Introduction

Nonunions pose complications in fracture management that can be treated using electrical stimulation (ES). Bone marrow mesenchymal stem cells (BMMSCs) are essential in fracture healing, although the effects of different clinical ES waveforms available in clinical practice on BMMSCs cellular activities is unknown.

Delayed facture repair and bony non-unions pose a clinical challenge. Understandably, novel methods to enhance bone healing have been studied by researchers worldwide. Electrical stimulation (ES) has shown to be effective in enhancing bone healing, however the best wave form and mechanism by which it stimulates osteoblasts remains unknown. Interestingly, it is considered that osteoblast activity depends on specific waveforms applied. Therefore, the aim of this study was to evaluate whether particular waveforms have a differential effect on osteoblast activity.

An osteoblast cell line was electrically stimulated with either capacitive coupling (CC) or a novel degenerate wave (DW) using a unique in vitro ES system. Following application of both waveforms, the extent of cytotoxicity, proliferation, differentiation and mineralisation of the osteoblasts were assessed using various assays. Differentiation and mineralisation were further analysed using quantitative real-time PCR (qRT PCR) and immunocytochemistry (ICC).

DW stimulation significantly enhanced the differentiation of the osteoblasts compared to CC stimulation, with increased protein and gene expression of alkaline phosphatase and type 1 collagen at 28 hours (p < 0.01). DW significantly enhanced the mineralisation of the osteoblasts compared to CC with greater Alizarin Red S staining and gene expression of osteocalcin, osteonectin, osteopontin and bone sialoprotein at 28 hours (p < 0.05). Moreover, immunocytochemical assays showed higher osteocalcin expression after DW stimulation compared to CC at 28 hours.

The Osteoprotegerin/RANK/RANKL system has been implicated in the biological cascade of events initiated by particulate wear debris and bacterial infection resulting in periprosthetic bone loss around loosened total hip arthroplasties (THA). Individual responses to such stimuli may be dictated by genetic variation and we have studied the effect of single nucleotide polymorphisms (SNPs) within these genes.

We performed a case control study of the Osteoprotegerin, RANK and RANKL genes for possible association with deep sepsis or aseptic loosening. All patients included in the study were Caucasian and had had a cemented Charnley THA and polyethylene acetabular cup. Cases consisted of 91 patients with early aseptic loosening and 71 patients with microbiological evidence at surgery of deep infection. Controls consisted of 150 THAs that were clinically asymptomatic for over 10 years and demonstrated no radiographic features of aseptic loosening. DNA samples from all individuals were genotyped using Taqman allelic discrimination.

The A allele (p<0.001) and homozygous genotype A/A (p<0.001) for the OPG-163 SNP were highly associated with aseptic failure. Additionally, the RANK-575 (C/T SNP) T allele (p=0.004) and T/T genotype (p=0.008) frequencies were associated with aseptic failure. No statistically significant relationship was found between aseptic loosening and the OPG- 245 or OPG-1181 SNPs.

When the septic group was compared to controls, the frequency of the A allele (p<0.001) and homozygous genotype A/A (p<0.001) for the OPG-163 SNP were statistically significant. No statistically significant relationship was found between septic failure and the OPG- 245, OPG-1181 or RANK-575 SNPs.

Aseptic loosening and possibly deep infection of THA may be under genetic influence to candidate susceptibility genes. SNP markers may serve as predictors of implant survival and aid pharmacogenomic prevention of THA failure.

Failure of bone repair is a challenging problem in the management of fractures. There is a limited supply of autologous bone grafts for treating nonunions, with associated morbidity after harvesting. There is need for a better source of cells for repair. Mesenchymal stem cells (MSCs) hold promise for healing of bone because of their capacity to differentiate into osteoblasts and their availability from a wide variety of sources. Our review aims to evaluate the available clinical evidence and recent progress in strategies which attempt to use autologous and heterologous MSCs in clinical practice, including genetically-modified MSCs and those grown on scaffolds. We have compared various procedures for isolating and expanding a sufficient number of MSCs for use in a clinical setting.

There are now a number of clinical studies which have shown that implantation of MSCs is an effective, safe and durable method for aiding the repair and regeneration of bone.

Introduction: In attempting to unravel the complex cellular responses leading to prosthetic loosening investigators have been limited to studying gene expression of extracellular molecules about which most is known whereas new microarray technology allows simultaneous expression profiling of thousands of genes from a complex sample such as the membrane formed around loosened hip prostheses.

Methods: Two groups of 8 patients were recruited who have undergone primary total hip arthroplasty for osteoarthritis and subsequently developed either septic or aseptic loosening +/− osteolysis. The control group consisted of one group of 5 patients with the same initial diagnosis who had undergone identical procedures, developed no clinical or radiological signs of aseptic or septic loosening, but had come to revision surgery for other complications as defined by the Swedish Hip register: fracture without previous osteolysis, dislocation, technical error, implant fracture, polyethylene wear or pain. Periprosthetic membrane was harvested at the time of revision surgery and subjected to RNA extraction. cDNA was then synthesized and hybridised to a Human Genome u95 Genechip ® array which contains a complete set of known human genes. Data normalisation, data filtering and pattern identification was performed using Genechip®3.1 software (Affymetrix, Santa Clara, CA).

Results: This has revealed the involvement of a large number of genes coding for transcriptional regulators upstream from the extracellular and cell-cell signalling molecules already known to be involved in osteolysis and deep infection and which may ultimately control the responses to wear particles and bacterial challenge. Differential expression of genes involved in cell survival and death, cell growth regulation, cell metabolism, inflammation and immune response was found. Most interestingly pathways for control of local bone resorption and inflammatory response have been shown to be highly activated.

Conclusions: The identification of these new pathogenetic mechanisms of total hip replacement failure make new indicators of disease susceptibility and prognosis plus new drug targets direct possibilities.

Aims: Tumour necrosis factor-alpha is a proinflammatory cytokine that has been implicated in the inflammatory response to bacterial infection and wear debris particles around loosened total hip replacements (THR). Individual TNF responses to such stimuli may be dictated by genetic variation and we have studied the effect of single nucleotide polymorphisms (SNPs) within the TNF gene.

Methods: We performed a case control study of 9 SNPs (−1031, −863, −857, −376, −308, −238, +489, +851 and +1304) for possible association with deep sepsis or aseptic loosening. All patients included in the study were Caucasian and had had a cemented Charnley THR. Cases consisted of 44 patients with early aseptic loosening and 30 patients with microbiological evidence at surgery of deep infection. Controls consisted of 85 THRs that were clinically asymptomatic for over 10 years and demonstrated no radiographic features of aseptic loosening. DNA was extracted from venous blood and genotyped by Snapshot assay.

Results: Genotype and allele frequencies for all SNPs were in Hardy-Weinberg equilibrium between THR controls and a random sample of UK Caucasians. A significant association was found for the -863 SNP and aseptic loosening (p< 0.05; OR=2.36; 95% CI: 0.976 – 5.71). A trend towards association was found between the -863A SNP and deep infection (p=0.80; OR=2.42; CI: 0.800 – 7.34).

Conclusions: Genetic polymorphism of TNF-alpha may play a significant role in THR aseptic loosening and possibly in deep infection. SNP markers may serve as predictors of implant survival and response to therapy such as anti-TNF treatment.

Purpose: In attempting to unravel the complex cellular responses leading to prosthetic loosening investigators have been limited to studying gene expression of extracellular molecules about which most is known whereas new microarray technology allows simultaneous expression profiling of thousands of genes from a complex sample such as the membrane formed around loosened hip prostheses.

Methods: Two groups of 8 patients were recruited who have undergone primary total hip arthroplasty for osteoarthritis and subsequently developed either septic or aseptic loosening +/− osteolysis. The control group consisted of one group of 5 patients with the same initial diagnosis who had undergone identical procedures, developed no clinical or radiological signs of aseptic or septic loosening, but had come to revision surgery for other complications as defined by the Swedish Hip register: fracture without previous osteolysis, dislocation, technical error, implant fracture, polyethylene wear or pain. Peri-prosthetic membrane was harvested at the time of revision surgery and subjected to RNA extraction. cDNA was then synthesized and hybridised to a Human Genome u95 Genechip ® array which contains a complete set of known human genes. Data normalisation, data filtering and pattern identification was performed using Genechip®3.1 software (Affymetrix, Santa Clara, CA).

Results: This has revealed the involvement of a large number of genes coding for transcriptional regulators upstream from the extracellular and cell-cell signalling molecules already known to be involved in osteolysis and deep infection and which may ultimately control the responses to wear particles and bacterial challenge. Differential expression of genes involved in cell survival and death, cell growth regulation, cell metabolism, inflammation and immune response was found. Most interestingly pathways for control of local bone resorption and inflammatory response have been shown to be highly activated.

Conclusions: The identification of these new pathogenetic mechanisms of total hip replacement failure make new indicators of disease susceptibility and prognosis plus new drug targets direct possibilities.

Dupuytren’s disease is a benign fibroproliferative disease of unknown aetiology. It is often familial and commonly affects Northern European Caucasian men, but genetic studies have yet to identify the relevant genes.

Transforming growth factor beta one (TGF-β1) is a multifunctional cytokine which plays a central role in wound healing and fibrosis. It stimulates the proliferation of fibroblasts and the deposition of extracellular matrix. Previous studies have implicated TGF-β1 in Dupuytren’s disease, suggesting that it may represent a candidate susceptibility gene for this condition.

We have investigated the association of four common single nucleotide polymorphisms in TGF-β1 with the risk of developing Dupuytren’s disease. A polymerase chain reaction-restriction fragment length polymorphism method was used for genotyping TGF-β1 polymorphisms. DNA samples from 135 patients with Dupuytren’s disease and 200 control subjects were examined.

There was no statistically significant difference in TGF-β1 genotype or allele frequency distributions between the patients and controls for the codons 10, 25, −509 and −800 polymorphisms.

Our observations suggest that common TGF-β1 polymorphisms are not associated with a risk of developing Dupuytren’s disease. These data should be interpreted with caution since the lack of association was shown in only one series of patients with only known, common polymorphisms of TGF-β1. To our knowledge, this is the first report of a case-control association study in Dupuytren’s disease using single nucleotide polymorphisms in TGF-β1.