Biomechanical investigation of dynamic hip screw and wire fixation on an unstable intertrochanteric fracture.

BACKGROUND: Although use of a dynamic hip screw (DHS) for stable intertrochanteric hip fracture fixation has been successfully applied in fracture healing for more than 20 years, DHS fixation on unstable intertrochanteric fractures still has a high failure rate, especially in patients with osteoporosis. Although the wire fixation is usually incorporated with orthopedic device to treat fracture, the wiring techniques are developed through experiences. Thus, this study is objective to investigate the biomechanical property of different wire fixation methods incorporated with DHS system to provide the lesser trochanter fragment stable fixation on osteoporotic TypeA2.1 fracture for enhancing stability after bone reduction. RESULTS: Sawbone testing results demonstrated higher maximum load, stiffness, and energy in a DHS with wire fixation compared with DHS fixation only. In static biomechanical testing of a cadaver femur, we compared the stiffness of five fixation models and then tested a fatigue failure model in cycle loading with DHS fixation only. Wiring fixation can enhance stability and the cut-out failure model in the fatigue test was identical to the clinical failure model. CONCLUSIONS: Lesser trochanteric fragment fixation is a crucial concern in the stability of an A2.1 unstable fracture, and the combination of a wiring technique with a DHS seems beneficial for achieving better stability. The addition of an antirotational greater trochanter is likely to enhance stability through wiring of the greater trochanter.

(-)-Epigallocatechin-3-Gallate (EGCG) Enhances Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells.

Osteoporosis is the second most-prevalent epidemiologic disease in the aging population worldwide. Cross-sectional and retrospective evidence indicates that tea consumption can mitigate bone loss and reduce risk of osteoporotic fractures. Tea polyphenols enhance osteoblastogenesis and suppress osteoclastogenesis in vitro. Previously, we showed that (-)-epigallocatechin-3-gallate (EGCG), one of the green tea polyphenols, increased osteogenic differentiation of murine bone marrow mesenchymal stem cells (BMSCs) by increasing the mRNA expression of osteogenesis-related genes, alkaline phosphatase activity and, eventually, mineralization. We also found that EGCG could mitigate bone loss and improve bone microarchitecture in ovariectomy-induced osteopenic rats, as well as enhancing bone defect healing partially via bone morphogenetic protein 2 (BMP2). The present study investigated the effects of EGCG in human BMSCs. We found that EGCG, at concentrations of both 1 and 10 µmol/L, can increase mRNA expression of BMP2, Runx2, alkaline phosphatase (ALP), osteonectin and osteocalcin 48 h after treatment. EGCG increased ALP activity both 7 and 14 days after treatment. Furthermore, EGCG can also enhance mineralization two weeks after treatment. EGCG without antioxidants also can enhance mineralization. In conclusion, EGCG can increase mRNA expression of BMP2 and subsequent osteogenic-related genes including Runx2, ALP, osteonectin and osteocalcin. EGCG further increased ALP activity and mineralization. Loss of antioxidant activity can still enhance mineralization of human BMSCs (hBMSCs).

Suppression of discoidin domain receptor 1 expression enhances the chondrogenesis of adipose-derived stem cells.

Effectively directing the chondrogenesis of adipose-derived stem cells (ADSCs) to engineer articular cartilage represents an important challenge in ADSC-based articular cartilage tissue engineering. The discoidin domain receptor 1 (DDR1) has been shown to affect cartilage homeostasis; however, little is known about the roles of DDR1 in ADSC chondrogenesis. In this study, we used the three-dimensional culture pellet culture model system with chondrogenic induction to investigate the roles of DDR1 in the chondrogenic differentiation of human ADSCs (hADSCs). Real-time polymerase chain reaction and Western blot were used to detect the expression of DDRs and chondrogenic genes. Sulfated glycosaminoglycan (sGAG) was detected by Alcian blue and dimethylmethylene blue (DMMB) assays. Terminal deoxy-nucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining was used to assess cell death. During the chondrogenesis of hADSCs, the expression of DDR1 but not DDR2 was significantly elevated. The depletion of DDR1 expression in hADSCs using short hairpin RNA increased the expression of chondrogenic genes (SOX-9, collagen type II, and aggrecan) and cartilaginous matrix deposition (collagen type II and sGAG) and only slightly increased cell death (2-8%). DDR1 overexpression in hADSCs decreased the expression of chondrogenic genes (SOX-9, collagen type II, and aggrecan) and sGAG and enhanced hADSC survival. Moreover, DDR1-depleted hADSCs showed decreased expression of the terminal differentiation genes runt-related transcription factor 2 (Runx2) and matrix metalloproteinase 13 (MMP-13). These results suggest that DDR1 suppression may enhance ADSC chondrogenesis by enhancing the expression of chondrogenic genes and cartilaginous matrix deposition. We proposed that the suppression of DDR1 in ADSCs may be a candidate strategy of genetic modification to optimize ADSC-based articular cartilage tissue engineering.

Single-pulsed electromagnetic field therapy increases osteogenic differentiation through Wnt signaling pathway and sclerostin downregulation.

Pulsed electromagnetic field (PEMF) therapy has been used for more than three decades to treat bone diseases. The main complaint about using PEMF is that it is time-consuming. Previously, we showed single-pulsed electromagnetic field (SPEMF) applied for 3 min daily increased osteogenic differentiation of mesenchymal stem cells and accelerated bone growth in a long bone defect model. In the current study, we investigated the mechanism of SPEMF to increase osteogenic differentiation in osteoblastic cells. We found that both short-term (SS) and long-term (SL) SPEMF treatment increased mineralization, while alkaline phosphatase (ALP) activity increased during the first 5 days of SPEMF treatment. SS treatment increased gene expression of Wnt1, Wnt3a, Wnt10b, Fzd9, ALP, and Bmp2. Also, SPEMF inhibited sclerostin after 5 days of treatment, and that inhibition was more significant with SL treatment. SL SPEMF increased expression of parathyroid hormone-related protein (PTHrP) but decreased expression of Sost gene, which encodes sclerostin. Together, the early osteogenic effect of SPEMF utilizes the canonical Wnt signaling pathway while the inhibitory effect of long-term SPEMF on sclerostin may be attributable to PTHrP upregulation. This study enhances our understanding of cellular mechanisms to support the previous finding and may provide new insight for clinical applications.

PPARγ silencing enhances osteogenic differentiation of human adipose-derived mesenchymal stem cells.

Peroxisome proliferator-activated receptor gamma (PPARγ) is the master regulator of adipogenesis, and has been indicated as a potential therapeutic target to promote osteoblast differentiation. However, recent studies suggest that suppression of PPARγ inhibits adipogenesis, but does not promote osteogenic differentiation in human bone marrow-derived mesenchymal stem cells (hBMSCs). It was reasoned that the osteogenic effect of PPARγ suppression may be masked by the strong osteogenesis-inducing condition commonly used, resulting in a high degree of matrix mineralization in both control and experimental groups. This study investigates the role of PPARγ in the lineage commitment of human adipose-derived mesenchymal stem cells (hADSCs) by interfering with the function of PPARγ mRNA through small interfering RNAs (siRNAs) specific for PPARγ2. By applying an osteogenic induction condition less potent than that used conventionally, we found that PPARγ silencing led to retardation of adipogenesis and stimulated a higher level of matrix mineralization. The mRNA level of PPARγ decreased to 47% of control 2 days after treatment with 50 nmol/l PPARγ2 siRNA, while its protein expression was 60% of mock control. In the meantime, osteogenic marker genes, including bone morphogenic protein 2 (BMP2), runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP) and osteocalcin (OC), were up-regulated under PPARγ silencing. Our results suggest that transient suppression of PPARγ promotes the onset of osteogenesis, and may be considered a new strategy to stimulate bone formation in bone tissue engineering using hADSCs.

Exogenous polyamines promote osteogenic differentiation by reciprocally regulating osteogenic and adipogenic gene expression.

Polyamines are naturally occurring organic polycations that are ubiquitous in all organisms, and are essential for cell proliferation and differentiation. Although polyamines are involved in various cellular processes, their roles in stem cell differentiation are relatively unexplored. In this study, we found that exogenous polyamines, putrescine, spermidine, and spermine, promoted osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) without inducing cell death or apoptosis. Alkaline phosphatase (ALP) activity and the mRNA level of osteogenic genes, including Runx2, ALP, osteopontin, and osteocalcin, were up-regulated by exogenous polyamines. When hBMSCs were cultured at high cell density favoring adipocyte formation, exogenous polyamines resulted in down-regulation of adipogenic genes such as PPARγ, aP2, and adipsin. Extracellular matrix mineralization, a marker for osteoblast maturation, was enhanced in the presence of exogenous polyamines, while lipid accumulation, an indication of adipogenic differentiation, was attenuated. Exogenous polyamines increased the mRNA expression of polyamine-modulated factor 1 (PMF-1) and its downstream effector, spermidine/spermine N(1)-acetyltransferase (SSAT), while that of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, was suppressed. These results lead to possible connections between polyamine metabolism and osteogenic differentiation pathways. To summarize, this study provides evidence for the involvement of polyamines in osteogenic differentiation of hBMSCs, and is the first to demonstrate that osteogenic and adipogenic differentiation are reciprocally regulated by exogenous polyamines.

Alendronate in the prevention of collapse of the femoral head in nontraumatic osteonecrosis: a two-year multicenter, prospective, randomized, double-blind, placebo-controlled study.

OBJECTIVE: Osteonecrosis is one of the major debilitating skeletal disorders. Most patients with osteonecrosis of the femoral head eventually need surgery, usually total hip arthroplasty (THA), within a few years of onset. Previous studies showed that alendronate has a pharmacologic effect in reducing osteoclast activity and that it significantly reduced the incidence of collapse of the femoral head in the osteonecrotic hip. The purpose of this study was to determine the cumulative incidence of THA in patients with osteonecrosis of the femoral head and the time-to-event after treatment with alendronate versus placebo during the study period. METHODS: A 2-year multicenter, prospective, randomized, double-blind study was performed. From June 2005 to December 2006, 64 patients were enrolled and randomly assigned to the alendronate or placebo group. In patients with bilateral hip osteonecrosis who met the inclusion criteria, both hips were counted in the analyses. Five patients were excluded from the analysis because they did not comply with any of the study regimens. Seven patients were ineligible because they were not diagnosed as having stage IIC or stage IIIC disease according to the University of Pennsylvania system. Thus, a total of 52 patients (65 hips) were assessed in this study. Disease progression was evaluated by radiography and magnetic resonance imaging (MRI). The Harris Hip Score and the Short Form 36 health survey were used to rate hip function and quality of life, respectively. RESULTS: There was no significant difference in radiographic and MRI data between the 2 study groups. Four of 32 hips in the alendronate treatment group underwent THA, while 5 of 33 hips in the placebo group had THA (P = 0.837). No differences were noted in disease progression, Harris Hip Scores, or Short Form 36 scores between the 2 groups. CONCLUSION: Alendronate has no obvious effect on preventing the necessity for THA, reducing disease progression, or improving life quality.

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 ß-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.

Microporation is a valuable transfection method for gene expression in human adipose tissue-derived stem cells.

Stem cells are a promising resource for gene therapy. Adipose tissue-derived stem cells (ADSCs) offer advantages because of their abundance and ease of isolation. However, it is difficult to transduce genes into ADSCs by common transfection methods, especially nonviral methods. We report here the use of a new electroporation method, termed "microporation," to transduce plasmids into human ADSCs (hADSCs). We determined optimal conditions that led to efficient transfection of >76.1% of the microporated hADSCs with only minimal cell damage or cytotoxicity. We demonstrated the expression of both enhanced green fluorescent protein (EGFP) and luciferase from different promoters in microporated hADSCs. More important, the microporated hADSCs retained their multipotency and reporter gene expression was maintained for >2 weeks in vitro and in vivo. We further showed that a Tet-ON-inducible gene expression system could be microporated into hADSCs and that this system was functional following transplantation of the microporated cells into nude mice. Taken together, our data demonstrate that microporation allows a highly efficient transfection of hADSCs, without impairing their stem cell properties.

Single pulsed electromagnetic field restores bone mass and microarchitecture in denervation/disuse osteopenic mice.

Pulsed electromagnetic fields (PEMFs) have been proposed to treat bone loss. However, time-consuming is the main complaint. A time-saving and effective treatment is of expectation. Previously, we showed a 3 min daily of single pulsed electromagnetic field (SPEMF) accelerated bone formation of long bone defect in mice. Here we compared the effect of SPEMF with PEMF for treating denervation/disuse osteopenic mice. Healthy mice were divided into 3 groups: intact mice (INT), INT + PEMF, and INT + SPEMF. Induced osteopenic mice were divided to osteopenia (IOP), IOP + PEMF, and IOP + SPEMF groups. The PEMF treated groups were subjected to daily 8 h PEMF(15 Hz, 18 G) exposure, while SPEMF treated groups were daily 3 min SPEMF(0.2 Hz, 1 T) exposure. BMD was evaluated every two weeks during the 12 weeks of treatment. Microarchitecture was evaluated on week 12. SPEMF significantly reversed bone loss in IOP mice as early as 6 weeks post-treatment, while PEMF reversed bone loss after 8 weeks. Bone volume was significantly increased in the IOP + PEMF and IOP + SPEMF group. Besides, bone volume and trabecular number of IOP + SPEMF mice were restored to the levels of INT mice in 12 weeks. Our finding suggests SPEMF increased BMD and restored microarchitecture of disuse osteopenic mice to healthy level.

A single-pulsed electromagnetic field enhances collagen synthesis in tendon cells.

Tendinopathy is a progressive pathology of tendon that is characteristic of imbalance between matrix synthesis and degeneration and is often caused by failure to adapt to mechanical loading. Non-steroidal anti-inflammatory medications (NSAIDS) are used as a conventional treatment to alleviate pain and swelling in the short term, but the ideal treatment for tendinopathy remains unclear. Here, we show a single pulsed electromagnetic field (SPEMF, 0.2 Hz) that up-regulated tenogenic gene expression (Col1a1, Col3a1, Scx, Dcn) and down-regulated inflammatory gene expression (Mmp1) in vitro. After five days of SPEMF stimulation (3 min/day), the collagen type I and total collagen synthesis protein levels were significantly increased. Under pro-inflammatory cytokine (IL-1ß) irritation, the decreased expression of Col1a1/Col3a1 was up-regulated by SPEMF treatment, and the increased expression of Mmp1 was also reversed. From the above, it can be inferred that SPEMF that enhances matrix synthesis and reduces matrix degeneration may counteract the imbalance in tendinopathy. SPEMF application may be developed as a potential future strategy for therapeutic intervention in tendon disorders.

(-)-Epigallocatechin gallate inhibition of osteoclastic differentiation via NF-kappaB.

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.

Anti-inflammatory drugs suppress proliferation and induce apoptosis through altering expressions of cell cycle regulators and pro-apoptotic factors in cultured human osteoblasts.

It has been reported that anti-inflammatory drugs (AIDs) inhibited bone repair in animal studies, and suppressed proliferation and induced cell death in rat osteoblast cultures. In this study, we further investigated the molecular mechanisms of AID effects on proliferation and cell death in human osteoblasts (hOBs). We examined the effects of dexamethasone (10(-7) and 10(-6)M), non-selective non-steroidal anti-inflammatory drugs (NSAIDs): indomethacin, ketorolac, piroxicam and diclofenac (10(-5) and 10(-4)M), and COX-2 inhibitor: celecoxib (10(-6) and 10(-5)M) on proliferation, cytotoxicity, cell death, and mRNA and protein levels of cell cycle and apoptosis-related regulators in hOBs. All the tested AIDs significantly inhibited proliferation and arrested cell cycle at G0/G1 phase in hOBs. Celecoxib and dexamethasone, but not non-selective NSAIDs, were found to have cytotoxic effects on hOB, and further demonstrated to induce apoptosis and necrosis (at higher concentration) in hOBs. We further found that indomethacin, celecoxib and dexamethasone increased the mRNA and protein expressions of p27(kip1) and decreased those of cyclin D2 and p-cdk2 in hOBs. Bak expression was increased by celecoxib and dexamethasone, while Bcl-XL level was declined only by dexamethasone. Furthermore, the replenishment of PGE1, PGE2 or PGF2alpha did not reverse the effects of AIDs on proliferation and expressions of p27(kip1) and cyclin D2 in hOBs. We conclude that the changes in expressions of regulators of cell cycle (p27(kip1) and cyclin D2) and/or apoptosis (Bak and Bcl-XL) by AIDs may contribute to AIDs caused proliferation suppression and apoptosis in hOBs. This effect might not relate to the blockage of prostaglandin synthesis by AIDs.

Different differentiation of stroma cells from patients with osteonecrosis: a pilot study.

Osteonecrosis (ON) is commonly caused by high doses of glucocorticoids or ethanol intake. We previously reported suppression of BMP-2 by dexamethasone was more pronounced and enhancement by lovastatin was less pronounced in bone marrow stromal cells (BMSCs) from patients with ON than from patients without ON. We therefore presumed the BMSCs might be dysfunctional in patients with ON and performed this pilot study. We obtained BMSCs from 10 patients with ethanol-induced ON, 10 patients with glucocorticoid-induced ON, and 12 patients without ON as control subjects, checking third passage cells for osteogenic and adipogenic gene expression and differentiation ability. BMSCs from patients with glucocorticoid-induced ON possessed less osteogenic gene expression and less osteogenic differentiation, whereas BMSCs from patients with ethanol-induced ON possessed more adipogenic gene expression and more adipogenic differentiation. Dysfunction of BMSCs may be one of the causes of ON, with differing dysfunction in ethanol-induced ON and glucocorticoid-induced ON. Glucocorticoids may possess more of a suppressive effect on osteogenesis than ethanol, whereas ethanol may possess a more potent adipogenic effect than glucocorticoids on BMSCs.

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

Enhancement of chondrogenesis of adipose-derived stem cells in HA-PNIPAAm-CL hydrogel for cartilage regeneration in rabbits.

Injectable thermoresponsive hydrogels have the advantages of effective cell delivery and minimal invasion for tissue engineering applications. In this study, we investigated the chondroinductive potential of newly developed hyaluronic acid (HA)-modified thermoresponsive poly(N-isopropylacrylamide) (HA-PNIPAAm-CL) hydrogels on enhancing rabbit ADSC (rADSC) chondrogenesis in vitro and in the synovial cavity of rabbit. The HA-mixed PNIPAAm (HA-PNIPAAm-CP) and HA-cross-linked PNIPAAm (HA-PNIPAAm-CL) were fabricated using physical interaction and chemical cross-linking methods, respectively. The in vitro results showed that, compared to unmodified PNIPAAm, both HA-modified hydrogels significantly increased cell viability, chondrogenic marker gene (aggrecan and type II collagen) expression and sulfide glycosaminoglycan (sGAG) formation in embedded rADSCs. However, HA-PNIPAAm-CL showed the highest rADSC viability and chondrogenesis. The chondrogenic effects of HA-modified hydrogels on rADSCs were confirmed in vivo by the intraarticular injection of hydrogel-embedded rADSC constructs into rabbit synovial cavities for 3 weeks and tracing with CM-DiI labeling. Neocartilage formation in the hydrogels was determined by histomorphological staining of GAG and type II collagen. In vivo injected rADSC/HA-PNIPAAm-CL constructs showed more hyaline cartilage formation than that of rADSC/HA-PNIPAAm-CP and rADSC/PNIPAAm constructs in the synovial cavity of rabbit. These results suggest that the HA-PNIPAAm-CL provides a suitable microenvironment to enhance ADSC chondrogenesis for articular cartilage tissue engineering applications.

Effects of anti-inflammatory drugs on proliferation, cytotoxicity and osteogenesis in bone marrow mesenchymal stem cells.

Nonsteroidal anti-inflammatory drugs (NSAIDs) were found to suppress proliferation and induce cell death in cultured osteoblasts, and steroids were found to decrease the osteogenesis potential of mesenchymal stem cells. In this study, we further tested the effects of anti-inflammatory drugs (AIDs) on the functions of bone marrow mesenchymal stem cells (BMSCs). The BMSCs from mice (D1-cells) and humans (hBMSCs) were treated with dexamethasone (10(-7) to 10(-6) M), cyclooxygenase-2 (COX-2) selective NSAIDs (10(-6) to 10(-5) M) and non-selective NSAIDs (10(-5) to 10(-4) M). Drug effects on proliferation, cell cycle kinetics, cytotoxicity and mRNA and protein expressions of cell cycle regulators were tested. The osteogenesis potential of D1-cells were evaluated by testing mRNA expressions of type Ialpha collagen and osteocalcin 2-8 days after treatments, and testing mineralization 1-3 weeks after treatments. The results showed that all the tested drugs suppressed proliferation and arrested cell cycle of D1-cells, but no significant cytotoxic effects was found. Prostaglandin E1, E2 and F2alpha couldn't rescue the effects of AIDs on proliferation. The p27kip1 expression was up-regulated by indomethacin, celecoxib and dexamethasone in both D1-cells and hBMSCs. Higher concentrations of indomethacin and dexamethasone also up-regulated p21Cip1/Waf1 expression in hBMSCs, and so did celecoxib on D1-cells. Expressions of cyclin E1 and E2 were down-regulated by these AIDs in D-cells, while only cyclin E2 was down-regulated by dexamethasone in hBMSCs. All the tested NSAIDs revealed no obvious detrimental effects on osteogenic differentiation of D1-cells. These results suggest that the proliferation suppression of AIDs on BMSCs may act via affecting expressions of cell cycle regulators, but not prostaglandin-related mechanisms.

Minimally invasive total hip arthroplasty using a posterolateral approach: technique and preliminary results.

Minimally invasive total hip arthroplasty (THA) has become popular over the past few years. The advantages of this technique include reduced soft tissue damage. On the other hand, there are new risks related to reduced visualization. The widespread introduction of minimally invasive THA is still controversial. Here, we present our experiences and early results with a posterolateral approach to minimally invasive THA. Between August 2005 and July 2006, 85 hips from 79 consecutive patients were operated on using posterolateral minimally invasive THA. The outcomes were assessed on the basis of clinical and radiographic parameters. The mean operative time was 55 minutes. The mean length of hospital stay was 5.3 days. Average postoperative Harris hip score was 92.0 at 3 months postoperatively. Complications included only one (1.18%) intraoperative nondisplaced calcar split. There were no cases of dislocation, neurovascular injury or postoperative infection. Our study indicates an early result of low complication rate and good functional recovery following minimally invasive THA using a posterolateral approach. This minimally invasive THA technique provides short-term safety and efficacy.

Effects of non-steroidal anti-inflammatory drugs on cell proliferation and death in cultured epiphyseal-articular chondrocytes of fetal rats.

Previous reports indicated that non-steroidal anti-inflammatory drugs (NSAIDs) suppress bone repair. Our previous study further found that ketorolac delayed the endochondral bone formation, and the critical effective timing was at the early stage of repair. Furthermore, we found that NSAIDs suppressed proliferation and induced cell death of cultured osteoblasts. In this study, we hypothesized that chondrocytic proliferation and death, which plays an important role at the early stage of endochondral bone formation, might be affected by NSAIDs. Non-selective NSAIDs, indomethacin, ketorolac, diclofenac and piroxicam; cyclooxygenase-2 (COX-2) selective NSAIDs, celecoxib and DFU (an analog of rofecoxib); prostaglandins (PGs), PGE1, PGE2 and PGF2alpha; and each NSAID plus each PG were tested. The effects of NSAIDs on proliferation, cell cycle kinetics, cytotoxicity and cell death of epiphyseal-articular chondrocytes of fetal rats were examined. The results showed that all the tested NSAIDs, except DFU, inhibited thymidine incorporation of chondrocytes at a concentration range (10(-8) to 10(-4)M) covering the theoretic therapeutic concentrations. Cell cycle was arrested by NSAIDs at the G(0)/G(1) phase. Upon a 24h treatment, LDH leakage and cell death (both apoptosis and necrosis) were significantly induced by the four non-selective NSAIDs in chondrocyte cultures. However, COX-2 inhibitors revealed non-significant effects on cytotoxicity of chondrocytes except higher concentration of celecoxib (10(-4)M). Replenishments of PGE1, PGE2 or PGF2alpha could not reverse the effects of NSAIDs on chondrocytic proliferation and cytotoxicity. In this study, we found that therapeutic concentrations of non-selective NSAIDs caused proliferation suppression and cell death of chondrocytes, suggesting these adverse effects may be one of the reasons that NSAIDs delay the endochondral ossification during bone repair found in previous studies. Furthermore, these effects of NSAIDs may act via PG-independent mechanisms. COX-2 selective NSAIDs showed less deleterious effects on chondrocytic proliferation and death.

Alcohol-induced adipogenesis in a cloned bone-marrow stem cell.

BACKGROUND: Alcohol has been shown to be associated with osteoporosis and osteonecrosis in patients and in animal models. Recent studies have demonstrated that alcohol contributes to abnormal lipid metabolism in the stromal cells of bone marrow, but the mechanisms have not been defined. The purpose of this study was to evaluate the effects of alcohol on the differentiation of a stem cell that was cloned from bone marrow. METHODS: D1 cells (cloned bone-marrow stem cells from a BALB/c mouse) were treated either with increasing concentrations of ethanol (0.09, 0.15, and 0.21 mol/L) or without alcohol to serve as controls. Morphologic features of the cells were monitored with use of a phase-contrast microscope. Alkaline phosphatase activity was determined with use of a colorimetric assay. The expression of genes that are indicators of adipogenesis [422(aP2), PPARgamma] and osteogenesis (osteocalcin) was evaluated using Northern blot and reverse transcription-polymerase chain reaction assays. RESULTS: The cells treated with ethanol started to accumulate triglyceride vesicles at day seven. The number of adipocytes and the percentage of the area that contained the cells with fat vesicles increased significantly (p < 0.05), and the level of alkaline phosphatase activity diminished with longer durations of exposure to ethanol and with higher concentrations. Analysis of gene expression showed diminished expression of osteocalcin. This occurred without a significant increase in the expression of either the fat-cell-specific gene 422(aP2) or PPARgamma in cells treated with ethanol, suggesting that adipogenesis may occur at a point downstream in the fatty-acid-metabolism pathway. CONCLUSIONS: Alcohol treatment decreases osteogenesis while enhancing adipogenesis in a cloned bone-marrow stem cell, indicating that alcohol abuse may be one of the mechanisms leading to osteoporosis and osteonecrosis. This finding explains the clinical observation that there is increased adipogenesis in alcohol-induced osteoporosis and osteonecrosis. CLINICAL RELEVANCE: The inhibition of bone-marrow adipogenesis and the concomitant enhancement of osteogenesis may provide a novel approach to the prevention or treatment of osteonecrosis and osteoporosis.