In relation to regenerative therapies in osteoarthritis and cartilage repair, mesenchymal stromal cells (MSCs) have immunomodulatory functions and influence macrophage behaviour. Macrophages exist as a spectrum of pro-(M1) and anti-(M2) inflammatory phenotypic subsets. In the context of cartilage repair, we investigated MSC-macrophage crosstalk, including specifically the priming of cartilage cells by macrophages to achieve a regenerative rather than fibrotic outcome. Human monocytes were isolated from blood cones and differentiated towards M1 and M2 macrophages. Monocytes (Mo), M1 and M2 macrophages were cultured directly and indirectly (trans-well system) with human bone marrow derived MSCs. MSCs were added during M1 polarisation and separately to already induced M1 cells. Outcomes (M1/M2 markers and ligands/receptors) were evaluated using RT-qPCR and flow cytometry. Influence on chondrogenesis was assessed by applying M1 and M2 macrophage conditioned media (CM) sequentially to cartilage derived cells (recapitulating an acute injury environment). RT-qPCR was used to evaluate chondrogenic/fibrogenic gene transcription. The ratio of M2 markers (CD206 or CD163) to M1 markers (CD38) increased when MSCs were added to Mo/M1 macrophages, regardless of culture system used (direct or indirect). Pro-inflammatory markers (including TNFβ) decreased. CXCR2 expression by both M1 macrophages and MSCs decreased when MSCs were added to differentiated M1 macrophages in transwell. When adding initially M1 CM (for 12 hours) followed by M2 CM (for 12 hours) sequentially to chondrocytes, there was a significant increase of Aggrecan and Collagen type 2 gene expression and decrease in fibroblastic cell surface markers (PDPN/CD90). Mo/M1 macrophages cultured with MSCs, directly or indirectly, are shifted towards a more M2 phenotype. Indirect culture suggests this effect can occur via soluble signaling mediators. Sequential exposure of M1CM followed by M2CM to chondrocytes resulted in increased chondrogenic and reduced fibrotic gene expression, suggesting that an acute pro-inflammatory stimulus may prime chondrocytes before repair.

Cell culture on tissue culture plastic (TCP) is widely used across biomedical research to understand the in vivo environment of a targeted biological system. However, growing evidence indicates that the characteristics of cells investigated in this way differ substantially from their characteristics in the human body. The limitations of TCP monolayer cell cultures are especially relevant for chondrocytes, the cell population responsible for producing cartilage matrix, because their zonal organization in hyaline cartilage is not preserved in a flattened monolayer assay. Here, we contrast the response of primary human chondrocytes to inflammatory cytokines, tumor necrosis factor-alpha and interferon-gamma, via transcriptional, translational, and histological profiling, when grown either on TCP or within a 3D cell pellet (scaffold-less). We focus on anti-apoptotic (Bcl2), pro-apoptotic (Bax, Mff, Fis1), and senescent (MMP13, MMP1, PCNA, p16, p21) markers. We find that the 3D environment of the chondrocyte has a profound effect on the behavior and fate of the cell; in TCP monolayer cultures, chondrocytes become anti-apoptotic and undergo senescence in response to inflammatory cytokines, whereas in 3D cell pellet cultures, they exhibit a pro-apoptotic response. Our findings demonstrate that chondrocyte culture environment plays a pivotal role in cell behavior, which has important implications for the clinical applicability of in vitro research of cartilage repair. Although there are practical advantages to 2D cell cultures, our data suggest researchers should be cautious when drawing conclusions if they intend to extrapolate findings to in vivo phenomena. Our data demonstrates opposing chondrocyte responses in relation to apoptosis and senescence, which appear to be solely reliant on the environment of the culture system. This biological observation highlights that proper experimental design is crucial to increase the clinical utility of cartilage repair experiments and streamline their translation to therapy development.

Abstract

Abstract

Abstract

Osteoarthritis is a common articular cartilage disorder and causes a significant global disease burden. Articular cartilage has a limited capacity of repair and there is increasing interest in the use of cell-based therapies to facilitate repair including the use of Mesenchymal Stromal Cells (MSCs). There is some evidence in the literature that suggests that advancing age is associated with declining MSC function, including reduced proliferation and differentiation potential, and greater cellular apoptosis. In our study, we first performed a systematic review of the literature to determine the effects of chronological age on the in vitro properties of MSCs, and then performed a laboratory study to investigate these properties.

We initially conducted a PRISMA systematic review of the literature to review the evidence base for the effects of chronological age on the in vitro properties of MSCs including cell numbers, expansion, cell surface characterization and differentiation potential. This was followed by laboratory based experiments to assess these properties. Tissue from patients undergoing total knee replacement surgery was used to isolate MSCs from the bone fragments using a method developed in our laboratory. The growth kinetics was determined by calculating the population doublings per day. Following expansion in culture, MSCs at P2 were characterised for a panel of cell surface markers using flow cytometry. The cells were positive for CD73, CD90 and CD105, and negative for CD34 and CD45. The differentiation potential of the MSCs was assessed through tri-lineage differentiation assays. Clear differences between the younger and older patients were indicated.

Chronological age-related changes in MSC function have important implications on the use of these cells in clinical applications for an ageing population. The results from this study will be used to plan further work looking at the effects of chronological age on cellular senescence and identify pathways that could be targeted to potentially reverse any age-related changes.

The use of mesenchymal stromal cells (MSCs) in regenerative medicine and tissue engineering is well established, given their properties of self-renewal and differentiation. However, several studies have shown that these properties diminish with age, and understanding the pathways involved are important to provide regenerative therapies in an ageing population.

In this PRISMA systematic review, we investigated the effects of chronological donor ageing on the senescence of MSCs. We identified 3023 studies after searching four databases including PubMed, Web of Science, Cochrane, and Medline. Nine studies met the inclusion and exclusion criteria and were included in the final analyses.

These studies showed an increase in the expression of p21, p53, p16, ROS, and NF- B with chronological age. This implies an activated DNA damage response (DDR), as well as increased levels of stress and inflammation in the MSCs of older donors. Additionally, highlighting the effects of an activated DDR in cells from older donors, a decrease in the expression of proliferative markers including Ki67, MAPK pathway elements, and Wnt/ -catenin pathway elements was observed. Furthermore, we found an increase in the levels of SA- -galactosidase, a specific marker of cellular senescence.

Together, these findings support an association between chronological age and MSC senescence. The precise threshold for chronological age where the reported changes become significant is yet to be defined and should form the basis for further scientific investigations. The outcomes of this review should direct further investigations into reversing the biological effects of chronological age on the MSC senescence phenotype.

Abstract

Osteoarthritis is a common articular cartilage disorder and causes a significant global disease burden. Articular cartilage has a limited capacity of repair and there is increasing interest in the use of cell-based therapies to facilitate repair including the use of Mesenchymal Stromal Cells (MSCs). There is some evidence in the literature that suggests that advancing age is associated with declining MSC function, including reduced proliferation and differentiation potential, and greater cellular apoptosis. In our study, we first performed a systematic review of the literature to determine the effects of chronological age on the in vitro properties of MSCs, and then performed a laboratory study to investigate these properties. We initially conducted a PRISMA systematic review of the literature to review the evidence base for the effects of chronological age on the in vitro properties of MSCs including cell numbers, expansion, cell surface characterization and differentiation potential. This was followed by laboratory based experiments to assess these properties. Tissue from patients undergoing total knee replacement surgery was used to isolate MSCs from the infrapatellar fat pad using a method developed in our laboratory. The growth kinetics was determined by calculating the population doublings per day. Following expansion in culture, MSCs at P2 were characterised for a panel of cell surface markers using flow cytometry. The cells were positive for CD73, CD90 and CD105, and negative for CD34 and CD45. The differentiation potential of the MSCs was assessed through tri-lineage differentiation assays. Chronological age-related changes in MSC function have important implications on the use of these cells in clinical applications for an ageing population. The results from this study will be used to plan further work looking at the effects of chronological age on cellular senescence and identify pathways that could be targeted to potentially reverse any age-related changes.

Abstract

Focal articular cartilage defects do not heal and, left untreated, progress to more widespread degenerative changes. A promising new approach for the repair of articular cartilage defects is the application of cell-based regenerative therapies using mesenchymal stromal cells (MSCs). MSCs are however present in a number of tissues and studies suggest that they vary in their proliferation, cell surface characterisation and differentiation. As the phenotypic properties of MSCs vary depending on tissue source, a systematic comparison of the transcriptomic signature would allow a better understanding of these differences between tissues, and allow the identification of markers specific to a MSC source that is best suited for clinical application. Tissue was used from patients undergoing total knee replacement surgery for osteoarthritis following ethical approval and informed consent. MSCs were isolated from bone, cartilage, synovium and infrapatellar fat pad. MSC number and expansion were quantified. Following expansion in culture, MSCs were characterised using flow cytometry with several cell surface markers; the cells from all sources were positive for CD44, CD90 and CD105. Their differentiation potential was assessed through tri-lineage differentiation assays. In addition, bulk mRNA-sequencing was used to determine the transcriptomic signatures. Differentially expressed (DE) genes were predicted. An enrichment analysis focused on the DE genes, against GO and pathway databases (KEGG and Reactome) was performed; protein-protein interaction networks were also inferred (Metascape, Reactome, Cytoscape). Optimal sourcing of MSCs will amplify their cartilage regeneration potential. This is imperative for assessing future therapeutic transplantation to maximise the chance of successful cartilage repair. A better understanding of differences in MSCs from various sources has implications beyond cartilage repair.

Abstract

Abstract

Osteoarthritis (OA) is a degenerative disorder associated with cartilage loss and is a leading cause of disability around the world. In old age, the capacity of cartilage to regenerate is diminished. With an aging population, the burden of OA is set to rise. Currently, there is no definitive treatment for OA. However, cell-based therapies derived from adipose tissue are promising. A PRISMA systematic review was conducted employing four databases (MEDLINE, EMBASE, Cochrane, Web of Science) to identify all clinical studies that utilized adipose tissue derived mesenchymal stem cells (AMSCs) or stromal vascular fraction (SVF) for the treatment of knee OA. Eighteen studies were included, which met the inclusion criteria. Meta-analyses were conducted on fourteen of these studies, which all documented WOMAC scores after the administration of AMSCs. Pooled analysis revealed that cell-based treatments definitively improve WOMAC scores, post treatment. These improvements increased with time. The studies in this meta-analysis have established the safety and efficacy of both AMSC therapy and SVF therapy for knee OA in old adults and show that they reduce pain and improve knee function in symptomatic knee OA suggesting that they may be effective therapies to improve mobility in an aging population.

Aims

To investigate factors that contribute to patient decisions regarding attendance for arthroplasty during the COVID-19 pandemic.

Abstract

Abstract

The current standard of practice following knee arthroplasty is to demonstrate the appropriate alignment of knee replacements using knee radiographs. Recent studies have suggested that standard knee radiographs provide adequate accuracy for tibial prosthesis alignment assessment as compared with long knee view radiographs which are more technically demanding and carry greater radiation exposure. In this study, we aim to address whether alignment measured on standard knee radiographs are reliable and reproducible over time.

We examined a cohort of 80 patients 37 male (46%), 43 females (54%), mean age = 68 years) who underwent total knee arthroplasty (TKA). Standard knee anteroposterior radiographs performed within 2 days following surgery were compared to standard knee anteroposterior radiographs taken 1 year following the surgery in patients with well-functioning prosthesis. Tibial prosthesis alignment angles between the longitude of the tibial shaft and the tibial baseplate were calculated using Centricity Enterprise Web V3.0 software. The data was examined using R software.

In well-functioning primary knee arthroplasties, tibial prosthesis alignment angles measured in the 1-year follow-up standard view knee radiographs were found to deviate from measurements obtained with the same radiographic specifications in the immediate post-operative period. A significant mean percentage difference was found between the two radiographs.

Long knee view radiographs may be required in order to accurately assess tibial prothesis alignment following total knee arthroplasty.

Abstract

Companies manufacturing total knee arthroplasty (TKA) prostheses produce a variety of tibial and femoral components of different dimensions denoted by numbers or letters. Surgeons frequently implant components that are compatible but not of the same size on the femur and tibia. Recent studies suggest that equally sized femoral and tibial components produce better outcomes compared to size-mismatched components. In our study, we aim to explore the relationship between component size and outcome measured by oxford knee score at six weeks and one year following TKA.

A cohort of twenty-four patients who underwent TKA and had well-functioning prosthesis were studied. Thirteen (54%) had equally sized TKA components implanted, seventy-four patients (42%) had components that were mismatched by one size, and one (4%) had components that were mismatched by more than one size. The Oxford Knee Score (OKS) obtained preoperatively, at six weeks and one year postoperatively were retrieved from an electronic database. All data were analysed using R software.

A significant improvement in pre-operative and one-year postoperative OKS was observed. Patients who received one-size mismatched tibial and femoral components demonstrated a less pronounced improvement in OKS as compared with patients who received equally sized components.

When possible, it may be best to utilise equally sized prosthetic tibial and femoral components when performing total knee arthroplasty. Manufacturers may be able to produce better patient outcomes by including prostheses that are between sizes as part of their production line.

At first-stage revision surgery for infection of total knee arthroplasties, antibiotic-impregnated cement spacers are frequently implanted. Two types of cement spacers are commonly used, “static” and “articulating” cement spacers. Advocates of cement spacers state that they deliver high doses of antibiotics locally, increase patient comfort, allow mobility and provide joint stability. They also minimize contracture of collateral ligaments, thereby facilitating re-implantation of a definitive prosthesis at a later stage. The use of these cement spacers, however, are not without significant complications, including patella tendon injuries.

We describe a series of three patients who sustained patella tendon injuries in infected total knee arthroplasties following the use of a static cement spacer at first-stage knee revision.

The patella tendon injuries resulted in significant compromise to wound healing and knee stability requiring multiple surgeries. The mid-term function was poor with an Oxford score at 24 months ranging from 12–20

Based on our experience, we advise caution in the use of static cement spacer blocks. If they are to be used, we recommend that they should be keyed in the bone to prevent patella tendon injuries.

Honey has been used as a topical antiseptic for at least 5,000 years. SurgiHoney is a CE licensed sterile product, which has been proven to be non-toxic and effective when used topically in the treatment of chronically infected wounds. The key difference from other medical grade honey is the broad spectrum antimicrobial characteristics with activity against Gram +ve, Gram –ve and multi-resistant organisms. Its novel role against the bacterial bioburden and biofilm associated with periprosthetic infections around total knee arthroplasties (TKA's) is therefore considered.

SurgiHoney was used as an implant coating immediately prior to wound closure after implantation of salvage endoprosthesis for multiply revised, infected TKA's undergoing staged reconstruction.

We report a consecutive series of multi-revised, infected revision TKA's where SurgiHoney was used as an active antimicrobial coating. We discuss its intra-operative application and early clinical outcomes.

The use of Surgihoney as a novel anti-microbial is established in the management of complex wound infections. This is the first reported use of SurgiHoney as a deep, implant coating in the salvage of prosthetic joint infection.

Joint degeneration may make a total knee arthroplasty (TKA) a requirement for pain relief and function. However, the presence of ipsilateral limb osteomyelitis (OM) makes surgical management extremely challenging.

We report the experience of a high volume revision knee surgeon managing ipsilateral limb multi resistant OM and the outcome of subsequent TKA.

Four consecutive patients were identified who had either ipsilateral femoral or tibial chronic osteomyelitis treated prior to undergoing TKA. Surgery to eradicate the osteomyelitis involved a Lautenbach compartmental debridement, and where necessary, healing by secondary intention. The decision to proceed to a TKA was based on history, clinical examination and radiological findings of advanced osteoarthritic change.

The patients had a mean age of 50 years. They had a background of multi-organism OM and underwent single-stage TKAs at an average of 63 months following eradication of the underlying OM.

Three patients did well but had complications associated with poor skin and soft tissues, and abnormal bone anatomy. One patient developed an infection and following a re-revision had an arthrodesis.

The results for the four cases are summarised in Table 1.

We have highlighted that patients with ipsilateral limb multi resistant OM are a difficult cohort to manage.

A common step to revision surgery for infected total knee replacement (TKR) is a thorough debridement. Whilst surgical and mechanical debridement are established as the gold standard, we investigate a novel adjuvant chemical debridement using an Acetic Acid (AA) soak that seeks to create a hostile environment for organisms, further degradation of biofilm and death of the bacteria.

We report the first orthopaedic in vivo series using AA soak as an intra-operative chemical debridement agent for treating infected TKR's. We also investigate the in vitro efficacy of AA against bacteria isolated from infected TKR's.

A prospective single surgeon consecutive series of patients with infected TKR were treated according to a standard debridement protocol. Patients in the series received sequential debridement of surgical, mechanical and finally chemical debridement with a 10 minute 3% AA soak.

In parallel, we isolated, cultured and identified bacteria from infected TKR's and assessed the in vitro efficacy of AA. Susceptibility testing was performed with AA solutions of different concentrations as well as with a control of a gentamicin sulphate disc. The effect of AA on the pH of tryptone soya was also monitored in an attempt to understand its potential mechanism of action.

Physiological responses during the AA soak were unremarkable. Intraoperatively, there were no tachycardic or arrythmic responses, any increase in respiratory rate or changes in blood pressure. This was also the case when the tourniquet was released. In addition, during the post-operative period no increase in analgesic requirements or wound complications was noted. Wound and soft tissue healing was excellent and there have not been any early recurrent infections at mean of 18 months follow up.

In vitro, zones of inhibition were formed on less than 40% of the organisms, demonstrating that AA was not directly bactericidal against the majority of the clinical isolates. However, when cultured in a bacterial suspension, AA completely inhibited the growth of the isolates at concentrations as low as 0.19%v/v.

This study has shown that the use of 3% AA soak, as part of a debridement protocol, is safe. Whilst the exact mechanism of action of acetic acid is yet to be determined, we have demonstrated that concentrations as low as 0.19%v/v in solution in vitro is sufficient to completely inhibit bacterial growth from infected TKR's.

Introduction

Mesenchymal stem cells (MSCs) are a potential source of cells for the repair of articular cartilage and osteochondral defects (OCD) in the ankle. Synovial tissue has been shown to be a rich source of MSCs with the ability to undergo chondrogenic differentiation. Although these cells represent a heterogenous population, clonal populations have not been previously studied.

In recent years, there has been an increase in using self- admistrated questionnaires to accurately assess intervention outcomes in hand surgery to determine the quality of healthcare. This study aims to evaluate whether the Manchester Modified Disabilities of the Arm, Shoulder and Hand (M2DASH) questionnaire is a valid, reliable, responsive, and unbiased outcome measure for Carpal Tunnel syndrome compared to the Disability of Arm, Shoulder, and Hand (DASH) questionnaire, Boston questionnaire (BQ), and Nerve Conduction Studies (NCS).

Introduction

Patients undergoing limb reconstruction with the Taylor Spatial Frame (TSF) often perceive that their frame is loose due to the rattle they hear when mobilising. Our aim was to determine how much and where this movement is in the various frame/bone constructs currently on the market.

Background & Objectives

Sensory and motor manifestations in carpal tunnel syndrome (CTS) are well documented, whereas the associated autonomic dysfunction is often overlooked. The aim of this study is to demonstrate that autonomic dysfunction of the CTS hands can be quantified by measuring skin capacitance.

INTRODUCTION

Bone marrow derived mesenchymal stem cells are a potential source of cells for the repair of articular cartilage defects. Hypoxia has been shown to improve chondrogenesis in adult stem cells. In this study we characterised bone marrow derived stem cells and investigated the effects of hypoxia on gene expression changes and chondrogenesis.

Introduction

Mesenchymal stem cells are a potential source of cells for the repair of articular cartilage defects. We have previously demonstrated that the infrapatellar synovial fat pad is a rich source of mesenchymal stem cells and these cells are able to undergo chondrogenic differentiation. Although synovial fat pad derived mesenchymal stem cells may represent a heterogenous population, clonal populations derived from the synovial fat pad have not previously been studied.

INTRODUCTION

Bone marrow derived mesenchymal stem cells are a potential source of cells for the repair of articular cartilage defects. Hypoxia has been shown to improve chondrogenesis in adult stem cells. In this study we characterised bone marrow derived stem cells and investigated the effects of hypoxia on gene expression changes and chondrogenesis.

Introduction

Mesenchymal stem cells are a potential source of cells for the repair of articular cartilage defects. We have previously demonstrated that the infrapatellar synovial fat pad is a rich source of mesenchymal stem cells and these cells are able to undergo chondrogenic differentiation. Although synovial fat pad derived mesenchymal stem cells may represent a heterogenous population, clonal populations derived from the synovial fat pad have not previously been studied.

Introduction: Bone marrow derived mesenchymal stem cells are a potential source of cells for the repair of articular cartilage defects. Hypoxia has been shown to improve chondrogenesis in adult stem cells. In this study we characterised bone marrow derived stem cells and investigated the effects of hypoxia on gene expression changes and chondrogenesis.

Material and Methods: Adherent colony forming cells were isolated and cultured from the stromal component of bone marrow. The cells at passage 2 were characterised for stem cell surface epitopes, and then cultured as cell aggregates in chondrogenic medium under normoxic (20% oxygen) or hypoxic (5% oxygen) conditions for 14 days. Gene expression analysis, glycosoaminoglycan and DNA assays, and immunohistochemical staining were determined to assess chondrogenesis.

Results: Bone marrow derived adherent colony forming cells stained strongly for markers of adult mesenchymal stem cells including CD44, CD90 and CD105, and they were negative for the haematopoietic cell marker CD34 and for the neural and myogenic cell marker CD56. Interestingly, a high number of cells were also positive for the pericyte marker 3G5. Cell aggregates showed a chondrogenic response and in lowered oxygen there was increased matrix accumulation of proteoglycan, but less cell proliferation, which resulted in 3.2-fold more glycosoaminoglycan per DNA after 14 days of culture. In hypoxia there was increased expression of key transcription factor SOX6, and the expression of collagens II and XI, and aggrecan was also increased.

Discussion: Pericytes are a candidate stem cell in many tissue and our results show that bone marrow derived mesenchymal stem cells express the pericyte marker 3G5. The response to chondrogenic culture in these cells was enhanced by lowered oxygen tension, which up-regulated SOX6 and increased the synthesis and assembly of matrix during chondrogenesis. This has important implications for tissue engineering applications of bone marrow derived stem cells.

Introduction: There is an ever-increasing clinical need for the regeneration and replacement of tissue to replace soft tissue lost due to trauma, disease and cosmetic surgery. A potential alternative to the current treatment modalities is the use of tissue engineering applications using mesenchymal stem cells that have been identified in many tissues including the fat pad. In this study, stem cells isolated from the fat pad were characterised and their differentiation potential assessed.

Materials and Methods: The infrapatellar fat pad was obtained from total knee replacement for osteoarthritis. Cells were isolated, expanded and stained for a number of stem cell markers. For adipogenic differentiation, cells were cultured in adipogenic inducing medium (10ug/ml insulin, 1uM dexamthasone, 100uM indomethacin and 500uM 3-isobutyl-1-methyl xanthine). Gene expression analyses and Oil red O staining was performed to assess adipogenesis.

Results: Cells at passage 2 stained strongly for CD13, CD29, CD44, CD90 and CD105 (mesenchymal stem cell markers). The cells stained sparsely for 3G5 (peri-cyte marker). On gene expression analyses, the cells cultured under adipogenic conditions had almost a 1,000 fold increase in expression of peroxisome proliferator-activated receptor gamma-2 (PPAR gamma-2) and 1,000,000 fold increase in expression of lipoprotein lipase (LPL). Oil red O staining revealed triglyceride accumulation within typical adipogenic morphology, confirming the adipogenic nature of the observed vacuoles, and showed failure of staining in control cells.

Discussion: Fat pad derived stem cells expressed a cell surface epitope profile of mesenchymal stem cells, and exhibited the potential to undergo adipogenic differentiation. Our results show that the human fat pad is a viable potential autogeneic source for mesenchymal stem cells capable of adipogenic differentiation as well as previously documented ostegenic and chondrogenic differentiation. This cell source has potential use in tissue engineering applications.

Bone marrow derived mesenchymal stem cells are a potential source of cells for the repair of articular cartilage defects. Hypoxia has been shown to improve chondrogenesis in adult stem cells. In this study we characterised bone marrow derived stem cells and investigated the effects of hypoxia on gene expression changes and chondrogenesis.

Adherent colony forming cells were isolated and cultured from the stromal component of bone marrow. The cells at passage 2 were characterised for stem cell surface epitopes, and then cultured as cell aggregates in chondrogenic medium under normoxic (20% oxygen) or hypoxic (5% oxygen) conditions for 14 days. Gene expression analysis, glycosoaminoglycan and DNA assays, and immunohistochemical staining were determined to assess chondrogenesis.

Bone marrow derived adherent colony forming cells stained strongly for markers of adult mesenchymal stem cells including CD44, CD90 and CD105, and they were negative for the haematopoietic cell marker CD34 and for the neural and myogenic cell marker CD56. Interestingly, a high number of cells were also positive for the pericyte marker 3G5. Cell aggregates showed a chondrogenic response and in lowered oxygen there was increased matrix accumulation of proteoglycan, but less cell proliferation, which resulted in 3.2-fold more glycosoaminoglycan per DNA after 14 days of culture. In hypoxia there was increased expression of key transcription factor SOX6, and the expression of collagens II and XI, and aggrecan was also increased.

Pericytes are a candidate stem cell in many tissue and our results show that bone marrow derived mesenchymal stem cells express the pericyte marker 3G5. The response to chondrogenic culture in these cells was enhanced by lowered oxygen tension, which up-regulated SOX6 and increased the synthesis and assembly of matrix during chondrogenesis. This has important implications for tissue engineering applications of bone marrow derived stem cells.

The aim of this study was two-fold; firstly, to investigate the construct validity of the Disability of the Arm, Shoulder and Hand (DASH) score in patients following injuries to the upper and lower limbs, and to confirm that DASH score does not measure disability solely attributed to the upper limb. Secondly, to create a modified DASH questionnaire (M2 DASH) with fewer questions that can discriminate clearly between disabilities due to problems at the upper limb, and is more specific to the upper limb.

Patients were asked to fill in the DASH questionnaire in a fracture clinic following ethical approval. This included upper limb injuries (79), lower limb injuries (61) and control subjects (52). The median DASH scores for the three groups were 57, 16 and one respectively. The DASH scores varied significantly between the three groups (Kruskal-Wallis: p< 0.001); the scores for the upper limb group were higher than the lower limb group, and the scores for the lower limb group was higher than the control group (Mann-Whitney: p< 0.001). The M2 DASH questionnaire was developed using questions specific to the upper limb and included questions 1–4, 6, 13–17, 21–23 and 26–30. The median M2 DASH scores for the three groups were 50, 7 and 0 respectively. The revised questionnaire score was then calculated for the upper limb group and a correlation study showed good correlation between the two questionnaires.

Our study shows that the original DASH questionnaire is not specific for the upper limb. This has important implications in measuring response in injuries and disease that involve both upper and lower limbs. We have devised a revised questionnaire that we suggest is referred to as M2 DASH questionnaire. The M2 DASH questionnaire has the advantage of being more specific for the upper limb than the DASH questionnaire.

Bone marrow derived mesenchymal stem cells are a potential source of cells for the repair of articular cartilage defects. Hypoxia has been shown to improve chondrogenesis in adult stem cells. In this study we characterised bone marrow derived stem cells and investigated the effects of hypoxia on gene expression changes and chondrogenesis.

Adherent colony forming cells were isolated and cultured from the stromal component of bone marrow. The cells at passage 2 were characterised for stem cell surface epitopes, and then cultured as cell aggregates in chondrogenic medium under normoxic (20% oxygen) or hypoxic (5% oxygen) conditions for 14 days. Gene expression analysis, glycosoaminoglycan and DNA assays, and immunohistochemical staining were determined to assess chondrogenesis.

Bone marrow derived adherent colony forming cells stained strongly for markers of adult mesenchymal stem cells including CD44, CD90 and CD105, and they were negative for the haematopoietic cell marker CD34 and for the neural and myogenic cell marker CD56. Interestingly, a high number of cells were also positive for the pericyte marker 3G5. Cell aggregates showed a chondrogenic response and in lowered oxygen there was increased matrix accumulation of proteoglycan, but less cell proliferation, which resulted in 3.2-fold more glycosoaminoglycan per DNA after 14 days of culture. In hypoxia there was increased expression of key transcription factor SOX6, and the expression of collagens II and XI, and aggrecan was also increased.

Pericytes are a candidate stem cell in many tissue and our results show that bone marrow derived mesenchymal stem cells express the pericyte marker 3G5. The response to chondrogenic culture in these cells was enhanced by lowered oxygen tension, which up-regulated SOX6 and increased the synthesis and assembly of matrix during chondrogenesis. This has important implications for tissue engineering applications of bone marrow derived stem cells.

There is an ever-increasing clinical need for the regeneration and replacement of tissue to replace soft tissue lost due to trauma, disease and cosmetic surgery. A potential alternative to the current treatment modalities is the use of tissue engineering applications using mesenchymal stem cells that have been identified in many tissue including the infrapatellar fat pad. In this study, stem cells isolated from the infrapatellar fat pad were characterised to ascertain their origin, and allowed to undergo adipogenic differentiation to confirm multilineage differentiation potential.

The infrapatellar fat pad was obtained from total knee replacement for osteoarthritis. Cells were isolated and expanded in monolayer culture. Cells at passage 2 stained strongly for CD13, CD29, CD44, CD90 and CD105 (mesenchymal stem cell markers). The cells stained poorly for LNGFR and STRO1 (markers for freshly isolated bone marrow derived stem cells), and sparsely for 3G5 (pericyte marker). Staining for CD34 (haematopoetic marker) and CD56 (neural and myogenic lineage marker) was negative. {BR}For adipogenic differentiation, cells were cultured in adipogenic inducing medium consisting of basic medium with 10ug/ml insulin, 1uM dexamthasone, 100uM indomethacin and 500uM 3-isobutyl-1-methyl xanthine. By day 16, many cells had lipid vacuoles occupying most of the cytoplasm. On gene expression analyses, the cells cultured under adipogenic conditions had almost a 1,000 fold increase in expression of peroxisome proliferator-activated receptor gamma-2 (PPAR gamma-2) and 1,000,000 fold increase in expression of lipoprotein lipase (LPL). Oil red O staining confirmed the adipogenic nature of the observed vacuoles and showed failure of staining in control cells.

Our results show that the human infrapatellar fat pad is a viable potential autogeneic source for mesenchymal stem cells capable of adipogenic differentiation as well as previously documented ostegenic and chondrogenic differentiation. This cell source has potential use in tissue engineering applications.

Introduction: Autologous chondrocytes harvested from articular cartilage are being used for the repair of focal cartilage defects. The procedure involves injury to the cartilage and alternative sources of stem cells for use in repair are being explored. Stem cells have been found in many tissue including bone marrow and the infrapa-tellar fat pad. Infrapatellar fat pad derived stem cells present a viable and easily accessible source of stem cells for the repair of cartilage defects and tissue engineering applications.

Hypoxia has been shown to improve chondrogenesis in stem cells derived from the bone marrow. We explore the hypothesis that this effect would also apply to stem cells derived from the infrapatellar fat pad.

Materials and methods: Cell aggregates from early passage stem cells isolated from the infrapatellar fat pad were placed in chondrogenic media for 14 days either in a normoxic (20% oxygen) or hypoxic (5% oxygen) environment. Gene expression analysis, DNA and glycosoaminoglycan assays and immunohistochemi-cal studies were performed on the aggregates to assess chondrogenesis.

Results: Cells grown under hypoxic conditions showed significantly improved chondrogenesis as determined by relatively higher gene expression of proteoglycans, collagens and SOX genes. The cell aggregates also had a higher glycosoaminoglycan content and glycosoamino-glycan content per DNA. Immunohistochemical studies confirm enhanced production of collagen types I and II and aggrecan.

Discussion: These findings confirm the previously documented effects of hypoxic culture conditions on stem cells and extend the findings to include infrapatellar fat pad derived stem cells. Our findings suggest that oxygen tension has a role in regulating the function of stem cells as they undergo chondrogenesis. In culture these cells appear to function optimally in an atmosphere of reduced oxygen that more closely approximates documented in vivo oxygen tension. This has important implications in future tissue engineering applications of these cells.

Introduction: In this study the optimal angle of fixation or splintage for partially weight bearing fractures of the patella was determined by a gait analysis measurement system.

Subjects and Methods: A knee brace was applied to eight subjects and locked at 0, 10, 20 and 30 degrees. Measurements were also taken for an unlocked brace and in the absence of a brace. The subjects were instructed on partial weight bearing mobilisation. Three dimensional motion analyses were performed using an infrared 8-camera system. The ground reaction force was recorded by two 3-dimensional force plates embedded in the walkway. Kinematic and kinetic data was collected and the data was transferred to a computer programme for further analysis and the forces acting on the patella were calculated.

Results: The results showed that the forces acting on the patella were directly proportional to the knee flexion angle. The results also showed that the knee flexion angle does not always correspond with the angle set at the knee brace; however they did exhibit a direct relationship.

Conclusion: Our findings show that, for partially weight bearing patella fractures, the optimum form of splintage corresponds with a low knee flexion angle.

Patella and extensor mechanism injuries are common injuries and are generally managed with some degree of immobilisation and partial weight bearing to facilitate healing. The aim of this project was to determine the type of immobilisation or splintage during partial weight bearing that results in minimal forces acting through the extensor mechanism.

Gait analysis studies were performed on eight healthy male subjects mobilising partially weight bearing. Measurements were taken for six types of immobilisation: locked at 0, 10, 20, 30 degrees and unlocked in an orthotic knee brace, and without a brace. The ground reaction force, knee joint angle and the knee flexion moment were measured using Qualisys Track Manager and Visual 3D Software. The extensor mechanism moment and the extensor mechanism force were calculated using static equilibrium equations and documented data. A one-way analysis of variance statistical test was performed to determine the statistical significance of the differences between the six types of immobilisation.

There was a direct relationship between the knee flex-ion angle and the extensor mechanism force. The extensor mechanism force at 0 degrees of immobilisation was significantly lower than that for 20 and 30 degrees (p< 0.05). The increase in the extensor mechanism moment arm with increasing knee flexion was not suf-ficient to offset the increase in the extensor mechanism force caused by the increase in the knee flexion moment. The results also showed that the knee flexion angle does not always correspond with the angle set at the knee brace; however they did exhibit a direct relationship.

These results have important implications for the management of patients with patella and extensor mechanism injuries. The results suggest that improvements in knee brace design to allow 0 degrees of knee flexion, rather than the 10 degrees as seen in this study, are likely to result in significantly reduced extensor mechanism tensile forces.

Introduction: In this study infrapatellar fat pad (IPFP) derived stem cells were expanded with and without Fibroblast Growth Factor-2 (FGF-2) supplementation and were compared with regards to their ability to proliferate and differentiate into chondrocytes.

Materials and Methods: Cells were isolated from the IPFP tissue and expanded in monolayer culture with and without rhFGF-2 supplementation (final concentration 10ng/ ml). Cell aggregates were placed in chondrogenic media for two weeks. Gene expression studies were carried out using quantitative real time PCR. Immunohistochemical labelling was performed with antibody localisation determined by an immunoperoxidase procedure. The pellets were also weighed and digested in papain for DNA and glycosoaminoglycan (GAG) analysis.

Results: Cells expanded in FGF-2 supplemented media were smaller and proliferated more rapidly. The FGF-2 supplemented cell aggregates also showed 100 times higher expression of collagen type II (COL2A1). Immunohistochemical studies showed that pellets made from FGF-2 treated cells stained more strongly for collagen II and more weakly for collagen I. Pellets made with FGF-2 treated cells were larger, continued with enhanced proliferation and contained more proteoglycan.

Conclusion: Our findings show enhanced proliferation and chondrogenic differentiation in IPFP derived stem cells expanded in FGF-2 supplemented media.