Limb salvage involving wide resection and reconstruction is now well established for managing musculoskeletal sarcomas. However, involvement of major nerves and vessels with a large volume of muscle and skin may result in a useless limb, contributing to depression and a low quality of life. We have been studying alternative treatments for musculoskeletal sarcoma since 1990, and have recently established a regime using photodynamic surgery with cells labelled with acridine orange, photodynamic therapy with cells treated similarly and radiodynamic treatment using the effect of X-rays on such cells.

These techniques have been used after marginal or intralesional resection of tumours since 1999 and have enabled maintenance of excellent limb function in patients with sarcomas.

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.

The belief that an intervertebral disc must degenerate before it can herniate has clinical and medicolegal significance, but lacks scientific validity. We hypothesised that tissue changes in herniated discs differ from those in discs that degenerate without herniation. Tissues were obtained at surgery from 21 herniated discs and 11 non-herniated discs of similar degeneration as assessed by the Pfirrmann grade. Thin sections were graded histologically, and certain features were quantified using immunofluorescence combined with confocal microscopy and image analysis. Herniated and degenerated tissues were compared separately for each tissue type: nucleus, inner annulus and outer annulus.

Herniated tissues showed significantly greater proteoglycan loss (outer annulus), neovascularisation (annulus), innervation (annulus), cellularity/inflammation (annulus) and expression of matrix-degrading enzymes (inner annulus) than degenerated discs. No significant differences were seen in the nucleus tissue from herniated and degenerated discs. Degenerative changes start in the nucleus, so it seems unlikely that advanced degeneration caused herniation in 21 of these 32 discs. On the contrary, specific changes in the annulus can be interpreted as the consequences of herniation, when disruption allows local swelling, proteoglycan loss, and the ingrowth of blood vessels, nerves and inflammatory cells.

In conclusion, it should not be assumed that degenerative changes always precede disc herniation.

Cite this article: Bone Joint J 2013;95-B:1127–33.

The October 2012 Research Roundup³⁶⁰ looks at: whether you can escape your genes; oral prophylaxis for DVT; non-responders and the internet; metal-on-metal, mice and damaged livers; sleeping on the job; cartilage contact stress in the normal human hip; and a perfect reason to subscribe to 360.

The April 2012 Research Roundup³⁶⁰ looks at who is capable of being an arthroscopist, bupivacaine, triamcinolone and chondrotoxicity, reducing scarring in injured skeletal muscle, horny Goat Weed and the repair of osseous defects, platelet-derived growth factor and fracture healing, the importance of the reserve zone in a child’s growth plate, coping with advanced arthritis, hydroxyapatite and platelet-rich plasma for bone defects, and calcium phosphate and bone regeneration

We have developed an animal model to examine the formation of heterotopic ossification using standardised muscular damage and implantation of a beta-tricalcium phosphate block into a hip capsulotomy wound in Wistar rats. The aim was to investigate how cells originating from drilled femoral canals and damaged muscles influence the formation of heterotopic bone. The femoral canal was either drilled or left untouched and a tricalcium phosphate block, immersed either in saline or a rhBMP-2 solution, was implanted. These implants were removed at three and 21 days after the operation and examined histologically, histomorphometrically and immunohistochemically.

Bone formation was seen in all implants in rhBMP-2-immersed, whereas in those immersed in saline the process was minimal, irrespective of drilling of the femoral canals. Bone mineralisation was somewhat greater in the absence of drilling with a mean mineralised volume to mean total volume of 18.2% (sd 4.5) versus 12.7% (sd 2.9, p < 0.019), respectively.

Our findings suggest that osteoinductive signalling is an early event in the formation of ectopic bone. If applicable to man the results indicate that careful tissue handling is more important than the prevention of the dissemination of bone cells in order to avoid heterotopic ossification.

Objectives

Effects of insulin-like growth factor 1 (IGF1), fibroblast growth factor 2 (FGF2) and bone morphogenetic protein 2 (BMP2) on the expression of genes involved in the proliferation and differentiation of osteoblasts in culture were analysed. The best sequence of growth factor addition that induces expansion of cells before their differentiation was sought.

We attempted to characterise the biological quality and regenerative potential of chondrocytes in osteochondritis dissecans (OCD). Dissected fragments from ten patients with OCD of the knee (mean age 27.8 years (16 to 49)) were harvested at arthroscopy. A sample of cartilage from the intercondylar notch was taken from the same joint and from the notch of ten patients with a traumatic cartilage defect (mean age 31.6 years (19 to 52)). Chondrocytes were extracted and subsequently cultured. Collagen types 1, 2, and 10 mRNA were quantified by polymerase chain reaction. Compared with the notch chondrocytes, cells from the dissecate expressed similar levels of collagen types 1 and 2 mRNA. The level of collagen type 10 message was 50 times lower after cell culture, indicating a loss of hypertrophic cells or genes. The high viability, retained capacity to differentiate and metabolic activity of the extracted cells suggests preservation of the intrinsic repair capability of these dissecates. Molecular analysis indicated a phenotypic modulation of the expanded dissecate chondrocytes towards a normal phenotype. Our findings suggest that cartilage taken from the dissecate can be reasonably used as a cell source for chondrocyte implantation procedures.

Objectives

Our objective in this article is to test the hypothesis that type 2 diabetes mellitus (T2DM) is a factor in the onset and progression of osteoarthritis, and to characterise the quality of the articular cartilage in an appropriate rat model.

Failure of bone repair is a challenging problem in the management of fractures. There is a limited supply of autologous bone grafts for treating nonunions, with associated morbidity after harvesting. There is need for a better source of cells for repair. Mesenchymal stem cells (MSCs) hold promise for healing of bone because of their capacity to differentiate into osteoblasts and their availability from a wide variety of sources. Our review aims to evaluate the available clinical evidence and recent progress in strategies which attempt to use autologous and heterologous MSCs in clinical practice, including genetically-modified MSCs and those grown on scaffolds. We have compared various procedures for isolating and expanding a sufficient number of MSCs for use in a clinical setting.

There are now a number of clinical studies which have shown that implantation of MSCs is an effective, safe and durable method for aiding the repair and regeneration of bone.

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

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

Tendinopathy is a debilitating musculoskeletal condition which can cause significant pain and lead to complete rupture of the tendon, which often requires surgical repair. Due in part to the large spectrum of tendon pathologies, these disorders continue to be a clinical challenge. Animal models are often used in this field of research as they offer an attractive framework to examine the cascade of processes that occur throughout both tendon pathology and repair. This review discusses the structural, mechanical, and biological changes that occur throughout tendon pathology in animal models, as well as strategies for the improvement of tendon healing.

Cite this article: Bone Joint Res 2014;3:193–202.

We investigated the clinical outcome of internal fixation for pathological fracture of the femur after primary excision of a soft-tissue sarcoma that had been treated with adjuvant radiotherapy.

A review of our database identified 22 radiation-induced fractures of the femur in 22 patients (seven men, 15 women). We noted the mechanism of injury, fracture pattern and any complications after internal fixation, including nonunion, hardware failure, secondary fracture or deep infection.

The mean age of the patients at primary excision of the tumour was 58.3 years (39 to 86). The mean time from primary excision to fracture was 73.2 months (2 to 195). The mean follow-up after fracture fixation was 65.9 months (12 to 205). Complications occurred in 19 patients (86%). Nonunion developed in 18 patients (82%), of whom 11 had a radiological nonunion at 12 months, five a nonunion and hardware failure and two an infected nonunion. One patient developed a second radiation-associated fracture of the femur after internal fixation and union of the initial fracture. A total of 13 patients (59%) underwent 24 revision operations.

Internal fixation of a pathological fracture of the femur after radiotherapy for a soft-tissue sarcoma has an extremely high rate of complication and requires specialist attention.

Cite this article: Bone Joint J 2013;95-B:1144–8.

The aim of this study was to investigate the occurrence of tissue hypoxia and apoptosis at different stages of tendinopathy and tears of the rotator cuff.

We studied tissue from 24 patients with eight graded stages of either impingement (mild, moderate and severe) or tears of the rotator cuff (partial, small, medium, large and massive) and three controls. Biopsies were analysed using three immunohistochemical techniques, namely antibodies against HIF-1α (a transcription factor produced in a hypoxic environment), BNip3 (a HIF-1α regulated pro-apoptotic protein) and TUNEL (detecting DNA fragmentation in apoptosis).

The HIF-1α expression was greatest in mild impingement and in partial, small, medium and large tears. BNip3 expression increased significantly in partial, small, medium and large tears but was reduced in massive tears. Apoptosis was increased in small, medium, large and massive tears but not in partial tears.

These findings reveal evidence of hypoxic damage throughout the spectrum of pathology of the rotator cuff which may contribute to loss of cells by apoptosis. This provides a novel insight into the causes of degeneration of the rotator cuff and highlights possible options for treatment.

Our understanding of the origin of hip pain in degenerative disorders of the hip, including primary osteoarthritis, avascular necrosis and femoroacetabular impingement (FAI), is limited. We undertook a histological investigation of the nociceptive innervation of the acetabular labrum, ligamentum teres and capsule of the hip, in order to prove pain- and proprioceptive-associated marker expression. These structures were isolated from 57 patients who had undergone elective hip surgery (44 labral samples, 33 ligamentum teres specimens, 34 capsular samples; in 19 patients all three structures were harvested). A total of 15 000 histological sections were prepared that were investigated immunohistochemically for the presence of protein S-100, 68 kDa neurofilament, neuropeptide Y, nociceptin and substance P. The tissues were evaluated in six representative areas.

Within the labrum, pain-associated free nerve ending expression was located predominantly at its base, decreasing in the periphery. In contrast, the distribution within the ligamentum teres showed a high local concentration in the centre. The hip capsule had an almost homogeneous marker expression in all investigated areas.

This study showed characteristic distribution profiles of nociceptive and pain-related nerve fibres, which may help in understanding the origin of hip pain.

Cite this article: Bone Joint J 2013;95-B:770–6.

Mesenchymal stem-cell based therapies have been proposed as novel treatments for intervertebral disc degeneration, a prevalent and disabling condition associated with back pain. The development of these treatment strategies, however, has been hindered by the incomplete understanding of the human nucleus pulposus phenotype and by an inaccurate interpretation and translation of animal to human research. This review summarises recent work characterising the nucleus pulposus phenotype in different animal models and in humans and integrates their findings with the anatomical and physiological differences between these species. Understanding this phenotype is paramount to guarantee that implanted cells restore the native functions of the intervertebral disc.

Cite this article: Bone Joint Res 2013;2:169–78.

Objectives

Lower limb muscle power is thought to influence outcome following total knee replacement (TKR). Post-operative deficits in muscle strength are commonly reported, although not explained. We hypothesised that post-operative recovery of lower limb muscle power would be influenced by the number of satellite cells in the quadriceps muscle at time of surgery.

Heterotopic ossification (HO) is perhaps the single most significant obstacle to independence, functional mobility, and return to duty for combat-injured veterans of Operation Enduring Freedom and Operation Iraqi Freedom. Recent research into the cause(s) of HO has been driven by a markedly higher prevalence seen in these wounded warriors than encountered in previous wars or following civilian trauma. To that end, research in both civilian and military laboratories continues to shed light onto the complex mechanisms behind HO formation, including systemic and wound specific factors, cell lineage, and neurogenic inflammation. Of particular interest, non-invasive in vivo testing using Raman spectroscopy may become a feasible modality for early detection, and a wound-specific model designed to detect the early gene transcript signatures associated with HO is being tested. Through a combined effort, the goals of early detection, risk stratification, and development of novel systemic and local prophylaxis may soon be attainable.

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.

Conventional amputation prostheses rely on the attachment of the socket to the stump, which may lead to soft-tissue complications. Intraosseous transcutaneous amputation prostheses (ITAPs) allow direct loading of the skeleton, but their success is limited by infection resulting from breaching of the skin at the interface with the implant. Keratinocytes provide the skin’s primary barrier function, while hemidesmosomes mediate their attachment to natural ITAP analogues. Keratinocytes must attach directly to the surface of the implant. We have assessed the proliferation, morphology and attachment of keratinocytes to four titaniumalloy surfaces in order to determine the optimal topography in vitro. We used immunolocalisation of adhesion complex components, scanning electron microscopy and transmission electron microscopy to assess cell parameters.

We have shown that the proliferation, morphology and attachment of keratinocytes are affected by the surface topography of the biomaterials used to support their growth. Smoother surfaces improved adhesion. We postulate that a smooth topography at the point of epithelium-ITAP contact could increase attachment in vivo, producing an effective barrier of infection.