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Hyaluronan (HA) is an important extracellular matrix component in the early stage of chondrogenesis. This study aimed to investigate the application of an HA microenvironment for human adipose-derived stem cells (hADSCs)-based articular cartilage regeneration. HA-enriched fibrin (HA/Fibrin) hydrogels were synthesized and characterized for use as HA microenvironments. The cell viability and chondrogenic gene expression of hADSCs cultured in HA/Fibrin (HA/Fibrin/hADSC) and Fibrin (Fibrin/hADSC) hydrogels were tested in vitro. A chondral defect created in osteochondral core explants ex vivo was used to test chondral defect regeneration by HA/Fibrin/hADSC or Fibrin/hADSC hydrogels. The results showed that HA/Fibrin hydrogels exhibited an increased swelling ratio and matrix stiffness and a smoother surface with more interconnected pores than in Fibrin hydrogels. The viability of hADSCs in HA/Fibrin/hADSC hydrogels was not altered, but they exhibited higher chondrogenic gene expression than those in Fibrin/hADSC hydrogels. For chondral defect regeneration, the HA/Fibrin/hADSC hydrogels exhibited the most cartilaginous tissue neo-formation, chondral integration and sGAG content in the surrounding tissue. This study demonstrated that an HA microenvironment enhances hADSC-mediated cartilage regeneration in chondral defects and thus may be used for ADSC-based articular cartilage tissue engineering., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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.

Matrix stiffness plays an important role in stem cell differentiation. This study reports the synthesis of methacrylated hyaluronan (MeHA) with different degrees of methacrylation, ranging from 15 to 140% per disaccharide unit, which corresponds to a matrix stiffness ranging from 1.5 to 8 KPa. The swelling ratio was inversely proportional to the matrix stiffness, but the water content remained constant at >97% of the hydrogel mass. A fibril-like surface morphology and larger pore size were observed in lyophilized MeHA hydrogel with a lower stiffness. The matrix stiffness also affected the degradability of the MeHA hydrogel, where softer MeHA hydrogels (MeHA 15 and MeHA 30 ) were completely degraded within 6 days and a stiffer MeHA hydrogel (MeHA 140 ) was able to retain ∼25% of its initial mass after 30 days. Subsequently, the crosslinked MeHA hydrogel was used as a scaffold to encapsulate human adipose-derived stem cells (hADSCs). The embedded cells remained viable and expressed ∼11-fold higher levels of aggrecan and 42-fold higher levels of collagen type II in MeHA 140 compared with ADSCs cultured in HA-coated wells. In addition, cells grown in MeHA 140 exhibited the highest rates of glycosaminoglycan and collagen type II synthesis of ∼5 ng/DNA and 0.4 ng/DNA, respectively. Immunofluorescence staining showed an increase of collagen type II synthesis in MeHA 65 , MeHA 85 and MeHA 140 . This study showed that the matrix stiffness of a hydrogel can be modulated by the degree of methacrylation, thus affecting the efficacy of chondrogenesis in hADSCs. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 808-816, 2018., (© 2017 Wiley Periodicals, Inc.)

Hyaluronan (HA) is a natural linear polymer that is one of the main types of extracellular matrix during the early stage of chondrogenesis. We found that the chondrogenesis of adipose-derived stem cells (ADSCs) can be initiated and promoted by the application of HA to mimic the chondrogenic niche. The aim of this study is to investigate the optimal HA molecular weight (Mw) for chondrogenesis of ADSCs and the detailed mechanism. In this study, we investigated the relationships among HA Mw, CD44 clustering, and the extracellular signal-regulated kinase (ERK)/SOX-9 pathway during chondrogenesis of ADSCs. Human ADSCs (hADSCs) and rabbit ADSCs (rADSCs) were isolated and expanded. Chondrogenesis was induced in rADSCs by culturing cells in HA-coated wells (HA Mw: 80 kDa, 600 kDa and 2000 kDa) and evaluated by examining cell aggregation, chondrogenic gene expression (collagen type II and aggrecan) and sulfated glycosaminoglycan (sGAG) deposition in vitro. Cartilaginous tissue formation in vivo was confirmed by implanting HA/rADSCs into joint cavities. CD44 clustering, ERK phosphorylation, SOX-9 expression and SOX-9 phosphorylation in cultured hADSCs were further evaluated. Isolated and expanded rADSCs showed multilineage potential and anchorage-independent growth properties. Cell aggregation, chondrogenic gene expression, and sGAG deposition increased with increasing HA Mw in rADSCs. The 2000 kDa HA had the most pronounced chondrogenic effect on rADSCs in vitro, and implanted 2000 kDa HA/rADSCs exhibited marked cartilaginous tissue formation in vivo. CD44 clustering and cell aggregation of hADSCs were enhanced by an increase in HA Mw. In addition, higher HA Mws further enhanced CD44 clustering, ERK phosphorylation, and SOX-9 expression and phosphorylation in hADSCs. Inhibiting CD44 clustering in hADSCs reduced HA-induced chondrogenic gene expression. Inhibiting ERK phosphorylation also simultaneously attenuated HA-induced SOX-9 expression and phosphorylation and chondrogenic gene expression in hADSCs. Our results indicate that HA initiates ADSC chondrogenesis and that higher Mw HAs exhibit stronger effects, with 2000 kDa HA having the strongest effect. These effects may be mediated through increased CD44 clustering and the ERK/SOX-9 signaling pathway., Statement of Significance: HA-based biomaterials have been studied in stem cell-based articular cartilage tissue engineering. However, little is known about the optimal HA size for stem cell chondrogenesis and the mechanism of how HA size modulates stem cell chondrogenesis. Accordingly, we used HAs with various Mws (80-2000 kDa) as culture substrates and tested their chondrogenic effect on ADSCs. Our results demonstrated that HAs with a Mw of 2000 kDa showed the optimal effect for chondrogenesis of ADSCs. Moreover, we found that HA size can regulate ADSC chondrogenesis via the CD44/ERK/SOX-9 pathway. This finding provides new information regarding the biochemical control of chondrogenesis by HA substrates that may add value to the development of HA-based biomaterials for articular cartilage regeneration., (Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

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., (Copyright © 2015 the American Physiological Society.)

Background Aims: Human adipose-derived stem cells (hADSCs) have become a popular stem cell source because of their abundant supplies, high differentiation ability and the fact that they present few ethical concerns. Suspension culture, a type of three-dimensional culture, is a more suitable model for mimicking cell-cell and cell-extracellular matrix interactions than is two-dimensional monolayer culture. The aim of this study was to determine the effects of suspension culture on the viability and differentiation potential of hADSCs., Methods: Different densities of hADSCs were cultured in ultra-low-attachment surface plates. The morphology and mean diameter of the resultant aggregates were determined by means of microscopy. The viability of the aggregates was evaluated with the use of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt, lactate dehydrogenase and live/dead assays. To detect osteogenesis, chondrogenesis and adipogenesis in hADSCs in suspension culture, cell aggregates were stained to determine cell function, and the expression of specific markers was evaluated through the use of real-time reverse transcriptase-polymerase chain reaction., Results: The hADSCs remained viable in suspension culture and formed cell aggregates. The diameter of the majority of the aggregates was in the range of 50-200 μm, regardless of cell density. The aggregation of the hADSCs served to maintain cell survival. In addition, the results of the histomorphometric and gene expression analyses showed that the hADSCs were more efficiently induced to differentiate into osteoblasts, chondrocytes and adipocytes in suspension culture than in two-dimensional monolayer culture., Conclusions: Suspension culture can be used to maintain cell viability and contributes to the effective differentiation of hADSCs, providing an alternative cell growth strategy for application to stem cell-based regenerative medicine., (Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

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.

We tested the hypothesis that electromagnetic field (EMF) stimulation enhances chondrogenesis in human adipose-derived stem cells (ADSCs) in a chondrogenic microenvironment. A two-dimensional hyaluronan (HA)-coated well (2D-HA) and a three-dimensional pellet culture system (3D-pellet) were used as chondrogenic microenvironments. The ADSCs were cultured in 2D-HA or 3D-pellet, and then treated with clinical-use pulse electromagnetic field (PEMF) or the innovative single-pulse electromagnetic field (SPEMF) stimulation. The cytotoxicity, cell viability, and chondrogenic and osteogenic differentiations were analyzed after PEMF or SPEMF treatment. The modules of PEMF and SPEMF stimulations used in this study did not cause cytotoxicity or alter cell viability in ADSCs. Both PEMF and SPEMF enhanced the chondrogenic gene expression (SOX-9, collagen type II, and aggrecan) of ADSCs cultured in 2D-HA and 3D-pellet. The expressions of bone matrix genes (osteocalcin and collagen type I) of ADSCs were not changed after SPEMF treatment in 2D-HA and 3D-pellet; however, they were enhanced by PEMF treatment. Both PEMF and SPEMF increased the cartilaginous matrix (sulfated glycosaminoglycan) deposition of ADSCs. However, PEMF treatment also increased mineralization of ADSCs, but SPEMF treatment did not. Both PEMF and SPEMF enhanced chondrogenic differentiation of ADSCs cultured in a chondrogenic microenvironment. SPEMF treatment enhanced ADSC chondrogenesis, but not osteogenesis, when the cells were cultured in a chondrogenic microenvironment. However, PEMF enhanced both osteogenesis and chondrogenesis under the same conditions. Thus the combination of a chondrogenic microenvironment with SPEMF stimulation can promote chondrogenic differentiation of ADSCs and may be applicable to articular cartilage tissue engineering.

Mesenchymal stem cell (MSC)-based tissue regeneration is a promising therapeutic strategy for treating damaged tissues. However, the inflammatory microenvironment that exists at a local injury site might restrict reconstruction. Low-power laser irradiation (LPLI) has been widely applied to retard the inflammatory reaction. The purpose of this study was to investigate the anti-inflammatory effect of LPLI on human adipose-derived stem cells (hADSCs) in an inflammatory environment. We showed that the hADSCs expressed Toll-like Receptors (TLR) 1, TLR2, TLR3, TLR4, and TLR6 and that lipopolysaccharide (LPS) significantly induced the production of pro-inflammatory cytokines (Cyclooxygenase-2 (Cox-2), Interleukin-1β (IL-1β), Interleukin-6 (IL-6), and Interleukin-8 (IL-8)). LPLI markedly inhibited LPS-induced, pro-inflammatory cytokine expression at an optimal dose of 8 J/cm². The inhibitory effect triggered by LPLI might occur through an increase in the intracellular level of cyclic AMP (cAMP), which acts to down-regulate nuclear factor kappa B (NF-κB) transcriptional activity. These data collectively provide insight for further investigations of the potential application of anti-inflammatory treatment followed by stem cell therapy.

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., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

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.

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.

Adipose-derived stem cells (ADSCs) are a type of mesenchymal stem cell that is investigated in bone tissue engineering (BTE). Osteoblasts are the main cells responsible for bone formation in vivo and directing ADSCs to form osteoblasts through osteogenesis is a research topic in BTE. In addition to the osteogenesis of ADSCs into osteoblasts, the crosstalk of ADSCs with osteoblasts through the secretion of extracellular vesicles (EVs) may also contribute to bone formation in ADSC-based BTE. We investigated the effect of ADSC-secreted EVs (ADSC-EVs) on osteoblast function. ADSC-EVs (size ≤ 1000 nm) were isolated from the culture supernatant of ADSCs through ultracentrifugation. The ADSC-EVs were observed to be spherical under a transmission electron microscope. The ADSC-EVs were positive for CD9, CD81, and Alix, but β-actin was not detected. ADSC-EV treatment did not change survival but did increase osteoblast proliferation and activity. The 48 most abundant known microRNAs (miRNAs) identified within the ADSC-EVs were selected and then subjected to gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. The GO analysis revealed that these miRNAs are highly relevant to skeletal system morphogenesis and bone development. The KEGG analysis indicated that these miRNAs may regulate osteoblast function through autophagy or the mitogen-activated protein kinase or Ras-related protein 1 signaling pathway. These results suggest that ADSC-EVs enhance osteoblast function and can contribute to bone regeneration in ADSC-based BTE. [ABSTRACT FROM AUTHOR]

Both stem cell therapy and physical treatments have been shown to be beneficial in accelerating bone healing. However, the efficacy of combined treatment with stem cells and physical stimuli for large bone defects remains uncertain. The aim of this study was to evaluate the bone regeneration effects of low-power laser irradiation (LPLI) and human adipose-derived stem cell (ADSC) treatments during fracture repair using a comparative rat calvarial defect model. We evaluated the viability of human ADSCs, which were cultured on a porous PLGA scaffold using an MTS assay. The critical-sized calvarial bone defect rats were divided into 4 groups: control group, LPLI group, ADSC group, and ADSC+LPLI group. Bone formation was evaluated using micro-CT. New bone formation areas and osteogenic factor expression levels were then examined by histomorphological analysis and immunohistochemical staining. Our data showed that PLGA had no cytotoxic effect on human ADSCs. Micro-CT analyses revealed that both the LPLI and ADSC groups showed improved calvarial bone defect healing compared to the control group. In addition, the ADSC+LPLI group showed significantly increased bone volume at 16 weeks after surgery. The area of new bone formation ranked as follows: control group < LPLI group < ADSC group < ADSC+LPLI group. There were significant differences between the groups. In addition, both ADSC and ADSC+LPLI groups showed strong signals of vWF expression. ADSC and LPLI treatments improved fracture repair in critical-sized calvarial defects in rats. Importantly, the combined treatment of ADSCs and LPLI further enhances the bone healing process. [ABSTRACT FROM AUTHOR]

Recent studies indicated that alendronate enhanced osteogenesis in osteoblasts and human bone marrow-derived stem cells. However, the time- and dose-dependent effects of Aln on ostegenic differentiation and cytotoxicity of hBMSCs remain undefined. In present study, we investigated the effective dose range and timing of hBMSCs. hBMSCs were treated with various Aln doses (1, 5 and 10 µM) according to the following groups: group A was treated with Aln during the first five days of bone medium, groups B, C and D were treated during the first, second, and final five days of osteo-induction medium and group E was treated throughout the entire experiment. The mineralization level and cytotoxicity were measured by quantified Alizarin Red S staining and MTT assay. In addition, the reversal effects of farnesyl pyrophosphate and geranylgeranyl pyrophosphate replenishment in group B were also investigated. The results showed that Aln treatment in groups A, B and E enhanced hBMSC mineralization in a dose-dependent manner, and the most pronounced effects were observed in groups B and E. The higher dose of Aln simultaneously enhanced mineralization and caused cytotoxicity in groups B, C and E. Replenishment of FPP or GGPP resulted in partial or complete reverse of the Aln-induced mineralization respectively. Furthermore, the addition of FPP or GGPP also eliminated the Aln-induced cytotoxicity. We demonstrated that hBMSCs are susceptible to 5 µM Aln during the initiation stage of osteogenic differentiation and that a 10 µM dose is cytotoxic. [ABSTRACT FROM AUTHOR]

Directing adipose-derived stem cells (ADSCs) toward chondrogenesis is critical for ADSC-based articular cartilage regeneration. Simvastatin (SIM) was reported to promote both chondrogenic and osteogenic differentiation of ADSCs by upregulating bone morphogenetic protein-2 (BMP-2). We previously found that ADSC chondrogenesis is initiated and promoted in a hyaluronan (HA) microenvironment (HAM). Here, we further hypothesized that SIM augments HAM-induced chondrogenesis but not osteogenesis of ADSCs. ADSCs were treated with SIM in a HAM (SIM plus HAM) by HA-coated wells or HA-enriched fibrin (HA/Fibrin) hydrogel, and chondrogenic differentiation of ADSCs was evaluated. SIM plus HAM increased chondrogenesis more than HAM or SIM alone, including cell aggregation, chondrogenic gene expression (collagen type II and aggrecan) and cartilaginous tissue formation (collagen type II and sulfated glycosaminoglycan). In contrast, SIM-induced osteogenesis in ADSCs was reduced in SIM plus HAM, including mRNA expression of osteogenic genes, osteocalcin and alkaline phosphatase (ALP), ALP activity and mineralization. SIM plus HAM also showed the most effective increases in the mRNA expression of BMP-2 and transcription factors of SOX-9 and RUNX-2 in ADSCs, while these effects were reversed by CD44 blockade. HAM suppressed the levels of JNK, p-JNK, P38 and p-P38 in ADSCs, and SIM plus HAM also decreased SIM-induced phosphorylated JNK and p38 levels. In addition, SIM enhanced articular cartilage regeneration, as demonstrated by implantation of an ADSCs/HA/Fibrin construct in an ex vivo porcine articular chondral defect model. The results from this study indicate that SIM may be an enhancer of HAM-initiated MSC-based chondrogenesis and avoid osteogenesis. [ABSTRACT FROM AUTHOR]