(D,L-lactide-co-glycolide) (α-TCP/PLGA) nanocomposite and study its release from the composite.

Using a rat model the characteristics of the sensory neurones of the dorsal-root ganglia (DRG) innervating the hip were investigated by retrograde neurotransport and immunohistochemistry.

Fluoro-Gold solution (FG) was injected into the left hip of ten rats. Seven days later the DRG from both sides between T12 and L6 were harvested. The number of FG-labelled calcitonin gene-related peptide-immunoreactive or isolectin B4-binding neurones were counted.

The FG-labelled neurones were distributed throughout the left DRGs between T13 and L5, primarily at L2, L3, and L4. Few FG-labelled isolectin B4-binding neurones were present in the DRGs of either side between T13 and L5, but calcitonin gene-related peptide-immunoreactive neurones made up 30% of all FG-labelled neurones.

Our findings may explain the referral of pain from the hip to the thigh or lower leg corresponding to the L2, L3 and L4 levels. Since most neurones are calcitonin gene-related peptide-immunoreactive peptide-containing neurones, they may have a more significant role in the perception of pain in the hip as peptidergic DRG neurones.

Peri-tendinous injection of local anaesthetic, both alone and in combination with corticosteroids, is commonly performed in the treatment of tendinopathies. Previous studies have shown that local anaesthetics and corticosteroids are chondrotoxic, but their effect on tenocytes remains unknown. We compared the effects of lidocaine and ropivacaine, alone or combined with dexamethasone, on the viability of cultured bovine tenocytes. Tenocytes were exposed to ten different conditions: 1) normal saline; 2) 1% lidocaine; 3) 2% lidocaine; 4) 0.2% ropivacaine; 5) 0.5% ropivacaine; 6) dexamethasone (dex); 7) 1% lidocaine+dex; 8) 2% lidocaine+dex; 9) 0.2% ropivacaine+dex; and 10) 0.5% ropivacaine+dex, for 30 minutes. After a 24-hour recovery period, the viability of the tenocytes was quantified using the CellTiter-Glo viability assay and fluorescence-activated cell sorting (FACS) for live/dead cell counts. A 30-minute exposure to lidocaine alone was significantly toxic to the tenocytes in a dose-dependent manner, but a 30-minute exposure to ropivacaine or dexamethasone alone was not significantly toxic.

Dexamethasone potentiated ropivacaine tenocyte toxicity at higher doses of ropivacaine, but did not potentiate lidocaine tenocyte toxicity. As seen in other cell types, lidocaine has a dose-dependent toxicity to tenocytes but ropivacaine is not significantly toxic. Although dexamethasone alone is not toxic, its combination with 0.5% ropivacaine significantly increased its toxicity to tenocytes. These findings might be relevant to clinical practice and warrant further investigation.

When transferring tissue regenerative strategies involving skeletal stem cells to human application, consideration needs to be given to factors that may affect the function of the cells that are transferred. Local anaesthetics are frequently used during surgical procedures, either administered directly into the operative site or infiltrated subcutaneously around the wound. The aim of this study was to investigate the effects of commonly used local anaesthetics on the morphology, function and survival of human adult skeletal stem cells.

Cells from three patients who were undergoing elective hip replacement were harvested and incubated for two hours with 1% lidocaine, 0.5% levobupivacaine or 0.5% bupivacaine hydrochloride solutions. Viability was quantified using WST-1 and DNA assays. Viability and morphology were further characterised using CellTracker Green/Ethidium Homodimer-1 immunocytochemistry and function was assessed by an alkaline phosphatase assay. An additional group was cultured for a further seven days to allow potential recovery of the cells after removal of the local anaesthetic.

A statistically significant and dose dependent reduction in cell viability and number was observed in the cell cultures exposed to all three local anaesthetics at concentrations of 25% and 50%, and this was maintained even following culture for a further seven days.

This study indicates that certain local anaesthetic agents in widespread clinical use are deleterious to skeletal progenitor cells when studied in vitro; this might have relevance in clinical applications.

We stably transfected early passage chondrocytes with an anti-apoptotic Bcl-2 gene in vitro using a retrovirus vector. Samples of articular cartilage were obtained from 11 patients with a mean age of 69 years (61 to 75) who were undergoing total knee replacement for osteoarthritis. The Bcl-2-gene-transfected chondrocytes were compared with non-transfected and lac-Z-gene-transfected chondrocytes, both of which were used as controls. All three groups of cultured chondrocytes were incubated with nitric oxide (NO) for ten days. Using the Trypan Blue exclusion assay, an enzyme-linked immunosorbent assay and flow cytometric analysis, we found that the number of apoptotic chondrocytes was significantly higher in the non-transfected and lac-Z-transfected groups than in the Bcl-2-transfected group (p < 0.05). The Bcl-2-transfected chondrocytes were protected from NO-induced impairment of proteoglycan synthesis.

We conclude that NO-induced chondrocyte death involves a mechanism which appears to be subject to regulation by an anti-apoptotic Bcl-2 gene. Therefore, Bcl-2 gene therapy may prove to be of therapeutic value in protecting human articular chondrocytes.

Systemic factors are believed to be pivotal for the development of heterotopic ossification in severely-injured patients. In this study, cell cultures of putative target cells (human fibroblastic cells, osteoblastic cells (MG-63), and bone-marrow stromal cells (hBM)) were incubated with serum from ten consecutive polytraumatised patients taken from post-traumatic day 1 to day 21 and with serum from 12 healthy control subjects.

The serum from the polytraumatised patients significantly stimulated the proliferation of fibroblasts, MG-63 and of hBM cells. The activity of alkaline phosphatase in MG-63 and hBM cells was significantly decreased when exposed to the serum of the severely-injured patient. After three weeks in 3D cell cultures, matrix production and osteogenic gene expression of hBM cells were equal in the patient and control groups. However, the serum from the polytraumatised patients significantly decreased apoptosis of hBM cells compared with the control serum (4.3% vs 19.1%, p = 0.031).

Increased proliferation of osteoblastic cells and reduced apoptosis of osteoprogenitors may be responsible for increased osteogenesis in severely-injured patients.

The biomechanics of the patellofemoral joint can become disturbed during total knee replacement by alterations induced by the position and shape of the different prosthetic components. The role of the patella and femoral trochlea has been well studied. We have examined the effect of anterior or posterior positioning of the tibial component on the mechanisms of patellofemoral contact in total knee replacement. The hypothesis was that placing the tibial component more posteriorly would reduce patellofemoral contact stress while providing a more efficient lever arm during extension of the knee.

We studied five different positions of the tibial component using a six degrees of freedom dynamic knee simulator system based on the Oxford rig, while simulating an active knee squat under physiological loading conditions. The patellofemoral contact force decreased at a mean of 2.2% for every millimetre of posterior translation of the tibial component. Anterior positions of the tibial component were associated with elevation of the patellofemoral joint pressure, which was particularly marked in flexion > 90°.

From our results we believe that more posterior positioning of the tibial component in total knee replacement would be beneficial to the patellofemoral joint.

We split 100 porcine flexor tendons into five groups of 20 tendons for repair. Three groups were repaired using the Pennington modified Kessler technique, the cruciate or the Savage technique, one using one new device per tendon and the other with two new devices per tendon. Half of the tendons received supplemental circumferential Silfverskiöld type B cross-stitch. The repairs were loaded to failure and a record made of their bulk, the force required to produce a 3 mm gap, the maximum force applied before failure and the stiffness. When only one device was used repairs were equivalent to the Pennington modified Kessler for all parameters except the force to produce a 3 mm gap when supplemented with a circumferential repair, which was equivalent to the cruciate.

When two devices were used the repair strength was equivalent to the cruciate repair, and when the two-device repair was supplemented with a circumferential suture the force to produce a 3 mm gap was equivalent to that of the Savage six-strand technique.

We investigated the effect of mitomycin-C on the reduction of the formation of peritendinous fibrous adhesions after tendon repair. In 20 Wistar albino rats the tendo Achillis was cut and repaired using a modified Kessler technique. The rats were divided into two equal groups. In group 1, an injection of mitomycin-C was placed between the tendon and skin of the right leg. In group 2, an identical volume of sterile normal saline was injected on the left side in a similar fashion. All the rats received mitomycin-C or saline for four weeks starting from the day of operation. The animals were killed after 30 days. The formation of peritendinous fibrous tissue, the inflammatory reaction and tendon healing were evaluated. The tensile strength of the repaired tendons was measured biomechanically. Microscopic evidence of the formation of adhesions and inflammation was less in group 1. There was no significant difference in the tensile load required to rupture the repaired tendons in the two groups.

Mitomycin-C may therefore provide a simple and inexpensive means of preventing of post-operative adhesions.

Gene therapy with insulin-like growth factor-1 (IGF-1) increases matrix production and enhances chondrocyte proliferation and survival in vitro. The purpose of this study was to determine whether arthroscopically-grafted chondrocytes genetically modified by an adenovirus vector encoding equine IGF-1 (AdIGF-1) would have a beneficial effect on cartilage healing in an equine femoropatellar joint model.

A total of 16 horses underwent arthroscopic repair of a single 15 mm cartilage defect in each femoropatellar joint. One joint received 2 × 10⁷ AdIGF-1 modified chondrocytes and the contralateral joint received 2 × 10⁷ naive (unmodified) chondrocytes. Repairs were analysed at four weeks, nine weeks and eight months after surgery. Morphological and histological appearance, IGF-1 and collagen type II gene expression (polymerase chain reaction, in situ hybridisation and immunohistochemistry), collagen type II content (cyanogen bromide and sodium dodecyl sulphate-polyacrylamide gel electrophoresis), proteoglycan content (dimethylmethylene blue assay), and gene expression for collagen type I, matrix metalloproteinase (MMP)-1, MMP-3, MMP-13, aggrecanase-1, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and TIMP-3 were evaluated.

Genetic modification of chondrocytes significantly increased IGF-1 mRNA and ligand production in repair tissue for up to nine weeks following transplantation. The gross and histological appearance of IGF-1 modified repair tissue was improved over control defects. Gross filling of defects was significantly improved at four weeks, and a more hyaline-like tissue covered the lesions at eight months. Histological outcome at four and nine weeks post-transplantation revealed greater tissue filling of defects transplanted with genetically modified chondrocytes, whereas repair tissue in control defects was thin and irregular and more fibrous. Collagen type II expression in IGF-1 gene-transduced defects was increased 100-fold at four weeks and correlated with increased collagen type II immunoreaction up to eight months.

Genetic modification of chondrocytes with AdIGF-1 prior to transplantation improved early (four to nine weeks), and to a lesser degree long-term, cartilage healing in the equine model.

The equine model of cartilage healing closely resembles human clinical cartilage repair. The results of this study suggest that cartilage healing can be enhanced through genetic modification of chondrocytes prior to transplantation.

The nervous system is known to be involved in inflammation and repair. We aimed to determine the effect of physical activity on the healing of a muscle injury and to examine the pattern of innervation. Using a drop-ball technique, a contusion was produced in the gastrocnemius in 20 rats. In ten the limb was immobilised in a plaster cast and the remaining ten had mobilisation on a running wheel. The muscle and the corresponding dorsal-root ganglia were studied by histological and immunohistochemical methods.

In the mobilisation group, there was a significant reduction in lymphocytes (p = 0.016), macrophages (p = 0.008) and myotubules (p = 0.008) between three and 21 days. The formation of myotubules and the density of nerve fibres was significantly higher (both p = 0.016) compared with those in the immobilisation group at three days, while the density of CGRP-positive fibres was significantly lower (p = 0.016) after 21 days.

Mobilisation after contusional injury to the muscle resulted in early and increased formation of myotubules, early nerve regeneration and progressive reduction in inflammation, suggesting that it promoted a better healing response.

We hypothesised that meniscal tears treated with mesenchymal stem cells (MSCs) together with a conventional suturing technique would show improved healing compared with those treated by a conventional suturing technique alone. In a controlled laboratory study 28 adult pigs (56 knees) underwent meniscal procedures after the creation of a radial incision to represent a tear. Group 1 (n = 9) had a radial meniscal tear which was left untreated. In group 2 (n = 19) the incision was repaired with sutures and fibrin glue and in group 3, the experimental group (n = 28), treatment was by MSCs, suturing and fibrin glue.

At eight weeks, macroscopic examination of group 1 showed no healing in any specimens. In group 2 no healing was found in 12 specimens and incomplete healing in seven. The experimental group 3 had 21 specimens with complete healing, five with incomplete healing and two with no healing. Between the experimental group and each of the control groups this difference was significant (p < 0.001).

The histological and macroscopic findings showed that the repair of meniscal tears in the avascular zone was significantly improved with MSCs, but that the mechanical properties of the healed menisci remained reduced.

Wear of polyethylene is associated with aseptic loosening of orthopaedic implants and has been observed in hip and knee prostheses and anatomical implants for the shoulder. The reversed shoulder prostheses have not been assessed as yet. We investigated the volumetric polyethylene wear of the reversed and anatomical Aequalis shoulder prostheses using a mathematical musculoskeletal model. Movement and joint stability were achieved by EMG-controlled activation of the muscles. A non-constant wear factor was considered. Simulated activities of daily living were estimated from in vivo recorded data.

After one year of use, the volumetric wear was 8.4 mm³ for the anatomical prosthesis, but 44.6 mm³ for the reversed version. For the anatomical prosthesis the predictions for contact pressure and wear were consistent with biomechanical and clinical data. The abrasive wear of the polyethylene in reversed prostheses should not be underestimated, and further analysis, both experimental and clinical, is required.

Human articular cartilage samples were retrieved from the resected material of patients undergoing total knee replacement. Samples underwent automated controlled freezing at various stages of preparation: as intact articular cartilage discs, as minced articular cartilage, and as chondrocytes immediately after enzymatic isolation from fresh articular cartilage. Cell viability was examined using a LIVE/DEAD assay which provided fluorescent staining. Isolated chondrocytes were then cultured and Alamar blue assay was used for estimation of cell proliferation at days zero, four, seven, 14, 21 and 28 after seeding. The mean percentage viabilities of chondrocytes isolated from group A (fresh, intact articular cartilage disc samples), group B (following cryopreservation and then thawing, after initial isolation from articular cartilage), group C (from minced cryopreserved articular cartilage samples), and group D (from cryopreserved intact articular cartilage disc samples) were 74.7% (95% confidence interval (CI) 73.1 to 76.3), 47.0% (95% CI 43 to 51), 32.0% (95% CI 30.3 to 33.7) and 23.3% (95% CI 22.1 to 24.5), respectively. Isolated chondrocytes from all groups were expanded by the following mean proportions after 28 days of culturing: group A ten times, group B 18 times, group C 106 times, and group D 154 times.

This experiment demonstrated that it is possible to isolate viable chondrocytes from cryopreserved intact human articular cartilage which can then be successfully cultured.

The gelatin-based haemostyptic compound Spongostan was tested as a three-dimensional (3D) chondrocyte matrix in an in vitro model for autologous chondrocyte transplantation using cells harvested from bovine knees. In a control experiment of monolayer cultures, the proliferation or de-differentiation of bovine chondrocytes was either not or only marginally influenced by the presence of Spongostan (0.3 mg/ml).

In monolayers and 3-D Minusheet culture chambers, the cartilage-specific differentiation markers aggrecan and type-II collagen were ubiquitously present in a cell-associated fashion and in the pericellular matrix. The Minusheet cultures usually showed a markedly higher mRNA expression than monolayer cultures irrespective of whether Spongostan had been present or not during culture. Although the de-differentiation marker type-I collagen was also present, the ratio of type-I to type-II collagen or aggrecan to type-I collagen remained higher in Minusheet 3-D cultures than in monolayer cultures irrespective of whether Spongostan had been included in or excluded from the monolayer cultures. The concentration of GAG in Minusheet cultures reached its maximum after 14 days with a mean of 0.83 ± 0.8 μg/10⁶ cells; mean ±, sem, but remained considerably lower than in monolayer cultures with/without Spongostan.

Our results suggest that Spongostan is in principle suitable as a 3-D chondrocyte matrix, as demonstrated in Minusheet chambers, in particular for a culture period of 14 days. Clinically, differentiating effects on chondrocytes, simple handling and optimal formability may render Spongostan an attractive 3-D scaffold for autologous chondrocyte transplantation.

The aim of our study was to investigate the effect of platelet-rich plasma on the proliferation and differentiation of rat bone-marrow cells and to determine an optimal platelet concentration in plasma for osseous tissue engineering. Rat bone-marrow cells embedded in different concentrations of platelet-rich plasma gel were cultured for six days. Their potential for proliferation and osteogenic differentiation was analysed. Using a rat limb-lengthening model, the cultured rat bone-marrow cells with platelet-rich plasma of variable concentrations were transplanted into the distraction gap and the quality of the regenerate bone was evaluated radiologically.

Cellular proliferation was enhanced in all the platelet-rich plasma groups in a dose-dependent manner. Although no significant differences in the production and mRNA expression of alkaline phosphatase were detected among these groups, mature bone regenerates were more prevalent in the group with the highest concentration of platelets.

Our results indicate that a high platelet concentration in the platelet-rich plasma in combination with osteoblastic cells could accelerate the formation of new bone during limb-lengthening procedures.

An understanding of the remodelling of tendon is crucial for the development of scientific methods of treatment and rehabilitation. This study tested the hypothesis that tendon adapts structurally in response to changes in functional loading. A novel model allowed manipulation of the mechanical environment of the patellar tendon in the presence of normal joint movement via the application of an adjustable external fixator mechanism between the patella and the tibia in sheep, while avoiding exposure of the patellar tendon itself. Stress shielding caused a significant reduction in the structural and material properties of stiffness (79%), ultimate load (69%), energy absorbed (61%), elastic modulus (76%) and ultimate stress (72%) of the tendon compared with controls. Compared with the material properties the structural properties exhibited better recovery after re-stressing with stiffness 97%, ultimate load 92%, energy absorbed 96%, elastic modulus 79% and ultimate stress 80%. The cross-sectional area of the re-stressed tendons was significantly greater than that of stress-shielded tendons.

The remodelling phenomena exhibited in this study are consistent with a putative feedback mechanism under strain control. This study provides a basis from which to explore the interactions of tendon remodelling and mechanical environment.

The biological significance of cobalt-chromium wear particles from metal-on-metal hip replacements may be different to the effects of the constituent metal ions in solution. Bacteria may be able to discriminate between particulate and ionic forms of these metals because of a transmembrane nickel/cobalt-permease. It is not known whether wear particles are bacteriocidal.

We compared the doubling time of coagulase negative staphylococcus, Staphylococcus aureus and methicillin resistant S. aureus when cultured in either wear particles from a metal-on-metal hip simulator, wear particles from a metal-on-polyethylene hip simulator, metal ions in solution or a control.

Doubling time halved in metal-on-metal (p = 0.003) and metal-on-polyethylene (p = 0.131) particulate debris compared with the control.

Bacterial nickel/cobalt-transporters allow metal ions but not wear particles to cross bacterial membranes. This may be useful for testing the biological characteristics of different wear debris. This experiment also shows that metal-on-metal hip wear debris is not bacteriocidal.

The outcome of a cemented hip arthroplasty is partly dependent on the type of cement which is used. The production of an interface gap between the stem and the cement mantle as a result of shrinkage of the cement, may be a factor involved. Palacos R, Palacos LV (both with gentamicin), CMW 1, CMW 2, CMW Endurance (CMWE) and Simplex were prepared under vacuum and allowed to cure overnight in similar cylinders. The next day this volume was determined by the displacement of water. Shrinkage varied between 3.82% and 7.08% with CMWE having the lowest and Palacos LV the highest. This could be a factor to consider when choosing a cement for a shape-closed stem.

The weight-bearing status of articular cartilage has been shown to affect its biochemical composition. We have investigated the topographical variation of sulphated glycosaminoglycan (GAG) relative to the DNA content of the chondrocyte in human distal femoral articular cartilage.

Paired specimens of distal femoral articular cartilage, from weight-bearing and non-weight-bearing regions, were obtained from 13 patients undergoing above-knee amputation. After papain enzyme digestion, spectrophotometric GAG and fluorometric DNA assays assessed the biochemical composition of the samples. The results were analysed using a paired t-test.

Although there were no significant differences in cell density between the regions, the weight-bearing areas showed a significantly higher concentration of GAG relative to DNA when compared with non-weight-bearing areas (p = 0.02).

We conclude that chondrocytes are sensitive to their mechanical environment, and that local loading conditions influence the metabolism of the cells and hence the biochemical structure of the tissue.

We have studied the effects of bupivacaine on human and bovine articular chondrocytes in vitro. Time-lapse confocal microscopy of human articular chondrocytes showed > 95% cellular death after exposure to 0.5% bupivacaine for 30 minutes. Human and bovine chondrocytes exposed to 0.25% bupivacaine had a time-dependent reduction in viability, with longer exposure times resulting in higher cytotoxicity. Cellular death continued even after removal of 0.25% bupivacaine. After exposure to 0.25% bupivacaine for 15 minutes, flow cytometry showed bovine chondrocyte viability to be 41% of saline control after seven days. After exposure to 0.125% bupivacaine for up to 60 minutes, the viability of both bovine and human chondrocytes was similar to that of control groups.

These data show that prolonged exposure 0.5% and 0.25% bupivacaine solutions are potentially chondrotoxic.

We investigated the effect of progesterone on the nerve during lengthening of the limb in rats. The sciatic nerves of rats were elongated by leg lengthening for ten days at 3 mm per day. On alternate days between the day after the operation and nerve dissection, the progesterone-treated group received subcutaneous injections of 1 mg progesterone in sesame oil and the control group received oil only. On the fifth, tenth and 17th day, the sciatic nerves were excised at the midpoint of the femur and the mRNA expression level of myelin protein P0 was analysed by quantitative real time polymerase chain reaction. On day 52 nodal length was examined by electron microscopy, followed by an examination of the compound muscle action potential (C-MAP) amplitude and the motor conduction velocity (MCV) of the tibial nerve on days 17 and 52. The P0 (a major myelin glycoprotein) mRNA expression level in the progesterone-treated group increased by 46.6% and 38.7% on days five and ten, respectively. On day 52, the nodal length in the progesterone-treated group was smaller than that in the control group, and the MCV of the progesterone-treated group had been restored to normal.

Progesterone might accelerate the restoration of demyelination caused by nerve elongation by activating myelin synthesis.

We have undertaken a prospective study in patients with a fracture of the femoral shaft requiring intramedullary nailing to test the hypothesis that the femoral canal could be a potential source of the second hit phenomenon. We determined the local femoral intramedullary and peripheral release of interleukin-6 (IL-6) after fracture and subsequent intramedullary reaming.

In all patients, the fracture caused a significant increase in the local femoral concentrations of IL-6 compared to a femoral control group. The concentration of IL-6 in the local femoral environment was significantly higher than in the patients own matched blood samples from their peripheral circulation. The magnitude of the local femoral release of IL-6 after femoral fracture was independent of the injury severity score and whether the fracture was closed or open.

In patients who underwent intramedullary reaming of the femoral canal a further significant local release of IL-6 was demonstrated, providing evidence that intramedullary reaming can cause a significant local inflammatory reaction.

Post-traumatic arthritis is a frequent consequence of articular fracture. The mechanisms leading to its development after such injuries have not been clearly delineated. A potential contributing factor is decreased viability of the articular chondrocytes. The object of this study was to characterise the regional variation in the viability of chondrocytes following joint trauma. A total of 29 osteochondral fragments from traumatic injuries to joints that could not be used in articular reconstruction were analysed for cell viability using the fluorescence live/dead assay and for apoptosis employing the TUNEL assay, and compared with cadaver control fragments.

Chondrocyte death and apoptosis were significantly greater along the edge of the fracture and in the superficial zone of the osteochondral fragments. The middle and deep zones demonstrated significantly higher viability of the chondrocytes. These findings indicate the presence of both necrotic and apoptotic chondrocytes after joint injury and may provide further insight into the role of chondrocyte death in post-traumatic arthritis.

Conventional amputation prostheses rely on the attachment of the socket to the stump, which may lead to soft-tissue complications. Intraosseous transcutaneous amputation prostheses (ITAPs) allow direct loading of the skeleton, but their success is limited by infection resulting from breaching of the skin at the interface with the implant. Keratinocytes provide the skin’s primary barrier function, while hemidesmosomes mediate their attachment to natural ITAP analogues. Keratinocytes must attach directly to the surface of the implant. We have assessed the proliferation, morphology and attachment of keratinocytes to four titaniumalloy surfaces in order to determine the optimal topography in vitro. We used immunolocalisation of adhesion complex components, scanning electron microscopy and transmission electron microscopy to assess cell parameters.

We have shown that the proliferation, morphology and attachment of keratinocytes are affected by the surface topography of the biomaterials used to support their growth. Smoother surfaces improved adhesion. We postulate that a smooth topography at the point of epithelium-ITAP contact could increase attachment in vivo, producing an effective barrier of infection.

We have investigated in vitro the release kinetics and bioactivity of fibroblast growth factor-2 (FGF-2) released from a carrier of fibrin sealant. In order to evaluate the effects of the FGF-2 delivery mechanism on the repair of articular cartilage, full-thickness cylindrical defects, 5 mm in diameter and 4 mm in depth, which were too large to undergo spontaneous repair, were created in the femoral trochlea of rabbit knees. These defects were then filled with the sealant.

Approximately 50% of the FGF-2 was released from the sealant within 24 hours while its original bioactivity was maintained. The implantation of the fibrin sealant incorporating FGF-2 successfully induced healing of the surface with hyaline cartilage and concomitant repair of the subchondral bone at eight weeks after the creation of the defect.

Our findings suggest that this delivery method for FGF-2 may be useful for promoting regenerative repair of full-thickness defects of articular cartilage in humans.

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.

The purpose of this study was to examine the effects of hyaluronic acid supplementation on chondrocyte metabolism in vitro. The clinical benefits of intra-articular hyaluronic acid injections are thought to occur through improved joint lubrication. Recent findings have shown that exogenous hyaluronic acid is incorporated into articular cartilage where it may have a direct biological effect on chondrocytes through CD44 receptors.

Bovine articular chondrocytes were isolated and seeded into alginate constructs. These were cultured in medium containing hyaluronic acid at varying concentrations. Samples were assayed for biochemical and histological changes.

There was a dose-dependent response to the exposure of hyaluronic acid to bovine articular chondrocytes in vitro. Low concentrations of hyaluronic acid (0.1 mg/mL and 1 mg/mL) significantly increase DNA, sulphated glycosaminoglycan and hydroxyproline synthesis. Immunohistology confirmed the maintenance of cell phenotype with increased matrix deposition of chondroitin-6-sulphate and collagen type II. These findings confirm a stimulatory effect of hyaluronic acid on chondrocyte metabolism.

Bovine and human articular chondrocytes were seeded in 2% alginate constructs and cultured for up to 19 days in a rotating-wall-vessel (RWV) and under static conditions. Culture within the RWV enhanced DNA levels for bovine chondrocyte-seeded constructs when compared with static conditions but did not produce enhancement for human cells. There was a significant enhancement of glycosaminoglycans and hydroxyproline synthesis for both bovine and human chondrocytes. In all cases, histological analysis revealed enhanced Safranin-O staining in the peripheral regions of the constructs compared with the central region. There was an overall increase in staining intensity after culture within the RWV compared with static conditions. Type-II collagen was produced by both bovine and human chondrocytes in the peripheral and central regions of the constructs and the staining intensity was enhanced by culture within the RWV. A capsule of flattened cells containing type-I collagen developed around the constructs maintained under static conditions when seeded with either bovine or human chondrocytes, but not when cultured within the RWV bioreactor.