Short intense electrical pulses transiently increase the permeability of the cell membrane, an effect known as electroporation. This can be combined with antiblastic drugs for ablation of tumours of the skin and subcutaneous tissue. The aim of this study was to test the efficacy of electroporation when applied to bone and to understand whether the presence of mineralised trabeculae would affect the capability of the electric field to porate the membrane of bone cells.

Different levels of electrical field were applied to the femoral bone of rabbits. The field distribution and modelling were simulated by computer. Specimens of bone from treated and control rabbits were obtained for histology, histomorphometry and biomechanical testing.

After seven days, the area of ablation had increased in line with the number of pulses and/or with the amplitude of the electrical field applied. The osteogenic activity in the ablated area had recovered by 30 days. Biomechanical testing showed structural integrity of the bone at both times.

Electroporation using the appropriate combination of voltage and pulses induced ablation of bone cells without affecting the recovery of osteogenic activity. It can be an effective treatment in bone and when used in combination with drugs, an option for the treatment of metastases.

Corticosteroids are prescribed for the treatment of many medical conditions and their adverse effects on bone, including steroid-associated osteoporosis and osteonecrosis, are well documented. Core decompression is performed to treat osteonecrosis, but the results are variable. As steroids may affect bone turnover, this study was designed to investigate bone healing within a bone tunnel after core decompression in an experimental model of steroid-associated osteonecrosis. A total of five 28-week-old New Zealand rabbits were used to establish a model of steroid-induced osteonecrosis and another five rabbits served as controls. Two weeks after the induction of osteonecrosis, core decompression was performed by creating a bone tunnel 3 mm in diameter in both distal femora of each rabbit in both the experimental osteonecrosis and control groups. An in vivo micro-CT scanner was used to monitor healing within the bone tunnel at four, eight and 12 weeks postoperatively. At week 12, the animals were killed for histological and biomechanical analysis.

In the osteonecrosis group all measurements of bone healing and maturation were lower compared with the control group. Impaired osteogenesis and remodelling within the bone tunnel was demonstrated in the steroid-induced osteonecrosis, accompanied by inferior mechanical properties of the bone.

We have confirmed impaired bone healing in a model of bone defects in rabbits with pulsed administration of corticosteroids. This finding may be important in the development of strategies for treatment to improve the prognosis of fracture healing or the repair of bone defects in patients receiving steroid treatment.

Platelet-leucocyte gel (PLG), a new biotechnological blood product, has hitherto been used primarily to treat chronic ulcers and to promote soft-tissue and bone regeneration in a wide range of medical fields. In this study, the antimicrobial efficacy of PLG against Staphylococcus aureus (ATCC 25923) was investigated in a rabbit model of osteomyelitis. Autologous PLG was injected into the tibial canal after inoculation with Staph. aureus. The prophylactic efficacy of PLG was evaluated by microbiological, radiological and histological examination. Animal groups included a treatment group that received systemic cefazolin and a control group that received no treatment.

Treatment with PLG or cefazolin significantly reduced radiological and histological severity scores compared to the control group. This result was confirmed by a significant reduction in the infection rate and the number of viable bacteria. Although not comparable to cefazolin, PLG exhibited antimicrobial efficacy in vivo and therefore represents a novel strategy to prevent bone infection in humans.

We used demineralised bone matrix (DBM) to augment re-attachment of tendon to a metal prosthesis in an in vivo ovine model of reconstruction of the extensor mechanism at the knee. We hypothesised that augmentation of the tendon-implant interface with DBM would enhance the functional and histological outcomes as compared with previously reported control reconstructions without DBM. Function was assessed at six and 12 weeks postoperatively, and histological examination was undertaken at 12 weeks.

A significant increase of 23.5% was observed in functional weight-bearing at six weeks in the DBM-augmented group compared with non-augmented controls (p = 0.004). By 12 weeks augmentation with DBM resulted in regeneration of a more direct-type enthesis, with regions of fibrocartilage, mineralised fibrocartilage and bone. In the controls the interface was predominantly indirect, with the tendon attached to the bone graft-hydroxyapatite base plate by perforating collagen fibres.

Fractures of the proximal humerus with concomitant vascular injury are rare in children. We describe the presentation, diagnosis, and treatment of a fracture of the proximal humerus in association with an axillary artery injury in a child.

In a rabbit model we investigated the efficacy of a silk fibroin/hydroxyapatite (SF/HA) composite on the repair of a segmental bone defect. Four types of porous SF/HA composites (SF/HA-1, SF/HA-2, SF/HA-3, SF/HA-4) with different material ratios, pore sizes, porosity and additives were implanted subcutaneously into Sprague-Dawley rats to observe biodegradation. SF/HA-3, which had characteristics more suitable for a bone substitite based on strength and resorption was selected as a scaffold and co-cultured with rabbit bone-marrow stromal cells (BMSCs). A segmental bone defect was created in the rabbit radius. The animals were randomised into group 1 (SF/HA-3 combined with BMSCs implanted into the bone defect), group 2 (SF/HA implanted alone) and group 3 (nothing implanted). They were killed at four, eight and 12 weeks for visual, radiological and histological study.

The bone defects had complete union for group 1 and partial union in group 2, 12 weeks after operation. There was no formation of new bone in group 3. We conclude that SF/HA-3 combined with BMSCs supports bone healing and offers potential as a bone-graft substitute.

The response of the muscle is critical in determining the functional outcome of limb lengthening. We hypothesised that muscle response would vary with age and therefore studied the response of the muscles during tibial lengthening in ten young and ten mature rabbits. A bromodeoxyuridine technique was used to identify the dividing cells.

The young rabbits demonstrated a significantly greater proliferative response to the distraction stimulus than the mature ones. This was particularly pronounced at the myotendinous junction, but was also evident within the muscle belly.

Younger muscle adapted better to lengthening, suggesting that in patients in whom a large degree of muscle lengthening is required it may be beneficial to carry out this procedure when they are young, in order to achieve the optimal functional result.

We compared time-dependent changes in the biomechanical properties of single-and double-row repair of a simulated acute tear of the rotator cuff in rabbits to determine the effect of the fixation techniques on the healing process.

A tear of the supraspinatus tendon was created in 80 rabbits which were separated into two equal groups. A single-row repair with two suture anchors was conducted in group 1 and a double-row repair with four suture anchors in group 2. A total of ten intact contralateral shoulder joints was used as a control group. Biomechanical testing was performed immediately post-operatively and at four and eight weeks, and histological analysis at four and eight weeks.

The mean load to failure in group 2 animals was greater than in group 1, but both groups remained lower than the control group at all intervals. Histological analysis showed similar healing properties at four and eight weeks in both groups, but a significantly larger number of healed tendon-bone interfaces were identified in group 2 than in group 1 at eight weeks (p < 0.012).

The ultimate load to failure increased with the number of suture anchors used immediately post-operatively, and at four and eight weeks. The increased load to failure at eight weeks seemed to be related to the increase in the surface area of healed tendon-to-bone in the double-row repair group.

To clarify the pathomechanisms of discogenic low back pain, the sympathetic afferent discharge originating from the L5-L6 disc via the L2 root were investigated neurophysiologically in 31 Lewis rats. Sympathetic afferent units were recorded from the L2 root connected to the lumbar sympathetic trunk by rami communicantes. The L5-L6 discs were mechanically probed, stimulated electrically to evoke action potentials and, finally, treated with chemicals to produce an inflammatory reaction. We could not obtain a response from any units in the L5-L6 discs using mechanical stimulation, but with electrical stimulation we identified 42 units consisting mostly of A-delta fibres. In some experiments a response to mechanical probing of the L5-L6 disc was recognised after producing an inflammatory reaction. This study suggests that mechanical stimulation of the lumbar discs may not always produce pain, whereas inflammatory changes may cause the disc to become sensitive to mechanical stimuli, resulting in nociceptive information being transmitted as discogenic low back pain to the spinal cord through the lumbar sympathetic trunk. This may partly explain the variation in human symptoms of degenerate discs.

An understanding of the remodelling of tendon is crucial for the development of scientific methods of treatment and rehabilitation. This study tested the hypothesis that tendon adapts structurally in response to changes in functional loading. A novel model allowed manipulation of the mechanical environment of the patellar tendon in the presence of normal joint movement via the application of an adjustable external fixator mechanism between the patella and the tibia in sheep, while avoiding exposure of the patellar tendon itself. Stress shielding caused a significant reduction in the structural and material properties of stiffness (79%), ultimate load (69%), energy absorbed (61%), elastic modulus (76%) and ultimate stress (72%) of the tendon compared with controls. Compared with the material properties the structural properties exhibited better recovery after re-stressing with stiffness 97%, ultimate load 92%, energy absorbed 96%, elastic modulus 79% and ultimate stress 80%. The cross-sectional area of the re-stressed tendons was significantly greater than that of stress-shielded tendons.

The remodelling phenomena exhibited in this study are consistent with a putative feedback mechanism under strain control. This study provides a basis from which to explore the interactions of tendon remodelling and mechanical environment.

We used an in vivo model to assess the use of an autogenous cancellous bone block and marrow graft for augmenting tendon reattachment to metallic implants. We hypothesised that augmentation of the tendon-implant interface with a bone block would enable retention of the graft on the implant surface, enhance biological integration, and result in more consistent functional outcomes compared with previously reported morcellised graft augmentation techniques.

A significant improvement in functional weight-bearing was observed between six and 12 weeks. The significant increase in ground reaction force through the operated limb between six and 12 weeks was greater than that reported previously with morcellised graft augmented reconstructions. Histological appearance and collagen fibre orientation with bone block augmentation more closely resembled that of an intact enthesis compared with the morcellised grafting technique. Bone block augmentation of tendon-implant interfaces results in more reliable functional and histological outcomes, with a return to pre-operative levels of weight-bearing by 24 weeks.

Little is known about the increase in length of tendons in postnatal life or of their response to limb lengthening procedures. A study was carried out in ten young and nine adult rabbits in which the tibia was lengthened by 20% at two rates 0.8 mm/day and 1.6 mm/day.

The tendon of the flexor digitorum longus (FDL) muscle showed a significant increase in length in response to lengthening of the tibia. The young rabbits exhibited a significantly higher increase in length in the FDL tendon compared with the adults. There was no difference in the amount of lengthening of the FDL tendon at the different rates. Of the increase in length which occurred, 77% was in the proximal half of the tendon.

This investigation demonstrated that tendons have the ability to lengthen during limb distraction. This occurred to a greater extent in the young who showed a higher proliferative response, suggesting that there may be less need for formal tendon lengthening in young children.

We studied the effect of vitamin C on fracture healing in the elderly. A total of 80 elderly Osteogenic Disorder Shionogi rats were divided into four groups with different rates of vitamin C intake. A closed bilateral fracture was made in the middle third of the femur of each rat. Five weeks after fracture the femora were analysed by mechanical and histological testing. The groups with the lower vitamin C intake demonstrated a lower mechanical resistance of the healing callus and a lower histological grade. The vitamin C levels in blood during healing correlated with the torque resistance of the callus formed (r = 0.525). Therefore, the supplementary vitamin C improved the mechanical resistance of the fracture callus in elderly rats. If these results are similar in humans, vitamin C supplementation should be recommended during fracture healing in the elderly.

The feasibility of bone transport with bone substitute and the factors which are essential for a successful bone transport are unknown. We studied six groups of 12 Japanese white rabbits. Groups A to D received cylindrical autologous bone segments and groups E and F hydroxyapatite prostheses. The periosteum was preserved in group A so that its segments had a blood supply, cells, proteins and scaffold. Group B had no blood supply. Group C had proteins and scaffold and group D had only scaffold. Group E received hydroxyapatite loaded with recombinant human bone morphogenetic protein-2 and group F had hydroxyapatite alone.

Distraction osteogenesis occurred in groups A to C and E which had osteo-conductive transport segments loaded with osteo-inductive proteins. We conclude that scaffold and proteins are essential for successful bone transport, and that bone substitute can be used to regenerate bone.

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.

This study investigates the use of porous biphasic ceramics as graft extenders in impaction grafting of the femur during revision hip surgery.

Impaction grafting of the femur was performed in four groups of sheep. Group one received pure allograft, group two 50% allograft and 50% BoneSave, group three 50% allograft and 50% BoneSave type 2 and group four 10% allograft and 90% BoneSave as the graft material. Function was assessed using an index of pre- and post-operative peak vertical ground reaction force ratios. Changes in bone mineral density were measured by dual energy X ray absorptiometry (DEXA) scanning. Loosening and subsidence were assessed radiographically and by histological examination of the explanted specimens.

There was no statistically significant difference between the four groups after 18 months of unrestricted functional loading for all outcome measures.

We examined the mechanical properties of Vicryl (polyglactin 910) mesh in vitro and assessed its use in vivo as a novel biomaterial to attach tendon to a hydroxyapatite-coated metal implant, the interface of which was augmented with autogenous bone and marrow graft. This was compared with tendon re-attachment using a compressive clamp device in an identical animal model. Two- and four-ply sleeves of Vicryl mesh tested to failure under tension reached 5.13% and 28.35% of the normal ovine patellar tendon, respectively. Four-ply sleeves supported gait in an ovine model with 67.05% weight-bearing through the operated limb at 12 weeks, without evidence of mechanical failure.

Mesh fibres were visible at six weeks but had been completely resorbed by 12 weeks, with no evidence of chronic inflammation. The tendon-implant neoenthesis was predominantly an indirect type, with tendon attached to the bone-hydroxyapatite surface by perforating collagen fibres.

Despite worldwide clinical use of bio-absorbable devices for internal fixation in orthopaedic surgery, the degradation behaviour and tissue replacement of these implants are not fully understood.

In a long-term experimental study, we have determined the patterns of tissue restoration 36 and 54 months after implantation of polyglycolic acid and poly-laevo-lactic acid screws in the distal femur of the rabbit.

After 36 months in the polyglycolic acid group the specimens showed no remaining polymer and loose connective tissue occupied 80% of the screw track. Tissue restoration remained poor at 54 months, the amounts of trabecular bone and haematopoietic elements being significantly lower than those in the intact control group. The amount of trabecular bone within the screw track at 54 months in the polyglycolic acid group was less than in the empty drill holes (p = 0.04). In the poly-laevo-lactic acid group, polymeric material was present in abundance after 54 months, occupying 60% of the cross-section of the core area of the screw track.

When using absorbable internal fixation implants we should recognise that the degradation of the devices will probably not be accompanied by the restoration of normal trabecular bone.

The re-establishment of vascularity is an early event in fracture healing; upregulation of angiogenesis may therefore promote the formation of bone. We have investigated the capacity of vascular endothelial growth factor (VEGF) to stimulate the formation of bone in an experimental atrophic nonunion model.

Three groups of eight rabbits underwent a standard nonunion operation. This was followed by interfragmentary deposition of 100 μg VEGF, carrier alone or autograft.

After seven weeks, torsional failure tests and callus size confirmed that VEGF-treated osteotomies had united whereas the carrier-treated osteotomies failed to unite. The biomechanical properties of the groups treated with VEGF and autograft were identical. There was no difference in bone blood flow.

We considered that VEGF stimulated the formation of competent bone in an environment deprived of its normal vascularisation and osteoprogenitor cell supply. It could be used to enhance the healing of fractures predisposed to nonunion.

Critical size defects in ovine tibiae, stabilised with intramedullary interlocking nails, were used to assess whether the addition of carboxymethylcellulose to the standard osteogenic protein-1 (OP-1/BMP-7) implant would affect the implant’s efficacy for bone regeneration. The biomaterial carriers were a ‘putty’ carrier of carboxymethylcellulose and bovine-derived type-I collagen (OPP) or the standard with collagen alone (OPC). These two treatments were also compared to “ungrafted” negative controls. Efficacy of regeneration was determined using radiological, biomechanical and histological evaluations after four months of healing. The defects, filled with OPP and OPC, demonstrated radiodense material spanning the defect after one month of healing, with radiographic evidence of recorticalisation and remodelling by two months. The OPP and OPC treatment groups had equivalent structural and material properties that were significantly greater than those in the ungrafted controls. The structural properties of the OPP- and OPC-treated limbs were equivalent to those of the contralateral untreated limb (p > 0.05), yet material properties were inferior (p < 0.05). Histopathology revealed no residual inflammatory response to the biomaterial carriers or OP-1. The OPP- and OPC-treated animals had 60% to 85% lamellar bone within the defect, and less than 25% of the regenerate was composed of fibrous tissue. The defects in the untreated control animals contained less than 40% lamellar bone and more than 60% was fibrous tissue, creating full cortical thickness defects. In our studies carboxymethylcellulose did not adversely affect the capacity of the standard OP-1 implant for regenerating bone.