Adipose Tissue-Derived Stem Cells Retain Their Adipocyte Differentiation Potential in Three-Dimensional Hydrogels and Bioreactors (original) (raw)
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Materials, 2014
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Journal of Nanomaterials, 2016
Skin wound repair requires the development of different kinds of biomaterials that must be capable of restoring the damaged tissue. Type I collagen and chitosan have been widely used to develop scaffolds for skin engineering because of their cell-related signaling properties such as proliferation, migration, and survival. Collagen is the major component of the skin extracellular matrix (ECM), while chitosan mimics the structure of the native polysaccharides and glycosaminoglycans in the ECM. Chitosan and its derivatives are also widely used as drug delivery vehicles since they are biodegradable and noncytotoxic. Regulation of the inflammatory response is crucial for wound healing and tissue regeneration processes; and, consequently, the development of biomaterials such as hydrogels with anti-inflammatory properties is very important and permissive for the growth of cells. In the last years, it has been shown that mesenchymal stem cells have clinical importance in the treatment of di...
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2014
An injectable tissue-engineered adipose substitute that could be used to deliver adipose-derived stem cells (ASCs), filling irregular defects and stimulating natural soft tissue regeneration, would have significant value in plastic and reconstructive surgery. With this focus, the primary aim of the current study was to characterize the response of human ASCs encapsulated within three-dimensional bioscaffolds incorporating decellularized adipose tissue (DAT) as a bioactive matrix within photo-cross-linkable methacrylated glycol chitosan (MGC) or methacrylated chondroitin sulphate (MCS) delivery vehicles. Stable MGC-and MCS-based composite scaffolds were fabricated containing up to 5 wt% cryomilled DAT through initiation with long-wavelength ultraviolet light. The encapsulation strategy allows for tuning of the 3-D microenvironment and provides an effective method of cell delivery with high seeding efficiency and uniformity, which could be adapted as a minimally-invasive in situ approach. Through in vitro cell culture studies, human ASCs were assessed over 14 days in terms of viability, glycerol-3-phosphate dehydrogenase (GPDH) enzyme activity, adipogenic gene expression and intracellular lipid accumulation. In all of the composites, the DAT functioned as a cell-supportive matrix that enhanced ASC viability, retention and adipogenesis within the gels. The choice of hydrogel also influenced the cell response, with significantly higher viability and adipogenic differentiation observed in the MCS composites containing 5 wt% DAT. In vivo analysis in a subcutaneous Wistar rat model at 1, 4 and 12 weeks showed superior implant integration and adipogenesis in the MCS-based composites, with allogenic ASCs promoting cell infiltration, angiogenesis and ultimately, fat formation.
Impact of hydrogel stiffness on differentiation of human adipose-derived stem cell microspheroids
Tissue Engineering Part A
Hydrogels represent an attractive material platform for realization of three-dimensional (3D) tissue-engineered constructs, as they have tunable mechanical properties, are compatible with different types of cells, and resemble elements found in natural extracellular matrices. So far, numerous hydrogel-cartilage/bone tissue engineering (TE)-related studies were performed by utilizing a single cell encapsulation approach. Although multicellular spheroid cultures exhibit advantageous properties for cartilage or bone TE, the chondrogenic or osteogenic differentiation potential of stem cell microspheroids within hydrogels has not been investigated much. This study explores, for the first time, how stiffness of gelatin-based hydrogels (having a storage modulus of 538, 3584, or 7263 Pa) affects proliferation and differentiation of microspheroids formed from telomerase-immortalized human adipose-derived stem cells (hASC/hTERT). Confocal microscopy indicates that all tested hydrogels supported cell viability during their 3-to 5-week culture period in the control, chondrogenic, or osteogenic medium. Although in the softer hydrogels cells from neighboring microspheroids started outgrowing and interconnecting within a few days, their protrusion was slower or limited in stiffer hydrogels or those cultured in chondrogenic medium, respectively. High expressions of chondrogenic markers (SOX9, ACAN, COL2A1), detected in all tested hydrogels, proved that the chondrogenic differentiation of hASC/hTERT microspheroids was very successful, especially in the two softer hydrogels, where superior cartilage-specific properties were confirmed by Alcian blue staining. These chondrogenically induced samples also expressed COL10A1, a marker of chondrocyte hypertrophy. Interestingly, the hydrogel itself (with no differentiation medium) showed a slight chondrogenic induction. Regardless of the hydrogel stiffness, in the samples stimulated with osteogenic medium, the expression of selected markers RUNX2, BGLAP, ALPL, and COL1A1 was not conclusive. Nevertheless, the von Kossa staining confirmed the presence of calcium deposits in osteogenically stimulated samples in the two softer hydrogels, suggesting that these also favor osteogenesis. This observation was also confirmed by Alizarin red quantification assay, with which higher amounts of calcium were detected in the osteogenically induced hydrogels than in their controls. The presented data indicate that the encapsulation of adipose-derived stem cell microspheroids in gelatinbased hydrogels show promising potential for future applications in cartilage or bone TE.
Methods and Protocols
Adipose-derived mesenchymal stem cells (ASCs) are a promising source for clinical application in regenerative orthopedics. ASCs derived from the infra-patellar fat pad (IFP)—a distinct adipose structure in the knee—show superior regenerative potential compared to subcutaneous-fat-derived cells. Furthermore, it has been shown that blood products enhance ASCs’ viability. A major challenge for clinical translation of both ASCs and blood products is the low comparability of obtained data due to non-standardized harvesting, isolation and preparation methods. The aim of this method-paper is to provide reproducible protocols to help standardize basic research in the field to build a sound basis for clinical translation with an emphasize on practicability. The presented protocols include (i) ASC isolation from the IFP, (ii) blood product preparation and (iii) ASC incubation with blood products.
Eskişehir Technical University Journal of Science and Technology A - Applied Sciences and Engineering, 2021
Mesenchymal stem cells (MSCs) are multipotent stem cells that can support various tissues including bone marrow, adipose tissue, and synovial fluids, from which they can be readily isolated. The objective of this study is to harness the advantages of microfluidic systems for controlling and enhancing the maintenance and viability, and regenerative properties of MSCs by providing a 3D culture microenvironment with gelatin methacrylate (GelMA) hydrogel and exposing the cells to a slow fluid flow and low shear stress conditions. GelMA has methacryloyl groups and can be crosslinked by a photocuring process using biocompatible photoinitiators. The most common used photoinitiator for cellular encapsulation within hydrogels is the ultraviolet (UV) initiator 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959 or I2959), but due to its low water solubility and the necessity of using a shorter wavelength light (365 nm), it can lead to cellular phototoxic and genotoxic effects. To overcome these limitations, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) have recently been used with GelMA as an alternative photoinitiator. Because LAP is highly water soluble and has a 10 times faster polymerization rate, and it requires a visible light (λ = 405 nm) which makes it much safer for the cells, we use 10% GelMA together with 0.05% LAP photoinitiator for bioprinting human adipose tissue derived MSCs (hAT-MSCs) onto a membrane that has a 40 µm mesh size. To demonstrate a microfluidic culture advancement for improving the biological activities and regenerative capacity of the cells including cell adhesion, growth, viability and proliferation capacity as ultimate goals of this study, the membrane carrying the bioprinted construct was placed in a PDMS microchannel and exposed to the fluid to obtain dynamic microenvironments found in the human body. As a result, the cells were successfully maintained in the microfluidic 3D cell culture for two days, with a high cell viability of 99%.
Biomaterials, 2008
Due to increasing clinical demand for adipose tissue, a suitable scaffold for engineering adipose tissue constructs is needed. In this study, we have developed a three-dimensional (3-D) culture system using bone marrow-derived mesenchymal stem cells (BM-MSC) and a Pluronic F-127 hydrogel scaffold as a step towards the in vitro tissue engineering of fat. BM-MSC were dispersed into a Pluronic F-127 hydrogel with or without type I collagen added. The adipogenic differentiation of the BM-MSC was assessed by cellular morphology and further confirmed by Oil Red O staining. The BM-MSC differentiated into adipocytes in Pluronic F-127 in the presence of adipogenic stimuli over a period of 2 weeks, with some differentiation present even in absence of such stimuli. The addition of type I collagen to the Pluronic F-127 caused the BM-MSC to aggregate into clumps, thereby generating an uneven adipogenic response, which was not desirable. Crown
Gene reports, 2018
The poor healing potential of intra-articular ligament injuries drives a need for the development of novel, viable 'neo-ligament' alternatives. Ex vivo approaches combining stem cell engineering, 3-dimensional biocompatible scaffold design and enhancement of biological and biomechanical functionality via the introduction of key growth factors and morphogens, represent a promising solution to ligament regeneration. We investigated growth, differentiation and extracellular matrix (ECM) protein production of human adipose-derived mesenchymal stem/stromal cells (MSCs), cultured in 5% human platelet lysate (PL) and seeded on three-dimensional polycaprolactone (PCL) scaffolds, in response to the connective-tissue related ligands fibroblast growth factor 2 (basic) (FGF2) and growth and differentiation factor-5 (GDF5). Phenotypic alterations of MSCs under different biological conditions were examined using cell viability assays, real time qPCR analysis of total RNA, as well as immun...