Ischaemia-reperfusion injury (IRI) is caused by endothelial and subendothelial damage by neutrophil-derived oxidants. Vitamin C is an antioxidant which attenuates endothelial injury after IRI. Our aim was to evaluate the effect of oral vitamin C in the prevention of IRI in skeletal muscle. We used a model of cross-clamping (3 hours) and reperfusion (1 hour) of the cremaster muscle in rats. Muscle function was assessed electrophysiologically by electrical field stimulation. Infiltration by neutrophils was determined by the activity of tissue myeloperoxidase (MPO) and tissue oedema by the wet-to-dry ratio. Neutrophil respiratory burst activity was measured in control animals and groups pretreated with vitamin C. IRI significantly decreased muscle function and increased muscle neutrophil MPO activity and muscle oedema. Pretreatment with vitamin C preserved muscle function and reduced tissue oedema and neutrophil infiltration. Neutrophil respiratory burst activity was reduced in the group treated with vitamin C compared with the control group. We conclude that pretreatment with oral vitamin C protects against acute muscle IRI, possibly by attenuating neutrophil respiratory burst activity

Compartment syndrome is a unique form of ischaemia of skeletal muscle which occurs despite patency of the large vessels. Decompression allows the influx of activated leucocytes which cause further injury. Vitamin C is a powerful antioxidant which concentrates preferentially in leucocytes and attenuates reperfusion-induced muscle injury. We have evaluated the use of pretreatment with oral vitamin C in the prevention of injury caused by compartment syndrome in a rat cremasteric muscle model. Acute and delayed effects of pretreatment with vitamin C were assessed at one and 24 hours after decompression of compartment syndrome. Muscle function was assessed electrophysiologically. Vascular, cellular and tissue inflammation was assessed by staining of intercellular adhesion molecule-1 (ICAM-1) and by determination of the activity of myeloperoxidase (MPO) in neutrophils and tissue oedema. Compartment syndrome impaired skeletal muscle function and increased the expression of ICAM-1, activity of MPO and muscle weight increased significantly. Pretreatment with vitamin C preserved muscle function and reduced the expression of ICAM-1, infiltration of the neutrophils and oedema

It has been suggested that reamed intramedullary nailing of the femur should be avoided in some patients with multiple injuries. We have studied prospectively the effect of femoral reaming on the inflammatory process as implicated in the pathogenesis of acute respiratory distress syndrome (ARDS) and multiple-organ failure (MOF). We studied changes in the levels of serum interleukin-6 (IL-6) (proinflammatory cytokine), neutrophil CD11b (C3) receptor expression (activated neutrophil adhesion molecule), serum soluble intracellular adhesion molecule (s-ICAM-1), serum soluble E-selectin (the soluble products of endothelial adhesion molecules) and plasma elastase (neutrophil protease) in a series of patients with femoral fractures treated by nailing. We have also compared reamed nailing with unreamed nailing. We found that the levels of serum IL-6 and elastase rose significantly during the nailing procedure indicating a measurable ‘second hit’. There was no clear response in leukocyte activation and no difference in the release of endothelial adhesion molecule markers. There was no significant difference between groups treated by reamed and unreamed nailing. Although clinically unremarkable, the one patient who died from ARDS was shown to be hyperstimulated after injury and again after nailing, suggesting the importance of an excessive inflammatory reaction in the pathogenesis of these serious problems. Our findings have shown that there is a second hit associated with femoral nailing and suggest that the degree of the inflammatory reaction may be important in the pathogenesis of ARDS and MOF

We have previously shown that prior exposure of rat hind limbs to ischaemia for five minutes and reperfusion for five minutes reduced the structural damage to skeletal muscle which followed a subsequent period of ischaemia for four hours and reperfusion for one hour. We have now examined the potential mechanisms by which this ischaemic preconditioning protocol may be effective in reducing damage to skeletal muscle induced by prolonged ischaemia and reperfusion. Prior exposure of the hindlimb to ischaemia for five minutes and reperfusion for five minutes did not prevent the fall in the ATP content of tibialis anterior which occurred after a subsequent period of ischaemia for four hours and reperfusion for one hour. Similarly, no effect of the preconditioning protocol was seen on the elevated muscle myeloperoxidase, indicative of an elevated neutrophil content, or abnormal muscle cation content. Reperfused ischaemic muscle was also found to have an increased content of heat-shock protein (HSP) 72, but the preconditioning protocol did not further increase the content of this or other HSPs indicating that it was not acting by increasing the expression of these cytoprotective proteins. The protective effects of preconditioning appeared to be mimicked by the infusion of adenosine to animals immediately before exposure to the four-hour period, indicating a potential mechanism by which skeletal muscle may be preconditioned to maintain structural viability

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.

In order to clarify the role of cytokines in the remodelling of the grafted tendon for ligament reconstruction we compared the responses to interleukin (IL)-1β, platelet-derived growth factor (PDGF)-BB and transforming growth factor (TGF)-β1 of extrinsic fibroblasts infiltrating the frozen-thawed patellar tendon in rats with that of the normal tendon fibroblasts, in regard to the gene expression of matrix metalloproteinase (MMP)-13, using Northern blot analysis. We also examined, immunohistologically, the local expression of IL-1β, PDGF-BB, and TGF-β1 in fibroblasts infiltrating the frozen-thawed patellar tendon.

Northern blot analysis showed that fibroblasts derived from the patellar tendon six weeks after the freeze-thaw procedure in situ showed less response to IL-1β than normal tendon fibroblasts with respect to MMP-13 mRNA gene expression. The immunohistological findings revealed that IL-1β was over-expressed in extrinsic fibroblasts which infiltrated the patellar tendon two and six weeks after the freeze-thaw procedure in situ, but neither PDGF-BB nor TGF-β1 was over-expressed in these extrinsic fibroblasts. Our findings indicated that IL-1β had a close relationship to matrix remodelling of the grafted tendon for ligament reconstruction, in addition to the commencement of inflammation during the tissue-healing process.

Ciprofloxacin hydrochloride-loaded microspheres were prepared by a spray-drying method using pectin and chitosan. The effects of different polymers and drug ratios were investigated.

The most appropriate carriers were selected by in vitro testing. A rat methicillin-resistant Staphylococcus aureus osteomyelitis model was used to evaluate the effects of the loaded microspheres.

The drug was released rapidly from the pectin carrier but this was more sustained in the chitosan formulation.

Chitosan microspheres loaded with ciprofloxacin hydrochloride were more effective for the treatment of osteomyelitis than equivalent intramuscular antibiotics.

We evaluated the possible induction of a systemic immune response to increase anti-tumour activity by the re-implantation of destructive tumour tissue treated by liquid nitrogen in a murine osteosarcoma (LM8) model. The tumours were randomised to treatment by excision alone or by cryotreatment after excision. Tissue from the tumour was frozen in liquid nitrogen, thawed in distilled water and then re-implanted in the same animal. In addition, some mice received an immunological response modifier of OK-432 after treatment. We measured the levels of interferon-gamma and interleukin-12 cytokines and the cytotoxicity activity of splenocytes against murine LM8 osteosarcoma cells. The number of lung and the size of abdominal metastases were also measured.

Re-implantation of tumour tissue after cryotreatment activated immune responses and inhibited metastatic tumour growth. OK-432 synergistically enhanced the anti-tumour effect. Our results suggest that the treatment of malignant bone tumours by reconstruction using autografts containing tumours which have been treated by liquid nitrogen may be of clinical value.

Platelet-rich plasma is a new inductive therapy which is being increasingly used for the treatment of the complications of bone healing, such as infection and nonunion. The activator for platelet-rich plasma is a mixture of thrombin and calcium chloride which produces a platelet-rich gel.

We analysed the antibacterial effect of platelet-rich gel in vitro by using the platelet-rich plasma samples of 20 volunteers. In vitro laboratory susceptibility to platelet-rich gel was determined by the Kirby-Bauer disc-diffusion method. Baseline antimicrobial activity was assessed by measuring the zones of inhibition on agar plates coated with selected bacterial strains.

Zones of inhibition produced by platelet-rich gel ranged between 6 mm and 24 mm (mean 9.83 mm) in diameter. Platelet-rich gel inhibited the growth of Staphylococcus aureus and was also active against Escherichia coli. There was no activity against Klebsiella pneumoniae, Enterococcus faecalis, and Pseudomonas aeruginosa. Moreover, platelet-rich gel seemed to induce the in vitro growth of Ps. aeruginosa, suggesting that it may cause an exacerbation of infections with this organism. We believe that a combination of the inductive and antimicrobial properties of platelet-rich gel can improve the treatment of infected delayed healing and nonunion.

A retrospective series of 45 cases of chronic osteomyelitis collected over a period of 14 years was histologically classified into tuberculous osteomyelitis (25) and chronic non-granulomatous osteomyelitis (20). The tuberculous osteomyelitis group was divided into three subgroups: a) typical granulomas (13 cases); b) ill-defined granulomas (seven cases), and c) suspected granulomas (five cases). An in-house polymerase chain reaction amplifying the 245 bp nucleotide sequence, and capable of detecting 10 fg of DNA of Mycobacterium tuberculosis, was used on the DNA extracted from the paraffin blocks. The polymerase chain reaction was positive in 72% of cases (18) of tuberculous osteomyelitis, but when typical cases of tuberculous osteomyelitis with confirmed granulomas were considered (13), this increased to 84.6% (11). The chronic non-granulomatous osteomyelitis group gave positive polymerase chain reaction results in 20% of the cases (4).

Our preliminary study on tuberculous osteomyelitis shows that the polymerase chain reaction can be a very useful diagnostic tool, since a good correlation was seen between typical granulomas and polymerase chain reaction with a sensitivity of 84.6% and a specificity of 80%. In addition, our study shows that tuberculous osteomyelitis can be diagnosed in formalin-fixed paraffin-embedded tissues in the absence of typical granulomas.

The stress response to trauma is the summation of the physiological response to the injury (the ‘first hit’) and by the response to any on-going physiological disturbance or subsequent trauma surgery (the ‘second hit’).

Our animal model was developed in order to allow the study of each of these components of the stress response to major trauma. High-energy, comminuted fracture of the long bones and severe soft-tissue injuries in this model resulted in a significant tropotropic (depressor) cardiovascular response, transcardiac embolism of medullary contents and activation of the coagulation system. Subsequent stabilisation of the fractures using intramedullary nails did not significantly exacerbate any of these responses.

Systemic factors are believed to be pivotal for the development of heterotopic ossification in severely-injured patients. In this study, cell cultures of putative target cells (human fibroblastic cells, osteoblastic cells (MG-63), and bone-marrow stromal cells (hBM)) were incubated with serum from ten consecutive polytraumatised patients taken from post-traumatic day 1 to day 21 and with serum from 12 healthy control subjects.

The serum from the polytraumatised patients significantly stimulated the proliferation of fibroblasts, MG-63 and of hBM cells. The activity of alkaline phosphatase in MG-63 and hBM cells was significantly decreased when exposed to the serum of the severely-injured patient. After three weeks in 3D cell cultures, matrix production and osteogenic gene expression of hBM cells were equal in the patient and control groups. However, the serum from the polytraumatised patients significantly decreased apoptosis of hBM cells compared with the control serum (4.3% vs 19.1%, p = 0.031).

Increased proliferation of osteoblastic cells and reduced apoptosis of osteoprogenitors may be responsible for increased osteogenesis in severely-injured patients.

We investigated the effect of stimulation with a pulsed electromagnetic field on the osseointegration of hydroxyapatite in cortical bone in rabbits. Implants were inserted into femoral cortical bone and were stimulated for six hours per day for three weeks.

Electromagnetic stimulation improved osseointegration of hydroxyapatite compared with animals which did not receive this treatment in terms of direct contact with the bone, the maturity of the bone and mechanical fixation. The highest values of maximum push-out force (F_max) and ultimate shear strength (σ_u) were observed in the treated group and differed significantly from those of the control group at three weeks (F_max; p < 0.0001; σ_u, p < 0.0005).