Carbonate-substituted hydroxyapatite (CHA) is more osteoconductive and more resorbable than hydroxyapatite (HA), but the underlying mode of its action is unclear. We hypothesised that increased resorption of the ceramic by osteoclasts might subsequently upregulate osteoblasts by a coupling mechanism, and sought to test this in a large animal model.

Defects were created in both the lateral femoral condyles of 12 adult sheep. Six were implanted with CHA granules bilaterally, and six with HA. Six of the animals in each group received the bisphosphonate zoledronate (0.05 mg/kg), which inhibits the function of osteoclasts, intra-operatively.

After six weeks bony ingrowth was greater in the CHA implants than in HA, but not in the animals given zoledronate. Functional osteoclasts are necessary for the enhanced osteoconduction seen in CHA compared with HA.

Objective

Excessive mechanical stress on synovial joints causes osteoarthritis (OA) and results in the production of prostaglandin E2 (PGE2), a key molecule in arthritis, by synovial fibroblasts. However, the relationship between arthritis-related molecules and mechanical stress is still unclear. The purpose of this study was to examine the synovial fibroblast response to cyclic mechanical stress using an in vitro osteoarthritis model.

The ability of mesenchymal stem cells (MSCs) to differentiate in vitro into chondrocytes, osteocytes and myocytes holds great promise for tissue engineering. Skeletal defects are emerging as key targets for treatment using MSCs due to the high responsiveness of bone to interventions in animal models. Interest in MSCs has further expanded in recognition of their ability to release growth factors and to adjust immune responses.

Despite their increasing application in clinical trials, the origin and role of MSCs in the development, repair and regeneration of organs have remained unclear. Until recently, MSCs could only be isolated in a process that requires culture in a laboratory; these cells were being used for tissue engineering without understanding their native location and function. MSCs isolated in this indirect way have been used in clinical trials and remain the reference standard cellular substrate for musculoskeletal engineering. The therapeutic use of autologous MSCs is currently limited by the need for ex vivo expansion and by heterogeneity within MSC preparations. The recent discovery that the walls of blood vessels harbour native precursors of MSCs has led to their prospective identification and isolation. MSCs may therefore now be purified from dispensable tissues such as lipo-aspirate and returned for clinical use in sufficient quantity, negating the requirement for ex vivo expansion and a second surgical procedure.

In this annotation we provide an update on the recent developments in the understanding of the identity of MSCs within tissues and outline how this may affect their use in orthopaedic surgery in the future.

Cite this article: Bone Joint J 2014;96-B:291–8.

We compared the biological characteristics of extrinsic fibroblasts infiltrating the patellar tendon with those of normal, intrinsic fibroblasts in the normal tendon in vitro. Infiltrative fibroblasts were isolated from the patellar tendons of rabbits six weeks after an in situ freeze-thaw treatment which killed the intrinsic fibroblasts. These intrinsic cells were also isolated from the patellar tendons of rabbits which had not been so treated.

Proliferation and invasive migration into the patellar tendon was significantly slower for infiltrative fibroblasts than for normal tendon fibroblasts. Flow-cytometric analysis indicated that expression of α5β1 integrin at the cell surface was significantly lower in infiltrative fibroblasts than in normal tendon fibroblasts. The findings suggest that cellular proliferation and invasive migration of fibroblasts into the patellar tendon after necrosis are inferior to those of the normal fibroblasts. The inferior intrinsic properties of infiltrative fibroblasts may contribute to a slow remodelling process in the grafted tendon after ligament reconstruction.

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.

We hypothesised that cells obtained via a Reamer–Irrigator–Aspirator (RIA) system retain substantial osteogenic potential and are at least equivalent to graft harvested from the iliac crest. Graft was harvested using the RIA in 25 patients (mean age 37.6 years (18 to 68)) and from the iliac crest in 21 patients (mean age 44.6 years (24 to 78)), after which ≥ 1 g of bony particulate graft material was processed from each. Initial cell viability was assessed using Trypan blue exclusion, and initial fluorescence-activated cell sorting (FACS) analysis for cell lineage was performed. After culturing the cells, repeat FACS analysis for cell lineage was performed and enzyme-linked immunosorbent assay (ELISA) for osteocalcin, and Alizarin red staining to determine osteogenic potential. Cells obtained via RIA or from the iliac crest were viable and matured into mesenchymal stem cells, as shown by staining for the specific mesenchymal antigens CD90 and CD105. For samples from both RIA and the iliac crest there was a statistically significant increase in bone production (both p < 0.001), as demonstrated by osteocalcin production after induction.

Medullary autograft cells harvested using RIA are viable and osteogenic. Cell viability and osteogenic potential were similar between bone grafts obtained from both the RIA system and the iliac crest.

Cite this article: Bone Joint J 2013;95-B:1269–74.

Human articular cartilage samples were retrieved from the resected material of patients undergoing total knee replacement. Samples underwent automated controlled freezing at various stages of preparation: as intact articular cartilage discs, as minced articular cartilage, and as chondrocytes immediately after enzymatic isolation from fresh articular cartilage. Cell viability was examined using a LIVE/DEAD assay which provided fluorescent staining. Isolated chondrocytes were then cultured and Alamar blue assay was used for estimation of cell proliferation at days zero, four, seven, 14, 21 and 28 after seeding. The mean percentage viabilities of chondrocytes isolated from group A (fresh, intact articular cartilage disc samples), group B (following cryopreservation and then thawing, after initial isolation from articular cartilage), group C (from minced cryopreserved articular cartilage samples), and group D (from cryopreserved intact articular cartilage disc samples) were 74.7% (95% confidence interval (CI) 73.1 to 76.3), 47.0% (95% CI 43 to 51), 32.0% (95% CI 30.3 to 33.7) and 23.3% (95% CI 22.1 to 24.5), respectively. Isolated chondrocytes from all groups were expanded by the following mean proportions after 28 days of culturing: group A ten times, group B 18 times, group C 106 times, and group D 154 times.

This experiment demonstrated that it is possible to isolate viable chondrocytes from cryopreserved intact human articular cartilage which can then be successfully cultured.

The mammalian growth plate is a complex structure which is essential for the elongation of long bones. However, an understanding of how the growth plate functions at the cellular level is lacking. This review, summarises the factors involved in growth-plate regulation, its failure and the consequence of injury. We also describe some of the cellular mechanisms which underpin the increase in volume of the growth-plate chondrocyte which is the major determinant of the rate and extent of bone lengthening. We show how living in situ chondrocytes can be imaged using 2-photon laser scanning microscopy to provide a quantitative analysis of their volume. This approach should give better understanding of the cellular control of bone growth in both healthy and failed growth plates.

Bone cements produced by different manufacturers vary in their mechanical properties and antibiotic elution characteristics. Small changes in the formulation of a bone cement, which may not be apparent to surgeons, can also affect these properties. The supplier of Palacos bone cement with added gentamicin changed in 2005. We carried out a study to examine the mechanical characteristics and antibiotic elution of Schering-Plough Palacos, Heraeus Palacos and Depuy CMW Smartset bone cements.

Both Heraeus Palacos and Smartset bone cements performed significantly better than Schering-Plough Palacos in terms of mechanical characteristics, with and without additional vancomycin (p < 0.001). All cements show a deterioration in flexural strength with increasing addition of vancomycin, albeit staying above ISO minimum levels. Both Heraeus Palacos and Smartset elute significantly more gentamicin cumulatively than Schering-Plough Palacos. Smartset elutes significantly more vancomycin cumulatively than Heraeus Palacos.

The improved antibiotic elution characteristics of Smartset and Heraeus Palacos are not associated with a deterioration in mechanical properties. Although marketed as the ‘original’ Palacos, Heraeus Palacos has significantly altered mechanical and antibiotic elution characteristics compared with the most commonly-used previous version.

Curettage and packing with polymethylmethacrylate cement is a routine treatment for giant-cell tumour (GCT) of bone. We performed an in vitro evaluation of the cytotoxic effect of a combination of cement and methotrexate, doxorubicin and cisplatin on primary cell cultures of stromal GCT cells obtained from five patients. Cement cylinders containing four different concentrations of each drug were prepared, and the effect of the eluted drugs was examined at three different time intervals.

We found that the cytotoxic effect of eluted drugs depended on their concentration and the time interval, with even the lowest dose of each drug demonstrating an acceptable rate of cytotoxicity. Even in low doses, cytotoxic drugs mixed with polymethylmethacrylate cement could therefore be considered as effective local adjuvant treatment for GCTs.

Periosteum is important for bone homoeostasis through the release of bone morphogenetic proteins (BMPs) and their effect on osteoprogenitor cells. Smoking has an adverse effect on fracture healing and bone regeneration. The aim of this study was to evaluate the effect of smoking on the expression of the BMPs of human periosteum. Real-time polymerase chain reaction was performed for BMP-2,-4,-6,-7 gene expression in periosteal samples obtained from 45 fractured bones (19 smokers, 26 non-smokers) and 60 non-fractured bones (21 smokers, 39 non-smokers). A hierarchical model of BMP gene expression (BMP-2 > BMP-6 > BMP-4 > BMP-7) was demonstrated in all samples. When smokers and non-smokers were compared, a remarkable reduction in the gene expression of BMP-2, -4 and -6 was noticed in smokers. The comparison of fracture and non-fracture groups demonstrated a higher gene expression of BMP-2, -4 and -7 in the non-fracture samples. Within the subgroups (fracture and non-fracture), BMP gene expression in smokers was either lower but without statistical significance in the majority of BMPs, or similar to that in non-smokers with regard to BMP-4 in fracture and BMP-7 in non-fracture samples. In smokers, BMP gene expression of human periosteum was reduced, demonstrating the effect of smoking at the molecular level by reduction of mRNA transcription of periosteal BMPs. Among the BMPs studied, BMP-2 gene expression was significantly higher, highlighting its role in bone homoeostasis.

We isolated multilineage mesenchymal progenitor cells from haematomas collected from fracture sites. After the haematoma was manually removed from the fracture site it was cut into strips and cultured. Homogenous fibroblastic adherent cells were obtained. Flow cytometry revealed that the adherent cells were consistently positive for mesenchymal stem-cell-related markers CD29, CD44, CD105 and CD166, and were negative for the haemopoietic markers CD14, CD34, CD45 and CD133 similar to bone-marrow-derived mesenchymal stem cells. In the presence of lineage-specific induction factors the adherent cells could differentiate in vitro into osteogenic, chondrogenic and adipogenic cells.

Our results indicate that haematomas found at a fracture site contain multilineage mesenchymal progenitor cells and play an important role in bone healing. Our findings imply that to enhance healing the haematoma should not be removed from the fracture site during osteosynthesis.

Objectives

There is increasing application of bone morphogenetic proteins (BMPs) owing to their role in promoting fracture healing and bone fusion. However, an optimal delivery system has yet to be identified. The aims of this study were to synthesise bioactive BMP-2, combine it with a novel α-tricalcium phosphate/poly(D,L-lactide-co-glycolide) (α-TCP/PLGA) nanocomposite and study its release from the composite.

Allograft bone is widely used in orthopaedic surgery, but peri-operative infection of the graft remains a common and disastrous complication. The efficacy of systemic prophylactic antibiotics is unproven, and since the graft is avascular it is likely that levels of antibiotic in the graft are low.

Using an electrical potential to accelerate diffusion of antibiotics into allograft bone, high levels were achieved in specimens of both sheep and human allograft. In human bone these ranged from 187.1 mg/kg in endosteal (sd 15.7) to 124.6 (sd 46.2) in periosteal bone for gentamicin and 31.9 (sd 8.9) in endosteal and 2.9 (sd 1.1) in periosteal bone for flucloxacillin. The antibiotics remained active against bacteria in vitro after iontophoresis and continued to elute from the allograft for up to two weeks.

Structural allograft can be supplemented directly with antibiotics using iontophoresis. The technique is simple and inexpensive and offers a potential means of reducing the rate of peri-operative infection in allograft surgery. Iontophoresis into allograft bone may also be applicable to other therapeutic compounds.

The success of long-term transcutaneous implants depends on dermal attachment to prevent downgrowth of the epithelium and infection. Hydroxyapatite (HA) coatings and fibronectin (Fn) have independently been shown to regulate fibroblast activity and improve attachment. In an attempt to enhance this phenomenon we adsorbed Fn onto HA-coated substrates. Our study was designed to test the hypothesis that adsorption of Fn onto HA produces a surface that will increase the attachment of dermal fibroblasts better than HA alone or titanium alloy controls.

Iodinated Fn was used to investigate the durability of the protein coating and a bioassay using human dermal fibroblasts was performed to assess the effects of the coating on cell attachment. Cell attachment data were compared with those for HA alone and titanium alloy controls at one, four and 24 hours. Protein attachment peaked within one hour of incubation and the maximum binding efficiency was achieved with an initial droplet of 1000 ng. We showed that after 24 hours one-fifth of the initial Fn coating remained on the substrates, and this resulted in a significant, three-, four-, and sevenfold increase in dermal fibroblast attachment strength compared to uncoated controls at one, four and 24 hours, respectively.

It has been proposed that intervertebral disc degeneration might be caused by low-grade infection. The purpose of the present study was to assess the incidence of herpes viruses in intervertebral disc specimens from patients with lumbar disc herniation. A polymerase chain reaction based assay was applied to screen for the DNA of eight different herpes viruses in 16 patients and two controls. DNA of at least one herpes virus was detected in 13 specimens (81.25%). Herpes Simplex Virus type-1 (HSV-1) was the most frequently detected virus (56.25%), followed by Cytomegalovirus (CMV) (37.5%). In two patients, co-infection by both HSV-1 and CMV was detected. All samples, including the control specimens, were negative for Herpes Simplex Virus type-2, Varicella Zoster Virus, Epstein Barr Virus, Human Herpes Viruses 6, 7 and 8. The absence of an acute infection was confirmed both at the serological and mRNA level.

To our knowledge this is the first unequivocal evidence of the presence of herpes virus DNA in intervertebral disc specimens of patients with lumbar disc herniation suggesting the potential role of herpes viruses as a contributing factor to the pathogenesis of degenerative disc disease.

Various chemicals are commonly used as adjuvant treatment to surgery for giant-cell tumour (GCT) of bone. The comparative effect of these solutions on the cells of GCT is not known. In this study we evaluated the cytotoxic effect of sterile water, 95% ethanol, 5% phenol, 3% hydrogen peroxide (H₂O₂) and 50% zinc chloride (ZnCI₂) on GCT monolayer tumour cultures which were established from six patients. The DNA content, the metabolic activity and the viability of the cultured samples of tumour cells were assessed at various times up to 120 hours after their exposure to these solutions.

Equal cytotoxicity to the GCT monolayer culture was observed for 95% ethanol, 5% phenol, 3% H₂O₂ and 50% ZnCI₂. The treated samples showed significant reductions in DNA content and metabolic activity 24 hours after treatment and this was sustained for up to 120 hours. The samples treated with sterile water showed an initial decline in DNA content and viability 24 hours after treatment, but the surviving cells were viable and had proliferated. No multinucleated cell formation was seen in these cultures.

These results suggest that the use of chemical adjuvants other than water could help improve local control in the treatment of GCT of bone.

The aim of this study was to determine whether exposure of human articular cartilage to hyperosmotic saline (0.9%, 600 mOsm) reduces in situ chondrocyte death following a standardised mechanical injury produced by a scalpel cut compared with the same assault and exposure to normal saline (0.9%, 285 mOsm). Human cartilage explants were exposed to normal (control) and hyperosmotic 0.9% saline solutions for five minutes before the mechanical injury to allow in situ chondrocytes to respond to the altered osmotic environment, and incubated for a further 2.5 hours in the same solutions following the mechanical injury.

Using confocal laser scanning microscopy, we identified a sixfold (p = 0.04) decrease in chondrocyte death following mechanical injury in the superficial zone of human articular cartilage exposed to hyperosmotic saline compared with normal saline.

These data suggest that increasing the osmolarity of joint irrigation solutions used during open and arthroscopic articular surgery may reduce chondrocyte death from surgical injury and could promote integrative cartilage repair.

We used a goat model of a contaminated musculoskeletal defect to determine the effectiveness of rapidly-resorbing calcium-sulphate pellets containing amikacin to reduce the local bacterial count. Our findings showed that this treatment eradicated the bacteria quickly, performed as well as standard polymethylmethacrylate mixed with an antibiotic and had many advantages over the latter. The pellets were prepared before surgery and absorbed completely. They released all of the antibiotic and did not require a subsequent operation for their removal. Our study indicated that locally administered antibiotics reduced bacteria within the wound rapidly. This method of treatment may have an important role in decreasing the rate of infection in contaminated wounds.

We examined the usefulness of neutrophil CD64 expression in detecting local musculoskeletal infection and the impact of antibiotics on its expression. Of 141 patients suspected of musculoskeletal infection, 46 were confirmed by microbiological culture to be infected and 95 had infection excluded. The median CD64 count of patients with localised infection was 2230 molecules per cell (interquartile range (IQR) 918 to 4592) and that of the patients without infection was 937 molecules per cell (IQR 648 to 1309) (p < 0.001). The level of CD64 correlated with the CRP level in patients with infection, but not in those without infection (r = 0.59, p < 0.01). Receiver operator characteristic curve analysis revealed that CD64 was a good predictor of local infection. When the patients were subdivided into two groups based on the administration of antibiotics at the time of CD64 sampling, the sensitivity for detecting infection was better in those who had not received antibiotics.

These results suggest that measurement of CD64 expression is a useful marker for local musculoskeletal infection.