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Biomedical subjects

M H Zheng

Publications and source records attributed to M H Zheng.

At least 19 recordsLinked to original sources
PubMed2006

Bone allograft non-union is related to excessive osteoclastic bone resorption: a sheep model study.

Using a sheep femoral allograft model we have investigated the cellular and molecular mechanisms associated with non-union of bone allografts. Histomorphometric analysis revealed that allograft non-unions featured both marked increases in osteoclast (OC) numbers and total eroded bone surface as compared to allografts which had undergone direct union. Three distinct cellular layers lying adjacent to the allograft bone surface were identified in all non-union cases. The outer or fibroblastic layer contained an abundance of fibroblasts and connective tissue. Circumscribing this layer was a band of synovial-like cells consisting mainly of large spindle-shaped mononuclear cells mixed with scattered round-shaped mononuclear cells. The third layer, which was directly juxtaposed to the allograft bone surface, consisted predominantly of multinuclear OCs which were positively identified by calcitonin receptor immunohistochemistry. Interestingly, in-situ hybridisation revealed that surrounding synovial-like cells in non-union allografts, expressed abundant gene transcripts for receptor activator NF-kappaB ligand (RANKL), a membrane bound factor critical for both the induction of OC activity and osteoclastogenesis. We propose that excessive bone resorption by host OCs contributes, at least partially, to the failure of bone allografts. The production of RANKL by synovial-like fibroblasts may be the driving force responsible for the elevated generation and activation of OCs. Based on such evidence novel therapeutic strategies for the treatment of non-union bone allografts using anti-bone resorbing agents may be devised.

Animals↗
PubMed2006

Evidence of circulating donor genetic material in bone allotransplantation.

Musculoskeletal allotransplantion is the most common form of human tissue transplantation. Unlike solid organ transplants, bone allotransplants undergo rigorous processing and are considered non-viable tissue. In this study, we propose that donor genetic material may exist in circulation after bone allotransplantation. Fifty-one female patients who received bone allotransplants from male donors were assessed. Blood plasma samples were analyzed using real-time quantitative polymerase chain reaction (PCR) with dual labeled fluorogenic probes for the presence of the SRY gene on the Y chromosome. Of the total 51 patients, the SRY sequence was detected in 6 patients. Five were positive at day 1 postoperatively and negative thereafter, with the remaining patient positive at 3 months post-transplantation. Our results document, for the first time, the presence of donor DNA in the circulation of recipients after bone allotransplantation. Our findings suggest a potential new investigative tool to assess the postoperative status of bone allotransplants.

Bone Remodeling↗
PubMed2005

Porcine small intestine submucosa (SIS) is not an acellular collagenous matrix and contains porcine DNA: possible implications in human implantation.

Porcine small intestinal submucosa (SIS) has been recommended as a cell-free, biocompatible biomaterial for the repair of rotator cuff tendon tear. However, we have observed noninfectious edema and severe pain in patients who have undergone SIS implantation for tendon repair. The aim of this study was to conduct an independent assessment of the safety and efficacy of Restore SIS membrane. The Restore orthobiologic implant was examined by histology and the nested PCR technique using porcine immunoreceptor DAP12 gene to examine if SIS membrane contained porcine cells or DNA, respectively. The material was also implanted into mice and rabbits for the evaluation of biological reaction and inflammatory response. Restore SIS was found to contain multiple layers of porcine cells. Chloroacetate esterase staining showed that some of these cells were mast cells. Nested PCR of the DAP12 gene demonstrated that Restore SIS contained porcine DNA material. Subcutaneous implantation of Restore SIS membrane in mice, and in rabbits for rotator cuff tendon repair, showed that the membrane caused an inflammatory reaction characterized by massive lymphocyte infiltration. In conclusion, Restore SIS is not an acellular collagenous matrix, and contains porcine DNA. Our results contradict the current view that Restore SIS is a cell-free biomaterial, and that no inflammatory response is elicited by its implantation. We suggest that further studies should be conducted to evaluate the clinical safety and efficacy of SIS implant biomaterials.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Confocal laser scanning microscopy in orthopaedic research.

Confocal laser scanning microscopy (CLSM) is a type of high-resolution fluorescence microscopy that overcomes the limitations of conventional widefield microscopy and facilitates the generation of high-resolution 3D images from relatively thick sections of tissue. As a comparatively non-destructive imaging technique, CLSM facilitates the in situ characterization of tissue microstructure. Images generated by CLSM have been utilized for the study of articular cartilage, bone, muscle, tendon, ligament and menisci by the foremost research groups in the field of orthopaedics including those teams headed by Bush, Errington, Guilak, Hall, Hunziker, Knight, Mow, Poole, Ratcliffe and White. Recent evolutions in techniques and technologies have facilitated a relatively widespread adoption of this imaging modality, with increased "user friendliness" and flexibility. Applications of CLSM also exist in the rapidly advancing field of orthopaedic implants and in the investigation of joint lubrication.

Animals↗
PubMed2004

Bisphosphonates induce apoptosis of stromal tumor cells in giant cell tumor of bone.

Giant cell tumour of bone (GCT) is an aggressive primary neoplasm that results in the production of osteolytic lesions. Stromal cells, which form the main neoplastic component of this tumor, regulate the formation of osleoclast-like giant cells that are ultimately responsible for bone destruction. Bisphosphonates prevent bone resorption by inhibiting osteoclast activity and promoting osteoclast apoptosis, and they have been known to induce apoptosis of primary neoplastic cells such as those in breast and prostate cancers. We hypothesized that in bisphosphonates may induce apoptosis not only in osteoclast-like giant cells but also in neoplastic stromal cells of GCT both in vitro and in vivo. Twelve patients with GCT were treated with weekly injections of pamidronate for a period of 6 weeks prior to surgery. GCT specimens were collected at the time of biopsy and during definitive surgery. TUNEL assay was used to evaluate apoptotic DNA fragmentation in cells. In addition, twelve GCT primary cultures from these patients were treated with zoledronate, pamidronate, or alendronate for 48 hours at different doses (3, 30, or 150 microM) and subjected to apoptosis assay by flow cytometry following fluorescent Annexin-V labeling. The results showed that pamidronate significantly induced apoptosis in both osteoclast-like giant cells and stromal tumor cells, in vivo. All three bisphosphonates caused substantial apoptosis of stromal tumor cells in cultures. Zoledronate was the most potent reagent, resulting in an average cell death of 27.41% at 150 microM, followed by pamidronate (22.23%) and alendronate (15.3%). Our observations suggest that these drugs may be considered as potential adjuvants in the treatment of GCT.

Alendronate↗
PubMed2004

Interaction between osteoblast and osteoclast: impact in bone disease.

The intercellular communication between osteoblasts and osteoclasts is crucial to bone homeostasis. Since Rodan and Martin proposed the control of osteoclasts by osteoblasts in the 1980s, many factors have been isolated from osteoblasts and shown to regulate the differentiation and function of osteoclasts. However, the mechanism by which osteoblasts regulate osteoclasts during bone remodelling is still unclear. On the other hand, it is well accepted that many metabolic bone diseases are associated with the disruption of the communication between osteoblast and osteoclasts. Thus, this review focuses on the cross-talk between osteoblasts and osteoclasts and its impact in bone disease.

Animals↗
PubMed2004

Molecular characterisation of chondrocytes in autologous chondrocyte implantation.

Autologous chondrocyte implantation (ACI) relies on the use of cultured cells. However, the biosynthetic profile of cultured chondrocytes is shown to be altered during in vitro expansion. The purpose of this study therefore, was to examine the cellular phenotype of chondrocytes cultured for ACI and to determine the apoptotic index of cells implanted into patients. Using electron microscopy, immunohistochemistry, RT-PCR and flow cytometry analyses, we have investigated protein and gene expression of several chondrocyte-specific, or associated markers in cultured cells used for implantation in patients. They included S-100, type I and II collagen, aggrecan, transforming growth factor beta, glucocorticoid receptor alpha and beta and vitamin D3 receptor. We have also examined the apoptotic index of chondrocytes. Our results demonstrated that cultured cells for ACI display the characteristics of chondrocytes. These cells are round in shape, contain numerous small surface processes of cytoplasmic membrane and have an accumulation of glycogen within the cytoplasm. They express S-100, aggrecan TGF-beta, glucocorticoid receptor alpha and vitamin D3 receptor as evidenced by either immunohistochemistry or RT-PCR, however, there is variation in the expression of type I, type II collagen glucocorticoid receptor beta between cases. Chondrocyte used for implantation has relatively low level of apoptosis (<11%). In conclusion, although there was variation in the level of expression of these genetic markers, our data indicate that cultured cells used for ACI were of chondrocytic lineage cells and have low level of apoptotic cells.

Adult↗
PubMed2003

Expression of VEGF and MMP-9 in giant cell tumor of bone and other osteolytic lesions.

This study aims to investigate the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) in giant cell tumor of bone (GCT) and other osteolytic lesions in bone. By using semi-quantitative RT-PCR, we showed that three major isoforms of VEGF (121, 165 and 189) were expressed in GCTs, with isoform 121 being the most abundant. The expression levels of VEGF and MMP-9 mRNA were significantly higher in advanced GCTs (stage II/III) than in stage I GCTs. We further elucidated the cellular localization of VEGF and MMP-9 gene transcripts in GCT and other osteolytic lesions using an in situ hybridization assay. The results showed that stromal tumor cells and osteoclast-like giant cells of GCT, fibrous stromal cells in anuerysmal bone cysts and fibrous dysplasia, and Langerhans-type giant cells as well as histocytes in eosinophillic granuloma, were all strongly positive for VEGF and MMP-9 mRNA expression. In a prospective study, we performed VEGF and MMP-9 immuno-staining on paraffin sections of pathological tissues harvested from 48 patients (14 GCT, 10 anuerysmal bone cysts, 10 eosinophillic granuloma, 4 fibrous dysplasia, 2 simple bone cyst, 2 osteomyelitis and 6 patients with fractured femoral head as control). The results showed that the differences in VEGF and MMP-9 expression between Stage I and other advanced Stages (II, III and recurrent) were highly significant (p<0.001), with advanced stages showing a higher mean expression. The difference between recurrent and Stage II and III lesions, was also statistically significant (p=0.03 for VEGF, and p=0.01 for MMP-9 expression), with recurrent lesions showing a higher mean expression of both VEGF and MMP-9. In conclusion, VEGF and MMP-9 expression in osteolytic lesions of bone co-relates well with the extent of bone destruction and local recurrence. Their expression may therefore provide some prognostic indication of the possible aggressive behavior of the underlying pathology.

Bone Cysts↗
PubMed2003

Receptor activator of NF-kappaB ligand (RANKL) is expressed in chondroblastoma: possible involvement in osteoclastic giant cell recruitment.

AIMS: Chondroblastoma is a rare, locally aggressive bone tumour that causes osteolytic destruction at the epiphyseal end of the affected bone. It is possible that tumour cells may stimulate osteoclastogenesis and osteolytic destruction through the production of receptor activator of NF-kappaB ligand (RANKL), which is a key molecule essential for regulating osteoclast formation and activity. Therefore, the expression of RANKL at both the mRNA and the protein level was investigated in chondroblastoma tumour tissue obtained from patients. METHODS: The expression of RANKL gene transcripts was analysed by the reverse transcription-polymerase chain reaction (RT-PCR), and the cellular localisation of RANKL mRNA and protein was demonstrated by means of in situ hybridisation and immunohistochemistry. RESULTS: RT-PCR analysis indicated that RANKL mRNA was present in all chondroblastoma specimens and normal cancellous bone samples, but not in normal articular cartilage and chondrosarcoma tissues. In contrast, gene transcripts of osteoprotegerin (OPG), the decoy receptor of RANKL, were detected in all types of tissues. The chondroid origin of neoplastic mononuclear cells in chondroblastoma was confirmed by positive S-100 immunohistochemical staining. Both RANKL mRNA and protein were exclusively expressed in these neoplastic mononuclear cells. CONCLUSIONS: These findings suggest that RANKL may be involved in the tumour cell induced recruitment of osteoclast-like cells and consequent osteolytic bone destruction in chondroblastoma.

Adolescent↗
PubMed2003

Expression of RANKL and OPG mRNA in periodontal disease: possible involvement in bone destruction.

Periodontitis is a complex, multifactorial process affected by bacterial plaque-components and host defense mechanisms. Inflammation of the periodontitium may lead the destruction of the underlying ligament and alveolar bone. Receptor activator of NF-kappaB ligand (RANKL), a novel TNF receptor-related protein is an important factor for osteoclast differentiation and activation. Given osteolysis by osteoclast has been demonstrated in periodontitis, we hypothesized that RANKL expression may be associated with bone destruction in periodontitis. We used semi-quantitative RT-PCR to compare the gene expression of RANKL and osteoprogerin (OPG), a decoy receptor of RANKL, between moderate and advanced periodontitis, and healthy subjects. The level of RANKL mRNA was highest in advanced periodontitis. In contrast, the level of OPG mRNA in both advanced and moderate periodontitis was lower than that in the healthy group. It appears that the ratio of RANKL to OPG mRNA in periodontitis has increased. To determine the localization of RANKL gene transcripts in gingival tissue at the cellular level, in situ hybridization was performed using digoxigenin-labeled specific riboprobes. RANKL mRNA was expressed in inflammatory cells, mainly lymphocyte and macrophages. In addition, proliferating epithelium in the vicinity of inflammatory cells expressed high levels of RANKL mRNA. In short, our data suggest that up regulation of RANKL mRNA in both inflammatory cells and epithelium may be associated with the activation of osteoclastic bone destruction in periodontitis.

Adult↗
PubMed2002

Rab3D: a regulator of exocytosis in non-neuronal cells.

Rab3D, a small Ras-like GTPase, is a key regulator of intracellular vesicle transport during exocytosis. It has been shown that Rab3 GTPases are abundant in cells with regulated secretory pathways and are thought to confer the specificity of docking and fusion during regulated exocytosis. Unlike other Rab3 isoforms, Rab3D is enriched in a number of non-neuronal tissues and is localised to secretory granules in the cytoplasm of these cells. The structure of Rab3D exhibits all of the conserved domains from the Rab family and also contains hypervariable N- and C-terminal regions. Rab3D undergoes post-translational isoprenylation and cycles between GDP- and GTP-bound forms. Apart from the factors involved in the Rab activation cycle, few Rab3D effector proteins have been identified to date. Nevertheless, it has long been suggested that Rab3D plays a role in regulated exocytotic processes as well as apically directed transcytosis. This review summarises the recent work on the biological function, structural integrity and molecular interactions of Rab3D in non-neuronal cells.

Amino Acid Sequence↗
PubMed2001

Gene expression of glucocorticoid receptor alpha and beta in giant cell tumour of bone: evidence of glucocorticoid-stimulated osteoclastogenesis by stromal-like tumour cells.

Glucocorticoids have been shown to increase bone resorption in vitro and in vivo, however, the mechanism(s) of this action are not fully understood. Given that human giant cell tumour of bone (GCT) is considered to arise from mesenchymal stromal cells and has the capacity to recruit and harbour macrophages and multinucleated osteoclasts, we have used GCT as a model for studying the effect of glucocorticoids on osteoclast formation. We have demonstrated, by RT-PCR and fluorescence in-situ hybridisation, that both glucocorticoid receptor alpha and beta (GRalpha and GRbeta) gene transcripts were present in the stromal-like tumour cells, macrophage-like cells (putative osteoclast precursors) and multinucleated osteoclast-like cells. Moreover, in the presence of 1,25(OH)(2)D(3), dexamethasone dose-dependently stimulated the formation of osteoclast-like cells from GCT-derived co-culture system of stromal-like tumour cells and macrophage-like cells. The stimulation of osteoclastogenesis by dexamethasone was coincident with the up-regulation of receptor activator of NF-kappaB ligand (RANKL) but down-regulation of osteoprotegerin (OPG) gene expression in stromal-like tumour cells. These data are consistent with the hypothesis that glucocorticoids increase bone resorption by promoting osteoclastogenesis which is at least in part due to the stimulation of RANKL and inhibition of OPG production in bone stromal cells.

Cells, Cultured↗
PubMed2001

Establishment of bee venom-induced contralateral heat hyperalgesia in the rat is dependent upon central temporal summation of afferent input from the site of injury.

The present investigation was designed to study whether central sensitization is determined by a time window of central summation of ongoing primary afferent input from a peripheral injury site. Sensitization was assessed behaviorally in the rat as contralateral heat hyperalgesia induced by injection of bee venom (BV) in the hind paw. The sciatic nerve was transected at various time points following intraplantar BV injection to analyze the time window for contralateral hyperalgesia. The results show that after a dose of 0.2 mg BV, axotomy at 5 min completely prevented contralateral heat hyperalgesia but was without effect at 10 min, whereas after a dose of 0.1 mg BV, axotomy at 10 min was able to prevent the contralateral heat hyperalgesia but remained without effect at 20 min. These findings suggest an important role of the amount of ipsilateral ongoing primary afferent in establishing the contralateral heat hyperalgesia. Moreover, by counting the total amount of paw flinches that is believed to be mediated by ongoing primary afferent input, it was shown that 87.35+/-5.36, 170.50+/-9.15 and 305.80+/-20.13 flinches were induced by 0.2 mg BV for a period of 5, 10 and 20 min, respectively. At the lower dose of 0.1 mg BV significant fewer flinches were elicited with 59.17+/-13.54, 133.00+/-22.33 and 234.00+/-36.42 within the three corresponding time windows before sciatic nerve transection. The results suggest that the amount of primary afferent input determines the time window required to establish central changes that are independent of further afferent input.

Afferent Pathways↗