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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.

A rat model of lumbar root constriction with an additional sympathectomy in some animals was used to assess whether the sympathetic nerves influenced radicular pain. Behavioural tests were undertaken before and after the operation.

On the 28th post-operative day, both dorsal root ganglia and the spinal roots of L4 and L5 were removed, frozen and sectioned on a cryostat (8 μm to 10 μm). Immunostaining was then performed with antibodies to tyrosine hydroxylase (TH) according to the Avidin Biotin Complex method. In order to quantify the presence of sympathetic nerve fibres, we counted TH-immunoreactive fibres in the dorsal root ganglia using a light microscope equipped with a micrometer graticule (10 x 10 squares, 500 mm x 500 mm). We counted the squares of the graticule which contained TH-immunoreactive fibres for each of five randomly-selected sections of the dorsal root ganglia.

The root constriction group showed mechanical allodynia and thermal hyperalgesia. In this group, TH-immunoreactive fibres were abundant in the ipsilateral dorsal root ganglia at L5 and L4 compared with the opposite side. In the sympathectomy group, mechanical hypersensitivity was attenuated significantly.

We consider that the sympathetic nervous system plays an important role in the generation of radicular pain.

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.