Stiffness-mediated mesenchymal stem cell fate decision in 3D-bioprinted hydrogels.
ABSTRACT: Background:Hydrogels with tuneable mechanical properties are an attractive material platform for 3D bioprinting. Thus far, numerous studies have confirmed that the biophysical cues of hydrogels, such as stiffness, are known to have a profound impact on mesenchymal stem cell (MSC) differentiation; however, their differentiation potential within 3D-bioprinted hydrogels is not completely understood. Here, we propose a protocol for the exploration of how the stiffness of alginate-gelatin (Alg-Gel) composite hydrogels (the widely used bioink) affects the differentiation of MSCs in the presence or absence of differentiation inducing factors. Methods:Two types of Alg-Gel composite hydrogels (Young's modulus: 50 kPa vs. 225 kPa) were bioprinted independently of porosity. Then, stiffness-induced biases towards adipogenic and osteogenic differentiation of the embedded MSCs were analysed by co-staining with alkaline phosphatase (ALP) and oil red O. The expression of specific markers at the gene level was detected after a 3-day culture. Results:Confocal microscopy indicated that all tested hydrogels supported MSC growth and viability during the culture period. Higher expression of adipogenic and osteogenic markers (ALP and lipoprotein lipase (LPL)) in stiffer 3D-bioprinted matrices demonstrated a more significant response of MSCs to stiffer hydrogels with respect to differentiation, which was more robust in differentiation-inducing medium. However, the LPL expression in stiffer 3D-bioprinted constructs was reduced at day 3 regardless of the presence of differentiation-inducing factors. Although MSCs embedded in softer hydrogels to some extent proceeded toward adipogenic and osteogenic lineages within a few days, their differentiation seemed to be slower and more limited. Interestingly, the hydrogel itself (without differentiation-inducing factors) exhibited a slight effect on whether MSCs differentiated towards an adipogenic or an osteogenic fate. Considering that the mechano-regulated protein Yes-associated protein (YAP) is involved in MSC fate decisions, we further found that inhibition of YAP significantly downregulated the expression of ALP and LPL in MSCs in stiffer constructs regardless of the induced growth factors present. Conclusions:These results demonstrate that the differentiation of MSCs in 3D-bioprinted matrices is dependent on hydrogel stiffness, which emphasizes the importance of biophysical cues as a determinant of cellular behaviour.
Project description:Alginate is a commonly used bioink in 3D bioprinting. Matrix stiffness is a key determinant of mesenchymal stem cell (MSC) differentiation, suggesting that modulation of alginate bioink mechanical properties represents a promising strategy to spatially regulate MSC fate within bioprinted tissues. In this study, we define a printability window for alginate of differing molecular weight (MW) by systematically varying the ratio of alginate to ionic crosslinker within the bioink. We demonstrate that the MW of such alginate bioinks, as well as the choice of ionic crosslinker, can be tuned to control the mechanical properties (Young's Modulus, Degradation Rate) of 3D printed constructs. These same factors are also shown to influence growth factor release from the bioinks. We next explored if spatially modulating the stiffness of 3D bioprinted hydrogels could be used to direct MSC fate inside printed tissues. Using the same alginate and crosslinker, but varying the crosslinking ratio, it is possible to bioprint constructs with spatially varying mechanical microenvironments. Moreover, these spatially varying microenvironments were found to have a significant effect on the fate of MSCs within the alginate bioinks, with stiffer regions of the bioprinted construct preferentially supporting osteogenesis over adipogenesis.
Project description:Mesenchymal stromal cells (MSCs) are a heterogeneous population of cells that possess multilineage differentiation potential and extensive immunomodulatory properties. In mice and rats, MSCs produce nitric oxide (NO), as immunomodulatory effector molecule that exerts an antiproliferative effect on T cells, while the role of NO in differentiation was less clear. Here, we investigated the role of NO synthase 2 (NOS2) on adipogenic and osteogenic differentiation of rat MSCs. MSCs isolated from NOS2-null (NOS2<sup>-/-</sup>) and wild type (WT) Sprague-Dawley (SD) rats exhibited homogenous fibroblast-like morphology and characteristic phenotypes. However, after induction, adipogenic differentiation was found significantly promoted in NOS2<sup>-/-</sup> MSCs compared to WT MSCs, but not in osteogenic differentiation. Accordingly, qRT-PCR revealed that the adipogenesis-related genes PPAR-γ, C/EBP-α, LPL and FABP4 were markedly upregulated in NOS2<sup>-/-</sup> MSCs, but not for osteogenic transcription factors or marker genes. Further investigations revealed that the significant enhancement of adipogenic differentiation in NOS2<sup>-/-</sup> MSCs was due to overactivation of the STAT3 signaling pathway. Both AG490 and S3I-201, small molecule inhibitors that selectively inhibit STAT3 activation, reversed this adipogenic effect. Furthermore, after high-fat diet (HFD) feeding, knockout of NOS2 in rat MSCs resulted in significant obesity. In summary, NOS2 is involved in the regulation of rat MSC adipogenic differentiation <i>via</i> the STAT3 signaling pathway.
Project description:Proliferation and differentiation of cells are known to be influenced by the physical properties of the extracellular environment. Previous studies examining biophysics underlying cell response to matrix stiffness utilized a two-dimensional (2D) culture format, which is not representative of the three-dimensional (3D) tissue environment in vivo. We report on the effect of 3D matrix modulus on human bone marrow stromal cell (hBMSC) differentiation. hBMSCs underwent osteogenic differentiation in poly(ethylene glycol) hydrogels of all modulus (300-fold modulus range, from 0.2 kPa to 59 kPa) in the absence of osteogenic differentiation supplements. This osteogenic differentiation was modulus-dependent and was enhanced in stiffer gels. Osteogenesis in these matrices required integrin-protein ligation since osteogenesis was inhibited by soluble Arginine-Glycine-Aspartate-Serine peptide, which blocks integrin receptors. Immunostained images revealed lack of well-defined actin filaments and microtubules in the encapsulated cells. Disruption of mechanosensing elements downstream of integrin binding that have been identified from 2D culture such as actin filaments, myosin II contraction, and RhoA kinase did not abrogate hBMSC material-driven osteogenic differentiation in 3D. These data show that increased hydrogel modulus enhanced osteogenic differentiation of hBMSCs in 3D scaffolds but that hBMSCs did not use the same mechanosensing pathways that have been identified in 2D culture.
Project description:Dimensionality can have a profound impact on stiffness-mediated differentiation of mesenchymal stem cells (MSCs). However, while we have begun to understand cellular response when encapsulated within 3D substrates, the behavior of cells within macro-porous substrates is relatively underexplored. The goal of this study was to determine the influence of macro-porous topographies on stiffness-mediated differentiation of MSCs. We developed macro-porous recombinant elastin-like protein (ELP) substrates that allow independent control of mechanical properties and ligand chemistry. We then used computational modeling to probe the impact of pore topography on the mechanical stimulus that cells are exposed to within these substrates, and finally we investigated stiffness induced biases towards adipogenic and osteogenic differentiation of MSCs within macro-porous substrates. Computational modeling revealed that there is significant heterogeneity in the mechanical stimuli that cells are exposed to within porous substrates and that this heterogeneity is predominantly due to the wide range of possible cellular orientations within the pores. Surprisingly, MSCs grown within 3D porous substrates respond to increasing substrate stiffness by up-regulating both osteogenesis and adipogenesis. These results demonstrate that within porous substrates the behavior of MSCs diverges from previously observed responses to substrate stiffness, emphasizing the importance of topography as a determinant of cellular behavior.
Project description:Directed differentiation of induced pluripotent stem cells (iPSCs) towards specific lineages remains a major challenge in regenerative medicine, while there is a growing perception that this process can be influenced by the three-dimensional environment. In this study, we investigated whether iPSCs can differentiate towards mesenchymal stromal cells (MSCs) when embedded into fibrin hydrogels to enable a one-step differentiation procedure within a scaffold. Differentiation of iPSCs on tissue culture plastic or on top of fibrin hydrogels resulted in a typical MSC-like phenotype. In contrast, iPSCs embedded into fibrin gel gave rise to much smaller cells with heterogeneous growth patterns, absence of fibronectin, faint expression of CD73 and CD105, and reduced differentiation potential towards osteogenic and adipogenic lineage. Transcriptomic analysis demonstrated that characteristic genes for MSCs and extracellular matrix were upregulated on flat substrates, whereas genes of neural development were upregulated in 3D culture. Furthermore, the 3D culture had major effects on DNA methylation profiles, particularly within genes for neuronal and cardiovascular development, while there was no evidence for epigenetic maturation towards MSCs. Taken together, iPSCs could be differentiated towards MSCs on tissue culture plastic or on a flat fibrin hydrogel. In contrast, the differentiation process was heterogeneous and not directed towards MSCs when iPSCs were embedded into the hydrogel.
Project description:Calcific aortic valve disease (CAVD) progression is a highly dynamic process whereby normally fibroblastic valve interstitial cells (VIC) undergo osteogenic differentiation, maladaptive extracellular matrix (ECM) composition, structural remodeling, and tissue matrix stiffening. However, how VIC with different phenotypes dynamically affect matrix properties and how the altered matrix further affects VIC phenotypes in response to physiological and pathological conditions have not yet been determined. In this study, we develop 3D hydrogels with tunable matrix stiffness to investigate the dynamic interplay between VIC phenotypes and matrix biomechanics. We find that VIC populated within hydrogels with valve leaflet like stiffness differentiate towards myofibroblasts in osteogenic media, but surprisingly undergo osteogenic differentiation when cultured within lower initial stiffness hydrogels. VIC differentiation progressively stiffens the hydrogel microenvironment, which further upregulates both early and late osteogenic markers. These findings identify a dynamic positive feedback loop that governs acceleration of VIC calcification. Temporal stiffening of pathologically lower stiffness matrix back to normal level, or blocking the mechanosensitive RhoA/ROCK signaling pathway, delays the osteogenic differentiation process. Therefore, direct ECM biomechanical modulation can affect VIC phenotypes towards and against osteogenic differentiation in 3D culture. These findings highlight the importance of the homeostatic maintenance of matrix stiffness to restrict pathological VIC differentiation.We implement 3D hydrogels with tunable matrix stiffness to investigate the dynamic interaction between valve interstitial cells (VIC, major cell population in heart valve) and matrix biomechanics. This work focuses on how human VIC responses to changing 3D culture environments. Our findings identify a dynamic positive feedback loop that governs acceleration of VIC calcification, which is the hallmark of calcific aortic valve disease. Temporal stiffening of pathologically lower stiffness matrix back to normal level, or blocking the mechanosensitive signaling pathway, delays VIC osteogenic differentiation. Our findings provide an improved understanding of VIC-matrix interactions to aid in interpretation of VIC calcification studies in vitro and suggest that ECM disruption resulting in local tissue stiffness decreases may promote calcific aortic valve disease.
Project description:Extracellular matrix (ECM) proteins are structural elements of tissue and also potent signaling molecules. Previously, our laboratory showed that ECM of 2D coatings can trigger differentiation of bone marrow-derived mesenchymal stem cells (MSCs) into mesodermal lineages in an ECM-specific manner over 14 days, in some cases comparable to chemical induction. To test whether a similar effect was possible in a 3D, tissue-like environment, we designed a synthetic-natural biomaterial composite. The composite can present whole-molecule ECM proteins to cells, even those that do not spontaneously form hydrogels ex vivo, in 3D. To this end, we entrapped collagen type I, laminin-111, or fibronectin in ECM composites with MSCs and directly compared markers of mesodermal differentiation including cardiomyogenic (ACTC1), osteogenic (SPP1), adipogenic (PPARG), and chondrogenic (SOX9) in 2D versus 3D. We found the 3D condition largely mimicked the 2D condition such that the addition of type I collagen was the most potent inducer of differentiation to all lineages tested. One notable difference between 2D and 3D was pronounced adipogenic differentiation in 3D especially in the presence of exogenous collagen type I. In particular, PPARG gene expression was significantly increased ?16-fold relative to chemical induction, in 3D and not in 2D. Unexpectedly, 3D engagement of ECM proteins also altered immunomodulatory function of MSCs in that expression of IL-6 gene was elevated relative to basal levels in 2D. In fact, levels of IL-6 gene expression in 3D composites containing exogenously supplied collagen type I or fibronectin were statistically similar to levels attained in 2D with tumor necrosis factor-? (TNF-?) stimulation and these levels were sustained over a 2-week period. Thus, this novel biomaterial platform allowed us to compare the biochemical impact of whole-molecule ECM proteins in 2D versus 3D indicating enhanced adipogenic differentiation and IL-6 expression of MSC in the 3D context. Exploiting the biochemical impact of ECM proteins on MSC differentiation and immunomodulation could augment the therapeutic utility of MSCs.
Project description: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 gelatin-based hydrogels show promising potential for future applications in cartilage or bone TE. Impact Statement Osteochondral defects represent one of the leading causes of disability in the world. Although numerous tissue engineering (TE) approaches have shown success in cartilage and bone tissue regeneration, achieving native-like characteristics of these tissues remains challenging. This study demonstrates that in the presence of a corresponding differentiation medium, gelatin-based hydrogels support moderate osteogenic and excellent chondrogenic differentiation of photo-encapsulated human adipose-derived stem cell microspheroids, the extent of which depends on hydrogel stiffness. Because photosensitive hydrogels are a convenient material platform for creating stiffness gradients in three dimensions, the presented microspheroid-hydrogel encapsulation strategy holds promise for future strategies of cartilage or bone TE.
Project description:Bone marrow mesenchymal stem cells (MSCs) are a valuable cell source for tissue engineering and regenerative medicine. Transforming growth factor ? (TGF-?) can promote MSC differentiation into either smooth muscle cells (SMCs) or chondrogenic cells. Here we showed that the stiffness of cell adhesion substrates modulated these differential effects. MSCs on soft substrates had less spreading, fewer stress fibers and lower proliferation rate than MSCs on stiff substrates. MSCs on stiff substrates had higher expression of SMC markers ?-actin and calponin-1; in contrast, MSCs on soft substrates had a higher expression of chondrogenic marker collagen-II and adipogenic marker lipoprotein lipase (LPL). TGF-? increased SMC marker expression on stiff substrates. However, TGF-? increased chondrogenic marker expression and suppressed adipogenic marker expression on soft substrates, while adipogenic medium and soft substrates induced adipogenic differentiation effectively. Rho GTPase was involved in the expression of all aforementioned lineage markers, but did not account for the differential effects of substrate stiffness. In addition, soft substrates did not significantly affect Rho activity, but inhibited Rho-induced stress fiber formation and ?-actin assembly. Further analysis showed that MSCs on soft substrates had weaker cell adhesion, and that the suppression of cell adhesion strength mimicked the effects of soft substrates on the lineage marker expression. These results provide insights of how substrate stiffness differentially regulates stem cell differentiation, and have significant implications for the design of biomaterials with appropriate mechanical property for tissue regeneration.
Project description:A theoretical inverse relationship has long been postulated for osteogenic and adipogenic differentiation (bone versus adipose tissue differentiation). This inverse relationship in theory at least partially underlies the clinical entity of osteoporosis, in which marrow mesenchymal stem cells (MSCs) have a predilection for adipose differentiation that increases with age. In the present study, we assayed the potential anti-adipogenic effects of Nell-1 protein (an osteoinductive molecule). Using 3T3-L1 (a human preadipocyte cell line) cells and human adipose-derived stromal cells (ASCs), we observed that adenoviral delivered (Ad)-Nell-1 or recombinant NELL-1 protein significantly reduced adipose differentiation across all markers examined (Oil red O staining, adipogenic gene expression [Pparg, Lpl, Ap2]). In a prospective fashion, Hedgehog signaling was assayed as potentially downstream of Nell-1 signaling in regulating osteogenic over adipogenic differentiation. In comparison to Ad-LacZ control, Ad-Nell-1 increased expression of hedgehog signaling markers (Ihh, Gli1, Ptc1). These studies suggest that Nell-1 is a potent anti-adipogenic agent. Moreover, Nell-1 signaling may inhibit adipogenic differentiation via a Hedgehog dependent mechanism.