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.

Purpose: To determine if administration of recombinant bFGF in an alginate gel would increase early healing mechanical parameters in acutely injured rat rotator cuff tendon at specific time points.

Method: Sprague Dawley rats were randomly divided into 2 groups and had surgically created 1mm (half tendon width) full thickness injuries at exactly 2mm from insertion site of Infraspinatus on the humerus. 200ng of bFGF or vehicle control was administered to randomly chosen rats. Tendons were harvested at 1 week, 2 weeks and 4 weeks. In both groups, the Infraspinatus tendon was dissected, and left attached to the humerus. At the time of testing, the intact portion of the injured tendon was divided sharply across tendon fibers at the level of the injury leaving only the healing tissue callus in continuity with the remaining proximal and distal portions of the tendon and loaded to failure.

Results: At 1 week the injury group’s average load to failure was 0.60N versus 0.61N in the bFGF injury group P = 1.000. At 2 weeks the injury group’s average load to failure increased to 1.03N versus 2.08N in the bFGF injury group P = 0.440 At 4 weeks the injury group’s average load to failure increased to 3.93N versus 5.56N in the bFGF injury group P = 0.008 representing a 41% increase in ultimate load. At 4 weeks, callus size of the injury group was 0.4mm2 versus 2.7mm2 in the bFGF injury group P < 0.001. Stiffness at 4 weeks for the injury tendons was 2.15 N/mm versus 3.54 N/mm in the bFGF group P = 0.008.

Conclusion: At 4 weeks healing tissue of acutely injured rotator cuff exposed to bFGF has an increase in ultimate load to failure (41% compared to control), increase in tendon callus size and stiffness. Our findings suggest a role of bFGF or similar growth factors in accelerating the healing of injured rotator cuff tendon.

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 Ą 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 kinases³ 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.

Introduction Bone morphogenetic protein-7 (BMP-7) is known to stimulate both cellular proliferation and extracellular matrix synthesis in the intervertebral disc but its protective role in apoptosis is unknown. The aim of this study was to determine whether BMP-7 protect cultured intervertebral disc cells following stimulation of apoptosis.

Methods Nucleus pulposus tissues were obtained from consent individuals under surgical procedures and digested with collagenase prior to culturing. Cellular apoptosis was achieved by either tumor necrosis factor-alpha (TNF-β) or hydrogen peroxide (H2O2) incubation. BMP-7 (Stryker) was used at 100ng/ml, 5 hours prior to the addition of apoptotic stimulation. Cellular apoptosis was detected by TUNEL assay, caspase-3 activity and caspase-3 protein expression. Cellular proliferation and viability was assayed by H3-thymidine incorporation and MTS assay respectively. Collagen II and aggrecan protein levels were measured using western blots and immunostaining. Proteoglycan synthesis was determined by (35)S-sulfate incorporation method. Nitric oxide and alkaline phosphatase activity were measured.

Results Both extrinsic and intrinsic apoptotic pathways were induced by TNF-β or hydrogen peroxide with increased proteolytic activity of caspase-3 as well as cellular shrinkage and nuclear condensation. Addition of BMP-7 prior to stimulation of apoptosis resulted in complete block of the apoptotic effects of both inducers as well as the cellular nitric oxide induced by TNF-β and BMP-7 increases cellular viability, proliferation and extracellular matrix production in an apoptotic environment with no osteoblastic activity induction of discal cells.

Discussion BMP-7 prevents apoptosis of cultured human disc cells induced by either tumor necrosis factor-alpha (TNF-β) or hydrogen peroxide. Induction of apoptosis led to down regulation of extracellular matrix proteins, decreased cell viability, morphological changes and activation of caspase-3, however addition of BMP-7 alone prevented the effects observed. One possible mechanism of the anti-apoptotic effects of BMP-7 was shown by its retardation of the elevated levels of TNF-β induced nitric oxide.

Introduction The present experiment is undertaken to determine if a single dose addition of OP-1 device (rhBMP-7 and TCP-CMC) will enhance posterolateral spinal fusion in an osteoporotic rat mode (estrogen deficiency). Posterolateral intertransverse process spinal fusion using recombinant human osteogenic protein (rhBMP-7) was performed in ovariectomised female rats. OP-1 can be manipulated to enhance fusion rates and fracture healing with or without osteoporosis. Osteoporosis is characterised by low bone mass and micro-architectural deterioration of bone structure, resulting in bone fragility and an increase in susceptibility to fracture. Ovariectomised rats have been used as an osteoporotic model for posterolateral intertransverse process fusion in BMP experimental studies. Many studies have shown rhBMP-7 promotes spinal fusions in posterolateral fusion animal models. Not only is OP-1 able to promote spinal fusion in a standard animal model, but also it has been shown to overcome the inhibitory effects of nicotine in a rabbit posterolateral spinal fusion model. OP-1 Putty (Stryker) is an osteoinductive and osteoconductive bone graft material which consists of the recombinant human Osteogenic Protein (rhBMP-7), and TCP putty containing carboxymethylcellulose sodium (CMC) and tricalcium phosphate. This standard OP-1 device is somewhat different from the one Moazzaz et al used (1). The implication of OP-1 in osteoporotic model will open a new therapeutic window for osteoporotic or osteopaenial patients for the requirements of spinal fusion.

Methods In present study, a total of 42 ovariectomised Sprague-Dawley female rats were randomly assigned to groups receiving 30 μg lactose + 400mg TCP-CMC, 90 μg lactose + 400 mg TCP-CMC, 30 μg rhBMP-7 + 400 mg TCP-CMC and 90 μg rhBMP-7 + 400 mg TCP-CMC. There was a group of rats receiving 400 mg TCP-CMC alone. Spinal fusion was evaluated by manual motion testing at each lumbar segment, Faxitron digital X-ray evaluation using the Lenke grading system, CT scans, DEXA scans and histology.

Results Ovariectomized rats receiving 30 μg lactose + 400mg TCP-CMC, 90 μg lactose + 400 mg TCP-CMC, and 400 mg TCP-CMC alone did not show spinal fusion. OVX rats receiving 90 μg rhBMP-7 + 400 mg TCP-CMC showed significantly higher fusion rates than other groups (P < 0.0001). However, the rats receiving 30 μg rhBMP-7 + 400 mg TCP-CMC did not show solid fusion either radiologically and histologically.

Discussion Therefore rhBMP-7, in dose of 90 μg, is able to overcome the inhibitory effects of estrogen deficiency on posterolateral spinal fusion and generate a relatively robust fusion. The effect of the OP-1 on osteoporotic spine is dose-dependent with/without carrier-dependent.

Introduction Intervertebral disc degeneration may cause chronic low back pain. Disc degeneration is characterized by dysfunctional cells and a decrease in extra-cellular components. Bone marrow derived mononuclear cells are a heterogeneous cell population which contains mesenchymal stem cells. Transplantation of stem cells and progenitor cells may provide a new approach to treat disc degeneration, but it is unclear if transplanted cells can survive and differentiate in the non-vascularized disc.

Methods Bone marrow was collected from syngeneic Sprague-Dawley rats and mononuclear cells were isolated. The cells were labelled with a fluorescence dye (Cell Tracker Orange) and suspended in PBS. 10–20μl of the cell suspension (1–2x10⁵ cells/disc) was transplanted into coccygeal discs in 12 syngeneic rats. For each rat two discs were cell transplanted and one disc served as control. The rats were sacrificed after 0, 7, 14 or 21 days. For each time point the discs from one animal were saved for routine histological staining. The cell transplanted discs of the other animals (n=4 discs per time point) were formalin-fixed, frozen and sectioned together with the control discs. Frozen disc sections were visualized with fluorescence microscopy and the number of transplanted cells assessed. Expression of collagen II, a marker of chondrocytes and chondrocyte-like cells in the disc, was assessed in the transplanted cells using immunofluorescence technique.

Results All cell-suspension injected discs contained transplanted bone-marrow cells. The discs within each time-group demonstrated a large variation in number of detected cells. There was a decrease in detected cells at 7, 14 and 21 days compared to day 0. Transplanted cells expressed collagen II after 21 days but not after 7 and 14 days.

Discussion The results suggest that transplanted bone marrow-derived mononuclear cells can survive and differentiate within the intervertebral disc. Further studies in models of disc degeneration are warranted to investigate the regenerative potential of the disc following cell transplantation.