Osteogenic differentiation of human mesenchymal stem cells through alginate-graft-poly(ethylene glycol) microsphere-mediated intracellular growth factor delivery.
ABSTRACT: The intracellular delivery of growth factors increases opportunities for controlling cell behavior and maintaining tissue homeostasis. Recently, VEGFA was reported to enhance osteogenic differentiation of mesenchymal stem cells (MSCs) through an intracrine mechanism, suggesting a new strategy to promote bone tissue formation in osteoporotic patients. The goal of this study was to design and fabricate ligand-conjugated alginate-graft-poly(ethylene glycol) microspheres for intracellular delivery and release of VEGFA in primary human MSCs to enhance osteogenic differentiation as a potential therapeutic. Three types of microspheres were synthesized and characterized by scanning electron microscopy, in vitro drug release kinetics, MSC uptake and internalization: alginate alone (Alg), alginate-graft-poly(ethylene glycol) (Alg-g-PEG) and alginate-graft-poly(ethylene glycol)-S-S-arginine-glycine-aspartic acid (Alg-g-RGD). Each of the different microsphere formulations successfully transported bioactive VEGFA into primary human MSCs within 48h of culture, and significantly enhanced osteogenic differentiation compared to control treatments with empty microspheres (intracellular control) or non-encapsulated VEGFA (extracellular control). Adipogenic differentiation was not affected by the presence of VEGFA intracellularly or extracellularly. These results demonstrating the internalization of alginate-based microspheres and intracellular delivery of VEGFA support the efficacy of using this drug delivery and intracrine mechanism to control the fate of human MSCs and enhance osteogenic differentiation.
Project description:The progress of medical therapies, which rely on the transplantation of microencapsulated living cells, depends on the quality of the encapsulating material. Such material has to be biocompatible, and the microencapsulation process must be simple and not harm the cells. Alginate-poly(ethylene glycol) hybrid microspheres (alg-PEG-M) were produced by combining ionotropic gelation of sodium alginate (Na-alg) using calcium ions with covalent crosslinking of vinyl sulfone-terminated multi-arm poly(ethylene glycol) (PEG-VS). In a one-step microsphere formation process, fast ionotropic gelation yields spherical calcium alginate gel beads, which serve as a matrix for simultaneously but slowly occurring covalent cross-linking of the PEG-VS molecules. The feasibility of cell microencapsulation was studied using primary human foreskin fibroblasts (EDX cells) as a model. The use of cell culture media as polymer solvent, gelation bath, and storage medium did not negatively affect the alg-PEG-M properties. Microencapsulated EDX cells maintained their viability and proliferated. This study demonstrates the feasibility of primary cell microencapsulation within the novel microsphere type alg-PEG-M, serves as reference for future therapy development, and confirms the suitability of EDX cells as control model.
Project description:A great interest has been shown in the injectable scaffolds for cartilage tissue regeneration because it can fill irregularly shaped defects easily through minimally invasive surgical treatments. Herein, we developed a new injectable three-dimensional (3D) alginate hydrogel loaded with biodegradable porous poly(?-caprolactone)-b-poly(ethylene glycol)-b-poly(?-caprolactone) microspheres (MPs/Alg) as the calcium gluconate container to cross-link alginate. Suspensions of chondrocytes/alginate and porous microspheres turned into a gel because of the release of calcium gluconate; thus, the injectable composite hydrogels give a 3D scaffold to fit the defects perfectly and integrate the extracellular-matrix-mimicking architecture to efficiently accommodate cartilage cells in situ. Tissue repair in a full-thickness cartilage defect model was controlled at 6, 12, and 18 weeks after the implant by micro-CT and immunohistochemistry to evaluate the healing status. The defect in the MPs/Alg+ cells group achieved an almost complete repair at 18 weeks, and the repaired chondrocytes regained a normal tissue structure. Moreover, the MPs/Alg+ cells-treated group increased the quality of tissue formed, including the accumulated glycosaminoglycan and the uniformly deposited type II collagen. The results point out the promising application of the injectable MPs/Alg-chondrocytes system for cartilage tissue engineering.
Project description:Platelet-rich plasma (PRP) consists of platelet-derived growth factor and transforming growth factor-? that increase proliferation of mesenchymal stem cells (MSCs), whereas bone morphogenetic protein-2 (BMP2) promotes osteogenic differentiation of MSCs. However, the high degradation rate of fibrin leads to the dissociation of cytokines even before the process of bone regeneration begins. To the best of the authors' knowledge, this is the first study to examine the combined effect of sustained release of PRP from alginate beads on BMP2-modified MSC osteogenic differentiation in vitro and sustained release of PRP alone on a fracture defect model ex vivo as well as its effect on calvarial suture closure.After optimizing the alginate concentration for microspheres, the combined osteogenic and mineralization effect of PRP and BMP2 on MSCs was studied. Self-setting alginate hydrogel carrying PRP was tested on a femur defect model ex vivo. The effect of PRP at day 15 on the closure of the embryonic mouse calvaria sutures ex vivo was also studied.Increase of PRP concentration promoted proliferation of MSCs, and 2.5% to 10% of PRP gradually increased alkaline phosphatase (ALP) activity in the cells in a dose-dependent manner. Sustained release of PRP and BMP2 demonstrated significantly higher ALP and mineralization activity (P <0.05). Radiographs of alginate hydrogel with PRP-treated bone demonstrated nearly complete healing of the fracture, and histologic sections of the embryonic calvaria revealed that PRP leads to suture fusion.Sustained release of PRP along with BMP2-modified MSCs can significantly promote bone regeneration.
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:The efficacy of cell-based therapies as an alternative to autologous bone grafts requires biomaterials to localize cells at the defect and drive osteogenic differentiation. Hydrogels are ideal cell delivery vehicles that can provide instructional cues via their composition or mechanical properties but commonly lack osteoconductive components that nucleate mineral. To address this challenge, we entrapped mesenchymal stromal cells (MSCs) in a composite hydrogel based on two naturally-derived polymers (alginate and hyaluronate) containing biomineralized polymeric microspheres. Mechanical properties of the hydrogels were dependent upon composition. The presentation of the adhesive tripeptide Arginine-Glycine-Aspartic Acid (RGD) from both polymers induced greater osteogenic differentiation of ovine MSCs in vitro compared to gels formed of RGD-alginate or RGD-alginate/hyaluronate alone. We then evaluated the capacity of this construct to stimulate bone healing when transplanting autologous, culture-expanded MSCs into a surgical induced, critical-sized ovine iliac crest bone defect. At 12 weeks post-implantation, defects treated with MSCs transplanted in composite gels exhibited significant increases in blood vessel density, osteoid formation, and bone formation compared to acellular gels or untreated defects. These findings demonstrate the capacity of osteoconductive hydrogels to promote bone formation with autologous MSCs in a large animal bone defect model and provide a promising vehicle for cell-based therapies of bone healing.
Project description:<h4>Background</h4>Hydrogels based on organic/inorganic composites have been at the center of attention for the fabrication of engineered bone constructs. The establishment of a straightforward 3D microenvironment is critical to maintaining cell-to-cell interaction and cellular function, leading to appropriate regeneration. Ionic cross-linkers, Ca<sup>2+</sup>, Ba<sup>2+</sup>, and Sr<sup>2+</sup>, were used for the fabrication of Alginate-Nanohydroxyapatite-Collagen (Alg-nHA-Col) microspheres, and osteogenic properties of human osteoblasts were examined in in vitro and in vivo conditions after 21 days.<h4>Results</h4>Physicochemical properties of hydrogels illustrated that microspheres cross-linked with Sr<sup>2+</sup> had reduced swelling, enhanced stability, and mechanical strength, as compared to the other groups. Human MG-63 osteoblasts inside Sr<sup>2+</sup> cross-linked microspheres exhibited enhanced viability and osteogenic capacity indicated by mineralization and the increase of relevant proteins related to bone formation. PCR (Polymerase Chain Reaction) array analysis of the Wnt (Wingless-related integration site) signaling pathway revealed that Sr<sup>2+</sup> cross-linked microspheres appropriately induced various signaling transduction pathways in human osteoblasts leading to osteogenic activity and dynamic growth. Transplantation of Sr<sup>2+</sup> cross-linked microspheres with rat osteoblasts into cranium with critical size defect in the rat model accelerated bone formation analyzed with micro-CT and histological examination.<h4>Conclusion</h4>Sr<sup>2+</sup> cross-linked Alg-nHA-Col hydrogel can promote functionality and dynamic growth of osteoblasts.
Project description:A new microsphere consisting of inorganic mesoporous silica nanoparticles (MSNs) and organic alginate (denoted as MSN@Alg) was successfully synthesized by air-dynamic atomization and applied to the intracellular drug delivery systems (DDS) of liver cancer cells with sustained release and specific targeting properties. MSN@Alg microspheres have the advantages of MSN and alginate, where MSN provides a large surface area for high drug loading and alginate provides excellent biocompatibility and COOH functionality for specific targeting. Rhodamine 6G was used as a model drug, and the sustained release behavior of the rhodamine 6G-loaded MSN@Alg microspheres can be prolonged up to 20 days. For targeting therapy, the anticancer drug doxorubicin was loaded into MSN@Alg microspheres, and the (lysine)4-tyrosine-arginine-glycine-aspartic acid (K4YRGD) peptide was functionalized onto the surface of MSN@Alg for targeting liver cancer cells, hepatocellular carcinoma (HepG2). The results of the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay and confocal laser scanning microscopy indicate that the MSN@Alg microspheres were successfully uptaken by HepG2 without apparent cytotoxicity. In addition, the intracellular drug delivery efficiency was greatly enhanced (ie, 3.5-fold) for the arginine-glycine-aspartic acid (RGD)-labeled, doxorubicin-loaded MSN@Alg drug delivery system compared with the non-RGD case. The synthesized MSN@Alg microspheres show great potential as drug vehicles with high biocompatibility, sustained release, and targeting features for future intracellular DDS.
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:Bone tissue engineering is a widely growing field that requires the combination of cells, scaffolds and signaling molecules. Adipose derived stem cells (ADSCs) are an accessible and abundant source of mesenchymal stem cells with high plasticity. Polycaprolactone/alginate (PCL/Alg) composite scaffolds have been used in bone regeneration and nano-hydroxyapatite (n-HA) is used as a reinforcing, osteoconductive component in scaffold fabrication. This study was conducted to assess the ability of three different PCL/Alg based scaffolds to induce osteogenic differentiation of ADSCs and to compare between them. Methods:The study comprised 5 groups; negative control group with ADSCs cultured in complete culture media, positive control group with ADSCs cultured in osteogenic differentiation media, and 3 experimental groups with ADSCs seeded onto 3 scaffolds: S1 (PCL/Alg), S2 (PCL/Alg/Ca) and S3 (PCL/Alg/Ca/n-HA) respectively and cultured in osteogenic media. Mineralization and gene expression were assessed by Alizarin red S (ARS) staining and real time quantitative polymerase chain reaction (RT-qPCR). Evaluation was done at 7, 14 and 21 days. Results:ARS staining reflected a time dependent increase through days 7, 14 and 21, with S3 (PCL/Alg/Ca/n-HA) group showing the highest mineralization levels. RT-qPCR detected upregulation of ALP gene expression at day 7 and decline thereafter. S2 (PCL/Alg/Ca) and S3 (PCL/Alg/Ca/n-HA) groups showed significantly higher gene expression levels than S1 (PCL/Alg). Conclusions:ADSCs and PCL/Alg-based scaffolds compose a good tissue engineering complex for bone regeneration. Addition of n-HA to PCL/Alg scaffolds and crosslinking with CaCl2 efficiently improve the osteogenic potential of ADSCs.
Project description:Purpose: Today, there is an urgent need to develop a three-dimentional culture systems mimicking native in vivo condition in order to screen potency of drugs and possibly any genetic alterations in tumor cells. Due to the existence of limitations in animal models, the development of three dimensional systems is highly recommended. To this end, we encapsulated human colon adenocarcinoma cell line HT29 with alginate-poly-L-lysine (Alg-PLL) microspheres and the rate of epithelial-mesenchymal transition was monitored. Methods: Cells were randomly divided into three groups; control, alginate and Alg-PLL. To encapsulate cells, we mixed HT-29 cells (1 × 106 ) with 1 mL of 0.05% PLL and 1% Alg mixture and electrosprayed into CaCl2 solution by using a high-voltage power. Cells from all groups were maintained at 37?C in a humidified atmosphere containing 5% CO2 for 7 days. Cell viability was assessed by MTT assay. To monitor the stemness feature, we measured the transcription of genes such as Snail, Zeb, and Vimentin by using real-time PCR analysis. Results: Addition of PLL to Alg in a hallowed state increased the cell survival rate compared to the control and Alg groups (P<0.05). Cells inside Alg-PLL tended to form microcellular aggregates while in Alg microspheres an even distribution of HT-29 cells was found. Real-time PCR analysis showed the up-regulation of Snail, Zeb, and Vimentin in Alg-PLL microspheres compared to the other groups, showing the acquisition of stemness feature (P<0.05). Conclusion: This study showed that hallow Alg-PLL microspheres increased the epithelialmesenchymal transition rate after 7 days in in vitro condition. Such approaches could be touted as appropriate in vitro models for drug screening.