Stem cell migration and mechanotransduction on linear stiffness gradient hydrogels.

The spatial presentation of mechanical information is a key parameter for cell behavior. We have developed a method of polymerization control in which the differential diffusion distance of unreacted cross-linker and monomer into a prepolymerized hydrogel sink results in a tunable stiffness gradient at the cell-matrix interface. This simple, low-cost, robust method was used to produce polyacrylamide hydrogels with stiffness gradients of 0.5, 1.7, 2.9, 4.5, 6.8, and 8.2 kPa/mm, spanning the in vivo physiological and pathological mechanical landscape. Importantly, three of these gradients were found to be nondurotactic for human adipose-derived stem cells (hASCs), allowing the presentation of a continuous range of stiffnesses in a single well without the confounding effect of differential cell migration. Using these nondurotactic gradient gels, stiffness-dependent hASC morphology, migration, and differentiation were studied. Finally, the mechanosensitive proteins YAP, Lamin A/C, Lamin B, MRTF-A, and MRTF-B were analyzed on these gradients, providing higher-resolution data on stiffness-dependent expression and localization.

Interplay of matrix stiffness and protein tethering in stem cell differentiation.

Stem cells regulate their fate by binding to, and contracting against, the extracellular matrix. Recently, it has been proposed that in addition to matrix stiffness and ligand type, the degree of coupling of fibrous protein to the surface of the underlying substrate, that is, tethering and matrix porosity, also regulates stem cell differentiation. By modulating substrate porosity without altering stiffness in polyacrylamide gels, we show that varying substrate porosity did not significantly change protein tethering, substrate deformations, or the osteogenic and adipogenic differentiation of human adipose-derived stromal cells and marrow-derived mesenchymal stromal cells. Varying protein-substrate linker density up to 50-fold changed tethering, but did not affect osteogenesis, adipogenesis, surface-protein unfolding or underlying substrate deformations. Differentiation was also unaffected by the absence of protein tethering. Our findings imply that the stiffness of planar matrices regulates stem cell differentiation independently of protein tethering and porosity.

Getting physical: Material mechanics is an intrinsic cell cue.

Advances in biomaterial science have allowed for unprecedented insight into the ability of material cues to influence stem cell function. These material approaches better recapitulate the microenvironment, providing a more realistic ex vivo model of the cell niche. However, recent advances in our ability to measure and manipulate niche properties in vivo have led to novel mechanobiological studies in model organisms. Thus, in this review, we will discuss the importance of material cues within the cell niche, highlight the key mechanotransduction pathways involved, and conclude with recent evidence that material cues regulate tissue function in vivo.

Hydrogels with an embossed surface: An all-in-one platform for mass production and culture of human adipose-derived stem cell spheroids.

Stem cell spheroids have been studied extensively in organoid culture and therapeutic transplantation. Herein, hydrogels with an embossed surface (HES) were developed as an all-in-one platform that can enable the rapid formation and culture of a large quantity of size-controllable stem cell spheroids. The embossed structure on the hydrogel was adjustable according to the grit designation of the sandpaper. Human adipose-derived stem cells (hADSCs) were rapidly assembled into spheroids on the hydrogel, with their size distribution precisely controlled from 95 ±â€¯6 µm to 181 ±â€¯15 µm depending on surface roughness. The hADSC spheroids prepared from the HES demonstrated expression of stemness markers and differentiation capacity. In addition, HES-based spheroids showed significantly greater VEGF secretion than spheroids grown on a commercially available low-attachment culture plate. Exploiting those advantages, the HES-based spheroids were used for 3D bioprinting, and the spheroids within the 3D-printed construct showed improved retention and VEGF secretion compared to the same 3D structure containing single cell suspension. Collectively, HES would offer a useful platform for mass fabrication and culture of stem cell spheroids with controlled sizes for a variety of biomedical applications.

Microcontact printing of polydopamine on thermally expandable hydrogels for controlled cell adhesion and delivery of geometrically defined microtissues.

Scaffold-free harvest of microtissue with a defined structure has received a great deal of interest in cell-based assay and regenerative medicine. In this study, we developed thermally expandable hydrogels with spatially controlled cell adhesive patterns for rapid harvest of geometrically controlled microtissue. We patterned polydopamine (PD) on to the hydrogel via microcontact printing (µCP), in linear shapes with widths of 50, 100 and 200µm. The hydrogels facilitated formation of spatially controlled strip-like microtissue of human dermal fibroblasts (HDFBs). It was possible to harvest and translocate microtissues with controlled widths of 61.4±14.7, 104.3±15.6, and 186.6±22.3µm from the hydrogel to glass substrates by conformal contact upon expansion of the hydrogel in response to a temperature change from 37 to 4°C, preserving high viability, extracellular matrix, and junction proteins. Microtissues were readily translocated in vivo to the subcutaneous tissue of mouse. The microtissues were further utilized as a simple assay model for monitoring of contraction in response to ROCK1 inhibitor. Collectively, micro-sized patterning of PD on the thermally expandable hydrogels via µCP holds promise for the development of microtissue harvesting systems that can be employed to ex vivo tissue assay and cell-based therapy. STATEMENT OF SIGNIFICANCE: Harvest of artificial tissue with controlled cellular arrangement independently from external materials has been widely studied in cell-based assay and regenerative medicine. In this study, we developed scaffold-free harvest system of microtissues with anisotropic arrangement and controlled width by exploiting thermally expandable hydrogels with cell-adhesive patterns of polydopamine formed by simple microcontact printing. Cultured strips of human dermal fibroblasts on the hydrogels were rapidly delivered to various targets ranging from flat coverglass to mice subcutaneous tissue by thermal expansion of the hydrogel at 4°C for 10min. These were further utilized as a drug screening model responding to ROCK1 inhibitor, which imply its versatile applicability.

Adipose tissue engineering using mesenchymal stem cells attached to injectable PLGA spheres.

The reconstruction of soft tissue defects remains a challenge in plastic and reconstructive surgery, and a real clinical need exists for an adequate solution. This study was undertaken in order to differentiate mesenchymal stem cells (MSCs) into adipocytes, and to then assess the possibility of constructing adipose tissue via the attachment of MSCs to injectable PLGA spheres. We also designed injectable PLGA spheres for scar-free transplantation. In this study, MSCs and adipo-MSCs (MSCs cultured in adipogenic medium for 7 days) were attached to PLGA spheres and cultured for 7 days, followed by injection into nude mice for 2 weeks. As a result, the difference between lipid accumulation in adipo-MSCs at 1 and 7 days was much higher in vitro than in the MSCs. Two weeks after injection, a massive amount of new tissue was formed in the APLGA group, whereas only a small amount was formed in the MPLGA group. We verified that the newly formed tissue originated from the injected MSCs via GFP testing, and confirmed that the created tissue was actual adipose tissue by oil red O staining and Western blot (PPAR(gamma) and C/EBP(alpha) were expressed only in APLGA groups). Therefore, this study presents an efficient model of adipose tissue engineering using MSCs and injectable PLGA spheres.

Mesenchymal stem cell durotaxis depends on substrate stiffness gradient strength.

Mesenchymal stem cells (MSCs) respond to the elasticity of their environment, which varies between and within tissues. Stiffness gradients within tissues can result from pathological conditions, but also occur through normal variation, such as in muscle. MSC migration can be directed by shallow stiffness gradients before differentiating. Gradients with fine control over substrate compliance - both in range and rate of change (strength) - are needed to better understand mechanical regulation of MSC migration in normal and diseased states. We describe polyacrylamide stiffness gradient fabrication using three distinct systems, generating stiffness gradients of physiological (1 Pa/µm), pathological (10 Pa/µm), and step change (≥ 100Pa/µm) strength. All gradients spanned a range of physiologically relevant elastic moduli for soft tissues (1-12 kPa). MSCs migrated to the stiffest region on each gradient. Time-lapse microscopy revealed that migration velocity correlated directly with gradient strength. Directed migration was reduced in the presence of the contractile agonist lysophosphatidic acid (LPA) and cytoskeleton-perturbing drugs nocodazole and cytochalasin. LPA- and nocodazole-treated cells remained spread and protrusive on the substrate, while cytochalasin-treated cells did not. Nocodazole-treated cells spread in a similar manner to untreated cells, but exhibited greatly diminished traction forces. These data suggest that a functional actin cytoskeleton is required for migration whereas microtubules are required for directed migration. The data also imply that, in vivo, MSCs may preferentially accumulate in regions of high elastic modulus and make a greater contribution to tissue repairs in these locations.

The alignment and fusion assembly of adipose-derived stem cells on mechanically patterned matrices.

Cell patterning is typically accomplished by selectively depositing proteins for cell adhesion only on patterned regions; however in tissues, cells are also influenced by mechanical stimuli, which can also result in patterned arrangements of cells. We developed a mechanically-patterned hydrogel to observe and compare it to extracellular matrix (ECM) ligand patterns to determine how to best regulate and improve cell type-specific behaviors. Ligand-based patterning on hydrogels was not robust over prolonged culture, but cells on mechanically-patterned hydrogels differentially sorted based on stiffness preference: myocytes and adipose-derived stem cells (ASCs) underwent stiffness-mediated migration, i.e. durotaxis, and remained on myogenic hydrogel regions. Myocytes developed aligned striations and fused on myogenic stripes of the mechanically-patterned hydrogel. ASCs aligned and underwent myogenesis, but their fusion rate increased, as did the number of cells fusing into a myotube as a result of their alignment. Conversely, neuronal cells did not exhibit durotaxis and could be seen on soft regions of the hydrogel for prolonged culture time. These results suggest that mechanically-patterned hydrogels could provide a platform to create tissue engineered, innervated micro-muscles of neural and muscle phenotypes juxtaposed next to each other in order better recreate a muscle niche.

The effect of PLGA sphere diameter on rabbit mesenchymal stem cells in adipose tissue engineering.

To investigate the effect of injectable PLGA sphere's diameter on adipose tissue engineering, rabbit mesenchymal stem cells were attached to various diameters of injectable PLGA spheres (<75; 75-100; 100-150; 150-200; and 200-250 microm). These five groups were cultured in adipogenic media for 2 weeks in vitro and injected into necks of nude mice. Prior to in vivo study, cell proliferation and adipogenic differentiation were determined by hexosaminidase assay and Oil red O staining after 2 weeks. Group C (100-150 microm) showed the highest adipogenic differentiation and the proliferation capacity of Group B (75-100 microm) was significantly higher than that of any other group. We harvested newly formed tissues from necks of nude mice after 1 and 4 weeks. Although PLGA spheres have not been degraded and there was no significant histological difference among various sizes of spheres after 1 week, well-organized fat pads (PLGA spheres were completely degraded) could be observed, and the histology of the 100-150 microm groups resembled that of native tissue after 4 weeks. Based on these experiments, we could conclude that the optimal size of PLGA spheres for adipogenesis was 100-150 microm.

Preparation and characterization of biodegradable anti-adhesive membrane for peritoneal wound healing.

Postoperative adhesions remain a significant complication of abdominal surgery although the wide variety of physical barriers has been developed to reduce the incidence of adhesion. In this study, the bilayered composite membrane formed by the association of a methoxy poly (ethylene glycol)-poly (L-lactide-co-glycolide) (mPEG-PLGA) film and a crosslinked collagen-hyaluronic acid (Col-HA) membrane with fibronectin (FN) coating was prepared for promoting wound healing and providing tissue adhesion resistance simultaneously. In vitro adhesion test revealed that fibroblasts attached better on Col-HA membrane compared to those on mPEG-PLGA film, PLGA film or Interceed (oxidized cellulose) while mPEG-PLGA film had the lowest cell adhesive property. In confocal microscopic observation, the actin filaments were significantly further polymerized when 50 or 100 microg/cm(3) fibronectin was incorporated on the COL-HA membranes. After 7-day culture, fibroblasts penetrated throughout the Col-HA-FN network and the cell density increased whereas very few cells were found attached on the surface of the mPEG-PLGA film. In vivo evaluation test showed that the composite membrane could remain during the critical period of peritoneal healing and did not provoke any inflammation or adverse tissue reaction.

Adipogenic differentiation of adipose tissue derived adult stem cells in nude mouse.

We evaluated the use of a combination of adipose tissue derived adult stem cells (ADSCs) obtained from liposuction and injectable poly(lactic-co-glycolic acid) (PLGA) spheres for adipose tissue engineering. Adipogenesis was examined in nude mice injected subcutaneously with ADSCs (group I), PLGA spheres (group II), or ADSCs attached PLGA spheres (group III) cultured in adipogenic medium for 7 days. After 4 and 8 weeks, newly formed adipose tissue was observed in groups II and III but not in group I. Oil red O staining of newly formed tissue showed that there was substantially more tissue regeneration and adipogenic differentiation in group III than in group II. RT-PCR confirmed that, after 8 weeks, the PLGA-attached ADSCs had fully differentiated into adipocytes. This study provides significant evidence that ADSCs and PLGA spheres can be used in a clinical setting to generate adipose tissue as a noninvasive soft tissue filler.

Muscle regeneration by adipose tissue-derived adult stem cells attached to injectable PLGA spheres.

The [corrected] use of adult stem cells for cell-based tissue engineering and regeneration strategies represents a promising approach for skeletal muscle repair. We have evaluated the combination of adipose tissue-derived adult stem cells (ADSCs) obtained from autologous liposuction and injectable poly(lactic-co-glycolic acid) (PLGA) spheres for muscle regeneration. ADSCs attached to PLGA spheres and PLGA spheres alone were cultured in myogenic medium for 21 days and injected subcutaneously into the necks of nude mice. After 30 and 60 days, the mice were sacrificed, and newly formed tissues were analyzed by immunostaining, H and E staining, and RT-PCR. We found that ADSCs attached to PLGA spheres, but not PLGA spheres alone, were able to generate muscle tissue. These findings suggest that ADSCs and PLGA spheres are useful materials for muscle tissue engineering and that their combination can be used in clinical settings for muscle regeneration.