Shao- Hung

Proliferation and differentiation potential of human adipose‐derived mesenchymal stem cells isolated from elderly patients with osteoporotic fractures

Aging has less effect on adipose‐derived mesenchymal stem cells (ADSCs) than on bone marrow‐derived mesenchymal stem cells (BMSCs), but whether the fact holds true in stem cells from elderly patients with osteoporotic fractures is unknown. In this study, ADSCs and BMSCs of the same donor were harvested and divided into two age groups. Group A consisted of 14 young patients (36.4 ± 11.8 years old), and group B consisted of eight elderly patients (71.4 ± 3.6 years old) with osteoporotic fractures. We found that the doubling time of ADSCs from both age groups was maintained below 70 hrs, while that of BMSCs increased significantly with the number of passage. When ADSCs and BMSCs from the same patient were compared, there was a significant increase in the doubling time of BMSCs in each individual from passages 3 to 6. On osteogenic induction, the level of matrix mineralization of ADSCs from group B was comparable to that of ADSCs from group A, whereas BMSCs from group B produced least amount of mineral deposits and had a lower expression level of osteogenic genes. The p21 gene expression and senescence‐associated β‐galactosidase activity were lower in ADSCs compared to BMSCs, which may be partly responsible for the greater proliferation and differentiation potential of ADSCs. It is concluded that the proliferation and osteogenic differentiation of ADSCs were less affected by age and multiple passage than BMSCs, suggesting that ADSCs may become a potentially effective therapeutic option for cell‐based therapy, especially in elderly patients with osteoporosis.

Parathyroid hormone 1-34 inhibits terminal differentiation of human articular chondrocytes and osteoarthritis progression in rats.

OBJECTIVE Parathyroid hormone 1-34 (PTH[1-34]), a parathyroid hormone analog, shares the same receptor, PTH receptor 1, with parathyroid hormone-related peptide (PTHrP). This study was undertaken to address the hypothesis that PTH(1-34) inhibits terminal differentiation of articular chondrocytes and in turn suppresses the progression of osteoarthritis (OA). METHODS We studied the effect of PTH(1-34) on human articular chondrocytes with azacytidine (azaC)-induced terminal differentiation in vitro and on papain-induced OA in the knee joints of rats. In the in vitro study, we measured the levels of messenger RNA for SOX9, aggrecan, type II collagen, type X collagen, alkaline phosphatase (AP), Indian hedgehog (IHH), Bcl-2, and Bax by real-time polymerase chain reaction, levels of glycosaminoglycan (GAG) by dimethylmethylene blue assay, and rate of apoptosis by TUNEL staining. In the in vivo study, we evaluated the histologic changes in GAG, type II collagen, type X collagen, and chondrocyte apoptosis in the articular cartilage of rat knees. RESULTS AzaC induced terminal differentiation of human chondrocytes, including down-regulation of aggrecan, type II collagen, and GAG and up-regulation of type X collagen, alkaline phosphatase, and IHH. Apoptosis was reversed by 3-10 days of treatment with 10 nM PTH(1-34). SOX9 expression was not changed by either azaC or PTH(1-34) treatment. Bcl-2 and Bax were up-regulated on day 10 and day 14, respectively, after azaC induction of terminal differentiation, but PTH(1-34) treatment did not reverse this effect. Furthermore, PTH(1-34) treatment reversed papain-induced OA changes (decreasing GAG and type II collagen, and increasing type X collagen and chondrocyte apoptosis) in the knee joints of rats. CONCLUSION Our findings indicate that PTH(1-34) inhibits the terminal differentiation of human articular chondrocytes in vitro and inhibits progression of OA in rats in vivo, and may be used to treat OA.

Simvastatin increases osteoblasts and osteogenic proteins in ovariectomized rats

Background  Previous reports have indicated that statins could prevent bone loss in ovariectomized (OVX) rats and increase the expressions of osteogenic genes in cultured osteoblasts. In this study, we hypothesized that simvastatin might increase osteoblast number and protein expressions of osteogenic markers localized in bones in concomitance with the prevention of bone loss in OVX rats.

(–)–Epigallocatechin Gallate Inhibition of Osteoclastic Differentiation via NF-Κb

People who regularly drink tea have been found to have a higher bone mineral density (BMD) and to be at less risk of hip fractures than those who do not drink it. Green tea catechins such as (-)-epigallocatechin gallate (EGCG) have been reported to increase osteogenic functioning in mesenchymal stem cells. However, its effect on osteoclastogenesis remains unclear. In this study, we investigated the effect of EGCG on RANKL-activation osteoclastogenesis and NF-kappaB in RAW 264.7, a murine preosteoclast cell line. EGCG (10-100 microM) significantly suppressed the RANKL-induced differentiation of osteoclasts and the formation of pits in murine RAW 264.7 cells and bone marrow macrophages (BMMs). EGCG appeared to target osteoclastic differentiation at an early stage but had no cytotoxic effect on osteoclast precursors. In addition, it significantly inhibited RANKL-induced NF-kappaB transcriptional activity and nuclear translocation. We conclude that EGCG inhibits osteoclastogenesis through its activation of NF-kappaB.

Semitubular Plates for Acutely Displaced Midclavicular Fractures: A Retrospective Study of 111 Patients Followed for 2.5 to 6 Years

Objectives: We designed this study to determine the usefulness of semitubular plates for acute displaced or comminuted fractures of the midclavicle. Design: Nonrandomized retrospective study. Setting: A secondary transfer hospital specializing in orthopaedics. Patients: From May 1997 to July 2001, 121 patients were treated with a 92% (111) follow-up rate. The mean follow-up time was 3.5 years (range, 2.5 to 6 years). Intervention: Semitubular plates using 4.5-mm cortical or 6.5-mm cancellous screws and wire as necessary. Main Outcome Measurement: The functional result was evaluated by the Disabilities of the Arm, Shoulder and Hand (DASH) score at the time of admission for implant removal in 82 patients or at the end of follow-up by telephone in 29 patients. Results: Most (107 of 111) fractures healed within 6 months. Three patients with implant failure due to backing out of the screws healed after surgical revision. One patient had an infected nonunion with a poor result. The other 110 patients had good results. No implant breakage was noted. No other major complications were noted except for 1 deep infection. No bone graft was needed, even with comminution at the fracture site. Of the 107 patients with uneventful union, 82 had hardware removal. The other 25 were diagnosed as having union both radiographically and clinically and did not have their hardware removed. Conclusion: Overall, 95% of patients were satisfied with the surgical procedure. We suggest that a semitubular plate with 4.5-mm cortical and 6.5-mm cancellous screws with wire augmentation if necessary is a reliable procedure for acute severely displaced or comminuted midclavicular fractures.

Surgical treatment for ipsilateral fractures of the hip and femoral shaft

Concomitant ipsilateral femoral shaft and neck fractures are difficult to treat. There is still no consensus on the optimal treatment of these complex fractures. Forty-seven patients with these complex fractures were treated in Kaohsiung Medical University Hospital between the periods of 1982 and 1998. Our standard treatment protocol is plate fixation for femoral shaft fracture and lag screw or dynamic hip screw (DHS) fixation for hip fracture. Among 42 cases treated with this protocol, 34 were males and 8 were females with an average age of 36 years and average follow-up period of 55 months. We divided hip fractures into two groups: femoral neck fracture as group I and intertrochanteric fracture as group II. There were no non-union and osteonecrosis of the hip in either group. One diaphyseal non-union was observed in group I and four in group II. There were 92 and 76% good functional results in groups I and II, respectively. The result shows that our standard method can yield a reliable outcome in group I, but not in group II.

Pioglitazone and dexamethasone induce adipogenesis in D1 bone marrow stromal cell line, but not through the peroxisome proliferator-activated receptor-γ pathway

Osteoblasts and adipocytes share a common progenitor in bone marrow. Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) plays a critical role in adipogenesis. Using a mouse pluripotent mesenchymal cell, D1, as a model, several reports have demonstrated that dexamethasone, a glucocorticoid, can induce adipogenesis. We first examined whether adipogenesis induction in D1 cells is initiated by activation of PPAR-gamma. The results revealed that pioglitazone induces adipogenesis in D1 cells in a dose-dependent manner and decreases alkaline phosphatase activity in D1 cells. Interestingly, this adipogenesis was not blocked by bisphenol A diglycidyl ether, a PPAR-gamma antagonist. A PPAR-gamma-mediated reporter gene assay showed no response to pioglitazone. We then asked whether dexamethasone-induced adipogenesis can be repressed by mifepristone (RU486), an antagonist of glucocorticoid receptor. The results disclosed that mifepristone cannot counteract dexamethasone-induced adipogenesis, and mifepristone itself induced adipogenesis in D1 cells. Moreover, glucocorticoid receptor-mediated reporter gene assay was not responsive to dexamethasone or mifepristone. We concluded that the adipogenesis induced by pioglitazone and dexamethasone in D1 cells may not occur via a PPAR-gamma and glucocorticoid receptor pathway. Finally, we analyzed the gene expression profile of D1 by cDNA microarray after treatment with dexamethasone. We found that the expression of several adipogenesis-related genes is highly provoked by this agent.

Ankle Arthrodesis: Internal Non-Compression Arthrodesis versus Internal Compression Arthrodesis

Ankle arthrodesis is still considered to be the standard treatment for most disabling types of ankle arthritis, but fusion methods are varied. We report our experience of ankle arthrodesis and compare a group of 34 cases treated by Blairs non-compression arthrodesis to another group of 32 cases treated by internal compression arthrodesis using two crossed screws. The same surgeon performed all the operations. The Blairs non-compression arthrodesis group included 21 males and 13 females with an average age of 42 y/o (range 18-70 y/o) and an average follow up period of 38.6 months (range 26-62 months). The union rate was 91.2% and the average union time was 5.6 months (range 2-10 months). There were three cases of non-union. The cross-screw compression arthrodesis group included 20 males and 12 females with an average age of 45 y/o (range 20-86 y/o) and an average follow up period of 38.3 months (range 15-81 months). The union rate was 96.9% and the average union time was 2.7 months (range 1.5-4.4 months). There was one case of non-union. We conclude that our cross-screws compression arthrodesis with its shorter fusion time was found to be superior to the Blairs non-compression arthrodesis.

A novel terminal differentiation model of human articular chondrocytes in three-dimensional cultures mimicking chondrocytic changes in osteoarthritis

This study establishes a cell culture model mimicking the terminal differentiation occurring in osteoarthritic chondrocytes. Normal articular chondrocytes obtained from human knees treated with 5‐azacytidine (Aza‐C) were harvested 3, 7 and 14 days after treatment. Phenotypic and genetic changes of articular chondrocytes were detected. The results show that mRNA expression of collagen type II, a marker for normal functional articular chondrocytes, was significantly decreased after Aza‐C treatment in comparison to the control cultures, while those of collagen type X and ALP, markers for hypertrophic chondrocytes, were significantly increased. Cell size and apoptotic rate of articular chondrocytes showed significant increases compared to the control after 14 days of Aza‐C treatment. Terminal differentiation is shown by this model of three‐dimensional cultured human articular chondrocytes, which could apply to the studies of the cellular mechanisms of osteoarthritis.

Combining Paclitaxel with ABT-263 Has a Synergistic Effect on Paclitaxel Resistant Prostate Cancer Cells

We assessed the capability of paclitaxel, one of the taxanes, to induce death in two prostate cancer lines, LNCaP and PC3. Paclitaxel drove an apoptotic pathway in LNCaP, but not in PC3 cells, in response to G2/M arrest. An examination of the levels of anti-apoptotic proteins revealed that Bcl-xl was much higher in PC3 cells than in LNCaP cells and Bcl2 could be detected only in PC3 cells, not in LNCaP cells. Knocking down Bcl-xl enhanced paclitaxel-induced apoptosis in LNCaP cells, while we were unable to knock down Bcl-xl efficiently in PC3 cells. Significantly, a comparison of ABT-263, a specific inhibitor of Bcl2 and Bcl-xl, with ABT-199, a Bcl2 selective inhibitor, disclosed that only ABT-263, not ABT-199, could induce apoptosis in LNCaP and PC3 cells. The results indicate that Bcl-xl has a protective role against paclitaxel-induced apoptosis in LNCaP and PC3 cells, and its overexpression causes the paclitaxel resistance seen in PC3 cells. Interestingly, combined paclitaxel with ABT-263 to treat LNCaP and PC3 cells demonstrated synergistic apoptosis activation, indicating that ABT-263 could enhance paclitaxel-induced apoptosis in LNCaP cells and overcome Bcl-xl overexpression to trigger paclitaxel-induced apoptosis in PC3 cells. We also observed that the activation of apoptosis in LNCaP cells was more efficient than in PC3 cells in response to paclitaxel plus ABT-263 or to ABT-263 alone, suggesting that the apoptosis pathway in PC3 cells might have further differences from that in LNCaP cells even after Bcl-xl overexpression is accounted for.