Mei-Ling Ho

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.

BMP-2 plasmid loaded PLGA/HAp composite scaffolds for treatment of bone defects in nude mice

We studied three different types of scaffolds, encapsulating bone morphogenetic protein-2 (BMP-2) plasmid, in terms of their performances in bone regeneration in nude mice. The plasmid was loaded into fibrous matrices in three different ways: coating of naked DNA (Group A) or DNA/chitosan nanoparticles (Group B) onto scaffolds after fiber fabrication by dripping, and encapsulation of DNA/chitosan nanoparticles into scaffold by mixing them with PLGA/DCM solution before fiber fabrication (Group C). Their individual performances were examined by soft X-ray observation, histological analysis and immunostaining of bone tissue. In addition, the BMP-2 protein concentration and alkaline phosphatase (ALP) activity in serum were monitored. The results revealed that the bioactivity of BMP-2 plasmid released from all three kinds of scaffolds was well maintained; this eventually helped improve the healing of segmental defects in vivo. Interestingly, the three kinds of scaffolds released DNA or DNA nanoparticles in different modes and their performances in bone healing were diverse. These observations demonstrate that the in vivo performance of these newly developed DNA delivery devices correlates well with their in vitro release profiles.

Enhancement of chondrogenesis of human adipose derived stem cells in a hyaluronan-enriched microenvironment

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/cm(2)) showed enhanced aggregation and mRNA expressions (SOX-9, collagen type II, and aggrecan) after 24h, 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.

Effects of nonsteroidal anti-inflammatory drugs and prostaglandins on osteoblastic functions

It has been reported that nonsteroidal anti-inflammatory drugs (NSAIDs) suppress bone repair and bone remodeling but only mildly inhibit bone mineralization at the earlier stage of the repair process. We proposed that the proliferation and/or the earlier stage of differentiation of osteoblasts may be affected by NSAIDs. This study was designed to investigate whether NSAIDs affect the proliferation and/or differentiation of osteoblasts and whether these effects are prostaglandin (PG) mediated. The effects of PGE1 and PGE2, indomethacin, and ketorolac on thymidine incorporation, cell count, intracellular alkaline phosphatase (ALP) activity, and Type I collagen content in osteoblast-enriched cultures derived from fetal calvaria were evaluated. The results showed that both PGs and NSAIDs inhibited DNA synthesis and cell mitosis in a time- and concentration-dependent manner. However, intracellular ALP activity and Type I collagen content were stimulated at an earlier stage of differentiation in osteoblasts. These results suggested that (i) the inhibitory effect of ketorolac on osteoblastic proliferation contributes to its suppressive effects on bone repair and remodeling in vivo; (ii) PGEs and NSAIDs may be involved in matrix maturation and biologic bone mineralization in the earlier stage of osteoblast differentiation; and (iii) the effects of ketorolac and indomethacin on cell proliferation and differentiation may not be through the inhibition of the synthesis of PGE1 or PGE2.

Novel Biodegradable Porous Scaffold Applied to Skin Regeneration

Skin wound healing is an important lifesaving issue for massive lesions. A novel porous scaffold with collagen, hyaluronic acid and gelatin was developed for skin wound repair. The swelling ratio of this developed scaffold was assayed by water absorption capacity and showed a value of over 20 g water/g dried scaffold. The scaffold was then degraded in time- and dose-dependent manners by three enzymes: lysozyme, hyaluronidase and collagenase I. The average pore diameter of the scaffold was 132.5±8.4 µm measured from SEM images. With human skin cells growing for 7 days, the SEM images showed surface fractures on the scaffold due to enzymatic digestion, indicating the biodegradable properties of this scaffold. To simulate skin distribution, the human epidermal keratinocytes, melanocytes and dermal fibroblasts were seeded on the porous scaffold and the cross-section immunofluorescent staining demonstrated normal human skin layer distributions. The collagen amount was also quantified after skin cells seeding and presented an amount 50% higher than those seeded on culture wells. The in vivo histological results showed that the scaffold ameliorated wound healing, including decreasing neutrophil infiltrates and thickening newly generated skin compared to the group without treatments.

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.

The effect of the local delivery of alendronate on human adipose-derived stem cell-based bone regeneration.

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.

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.

Controlled-release of rhBMP-2 carriers in the regeneration of osteonecrotic bone.

Untreated osteonecrosis of the hip causes collapse of the femoral head and eventually leads to the development of premature degenerative arthritis. In order to reverse this late complication after the core decompression procedure, we studied three different types of carriers used to entrap recombinant human bone morphogenetic protein-2 (rhBMP-2) in terms of their performance in osteonecrosis regeneration and creeping substitution in Balb/C mice. The rhBMP-2 was loaded into PLGA-HAp microsphere in three different ways. We first verified the therapeutic dose in vitro using D1 and C2C12 cells. Then the individual performance of the three carrier preparations in vivo was examined by soft X-ray observation, histological analysis and immunostaining of bone tissue. In addition, the BMP-2 protein concentration activity in the serum was monitored. The results revealed that the bioactivity of rhBMP-2 released from a carrier with an ideal therapeutic dose was well maintained; this eventually helped to improve the healing and substitution of necrotic bone in vivo. These observations demonstrate that the in vivo performance of these newly developed rhBMP-2 delivery carriers correlates well with their in vitro release profiles. We concluded that sustained controlled-release of rhBMP-2 above a therapeutic dose could not only induce early callus wrapping of the necrotic bone but also produce neovascularization and substitution inside of the dead bone.

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.