Aims. Delayed postoperative inoculation of orthopaedic implants with persistent wound drainage or bacterial seeding of a haematoma can result in periprosthetic joint infection (PJI). The aim of this in vivo study was to compare the efficacy of vancomycin powder with vancomycin-eluting calcium sulphate beads in preventing PJI due to delayed inoculation. Methods. A mouse model of PJI of the knee was used. Mice were randomized into groups with intervention at the time of surgery (postoperative day (POD) 0): a sterile control (SC; n = 6); infected control (IC; n = 15); systemic vancomycin (SV; n = 9); vancomycin powder (VP; n = 21); and vancomycin bead (VB; n = 19) groups. Delayed inoculation was introduced during an arthrotomy on POD 7 with 1 × 10. 5. colony-forming units (CFUs) of a bioluminescent strain of Staphylococcus aureus. The bacterial burden was monitored using bioluminescence in vivo. All mice were killed on POD 21. Implants and soft-tissue were harvested and sonicated for analysis of the CFUs. Results. The mean in vivo bioluminescence in the VB group was significantly lower on POD 8 and POD 10 compared with the other groups. There was a significant 1.3-log. 10. (95%) and 1.5-log. 10. (97%) reduction in mean soft-tissue CFUs in the VB group compared with the VP and IC groups (3.6 × 10. 3. vs 7.0 × 10. 4. ; p = 0.022; 3.6 × 10. 3. vs 1.0 × 10. 5. ; p = 0.007, respectively) at POD 21. There was a significant 1.6-log. 10. (98%) reduction in mean implant CFUs in the VB group compared with the IC group (1.3 × 10. 0. vs 4.7 × 10. 1. , respectively; p = 0.038). Combined soft-tissue and implant infection was prevented in 10 of 19 mice (53%) in the VB group as opposed to 5 of 21 (24%) in the VP group, 3 of 15 (20%) in the IC group, and 0% in the SV group. Conclusion. In our in vivo mouse model, antibiotic-releasing calcium sulphate beads appeared to outperform vancomycin powder alone in lowering the bacterial burden and preventing soft-tissue and implant infections. Cite this article: Bone Joint J 2024;106-B(6):632–638

Aims. Demineralised bone matrix (DBM) is rarely used for the local delivery of prophylactic antibiotics. Our aim, in this study, was to show that a graft with a bioactive glass and DBM combination, which is currently available for clinical use, can be loaded with tobramycin and release levels of antibiotic greater than the minimum inhibitory concentration for Staphylococcus aureus without interfering with the bone healing properties of the graft, thus protecting the graft and surrounding tissues from infection. Materials and Methods. Antibiotic was loaded into a graft and subsequently evaluated for drug elution kinetics and the inhibition of bacterial growth. A rat femoral condylar plug model was used to determine the effect of the graft, loaded with antibiotic, on bone healing. Results. We found that tobramycin loaded into a graft composed of bioglass and DBM eluted antibiotic above the minimum inhibitory concentration for three days in vitro. It was also found that the antibiotic loaded into the graft produced no adverse effects on the bone healing properties of the DBM at a lower level of antibiotic. Conclusion. This antibiotic-loaded bone void filler may represent a promising option for the delivery of local antibiotics in orthopaedic surgery. Cite this article: Bone Joint J 2016;98-B:1126–31

Aims. Compartment syndrome results from increased intra-compartmental pressure (ICP) causing local tissue ischaemia and cell death, but the systemic effects are not well described. We hypothesised that compartment syndrome would have a profound effect not only on the affected limb, but also on remote organs. Methods. Using a rat model of compartment syndrome, its systemic effects on the viability of hepatocytes and on inflammation and circulation were directly visualised using intravital video microscopy. Results. We found that hepatocellular injury was significantly higher in the compartment syndrome group (192 PI-labelled cells/10. -1 . mm. 3. , standard error of the mean (. sem. ) 51) compared with controls (30 PI-labelled cells/10. -1 . mm. 3. , . sem . 12, p < 0.01). The number of adherent venular white blood cells was significantly higher for the compartment syndrome group (5 leukocytes/30s/10 000 μm. 2. , . sem 1. ) than controls (0.2 leukocytes/30 s/10 000 μm. 2. , . sem . 0.2, p < 0.01). Volumetric blood flow was not significantly different between the two groups, although there was an increase in the heterogeneity of perfusion. Conclusions. Compartment syndrome can be accompanied by severe systemic inflammation and end organ damage. This study provides evidence of the relationship between compartment syndrome in a limb and systemic inflammation and dysfunction in a remote organ. Cite this article: Bone Joint J 2016; 98-B:1132–7

Currently, there is no animal model in which to evaluate the underlying physiological processes leading to the heterotopic ossification (HO) which forms in most combat-related and blast wounds. We sought to reproduce the ossification that forms under these circumstances in a rat by emulating patterns of injury seen in patients with severe injuries resulting from blasts. We investigated whether exposure to blast overpressure increased the prevalence of HO after transfemoral amputation performed within the zone of injury. We exposed rats to a blast overpressure alone (BOP-CTL), crush injury and femoral fracture followed by amputation through the zone of injury (AMP-CTL) or a combination of these (BOP-AMP). The presence of HO was evaluated using radiographs, micro-CT and histology. HO developed in none of nine BOP-CTL, six of nine AMP-CTL, and in all 20 BOP-AMP rats. Exposure to blast overpressure increased the prevalence of HO. This model may thus be used to elucidate cellular and molecular pathways of HO, the effect of varying intensities of blast overpressure, and to evaluate new means of prophylaxis and treatment of heterotopic ossification. Cite this article: Bone Joint J 2015;97-B:572–6

A silver-containing hydroxyapatite (Ag-HA) coating has been developed using thermal spraying technology. We evaluated the osteoconductivity of this coating on titanium (Ti) implants in rat tibiae in relation to bacterial infection in joint replacement. At 12 weeks, the mean affinity indices of bone formation of a Ti, an HA, a 3%Ag-HA and a 50%Ag-HA coating were 97.3%, 84.9%, 81.0% and 40.5%, respectively. The mean affinity indices of bone contact of these four coatings were 18.8%, 83.7%, 77.2% and 40.5%, respectively. The indices of bone formation and bone contact around the implant of the 3%Ag-HA coating were similar to those of the HA coating, and no significant differences were found between them (bone formation, p = 0.99; bone contact, p = 0.957). However, inhibition of bone formation was observed with the 50%Ag-HA coating. These results indicate that the 3%Ag-HA coating has low toxicity and good osteoconductivity, and that the effect of silver toxicity on osteoconductivity depends on the dose

We developed a rat model of limb lengthening to study the basic mechanism of distraction osteogenesis, using a small monolateral external fixator. In 11-week-old male rats we performed a subperiosteal osteotomy in the midshaft of the femur with distraction at 0.25 mm every 12 hours from seven days after operation. Radiological and histological examinations showed a growth zone of constant thickness in the middle of the lengthened segment, with formation of new bone at its proximal and distal ends. Osteogenic cells were arranged longitudinally along the tension vector showing the origin and the fate of individual cells in a single section. Typical endochondral bone formation was prominent in the early stage of distraction, but intramembraneous bone formation became the predominant mechanism of ossification at later stages. We also showed a third mechanism of ossification, ‘transchondroid bone formation’. Chondroid bone, a tissue intermediate between bone and cartilage, was formed directly by chondrocyte-like cells, with transition from fibrous tissue to bone occurring gradually and consecutively without capillary invasion. In situ hybridisation using digoxigenin-11-UTP-labelled complementary RNAs showed that the chondroid bone cells temporarily expressed type-II collagen mRNA. They did not show the classical morphological characteristics of chondrocytes, but were assumed to be young chondrocytes undergoing further differentiation into bone-forming cells. We found at least three different modes of ossification during bone lengthening by distraction osteogenesis. We believe that this is the first report of such a rat model, and have shown the validity of in situ hybridisation techniques for the study of the cellular and molecular mechanisms involved in distraction osteogenesis

We examined whether enamel matrix derivative (EMD) could improve healing of the tendon–bone interface following reconstruction of the anterior cruciate ligament (ACL) using a hamstring tendon in a rat model. ACL reconstruction was performed in both knees of 30 Sprague-Dawley rats using the flexor digitorum tendon. The effect of commercially available EMD (EMDOGAIN), a preparation of matrix proteins from developing porcine teeth, was evaluated. In the left knee joint the space around the tendon–bone interface was filled with 40 µl of EMD mixed with propylene glycol alginate (PGA). In the right knee joint PGA alone was used. The ligament reconstructions were evaluated histologically and biomechanically at four, eight and 12 weeks (n = 5 at each time point). At eight weeks, EMD had induced a significant increase in collagen fibres connecting to bone at the tendon–bone interface (p = 0.047), whereas the control group had few fibres and the tendon–bone interface was composed of cellular and vascular fibrous tissues. At both eight and 12 weeks, the mean load to failure in the treated specimens was higher than in the controls (p = 0.009). EMD improved histological tendon–bone healing at eight weeks and biomechanical healing at both eight and 12 weeks. EMD might therefore have a human application to enhance tendon–bone repair in ACL reconstruction

Calcified matrix that is being absorbed has a characteristic appearance. At the junction of the epiphysis and metaphysis in the rat tibia this appearance can be seen near capillary endothelium as well as under osteoclasts. It is concluded that absorption can take place under the walls of capillaries without the presence of osteoclasts

In thirty-one rat tibiae, plugs of plain acrylic cement were inoculated with Staphylococcus aureus; these all remained contaminated at the end of two weeks when the animals were killed. Inoculation with known strains of Pseudomonas, Proteus and Gp. G Streptococcus resulted in 70 to 93 per cent persisting contamination. Gentamicin, to which the organisms were fully sensitive, was efficacious in controlling the infection (90 per cent plugs proving sterile after two weeks). Fucidin was less successful against Staphylococcus aureus although effective in vitro. Intravenous inoculation with a suspension of Staphylococcus aureus succeeded in contaminanting 70 per cent of sixty plain cement plugs when injected into the tail vein half an hour after closure of the leg wounds. Only 11 per cent of sixty-four plugs were so contaminanted when the injection was delayed for two weeks. This animal model is submitted as a possible future means of testing different antibiotic-cement combinations against infection

The passage of technetium-labelled methylene diphosphonate (99mTc-MDP) across rat bone was examined by autoradiography. The autoradiographs showed that shortly after an injection of the bone-seeking agent there was activity outside the bone, within the bone marrow and also adjacent to the highly vascular epiphysial plate; the distribution of the isotope in the incubated bone appeared to be non-uniform, a high concentration being seen adjacent to the epiphysial plate and also on the surfaces of the bone. The evidence suggested that a two-fold mechanism resulted in the uneven distribution of 99mTc-MDP. The first factor probably represented the regional distribution of blood flow with a transcapillary movement of the tracer from the capillary bed to the extravascular space; the subsequent incorporation of the tracer into bone appeared to depend on the nature of the bone matrix

For the treatment of ununited fractures, we developed a system of delivering magnetic labelled mesenchymal stromal cells (MSCs) using an extracorporeal magnetic device. In this study, we transplanted ferucarbotran-labelled and luciferase-positive bone marrow-derived MSCs into a non-healing femoral fracture rat model in the presence of a magnetic field. The biological fate of the transplanted MSCs was observed using luciferase-based bioluminescence imaging and we found that the number of MSC derived photons increased from day one to day three and thereafter decreased over time. The magnetic cell delivery system induced the accumulation of photons at the fracture site, while also retaining higher photon intensity from day three to week four. Furthermore, radiological and histological findings suggested improved callus formation and endochondral ossification. We therefore believe that this delivery system may be a promising option for bone regeneration

Growth at the proximal tibial epiphyseal plate of the rat has been measured following three different growth-stimulating procedures. These were proximal periosteal release, distal periosteal release and full periosteal stripping of the diaphysis. A new radiographic method using a photographic technique has made it possible to take accurate measurements of the rate of long-bone growth in small experimental animals. From the results of this animal series we conclude that proximal tibial periosteal division is likely to be the most effective of the three procedures when used to correct leg-length discrepancy in the growing child

The reduced stability of hydroxyapatite (HA)-coated implants in osteopenic conditions is considered to be a major problem. We therefore developed a model of a boosted cementless implantation in osteopenic rats. Twelve-week-old rats were either ovariectomised (OVX) or sham-operated (SO), and after 24 weeks plain or HA-coated implants were inserted. They were treated with either a prostaglandin EP4 receptor agonist (ONO-4819) or saline for one month. The EP4 agonist considerably improved the osteoporosis in the OVX group. Ultrastructural analysis and mechanical testing showed an improvement in the implant-bone attachment in the HA-coated implants, which was further enhanced by the EP4 agonist. Although the stability of the HA-coated implants in the saline-treated OVX rats was less than in the SO normal rats, the administration of the EP4 agonist significantly compensated for this shortage. Our results showed that the osteogenic effect of the EP4 agonist augmented the osteoconductivity of HA and significantly improved the stability of the implant-bone attachment in the osteoporotic rat model

1. Tetracycline labelling methods have been used to measure the rate of growth in length and the rate of growth in width of the tibia of the normal rat. 2. The main limitations of the tetracycline methods are that in very young animals the bands of labelled bone are indistinct and remodelling occurs quickly; in animals nearing maturity, the growth in width is very slow and periods of at least fourteen days are required to give reliable results. 3. The tetracycline labelling methods can be used also to determine changes in the basic processes of bone formation and bone resorption

Rat patellae were preincubated with culture medium M199 for one hour and then with either fresh culture medium or Ringer's solution, Ringer lactate, Ringer glucose, normal saline or Betadine for another hour. The rate of proteoglycan synthesis in the articular cartilage was then measured by uptake of 35SO4 for the next 16 hours. Cartilage metabolism was inhibited by all of the solutions even after a recovery time of 16 hours. The inhibition was by 5% for Ringer's solution, 10% for Ringer glucose (p < 0.01), 20% for saline and Ringer lactate (p < 0.001) and 55% for Betadine (p < 0.001). Ringer's solution is therefore the best choice for joint irrigation during arthroscopy or other procedures

The presence of the connective tissue components fibronectin and the different types of collagen was demonstrated by histological and immunohistological methods in the granulation and scar tissue of a healing injury in rat muscle. The effects of physical activity on granulation tissue production, scar formation and muscle regeneration at various stages of healing were studied. It was shown that immobilisation after injury accelerates granulation tissue production, but if continued too long, leads to contraction of the scar and to poor structural organisation of the components of regenerating muscle and scar tissue. However, a certain period of immobilisation, about five days for rat muscle, is required to allow newly-formed granulation tissue to cover the injured area and to have sufficient tensile strength to withstand subsequent mobilisation. This mobilisation, at the correct interval, seems essential for the quicker resorption of scar tissue and the better structural organisation of the muscle

The effects of extracorporeal shock waves (ESWT) on tendon healing were assessed by observing histological and biomechanical parameters in a rat model of injury to the tendo Achillis. The injury was created by inserting an 18-G needle through tendo Achillis in 48 adult Wistar albino rats. The animals were divided into three groups. The first group received radiation only after the operation. The second received no shock waves and the third had 500 15 KV shocks on the second post-operative day. All the rats were killed on the 21st day after surgery. Histopathological analysis showed an increase in the number of capillaries and less formation of adhesions in the study group compared with the control group (p = 0.03). A significantly greater force was required to rupture the tendon in the study group (p = 0.028). Our findings suggest a basis for clinical trials using ESWT

Intermittent treatment with parathyroid hormone (PTH) has an anabolic effect on both intact cancellous and cortical bone. Very little is known about the effect of the administration of PTH on the healing of fractures or the incorporation of orthopaedic implants. We have investigated the spontaneous ingrowth of callus and the formation of bone in a titanium chamber implanted at the medioproximal aspect of the tibial metaphysis of the rat. Four groups of ten male rats weighing approximately 350 g were injected with human PTH (1-34) in a dosage of 0, 15, 60 or 240 μg/kg/day, respectively, for 42 days from the day of implantation of the chamber. During the observation period the chamber became only partly filled with callus and bone and no difference in ingrowth distance into the chamber was found between the groups. The cancellous density was increased by 90%, 132% and 173% in the groups given PTH in a dosage of 15, 60 or 240 μg/kg/day, respectively. There was a linear correlation between bone density and the log PTH doses (r. 2. = 0.6). Our findings suggest that treatment with PTH may have a potential for enhancement of the incorporation of orthopaedic implants as well as a beneficial effect on the healing of fractures when it is given in low dosages

We studied the sensory afferent properties of normal, immobilised and inflamed rat knees by recording the activity of the medial articular nerve (MAN). When the knee was inflamed by kaolin-carrageenan or immobilised for six weeks, MAN activity significantly increased during rest and continuous passive motion (CPM). The maximal discharge rate tended to increase depending on the angular velocity of the CPM. When the knees were then rested for one hour before again starting CPM, activity was further increased at the initial CPM cycle, the ‘post-rest effect’. Analysis of the conduction velocity showed that 94% and 66% of spike units on the recorded discharge of the immobilised and inflamed knees, respectively, belonged to fine nerve fibres. Our findings show that the sensory receptors in the knee are sensitised in a similar manner by immobilisation and by inflammation, suggesting a relationship to pain. The post-rest effect may be related to a characteristic symptom of osteoarthritis called ‘starting pain’

When large daily doses of vitamin D were administered to rats endochondral growth was inhibited and bone resorption occurred; later in the process uncalcified matrix (osteoid) like that seen in rickets formed on trabecular margins. When vitamin D was given only for a short period and then discontinued, little resorption of bone was seen during the withdrawal period and wide seams of osteoid material appeared which eventually calcified in an irregular manner. When normal endochondral growth was resumed a wide transverse band of dense bone with enclosed cartilaginous cores was left in the marrow cavity. If, after a few days, a second large dose of the vitamin was given resorption again occurred and calcification of osteoid material was accelerated, the first microscopic sign being a dense, wide, granular, deeply staining line at the junction of the bone and new osteoid. After a second withdrawal period a second layer of osteoid formed; eventually another transverse band appeared in the metaphysis. If this hypervitaminosis D cycle (+4 -12) was continued rats continued to form new bone with relatively little remodelling, so that after three such cycles bones became dense and hard. Histological study showed that little marrow cavity remained in either skull, vertebrae or epiphyses and a dense mass of bone enclosing cartilage cores filled the metaphysial part of the long bones. In addition, ankylosis ofteeth, calcification of spinal ligaments and widespread metastatic calcification were present. When hypervitaminosis D cycles (+1 -12, +1 -21) were adjusted to produce minimal resorptive changes a wide range of bone change was observed. This varied from uniform dense metaphysial bone containing abnormal cartilage matrix arranged in longitudinal striations, dense transverse bands parallel to the epiphysial cartilage, to remnants of dense trabeculae extending into the marrow cavity. Bone changes in osteopetrosis structurally closely resembled the induced bone changes in the rat. It is concluded that an important mechanism in the production of osteopetrosis is an accentuated rhythm of bone change like that shown experimentally to be produced in these animals. It is emphasised that these changes are but part of a range of bone disorders associated with abnormalities of cycles of resorption and deposition of bone, the type of change differing with the nature of the cycles

The effect of tenotomy and of immobilisation in lengthened and shortened positions on the intramuscular connective tissue of the calf muscles of the rat was studied morphometrically and with a scanning electron microscope. Both tenotomy and immobilisation produced a marked increase in both the endomysial and the perimysial collagen networks, with a simultaneous decrease in intramuscular capillary density. The increase in connective tissue volume was more pronounced and occurred more rapidly in the soleus, which consists mainly of type I, slow-twitch fibres than in the gastrocnemius, which is mainly of type II, fast-twitch fibres. The relative volume of connective tissue increased in parallel with the duration of immobilisation or after tenotomy. There was slightly more increase after immobilisation in a shortened rather than in a lengthened position

Bone marrow mesenchymal stromal cells were aspirated from immature male green fluorescent protein transgenic rats and cultured in a monolayer. Four weeks after the creation of the osteochondral defect, the rats were divided into three groups of 18: the control group, treated with an intra-articular injection of phosphate-buffered saline only; the drilling group, treated with an intra-articular injection of phosphate-buffered saline with a bone marrow-stimulating procedure; and the bone marrow mesenchymal stromal cells group, treated with an intra-articular injection of bone marrow mesenchymal stromal cells plus a bone marrow-stimulating procedure. The rats were then killed at 4, 8 and 12 weeks after treatment and examined.

The histological scores were significantly better in the bone marrow mesenchymal stromal cells group than in the control and drilling groups at all time points (p < 0.05). The fluorescence of the green fluorescent protein-positive cells could be observed in specimens four weeks after treatment.

Soaking bone grafts in a bisphosphonate solution before implantation can prevent their resorption and increase the local bone density in rats and humans. However, recent studies suggest that pre-treatment of allografts with bisphosphonate can prevent bone ingrowth into impaction grafts. We tested the hypothesis that excessive amounts of bisphosphonate would also cause a negative response in less dense grafts. We used a model where non-impacted metaphyseal bone grafts were randomised into three groups with either no bisphosphonate, alendronate followed by rinsing, and alendronate without subsequent rinsing, and inserted into bone chambers in rats. The specimens were evaluated histologically at one week, and by histomorphometry and radiology at four weeks. At four weeks, both bisphosphonate groups showed an increase in the total bone content, increased newly formed bone, and higher radiodensity than the controls. In spite of being implanted in a chamber with a limited opportunity to diffuse, even an excessive amount of bisphosphonate improved the outcome. We suggest that the negative results seen by others could be due to the combination of densely compacted bone and a bisphosphonate.

We suggest that bisphosphonates are likely to have a negative influence where resorption is a prerequisite to create space for new bone ingrowth.

The nervous system is known to be involved in inflammation and repair. We aimed to determine the effect of physical activity on the healing of a muscle injury and to examine the pattern of innervation. Using a drop-ball technique, a contusion was produced in the gastrocnemius in 20 rats. In ten the limb was immobilised in a plaster cast and the remaining ten had mobilisation on a running wheel. The muscle and the corresponding dorsal-root ganglia were studied by histological and immunohistochemical methods.

In the mobilisation group, there was a significant reduction in lymphocytes (p = 0.016), macrophages (p = 0.008) and myotubules (p = 0.008) between three and 21 days. The formation of myotubules and the density of nerve fibres was significantly higher (both p = 0.016) compared with those in the immobilisation group at three days, while the density of CGRP-positive fibres was significantly lower (p = 0.016) after 21 days.

Mobilisation after contusional injury to the muscle resulted in early and increased formation of myotubules, early nerve regeneration and progressive reduction in inflammation, suggesting that it promoted a better healing response.

In vitro studies on isolated bone cells were undertaken to investigate the presence of transplantation (histocompatibility) antigens. Bone cells were cultured with allogeneic lymphocytes and exposed to cytotoxic sera containing antibodies against transplantation antigens, to determine their antigenic profile. Preliminary results suggest that bone cells may not express lymphocyte stimulating antigens in an active form, at least after the isolation procedure performed. On the other hand, bone cells were killed by cytotoxic antibodies in a specific way, providing evidence for the presence of serologically defined (SD) transplantation antigens on the cell surface. Additional studies with absorbed sera suggest "sharing" of histocompatibility antigens between bone cells and lymphocytes. The relevance of the surface antigens of bone cells to clinical fields such as bone allotransplantation, susceptibility to various orthopaedic diseases and skeletal sarcomata is discussed.

A computerised image analyser has been used experimentally to determine its possible usefulness in quantifying bone density. Rats were prepared for this purpose and ash weight/volume readings were obtained on femora which had been radiographed and studied to estimate radiographic density by means of the image analyser. The findings indicated that rats can be prepared by administering oestradiol or by allowing an additional week's growth, both of which give a statistically significant increase of bone density. The computerised image analyser was able to detect these differences but the correlations, although real and linear, between ash weight/volume and radiographic density were not large. Further refinements in techniques will be needed before the system is applied clinically.

1. The effects of frozen irradiated homogenous bone grafts on the healing of circumscribed defects in the mandibles and femora of forty albino rats have been studied.

2. The grafts were well tolerated by the host animals and did not appear in any way either to stimulate or to inhibit osteogenesis. The rate of healing of the defects containing the homografts compared favourably with that of the control defects.

1. The effect of implanting heterogenous anorganic bone, homogenous organic bone, autogenous compact bone from the iliac crest, and autogenous bony callus into circumscribed defects in the femur of albino rats of the Wistar strain is described.

2. Neither heterogenous anorganic bone nor homogenous organic bone appeared to induce new bone formation in a healing defect.

3. Some of the osteogenic cells of autogenous callus implants survived transplantation to a bone defect and gave rise to new bone formation. This did not occur when compact bone from autogenous iliac crest was implanted.

4. Implants of autogenous callus, autogenous compact bone, homogenous organic bone and heterogenous anorganic bone all impeded the normal development of host bone trabeculae in a healing bone defect, seemingly because they acted as physical barriers to the proliferating host callus. None of the implant materials appeared to suppress the healing reaction ofthe host.

5. Implanted homogenous organic bone was removed and replaced by host bone more quickly than was implanted heterogenous anorganic bone, and it appears to be the better material for grafting into bone defects.

6. Autogenous callus or autogenous cancellous bone is a superior implant material to autogenous compact bone and is the bone graft material of choice.

7. The absorption of all the implant materials used in this investigation was associated with the presence of multinucleated giant cells.

8. The activity of multinucleated giant cells may be influenced by the organic matrix of the material which is to be absorbed.

9. Except when fresh autogenous callus was implanted into the defects, the rate of healing in the grafted defects was slower than that in the control defects. In the defects grafted with fresh autogenous callus the healing rates of the control and grafted defects were the same.

Using in situ hybridisation and the terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick end-labelling (TUNEL) reaction in rats with osteonecrosis of the femoral head we have studied the effect of ischaemia on the gene expression of the stress proteins oxygen-regulated protein 150 (ORP150) and haemoxygenase 1 (HO1) and the death mechanism of the cells involved in osteonecrosis. Both ORP150 and HO1 have been reported to have important roles in the successful adaptation to oxygen deprivation.

ORP150 and HO1 mRNA expression was induced by ischaemia in osteoblasts and osteocytes. In proliferative chondrocytes, these signals were detected constitutively. During the development of ischaemic osteonecrosis, the mechanism of cell death was apoptosis as indicated by DNA fragmentation and the presence of apoptotic bodies in osteocytes, chondrocytes and bone-marrow cells. After the initial ischaemic event, expression of ORP150 and HO1 mRNA, the TUNEL-positive reaction and empty lacunae were found sequentially. These findings were exclusive and may be considered to be markers for each stage in the development of osteonecrosis.

The aim of our study was to investigate the effect of platelet-rich plasma on the proliferation and differentiation of rat bone-marrow cells and to determine an optimal platelet concentration in plasma for osseous tissue engineering. Rat bone-marrow cells embedded in different concentrations of platelet-rich plasma gel were cultured for six days. Their potential for proliferation and osteogenic differentiation was analysed. Using a rat limb-lengthening model, the cultured rat bone-marrow cells with platelet-rich plasma of variable concentrations were transplanted into the distraction gap and the quality of the regenerate bone was evaluated radiologically. Cellular proliferation was enhanced in all the platelet-rich plasma groups in a dose-dependent manner. Although no significant differences in the production and mRNA expression of alkaline phosphatase were detected among these groups, mature bone regenerates were more prevalent in the group with the highest concentration of platelets. Our results indicate that a high platelet concentration in the platelet-rich plasma in combination with osteoblastic cells could accelerate the formation of new bone during limb-lengthening procedures

Aims. This study aims to assess first, whether mutations in the epidermal growth factor receptor (EGFR) and Kirsten rat sarcoma (kRAS) genes are associated with overall survival (OS) in patients who present with symptomatic bone metastases from non-small cell lung cancer (NSCLC) and secondly, whether mutation status should be incorporated into prognostic models that are used when deciding on the appropriate palliative treatment for symptomatic bone metastases. Patients and Methods. We studied 139 patients with NSCLC treated between 2007 and 2014 for symptomatic bone metastases and whose mutation status was known. The association between mutation status and overall survival was analysed and the results applied to a recently published prognostic model to determine whether including the mutation status would improve its discriminatory power. Results. The median OS was 3.9 months (95% confidence interval (CI) 2.1 to 5.7). Patients with EGFR (15%) or kRAS mutations (34%) had a median OS of 17.3 months (95% CI 12.7 to 22.0) and 1.8 months (95% CI 1.0 to 2.7), respectively. Compared with EGFR-positive patients, EGFR-negative patients had a 2.5 times higher risk of death (95% CI 1.5 to 4.2). Incorporating EGFR mutation status in the prognostic model improved its discriminatory power. Conclusion. Survival prediction models for patients with symptomatic bone metastases are used to determine the most appropriate (surgical) treatment for painful or fractured lesions. This study shows that NSCLC should not be regarded as a single entity in such models. Cite this article: Bone Joint J 2017;99-B:516–21

Prophylactic antibiotics are important in reducing the risk of periprosthetic joint infection (PJI) following total knee arthroplasty. Their effectiveness depends on the choice of antibiotic and the optimum timing of their administration, to ensure adequate tissue concentrations. Cephalosporins are typically used, but an increasing number of resistant organisms are causing PJI, leading to the additional use of vancomycin. There are difficulties, however, with the systemic administration of vancomycin including its optimal timing, due to the need for prolonged administration, and potential adverse reactions. Intraosseous regional administration distal to a tourniquet is an alternative and attractive mode of delivery due to the ease of obtaining intraosseous access. Many authors have reported the effectiveness of intraosseous prophylaxis in achieving higher concentrations of antibiotic in the tissues compared with intravenous administration, providing equal or enhanced prophylaxis while minimizing adverse effects. This annotation describes the technique of intraosseous administration of antibiotics and summarizes the relevant clinical literature to date.

Cite this article: Bone Joint J 2023;105-B(11):1135–1139.

Platelet-derived growth factor (PDGF) is known to stimulate osteoblast or osteoprogenitor cell activity. We investigated the effect of locally applied PDGF from poly-. d. ,l-lactide (PDLLA)-coated implants on fracture healing in a rat model. A closed fracture of the right tibia of four-month-old Sprague-Dawley rats (n = 40) was stabilised with implants coated with a biodegradable PDLLA versus implants coated with PDLLA and PDGF. Radiographs were taken throughout the study, and a marker of DNA activity, bromodeoxyuridine (BrdU), was injected before the rats were killed at three, seven and ten days. The radiographs showed consolidation of the callus in the PDGF-treated group compared with the control group at all three time points. In the PDGF-treated group, immunohistochemical staining of BrdU showed that the distribution of proliferating cells in all cellular events was higher after ten days compared with that at three and seven days. These results indicate that local application of PDGF from biodegradable PDLLA-coated implants significantly accelerates fracture healing in experimental animals. Further development may help fracture healing in the clinical situation.

Congenital pseudarthrosis of the tibia is an uncommon manifestation of neurofibromatosis type 1 (NF1), but one that remains difficult to treat due to anabolic deficiency and catabolic excess. Bone grafting and more recently recombinant human bone morphogenetic proteins (rhBMPs) have been identified as pro-anabolic stimuli with the potential to improve the outcome after surgery. As an additional pharmaceutical intervention, we describe the combined use of rhBMP-2 and the bisphosphonate zoledronic acid in a mouse model of NF1-deficient fracture repair. Fractures were generated in the distal tibiae of neurofibromatosis type 1-deficient (Nf1. +/−. ) mice and control mice. Fractures were open and featured periosteal stripping. All mice received 10 μg rhBMP-2 delivered in a carboxymethylcellulose carrier around the fracture as an anabolic stimulus. Bisphosphonate-treated mice also received five doses of 0.02 mg/kg zoledronic acid given by intraperitoneal injection. When only rhBMP but no zoledronic acid was used to promote repair, 75% of fractures in Nf1. +/−. mice remained ununited at three weeks compared with 7% of controls (p < 0.001). Systemic post-operative administration of zoledronic acid halved the rate of ununited fractures to 37.5% (p < 0.07). These data support the concept that preventing bone loss in combination with anabolic stimulation may improve the outcome following surgical treatment for children with congenital pseudarthoris of the tibia and NF1

Aims

Aseptic loosening is a leading cause of uncemented arthroplasty failure, often accompanied by fibrotic tissue at the bone-implant interface. A biological target, neutrophil extracellular traps (NETs), was investigated as a crucial connection between the innate immune system’s response to injury, fibrotic tissue development, and proper bone healing. Prevalence of NETs in peri-implant fibrotic tissue from aseptic loosening patients was assessed. A murine model of osseointegration failure was used to test the hypothesis that inhibition (through Pad4^-/- mice that display defects in peptidyl arginine deiminase 4 (PAD4), an essential protein required for NETs) or resolution (via DNase 1 treatment, an enzyme that degrades the cytotoxic DNA matrix) of NETs can prevent osseointegration failure and formation of peri-implant fibrotic tissue.

Using a rat model the characteristics of the sensory neurones of the dorsal-root ganglia (DRG) innervating the hip were investigated by retrograde neurotransport and immunohistochemistry. Fluoro-Gold solution (FG) was injected into the left hip of ten rats. Seven days later the DRG from both sides between T12 and L6 were harvested. The number of FG-labelled calcitonin gene-related peptide-immunoreactive or isolectin B4-binding neurones were counted. The FG-labelled neurones were distributed throughout the left DRGs between T13 and L5, primarily at L2, L3, and L4. Few FG-labelled isolectin B4-binding neurones were present in the DRGs of either side between T13 and L5, but calcitonin gene-related peptide-immunoreactive neurones made up 30% of all FG-labelled neurones. Our findings may explain the referral of pain from the hip to the thigh or lower leg corresponding to the L2, L3 and L4 levels. Since most neurones are calcitonin gene-related peptide-immunoreactive peptide-containing neurones, they may have a more significant role in the perception of pain in the hip as peptidergic DRG neurones

Most animal studies indicate that early irrigation and debridement reduce infection after an open fracture. Unfortunately, these studies often do not involve antibiotics. Clinical studies indicate that the timing of initial debridement does not affect the rate of infection but these studies are observational and fraught with confounding variables. The purpose of this study was to control these variables using an animal model incorporating systemic antibiotics and surgical treatment. We used a rat femur model with a defect which was contaminated with Staphylococcus aureus and treated with a three-day course of systemic cefazolin (5 mg/kg 12-hourly) and debridement and irrigation, both of which were initiated independently at two, six and 24 hour time points. After 14 days the bone and hardware were harvested for separate microbiological analysis. No animal that received antibiotics and surgery two hours after injury had detectable bacteria. When antibiotics were started at two hours, a delay in surgical treatment from two to six hours significantly increased the development of infection (p = 0.047). However, delaying surgery to 24 hours increase the rate of infection, but not significantly (p = 0.054). The timing of antibiotics had a more significant effect on the proportion of positive samples than earlier surgery. Delaying antibiotics to six or 24 hours had a profoundly detrimental effect on the infection rate regardless of the timing of surgery. These findings are consistent with the concept that bacteria progress from a vulnerable planktonic form to a treatment-resistant biofilm

Thrombin has many biological properties similar to those of growth factors. In a previous study, we showed that thrombin improves healing of the rat tendo Achillis. Low molecular weight heparin (LMWH) inhibits the activity and the generation of thrombin. We therefore considered that LMWH at a thromboprophylactic dose might inhibit tendon repair. Transection of the tendo Achillis was carried out in 86 rats and the healing tested mechanically. Low molecular weight heparin (dalateparin) was either injected a few minutes before the operation and then given continuously with an osmotic mini pump for seven days, or given as one injection before the operation. In another experiment ,we gave LMWH or a placebo by injection twice daily. The anti-factor Xa activity was analysed. Continuous treatment with LMWH impaired tendon healing. After seven days, this treatment caused a 33% reduction in force at failure, a 20% reduction in stiffness and a 67% reduction in energy uptake. However, if injected twice daily, LMWH had no effect on tendon healing. Anti-factor Xa activity was increased by LMWH treatment, but was normal between intermittent injections. Low molecular weight heparin delays tendon repair if given continuously, but not if injected intermittently, probably because the anti-factor Xa activity between injections returns to normal, allowing sufficient thrombin stimulation for repair. These findings indicate the need for caution in the assessment of long-acting thrombin and factor Xa inhibitors

The role of inflammatory cells and their products in tendinopathy is not completely understood. Pro-inflammatory cytokines are upregulated after oxidative and other forms of stress. Based on observations that increased cytokine expression has been demonstrated in cyclically-loaded tendon cells we hypothesised that because of their role in oxidative stress and apoptosis, pro-inflammatory cytokines may be present in rodent and human models of tendinopathy. A rat supraspinatus tendinopathy model produced by running overuse was investigated at the genetic level by custom micro-arrays. Additionally, samples of torn supraspinatus tendon and matched intact subscapularis tendon were collected from patients undergoing arthroscopic shoulder surgery for rotator-cuff tears and control samples of subscapularis tendon from ten patients with normal rotator cuffs undergoing arthroscopic stabilisation of the shoulder were also obtained. These were all evaluated using semiquantitative reverse transcription polymerase chain-reaction and immunohistochemistry. We identified significant upregulation of pro-inflammatory cytokines and apoptotic genes in the rodent model (p = 0.005). We further confirmed significantly increased levels of cytokine and apoptotic genes in human supraspinatus and subscapularis tendon harvested from patients with rotator cuff tears (p = 0.0008). These findings suggest that pro-inflammatory cytokines may play a role in tendinopathy and may provide a target for preventing tendinopathies

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

An injectable material consisting of calcium sulphate mixed with hydroxyapatite was investigated as a possible alternative to autograft in the restoration of bone defects. The material was studied both in vitro in simulated body fluid (SBF) and in vivo when implanted in rat muscles and into the proximal tibiae of rabbits. Variation in the strength and weight of the material during ageing in SBF was measured. Tissue response, material resorption and bone ingrowth were studied in the animal models. A good tissue response was observed in both the rat muscles and rabbit tibiae without inflammatory reactions or the presence of fibrous tissue. Ageing in SBF showed that during the first week carbonated hydroxyapatite precipitated on the surfaces of the material and this may enhance bone ingrowth

Compartment syndrome, a devastating consequence of limb trauma, is characterised by severe tissue injury and microvascular perfusion deficits. We hypothesised that leucopenia might provide significant protection against microvascular dysfunction and preserve tissue viability. Using our clinically relevant rat model of compartment syndrome, microvascular perfusion and tissue injury were directly visualised by intravital video microscopy in leucopenic animals. We found that while the tissue perfusion was similar in both groups (38.8% (standard error of the mean (. sem). 7.1). , 36.4. % (. sem. 5.7), 32.0% (. sem. 1.7), and 30.5% (. sem. 5.35) continuously-perfused capillaries at 45, 90, 120 and 180 minutes compartment syndrome, respectively versus 39.2% (. sem. 8.6), 43.5% (. sem. 8.5). , . 36.6% (. sem. 1.4) and 50.8% (. sem. 4.8) at 45, 90, 120 and 180 minutes compartment syndrome, respectively in leucopenia), compartment syndrome-associated muscle injury was significantly decreased in leucopenic animals (7.0% (. sem. 2.0), 7.0%, (. sem. 1.0), 9.0% (. sem. 1.0) and 5.0% (. sem. 2.0) at 45, 90, 120 and 180 minutes of compartment syndrome, respectively in leucopenia group versus 18.0% (. sem. 4.0), 23.0% (. sem. 4.0), 32.0% (. sem. 7.0), and 20.0% (. sem. 5.0) at 45, 90, 120 and 180 minutes of compartment syndrome in control, p = 0.0005). This study demonstrates that the inflammatory process should be considered central to the understanding of the pathogenesis of cellular injury in compartment syndrome. Cite this article: Bone Joint J 2015;97-B:539–43

We undertook a study of the anti-tumour effects of hyperthermia, delivered via magnetite cationic liposomes (MCLs), on local tumours and lung metastases in a mouse model of osteosarcoma. MCLs were injected into subcutaneous osteosarcomas (LM8) and subjected to an alternating magnetic field which induced a heating effect in MCLs. A control group of mice with tumours received MCLs but were not exposed to an AMF. A further group of mice with tumours were exposed to an AMF but had not been treated with MCLs. The distribution of MCLs and local and lung metastases was evaluated histologically. The weight and volume of local tumours and the number of lung metastases were determined. Expression of heat shock protein 70 was evaluated immunohistologically. Hyperthermia using MCLs effectively heated the targeted tumour to 45°C. The mean weight of the local tumour was significantly suppressed in the hyperthermia group (p = 0.013). The mice subjected to hyperthermia had significantly fewer lung metastases than the control mice (p = 0.005). Heat shock protein 70 was expressed in tumours treated with hyperthermia, but was not found in those tumours not exposed to hyperthermia. The results demonstrate a significant effect of hyperthermia on local tumours and reduces their potential to metastasise to the lung

It has been thought that lumbar intervertebral discs were innervated segmentally. We have previously shown that the L5-L6 intervertebral disc in the rat is innervated bilaterally from the L1 and L2 dorsal root ganglia through the paravertebral sympathetic trunks, but the pathways between the disc and the paravertebral sympathetic trunks were unknown. We have now studied the spines of 17 rats to elucidate the exact pathways. We examined serial sections of the lumbar spine using immunohistochemistry for calcitonin gene-related peptide, a sensory nerve marker. We showed that these nerve fibres from the intervertebral disc ran through the sinuvertebral nerve into the rami communicantes, not into the corresponding segmental spinal nerve. In the rat, sensory information from the lumbar intervertebral discs is conducted through rami communicantes. If this innervation pattern applies to man, simple decompression of the corresponding nerve root will not relieve discogenic pain. Anterior interbody fusion, with the denervation of rami communicantes, may be effective for such low back pain

Aims

The aim of this study was to develop a single-layer hybrid organic-inorganic sol-gel coating that is capable of a controlled antibiotic release for cementless hydroxyapatite (HA)-coated titanium orthopaedic prostheses.

Aims

The aims of this study were to develop an in vivo model of periprosthetic joint infection (PJI) in cemented hip hemiarthroplasty, and to monitor infection and biofilm formation in real-time.

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

This study was undertaken to elucidate the mechanism of biological repair at the tendon-bone junction in a rat model. The stump of the toe flexor tendon was sutured to a drilled hole in the tibia (tendon suture group, n = 23) to investigate healing of the tendon-bone junction both radiologically and histologically. Radiological and histological findings were compared with those observed in a sham control group where the bone alone was drilled (n = 19). The biomechanical strength of the repaired junction was confirmed by pull-out testing six weeks after surgery in four rats in the tendon suture group. Callus formation was observed at the site of repair in the tendon suture group, whereas in the sham group callus formation was minimal. During the pull-out test, the repaired tendon-bone junction did not fail because the musculotendinous junction always disrupted first. In order to understand the factors that influenced callus formation at the site of repair, four further groups were evaluated. The nature of the sutured tendon itself was investigated by analysing healing of a tendon stump after necrosis had been induced with liquid nitrogen in 16 cases. A proximal suture group (n = 16) and a partial tenotomy group (n = 16) were prepared to investigate the effects of biomechanical loading on the site of repair. Finally, a group where the periosteum had been excised at the site of repair (n = 16) was examined to study the role of the periosteum. These four groups showed less callus formation radiologically and histologically than did the tendon suture group. In conclusion, the sutured tendon-bone junction healed and achieved mechanical strength at six weeks after suturing, showing good local callus formation. The viability of the tendon stump, mechanical loading and intact periosteum were all found to be important factors for better callus formation at a repaired tendon-bone junction

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

Antibiotic resistance represents a threat to human health. It has been suggested that by 2050, antibiotic-resistant infections could cause ten million deaths each year. In orthopaedics, many patients undergoing surgery suffer from complications resulting from implant-associated infection. In these circumstances secondary surgery is usually required and chronic and/or relapsing disease may ensue. The development of effective treatments for antibiotic-resistant infections is needed. Recent evidence shows that bacteriophage (phages; viruses that infect bacteria) therapy may represent a viable and successful solution. In this review, a brief description of bone and joint infection and the nature of bacteriophages is presented, as well as a summary of our current knowledge on the use of bacteriophages in the treatment of bacterial infections. We present contemporary published in vitro and in vivo data as well as data from clinical trials, as they relate to bone and joint infections. We discuss the potential use of bacteriophage therapy in orthopaedic infections. This area of research is beginning to reveal successful results, but mostly in nonorthopaedic fields. We believe that bacteriophage therapy has potential therapeutic value for implant-associated infections in orthopaedics.

Cite this article: Bone Joint J 2021;103-B(2):234–244.

Aims

Periprosthetic joint infections (PJIs) are among the most devastating complications after joint arthroplasty. There is limited evidence on the efficacy of different antiseptic solutions on reducing biofilm burden. The purpose of the present study was to test the efficacy of different antiseptic solutions against clinically relevant microorganisms in biofilm.

Periprosthetic joint infection (PJI) is one of the most dreaded complications after arthroplasty surgery; thus numerous approaches have been undertaken to equip metal surfaces with antibacterial properties. Due to its antimicrobial effects, silver is a promising coating for metallic surfaces, and several types of silver-coated arthroplasty implants are in clinical use today. However, silver can also exert toxic effects on eukaryotic cells both in the immediate vicinity of the coated implants and systemically. In most clinically-used implants, silver coatings are applied on bulk components that are not in direct contact with bone, such as in partial or total long bone arthroplasties used in tumour or complex revision surgery. These implants differ considerably in the coating method, total silver content, and silver release rates. Safety issues, such as the occurrence of argyria, have been a cause for concern, and the efficacy of silver coatings in terms of preventing PJI is also controversial. The application of silver coatings is uncommon on parts of implants intended for cementless fixation in host bone, but this option might be highly desirable since the modification of implant surfaces in order to improve osteoconductivity can also increase bacterial adhesion. Therefore, an optimal silver content that inhibits bacterial colonization while maintaining osteoconductivity is crucial if silver were to be applied as a coating on parts intended for bone contact. This review summarizes the different methods used to apply silver coatings to arthroplasty components, with a focus on the amount and duration of silver release from the different coatings; the available experience with silver-coated implants that are in clinical use today; and future strategies to balance the effects of silver on bacteria and eukaryotic cells, and to develop silver-coated titanium components suitable for bone ingrowth.

Cite this article: Bone Joint J 2021;103-B(3):423–429.

Aims

Vitamin E-infused highly cross-linked polyethylene (E1) has recently been introduced in total knee arthroplasty (TKA). An in vitro wear simulator study showed that E1 reduced polyethylene wear. However there is no published information regarding in vivo wear. Previous reports suggest that newly introduced materials which reduce in vitro polyethylene wear do not necessarily reduce in vivo polyethylene wear. To assist in the evaluation of the newly introduced material before widespread use, we established an in vivo polyethylene wear particle analysis for TKA. The aim of this study was to compare in vivo polyethylene wear particle generation between E1 and conventional polyethylene (ArCom) in TKA.

Aims

The purpose of this study was to identify the changes in untreated long head of the biceps brachii tendon (LHBT) after a rotator cuff tear and to evaluate the factors related to the changes.

Cartilage formation was provoked in the skull vault of the young rat by making multiple incisions, and scraping the periosteum to reduce the blood supply to the injured area. The hypothesis that ischaemia induces osteogenic cells to produce cartilage in the course of fracture repair thus receives experimental support

1. The radiographs of paired living and dead rat tibiae, obtained in an experiment previously reported, have been examined by densitometry. 2. The dead bone became progressively less dense than the living bone as the duration of the implantation increased. 3. The change in density was related to the quantity, but not to the quality, of the bone tissue examined

1. A metabolic study in a case of myositis ossificans progressiva is reported. 2. The serum showed an increased power of calcification of rachitic rat cartilage. 3. Estimations of alkaline phosphatase showed slightly raised values. 4. Surgical removal of a bony bar was followed by prolonged administration of ACTH and cortisone, but no effect on the calcium-phosphorus balance or on the re-ossification within the area of operation was observed

1. The results of the present investigation indicate that in the foetal rat the juxta-epiphysial vascular bed consists of a dense irregular network of sinusoids in direct contact with the growth cartilage, supplied by end-arteries, and drained by a profusion of metaphysial sinusoids. 2. The circulation is a closed one–that is, the endothelium is unbroken in its continuity and microhaemorrhages do not occur against the cartilage. 3. It is possible that juxta-epiphysial endothelial cells or their derivatives are chondrolytic, and that they participate directly, together with other mesenchymal derivatives, in the removal of cartilage as a preparatory stage in enchondral bone formation

We studied the precise role of the fracture haematoma in healing by the experimental transplantation of the haematoma at two days and four days after fracture of the rat femur to subperiosteal and intramuscular sites. We used bone marrow and peripheral blood haematomas for control experiments. The transplanted two-day fracture haematoma produced new bone by endochondral ossification at the subperiosteal site, but not at the intramuscular site. Four-day fracture haematoma produced new bone formation at both subperiosteal and intramuscular sites. These results suggest that fracture haematoma has an inherent osteogenetic potential

1. The pattern of tritiated thymidine labelling in the cells of the epiphysial cartilage and metaphysis of the tibia in the rat is described for intervals of one hour to twenty-eight days after injection. 2. The region of dividing cells is defined and evidence given for a zone of reserve cells at the top of the cartilage columns. 3. The difficulties of quantitative grain count studies are discussed, and some approximate values are given for the generation time and mitotic cycle periods of the cartilage plate cells. 4. Some further evidence is given about the life cycles of the osteoblast and the osteoclast

We studied the calcium content and mechanical strength of cortical bone from rats and dogs after different periods of demineralisation, showing that the rate of demineralisation differed considerably between the species. Specimens from the rat were further treated by chemical extraction and autolysis and tested for osteoinductive properties. We showed that partially demineralised cortical bone retained adequate mechanical strength, while retaining the biological effects of completely demineralised bone. This shows that it is possible to prepare allografts which have adequate mechanical strength and still retain osteo-inductive properties

Spinal nerve roots often sustain compression injuries. We used a Wistar rat model of the cauda equina syndrome to investigate such injuries. Rapid transient compression of the cauda equina was produced using a balloon catheter. The results were assessed by daily neurological examination and somatosensory evoked potential (SEP) recording before surgery and ten weeks after decompression. Compression of the spinal nerves induced changes in the SEP which persisted for up to ten weeks after decompression, but it had no effect on the final neurological outcome. Our study shows the importance of early surgical decompression for cauda equina syndrome

A stump neuroma is caused by the disorganised growth of axon cylinders into proliferating granulation tissue, but this is stopped by an undamaged epineural sleeve. We report experiments in the rat in which the epineural sleeve of the stump of the sciatic nerve was freed from nerve fascicles for about 5 mm and then sealed with a synthetic tissue adhesive. Neuroma formation was largely prevented in comparison with the results of other methods. This new technique has been used to treat 68 painful neuromas in 36 patients. All but three of the patients were cured or improved and none were made worse

Based on a study using a retrograde neurotracer, we have previously found that the dorsal portion of the L5/6 disc in the rat is multisegmentally innervated by dorsal root ganglia (DRG) from the level of T13 to L6, and that sensory nerve fibres from DRG of T13, L1 and L2 pass through the paravertebral sympathetic trunks. In this study in newborn rats, we injected crystals of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylinedocarbocyanine perchlorate (DiI) into the DRG of T13, L1 and L2 and showed DiI-labelled sensory nerve fibres in the dorsal portion of the discs from the level of T13/L1 to L5/6. Our results show that the dorsal portion of the lumbar discs is innervated by the DRG from levels T13 to L2

Ischaemic preconditioning is a process by which exposure of a tissue to a short period of non-damaging ischaemic stress leads to resistance to the deleterious effects of a subsequent prolonged ischaemic stress. It has been extensively described in the heart, but few studies have examined the possibility that it can occur in skeletal muscle. We have used a rat model of ischaemia of one limb to examine this possibility. Exposure of the hind limb to a period of ischaemia of five minutes and reperfusion for five minutes significantly protected the tibialis anterior muscle against the structural damage induced by a subsequent period of limb ischaemia for four hours and reperfusion for one hour. This protection was evident on examination of the muscle by both light and electron microscopy. Longer or shorter times of prior ischaemia had no effect

1. Decalcified lyophilised rat bone matrix prepared by Urist's method acts as an inductor of cartilage and bone when implanted into animals of other species, namely mice, rabbits and gerbils. Induction in rabbits and gerbils was very much weaker than in the mouse. 2. The site of implantation affected the outcome; intramuscular implants induced cartilage and bone more strongly and regularly than subcutaneous or intraperitoneal implants. 3. Rabbit transitional epithelium, growing in cortisone-treated gerbils, caused bone induction, but in general, results with this species suggest that it responds poorly to bone-inducing stimuli. 4. Cortisone, used as an immunosuppressant, did not inhibit bone and cartilage induction

Synthetic bone substitutes provide an alternative to autograft but do not give equivalent clinical results. Their performance may be enhanced by adding osteogenic growth factors. In this study, TGFβ1 was absorbed on to a carrier of β tricalcium phosphate and Gelfoam® and used to fill a defect around a tibial implant in a rat model of revision arthoplasty. We added 0.0, 0.02 μg, 0.1 μg or 1.0 μg of TGFβ1 to the carrier and then implanted it around an hydroxyapatite-coated stainless-steel pin in the proximal tibia of rats. The tibiae were harvested at three, six or 26 weeks and the amount of bone formation and ceramic resorption were assessed. TGFβ1 had no effect on the amount of bone in the defect, the amount of fluorescent label incorporated or the rate of mineral apposition. The growth factor did not significantly affect the amount of β TCP remaining in the tissue at any of the time points

Using a rat model, we created a bone-to-titanium interface and applied phagocytosable high-density polyethylene pArticles between the bone and implant, either initially or when the interface had matured. No fibrous membrane developed and no bone resorption was found. If sliding movements were initiated at the interface after two weeks, there was formation of a fibrous membrane. The additional application of pArticles did not change the thickness of the membrane, and there were only minor qualitative changes. Creation of a membrane by movement followed by cessation of movement and the application of pArticles caused the membrane to persist, whereas in a pArticle-free control group bone-to-metal contact was re-established. Our findings suggest that mechanical stimuli are of primary importance for prosthetic loosening, and that pArticles may modulate the later stages of the loosening process

1. An apparatus was designed to determine the shearing strength of the upper tibial epiphysis in the rat. Observations were made with this instrumenton normal animals, on animals receiving growth-hormone, and on animals receiving oestrogen. 2. When the epiphysis separates from the diaphysis, the plane of cleavage is constant, passing through the third layer of the epiphysial plate. 3. Growth-hormone decreases and sex-hormone increases the shearing strength of the epiphysial plate. These changes are due to alterations produced by these two hormones in the thickness of the third layer of the epiphysial plate. 4. It is suggested that these findings may be of significance in providing an anatomical basis for slipping of the upper femoral epiphysis in man, especially when it is associated with the adiposo-genital syndrome or with rapid adolescent growth

Our aim was to develop a clinically relevant model of atrophic nonunion in the rat to test the hypothesis that the vessel density of atrophic nonunion reaches that of normal healing bone, but at a later time-point. Atrophic nonunion is usually attributed to impaired blood supply and is poorly understood. We determined the number of blood vessels at the site of an osteotomy using immunolocalisation techniques in both normally healing bones and in atrophic nonunion. At one week after operation there were significantly fewer blood vessels in the nonunion group than in the healing group. By eight weeks, the number in the atrophic nonunion group had reached the same level as that in the healing group. Our findings suggest that the number of blood vessels in atrophic nonunion reaches the same level as that in healing bone, but at a later time-point. Diminished vascularity within the first three weeks, but not at a later time-point, may prevent fractures from uniting

Little is known about the tissue reactions to various implant materials which coincide with an inflammatory reaction. We used the avridine arthritis rat model to evaluate the tissue response in the synovial, interstitial and subcutaneous tissues after implant insertion. Quantitative immunohistochemistry showed that normal joint synovial tissue is dominated by ED2-positive resident macrophages. Polyethylene implants induced a much stronger foreign-body reaction than titanium implants, as measured by the number of interfacial ED1-positive macrophages. The tissue response to titanium and polyethylene was also vastly different in arthritic synovial tissue compared with control tissue. It is likely that these biomaterials interact differently with inflammatory cells or intermediary compounds. It may be that arthritic synovial tissue produces reactive oxygen intermediates (free radicals) with which titanium has a unique anti-inflammatory interaction in vitro

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

1. The process of repair after fracture of the humerus of the growing rat has been studied by histological, histochemical and biochemical methods. 2. Both periosteal and surrounding mesenchymal cells take part in the process of repair. 3. The primary framework of collagen bridging the gap is mainly formed by the mesenchymal cells, while calcification and ossification of the framework is largely a function of the periosteum. 4. The mucopolysaccharide content rises rapidly in the first week after injury, and is followed by a rise in the collagen content during the second week. The deposition of calcium phosphate during the third and fourth weeks causes an apparent fall in the collagen content during that period. The collagen content tends to return to normal during the phase of remodelling in the fifth and sixth weeks. 5. The tensile strength of the healing bone bears a close relation to its collagen content

Immobilisation causes denervation-like changes in the motor endplates, decreases the content of IGF-I, and increases the number of IGF-I receptors in the spinal cord. In the rat we investigated whether similar changes occur after a fracture of the midshaft of the femur which had been treated by intramedullary fixation with adequate or undersized pins. A more pronounced reduction in muscle wet weight was seen after fixation by undersized pins as well as decreased ash density of the ipsilateral tibia which did not completely return to normal within the 12-week experimental period. The nicotinic cholinergic receptors in the motor endplates of tibialis anterior were increased (p < 0.01) and there was a significant increase (p < 0.02) in IGF-I receptors in the lumbar spinal cord ipsilateral to the fracture after treatment by undersized nails. These changes may be associated with the impaired proprioception, co-ordination and motor activity which are sometimes seen after fractures

An orientated substratum has been implicated in the development and regeneration of axons and synapses. We prepared a basement membrane matrix from autogenous striated muscle, used it to repair the sciatic nerve in rats, then investigated the results by histology and electrophysiology. When treated grafts were coaxially aligned with the nerve fascicles functional recovery appeared within 30 days, with good growth of axons into the distal nerve. Grafts with myotubes at right angles to the nerve fascicles supported nerve regeneration but at a slower rate. Grafts of coaxially aligned but untreated muscle allowed axon penetration only through naturally degenerated muscle fibres, with minimal axon penetration of the distal nerve. It is concluded that in the rat a treated graft with correctly orientated empty myotubes can facilitate and guide the regeneration of peripheral nerve after injury and so lead to recolonisation of the distal stump with functional recovery

A model of tourniquet ischaemia was developed in the hind limb of the rat, and the metabolic changes that occurred in the calf muscles were monitored by the non-invasive technique of phosphorus-31 nuclear magnetic resonance spectroscopy. During ischaemia the intramyocellular pH became acidic as the level of phosphocreatine declined and that of inorganic phosphate rose. Phosphocreatine was no longer detectable after approximately 2 hours and ATP was depleted after approximately 3.5 hours. Metabolic recovery was rapid (1 hour) if ATP was present when the tourniquet was released but was prolonged (3 or more hours) if ATP was depleted. Hourly release of the tourniquet for 10 minutes ensured the maintenance of ATP and rapid metabolic recovery. Release for intervals of only 5 minutes did not have the same protective effect and in fact worsened tissue pH during the period of tourniquet ischaemia. Heparin and corticosteroids were without effect during and after periods of tourniquet ischaemia

In 65 mature Wistar rats a Kirschner wire was introduced into the medullary cavity of each femur. A closed transverse mid-shaft fracture of one femur was produced by a three-point bending technique. Subsequently the mechanical characteristics of the healing fracture, including the torque and angle of twist required to take the callus to its yield point and to ultimate failure, were compared with those for the opposite femur of each rat. Controls were killed in groups at two, three, four, five and seven weeks. Test animals were given bovine growth hormone in a daily dose of five milligrams before being killed in groups at two, three and four weeks. A significant increase in torque index was found in the two-week group of test animals but not in subsequent groups. No evidence was found that growth hormone given alone could produce an overall shortening of the healing time in fresh fractures

We used a rat model in vivo to study the effects of the concentration of polyethylene particles on the bone-implant interface around stable implants in the proximal tibia. Intra-articular injections of 10. 4. , 10. 6. or 10. 8. high-density polyethylene (HDPE) particles per joint were given 8, 10 and 12 weeks after surgery. The animals were killed after 14 and 26 weeks and the response at the interface determined. Fibrous tissue was seen at the bone-implant interface when the head of the implant was flush with the top of the tibia but not when it was sunk below the tibial plateau. In the latter case the implant was completely surrounded by a shell of bone. The area of fibrous tissue and that of the gap between the implant and bone was related to the concentration of particles in the 14-week group (p < 0.05). Foreign-body granulomas containing HDPE particles were seen at the bone-implant interface in animals given 10. 8. particles. The pathology resembles that seen around prostheses with aseptic loosening and we suggest that this is a useful model by which to study this process

Extraskeletal bone formation can be induced in rodents by implantation of demineralised bone matrix and such implantation has been used to treat bone defects in man, but it is uncertain if induction or merely conduction occurs. We studied bone induction in primates by excising segments of the fibulae of adult squirrel monkeys, defatting and demineralising them before reimplanting them into the quadriceps of the same animal. As a control experiment, rat matrix was prepared in exactly the same way and implanted in rats. After six weeks the implants were harvested and either ashed and analysed for calcium content or prepared for histology. In the rats, the calcium content indicated that about 20% of the original matrix had been replaced by new bone. In the monkeys the calcium content was about the same as that in normal body fluid and no bone was seen in histological sections. This result casts doubt on the use of demineralised human bone matrix as a bone inductor, although it may function by other mechanisms

Abundant implant-derived biomaterial wear particles are generated in aseptic loosening and are deposited in periprosthetic tissues in which they are phagocytosed by mononuclear and multinucleated macrophage-like cells. It has been stated that the multinucleated cells which contain wear particles are not bone-resorbing osteoclasts. To investigate the validity of this claim we isolated human osteoclasts from giant-cell tumours of bone and rat osteoclasts from long bones. These were cultured on glass coverslips and on cortical bone slices in the presence of particles of latex, PMMA and titanium. Osteoclast phagocytosis of these particle types was shown by light microscopy, energy-dispersive X-ray analysis and SEM. Giant cells containing phagocytosed particles were seen to be associated with the formation of resorption lacunae. Osteoclasts containing particles were also calcitonin-receptor-positive and showed an inhibitory response to calcitonin. Our findings demonstrate that osteoclasts are capable of phagocytosing particles of a wide range of size, including particles of polymeric and metallic bio-materials found in periprosthetic tissues, and that after particle phagocytosis, they remain fully functional, hormone-responsive, bone-resorbing cells

Aim

Mesenchymal stem cells (MSCs) have several properties that may support their use as an early treatment option for osteoarthritis (OA). This study investigated the role of multiple injections of allogeneic bone marrow-derived stem cells (BMSCs) to alleviate the progression of osteoarthritic changes in the various structures of the mature rabbit knee in an anterior cruciate ligament (ACL)-deficient OA model.

We used a rat model in vivo to study the effects of particulate bone cements at the bone-implant interface. A ceramic pin was implanted into the tibiae of 48 rats. Three types of particle of clinically relevant size were produced from one bone-cement base without radio-opacifier, with zirconium dioxide (ZrO. 2. ) and with barium sulphate (BaSO. 4. ). The rats were randomly assigned to four groups to receive one of the three bone cements or normal saline with 2% v/v Sprague-Dawley serum as the control. A total of 10. 9. particles was injected into the knee at 8, 10 and 12 weeks after the original surgery. The animals were killed at 14 weeks and the tibiae processed for histomorphometry. The area of fibrous tissue and the gap between the implant and bone were measured using image analysis. All three types of particle were associated with a larger area of bone resorption than the control. Only in the case of the BaSO. 4. -containing cement did this reach statistical significance (p = 0.01). Particles of bone cement appear to promote osteolysis at the bone-implant interface and this effect is most marked when BaSO. 4. is used as the radiopaque agent

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

The intermittent administration of parathyroid hormone (PTH) increases the formation of bone by stimulating osteoblastic activity. Our study evaluates the possibility that intermittent treatment with PTH (1-34) may also enhance the implant-bone fixation of stainless-steel screws. Twenty-eight rats received one screw in either one (n = 8) or in both (n = 20) proximal tibiae. We administered either PTH (1-34) in a dosage of 60 μg/kg/day (n = 14) or vehicle (n = 14) over a period of four weeks. At the end of this time, the degree of fixation was assessed by measuring the removal torque on one screw in each rat (n = 28) and the pull-out strength on the contralateral screw (n = 20). PTH increased the mean removal torque from 1.1 to 3.5 Ncm (p = 0.001) and the mean pull-out strength from 66 to 145 N (p = 0.002). No significant differences in body-weight or ash weight of the femora were seen. Histological examination showed that both groups had areas of soft tissue at the implant-bone interface, but these appeared less in the PTH group. These results indicate that intermittent treatment with PTH may enhance the early fixation of orthopaedic implants

Colchicine is often used in the treatment of diseases such as familial Mediterranean fever (FMF) and gout. We have previously reported that patients with FMF who had colchicine on a daily basis and who had a total hip arthroplasty showed no heterotopic ossification after surgery. The mechanism by which colchicine causes this clinical phenomenon has never been elucidated. We therefore evaluated the effect of various concentrations of colchicine on cell proliferation and mineralisation in tissue culture, using rat and human cells with and without osteogenic potential. Cell proliferation was assessed by direct cell counts and uptake of (. 3. H)thymidine, and mineralisation by measuring the amount of staining by Alizarin Red. Our findings indicate that concentrations of colchicine of up to 3 ng/ml did not affect cell proliferation but inhibition was observed at 10 to 30 ng/ml. Mineralisation decreased to almost 50%, which was the maximum inhibition observed, at concentrations of colchicine of 2.5 ng/ml. These results indicate that colchicine at low concentrations, of up to 3 ng/ml, has the capacity to inhibit selectively bone-like cell mineralisation in culture, without affecting cell proliferation. Further clinical and laboratory studies are necessary to evaluate the effects of colchicine on biological processes involving the proliferation of osteoblasts and tissue mineralisation in vivo, such as the healing of fractures, the formation of heterotopic bone and neoplastic bone growth

Mononuclear osteoclast precursors are present in the wear-particle-associated macrophage infiltrate found in the membrane surrounding loose implants. These cells are capable of differentiating into osteoclastic bone-resorbing cells when co-cultured with the rat osteoblast-like cell line, UMR 106, in the presence of 1,25(OH). 2. vitamin D. 3. In order to develop an in vitro model of osteoclast differentiation which more closely parallels the cellular microenvironment at the bone-implant interface in situ, we determined whether osteoblast-like human bone-derived cells were capable of supporting the differentiation of osteoclasts from arthroplasty-derived cells and analysed the humoral conditions required for this to occur. Long-term co-culture of arthroplasty-derived cells and human trabecular-bone-derived cells (HBDCs) resulted in the formation of numerous tartrate-resistant-acid-phosphatase (TRAP) and vitronectin-receptor (VNR)-positive multinucleated cells capable of extensive resorption of lacunar bone. The addition of 1,25(OH). 2. vitamin D. 3. was not required for the formation of osteoclasts and bone resorption. During the formation there was release of substantial levels of M-CSF and PGE. 2. Exogenous PGE. 2. (10. −8. to 10. −6. M) was found to stimulate strongly the resorption of osteoclastic bone. Our study has shown that HBDCs are capable of supporting the formation of osteoclasts from mononuclear phagocyte precursors present in the periprosthetic tissues surrounding a loose implant. The release of M-CSF and PGE. 2. by activated cells at the bone-implant interface may be important for the formation of osteoclasts at sites of pathological bone resorption associated with aseptic loosening

Apart from preliminary notices of present work, previous reports of experimental and clinical trials of the effects of a high-peak pulsed electromagnetic field (PEMF) on degeneration and regeneration of peripheral nerves lacked statistical analysis. Therefore, we designed experiments with standardised operative, histological, cytological and morphometric techniques to assess the effect of PEMF on lesions of the common peroneal nerves in paired male rats matched for age, environmental conditions and level and type of lesion. One of two types of lesion was induced in the left common peroneal nerve: in 12 pairs of rats the nerve was crushed just above the knee and in the remaining 12 pairs the nerve was cut and immediately sutured at the same level. The right common peroneal nerve of each rat served as a control. Animals received 15 minutes of PEMF produced by a Diapulse machine or sham treatment daily for periods ranging from three and a half days to eight weeks after injury. Healthy nerves were unaffected, but after damage there were statistically significant differences between PEMF treated and sham treated rats. PEMF accelerated the recovery of injured limbs and the degeneration, regeneration and maturation of myelinated axons; epineural, perineural and intraneural fibrosis was reduced; and the luminal cross-sectional area of intraneural vessels increased after both types of lesion. Findings are discussed and the need for clinical trials is stressed

1 . Implants of heterogenous cartilage are known to excite a foreign body reaction in host tissues. In these experiments the way such implants hinder the spread of ossification across a fracture gap was studied. A segment of bone and periosteum was excised from both fibulae in twelve young grivet monkeys, and epiphysial cartilage from a four-day- old rat was implanted in the gap on the left side. The repair processes were investigated at intervals up to twenty-eight weeks. 2. On both sides the gaps were filled by fibrous tissue growing in from the adjacent muscle, and four weeks elapsed before callus started to form. Thereafter ossification across the gap was active on the right side, with bony union in seven or eight weeks. 3. On the left side the implant was slowly resorbed by macrophages and giant cells. Ossification made little headway in the gap after the seventh week. Remains of the implant were found up to the end of the period covered by the experiment. The ends of the fragments were united by fibrous tissue. 4. The fifth to the eighth week seemed to be a critical period, during which the implant and its surrounding inflammatory cells hindered chondrification and ossification and prevented fusion of the masses of callus at the ends of the fragments. 5. It is concluded that anything that impedes callus formation across the fracture line during this critical period may lead to non-union

1. The effects of heavy training on a skeletal muscle have been studied in the rat. After denervation of the triceps surae muscle the tendon of the plantaris muscle was implanted into the tuberosity of the calcaneum. It was then possible to demand an unusual performance of the plantaris, the weight of which is only 18 per cent of the weight of the triceps surae. 2. Formation of new muscle fibres was observed after prolonged heavy training. This is incontrast to the opinion of most investigators, who have seen no new fibres formed after training. Degenerative changes followed by regeneration were also seen. 3. The trained muscle could almost double its weight, and treble its force. Paradoxically, the supposedly non-contractile sarcoplasm was seen to have increased after training. 4. Training induced a strong protein synthesis in muscle. In normal muscle protein synthesis can hardly be demonstrated. 5. Connective tissue grew between single muscle fibres in the heavily trained muscle. Its distribution was unequal. 6. Heavy exercise caused marked swelling of an untrained muscle. 7. Functional recovery was satisfactory after the operation. This showed that a muscle can be replaced by one only one-fifth its weight, provided the latter is trained adequately. 8. Not even the most arduous training could inflict permanent damage on the muscle

1. The effect of immobilisation on bone formation has been investigated in the rat. As chlortetracycline has the property of being deposited preferentially in areas of newly deposited bone, its uptake can be used as a reflection of osseous formation. 2. One hind limb of albino rats was immobilised either by section of the second, third and fourth lumbar nerve trunks or by section of tendo calcaneus and ligamentum patellae. The incorporation of chlortetracycline was determined quantitatively in the femur and tibia of both hind limbs at intervals after immobilisation. 3. Tetracycline uptake is expressed in terms of bone weight, this being most important in order to obtain correct values. 4. A comparison between the tetracycline uptake and the weight of the bones gives information about the rate of bone destruction. 5. In animals immobilised by nerve section three phases can be distinguished: a first phase with diminished bone formation, a second with increased formation and increased destruction and a third phase with diminished bone formation. Both mechanisms, decreased formation and increased destruction, are therefore important in the production of immobilisation osteoporosis; their relative importance depending upon the duration of the immobilisation. 6. In animals immobilised by tendon section the mechanical function becomes restored in the second week as a result of healing: this explains the rapid compensation for the initial loss of weight of the bones accompanied by a marked increase in tetracycline uptake. 7. These results are discussed and compared with information in the literature. Variations in bone formation and destruction rates with time could explain the varied results reported by other authors

The multifunctional adhesion molecule CD44 is a major cell-surface receptor for hyaluronic acid (HUA). Recent data suggest that it may also bind the ubiquitous bone-matrix protein, osteopontin (OPN). Because OPN has been shown to be a potentially important protein in bone remodelling, we investigated the hypothesis that OPN interactions with the CD44 receptor on bone cells participate in the regulation of the healing of fractures. We examined the spatial and temporal patterns of expression of OPN and CD44 in healing fractures of rat femora by in situ hybridisation and immunohistochemistry. We also localised HUA in the fracture callus using biotinylated HUA-binding protein. OPN was expressed in remodelling areas of the hard callus and was found in osteocytes, osteoclasts and osteoprogenitor cells, but not in cuboidal osteoblasts which were otherwise shown to express osteocalcin. The OPN signal in osteocytes was not uniformly distributed, but was restricted to specific regions near sites where OPN mRNA-positive osteoclasts were attached to bone surfaces. In the remodelling callus, intense immunostaining for CD44 was detected in osteocyte lacunae, along canaliculi, and on the basolateral plasma membrane of osteoclasts, but not in the cuboidal osteoblasts. HUA staining was detected in fibrous tissues but little was observed in areas of hard callus where bone remodelling was progressing. Our findings suggest that OPN, rather than HUA, is the major ligand for CD44 on bone cells in the remodelling phase of healing of fractures. They also raise the possibility that such interactions may be involved in the communication of osteocytes with each other and with osteoclasts on bone surfaces. The interactions between CD44 and OPN may have important clinical implications in the repair of skeletal tissues

Aims

It is increasingly appreciated that coordinated regulation of angiogenesis and osteogenesis is needed for bone formation. How this regulation is achieved during peri-implant bone healing, such as osseointegration, is largely unclear. This study examined the relationship between angiogenesis and osteogenesis in a unique model of osseointegration of a mouse tibial implant by pharmacologically blocking the vascular endothelial growth factor (VEGF) pathway.

Extracorporeal shock-wave (ESW) treatment hasbeen shown to be effective in promoting the healing of fractures. We aimed to determine whether ESW could enhance the growth of bone-marrow osteoprogenitor cells. We applied ESW to the left femur of rats 10 mm above the knee at 0.16 mJ/mm. 2. in a range of between 250 and 2000 impulses. Bone-marrow cells were harvested after ESW for one day and subjected to assessment of colony-forming unit (CFU) granulocytes, monocytes, erythocytes, megakaryocytes (CFU-Mix), CFU-stromal cells (CFU-S) and CFU-osteoprogenitors (CFU-O). We found that the mean value for the CFU-O colonies after treatment with 500 impulses of ESW was 168.2 CFU-O/well (. sem. 11.3) compared with 88.2 CFU-O/well (. sem. 7.2) in the control group. By contrast, ESW treatment did not affect haematopoiesis as shown by the CFU-Mix (p = 0.557). Treatment with 250 and 500 impulses promoted CFU-O, but not CFU-Mix formations whereas treatment with more than 750 impulses had an inhibiting effect. Treatment with 500 impulses also enhanced the activity of bone alkaline phosphatase in the subculture of CFU-O (p< 0.01), indicating a selective promotion of growth of osteoprogenitor cells. Similarly, formation of bone nodules in the long-term culture of bone-marrow osteoprogenitor cells was also significantly enhanced by ESW treatment with 500 impulses. The mean production of TGF-β1 was 610 pg/ml (. sem. 84.6) in culture supernatants from ESW-treated rats compared with 283 pg/ml (. sem. 36.8) in the control group. Our findings suggest that optimal treatment with ESW could enhance rat bone-marrow stromal growth and differentiation towards osteoprogenitors presumably by induction of TGF-β1

1 . Fresh bone autografts to a muscle bed in the rat gave rise to vigorous new bone formation from about the fourth day. The graft took the form of a hollow ossicle with central bone marrow at eighteen days: it became progressively more regular in outline and was still present at six months. 2. Fresh bone homografts produced two separate phases of new bone formation–early and late. In the early phase non-lamellar woven bone appeared at about the fourth day, continued to grow until eight days, and subsequently died. It arose from osteogenic cells of the homograft. In the late phase, which developed in relation to a few grafts after four weeks, the new bone was lamellar in character, and remained closely applied to the graft surface. Evidence is presented that this bone arose by metaplasia of the host connective tissues at the graft site. There was a local inflammatory response to the bone homograft. 3. Both phases of homograft new bone formation were abolished if the animal was prepared by a skin homograft from the same donor four weeks before, but not if four months elapsed between the two grafting procedures. 4. Freeze-dried bone homografts did not give rise to the early phase of homograft new bone but produced a few examples of the late phase after five months. The inflammatory response was less intense with freeze-dried homografts than with fresh homografts. 5. Skin homografts three weeks after fresh bone homografts from the same donor underwent an early rejection at five to six days. 6. Skin homografts three weeks after freeze-dried bone homografts from the same donor had a mean survival time of twelve days, which was significantly longer than the mean survival time of l0·9 days in normal rats

Aims

The success of anterior cruciate ligament reconstruction (ACLR) depends on osseointegration at the graft-tunnel interface and intra-articular ligamentization. Our aim was to conduct a systematic review of clinical and preclinical studies that evaluated biological augmentation of graft healing in ACLR.

1. The present study is an attempt to analyse and apportion significance to the role of inductive mechanisms in bone transplantation. 2. The experimental model used in the present work is that of the composite homograftautograft of cancellous bone previously described (Burwell 1964a). 3. Iliac bone was removed from hooded rats and washed free from its marrow. The bone was then treated by various physical and chemical methods (some of which have been used by other workers to prepare bank bone), namely freezing (-20 degrees Centigrade, -79 degrees Centigrade, -196 degrees Centigrade); freeze-drying (without sterilisation, sterilisation with high energy radiation, sterilisation with ß-propiolactone); decalcification (with E.D.T.A.); irradiation (in the frozen state at a dose of 4 million rads); boiling in water; immersion in merthiolate solution; extraction of organic components with ethylenediamine: and calcining at 660 degrees Centigrade. The treated bone was then impregnated with fresh autologous marrow procured from the femoral shaft of the Wistar rat into which the treated composite graft was to be implanted. The grafts were inserted intramuscularly and removed for study after two, six and twelve weeks. 4. After fixation, serial sectioning and staining, each graft was examined microscopically, and the proportion of new bone/grafted bone scored using an arbitrary scale (0-4). The mean score (and the standard error of the mean score) was then plotted for each treated composite graft and also for several types of fresh cancellous bone grafts. 5. It was found (Fig. 2) that the various treated composite grafts formed a spectrum of bone-forming capacities–the maximum scores being attained by the frozen and freeze-dried composite grafts, the lowest scores by the "deproteinised" composite grafts. 6. The reasons for these differences are discussed. It is concluded that cancellous bone, after transplantation, has the property to induce and promote osteogenesis in marrow; moreover, that this property is contained in the organic components of bone. 7. From the standpoint of inductive mechanisms, cancellous bone treated by freezing or freeze-drying seems to be the most suitable devitalised bone for grafting purposes; bone which has been boiled or merthiolated less suitable; and "deproteinised" bone the least suitable. 8. Freeze-dried bone sterilised physically (by high energy radiation) or chemically (by ß-propiolactone) did not form significantly less new bone than did freeze-dried bone which had not been sterilised. 9. Remodelling mechanisms in bone transplantation are briefly discussed and attention drawn to the deficiencies of present knowledge. The quantitative studies of other workers have indicated that freeze-dried bone may be more rapidly remodelled than is frozen bone. 10. The importance of fresh red marrow in promoting osteogenesis in bone transplantation and in the healing of certain fractures, is emphasised. It seems likely that the interrelationship of bone and marrow revealed by experiment has wider significance not only in health and in response to injury but also in causation of certain idiopathic bone disorders