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

Our aim was to investigate whether nitric oxide synthase (NOS) isoforms, responsible for the generation of NO, are expressed during the healing of fractures. To localise the sites of expression compared with those in normal bone we made standardised, stabilised, unilateral tibial fractures in male Wistar rats. Immunostaining was used to determine the precise tissue localisation of the different NOS isoforms. Western blotting was used to assess expression of NOS isoform protein and L-citrulline assays for studies on NOS activity. Control tissue was obtained from both the contralateral uninjured limb and limbs of normal rats. Immunohistochemistry showed increased expression of endothelial NOS (eNOS) to be strongest in the cortical blood vessels and in osteocytes in the early phase of fracture repair. Western blot and image analysis confirmed this initial increase. Significantly elevated calcium-dependent NOS activity was observed at day 1 after fracture. Inducible NOS (iNOS) was localised principally in endosteal osteoblasts and was also seen in chondroblasts especially in the second week of fracture healing. Western blotting showed a reduction in iNOS during the early healing period. Significantly reduced calcium-independent NOS activity was also seen. No neuronal NOS was seen in either fracture or normal tissue. Increased eNOS in bone blood vessels is likely to mediate the increased blood flow recognised during fracture healing. eNOS expression in osteocytes may occur in response to changes in either mechanical or local fluid shear stress. The finding that eNOS is increased and iNOS reduced in early healing of fractures may be important in their successful repair

We studied bone-tendon healing using immunohistochemical methods in a rabbit model.

Reconstruction of the anterior cruciate ligament was undertaken using semitendinosus tendon in 20 rabbits. Immunohistochemical evaluations were performed at one, two, four and eight weeks after the operation. The expression of CD31, RAM-11, VEGF, b-FGF, S-100 protein and collagen I, II and III in the bone-tendon interface was very similar to that in the endochondral ossification. Some of the type-III collagen in the outer layer of the graft, which was deposited at a very early phase after the operation, was believed to have matured into Sharpey-like fibres. However, remodelling of the tendon grafted into the bone tunnel was significantly delayed when compared with this ossification process. To promote healing, we believe that it is necessary to accelerate remodelling of the tendon, simultaneously with the augmentation of the ossification.

We attempted to repair full-thickness defects in the articular cartilage of the trochlear groove of the femur in 30 rabbit knee joints using allogenic cultured chondrocytes embedded in a collagen gel. The repaired tissues were examined at 2, 4, 8, 12 and 24 weeks after operation using histological and histochemical methods. The articular defect filling index measurement was derived from safranin-O stained sections. Apoptotic cellular fractions were derived from analysis of apoptosis in situ using TUNEL staining, and was confirmed using caspase-3 staining along with quantification of the total cellularity. The mean articular defect filling index decreased with time. After 24 weeks it was 0.7 (sd 0.10), which was significantly lower than the measurements obtained earlier (p < 0.01). The highest mean percentage of apoptotic cells were observed at 12 weeks, although the total cellularity decreased with time. Because apoptotic cell death may play a role in delamination after chondrocyte transplantation, anti-apoptotic gene therapy may protect transplanted chondrocytes from apoptosis.

Sex hormones play important roles in the regulation of the proliferation, maturation and death of chondrocytes in the epiphyseal growth plate. We have investigated the effects of male castration on the cell kinetics of chondrocytes as defined by the numbers of proliferating and dying cells. The growth plates of normal rabbits and animals castrated at eight weeks of age were obtained at 10, 15, 20 and 25 weeks of age.

Our study suggested that castration led to an increase in apoptosis and a decrease in the proliferation of chondrocytes in the growth plate. In addition, the number of chondrocytes in the castrated rabbits was less than that of normal animals of the same age.

We examined osteochondral autografts, obtained at a mean of 19.5 months (3 to 48) following extracorporeal irradiation and re-implantation to replace bone defects after removal of tumours. The specimens were obtained from six patients (mean age 13.3 years (10 to 18)) and consisted of articular cartilage (five), subchondral bone (five), external callus (one) and tendon (one). The tumour cells in the grafts were eradicated by a single radiation dose of 60 Gy. In three cartilage specimens, viable chondrocytes were detected. The survival of chondrocytes was confirmed with S-100 protein staining. Three specimens from the subchondral region and a tendon displayed features of regeneration. Callus was seen at the junction between host and irradiated bone.