Project description:Interactions between cells and the extracellular matrix (ECM) are known to play critical roles in regulating cell phenotype. The identity of ECM ligands presented to mesenchymal stem cells (MSCs) has previously been shown to direct the cell fate commitment of these cells. To enhance osteogenic differentiation of MSCs, alginate hydrogels were prepared that present the DGEA ligand derived from collagen I. When presented from hydrogel surfaces in 2D, the DGEA ligand did not facilitate cell adhesion, while hydrogels presenting the RGD ligand derived from fibronectin did encourage cell adhesion and spreading. However, the osteogenic differentiation of MSCs encapsulated within alginate hydrogels presenting the DGEA ligand was enhanced when compared with unmodified alginate hydrogels and hydrogels presenting the RGD ligand. MSCs cultured in DGEA-presenting gels exhibited increased levels of osteocalcin production and mineral deposition. These data suggest that the presentation of the collagen I-derived DGEA ligand is a feasible approach for selectively inducing an osteogenic phenotype in encapsulated MSCs.
Project description:Tendon injuries are often associated with significant dysfunction and disability due to tendinous tissue's very limited self-repair capacity and propensity for scar formation. Dental-derived mesenchymal stem cells (MSCs) in combination with appropriate scaffold material present an alternative therapeutic option for tendon repair/regeneration that may be advantageous compared to other current treatment modalities. The MSC delivery vehicle is the principal determinant for successful implementation of MSC-mediated regenerative therapies. In the current study, a co-delivery system based on TGF-?3-loaded RGD-coupled alginate microspheres was developed for encapsulating periodontal ligament stem cells (PDLSCs) or gingival mesenchymal stem cells (GMSCs). The capacity of encapsulated dental MSCs to differentiate into tendon tissue was investigated in vitro and in vivo. Encapsulated dental-derived MSCs were transplanted subcutaneously into immunocompromised mice. Our results revealed that after 4 weeks of differentiation in vitro, PDLSCs and GMSCs as well as the positive control human bone marrow mesenchymal stem cells (hBMMSCs) exhibited high levels of mRNA expression for gene markers related to tendon regeneration (Scx, DCn, Tnmd, and Bgy) via qPCR measurement. In a corresponding in vivo animal model, ectopic neo-tendon regeneration was observed in subcutaneous transplanted MSC-alginate constructs, as confirmed by histological and immunohistochemical staining for protein markers specific for tendons. Interestingly, in our quantitative PCR and in vivo histomorphometric analyses, PDLSCs showed significantly greater capacity for tendon regeneration than GMSCs or hBMMSCs (P < 0.05). Altogether, these findings indicate that periodontal ligament and gingival tissues can be considered as suitable stem cell sources for tendon engineering. PDLSCs and GMSCs encapsulated in TGF-?3-loaded RGD-modified alginate microspheres are promising candidates for tendon regeneration.
Project description:The host immune system (T-lymphocytes and their pro-inflammatory cytokines) has been shown to compromise bone regeneration ability of mesenchymal stem cells (MSCs). We have recently shown that hydrogel, used as an encapsulating biomaterial affects the cross-talk among host immune cells and MSCs. However, the role of hydrogel elasticity and porosity in regulation of cross-talk between dental-derived MSCs and immune cells is unclear. In this study, we demonstrate that the modulus of elasticity and porosity of the scaffold influence T-lymphocyte-dental MSC interplay by regulating the penetration of inflammatory T cells and their cytokines. Moreover, we demonstrated that alginate hydrogels with different elasticity and microporous structure can regulate the viability and determine the fate of the encapsulated MSCs through modulation of NF-kB pathway. Our in vivo data show that alginate hydrogels with smaller pores and higher elasticity could prevent pro-inflammatory cytokine-induced MSC apoptosis by down-regulating the Caspase-3- and 8- associated proapoptotic cascades, leading to higher amounts of ectopic bone regeneration. Additionally, dental-derived MSCs encapsulated in hydrogel with higher elasticity exhibited lower expression levels of NF-kB p65 and Cox-2 in vivo. Taken together, our findings demonstrate that the mechanical characteristics and microarchitecture of the microenvironment encapsulating MSCs, in addition to presence of T-lymphocytes and their pro-inflammatory cytokines, affect the fate of encapsulated dental-derived MSCs. STATEMENT OF SIGNIFICANCE:In this study, we demonstrate that alginate hydrogel regulates the viability and the fate of the encapsulated dental-derived MSCs through modulation of NF-kB pathway. Alginate hydrogels with smaller pores and higher elasticity prevent pro-inflammatory cytokine-induced MSC apoptosis by down-regulating the Caspase-3- and 8- associated proapoptotic cascade, leading to higher amounts of ectopic bone regeneration. MSCs encapsulated in hydrogel with higher elasticity exhibited lower expression levels of NF-kB p65 and Cox-2 in vivo. These findings confirm that the fate of encapsulated MSCs are affected by the stiffness and microarchitecture of the encapsulating hydrogel biomaterial, as well as presence of T-lymphocytes/pro-inflammatory cytokines providing evidence concerning material science, stem cell biology, the molecular mechanism of dental-derived MSC-associated therapies, and the potential clinical therapeutic impact of MSCs.
Project description:Repair or regeneration of damaged nerves is still a challenging clinical task in reconstructive surgeries and regenerative medicine. Here, it is demonstrated that periodontal ligament stem cells (PDLSCs) and gingival mesenchymal stem cells (GMSCs) isolated from adult human periodontal and gingival tissues assume neuronal phenotype in vitro and in vivo via a subcutaneous transplantation model in nude mice. PDLSCs and GMSCs are encapsulated in a 3D scaffold based on alginate and hyaluronic acid hydrogels capable of sustained release of human nerve growth factor (NGF). The elasticity of the hydrogels affects the proliferation and differentiation of encapsulated MSCs within scaffolds. Moreover, it is observed that PDLSCs and GMSCs are stained positive for ?III-tubulin, while exhibiting high levels of gene expression related to neurogenic differentiation (?III-tubulin and glial fibrillary acidic protein) via quantitative polymerase chain reaction (qPCR). Western blot analysis shows the importance of elasticity of the matrix and the presence of NGF in the neurogenic differentiation of encapsulated MSCs. In vivo, immunofluorescence staining for neurogenic specific protein markers confirms islands of dense positively stained structures inside transplanted hydrogels. As far as it is known, this study is the first demonstration of the application of PDLSCs and GMSCs as promising cell therapy candidates for nerve regeneration.
Project description:Dental-derived mesenchymal stem cells (MSCs) are promising candidates for cartilage regeneration, with a high capacity for chondrogenic differentiation. This property helps make dental MSCs an advantageous therapeutic option compared to current treatment modalities. The MSC delivery vehicle is the principal determinant for the success of MSC-mediated cartilage regeneration therapies. The objectives of this study were to: (1) develop a novel co-delivery system based on TGF-?1 loaded RGD-coupled alginate microspheres encapsulating periodontal ligament stem cells (PDLSCs) or gingival mesenchymal stem cells (GMSCs); and (2) investigate dental MSC viability and chondrogenic differentiation in alginate microspheres. The results revealed the sustained release of TGF-?1 from the alginate microspheres. After 4 weeks of chondrogenic differentiation in vitro, PDLSCs and GMSCs as well as human bone marrow mesenchymal stem cells (hBMMSCs) (as positive control) revealed chondrogenic gene expression markers (Col II and Sox-9) via qPCR, as well as matrix positively stained by Toluidine Blue and Safranin-O. In animal studies, ectopic cartilage tissue regeneration was observed inside and around the transplanted microspheres, confirmed by histochemical and immunofluorescent staining. Interestingly, PDLSCs showed more chondrogenesis than GMSCs and hBMMSCs (p<0.05). Taken together, these results suggest that RGD-modified alginate microencapsulating dental MSCs make a promising candidate for cartilage regeneration. Our results highlight the vital role played by the microenvironment, as well as value of presenting inductive signals for viability and differentiation of MSCs.
Project description: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:Gelatin-methacryloyl (GelMA) is a semi-synthetic hydrogel which consists of gelatin derivatized with methacrylamide and methacrylate groups. These hydrogels provide cells with an optimal biological environment (e.g., RGD motifs for adhesion) and can be quickly photo-crosslinked, which provides shape fidelity and stability at physiological temperature. In the present work, we demonstrated how GelMA hydrogels can be synthesized with a specific degree of functionalization (DoF) and adjusted to the intended application as a three-dimensional (3D) cell culture platform. The focus of this work lays on producing hydrogel scaffolds which provide a cell promoting microenvironment for human adipose tissue-derived mesenchymal stem cells (hAD-MSCs) and are conductive to their adhesion, spreading, and proliferation. The control of mechanical GelMA properties by variation of concentration, DoF, and ultraviolet (UV) polymerization conditions is described. Moreover, hAD-MSC cell viability and morphology in GelMA of different stiffness was evaluated and compared. Polymerized hydrogels with and without cells could be digested in order to release encapsulated cells without loss of viability. We also demonstrated how hydrogel viscosity can be increased by the use of biocompatible additives, in order to enable the extrusion bioprinting of these materials. Taken together, we demonstrated how GelMA hydrogels can be used as a versatile tool for 3D cell cultivation.
Project description:Decellularized tissues have proven to be versatile matrices for the engineering of tissues and organs. These matrices usually consist of collagens, matrix-specific proteins, and a set of largely undefined growth factors and signaling molecules. Although several decellularized tissues have found their way to clinical applications, their use in the engineering of cartilage tissue has only been explored to a limited extent. We set out to generate hydrogels from several tissue-derived matrices, as hydrogels are the current preferred cell carriers for cartilage repair. Equine cartilage, meniscus, and tendon tissue was harvested, decellularized, enzymatically digested, and functionalized with methacrylamide groups. After photo-cross-linking, these tissue digests were mechanically characterized. Next, gelatin methacrylamide (GelMA) hydrogel was functionalized with these methacrylated tissue digests. Equine chondrocytes and mesenchymal stromal cells (MSCs) (both from three donors) were encapsulated and cultured in vitro up to 6 weeks. Gene expression (COL1A1, COL2A1, ACAN, MMP-3, MMP-13, and MMP-14), cartilage-specific matrix formation, and hydrogel stiffness were analyzed after culture. The cartilage, meniscus, and tendon digests were successfully photo-cross-linked into hydrogels. The addition of the tissue-derived matrices to GelMA affected chondrogenic differentiation of MSCs, although no consequent improvement was demonstrated. For chondrocytes, the tissue-derived matrix gels performed worse compared to GelMA alone. This work demonstrates for the first time that native tissues can be processed into crosslinkable hydrogels for the engineering of tissues. Moreover, the differentiation of encapsulated cells can be influenced in these stable, decellularized matrix hydrogels.
Project description:Nanodiamonds (NDs) have attracted considerable attention as drug delivery nanocarriers due to their low cytotoxicity and facile surface functionalization. Given these features, NDs have been recently investigated for the fabrication of nanocomposite hydrogels for tissue engineering. Here we report the synthesis of a hydrogel using photocrosslinkable gelatin methacrylamide (GelMA) and NDs as a three-dimensional scaffold for drug delivery and stem cell-guided bone regeneration. We investigated the effect of different concentration of NDs on the physical and mechanical properties of the GelMA hydrogel network. The inclusion of NDs increased the network stiffness, which in turn augmented the traction forces generated by human adipose stem cells (hASCs). We also tested the ability of NDs to adsorb and modulate the release of a model drug dexamethasone (Dex) to promote the osteogenic differentiation of hASCs. The ND-Dex complexes modulated gene expression, cell area, and focal adhesion number in hASCs. Moreover, the integration of the ND-Dex complex within GelMA hydrogels allowed a higher retention of Dex over time, resulting in significantly increased alkaline phosphatase activity and calcium deposition of encapsulated hASCs. These results suggest that conventional GelMA hydrogels can be coupled with conjugated NDs to develop a novel platform for bone tissue engineering.
Project description:Biophysical cues can potently direct a cell's or tissue's behavior. Cells interpret their biophysical surroundings, such as matrix stiffness or dynamic mechanical stimulation, through mechanotransduction. However, our understanding of the various aspects of mechanotransduction has been limited by the lack of proper analysis platforms capable of screening three-dimensional (3D) cellular behaviors in response to biophysical cues. Here, we developed a dynamic compression bioreactor to study the combinational effects of biomaterial composition and dynamic mechanical compression on cellular behavior in 3D hydrogels. The bioreactor contained multiple actuating posts that could apply cyclic compressive strains ranging from 0 to 42% to arrays of cell-encapsulated hydrogels. The bioreactor could be interconnected with other compressive bioreactors, which enabled the combinatorial screenings of 3D cellular behaviors simultaneously. As an application of the screening platform, cell spreading, and osteogenic differentiation of human mesenchymal stem cells (hMSCs) were characterized in 3D gelatin methacryloyl (GelMA) hydrogels. Increasing hydrogel concentration from 5 to 10% restricted the cell spreading, however, dynamic compressive strain increased cell spreading. Osteogenic differentiation of hMSCs was also affected by dynamic compressive strains. hMSCs in 5% GelMA hydrogel were more sensitive to strains, and the 42% strain group showed a significant increase in osteogenic differentiation compared to other groups. The interconnectable dynamic compression bioreactor provides an efficient way to study the interactions of cells and their physical microenvironments in three dimensions.