Chondrogenic Differentiation Of hASCs Research Articles

MiR-539-3p inhibited chondrogenic differentiation in human adipose stem cells by targeting Sox9

BackgroundMesenchymal stem cells (MSCs) have emerged as the attractive candidates for cell therapy for cartilage repair in clinical therapy of osteoarthritis (OA). MiR-539-3p was reported to differentially express during chondrogenic differentiation of adipose stem cells (ASCs) by miRNA microarrays. The aim of the study was to investigate the effects and underlying mechanisms of miR-539-3p on chondrogenic differentiation of ASCs.MethodsHuman ASCs (hASCs) were obtained from liposuction and transfected with miR-539-3p mimic or inhibitor. Then, the cells were cultured in chondrogenic differentiation medium including transforming growth factor-β1 (TGF-β1).ResultsOur results found that miR-539-3p was gradually down-regulated during chondrogenic differentiation of hASCs. MiR-539-3p overexpression inhibited TGF-β1-induced chondrogenic differentiation of hASCs, as supported by reducing the gene and protein expression of chondrogenic differentiation markers type II collagen alpha 1 (COL2A1), aggrecan (ACAN), and type II collagen. In contrast, miR-539-3p inhibitor significantly promoted the chondrogenic differentiation of hASCs. Dual luciferase reporter assay demonstrated that Sox9 was a direct target gene of miR-539-3p. The expression of SRY-box transcription factor 9 (Sox9) was up-regulated progressively over time during chondrogenic differentiation of hASCs. Additionally, Sox9 overexpression notably reversed chondrogenic differentiation of hASCs inhibited by miR-539-3p mimic, as demonstrated by the decreased expression of COL2A1, ACAN, and type II collagen.ConclusionsAltogether, miR-539-3p inhibited chondrogenic differentiation of hASCs by targeting Sox9. MiR-539-3p may have significant clinical applications for use as a targeted therapy of OA.

Fish cartilage: A promising source of biomaterial for biological scaffold fabrication in cartilage tissue engineering.

Here, engineered cartilage-like scaffold using an extracellular matrix (ECM) from sturgeon fish cartilage provided a chondroinductive environment to stimulate cartilaginous matrix synthesis in human adipose stem cells (hASCs). Three dimensional porous and degradable fish cartilage ECM-derived scaffold (FCS) was produced using a protocol containing chemical decellularization, enzymatic solubilization, freeze-drying and EDC-crosslinking treatments and the effect of different ECM concentrations (10, 20, 30, and 40 mg/ml) on prepared scaffolds was investigated through physical, mechanical and biological analysis. The histological and scanning electron microscopy analysis revealed the elimination of the cell fragments and a 3-D interconnected porous structure, respectively. Cell viability assay displayed no cytotoxic effects. The prepared porous constructs of fish cartilage ECM were seeded with hASCs for 21 days and compared to collagen (Col) and collagen-10% hyaluronic acid (Col-HA) scaffolds. Cell culture results evidenced that the fabricated scaffolds could provide a proper 3-D structure to support the adhesion, proliferation and chondrogenic differentiation of hASCs considering the synthesis of specific proteins of cartilage, collagen type II (Col II) and aggrecan (ACAN). Based on the results of the present study, it can be concluded that the porous scaffold derived from fish cartilage ECM possesses an excellent potential for cartilage tissue engineering.

Polymeric Gelatin Scaffolds Affect Mesenchymal Stem Cell Differentiation and Its Diverse Applications in Tissue Engineering.

Studies using polymeric scaffolds for various biomedical applications, such as tissue engineering, implants and medical substitutes, and drug delivery systems, have attempted to identify suitable material for tissue regeneration. This study aimed to investigate the biocompatibility and effectiveness of a gelatin scaffold seeded with human adipose stem cells (hASCs), including physical characteristics, multilineage differentiation in vitro, and osteogenic potential, in a rat model of a calvarial bone defect and to optimize its design. This functionalized scaffold comprised gelatin-hASCs layers to improve their efficacy in various biomedical applications. The gelatin scaffold exhibited excellent biocompatibility in vitro after two weeks of implantation. Furthermore, the gelatin scaffold supported and specifically regulated the proliferation and osteogenic and chondrogenic differentiation of hASCs, respectively. After 12 weeks of implantation, upon treatment with the gelatin-hASCs scaffold, the calvarial bone harboring the critical defect regenerated better and displayed greater osteogenic potential without any damage to the surrounding tissues compared to the untreated bone defect. These findings suggest that the present gelatin scaffold is a good potential carrier for stem cells in various tissue engineering applications.

Combinatory approach for developing silk fibroin scaffolds for cartilage regeneration

Several processing technologies and engineering strategies have been combined to create scaffolds with superior performance for efficient tissue regeneration. Cartilage tissue is a good example of that, presenting limited self-healing capacity together with a high elasticity and load-bearing properties. In this work, novel porous silk fibroin (SF) scaffolds derived from horseradish peroxidase (HRP)-mediated crosslinking of highly concentrated aqueous SF solution (16 wt%) in combination with salt-leaching and freeze-drying methodologies were developed for articular cartilage tissue engineering (TE) applications. The HRP-crosslinked SF scaffolds presented high porosity (89.3 ± 0.6%), wide pore distribution and high interconnectivity (95.9 ± 0.8%). Moreover, a large swelling capacity and favorable degradation rate were observed up to 30 days, maintaining the porous-like structure and β-sheet conformational integrity obtained with salt-leaching and freeze-drying processing. The in vitro studies supported human adipose-derived stem cells (hASCs) adhesion, proliferation, and high glycosaminoglycans (GAGs) synthesis under chondrogenic culture conditions. Furthermore, the chondrogenic differentiation of hASCs was assessed by the expression of chondrogenic-related markers (collagen type II, Sox-9 and Aggrecan) and deposition of cartilage-specific extracellular matrix for up to 28 days. The cartilage engineered constructs also presented structural integrity as their mechanical properties were improved after chondrogenic culturing. Subcutaneous implantation of the scaffolds in CD-1 mice demonstrated no necrosis or calcification, and deeply tissue ingrowth. Collectively, the structural properties and biological performance of these porous HRP-crosslinked SF scaffolds make them promising candidates for cartilage regeneration. Statement of SignificanceIn cartilage tissue engineering (TE), several processing technologies have been combined to create scaffolds for efficient tissue repair. In our study, we propose novel silk fibroin (SF) scaffolds derived from enzymatically crosslinked SF hydrogels processed by salt-leaching and freeze-drying technologies, for articular cartilage applications. Though these scaffolds, we were able to combine the elastic properties of hydrogel-based systems, with the stability, resilience and controlled porosity of scaffolds processed via salt-leaching and freeze-drying technologies. SF protein has been extensively explored for TE applications, as a result of its mechanical strength, elasticity, biocompatibility, and biodegradability. Thus, the structural, mechanical and biological performance of the proposed scaffolds potentiates their use as three-dimensional matrices for cartilage regeneration.

Comparison of bilayered structures of gellan gum with and without incorporation of gold nanorods for osteochondral tissue engineering

Event Abstract Back to Event Comparison of bilayered structures of gellan gum with and without incorporation of gold nanorods for osteochondral tissue engineering Raphael Canadas1, 2, Silvia Vieira1, 2, Stephanie Vial1, 2, Alexandra P. Marques1, 2, Rui L. Reis1, 2 and Joaquim M. Oliveira1, 2 1 University of Minho, 3B´s Research Group – Biomaterials, Biodegradables and Biomimetics, Portugal 2 University of Minho, ICVS/3B's - PT Government Associate Laboratory, Portugal Introduction: In orthopedics, the regeneration and repair of osteochondral defects after trauma, cancer, or metabolic disorders is still a major clinical challenge, usually leading to an osteoarthritic condition which is a major cause of disability during aging. By the age of 60, close to 100% of the population will have degeneration of knee cartilage[1]. In order to outperform current methods, novel tissue engineering approaches have been proposed to take advantage of the use of stem cells, growth factors and/or pre-programming the stem cells fate, bioreactors and multilayered scaffolds design. The need for simultaneous regenerate both cartilage and subchondral bone in the same scaffold, is a requirement in osteochondral defects. The incorporation of strategic cues to guide the cell fates towards chondro- and osteo-genesis, is an approach to be considered to regenerate both tissues. A previous study performed in our Group showed that the presence of gold nanorods may potentiate the effect of the osteogenic media towards osteocyte-like phenotype, with concomitant reduction of ALP activity and increased mineralization capacity[2]. In the herein presentes work gellan gum based multilayered structures were developed to recreate the bone, calcified cartilage and hyaline cartilage regions. Gold nanorods (AuNRs) and hydroxyapatite particles were incorporated as it is hypothesized its benefits for promoting the osteogenic differentiation of human fat pad adipose-derived stem cells. Materials and Methods: Bilayered sponge-like scaffolds were developed to act as a template for culturing human fat pad adipose-derived stem cells (hASCs). Bilayered spongy structures made of low acyl gellan gum (LAGG)-LAGG/hydroxyapatite (HAp) with and without incorporation of AuNRs were produced integrating cartilage- and bone-like layers (figure 1 represents the AuNRs formation and crosslinking with LAGG). The effect of AuNRs was evaluated and characterized for hASCs osteogenic differentiation following standard protocols. Likewise, the chondrogenic differentiation of hASCs in LAGG was assessed. Figure 1. AuNRs formation, with incorporation of cetyltrimethylammonium bromide (CTAB), poly(acrylic acid) (PAA) and poly(allylamine hydrochloride) (PAH) and further crosslinking with LAGG. Results and Discussion: The freeze-dried bilayered scaffolds composed of LAGG2%(w/v)-LAGG2%/HAp30% (w/w) and LAGG/Goldnanorod 0.25mM-LAGG/Gold nanorod 0.25mM/HAp30% (w/w) have a gradient of HAp in the bone-like layer that, unlike cartilage-like layer, presented a bioactive behavior. The bilayered structures possessed about 90% porosity, 500 µm of pore size and 85% interconnectivity as determined by Micro-CT analysis. Swelling and degradation tests revealed that the structures absorb about 120% of their weight and lost 10% of their mass after 30 days in phosphate buffered saline solution (figure 2). Figure 2. Bilayered scaffolds. A) Bilayer hydrogel after crosslinking in calcium solution and prior freeze-drying; B) X-ray and micro-CT (C,D) visualization of the structure of LAGG with HAp particles in the bone-like layer; E) Alizarin red staining of HAp particles. In vitro studies with isolated hASCs from Fat Pad are being performed to compare the osteogenic and chondrogenic outcomes in the bilayered structures and the effect of the AuNRs regarding alkaline phosphatase (ALP) activity and mineralization. Conclusions: The described structures present the potential for orthopedic applications, namely for osteochondral tissue regeneration. Although the incorporation of AuNRs was already proved to promote the mineralization process, the mechanism seems to be an independent pathway from ALP activity and is now under investigation in this work. The incorporation of the gold nanorods changed the hydrophilicity of the structure, which can be useful also for the calcified cartilage transition zone, since cartilage present a high hydrated phenotype. This study received funding from Portuguese Foundation for Science and Technology (FCT) through the project OsteoCart (PTDC/CTM-BPC/115977/2009) and from European Union’s Seventh Framework Programme (FP7/2007-2013) for REGPOT-CT2012-316331-POLARIS. The authors also thanks FCT for the PhD scholarship provided to R. F. Canadas (SFRH/BD/92565/2013).

Bioactive effects of graphene oxide cell culture substratum on structure and function of human adipose‐derived stem cells

Nanoscale topography of artificial substrates can greatly influence the fate of stem cells including adhesion, proliferation, and differentiation. Thus the design and manipulation of nanoscale stem cell culture platforms or scaffolds are of great importance as a strategy in stem cell and tissue engineering applications. In this report, we propose that a graphene oxide (GO) film is an efficient platform for modulating structure and function of human adipose-derived stem cells (hASCs). Using a self-assembly method, we successfully coated GO on glass for fabricating GO films. The hASCs grown on the GO films showed increased adhesion, indicated by a large number of focal adhesions, and higher correlation between the orientations of actin filaments and vinculin bands compared to hASCs grown on the glass (uncoated GO substrate). It was also found that the GO films showed the stronger affinity for hASCs than the glass. In addition, the GO film proved to be a suitable environment for the time-dependent viability of hASCs. The enhanced differentiation of hASCs included osteogenesis, adipogenesis, and epithelial genesis, while chondrogenic differentiation of hASCs was decreased, compared to tissue culture polystyrene as a control substrate. The data obtained here collectively demonstrates that the GO film is an efficient substratum for the adhesion, proliferation, and differentiation of hASCs.

Chondrogenic potential of injectable κ-carrageenan hydrogel with encapsulated adipose stem cells for cartilage tissue-engineering applications

Due to the limited self-repair capacity of cartilage, regenerative medicine therapies for the treatment of cartilage defects must use a significant amount of cells, preferably applied using a hydrogel system that can promise their delivery and functionality at the specific site. This paper discusses the potential use of κ-carrageenan hydrogels for the delivery of stem cells obtained from adipose tissue in the treatment of cartilage tissue defects. The developed hydrogels were produced by an ionotropic gelation method and human adipose stem cells (hASCs) were encapsulated in 1.5% w/v κ-carrageenan solution at a cell density of 5 × 10(6) cells/ml. The results from the analysis of the cell-encapsulating hydrogels, cultured for up to 21 days, indicated that κ-carrageenan hydrogels support the viability, proliferation and chondrogenic differentiation of hASCs. Additionally, the mechanical analysis demonstrated an increase in stiffness and viscoelastic properties of κ-carrageenan gels with their encapsulated cells with increasing time in culture with chondrogenic medium. These results allowed the conclusion that κ-carrageenan exhibits properties that enable the in vitro functionality of encapsulated hASCs and thus may provide the basis for new successful approaches for the treatment of cartilage defects.

Intermittent Hydrostatic Pressure Enhances Growth Factor‐Induced Chondroinduction of Human Adipose‐Derived Mesenchymal Stem Cells

Hydrostatic pressure (HP) plays an essential role in regulating function of chondrocytes and chondrogenic differentiation. The objective of this study was to examine effects of intermittent HP on chondrogenic differentiation of human adipose-derived mesenchymal stem cells (hASCs) in the presence or absence of chemical chondrogenic medium. Cells were isolated from abdominal fat tissue and confirmed for expression of ASC surface proteins and differentiation potential. Passage 3 pellets were treated with chemical (growth factor), mechanical (HP of 5 MPa and 0.5 Hz with duration of 4 h/day for 7 consecutive days), and combined chemical-mechanical stimuli. Using real-time polymerase chain reaction, the expression of Sox9, collagen II, and aggrecan as three major chondrogenic markers were quantified among three experimental groups and compared to those of stem cells and human cartilage tissue. In comparison to the chemical and mechanical groups, the chemical-mechanical group showed the highest expression for all three chondrogenic genes close to that of cartilage tissue. Results show the beneficial role of intermittent HP on chondrogenic differentiation of hASCs, and that this loading regime in combination with chondrogenic medium can be used in cartilage tissue engineering.