Introduction

Ilizarov fixator is an invaluable tool in managing complex orthopaedic problem. Many factors influence adverse events such as pin site infections and failure of treatment. Our study evaluated risk factors affecting patient compliance and unfavourable outcome of Ilizarov surgery.

Our unpublished data has indicated that the perivascular stem cells (PSCs) have increased chondrogenic potential compared to mesenchymal stem cells (MSCs) derived in culture. There has been a recent change in the theory that stem cells work by a paracrine effect rather than differentiation. There are minimal data demonstrating the persistence of implanted stem cells when used for engraftment. This study aimed to develop an autologous large animal model for perivascular stem cells as well as to determine if cells were retained in the articular cartilage defects.

The reactivity of anti-human and anti-ovine antibodies was ascertained using immunohistochemistry and fluorescence-activated cell sorting (FACS). A panel of antibodies were combined and used to identify and purify pericytes (CD34-CD45-CD146+) and adventitial cells (CD34+CD45-CD146-) using FACS. The purified cells were cultured and their identity checked using FACS. These cultured cells demonstrated osteogenic, adipogenic and chondrogenic potential.

Autologous ovine PSCs (oPSCs) were isolated, cultured and transfected using a GFP virus. The transfection rate was 88%. The cells were implanted into an articular cartilage defect on the medial femoral condyle using a hydrogel, four weeks following implantation the condyle was explanted and confocal laser scanning microscopy demonstrated the presence of oPSCs in the defect. Histology did not demonstrate any repair tissue at this early time point.

These data have confirmed the viability our large animal model and that the implanted stem cells were retained in the defect four weeks following implantation.

Approximately 5 – 10% of all bone fractures are associated with impaired healing. It is thought that regenerative medicine has the potential to improve on existing treatments for non-union fractures, and the European market for such treatments is projected to reach £2.2 billion in 2010. The use of scaffolds for the delivery of both growth factors and human Marrow Stromal Cells (hMSCs) is thought to be a promising approach. It may be desirable to promote proliferation and chemotaxis of hMSCs at the defect site shortly after implantation, and differentiation in the longer term. This is likely to require a dual delivery system, capable of releasing multiple drugs with different release profiles. Our aim has been to develop a polymer scaffold capable of releasing bioactive molecules that are able to direct the differentiation of primary hMSCs down the osteoblastic lineage. We have examined two mutually compatible drug delivery systems: collagen coating for short term release, and polymer encapsulation for longer term release.

Polymer scaffolds were manufactured and coated with Type I Collagen containing BMP-7. hMSCs from three different patient sources were exposed to the scaffolds for 14 days. The cells were then histochemically stained for Alkaline Phosphatase (ALP) and photographed. The areas of ALP staining were then normalised against the total cell count.

Normalised ALP expression was increased compared to the controls for three different patients (‘110 ± 39% SE, n=6, p=0.005’, ‘540 ± 270% SE, n=6, p=0.001’, and ‘32 ± 17% SE, n=6’). Scaffolds were also manufactured either with 1,25 Vitamin D3 (another active compound) in a coating of Collagen, or encapsulated using proprietary methodologies. It was found that both treatments significantly increased normalised Alkaline Phosphatase expression within the 14d experimental period demonstrating release of the active 1,25 Vitamin D3 (’88 ± 37% SE, n=6, p=0.012’ and ‘100 ± 32% SE, n=6, p=0.012’ respectively).

Our findings suggest that, subject to future testing and development, such bioactive scaffolds could form the basis for a dual drug delivery system, suitable for applications in bone regenerative medicine.

We have used human Embryonic Stem cells (hESC) and human Mesenchymal Stem Cells (hMSC) in rat models of bone repair in order to assess the efficacy of these cells for treatments of trauma and skeletal diseases. Graft survival is considered to be of key importance to efficacy of these treatments. Therefore the aim of this study was to develop a technique for identifying implanted cells in histological preparations without the need for genetic engineering of the implanted cells.

Methods: In our experiments hES and hMSC were pre-differentiated during cell culture towards the osteoblast lineage, and then implanted in a Demineralised Bone Matrix (DBM) carrier into an experimentally created full thickness calvarial bone lesion. The animals were sampled seven days and fourteen days after implantation into either immune deficient (RNU-Foxn1rnu) or immune competent (wild type) Sprague Dawley rats. Fluorescent In Situ Hybridisation (FISH) using whole human genome probes identified the human cells within the host lesion site.

Results: Our results have demonstrated that hESC and hMSC derived cells survive in both immune competent (wild type) and immune compromised (nude) animals for the initial seven days post implantation. On the other hand while both the hESC and hMSC derived cells are capable of surviving for at least 14 days in immune compromised animals they do not survive for this period of time in immune competent animals.

Discussion: It appears that the cell/DBM graft is not rejected within seven days even when exposed to the wild type hosts T cell response. However longer term survival required an immune deficient model that is lacking in a T cell response. This data points to interesting future studies regarding which components of the host response are responsible for xenogenic stem cell implant rejection.

Aim: To investigate the directed chondrogenic differentiation of human embryonic and adult stem cells in 3D alginate bead culture.

Introduction: Cartilage possesses limited self-renewal potential and current repair of damage due to trauma or disease involves removal of non-load bearing chon-drocytes from a healthy part of the joint, expansion of chondrocytes and subsequent surgery to replace damaged, load-bearing cartilage. We investigated the potential of human embryonic and adult stem cells as an alternative cell source for cartilage repair.

Experimental design: Human embryonic stem cells (hESC) and human adult marrow stromal cells (hMSCs) cells were cultured in alginate in a 3D bead format in control or chondrogenic media over a 21day period. Cells were subsequently released from their matrix for gene expression analysis or fixed within alginate beads and crytostat sections prepared for immunostaining and histology.

Cell types used: H9 human embryonic stem cells, bone-marrow derived hMSCs and HEK293 (human embryonic kidney epithelium cell line, used as a negative control).

Data: H9 and hMSC cells cultured in alginate beads bathed in control media have a denser matrix with no lacunae-like structures compared to those cultured in the presence of chondrogenic media. The presence of chondrogenic media results in a matrix containing cells within lacunae-like structures very similar to those seen in human cartilage. In contrast, HEK293 cells formed large highly cellular clusters which had clearly undergone significant proliferation. As both H9 and HEK293 cells are highly proliferative the reduction in the proliferative potential of the chondrogenic H9 derived cells is consistent with entry into a stable terminally differentiated state.

Immunostaining demonstrated that hMSCs and H9 cells express cartilage specific Collagen II and Collagen X.

Conclusion: 3D culture of adult hMSCs and hESC (H9) in alginate beads has resulted in stable directed differentiation down the chondrogenic lineage. These data point towards the future use of these human cell sources in cartilage repair.

Introduction: Emerging therapies for regenerating skeletal tissues are focused on the repair of pathologically altered tissue by the transplantation of functionally competent cells and supportive matrices. Stem cells have the potential to differentiate into musculoskeletal tissue and may be the optimal cell source for such therapies. In vitro studies have demonstrated the ability of adult bone marrow stromal cells (MSC) and human embryonic stem cells (hES) to generate bone, but little is known regarding their potential to repair bone in vivo. Preclinical studies in animal models will allow investigation into the extent that regenerated tissue resembles functional and healthy tissue, and its potential clinical application.

Aim: To assess whether adult and embryonic stem cells maintained their ability to form musculoskeletal tissues in vivo using diffusion chambers implanted into the peritoneal cavity of nude mice. Currently, ongoing experiments are assessing the use of MSCs and hES cells to regenerate bone in a rodent preclinical model.

Methods: MSC cells and embryoid body-derived H9 hES cells were prepared as previously described (Haynesworth et al Bone 1992; Sottile et al Cloning Stem Cells 2003). Groups of cells were left untreated or pre-treated with osteogenic (OS) media for 5 days. Study 1: Single cell suspensions of untreated or pre-treated cells were injected into diffusion chambers which were implanted intraperitonealy into nude mice and left for 79 days. Study 2: OS pre-treated cells were implanted into an experimentally created full thickness calvarial defect in adult male Wistar rats. The defect area was left empty or filled with demineralised bone matrix (DBM: Allosource®) alone or with DBM/MSCs or DBM/hES composite. Tissues were collected 4 weeks after surgery.

Analysis: Histological and immunochemical techniques were used to evaluate cell phenotypes and the contribution of transplanted cells to tissue repair.

Results: Study 1: Both hES (in 2/3 chambers) and MSC (3/3) cells pre-treated with OS media formed only mineralised bone. No cartilage was detected in these OS pre-treated cells. Untreated hES cells formed both mineralised bone and cartilage within the chambers (2/3). In contrast, untreated MSC cells (3/3) produced no mineralised bone or cartilage. Preliminary analysis demonstrated the absence of any other tissue type in the diffusion chambers. Study 2: Active bone regeneration was observed at the edges of the calvarial defect after 4 weeks, with a high density of cells present within the MSC or hES/DBM composite. No signs of local cellular immunological response were seen.

Summary: OS pre-treatment restricted differentiation towards the osteoblast lineage in both hES and MSC cells indicating successful directed differentiation in vivo. Untreated hES and MSC cells produce a different range of cell phenotypes suggesting that the two cell sources represent cells at a different stage of commitment in a common cell lineage or cells derived from two distinct cell lineages. New bone formation was seen at the site of the calvarial defect in the presence OS pre-treated MSC and hES cells suggesting that these cells may support in vivo bone repair in a preclinical model. Funded by Geron Corporation

Introduction: Treatment for ruptured Achilles tendon can be classified into operative (open or percutaneous) and non-operative (cast immobilisation or functional bracing); post-operative splintage can be with a rigid cast or functional brace. The aim was to identify and summarise the evidence from randomised trials of the effectiveness of different interventions.

Methods: We searched the Cochrane specialised register, MEDLINE, reference lists of articles and contacted trialists directly for all randomised and quasiran-domised trials comparing different treatment regimes for acute Achilles tendon ruptures.

Results: Fourteen trials involving 891 patients were included.

Open operative treatment compared with non-operative treatment was associated with a lower risk of re-rupture (odds ratio (OR) = 0.25, 95% confidence interval (CI) = 0.1–0.6, p=0.003) but a higher risk of other complications including infection, adhesions and disturbed sensibility (OR = 14.1, 95%CI = 6.3–31.7, p< 0.00001).

Open versus percutaneous operative surgical repair was associated with a longer operation duration and higher risk of infection (OR = 12.9, 95%CI = 1.6–105.6, p=0.02).

Patients splinted with a functional brace rather than a cast post-operatively tended to have a shorter in-patient stay, less time off work, quicker return to sporting activities and fewer reported complications (p=0.0003).

Because of the small number of patients involved no definitive conclusions could be made regarding different operative techniques and different non-operative regimes.

Conclusions: Open operative treatment significantly reduces the risk of re-rupture but has the drawback of a significantly higher risk of other complications, including wound infection. The latter may be reduced by performing surgery percutaneously. Post-operative splintage in a functional brace appears to reduce hospital stay and time off work and sports.

Aim of Study: The aim of the study was to investigate the effect of muscle retractors on intramuscular pressure in the posterior spinal muscles during posterior spinal surgery.

Methods: Twenty patients undergoing posterior spinal surgery were recruited into this study and recordings of intramuscular pressure during surgery were performed using a Stryker® compartment pressure monitoring system, prior to insertion of retractors, 5, 30 and 60 minutes into surgery and on removal of retractors. Prior to and following use of the retractors, muscle biopsies were taken from the erector spinae muscle for analysis.

Results: A significant increase in intramuscular pressure (p< 0.001) was observed during surgery, with pressure rising from 7.1±4.1 mmHg pre-operatively to 26.4±16.0 mmHg 30 minutes into the operation. On removal of retractors, this pressure returned to or near to the original value. Analysis of muscle biopsies using calcium-activated ATPase birefringence revealed a reduction in muscle function following prolonged use of self-retaining retractors.

Discussion: This study demonstrates a substantial rise in pressure in the erector spinae muscle during posterior spinal surgery. Following retraction, marked changes were noted in the function of the muscles. This could be an important factor in the generation of operative scar tissue and post-operative dysfunction of the spinal muscles, and therefore, may be a cause of persistent back pain frequently observed in post-operative patients. Currently, this work is being extended to investigate the relationship between loss of muscle function and duration of retraction, and to study the long term implications of loss of muscle function with respect to surgical outcome and chronic back pain.