Summary

Osteoporosis reduces particle-induced osteolysis in rat model.

Objective

The objective of this study was to evaluate the rotation and translation of each joint in the hindfoot and compare the load response in healthy feet with that in stage II posterior tibial tendon dysfunction (PTTD) flatfoot by analysing the reconstructive three-dimensional (3D) computed tomography (CT) image data during simulated weight-bearing.

Free vascularised fibular grafting has been reported to be successful for adult patients with osteonecrosis of the femoral head (ONFH). However, its benefit in teenage patients with post-traumatic ONFH has not been determined. We evaluated the effectiveness of free vascularised fibular grafting in the treatment of this condition in children and adolescents. We retrospectively analysed 28 hips in 28 patients in whom an osteonecrotic femoral head had been treated with free vascularised fibular grafting between 2002 and 2008. Their mean age was 16.3 years (13 to 19). The stage of the disease at time of surgery, and results of treatment including pre- and post-operative Harris hip scores, were studied. We defined clinical failure as conversion to total hip replacement. All patients were followed up for a mean of four years (2 to 7). The mean Harris hip score improved from 60.4 (37 to 84) pre-operatively to 94.2 (87 to 100) at final follow-up. At the latest follow-up we found improved or unchanged radiographs in all four initially stage II hips and in 23 of 24 stage III or IV hips. Only one hip (stage V) deteriorated. No patient underwent total hip replacement.

Free vascularised fibular grafting is indicated for the treatment of post-traumatic ONFH in teenage patients.

This study prospectively compared the efficacy of kyphoplasty using a Jack vertebral dilator and balloon kyphoplasty to treat osteoporotic compression fractures between T10 and L5. Between 2004 and 2009, two groups of 55 patients each underwent vertebral dilator kyphoplasty and balloon kyphoplasty, respectively. Pain, function, the Cobb angle, and the anterior and middle height of the vertebral body were assessed before and after operation. Leakage of bone cement was recorded. The post-operative change in the Cobb angle was significantly greater in the dilator kyphoplasty group than in the balloon kyphoplasty group (−9.51° (sd 2.56) vs −7.78° (sd 1.19), p < 0.001)). Leakage of cement was less in the dilator kyphoplasty group. No other significant differences were found in the two groups after operation, and both procedures gave equally satisfactory results in terms of all other variables assessed. No serious complications occurred in either group.

These findings suggest that vertebral dilator kyphoplasty can facilitate better correction of kyphotic deformity and may ultimately be a safer procedure in reducing leakage of bone cement.

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 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: 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 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.

A foreign-body-type host response can contribute to the induction and release of collagenolytic tissue-destructive enzymes of pathogenetic significance. Our aim was to analyse collagenase-3 in two conditions with putative involvement of foreign-body reactions. Synovial membrane-like tissue samples were obtained from cases of aseptic loosening of a total hip replacement (THR) and osteoarthritis (OA).

The reverse transcription polymerase chain reaction (RT-PCR) disclosed that all the samples from patients contained collagenase-3 mRNA compared with only three out of ten control samples. The identity of the RT-PCR amplification product was confirmed by nucleotide sequencing. Immunohistochemical staining showed that collagenase-3 was present in endothelial cells, macrophages and fibroblasts, including those found in the synovial lining. This finding was confirmed by avidin-biotin-peroxidase complex-alkaline phosphatase-anti-alkaline phosphatase double staining and the specificity of the staining by antigen preabsorption using recombinant human collagenase-3.

Collagenase-3 was released into the extracellular space and thus found in the synovial fluid in all patient samples as shown by Western blotting. The similar extent of collagenase-3 expression in aseptic loosening and OA compared with the low expression in control synovial membrane suggests involvement of a similar, foreign-body-based pathogenetic component in both. Comparative analysis of collagenase-3 and of foreign particles indicates that paracrine factors rather than phagocytosis per se are responsible for the induction of collagenase-3.

We suggest that due to its localisation and substrate specificity, collagenase-3 may play a significant pathogenetic role in accelerating tissue destruction in OA and in aseptic loosening of a THR.