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).
Abstract Recent studies have shown that alendronate (Aln) enhances the osteogenesis of osteoblasts and bone marrow mesenchymal stem cells. In this study, we hypothesize that Aln may act as an ...osteo-inductive factor to stimulate the osteogenic differentiation of human adipose-derived stem cells (hADSCs) for bone regeneration. The in vitro effect of Aln (1–10 μM) on the osteogenic ability of hADSCs was evaluated by examining mineralization and alkaline phosphatase (ALP) activity. Bone morphogenetic protein 2 (BMP2) expression was measured using a real-time polymerase chain reaction and western blot analysis. Our results indicated that 5 μM Aln was sufficient to enhance BMP2 expression, ALP activity and mineralization in hADSCs. The in vivo effect of locally administered Aln on bone repair was examined in a rat critical-sized (7-mm) calvarial defect that was implanted with a hADSC-seeded poly(lactic-co-glycolic acid) (PLGA) scaffold. Aln (5 μM/100 μl/day) was injected locally into the defect site for one week. New bone formation was evaluated by radiographic and histological analyses at 8 and 12 weeks post-implantation. The expression levels of human BMP2 (hBMP2) and hADSC localization in defect sites were examined using immunohistochemistry analysis and fluorescent in situ hybridization, respectively. Results showed that local treatment of Aln on hADSC-seeded PLGA scaffolds at week 12 had a maximal effect on bone regeneration, enhancing mineralization and bone matrix formation. In addition, hADSCs and hBMP2 were also detected at the defect sites. These results demonstrated that local delivery of Aln, a potent osteo-inductive factor, enhances hADSC osteogenesis and bone regeneration.
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.
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.
Cell-matrix adhesion is one of the important interactions that regulates stem cell survival, self-renewal, and differentiation. Our previous report (Wu SC, Chang JK, Wang CK, Wang GJ, Ho ML. ...Biomaterials 31: 631-640, 2010) indicated that a microenvironment enriched with hyaluronan (HA) initiated and enhanced chondrogenesis in human adipose-derived stem cells (hADSCs). We further hypothesize that HA-induced chondrogenesis in hADSCs is mainly due to the interaction of HA and CD44 (HA-CD44), a cell surface receptor of HA. The HA-CD44 interaction was tested by examining the mRNA expression of hyaluronidase-1 (Hyal-1) and chondrogenic marker genes (SOX-9, collagen type II, and aggrecan) in hADSCs cultured on HA-coated wells. Cartilaginous matrix formation, sulfated glycosaminoglycan, and collagen productions by hADSCs affected by HA-CD44 interaction were tested in a three-dimensional fibrin hydrogel. About 99.9% of hADSCs possess CD44. The mRNA expressions of Hyal-1 and chondrogenic marker genes were upregulated by HA in hADSCs on HA-coated wells. Blocking HA-CD44 interaction by anti-CD44 antibody completely inhibited Hyal-1 expression and reduced chondrogenic marker gene expression, which indicates that HA-induced chondrogenesis in hADSCs mainly acts through HA-CD44 interaction. A 2-h preincubation and coculture of cells with HA in hydrogel (HA/fibrin hydrogel) not only assisted in hADSC survival, but also enhanced expression of Hyal-1 and chondrogenic marker genes. Higher levels of sulfated glycosaminoglycan and total collagen were also found in HA/fibrin hydrogel group. Immunocytochemistry showed more collagen type II, but less collagen type X, in HA/fibrin than in fibrin hydrogels. Our results indicate that signaling triggered by HA-CD44 interaction significantly contributes to HA-induced chondrogenesis and may be applied to adipose-derived stem cell-based cartilage regeneration.
Abstract Microenvironment plays a critical role in guiding stem cell differentiation. We investigated the enhancing effect of a hyaluronan (HA)-enriched microenvironment on human adipose derived stem ...cell (hADSC) chondrogenesis for articular cartilage tissue engineering. The hADSCs were obtained from patients undergoing hip replacement. HA-coated wells and HA-modified poly-(lactic-co-glycolic acid) (HA/PLGA) scaffolds were used as the HA-enriched microenvironment. The mRNA expressions of chondrogenic (SOX-9, aggrecan and collagen type II), fibrocartilage (collagen type I), and hypertrophic (collagen type X) marker genes were quantified by real-time polymerase chain reaction. Sulfated glycosaminoglycan (sGAG) deposition was detected by Alcian blue, safranin-O staining, and dimethylmethylene blue (DMMB) assays. Localized collagen type II was detected by immunohistochemistry. The hADSCs cultured in HA-coated wells (0.005–0.5 mg/cm2 ) showed enhanced aggregation and mRNA expressions (SOX-9, collagen type II, and aggrecan) after 24 h, and sGAG content was also significantly increased after 9 days of culture. The HA-modified PLGA did not change the cell adherence and viability of hADSCs. The mRNA expressions of chondrogenic marker genes were significantly enhanced in hADSCs cultured in HA/PLGA rather than those cultured in the PLGA scaffold after 1, 3, and 5 days of culture. The hADSCs cultured in HA/PLGA produced higher levels of sGAG and collagen type II, compared to those in the PLGA scaffold after 4 weeks of cultures. Our results suggest that HA-enriched microenvironment induces chondrogenesis in hADSCs, which may be beneficial in articular cartilage tissue engineering.
Pulsed electromagnetic field (PEMF) has been successfully applied to accelerate fracture repair since 1979. Recent studies suggest that PEMF might be used as a nonoperative treatment for the early ...stages of osteonecrosis. However, PEMF treatment requires a minimum of ten hours per day for the duration of the treatment. In this study, we modified the protocol of the single-pulsed electromagnetic field (SPEMF) that only requires a 3-minute daily treatment. In the in vitro study, cell proliferation and osteogenic differentiation was evaluated in the hBMSCs. In the in vivo study, new bone formation and revascularization were evaluated in the necrotic bone graft. Results from the in vitro study showed no significant cytotoxic effects on the hBMSCs after 5 days of SPEMF treatment (1 Tesla, 30 pulses per day). hBMSC proliferation was enhanced in the SPEMF-treated groups after 2 and 4 days of treatment. The osteogenic differentiation of hBMSCs was significantly increased in the SPEMF-treated groups after 3-7 days of treatment. Mineralization also increased after 10, 15, 20, and 25 days of treatment in SPEMF-treated groups compared to the control group. The 7-day short-course treatment achieved similar effects on proliferation and osteogenesis as the 25-day treatment. Results from the in vivo study also demonstrated that both the 7-day and 25-day treatments of SPEMF increased callus formation around the necrotic bone and also increased new vessel formation and osteocyte numbers in the grafted necrotic bone at the 2nd and 4th weeks after surgery. In conclusion, the newly developed SPEMF accelerates osteogenic differentiation of cultured hBMSCs and enhances bone repair, neo-vascularization, and cell growth in necrotic bone in mice. The potential clinical advantage of the SPEMF is the short daily application and the shorter treatment course. We suggest that SPEMF may be used to treat fractures and the early stages of osteonecrosis.
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.
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.
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.
Abstract 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 p27kip1 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 PGF2α did not reverse the effects of AIDs on proliferation and expressions of p27kip1 and cyclin D2 in hOBs. We conclude that the changes in expressions of regulators of cell cycle (p27kip1 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.
Low-power laser irradiation (LPLI) has been found to induce various biological effects and cellular processes. Also, LPLI has been shown to promote fracture repair. Until now, it has been unclear how ...LPLI promotes bone formation and fracture healing. The aim of this study was to investigate the potential mechanism of LPLI-mediated enhancement of bone formation using mouse bone marrow mesenchymal stem cells (D1 cells). D1 cells were irradiated daily with a gallium-aluminum-arsenide (GaAlAs) laser at dose of 0, 1, 2, or 4 J/cm(2). The lactate dehydrogenase (LDH) assay showed no cytotoxic effects of LPLI on D1 cells, and instead, LPLI at 4 J/cm(2) significantly promoted D1 cell proliferation. LPLI also enhanced osteogenic differentiation in a dose-dependent manner and moderately increased expression of osteogenic markers. The neutralization experiments indicated that LPLI regulated insulin-like growth factor 1 (IGF1) and bone morphogenetic protein 2 (BMP2) signaling to promote cell proliferation and/or osteogenic differentiation. In conclusion, our study suggests that LPLI may induce IGF1 expression to promote both the proliferation and osteogenic differentiation of D1 cells, whereas it may induce BMP2 expression primarily to enhance osteogenic differentiation.
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