Adhesion, migration and mechanics of human adipose-tissue-derived stem cells on silk fibroin–chitosan matrix (original) (raw)
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Adhesion, Migration, and Mechanics of Adipose Tissue Derived Stem Cells
Silk fibroin-chitosan (SFCS) scaffold is a naturally derived biocompatible matrix with potential reconstructive surgical applications. In this study, human adipose-derived mesenchymal stem cells (ASCs) were seeded on SFCS scaffolds and cell attachment was characterized by fluorescence, confocal, timelapse, atomic force, and scanning electron microscopy (SEM) studies. Adhesion of ASCs on SFCS was 39.4 ± 4.8% at 15 min, increasing to 92.8 ± 1.5% at 120 min. ASC adhered at regions of architectural complexity and infiltrate into three-dimensional scaffold. Time-lapse confocal studies indicated a mean ASC speed on SFCS of 18.47 ± 2.7 lm h À1 and a mean persistence time of 41.4 ± 9.3 min over a 2.75 h study period. Cytokinetic and SEM studies demonstrated ASC-ASC interaction via microvillus extensions. The apparent elastic modulus was significantly higher (p < 0.0001) for ASCs seeded on SFCS (69.0 ± 9.0 kPa) than on glass (6.1 ± 0.4 kPa). Also, cytoskeleton F-actin fiber density was higher (p < 0.05) for ASC seeded on SFCS (0.42 ± 0.02 fibers lm À1 ) than on glass-seeded controls (0.24 ± 0.03 fibers lm À1 ). Hence, SFCS scaffold facilitates mesenchymal stem cell attachment, migration, three-dimensional infiltration, and cell-cell interaction. This study showed the potential use of SFCS as a local carrier for autologous stem cells for reconstructive surgery application.
Properties of Biologic Scaffolds and Their Response to Mesenchymal Stem Cells
Arthroscopy: The Journal of Arthroscopic & Related Surgery, 2014
The purpose of this study was to examine, in vitro, the cellular response of human mesenchymal stem cells (MSCs) to sample types of commercially available scaffolds in comparison with control, native tendon tissue (fresh-frozen rotator cuff tendon allograft). Methods: MSCs were defined by (1) colony-forming potential; (2) ability to differentiate into tendon, cartilage, bone, and fat tissue; and (3) fluorescence-activated cell sorting analysis (CD73, CD90, CD45). Samples were taken from fresh-frozen human rotator cuff tendon (allograft), human highly cross-linked collagen membrane (Arthroflex; LifeNet Health, Virginia Beach, VA), porcine nonecross-linked collagen membrane (Mucograft; Geistlich Pharma, Lucerne, Switzerland), a human platelet-rich fibrin matrix (PRF-M), and a fibrin matrix based on platelet-rich plasma (ViscoGel; Arthrex, Naples, FL). Cells were counted for adhesion (24 hours), thymidine assay for cell proliferation (96 hours), and live/dead stain for viability (168 hours). Histologic analysis was performed after 21 days, and the unloaded scaffolds were scanned with electron microscopy. Results: MSCs were successfully differentiated into all cell lines. A significantly greater number of cells adhered to both the nonecross-linked porcine collagen scaffold and PRF-M. Cell activity (proliferation) was significantly higher in the nonecrosslinked porcine collagen scaffold compared with PRF-M and fibrin matrix based on platelet-rich plasma. There were no significant differences found in the results of the live/dead assay. Conclusions: Significant differences in the response of human MSCs to biologic scaffolds existed. MSC adhesion, proliferation, and scaffold morphology evaluated by histologic analysis and electron microscopy varied throughout the evaluated types of scaffolds. Nonecross-linked porcine collagen scaffolds showed superior results for cell adhesion and proliferation, as well as on histologic evaluation. Clinical Relevance: This study enables the clinician and scientist to choose scaffold materials according to their specific interaction with MSCs.
Acta Biomaterialia, 2011
Silk fibroin protein is biodegradable and biocompatible, exhibiting excellent mechanical properties for various biomedical applications. However, porous 3D silk fibroin scaffolds, or silk sponges, usually fall short in matching the initial mechanical requirements for bone tissue engineering. In the present study, silk sponge matrices were reinforced with silk microparticles to generate proteinprotein composite scaffolds with desirable mechanical properties for in vitro osteogenic tissue formation. It was found that increasing the silk microparticle loading led to a substantial increase in the scaffold compressive modulus from 0.3 MPa (nonreinforced) to 1.9 MPa for 1:2 (matrix:particle) reinforcement loading by dry mass. Biochemical, gene expression, and histological assays were employed to study the possible effects of increasing composite scaffold stiffness, due to microparticle reinforcement, on in vitro osteogenic differentiation of human mesenchymal stem cells (hMSCs). Increasing silk microparticle loading increased the osteogenic capability of hMSCs in the presence of bone morphogenic protein-2 (BMP-2) and other osteogenic factors in static culture for up to six weeks. The calcium adsorption increased dramatically with increasing loading, as observed from biochemical assays, histological staining, and microCT (μCT) analysis. Specifically, calcium content in the scaffolds increased by 0.57, 0.71, and 1.27 mg (per μg of DNA) from 3 to 6 weeks for matrix to particle dry mass loading ratios of 1:0, 1:1 and 1:2, respectively. In addition, μCT imaging revealed that at 6 weeks, bone volume fraction increased from 0.78% for nonreinforced to 7.1% and 6.7% for 1:1 and 1:2 loading, respectively. Our results support the hypothesis that scaffold stiffness may strongly influence the 3D in vitro differentiation capabilities of hMSCs, providing a means to improve osteogenic outcomes.
Journal of Anatomy, 2008
The fabrication of biodegradable 3-D scaffolds enriched with multipotent stem cells seems to be a promising strategy for the repair of irreversibly injured tissues. The fine mechanisms of the interaction of rat mesenchymal stem cells (rMSCs) with a hyaluronan-based scaffold, i.e. HYAFF®11, were investigated to evaluate the potential clinical application of this kind of engineered construct. rMSCs were seeded (2 × 10 6 cells cm -2 ) on the scaffold, cultured up to 21 days and analysed using appropriate techniques. Light (LM), scanning (SEM) and transmission (TEM) electron microscopy of untreated scaffold samples showed that scaffolds have a highly porous structure and are composed of 15-μ m-thick microfibres having a rough surface. As detected by trypan blue stain, cell adhesion was high at day 1. rMSCs were viable up to 14 days as shown by CFDA assay and proliferated steadily on the scaffold as revealed by MTT assay. LM showed rMSCs in the innermost portions of the scaffold at day 3. SEM revealed a subconfluent cell monolayer covering 40 ± 10% of the scaffold surface at day 21. TEM of early culture showed rMSCs wrapping individual fibres with regularly spaced focal contacts, whereas confocal microscopy showed polarized expression of CD44 hyaluronan receptor; TEM of 14-day cultures evidenced fibronexus formation. Immunohistochemistry of 21-day cultures showed that fibronectin was the main matrix protein secreted in the extracellular space; decorin and versican were seen in the cell cytoplasm only and type IV collagen was minimally expressed. The expression of CD90, a marker of mesenchymal stemness, was found unaffected at the end of cell culture. Our results show that HYAFF®11 scaffolds support the adhesion, migration and proliferation of rMSCs, as well as the synthesis and delivery of extracellular matrix components under static culture conditions without any chemical induction. The high retention rate and viability of the seeded cells as well as their fine modality of interaction with the substrate suggest that such scaffolds could be potentially useful when wide tissue defects are to be repaired as in the case of cartilage repair, wound healing and large vessel replacement.
Attachment, Growth, and Detachment of Human Mesenchymal Stem Cells in a Chemically Defined Medium
Stem cells international, 2016
The manufacture of human mesenchymal stem cells (hMSCs) for clinical applications requires an appropriate growth surface and an optimized, preferably chemically defined medium (CDM) for expansion. We investigated a new protein/peptide-free CDM that supports the adhesion, growth, and detachment of an immortalized hMSC line (hMSC-TERT) as well as primary cells derived from bone marrow (bm-hMSCs) and adipose tissue (ad-hMSCs). We observed the rapid attachment and spreading of hMSC-TERT cells and ad-hMSCs in CDM concomitant with the expression of integrin and actin fibers. Cell spreading was promoted by coating the growth surface with collagen type IV and fibronectin. The growth of hMSC-TERT cells was similar in CDM and serum-containing medium whereas the lag phase of bm-hMSCs was prolonged in CDM. FGF-2 or surface coating with collagen type IV promoted the growth of bm-hMSCs, but laminin had no effect. All three cell types retained their trilineage differentiation capability in CDM and...
Journal of Biomedical Materials Research Part A, 2010
Regenerative tissue engineering requires biomaterials that would mimic structure and composition of the extracellular matrix to facilitate cell infiltration, differentiation, and vascularization. Engineered scaffolds composed of natural biomaterials silk fibroin (SF) and chitosan (CS) blend were fabricated to achieve fibrillar nano-structures aligned in threedimensions using the technique of dielectrophoresis. The effect of scaffold properties on adhesion and migration of human adipose-derived stem cells (hASC) and endothelial cells (HUVEC) was studied on SFCS (micro-structure, unaligned) and engineered SFCS (E-SFCS; nano-structure, aligned). E-SFCS constituted of a nano-featured fibrillar sheets, whereas SFCS sheets had a smooth morphology with unaligned micro-fibrillar extensions at the ends. Adhesion of hASC to either scaffolds occurred within 30 min and was higher than HUVEC adhesion. The percentage of moving cells and average speed was highest for hASC on SFCS scaffold as compared to hASC cocultured with HUVEC. HUVEC interactions with hASC appeared to slow the speed of hASC migration (in coculture) on both scaffolds. It is concluded that the guidance of cells for regenerative tissue engineering using SFCS scaffolds requires a fine balance between cell-cell interactions that affect the migration speed of cells and the surface characteristics that affects the overall adhesion and direction of migration. V
Biomaterials, 2010
Fibrocartilaginous tissues serve critical load-bearing functions in numerous joints throughout the body. As these structures are often injured, there exists great demand for engineered tissue for repair or replacement. This study assessed the ability of human marrow-derived MSCs to elaborate a mechanically functional, fibrocartilaginous matrix in a nanofibrous microenvironment. Nanofibrous scaffolds, composed of ultra-fine biodegradable polymer fibers, replicate the structural and mechanical anisotropy of native fibrous tissues and serve as a 3D micro-pattern for directing cell orientation and ordered matrix formation. Mesenchymal stem cells (MSC) were isolated from four osteoarthritic (OA) patients, along with meniscal fibrochondrocytes (FC) which have proven to be a potent cell source for engineering fibrocartilage. Cell-seeded nanofibrous scaffolds were cultured in a chemically-defined medium formulation and mechanical, biochemical, and histological features were evaluated over 9 weeks. Surprisingly, and contrary to previous studies with juvenile bovine cells, matrix assembly by adult human MSCs was dramatically hindered compared to donor-matched FCs cultured similarly. Unlike FCs, MSCs did not proliferate, resulting in sparsely colonized constructs. Increases in matrix content, and therefore changes in tensile properties, were modest in MSC-seeded constructs compared to FC counterparts, even when normalized to the lower cell number in these constructs. To rule out the influence of OA sourcing on MSCs, constructs from healthy young donors were generated; these constructs matured no differently than those formed with OA MSCs. Importantly, there was no difference in matrix production of MSCs and FCs when cultured in pellet form, highlighting the sensitivity of human MSCs to their 3D microenvironment.
Evaluation of Scaffolds for the Delivery of Mesenchymal Stem Cells to Wounds
Mesenchymal stem cells (MSCs) have been shown to improve tissue regeneration in several preclinical and clinical trials. These cells have been used in combination with three-dimensional scaffolds as a promising approach in the field of regenerative medicine. We compare the behavior of human adipose-derived MSCs (AdMSCs) on four different biomaterials that are awaiting or have already received FDA approval to determine a suitable regenerative scaffold for delivering these cells to dermal wounds and increasing healing potential. AdMSCs were isolated, characterized, and seeded onto scaffolds based on chitosan, fibrin, bovine collagen, and decellularized porcine dermis. In vitro results demonstrated that the scaffolds strongly influence key parameters, such as seeding efficiency, cellular distribution, attachment, survival, metabolic activity, and paracrine release. Chick chorioallantoic membrane assays revealed that the scaffold composition similarly influences the angiogenic potential of AdMSCs in vivo. The wound healing potential of scaffolds increases by means of a synergistic relationship between AdMSCs and biomaterial resulting in the release of proangiogenic and cytokine factors, which is currently lacking when a scaffold alone is utilized. Furthermore, the methods used herein can be utilized to test other scaffold materials to increase their wound healing potential with AdMSCs.
Data in Brief, 2019
The data showed how gelatin hydrogel and silk fibroin scaffolds could facilitate the growth of human Mesenchymal Stem Cells (hMSC). Gelatin hydrogel and silk fibroin are biodegradable materials. Gelatin hydrogel already has many uses in the medical field, especially in tissue engineering, but silk fibroin scaffold, which is made from the cocoon of silkworm by salt leaching, its role in facilitating growth of hMSC still needs to be proven. Data was obtained by characterization of hMSC, then growing hMSC on silk fibroin scaffolds with pore sizes of ±500 mm and ±900 mm and on gelatin hydrogel scaffolds as control. Testing was performed by counting cell growth on days 1, 3, 5, 7 and 14 with the MTT cytotoxicity assay protocol. The morphology of hMSC that grew on gelatin and silk fibroin scaffolds was observed with a Scanning Electron Microscope (SEM) on day 3. Characterization of the hMSC showed that it fulfilled the requirements of the International Society for Cellular Therapy (ISCT). The water content of the gelatin hydrogel scaffold was higher than the silk fibroin scaffold. Biocompatibility testing showed that the gelatin hydrogel scaffold could support cell growth until day 7, then decreased on day 14 compared to the silk fibroin scaffold based on absorbance on the MTT cytotoxicity assay, while growth on silk fibroin scaffold with