SOX genes comprise a family of transcription factors characterised by a conserved HMG-box domain that confer pleiotropic effects on cell fate and differentiation through binding to the minor groove of DNA. Paracrine regulation and contact-dependant Notch signalling has been suggested to modulate the induction of SOX gene expression. The objective of this study is to investigate the crosstalk between and preconditioning of mesenchymal stem cells (MSCs) with chondrocytes through comparing SOX gene expression in their co-culture and respective monocultures.

Our study adopted an in vitro autologous co-culture of p0 adipose-derived MSCs (AMSCs) and articular chondrocytes derived from Kellgren-Lawrence Grade III/IV osteoarthritic knee joints (n=7). Samples were handled according to the 2004 UK Human Tissue Act. Cells were purified and co-cultured with one AMSC for every chondrocyte at 5000 cells/cm². The AMSCs were characterised by a panel of MSC surface markers in flow cytometry and were allowed to undergo trilineage differentiation for subsequent histological investigation. SOX5, SOX6, and SOX9 expression of co-cultures and monoculture controls were quantified by TaqMan quantitative real-time PCR. Experiments were performed in triplicate.

AMSC phenotype was evidenced by the expression of CD105, CD73, CD90 & heterogenous CD34 but not CD45, CD14, CD19 & HLA-DR in flow cytometry, and also differentiation into chondrogenic, osteogenic and adipogenic lineages with positive Alcian blue, Alizarin Red and Oil Red O staining. The expression of SOX5, SOX6, and SOX9 were greater in observed co-cultures than would be expected from an expression profile modelled from monocultures.

The findings provides evidence for the upregulation of SOX family transcription factors expression during the co-culture of MSCs and chondrocytes, suggesting an active induction of chondrogenic differentiation and change of cell fate amidst a microenvironment that facilitates cell-contact and paracrine secretion. This provides insight into the chondrogenic potential and therapeutic effects of MSCs preconditioned by the chondrocyte secretome (or potentially chondrocytes reinvigorated by the MSC secretome), and ultimately, cartilage repair.

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.

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: Fractures of the tuberosity heal well irrespective of the treatment instituted. Fractures distal to the tuberosity have a high incidence of delayed union and non-union. This could be due to disruption of the vascular supply that enters the bone at the metaphyseal-diaphyseal region. It has also been reported that in these injuries, stress fractures occur at a different anatomic site that is more distal to acute fractures.

We present one of the largest reported series of such fractures in which we have explored the above statements.

Materials & Methods: A retrospective review of 300 closed fractures of the base of the fifth metatarsal- 268 were tuberosity fractures (group 1) and 32 were fractures distal to the tuberosity (group 2).

The patients were followed up in the outpatients clinic for a mean period of 2 months (group 1) and 16 months (group 2).

The distance of the fracture site from the proximal tip of the metatarsal was measured on the radiographs.

Results:

All group 1 fractures healed well following symptomatic management and none required surgical intervention.

Conclusion:

A standardized classification is important because there is great variability in the types of fractures and appropriate treatment.

Introduction: There has been an increasing use of orthotic knee braces in the management of knee injuries. To ensure the biomechanics of the knee are not adversely affected, it is important that orthotic knee braces accurately provide the desired angle of immobilisation. The objective of our study was to measure the actual knee flexion angles for a lockable orthotic knee brace, and measure the resulting knee flexion moment.

Materials and methods: Eight healthy male volunteers participated in the study looking at six different types of knee immobilisation: locked in 0, 10, 20, 30 degrees of knee flexion, with the brace unlocked, and without a brace. Force and 3-dimensional motion data were collected using a single Kistler force plate and an eight-camera Qualisys ProReflex motion analysis system.

Results: The kinematic knee flexion angles were significantly different when compared with the angles set at the orthotic knee brace for 0 degrees (p=0.001) and 10 degrees (p=0.011). The kinematic knee flexion angle when no brace was used was significantly different from the angle for the unlocked orthotic knee brace (p= 0.003). The knee flexion moment was directly proportional to the knee flexion angle. There was a statistically significant difference between the knee flexion moment for the six types of immobilisation (p< 0.001).

Discussion: The knee flexion angles measured using the kinematic data did not always correspond with the angle set at the orthotic knee brace. These findings highlight inadequacies in the design of lockable orthotic knee braces, especially at low flexion angles of 0 and 10 degrees. The resulting higher actual knee flexion angles were associated with greater knee flexion moments and joint reaction forces at the tibiofemoral and patellofemoral joints. This can, at best result in increased energy expenditure and decreased agility, and at worse potentially augment injuries to the knee.

Introduction: Articular cartilage is frequently damaged but only shows a limited capacity for repair. Autologous chondrocytes are being used for the repair of focal articular cartilage defects in the ankle but their use has limitations. The use of undifferentiated progenitor cells from other sources is limited by the fact that these cells loose there stem cell characterisation with passage in culture. The fat pad derived stem cells are a possible alternative that maintain multipotentiality at higher passages. We explore the hypothesis that their cell surface characterisation will resemble that of mesenchymal stem cells and will not alter with passage.

Materials and Methods: Cells were isolated from the human fat pad and expanded in monolayer culture. On confluence, they were harvested by digestion and replated at a ratio of 1:3. Cells from passage 2, 4, 6, 8 and 10 were stained and analysed using flow cytometry for a panel of stem cell surface antibodies.

Results: Fat pad derived cells stained strongly for CD13, 29, 44 and 90 (markers of mesenchymal stem cells). The cells stained poorly for 3G5 (pericyte marker), CD34 and CD56 (marker for haematopoetic lineage), and LNG FR and STRO 1 (markers of bone marrow stem cells). These results suggest that the fat pad cell population has surface expression characteristics of mesenchymal stem cells, but differ from bone marrow derived stem cells. It is also important to note that the expression of these cell-surface markers was maintained up to passage 10.

Conclusion: The consistent pattern of cell surface expression, with little change with passage, shows that the proliferation and expansion of the fat pad stem cell population does not lead to major changes in phenotype of these cells. This can potentially allow a significant increase in number sufficient for clinical applications without loosing their multipotentiality.

Proximal fifth metatarsal fractures distal to the tuberosity, also known as Jones’ fractures, are troublesome fractures to manage with a high incidence of delayed union and nonunion.

We conducted a retrospective study of 32 patients with fractures of the fifth metatarsal distal to the tuberosity over a three year period. The aim was to assess healing with non-weight bearing and variations of weight bearing mobilization including minimal, partial and full weight bearing. This is one of the largest reported series of such fractures. These fractures were classified as acute fractures (14 fractures), fractures with features of delayed union (15 fractures) and fractures with features of nonunion (three fractures) at presentation according to the radiological classification used by Torg in 1984. These patients were treated in a plaster cast and mobilised either non-weight bearing or with variations of weight bearing. These patients were followed up for a mean of 16 months.

Our findings correspond with those observed by Torg and we describe a correlation between the radiological appearance of the fracture at presentation and the clinical course. Prevailing guidelines for the management of these fractures are ambiguous. A standardized classification is important because there is great variability in the types of fractures and appropriate treatment. It is important that radiological features are correlated with clinical features and appropriate treatment instituted. The treatment of choice for acute fractures is immobilization of the limb in a below-knee non-weight bearing plaster for 6 to 8 weeks. Fractures with delayed union may eventually heal if treated non-operatively, although this may take up to 20 weeks. An active athlete will benefit from early surgery. Fractures with symptomatic nonunion require surgery.