Extracorporal shock wave therapy (ESWT) seems to be a promising new tool for the treatment of chronic pain due to tendinopathies such as tennis elbow or a painful heel. Mechanisms of ESWT-induced analgesia are still unknown. One major system for controlling pain is the endogenous opioid system that could be the biochemical basis of the ESWT-effects. The aim of the study was to investigate the possible influence of low energy ESWT on the endogenous opioid-system in the lumbar spinal cord of the rat. Immunohistochemical analysis of the expression of opioids Met-Enkephalin (MRGL), and dynorphin (Dyn) were performed in rats treated either once with 1000 impulses or three times with 1000 impulses with two different energy flux densities each (0.04 and 0.11 mJ/mm. 2. ) at 4 or 72 h after ESWT. No different immunoreactivity of MRGL and Dyn was seen after single ESWT treatment in comparison with the sham group. This result was not influenced by different energy flux doses or repetitive ESWT treatment. Met-Enk and Dyn expression was similar on ipsi- and contralateral side and was unchanged at later time points after ESWT treatment. Low energy ESWT had no influence on the opioid-systems and therefore does not trigger this endogenous anti-nociceptive system under basal conditions. Furthermore these results show that low energy ESWT had no side effects on rat spinal cord (e.g. neuronal destruction or enhanced permeability of the blood brain barrier for leukocytes) even after the application of 3 x 1000 impulses with the energy flux density as high as 0.11 mJ/mm. 2. Although applications in orthopaedics have outnumbered those in urology, there is no firm evidence of efficacy of ESWT in orthopaedics from well-designed randomised clinical trials and the molecular mechanisms of the of the anti-nociceptive effect of ESWT are still unknown

Frozen Shoulder (FS) is a debilitating musculoskeletal condition with an uncertain aetiology and poorly understood pathogenic mechanism. This study aimed to investigate the pathology of FS. We hypothesised that an altered expression of cytokines may disrupt the normal tissue remodeling process, leading to FS, which would be apparent histologically.

Patients undergoing arthroscopic treatment of FS were prospectively recruited, along with control patients being treated for subacromial impingement. Synovial biopsies were taken from all subjects. Synovial RNA levels were analysed using quantitative Polymerase Chain Reaction (qPCR). Inflammatory cytokines and growth factors thought to play a role in the pathogenesis of FS were assessed. These included metalloproteases (MMP, ADAMTS) involved in tissue remodeling and fibrosis, inflammatory cytokines such as interleukins (IL), and growth factors such as colony stimulating factors (MCSF, GMCSF, CSF1R). Samples underwent histological analysis, to assess inflammation and fibrosis.

Thirteen patients with FS and ten control patients with subacromial impingement were recruited. Arthroscopic inspection revealed greater levels of synovitis (2.63+ vs 0.40+, p< 0.01) and papillary proliferation (50% vs 10%, p=0.02) in FS patients compared with the control group, confirming the initial clinical diagnosis of FS. Histological analysis of the synovium revealed samples from the FS group were more likely to demonstrate a fibrotic, focally nodular collagen morphology (53.8% vs 10%, p=0.03). There were similar levels of chronic inflammatory cells present in those with FS and control patients (53.8% vs 30%, p=0.25). There was no evidence of acute inflammation in any of the samples. Immunohistochemical staining revealed a high level of AGEs present in the synovium and smooth muscle tissue in all samples. There was no observed difference between diabetic and non-diabetic samples. Cytogenetic analysis using qPCR revealed fibrogenic factors MMP3 (p=0.068), and ADAMTS4 (p=0.083) to be elevated in FS cases, as were inflammatory cytokines IL6 (p=0.062) and IL8 (p=0.075)

We have quantified the level of inflammatory cytokines and growth factors in FS, demonstrating that these factors are elevated in FS. This indicates that altered levels of inflammatory cytokines may be associated with the pathogenesis of inflammation evolving into fibrosis, the characteristic feature of FS. We have also shown the histology of this fibrosis to be different to that observed in normal synovium.

Objectives

This study aimed to investigate time-dependent gene expression of injured human anterior cruciate ligament (ACL), and to evaluate the histological changes of the ACL remnant in terms of cellular characterisation.

The poor prognosis of patients with soft-tissue sarcoma as not changed in the past several decades, highlighting the necessity for new therapeutic approaches. T-cell based immunotherapies are a promising alternative to traditional cancer treatments due to their ability to target only malignant cells, leaving benign cells unharmed. The development of successful immunotherapy requires the identification and characterization of targetable immunogenic tumor antigens. Cancer-testis antigens (CTA) are a group of highly immunogenic tumor-associated proteins that have emerged as potential targets for CD8+ T-cell recognition. In addition to identifying a targetable antigen, it is crucial to understand the tumor immune microenvironment. The level of immune infiltration and mechanisms of immune suppression within the tumor play important roles in the outcome of immunotherapy. The goal of this study is to identify targetable immunogenic antigens for T-cell based immunotherapy and to characterize the tumor immune microenvironment in human dedifferentiated liposarcoma (DDLS) by Nanostring and IHC. To assess the complexity of the human DDLS tumor immune microenvironment and to identify target antigens we used the nCounter NanoString platform to generate a gene expression profile for hundreds of genes from RNA obtained from 29 DDLS and 10 control fat FFPE samples. To classify inflammatory status of DDLS tumors, we performed hierarchical clustering based on expression levels of selected tumor inflammatory signature genes (CCL5, CD27, CD274, CD276, CD8A, CMKLR1, CXCL9, CXCR6, HLA-DQA1, HLA-E, IDO1, LAG3, PDCDILG2, PSMB10, STAT1, TIGIT). To confirm protein expression and distribution of identified antigens, we performed immunohistochemistry on human tissue micro-arrays encompassing DDLPS tumor tissues and matched normal control tissue from 63 patients. IHC for the cancer testis antigens PBK, SPA17, MAGE-A3, NY-ESO-1 and SSX2 was performed, and the staining results were scored by two authors based on maximal staining intensity on a scale of zero to three (absent=0, weak=1, moderate=2, or strong=3) and the percentage of tumor cells that stained. Hierarchical clustering of DDLS tumors based on expression of tumor inflammation signature genes revealed two distinct groups, consisting of 15 inflamed tumor and 14 non-inflamed tumors, demonstrating tumor heterogeneity within the DDLS sarcoma subtype. All antigens were found to be expressed in DDLS at an mRNA level. SPA17 was expressed at the highest levels in DDLS, however, this antigen was expressed at high levels in normal fat. Notably, antigens PBK and TTK had the largest fold change increase in expression in DDLS compared to normal fat controls. Immunohistochemical analysis of selected antigens revealed that PBK was found to be expressed in 96% (52/54) of DDLS samples at high levels. Other antigens were absent or expressed at low levels in DDLS; MAGEA3 in 15.87% (10/63) NY-ESO-1 in 6.35% (4/62) and SSX2 in 12.7% (8/63) and SPA17 in 5.5% (3/54). This data shows considerable inter-tumoral heterogeneity of inflammation, which should be taken into consideration when designing an immunotherapy for DDLS. To date, these results show promising expression of PBK antigen in DDLS, which may be used as a target in the future development of an immunotherapy for sarcoma

Cartilage neoangiogenesis holds a key role in the development of osteoarthritis (OA) by promoting cartilage degradation with proteoglycan loss, subchondral bone sclerosis, osteophyte formation and synovial hyperplasia. This study aimed to assess the in vivo efficacy of bevacizumab, an antibody against vascular endothelial growth factor (VEGF) in an OA animal model. 24 New Zealand white rabbits underwent anterior cruciate ligament transection in order to spontaneously develop knee OA. Animals were divided into four groups: one receiving a sham intraarticular knee injection (saline) and three groups treated with 5, 10, and 20 mg intraarticular bevacizumab injections. The biological effect of the antibody on cartilage and synovium was evaluated through histology and quantified with the Osteoarthritis Research Society International (OARSI) scores. Immunohistochemical analysis was conducted to investigate type 2 collagen, aggrecan, and matrix metalloproteinase 13 (MMP-13) expression in both cartilage and synovium. Intraarticular bevacizumab led to a significant reduction of cartilage degeneration and synovial OA alterations. Immunohistochemistry showed a significantly reduced MMP-13 expression in all experimental groups, with the one receiving 20 mg bevacizumab showing the lowest. Furthermore, the antibody showed to increment the production of aggrecan and type 2 collagen after administration of 5, 10, and 20 mg. The group treated with 20 mg showed the highest levels of type 2 collagen expression, while aggrecan content was even higher than in the healthy cartilage. Intraarticular bevacizumab has demonstrated to effectively arrest OA progression in our model, with 20 mg being the most efficacious dose. By inhibiting cartilage and synovial neoangiogenesis, bevacizumab may serve as a possible disease-modifying osteoarthritis drug (DMOAD) in the next future

The poor prognosis of patients with advanced bone and soft-tissue sarcoma has highlighted the necessity for new therapeutic approaches. T-cell based immunotherapies are a promising alternative to traditional cancer treatments due to their ability to target only malignant cells, leaving benign cells unharmed. The development of successful immunotherapy requires the identification of targetable immunogenic tumor antigens. Cancer-testis antigens (CTA) are a group of highly immunogenic tumor-associated proteins that have emerged as potential targets for CD8+ T-cell recognition. The goal of this study is to screen for CTA expression, HLA expression, and tumor T-cell infiltration in human dedifferentiated liposarcoma (DDLPS) and osteosarcoma (OS) to establish their immune profile and to identify targetable immunogenic antigens for T-cell based immunotherapy. Human tissue micro-arrays composed of 78 cores of OS and 50 cores of DDLPS were obtained, along with matched control tissues from the same patients. IHC for the cancer testis antigens NY-ESO-1, MAGE-A3, and SSX2, was performed, and the staining results were scored by two authors based on maximal staining intensity on a scale of zero to three (absent=0, weak=1, moderate=2, or strong=3) and the percentage of tumor cells that stained. IHC for CD8 and CD3 was also performed, and T-cell tumor infiltration was defined as either brisk, nonbrisk, or absent, as described in melanoma literature. Concurrently, evaluation of 38 human DDLPS specimens and 10 healthy human fat specimens by the Nanostring nCounter platform is underway for identification of novel antigen targets and to establish the immune profile of DDLPS. Immunohistochemical analysis of CTA expression showed considerable inter- and intra-tumoral heterogeneity. DDLPS showed relatively low expression of all CTAs tested, with only 22% of samples exhibiting MAGE-A3 and one sample each (3.1%) showing expression of SSX2 and NY-ESO-1 in low percentages of tumor cells. By contrast, in osteosarcoma, 74% of samples expressed MAGE-A3 and 68% expressed SSX, both with >80% of positive cases showing moderate to high expression. NY-ESO-1 was expressed in 41% of OS samples, predominantly at low levels. Brisk infiltration of CD8+ T cells was observed in over 70% of both sarcoma types tested. Furthermore, all sarcoma samples tested were positive for HLA expression. To date, these results show promising expression of CTAs MAGE-A3 and SSX in OS, which may be used as targets in the future development of an immunotherapy for sarcoma. DDLPS shows relatively low expression, highlighting the need for more exploratory study with NanoString. The data generated throughout this project will provide insight into the immune profile of DDLPS

Surgical failure, mainly caused by loosening implants, causes great mental and physical trauma to patients. Improving the physicochemical properties of implants to achieve favourable osseointegration will continue to be the focus of future research. Strontium (Sr), a trace element, is often incorporated into hydroxyapatite (HA) to improve its osteogenic activity. Our previous studies have shown that miR-21 can promote the osteogenic differentiation of mesenchymal stem cells by the PI3K/β-catenin pathway. The aim of this study is to fabricate a SrHA and miR-21 composite coating and it is expected to have a favorable bone healing capability. Ti discs (20 mm diameter and one mm thickness for the in vitro section) and rods (four mm diameter and seven mm length for the in vivo section) were prepared by machining pure Ti. The Ti cylinders were placed in a Teflon-lined stainless-steel autoclave for treating at 150°C for 24 h to form SrHA layer. The miR-21 was encapsulated in nanocapsules. The miR-21 nanocapsules were mixed with CMCS powder to form a gel-like sample and uniformly coated on the SrHA modifed Ti. Osteoblast-like MG63 cells were cultured on SrHA and miR-21 modified Ti, Cell proliferation activity and osteogenesis-related gene expression were evaluated. A bone defect model was established with mature New Zealand to evaluate the osseointegration. Cylindrical holes (four mm in diameter) were created at the distal femur and tibial plateau. Each rabbit was implanted with four of the aforementioned rods (distal femur and tibial plateau of the hind legs). After implantation for one, two and three months, the rabbits were observed by X-ray and scanned using u-CT. Histological and Immunohistochemical analysis were performed to examine the osteogenic markers. A biomechanical push-in test was used to assess the bone-implant bonding strength. Both SrHA nanoparticles with good superhydrophilicity and miR-21 nanocapsules with uniform sizes were distributed evenly on the surface of the Ti. In vitro experiments revealed that the composite coating was beneficial to osteoblast proliferation, differentiation and mineralization. In vivo evaluations demonstrated that this coating could not only promote the expression of angiogenic factor CD31 but also enhance the expression of osteoblastic genes to facilitate angio-osteogenesis. In addition, the composite coating also showed a decreased RANKL expression compared with the miR-21 coating. As a result, the SrHA/miR-21 composite coating promoted new bone formation and mineralization and thus enhanced osseointegration and bone-implant bonding strength. A homogeneous SrHA and miR-21 composite coating was fabricated by generating pure Ti through a hydrothermal process, followed by adhering miR-21 nanocapsules. This coating combined the favorable physicochemical properties of SrHA and miR-21 that synergistically promoted angiogenesis, osteogenesis, osseointegration, bone mineralization and thus bone-implant bonding strength. This study provided a new strategy for surface modification of biomedical implants

The purpose of this study is to identify clinical and radiographic results of 78 uncemented total hip arthroplasties using Metasul. ®. metal on metal bearings with Wagner standard cup and proximal hydroxyapatite coated CLS stem. Mean age was 39 years and average follow-up period was 11.7 years. Mean Harris hip score had improved from 51.4 points preoperatively to 95.2 points finally. There were 2 hips with progressive osteolysis around the acetabular cup. Of them, one hip was revised due to loosening of the cup, and the other was observed because of patient’s refusal to revise. In histopathologic findings on osteolytic area, a lot of macrophage phagocytizing metal debris and perivascular lymphocyte infiltration were found. Immunohistochemical analysis suggested delayed metal hypersensitivity. Serum cobalt levels in hips with osteolysis were not higher than those in hips without osteolysis. Early osteolysis with sudden onset of groin pain in few hips remains a concern

In the past, the clinical outcome of earlier types of resurfacing hip arthroplasty was often characterised by a high percentage of failures and early mobilisations. An implant retrieval of a Co-Cr head and UHMWPE cup cemented resurfacing hip prosthesis was analysed. The implant was in place 11 years, without any clinical problem for nearly 10 years. The cup was highly worn. There was a complete fracture of the interface between cement and bone at the base of the femural neck. A significant hyperplastic reaction was present at the level of the synovial membrane, with fibrin deposits, hyperplasia of lining cells and a cellular infiltrate formed mostly by macrophages, with occasional giant cells and localised groups of perivascular lymphocytes. Immunohistochemical analysis showed that all lymphocytes were of the T type and that the largest part of macrophages containing debris were not activated. Inside the prosthetic head there were only traces of ossified tissue. This picture indicated that for a long time no viable bone tissue had been in contact with the cement and bone rarefaction was massive. The study shows an important cause of the failure of earlier types of hip resurfacing arthroplasty, namely the abnormal stress distribution that caused the complete bone rarefaction

Purpose: To establish and validate animal model for Achilles tendon disease with subsequent examination of histology, biochemistry and biomechanics. Methods: Experimental rats were subjected to an over-exercise running regime. Achilles tendons were analyzed for histology, glycosaminoglycan content, collagen content, collagen subtype, collagen cross-linking (hydrothermal isometric tension testing), and mechanical properties. Results: Experimental rat Achilles tendons demonstrated: decreased semi-quantitative grade for collagen organization (2.9 vs. 3.7, p < 0.05), decreased semi-quantitative grade for collagen staining (1.9 vs 3.5, p < 0.05) and increased nuclear numbers per high-power field (527 vs. 392, p < 0.05). Immunohistochemical analysis revealed a predominance of by fibroblasts or endothelial cells. The total collagen content remained unchanged (84.3 vs. 89.0% p=0.38), while the glycosaminoglycan content was increased (17.5 vs. 9.0% p=0.02). Differences in collagen cross-linking were characterized by a greater proportion of reducible intrahelical crosslinks. These differences did not translate into a decrease in ultimate tensile failure during mechanical testing (UTS of 77.8 vs. 88.8 N, p=0.26). Conclusions: Over-exercise model produced Achilles tendons with histology and biochemistry consistent with the animal and human tendon disease and characteristic of a remodeling response not an inflammatory response

The study describes the changes of condrocytes and extracellular matrix occurring in Hip OA. 16 femoral heads were included in the study. Cartilage explants were removed from 3 anatomical sites over the surface of 14 OA and 2 non-OA patients. Cartilage sections were evaluated with histological (EE, Alcian Blu and Mallory-Azan stainings) and immuno-histochemichal (antibodies directed against fibronectin, tenascin, laminin, type I and type IV collagen, metallo-proteinase-1,-2,-7 and -7) analysis. Histological analysis of cartilage of central and per-hipheral biopsies from patients with severe OA showed significant reduced number of chondrocytes in both superficial and middle zones. In the lower cartilage layer with severe structural lesions a cospicous number of cartilagineous repair-islands were noticed. Immunohistochemical analysis showed high levels of tenascin in all cartilage layers of byopses showing structural damages. Frequently we observed an altered distribution of fibronectin. Metalloproteinase-2 (constitutive) is present in all stages during coxarthritis. Metalloproteinase-9 (not constitutive) is expressed at the final stages suggesting an important late role. Obtained results show that metalloproteinases have a peculiar behaviour during coxarthritis vs. other pathologies. Costitutive metal-loproteinases have a fundamental role in extracellular matrix remodelling, MMP-2 especially

Aims

The antidiabetic agent metformin inhibits fibrosis in various organs. This study aims to elucidate the effects of hyperglycaemia and metformin on knee joint capsule fibrosis in mice.

Study Design: Experimental in vivo study on New Zealand White Rabbits. Summary of Background Data: We have developed an in-vivo rabbit model of lumbar disc degeneration. This model provides a defined loading of one single disc. However, the molecular mechanism that leads to mechanically-induced disc degeneration remains unclear. Objective: To investigate the process of mechanically induced disc degeneration in New Zealand White Rabbits with respect to remodeling on the gene and the level of protein expression. Subjects: Seven animals were treated with an external compression-device applying 200N on segment L3/4. Eight animals underwent sham surgery. Outcome Measures: After 28 days discs were harvested and cut into two pieces in a sagittal plain. One piece was used for protein analysis utilizing immunohistochemical protocols for collagen I, II and aggrecan. The other half of the disc was used for quantitative real-time RT-PCR to determine gene expression of selected matrix genes. Results: In the compression group matrix genes were upregulated: collagen I (6.46x; p=0,018), collagen II (2.14x), biglycan (2.97x; p=0,049), decorin (4.64x; p=0,043), aggrecan (1.2x), osteonectin (2.03x), fibronectin (3.48x), fibromodulin (2.6x; p=0,037). The MMP-13 gene could only be detected in compressed discs. Gene transcripts of the metalloproteinase-inhibitor TIMP-1 were 4.5 times upregulated (p=0,007). Immunohistochemical analysis revealed a decrease of aggrecan and collagen I. Conclusions: In our animal model mechanical loading caused degradation of the matrix proteins collagen I and aggrecan. Metalloproteinases like MMP-13 trigger this degenerative process. The elevated expression of matrix genes and TIMP-1 transcripts may characterize a mechanism of compensation

Excessive apoptosis has been found in torn supraspinatus tendon1 and mechanically loaded tendon cells2. Following oxidative and other forms of stress, one family of proteins that is often unregulated are Heat Shock Proteins (HSPs). The purpose of this study was to determine if HSPs were unregulated in human and rat models of tendinopathy and to determine if this was associated with increased expression of regulators of apoptosis (cFLIP, Caspases 3& 8). A running rat supraspinatus tendinopathy overuse model 3 was used with custom microarrays consisting of 5760 rat oligonucleotides in duplicate. Seventeen torn supraspinatus tendon and matched intact subscapularis tendon samples were collected from patients undergoing arthroscopic shoulder surgery. Control samples of subscapularis tendon were collected from ten patients undergoing arthroscopic stabilisation surgery and evaluated using semiquantative RT-PCR and immunohistochemistry. Rat Microarray: Upregulation of HSP 27 (×3.4) & 70 (×2.5) and cFLIP (×2.2) receptor was noted in degenerative rat supraspinatus tendon subjected to daily treadmill running for 14 days compared to tendons of animals subject to cage activity only. Histological analysis: All torn human supraspinatus tendons exhibited changes consistent with marked tendinopathy. Matched subscapularis tendon showed appearances of moderate-advanced degenerative change. Apoptosis mRNA expression: The expression levels of caspase 3 & 8 and HSPs 27 & 70 were significantly higher in the torn edges of supraspinatus when compared to matched subscapularis tendon and control tendon (p< 0.01). cFLIP showed significantly greater (p< 0.001) expression in matched subscapularis compared to supraspinatus and control tendon. Immunohistochemical analysis: cFLIP, Caspase 3 & 8 and HSP 27 and 70 was confirmed in all samples of torn supraspinatus tendon. Significantly increased immunoactivity of Caspase 3& 8 and HSP 27 & 70 were found in torn supraspinatus (p< 0.001) compared to matched and normal subscapularis. The proteins were localized to tendon cells. The finding of significantly increased levels of Heat Shock Proteins in human and rat models of tendinopathy with the co-expression of other regulators of apoptosis suggests that Heat Shock Proteins play a role in the cascade of stress activated-programmed cell death and degeneration in tendinopathy

Aim: The purpose of this study was to evaluate the cytokine molecules present in a rat tendinopathy model and in the torn edge of human rotator cuff tendon in an attempt to understand their role in tendon degeneration. Methods: A rat tendon overuse model was used with custom microarrays consisting of 5760 rat oligonucleotide features in duplicate. Seventeen torn supraspinatus tendon and matched intact subscapularis tendon samples were collected from patients undergoing arthroscopic shoulder surgery.Control samples of subscapularis tendon were collected from ten patients undergoing arthroscopic stabilisation surgery.Specimens were analysed for the presence of interleukins 18, 15, 12, 11, 6, 2, macrophage inhibitory factor (MIF), and tumour necrosis factor ƒÑ by semiquantitative RT-PCR and immunohistochemistry. Tendinopathy was assessed on a basic histological scale. Results: Rat Microarray analysis: Upregulation of IL-6, IL-11 and IL18 receptor was noted in the degenerated rat supraspinatus tendon. Downregulation of IL-2 was noted. No other cytokine signal was expressed. Histological analysis: All torn human supraspinatus tendons changes consistent with marked tendinopathy. Matched subscapularis tendon showed appearances of moderate-advanced degenerative change. Cytokine mRNA expression: TNF-£\ mRNA expression was found to be significantly elevated (p< 0.01) in subscapularis tendon compared to torn supraspinatus samples. The expression levels of IL-18, IL-15, IL-6 and MIF was significantly higher in the torn edges of supraspinatus when compared to matched subscapularis tendon and normal control tendon (p< 0.001). Immunohistochemical analysis: Presence of IL-18, IL-15, Il-6, MIF and TNF-£\ was confirmed in all samples of torn supraspinatus tendon. Significantly increased levels of IL-18, IL-15, IL-6 and MIF were found in torn supraspinatus. (p< 0.01) compared to matched and normal subscapularis. Conclusions: Cytokines have been shown to promote the intensive production of reactive O2 metabolites . 1. and are potent agonists of protein kinases . 2. Our finding of significantly increased cytokine levels may suggest that these molecules when expressed during the degenerate and healing phases of tendon injury result in the subsequent production of reactive O2 species and protein kinases. 3. causing tendon damage or failure of the normal reparative process. Our finding of marked tendinopathy in matched subscapularis tendon may also provide a useful human tendinopathy model

Purpose/introduction: 80% of individuals experience low back pain in their lifetime. This is often due to disc injury or degeneration. Conservative treatment of discogenic pain is often unsuccessful whilst surgery with the use of spacers of fusion is non-physiological. The aim of this study was to develop an animal model to assess the viability of autologous disc cell therapy. Method: The Fat Sand Rat (Psammomys obesus obesus) was chosen due to its predisposition to the early development of spondylosis. Using microsurgical techniques fragments of annulus and nucleus were harvested from a single disc in 52 sand rats. Vascular clips were placed on the adjacent psoas muscle to mark the harvested level. Disc material was initially cultured in monolayer then transferred into a three dimensional culture media of agarose. This technique yields greater cellular proliferation and the development of cell growth in colonies. Cells were labelled with Bromodeoxyuridine for later immunohistochemical identification. 20 000 cells in a carrier media were then re-implanted at a second operation at an adjacent disc level in the same animal. The rat was subsequently euthanised and the histology of the disc space reviewed. Results: To date 52 primary disc harvests and 20 reimplantations have been performed. 15 rats have been euthanised and sectioned. Average age at primary surgery was 6.8 months reimplantation eight months and euthanisation 11.2 months. Cell colony viability was inversely related to rat age at harvest. Immunohistochemical analysis of colony extracellular matrix revealed production of type 1 and 2 collagen, chondroitin and keratin sulphate Two rats died prior to reimplantation. All histological specimens confirm the presence of viable transplanted disc cells. Transplanted cells did not alter the progression of degenerative changes on x-ray. Conclusion: Autologous disc cell transplantation can be performed in the rat. Further modification of these techniques may lead to the development of autologous disc cell therapy comparable to that currently successfully used in hyaline cartilage defects of synovial joints in humans

Introduction Osteochondral lesions of the talus are a common occurrence especially in sports injuries. The biomechanical nature of the ankle joint makes it susceptible to sprains which can cause damage not only to the capsulo-ligamentous structures, but also to the joint cartilage and subchondral bone. As it is known, joint cartilage is a highly specialized and multitask tissue. Because joint cartilage has poor reparative capability, damage may be irreversible and as a consequence, can also lead to osteoarthritis. The purpose of this study is to review the results of a series of patients treated with autologous chondrocytes implantation (A.C.I.) and to describe the evolution in surgical technique that we have been implemented in the last 8 years. Methods Thirty-nine patients with a mean age of 27 8 years affected by osteochondral lesions of the talus > 1.5 cm2, were treated by autologous chondrocyte implantation. All patients were checked clinically and by MRI up to 4 years follow-up. The first 9 patients received the ACI by open technique and the remaining 30, arthroscopically. In the last 10 patients the cartilage harvested from the detached osteochondral fragment was used for the colture. All patients were checked clinically (AOFAS score), radiographically and by MRI, before surgery, at 12 months and at follow-up. Eleven patients underwent a second arthroscopy with a bioptic cartilage harvest at 1 year follow-up. Samples were stained with Safranin-O and Alcian Blue. Immunohistochemical analysis for collagen type II was also performed. Results Before surgery the mean score was 48.4 17 points, at 12 months 90.9 12 (p< 0.0005), while at follow up was 93.8 8 (p< 0.0005) demonstrating an improvement over time. The histological and immunohistological analyses performed on the cartilage samples using Safranin-O, Alcian Blue staining and anti-human collagen type II antibody respectively showed a typical cartilage morphology, were positive for collagen type II and for proteoglycans expression. Conclusions The clinical and histological results have confirmed the validity of the technique utilized, with laboratory data confirming the newly formed cartilage was of hyaline type for all the cases evaluated

Lesions to articular cartilage have a poor capability of regeneration and by mechanical wear and enzymatic digestion they may progress to osteoarthritis. In Sweden more than 900 patients with chondral or osteochondral lesions have been treated with autologous chondrocyte transplantation (ACT) since 1987. Cartilage is harvested arthroscopically and the chondrocytes are isolated. After two weeks of culturing the chondrocytes are deposited in the cartilage lesion in a cell suspension. The chondrocytes start to produce matrix and gradually form new hyaline cartilage able to withstand the forces of the knee. Lesions to the femoral condyles have shown the most promising results when treated with ACT (90% Good/Excellent, n=57), osteochonditis dissecans showed 84% Good/Excellent results (n=32), multiple knee joint lesions 75% Good/Excellent (n=53) and femoral condyle lesions with anterior cruciate ligament reconstruction 74% Good/Excellent (n=−27) at a long term follow up (2–11 years). The outcome after patella lesions treated with ACT were initially not as good (2 of 7 patients were graded Good or Excellent at a mean follow-up of 36 months) but better understanding of the nature of patellar lesions and development of the surgical technique have improved the result (65% Good or Excellent, n=32). Patients treated with ACT for cartilage lesion to the femoral trochlea showed Good/Excellent results in 58% (n=12). At a second look arthroscopy biopsies were taken in 37 patients. In 80% of the biopsies the repair tissue was classified as hyaline like cartilage. Immunohistochemical analysis of collagen II, aggrecan and comp showed ++ to +++ for the hyaline like repair tissue compared to +++ for normal cartilage. There were also a strong correlation (0.73) between hyaline like repair and Good/ Excellent results. Other areas have been transplanted as well such as the tibial plateau, the talus and the head of the humerus, but due to the small numbers of patients and short follow-up ACT to these areas is not yet recommended. The clinical outcome after treating chondral and osteochondral lesions in the knee is good at a long term follow-up and the repair tissue is histological similar to normal articular cartilage

Introduction: Apoptosis, or secondary cell death, has been demonstrated in a number of neurological conditions, including Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis and brain ischaemia. It is well established from studies of acute spinal cord injury that apoptosis seems an important factor in secondary cell death and irreversible neurological deficit. It is only recently that studies have emerged analysing secondary cell death in chronic injury to the cord. In this study, the spatial and temporal expression of apoptotic cells was analysed in acute traumatic spinal cord injury (SCI) (n=6) and chronic myelopathies due to metastatic tumour (n=5), degenerative spondylosis (n=6) and syringomyelia (n=4). The study aimed to demonstrate apoptosis in compressive spinal cord injury and to analyse the spatial and temporal distribution of apoptosis in acute and chronic myelopathy. Method: Archival material from 21 spinal cords of patients with documented myelopathy during life and definitive evidence on post mortem examination were available for study. The spatial and temporal expression of apoptotic cells was analysed in acute traumatic spinal cord injury (SCI) (n=6) and chronic myelopathy due to metastatic tumour (n=5), degenerative spondylosis (n=6) and syringomyelia (n=4). Immunohistochemical analysis of each specimen was conducted using markers of apoptosis, as well as the biochemical apoptotic marker TUNEL. A total of 1800 histopathological slides were analysed. Specimens were also analysed using confocal microscopy to identify the immunopositive cell type. A combination of morphological, immunohistochemical and in situ end-labelling techniques were used to investigate the mechanism of cell death in this experiment. The analytical techniques employed were aimed at showing firstly the presence of apoptosis and secondly the size and position of the damaged regions. Results: Positivity for active Caspase-3, DNA-PKCS, PARP, TUNEL and active Caspase-9 was found in glia (oligodendrocytes and microglia) axons and neurons in both acute and chronic compression above, below and at the site of compression. In chronic compression, the severity of positivity for apoptotic immunological markers was positively correlated with the severity of white matter damage, as measured by APP immunostaining for axonal injury, and Wallerian degeneration. There was no correlation between the duration of chronic compression and immunopositivity for apoptotic markers. In acute SCI, axonal swellings were consistently positive for Caspases −9 and -3, suggesting mitochondrial activation of apoptotic pathways. Conclusion: Apoptosis occurs in both acute and chronic spinal cord injury. In acute compression, axonal injury is associated with apoptotic immunopositivity of glia and neurons. In chronic compression, apoptosis of oligodendrocytes and microglia correlates with demyelination of axons within the white matter

INTRODUCTION: Apoptosis, or secondary cell death, has been demonstrated in a number of neurological conditions, including Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis and brain ischaemia. It is well established from studies of acute spinal cord injury that apoptosis seems an important factor in secondary cell death and irreversible neurological deficit. It is only recently that studies have emerged analysing secondary cell death in chronic injury to the cord. In this study, the spatial and temporal expression of apoptotic cells was analysed in acute traumatic spinal cord injury (SCI) (n=6) and chronic myelopathies due to meta-static tumour (n=5), degenerative spondylosis (n=6) and syringomyelia (n=4). The study aimed to demonstrate apoptosis in compressive spinal cord injury and to analyse the spatial and temporal distribution of apoptosis in acute and chronic myelopathy. METHOD: Archival material from 21 spinal cords of patients with documented myelopathy during life and definitive evidence on post mortem examination were available for study. The spatial and temporal expression of apoptotic cells was analysed in acute traumatic spinal cord injury (SCI) (n=6) and chronic myelopathy due to metastatic tumour (n=5), degenerative spondylosis (n=6) and syringomyelia (n=4). Immunohistochemical analysis of each specimen was conducted using markers of apoptosis, as well as the biochemical apoptotic marker TUNEL. A total of 1800 histopathological slides were analysed. Specimens were also analysed using confocal microscopy to identify the immunopositive cell type. A combination of morphological, immunohistochemical and in situ end-labelling techniques were used to investigate the mechanism of cell death in this experiment. The analytical techniques employed were aimed at showing firstly the presence of apoptosis and secondly the size and position of the damaged regions. RESULTS: Positivity for active Caspase-3, DNA-PKCS, PARP, TUNEL and active Caspase-9 was found in glia (oligodendrocytes and microglia) axons and neurons in both acute and chronic compression above, below and at the site of compression. In chronic compression, the severity of positivity for apoptotic immunological markers was positively correlated with the severity of white matter damage, as measured by APP immunostaining for axonal injury, and Wallerian degeneration. There was no correlation between the duration of chronic compression and immunopositivity for apoptotic markers. In acute SCI, axonal swellings were consistently positive for Caspases −9 and -3, suggesting mitochon-drial activation of apoptotic pathways. CONCLUSION: Apoptosis occurs in both acute and chronic spinal cord injury. In acute compression, axonal injury is associated with apoptotic immunopositivity of glia and neurons. In chronic compression, apoptosis of oligodendrocytes and microglia correlates with demyelination of axons within the white matter