Tendinopathy is the most frequent musculoskeletal disease that requires medical attention. Mechanical overload has been considered as a key driver of its pathology. However, the underline mechanism on how overload induces tendinopathy and inflammation is unclear. Extracellular mitochondria (EM) are newly identified as cell-to-cell communicators. The aim of this study is to elucidate the role of mitochondria in overload-induced inflammation.

We performed three-dimensional uniaxial stretching to mouse tendon organoid in bioreactors. Cyclic strain of uniaxial loadings included underload, normal load, and overload, according to previous work. We then harvested microvesicles including EM, from the bioreactor by differential centrifugation and evaluated their characteristics by flow cytometry and super-resolution confocal microscopy. Raw 264.7 mouse macrophage cell line was used for chemotaxis assay in a Boyden Chamber System with Magnetic-Activated Cell Sorting Technology. EM induced cytokines secretion by macrophages was analyzed by a bead-based multiplex assay panel. N-Acetyl-L-cysteine (NAC) was used as the antioxidant to tendon organoid to regulate mitochondrial fitness.

We showed mechanical load induced tendon organoid to release microvesicles including mitochondria. The size of microvesicles is mainly in the range from 220nm to 880nm. More than 75% of microvesicles could be stained by PKH26, confirming they were with lipophilic membrane. Super-resolution confocal microscopy identified two forms of mitochondria, including mitochondria encapsulated in vesicles and free mitochondria. Overload led to the degeneration of the organoid and induced microvesicles release containing most EM. Chemotaxis assay showed that EM from overloaded tendon organoid induced macrophages chemotaxis. In addition, microvesicles extracted from overloaded tendon organoid induced the production of proinflammatory cytokines including IL-6, KC (Keratinocyte-Derived Chemokine) and IL-18. NAC treatment to tendon cells could attenuate overload-induced macrophage chemotaxis.

Overload induces EM releasing from tendon cells, which leads to chemotaxis of macrophages toward tendon, resulting in induction of inflammation.

To determine the risk of total knee replacement (TKR) for primary osteoarthritis (OA) associated with overweight/obesity in the Australian population.

This population-based study analyzed 191,723 cases of TKR collected by the Australian Orthopaedic Association National Joint Registry and population data from the Australian Bureau of Statistics. The time-trend change in incidence of TKR relating to BMI was assessed between 2015-2018. The influence of obesity on the incidence of TKR in different age and gender groups was determined. The population attributable fraction (PAF) was then calculated to estimate the effect of obesity reduction on TKR incidence.

The greatest increase in incidence of TKR was seen in patients from obese class III. The incidence rate ratio for having a TKR for obesity class III was 28.683 at those aged 18-54 years but was 2.029 at those aged >75 years. Females in obesity class III were 1.7 times more likely to undergo TKR compared to similarly classified males. The PAFs of TKR associated with overweight or obesity was 35%, estimating 12,156 cases of TKR attributable to obesity in 2018. The proportion of TKRs could be reduced by 20% if overweight and obese population move down one category.

Obesity has resulted in a significant increase in the incidence of TKR in the youngest population in Australia. The impact of obesity is greatest in the young and the female population. Effective strategies to reduce the national obese population could potentially reduce 35% of the TKR, with over 10,000 cases being avoided.

Though dentin matrix protein 1 (Dmp1) is known to play critical role in mediating bone mineralization, it has also been validated to be expressed in brain and helps maintain blood brain barrier (BBB). Our study aims to clarify the expression pattern of Dmp1 in mouse brain and explore whether intercellular mitochondrial transfer occurs between Dmp1 positive astrocytes (DPAs) and endothelial cells, and thus acting as a mechanism in maintaining BBB during aging.

Single cell RNA sequencing (scRNAseq) of 1 month, 6 month, and 20 month old mice brain (n=1, respectively) was employed to identify Dmp1 positive cell types. Dmp1^Cre-mGmT and Dmp1^Cre-COX8a fluorescent mice were generated to visualize DPAs and investigate their mitochondrial activities. A 3D noncontact coculture system and mitochondrial transplantation were applied to study the role of mitochondrial transfer between astrocytes and bEnd.3 endothelial cells. Dmp1^Cre-Mfn2^f/f mice were generated by depleting the ER-mitochondria tethering protein Mfn2 in DPAs.

Dmp1 was mainly expressed in astrocytes at different ages. GO analysis revealed that cell projection and adhesion of DPAs were upregulated. Confocal imaging on Dmp1^Cre-mGmT mice indicated that DPAs are a cluster of astrocytes that closely adhere to blood vessels (n=3). Bioinformatics analysis revealed that mitochondrial activity of DPAs were compromised during aging. Enriched scRNAseq of fluorescent cells from Dmp1^Cre-COX8a mice (n=2) and immunofluorescent imaging (n=3) validated the acquisition of extrinsic mitochondria in endothelial cells. 3D coculture of astrocytes and bEnd.3 and direct mitochondrial transplantation revealed the rescue effect of mitochondrial transfer on damaged bEnd.3. BBB was impaired after depleting Mfn2 in DPAs, expressing a similar phenotype with aging brain.

Astrocytes that express Dmp1 play a significant role in maintaining BBB via transferring mitochondria to vascular endothelial cells. Compromised mitochondrial transfer between DPAs and endothelial cells might be the potential mechanism of impaired BBB during aging.

Nerve transfer is an emerging treatment to restore upper limb function in people with tetraplegia. The objective of this study is to examine if a flexible collage sheet (FCS) can act as epineurial-like substitute to promote nerve repair in nerve transfer.

A preclinical study using FCS was conducted in a rat model of sciatic nerve transection. A prospective case series study of nerve transfer was conducted in patients with C5-C8 tetraplegia who received nerve transfer to restore upper limb function. Motor function in the upper limb was assessed pre-treatment, and at 6-,12-, and 24-months post-treatment.

Macroscopic assessment in preclinical model showed nerve healing by FCS without encapsulation or adhesions. Microscopic examination revealed that a new, vascularised epineurium-like layer was observed at the FCS treatment sites, with no evidence of inflammatory reaction or nerve compression. Treatment with FCS resulted in well-organised nerve fibres with dense neurofilaments distal to the coaptation site. Axon counts performed proximal and distal to the coaptation site showed that 97% of proximal axon count of myelinated axons regenerated across the coaptation site after treatment with CND. In the proof of concept clinical study 17 nerve transfers were performed in five patients. Nerve transfers included procedures to restore triceps function (N=4), wrist/finger/thumb extension (N=6) and finger flexion (N=7). Functional motor recovery (MRC ≥3) was achieved in 76% and 88% of transfers at 12 and 24 months, respectively.

The preclinical study showed that FCS mimics epineurium and enable to repair nerve resembled to normal nerve tissue. Clinical study showed that patients received nerve transfer with FCS experienced consistent and early return of motor function in target muscles. These results provide proof of concept evidence that CND functions as an epineurial substitute and is promising for use in nerve transfer surgery.

To address the current challenge of anterior cruciate ligament (ACL) reconstruction, this study is the first to fabricate a braided collagen rope (BCR) which mimics native hamstring for ACL reconstruction. The study aims to evaluate the biological and biomechanical properties of BCR both in vivo and vitro.

Rabbit ACL reconstruction model using collagen rope and autograft (hamstring tendon) was conducted. The histological and biomechanical evaluations were conducted at 6-, 12-, 18, 26-week post-operation. In vitro study included cell morphology analysis, cell function evaluation and RNA sequencing of the tenocytes cultured on BCR. A cadaver study was also conducted to verify the feasibility of BCR for ACL reconstruction.

BCR displays satisfactory mechanical strength similar to hamstring graft for ACL reconstruction in rabbit. Histological assessment showed BCR restore ACL morphology at 26 weeks similar to native ACL. The superior dynamic ligamentization in BCR over autograft group was evidenced by assessment of cell and collagen morphology and orientation. The in vitro study showed that the natural collagen fibres within BCR enables to signal the morphology adaptation and orientation of human tenocytes in bioreactor. BCR enables to enhance cell proliferation and tenogenic expression of tenocytes as compared to hydrolysed collagen. We performed an RNA-Sequencing (RNA-seq) experiment where RNA was extracted from tenocyte seeded with BCR. Analysis of enriched pathways of the up-regulated genes revealed that the most enriched pathways were the Hypoxia-inducible factor 1-alpha (HIF1A) regulated networks, implicating the possible mechanism BCR induced ACL regeneration. The subsequent cadaver study was conducted to proof the feasibility of BCR for ACL reconstruction.

This study demonstrated the proof-of-concept of bio-textile braided collagen rope for ACL reconstruction, and the mechanism by which BCR induces natural collagen fibres that positively regulate morphology and function of tenocytes.

Interstitial supraspinatus tears can cause persistent subacromial impingement symptoms despite non operative treatment. Autologous tendon cell injection (ATI) is a non-surgical treatment for tendinopathies and tear. We report a randomised controlled study of ATI compared to corticosteroid injection (CS) as treatment for interstitial supraspinatus tears and tendinopathy.

Inclusion criteria were patients with symptom duration > 6 months, MRI confirmed intrasubstance supraspinatus tear, and prior treatment with physiotherapy and ≥ one CS or PRP injection. Participants were randomised to receive ATI to the interstitial tear or corticosteroid injection to the subacromial bursa in a 2:1 ratio, under ultrasound guidance. Assessments of pain (VAS) and function (ASES) were performed at baseline, and 1, 3, 6 and 12 months post treatment.

30 participants (19 randomised to ATI) with a mean age of 50.5 years (10 females) and a mean duration of symptoms of 23.5 months. Baseline VAS pain and ASES scores were comparable between groups. While mean VAS pain scores improved in both groups at 3 months after treatment, pain scores were superior with ATI at 6 months (p=0.01). Mean ASES scores in the ATI group were superior to the CS group at 3 months (p=0.026) and 6 months (p=0.012). Seven participants in the CS group withdrew prior to 12 months due to lack of improvement. At 12 months, mean VAS pain in the ATI group was 1.6 ± 1.3. The improvements in mean ASES scores in the ATI group at 6 and 12 months were greater than the MCID (12.0 points). At 12 months, 95% of ATI participants had an ASES score > the PASS (patient acceptable symptom state).

This is the first level one study using ATI to treat interstitial supraspinatus tear. ATI results in a significant reduction in pain and improvement in shoulder function.

Metaphyseal fracture healing is important in joint-adjacent fractures and appears to differ from diaphyseal healing. We recently found that a biomaterial delivering bone morphogenic protein-2 (BMP-2) and zoledronic acid (ZA) healed the metaphyseal bone in a tibial defect but failed closing the cortical defect. In this study we added a BMP-2 soaked collagen membrane to study cortical healing from the muscle tissue surrounding the bone. We used SD rats and a 4.5 mm metaphyseal circular tibial defect. In group 1 (G1), a porous gelatin-calcium sulphate-hydroxyapatite (GCH) biomaterial containing rhBMP-2 and ZA was used to fill the defect (GCH+5 μg BMP-2+10 μg ZA). In group 2 (G2), we used a collagen membrane (2 μg BMP-2) to cover the GCH filled defect (GCH+3μg BMP+10 μg ZA). Group 3 (G3) was an empty control. Animals were sacrificed after 8-weeks and bone regeneration was evaluated with micro-CT and histology. In both G1 (P<0.001) and G2 (p<0.001) a significantly higher mineralized volume was found in the defect compared to empty G3. In G2 higher mineralized volume was found in the cortical region compared to both G1 (p<0.01) and G3 (p<0.001) as seen via micro-CT. Histologically, G1 and G2 showed islands of trabecular bone in the defect peripherally but only G2 showed cortical healing. G3 was empty in the middle but showed healed cortex. In conclusion, GCH can be used to deliver BMP-2 and ZA to promote metaphyseal bone growth. A membrane (CM) doped with low dose BMP-2 improved cortical regeneration.

Osteoarthritis (OA) is traditionally believed to affect the osteochondral unit by wear-and-tear from the superficial zone to the deep zone of cartilage and extended to subchondral plate. Obesity is commonly considered as a risk of OA development and hence total knee replacement (TKR), but the mechanism remains unclear. We hypothesized that obesity accelerated OA development by deteriorating tidemarks and increasing bone remodelling. 616,495 cases of TKR for OA from Australia and British joint replacement registries were collected, and data indicated that patients with higher BMI had TKR at earlier age. Specifically, patients with BMI ≤25kg/m² showed 8 years younger than patients with BMI ≥40kg/m² (P<0.0001) when they received TKR. We next examined tibia plateaus of 88 knee OA patients by micro-CT and histomorphometry. Linear regression showed that less cartilage degradation was associated with increased BMI in the load-bear compartment (p<0.05), while 58.3% of patients with BMI≥40kg/m² demonstrated a clear anatomical separation close to tidemarks filled with fibrosis, erythrocytes and bone fragments (compared to BMI ≤25kg/m² group: 7.7%, p<0.01). In subchondral bone, elevated bone formation was associated with increased BMI, as higher thickness of osteoid (p<0.01), percent osteoid volume (p<0.01), percent osteoid surface (p<0.01) were found in obese patients. However, no alteration of bone resorption and microstructural parameters was found to be associated with BMI. We suspected that the abnormal loading in knee joint due to high BMI led to the direct deterioration of binding site of osteochondral unit, which might be the mechanism of the rapid progression in obesity-related OA.

Mechanical loading plays an essential role in both tendon development and degradation. However, the underlying mechanism of how tendons sense and response to mechanical loading remains largely unknown. SPARC, a multifunctional extracellular matrix glycoprotein, modulates cell extracellular matrix contact, cell-cell interaction, ECM deposition and cell migration. Adult mice with SPARC deficiency exhibited hypoplastic tendons in load-bearing zone. By investigating tendon maturation in different stages, we found that hypoplastic tendons developed at around postnatal 3 weeks when the mice became actively mobile. The in vitro experiments on primary tendon derived stem cells demonstrated that mechanical loading induced SPARC production and AKT/S6K signalling activation, which was disrupted by deleting SPARC causing reduced collagen type I production, suggesting that mechanical loading was harmful to tendon homeostasis without SPARC. In vivo treadmill training further confirmed that increased loading led to reduced Achilles tendon size and eventually caused tendon rupture in SPARC-/− mice, whereas no abnormality was seen in WT mice after training. We then investigate whether paralysing the hindlimb of SPARC-/− mice using BOTOX from postnatal 2 weeks to 5 weeks would delay the hypoplastic tendon development. Increased patellar tendon thickness was shown in SPARC-/− mice by reducing mechanical loading, whereas opposite effect was seen in WT mice. Finally, we identified a higher prevalence of a missense SNP in the SPARC gene in patients who suffered from a rotator cuff tear. In conclusion, SPARC is a mechano-sensor that regulates tendon development and homeostasis.

Objectives

We have observed clinical cases where bone is formed in the overlaying muscle covering surgically created bone defects treated with a hydroxyapatite/calcium sulphate biomaterial. Our objective was to investigate the osteoinductive potential of the biomaterial and to determine if growth factors secreted from local bone cells induce osteoblastic differentiation of muscle cells.

To demonstrate the role of an antibiotic containing bone substitute, native bone active proteins and muscle transforming into bone.

Recurrent osteomyelitis was eradicated and filled with a gentamycin eluting bone substitute (Cerament™l G) consisting of sulphate and apatite phases and covered by a muscle flap.

A chronic, longstanding, fistulating osteomyelitis was operated with radical eradication and filling of the cavity with gentamycin eluting bone substitute. At one year, the patient had no leg pain and a healed wound. Significant bone was also seen in the overlaying muscle, at one month post-op disappearing after 6-months. Local delivery of gentamycin had a protective effect on bone formation.

Bone substitute with gentamycin leads to differentiation of mesenchymal cells into bone in-vitro.

Objective

To investigate the histological and immunohistochemical characteristics of revised and failed MACI repair tissues.

Autologous cell therapy using stem cells and progenitor cells is considered to be a popular approach in regenerative medicine for the repair and regeneration of tissue and organs. In orthopaedic practice, autologous cell therapy has become a major focus, particularly, as a feasible treatment for tendon injury.

Tendons are dense connective tissue that bridge bone to muscle and transmit forces between muscle and bone to maintain mechanical movement. Tendons are poorly vascularised and have very little capacity to self-regenerate. Degeneration of tendon is often caused by injury. The pathogenesis of tendon injury, commonly known as tendinosis, is not an inflammatory condition but is secondary to degenerative changes, including disruption of the collagen matrix, calcification, vascularisation and adipogenesis. The aetiology of tendinosis is considered to be multifactorial and the pathogenesis is still unclear. Intrinsic factors such as a lack of blood and nutrition supply and extrinsic factors such as acute trauma and overuse injury caused by repetitive strain, have been implicated as contributors to the pathogenesis of tendinosis. More recent studies suggest that programmed tendon cell death (tenocyte apoptosis) may play a major role in the development of tendinosis. Such cellular abnormalities may influence the capacity of tendon to maintain its integrity.

Traditional treatments such as anti-inflammatory drugs, steroid injections and physiotherapy are aimed at symptom relief and do not address the underlying pathological changes of degeneration. Here, we propose that autologous cell therapy may be an innovative and promising treatment for tendon injury. We will present evidence that suggest that autologous tendon cell therapy may be feasible to repair and regenerate tendon.

We will also present data summarising the preclinical evaluation of autologous tendon cell therapy in animal models and the safety and tolerability of autologous tendon cell therapy in humans in studies, which are currently conducted at the Centre for Orthopaedic Research at the University of Western Australia.

Osteoclastic bone resorption is a highly dynamic process that requires the tight ordering of intracellular trafficking events in order to maintain the structural and functional polarization of the ruffled border and basolateral domains. Rab3 proteins are a subfamily of GTPases, known to mediate membrane transport in eukaryotic cells and play a role in exocytosis. Our recent data indicates that Rab3D modulates a post-TGN trafficking step that is required for osteoclastic bone resorption (1). Here, to identify down-stream regulatory molecules of Rab3D, we have performed a yeast two-hybrid screen. Amongst several candidate Rab3D-interacting proteins identified, Rab3D was found to associate with calmodulin, an established regulator of osteoclastic bone resorption. As an initial effort to better define the interaction between Rab3D and calmodulin, we generated several mutants of Rab3D which interfere with the GDP/GTP nucleotide exchange (Rab3DQ81L, Rab3DN135I) and/or membrane attachment of Rab3D (Rab3D-CXC). By in vivo bioluminescence resonance energy transfer (BRET) assay, Calmodulin was found to associate equivalently with wild type Rab3D as well as Rab3DN135I and Rab-3DCXC variants. Over expression of constitutively active Rab3D (Rab3DQ81L) enhanced this interaction suggesting that the active form of Rab3D (i.e. GTP-bound) might recruit additional effector molecules which further potentiate it’s binding to calmodulin. In an attempt to address the impact of calmodulin activity on Rab3D-calmodulin interaction and osteoclastic bone resorption, we performed complementary BRET and in vitro bone resorption assays in the presence of the calmodulin inhibitor, calmidazolium chloride. Interestingly, we show that suppression of calmodulin activity via calmidazolium chloride impairs the association of Rab3D with calmodulin, an affect that correlates with a disruption in osteoclastic bone resorption. We propose that the recruitment of calmodulin by Rab3D might be an important requirement for osteoclast-mediated bone resorption.

Vacuolar adenosine triphosphatase (V-ATPase) proton pumps play an essential role in the acidification of the bone matrix during osteoclast-mediated bone resorption. Recently, mice lacking the V-ATPase d2 subunit have been shown to be osteopetrotic due to defective osteoclasts (Lee et al., Nature Med, 2006). Here, to investigate the transcriptional regulation of the d2 gene during RANKL-induced osteoclastogenesis, we have cloned and characterized its promoter region. By semi-quantitative RT-PCR, expression of d2 and NFATc1 was found to be strongly up-regulated by RANKL but not by other pro-osteoclastic factors including TNF, LPS and M-CSF. Bioinformatic analysis of the cloned 3 kb d2 promoter region revealed several candidate transcription factor binding sites including NFATc1, a key transcription factor for osteoclastogenesis.

To explore the influence of RANKL on d2 transcription, we generated a series of d2 promoter constructs using the pGL-3 reporter plasmid. Using luciferase assays, the d2 promoter was found to be induced by RANKL stimulation. Chromatin immunoprecipitation (ChIP) assays demonstrate that NFATc1 forms a complex with the d2 promoter. Using EMSA assays, we have defined a specific NFATc1 biding site between nucleotide − 555 to −561 upstream from the translation start site of d2 gene. Furthermore, targeted mutagenesis of the putative NFAT transcription binding site was found to significantly reduce the luciferase activity as induced by NFATc1 over expression. Addition of the NFAT inhibitor cyclosporin A was found to blunt the mRNA expression of d2 induced by RANKL in RAW264.7 cells.

We propose that NFATc1 is an important regulator of d2 transcription during RANKL-induced osteoclastogenesis.

Calcitonin has been recently shown to have a direct protective effect on articular cartilage against joint degenerative disease. It has been proposed that calcitonin might act through the calcitonin receptor (CTR) to activate the cyclic AMP pathway and protect type II collagen degradation. In this study, we examined the presence of the CTR in human articular cartilage and chondrocytes and investigated the potential pharmacological effects and transduction pathway of salmon calcitonin in human chondrocytes.

Five human articular cartilage samples were examined for the expression of the CTR by polymerase chain reaction (PCR), immunostaining and Western blotting. Cyclic AMP levels in human chondrocyte stimulated with salmon calcitonin were measured by ELISA. The effect of salmon calcitonin on the gene expression profiles, including aggrecan, type II collagen, matrix metalloproteinase (MMP)-1, MMP-3 and MMP-13, of human chondrocytes was also examined by Real-time PCR.

It was shown that CTR was not detectable in human cartilage and chondrocytes. The cAMP level in human chondrocytes in vitro was significantly increased by forskolin (100μM) by > 10 fold (P< 0.001), but was not induced by salmon calcitonin (10^-7M, 10^-8M, 10^-9M). Real-time PCR demonstrated that salmon calcitonin tended to reduce the gene expression of MMPs, yet without statistical significance. In contrast to previous reports, our data showed that human cartilage and chondrocytes do not express calcitonin receptors. There was no direct effect of salmon calcitonin on human chondrocytes.

The result suggests that the chondroprotective effect of calcitonin observed in vivo may be indirect via its effect on subchondral bone resorptive activity.

Large and retracted rotator cuff tendon tears fail to repair, or re-tear following surgical intervention. This study attempted to develop novel tissue engineering approaches using tenocytes-seeded bioscaffolds for tendon reconstruction of massive rotator cuff tendon defect in rabbits. Porcine small intestine submucosa (Restore™) and type I/III collagen bioscaffold (ACI-MaixTM) were chosen as bioscaffold carriers for autologous tenocytes. Biological characterization of autologous tenocytes was conducted prior to the implantation. The tenocyte-seeded bioscaffolds were implanted as interposition grafts to reconstruct massive rotator cuff tendon defects in rabbits. In situ re-implantation of the autologous rotator cuff tendon, excised during defect creation served as a positive control. Histological outcomes were analysed and semi-quantitatively graded at four and eight weeks after surgery.

The results demonstrate that at four weeks both tenocyte-seeded bioscaffolds display inflammatory reaction similar to bioscaffold-only cuff reconstruction and the histological grading were inferior to control repair. However, at eight weeks inflammatory reaction of both tenocyte-seeded bioscaffolds were dramatically reduced as compared to bioscaffold alone. In addition, bioscaf-folds seeded with tenocytes generated similar histological appearance to that of the positive control.

The implantation of autologous tenocytes on collagen-based bioscaffold offers improved rotator cuff tendon healing and remodelling compared to the implantation of bioscaffold alone.

Spinal fusion surgery is a common procedure for the treatment of various spinal diseases. Several growth factors, including bone morphogenic protein-2 (BMP-2) and osteogenic protein-1 (OP-1) have been used in spinal fusion for the induction of bone formation. But complications have been reported due to the lack of suitable carrier. Here we hypothesis that Insoluble Bone Gelatin (ISBG) may be a good carrier for OP-1 in the induction of bone formation during spinal fusion. The aim of this study is to examine the efficacy of osteoconductive carrier, ISBG, for OP-1 in rabbit lumbar inter-transverse process fusion model.

Adult New Zealand White rabbits (n=32) underwent bilateral lumbar intertransverse process fusion at L5-L6. The animals were divided into four groups based on the materials implanted:

Autograft group,

Spinal fusion masses were evaluated by manual palpation, biomechanical testing, radiographic examination, micro-CT Scanning, and histological analysis six weeks after surgery.

ISBG+OP-1 group demonstrated significantly higher fusion rates (7/7) than autograft (3/7), ISBG (2/8), and OP-1 groups (2/7) (P< 0.05) based on manual palpation. In biomechanical testing, given the same moment, the fusion masses of ISBG+OP-1 group had less range of motions than those of other groups (P< 0.05) in main direction motion. Radiographic examination and micro-CT demonstrated that continuous trabecular pattern within intertransverse process area in ISBG+OP-1 group than other groups, and radiographic scores and bone volume base on micro-CT were also higher than other groups. Mature new bone formation was observed covering the surface of transverse processes in all four groups in histological findings. Continuous trabeculae connected two transverse processes and endochondral bone formation was observed attached the surface of ISBG in ISBG+OP-1 group. However, in other three groups, obvious gaps were noted in fusion masses and fibrous tissue was filled in these gaps.

In conclusion, OP-1 carried by ISBG results in more effective spinal fusion in posterolateral lumbar transverse fusion in rabbit model than autograft, ISBG or OP-1 alone.

Introduction: There are no current estimates of the risk of transmission of HIV, HBV, HCV, or HTLV by musculoskeletal tissue transplantation. Such accurate data would be helpful to determine the effectiveness of current and proposed screening and processing procedures, and contribute to increased confidence in the use of musculoskeletal tissue products.

Methods: The prevalence rates of HIV, HBV, HCV, and HTLV were determined from 12.245 musculoskeletal tissue donors from three bone tissue banks across Australia from the period 1993 to 2004. The incidence rates among tissue donors were estimated by comparing the data with age-specific incidence rates of first-time blood donors. We estimated the probability of a tissue donor was within the window period when infection was undetected by serological screening procedures by the modified incidence-window period model. Further we calculated the projected probability of viremia with the addition of nucleic-acid amplification testing (NAT).

Results: The prevalence (per 100,000 persons) of confirmed positive tests among musculoskeletal tissue donors was 169.15 for HIV, 427.68 for HBV, 534.63 for HCV, and 121.66 for HTLV. This is greater than the prevalence among first-time blood donors during the same period (6.47 for HIV, 136.00 for HBV, 215.29 for HCV, and 3.46 for HTLV). The incidence rate among musculoskeletal donors were estimated to be 15.81, 0.68, 3.53, and 4.85 per 100,000 person-years, respectively. The estimated probability of viremia (per 100,000 persons) at the time of donation was 1.38 for HIV, 0.46 for HBV, 1.82 for HCV, and 0.85 for HTLV. These estimations would be even lower with the addition of NAT – 0.57, 0.23, and 0.20 respectively.

Conclusions: The prevalence and incidence of HIV, HBV, HCV, and HTLV among musculoskeletal tissue donors, although low are significantly higher than those of first-time blood donors. Current screening and processing measures are effective, though the probability of viremia can be reduced further by nucleic-acid amplification testing.

Introduction and aim: Early symptomatic osteoarthritis (OA) of the knee poses a difficult challenge to orthopaedic surgeons, particularly in the presence of malalignment. Most surgical options are palliative. Our aim was to assess combined high tibial osteotomy (HTO) and matrix-induced autologous chondrocyte implantation (MACI) as a curative option.

Methods Patients with localised medial compartment OA and varus malalignment were identified. Suitability for the above procedure was confirmed at arthroscopy and specimen taken for culture. HTO and MACI procedures were performed in one sitting by a single surgeon. Patients received three months rehabilitation and function was assessed preoperatively and at three-monthly intervals.

Results Twelve patients were identified: nine male; average age 46 years (27–58). Mean varus deformity was 6 degrees. Two patients also had evidence of osteochondritis dissecans, and two early patello-femoral OA. Eight patients had had previous surgery to the knee.

Eleven patients had a lateral closing wedge osteotomy; the medial opening wedge was performed in a case of leg shortening. Mean operation duration was 72 minutes (60–90). The graft was fixed with fibrin glue in all cases, and augmented with stitches or vicryl pins in five cases. Mean defect size was 6.2cm2 (2–12). There were three complications: one DVT, a haemarthrosis and a graft detachment.

Average follow-up was 16 months. MRI scans at three months show oedematous tissue at the defect sites, contrasting with the fluid filled defects seen preoperatively. Scans at one-year show hyaline-like cartilage infill with similar signal characteristics to native hyaline cartilage. Six minute walk test and knee injury and osteoarthritis outcome score indicate significantly improved functional capacity at six months and one year.

Conclusions Preliminary results suggest combined HTO and MACI is successful for young patients with early OA associated with malalignment.

Introduction and Aims: Fibrin-sealant has been recommended as a tissue glue for autologous chondrocyte implantation. It is known that the active compound of fibrin-sealant is thrombin, but its effect on chondrocytes is still unclear. The aims of this study are to examine if fibrin-sealant stimulates proliferation and survival of human chondrocytes.

Method: To determine if human chondrocytes express thrombin receptors, we have conducted immunoconfocal analyses and RT-PCR for the detection of PAR type I, II, III and IV. To examine if thrombin activates intracellular signalling of chondrocytes, we have examined the intracellular calcium signalling by thrombin. Proliferation of chondrocytes was also tested with various concentrations of thrombin. The migration of chondrocytes was monitored by co-culturing of the cells with fibrin-sealant for up to 15 days.

Results: Primary human chondrocytes express thrombin receptor PAR types I, II, II and IV as evidenced by immunohistochemistry and RT-PCR. Induction of intracellular calcium signals was evidenced in majority of chondrocytes at 100 seconds after addition of thrombin. To confirm if evaluation of calcium signal activation is by a specific PAR receptor, we have examined the effect of specific peptides, which mimic the receptor activation on calcium signalling. The result showed that expression of PAR I and II receptor in chondrocytes is responsible for the activation of intracellular calcium. When human chondrocytes were co-cultured with thrombin at a dose between 1u/mL to 10u/mL, there was no effect on cellular proliferation at 24 hours. However at 48 hours, thrombin stimulated proliferation and survival of chondrocytes in a dose-dependent manner. A maximum of threefolds induction was evidenced at a dose of 10u/mL (p< 0001). Co-culture of chondrocytes with fibrin-sealant showed that after 12 hours only a few cells had migrated from the membrane to the fibrin-sealant, but after 36 hours many cells had formed a layer on the surface of the fibrin-sealant. By 15 days of co-culture, it was evidenced that the majority of chondrocytes were migrating into the fibrin-sealant.

Conclusion: The results of this study show that human chondrocytes express thrombin receptor and fibrin-sealant is capable of inducing chondrocyte proliferation and migration.

Introduction and Aims: The aim of this study was to use biological, functional and radiographic evaluation to demonstrate that cultured autologous chondrocytes implanted using a type I/III collagen membrane leads to regeneration of hyaline-like articular cartilage in the knee.

Method: Approximately 70,000 knee arthroscopies are performed every year in Australia; 60% involve chondral surface defects. Three regenerative autologous cell therapy techniques have been used in Australia to treat full thickness chondral lesions:

periostial-covered autologous chondrocyte implantation (PACI);

The team at the University of Western Australia has concentrated on CACI and MACI techniques because of concerns over fibroblast formation and hypertrophy with PACI. Definitive evidence regarding the role of the membrane in enhancing chondrocyte-mediated cartilage regeneration is lacking.

Results: The series consists of a total of 71 patients who had failed previous surgical treatment prior to definitive collagen-covered ACI (32 implantations in 31 patients) or MACI (43 implantations in 40 patients). Biological, functional and radiographic evaluations were conducted pre-operatively, and post-operatively in order to determine the success of integration of implanted chondrocytes and categorise the level of restoration in knee joint function. Post-operative MRI scans at three months show oedematous tissue at the defect sites, contrasting with the fluid-filled defects seen pre-operatively. MRI scans at one, two and three years (collagen-covered) and one year (MACI) show normal cartilage signal. Apopototic test of chondrocytes before implantation showed that viability of chondrocytes was over 85% where apopototic rate of chondrocytes was less than 2%. Six-minute walk test and KOOS results indicate improved functional capacity following collagen covered and MACI.

Conclusion: Results from this clinical study indicate that the use of a type I/III collagen membrane in conjunction with ACI is a valid new approach for the treatment of chondral defects. Results from radiographic, functional and biological evaluations are encouraging. Ongoing follow-up will reveal the durability of reconstructions with CACI and MACI.

Introduction and Aims: Treatment of rotator cuff tendon tear presents a significant therapeutic challenge to surgeons. Porcine small intestinal submucosa (SIS) is a biomaterial approved by TGA and FDA for the repair of rotator cuff tendon tear. The aims of this study are to evaluate the safety and efficacy of SIS.

Method: SIS purchased from DePuy Johnson & Johnson was examined by histology and PCR technique. The material was also implanted into mice and rabbits for the evaluation of biological reaction and inflammatory response. Porcine immunoreceptor DAP12 gene was used to examine if the material contained porcine DNA.

Results: Fresh SIS membrane before implantation contains multiple layers of spindle-shaped cells mixed with a small population of round-shaped cells. Chloro-acetate esterase staining showed that the round-shaped cells are positive, indicating that they are mast cells. The tissue architecture of SIS mimics to tendon structure as evidenced by H& E staining. To further confirm if cells present in SIS material were porcine origin, nested PCR for the amplification of DAP12 gene was used. The result demonstrated that SIS membrane contain porcine DNA materials.

Conclusion: SIS contains porcine cells and nuclei acid, which contradicts with current views that SIS is a cell-free biomaterial. Although no foreign body reaction of SIS was observed, SIS implant may cause chronic inflammation. Further studies should be conducted to confirm the clinical efficacy of SIS implant.

Introduction and Aims: Autologous chondrocyte implantation (ACI) is emerging as a leading technique for the treatment of articular cartilage defects. However, there exists some debate regarding which ACI technique is best able to regenerate hyaline cartilage. To this end, the development of a non-invasive technique enabling the examination of microstructure after ACI is essential.

Method: In this study, we have developed a novel 2D Laser Scanning Confocal Arthroscope (LSCA) in the assessment of articular cartilage and examined the microstructure of knee articular cartilage from rabbits and patients with total knee arthroplasty. The LSCA system consists of the LSA handheld probe, a Launch and Detection Unit (LDU) with a built in 488nm–514nm Krypton Argon Laser and Master Control unit (MCU). Human and rabbit knee articular cartilage stained with Fluoroscein (5g/L) and Acriflavine (0.5g/L) were used to examine the microstructure of cartilage by LSCA.

Results: By LSCA we have generated optical histology images of normal human and rabbit articular cartilage from the femoral condyle. Optical histology of normal articular cartilage tissue reveals typically smooth surface texture with relatively homogenous sub-surface distribution of viable chondrocyte cells. The general orientation of collagen fibres is occasionally visible in surface images. Optical histology of arthritic cartilage of humans showed clusters of round-shaped chondrocytes mixed with spindle-shaped cells. Surface cracking typically indicative of tissue damage is also evident by LSCA observation. Examination of rabbit knee six weeks after ACI showed high density of chondrocytes and homogeneous matrix on the site of the defect.

Conclusion: In short, we have shown the efficacy of LSCA in the non-destructive assessment of articular cartilage in vivo. Further study is required to evaluate the clinical significance of optical histology of LSCA.

Introduction and Aims: Large or recurrent rotator cuff tendon tears are difficult to treat effectively. Collagen bio-scaffolds have become available to reinforce a tendon repair or as an interpositional graft. This study compares the suitability of two collagen bio-scaffolds for autologous tenocyte implantation, and assesses the in vivo rotator cuff healing response with these grafts in a rabbit model.

Method: Tenocytes were isolated from rabbit tendon, cultured and seeded onto the Restore patch (DePuy), or the Matricel (Verigen) collagen membrane. Serial scanning electron microscopy examined tenocyte integration with the bio-scaffold, and extra-cellular matrix synthesis over time. A rotator cuff tendon defect was created in 50 rabbits and repaired by either: a) direct suture to tuberosity; b) Matricel interposition graft; c) Matricel interposition with autologous tenocytes; d) Restore patch interposition graft; e) Restore patch interposition with autologous tenocytes. Gross and histological evaluation were performed at four weeks and eight weeks post-surgery.

Results: Scanning electron microscopy of the Matricel membrane showed a rough surface characterised by a loose arrangement of collagen fibres capable of cell adhesion. SEM at one, three and five days after cell seeding, showed progressive integration of tenocytes into the three-dimensional membrane structure with extra-cellular matrix neosynthesis in the spaces between the native collagen fibres. SEM of the Restore patch showed a relatively smooth surface of highly compacted collagen fibres. Serial SEM after cell seeding showed relatively less tenocyte integration onto the membrane surface though tenocyte replication and matrix neo-synthesis was observed. All 50 rabbits regained normal gait at two weeks post-surgery. At sacrifice, no tendon ruptures had occurred at either time point in any of the five groups. At four weeks, the Matricel and Restore bio-scaffold membranes were partially absorbed, and a florid lymphocytic inflammatory response was evident surrounding the remaining membrane. By eight weeks, graft tissue had been resorbed further, the inflammatory response had decreased, and the regenerating tendon showed progressive remodelling. Autologous tenocyte implantation on both membranes improved the reparative tendon histological grade at eight weeks compared to membranes without cell implantation, and was equivalent to the direct repair group.

Conclusion: Autologous tenocytes can be implanted onto both Matricel and Restore collagen bio-scaffolds. Though both Xeno grafts induce an anti-inflammatory response in vivo, membrane resorption subsequently occurs. The healing response of large rotator cuff defects treated with interpositional collagen grafts is improved with autologous tenocyte implantation in a rabbit model.

Introduction and Aims: Conventional histology requires the traumatic removal of tissue from its native environment. This is not only a destructive process but also leads to tissue preparation artefact. We report on a novel arthroscopic instrument, the laser scanning confocal arthroscope (LSCA), which can image tissues of the knee at depth without the need for a damaging tissue biopsy.

Method: The new confocal arthroscope contains 4.4mm diameter with a 90-degree lens. Using three knee joints from two adult Merino sheep we imaged muscle, cartilage, ligament, tendon, synovium, meniscus and loose connective tissue. The knees were separately injected with three fluorophores (Acridine Orange, Acriflavine/Calcein-AM or Fluorescein) prior to imaging. Using a medial para-patellar incision, the contents of the knee were exposed and the confocal arthroscope was held directly on the tissue of interest. A second operator captured the images on a computer.

Results: We were able to demonstrate the common histological features of normal sheep articular cartilage, meniscus, synovium, ligament, tendon and muscle. Tissues were imaged to depths of 200 microns. Articular cartilage was characterised by a layer of dense superficial cells surrounded by extracellular matrix. There was no visible orderly arrangement of cells in this layer. Meniscus was characterised by closely packed circumferential collagen fibres. Synovium demonstrated a dense collection of cells in a thin membrane, typical of this secretory tissue. Ligament and tendon were characterised by bundles of parallel collagen fibres interspersed by scattered cells. Muscle revealed a typical arrangement of muscle fibres surrounded by a loose connective tissue and separated by capillaries and nerves. Eccentric nuclei were seen, however striations were beyond the imaging resolution of the arthroscope.

Conclusion: This study demonstrates the use of a novel arthroscopic instrument for the non-destructive examination of the components of the sheep knee joint. We foresee that the laser scanning confocal arthroscope will have future application in the assessment of cartilage grafting techniques and arthritis modifying drugs.

Introduction Rotator cuff degeneration is considered to be a major factor in the pathogenesis of rotator cuff tendon tear. Degenerative weakening of the rotator cuff can result in irreversible complete cuff-tear arthropathy syndrome. Recently a porcine small intestinal submucosa (SIS) has been approved by TGA as biological implant for the repair of rotator cuff tendon tear. The aims of this study are to evaluate the safety and efficacy of SIS.

Methods A commercial brand of SIS was examined by histology and PCR technique. The material was implanted into mice and rabbits for the evaluation of biological reaction and inflammatory response. Next, we have used SIS to replace the rotator cuff tendon in rabbit (N=10) and compared to control (N=10). Histological examination was conducted at four and eight weeks after implantation. To further confirm if cells present in SIS material were of porcine origin, nested PCR for the amplification of DAP12 gene was used.

Results Fresh SIS membrane before implantation contain multiple layers of spindle-shaped cells mixed with a small population of round-shaped cells. Chloroacetate esterase staining showed that the round-shaped cells are positive, indicating that they are mast cells. The tissue architecture of SIS mimics tendon structure as evidenced by H & E staining. The SIS membrane contained porcine DNA materials. Subcutaneous implant of SIS in mice (by six) for up to seven days showed no obvious inflammatory response or foreign body reaction. The result demonstrated that SIS has remained in the region and mixed with regenerative fibrous tissue after eight weeks. In some cases there was a massive recruitment of lymphocytes along the surface of membrane. However, no foreign body reactive giant cells were observed.

Conclusions The result of this study indicated that SIS contains porcine cells and nucleic acid, which contradicts current views that SIS is a cell free biomaterial. Although no foreign body reaction of SIS was observed, SIS implant may cause chronic inflammation. Further studies should be conducted to confirm the clinical efficacy of SIS implant for rotator cuff tendon tear.

Introduction Fibrin-sealant has been widely used clinically for the protection of haemorrhage, wounds and tissue fluid leakage. Recently fibrin-sealant has been recommended as a tissue glue for autologous chondrocyte implantation. It is known that the active compound of fibrin-sealant is thrombin but its effect on chondro-cyte is still unclear. The aims of this study are to examine if fibrin-sealant stimulates proliferation and survival of human chondrocytes.

Methods Primary human chondrocytes derived from articular cartilage were used for the detection of thrombin receptors RAR type I, II, III and IV by immunohistochemistry and RT-PCR. To examine the effect of thrombin on chondrocytes, the changes in free intra-cellular calcium were monitored after the addition of thrombin. Proliferation of chondrocytes were also tested with various concentrations of thrombin. The survival of chondrocytes was monitored by co-culturing of the cells with fibrin-sealant for up to 15 days. Primary human chondrocytes express thrombin receptor RAR types I, II, III and IV as evidenced by immunohistochemistry and RT-PCR. However, the level of expression appears to be varied between cells. This has been reflected by the measurement of intracellular calcium signal in chondrocytes.

Results Induction of intracellular calcium signals was evidenced in the majority of chondrocytes at 100 seconds after addition of thrombin. When human chondrocytes were co-cultured with thrombin at a dose between 1u/ml to 10u/ml, there was no effect on cellular proliferation at 24 hours. However, at 48 hours thrombin stimulated proliferation and survival of chondrocytes in a dose dependent manner. A maximum of three folds induction was evidenced at a dose of 10u/ml (p< 0001). Co-culture of chondrocytes with fibrin-sealant showed that after 12 hours only a few cells had migrated from the membrane to the fibrin-sealant, but after 36 hours many cells had formed a layer on the surface of fibrin-sealant. By 15 days of co-culture, it was evidenced that majority of chondrocytes were migrating into the fibrin-sealant. Immunohistology study showed that these cells express type II collagen, suggesting that they maintain the phenotype of chondrocytes.

Conclusions The results of this study show that human chondrocytes express thrombin receptor and fibrin-sealant is capable of inducing chondrocyte proliferation and maintain the survival of chondrocytes.

In relation to the conduct of this study, one or more of the authors is in receipt of a research grant from a non-commercial source.

Successful reconstructive surgery with allografts is severely limited by a failure rate of 30 – 40%. Allograft failure is due to nonunion of the graft-host junction. The molecular mechanism by which this occurs is not yet fully elucidated. Using a sheep femoral allograft model, we have investigated the cellular and molecular mechanisms associated with nonunion of bone allografts. Five, from a total of twelve operations, resulted in the development of graft-host nonunion, reflecting a failure rate of 42%. Histological assessment revealed that allograft failure was due to the excessive accumulation of and resorption by, osteoclasts (Ocs) on the surface of the bone allograft. Three distinct layers, lying adjacent to the allograft bone surface, in the nonunion groups, were identified. The outer fibroblastic layer contained abundant fibroblasts and connective tissue. Underlying this layer were synovial-like cells and some multinuclear giant cells. The third layer, opposing the bone surface, consisted of Ocs and round mononuclear cells. Histomorphometric analysis showed that allograft unions, featured a large amount of newly formed bone on the surface, (OS/BS = 47.81%) with a small proportion of eroded surface (ES/BS = 20.59%). The number of osteoclasts associated with the allograft bone surface were few (Oc/B.Pm = 1.7190/mm) and activity (ES/BS = 46.68%) of Ocs with a reduced amount of new bone formation (OS = 6.35%). Both calcitonin receptor and H+ATPase mRNA, characteristically expressed by Ocs, were localised to the multinuclear giant cells, indicating that they were Ocs. Synovial-like cells in the histological layer above the Ocs, expressed gene transcript for the Osteoprotegrin Ligand (OPGL), a membrane bound factor that is critical for the induction of Oc activity and osteoclastogenesis. In conclusion, these findings suggest that failure of bone allografts is partially due to excessive resorption by host Ocs, accompanied by reduced bone formation. The production of OPGL by synovial-like cells, may be responsible for the recruitment and generation of Ocs.

Introduction: Autologous chondrocyte transplantation (ACT) has been shown to be a promising method for restoring hyaline cartilage defects. Since it was first reported by Brittberg et al nine years worth of clinical follow up studies indicate that ACT has provided an excellent outcome in the restoration of hyaline cartilage. As ACT relies on the use of cultured cells and the biosynthetic profile of cultured chondrocytes has been shown to be altered during in vitro expansion, cultivation of chondrocytes for ACT has presented many technical and quality control challenges.

Aim: To perform an assessment of the cellular phenotype of cultured chondrocytes, consistent with differentiation of articular hyaline cartilage, to ensure the delivery of ACT for restoration of hyaline cartilage.

Methods: Using RT-PCR and flow cytometry analyses, we characterised the cellular phenotype of culture chondrocytes used for ACT. We examined several transcriptional factors, cytokines and matrix proteins necessary for the differentiation of chondrocytes in a total of 15 cases of ACT. These included SOX9, Cbfa1, Indian Hedgehog (Ihh), TGF-b3, BMP-2, PTHrP, type I and type II collagen, aggrecan and alkaline phosphatase.

Results: The results demonstrated that there is a variety in the expression of these genetic makers but cultured cells used for ACT were within the programme of chondrocyte differentiation. Furthermore, there is variation in the level of apoptosis of chondrocytes between patients as evidenced by annexin V flow cytometry. As evidenced by MRI in two patient samples, apoptosis of chondrocytes greater than 8% was coincident with cases that could not restore hyaline cartilage three months after ACT.

Conclusions: Given that there is a medical need for ACT in the treatment of articular cartilage injury, a process for monitoring the quality of culture chondrocyte prior to implantation may provide a better clinical outcome of ACT.

Introduction: Rotator cuff tears are a common injury which affects both the young athlete and the sedentary elderly alike. This condition is commonly treated with glucocorticoid injections as part of initial management. The effects, however, of these injections on the histology of collagen and the metabolism of tendon fibroblasts are still controversial.

Materials and methods: In this study, samples from 19 patients with rotator cuff tears were taken during definitive surgery to manage these tears. There was a history of glucocorticoid injections in all of the patients. The samples were examined in terms of histopathology using light microscopy, in situ hybridization to detect the presence of glucocorticoid receptor mRNA and TUNEL assay to determine the incidence of apoptosis.

Results: Light microscopy of hematoxylin-eosin stained samples from the study group showed marked cellularity although there were no signs of inflammation. The nuclei were noted to be rounded and a significant number showed pyknosis. Angiogenesis was also noted in the sections, consistent with previous finding of angio-fibroblastic hyperplasia as a characteristic of tendinosis. Collagen structure was noted to be abnormal, with longitudinal clefts and focal areas of marked disorganization of fibers. In situ hybridization showed a strong signal for glucocorticoid receptor mRNA in all of the samples. TUNEL assay also showed a strong signal for apoptosis of the tendon fibroblasts in the study group as compared to the control group which showed almost no signal.

Conclusion: Our results suggest that although an overall picture of hypercellularity is seen in cases of tendinosis and tendon tears, a high percentage of these cells are undergoing apoptosis. This may reflect a natural high rate of turnover of cells during the process of repair or may be due to exogenous factors. Glucocorticoids almost certainly affect metabolism of tendon fibroblasts and subsequently collagen structure as seen by the abundant expression of the receptor mRNA. However, a causal relationship between glucocorticoids and apoptosis of tenocytes is yet to be established.

Articular cartilage defects of the knee occur commonly in sports injuries and trauma. Increasing evidence suggests that the only technique that enables the regeneration of articular hyaline cartilage in chondral defects is autologous chondrocyte implantation (ACI). Here we have reported our clinical experience of autologous chondrocyte implantation using biodegradable type I/III collagen membrane (CACI). A total of 26 patients (age range from 19 to 60 years, average 37 years) was conducted with CACI. Pre-operative magnetic resonance imaging (MRI) scans were performed on all patients. Post-operative MRI scans were planned for approximately three and 12 months after the surgery to determine the success of integration of implanted chondrocytes.

The results demonstrated that the initial post-operative MRI scans at three months showed the presence of oedematous tissue at the defect sites in 23 patients, contrasting with the fluid filled defects seen preoperatively and with and MRI signal differing from that of the surrounding normal hyaline articular cartilage. MRI scans in nine patients at 12 months after their operations showed maturation of cartilage graft in all patients. Apopototic testing of the chondrocytes using Annexin IV before implantation showed that the viability of the chondrocytes was over 85% where the apopototic rate of chondrocytes was less than 2%. One patient with an apopototic rate of over 10% has a delayed repair in cartilage defects as shown by MRI.

In conclusion, early phase clinical studies showed that autologous chondrocyte implantation remains promising for the treatment of chondral defects with restoration of hyaline cartilage. Longer clinical follow-up of the patients and better assessment of cellular phenotype of chondrocytes before implantation are required.