Multiprotein collagen/keratin hydrogel promoted myogenesis and angiogenesis of injured skeletal muscles in a mouse model.

Volumetric loss is one of the challenging issues in muscle tissue structure that causes functio laesa. Tissue engineering of muscle tissue using suitable hydrogels is an alternative to restoring the physiological properties of the injured area. Here, myogenic properties of type I collagen (0.5%) and keratin (0.5%) were investigated in a mouse model of biceps femoris injury. Using FTIR, gelation time, and rheological analysis, the physicochemical properties of the collagen (Col)/Keratin scaffold were analyzed. Mouse C2C12 myoblast-laden Col/Keratin hydrogels were injected into the injury site and histological examination plus western blotting were performed to measure myogenic potential after 15 days. FTIR indicated an appropriate interaction between keratin and collagen. The blend of Col/Keratin delayed gelation time when compared to the collagen alone group. Rheological analysis revealed decreased stiffening in blended Col/Keratin hydrogel which is favorable for the extrudability of the hydrogel. Transplantation of C2C12 myoblast-laden Col/Keratin hydrogel to injured muscle tissues led to the formation of newly generated myofibers compared to cell-free hydrogel and collagen groups (p < 0.05). In the C2C12 myoblast-laden Col/Keratin group, a low number of CD31+ cells with minimum inflammatory cells was evident. Western blotting indicated the promotion of MyoD in mice that received cell-laden Col/Keratin hydrogel compared to the other groups (p < 0.05). Despite the increase of the myosin cell-laden Col/Keratin hydrogel group, no significant differences were obtained related to other groups (p > 0.05). The blend of Col/Keratin loaded with myoblasts provides a suitable myogenic platform for the alleviation of injured muscle tissue.

Phenolated alginate hydrogel induced osteogenic properties of mesenchymal stem cells via Wnt signaling pathway.

Osteogenic properties of phenolated alginate (1.2 %) hydrogel containing collagen (0.5 %)/nano-hydroxyapatite (1 %) were studied on human mesenchymal stem cells in vitro. The phenolation rate and physical properties of the hydrogel were assessed using nuclear magnetic resonance (NMR), Fourier-transform infrared spectroscopy (FTIR), Scanning electron microscope (SEM), swelling ratio, gelation time, mechanical assay, and degradation rate. The viability of encapsulated cells was monitored on days 7, 14, and 21 using an MTT assay. Osteoblast differentiation was studied using western blotting, and real-time PCR. Using PCR array analysis, the role of the Wnt signaling pathway was also investigated. Data showed that the combination of alginate/collagen/nanohydroxyapatite yielded proper mechanical features. The addition of nanohydroxyapatite, and collagen reduced degradation, swelling rate coincided with increased stiffness. Elasticity and pore size were also diminished. NMR and FTIR revealed suitable incorporation of collagen and nanohydroxyapatite in the structure of alginate. Real-time PCR analysis and western blotting indicated the expression of osteoblast-related genes such as Runx2 and osteocalcin. PCR array revealed the induction of numerous genes related to Wnt signaling pathways during the maturation of human stem cells toward osteoblast-like cells. In vivo data indicated that transplantation of phenolated alginate/collagen/nanohydroxyapatite hydrogel led to enhanced de novo bone formation in rats with critical-sized calvarial defects. Phenolated alginate hydrogel can promote the osteogenic capacity of human amniotic membrane mesenchymal stem cells in the presence of nanohydroxyapatite and collagen via engaging the Wnt signaling pathway.

Combination of polyglycerol sebacate coated with collagen for vascular engineering.

Introduction: Here, we monitored the cytocompatibility of scaffolds consisting of poly (glycerol sebacate) (PGS) coated with collagen (Col) for endothelial cell activity after 72 hours. Methods: Human endothelial cells were allocated into Control, PGS, and PGS+Col groups. Scaffolds were characterized using FTIR and HNMR spectroscopy. Contact angel analysis and SEM were used to study wettability, surface morphology, and cell attachment. Cell survival was assessed using LDH leakage assay. Levels of Tie-1, Tie-2, VE-Cadherin, and VEGFR-2 were measured using western blotting and real-time PCR. Results: FTIR and HNMR analyses revealed the proper blending in PGS+Col group. SEM imaging exhibited a flat surface in the PGS group while thin Col fibers were detected in PGS+Col surface. The addition of Col to the PGS reduced the contract angle values from 97.3˚ to 81.1˚. Compared to PGS substrate alone, in PGS+Col group, cells appropriately attached to the surface. PGS and PGS+Col did not alter the leakage of LDH to the supernatant compared to control cells, showing the cytocopatiblity of PGS-based scaffolds. SOD and NO levels were increased significantly in PGS (p<0.05) and PGS+Col groups (p<0.001), respectively. We found that PGS+Col decreased Tie-1 content in endothelial cells whereas protein levels of Tie-2 and VE-Cadherin and expression of VEGFR-2 remained unchanged compared to PGS and control groups. Conclusion: Simultaneous application of Col and PGS can stimulate normal endothleial cell morphology without the alteration of tyrosine kinases receptors and cadherin.

Decellularized bovine ovarian niche restored the function of cumulus and endothelial cells.

OBJECTIVE: Recently, the decellularization technique is introduced as one of the tissue engineering procedures for the treatment of various deficiencies. Here, we aimed to assess the dynamic activity of CCs and HUVECs within decellularized bovine ovarian tissue transplanted subcutaneously in rats. Ovarian tissue was decellularized using a cocktail consisting of different chemicals, and the efficiency of decellularization was assessed using hematoxylin-eosin and DAPI staining. The cell survival was evaluated using an LDH leakage assay. Thereafter, decellularized samples were recellularized using HUVECs and CCs, encapsulated inside alginate (1.2%)-gelatin, (1%) hydrogel, and transplanted subcutaneously to rats. The existence of CD31- and estrogen-positive cells was assessed using immunohistochemistry staining. RESULTS: Bright-field imaging and DAPI staining revealed the lack of nuclei with naive matrix structure in ovarian tissue subjected to decellularization protocol. SEM imaging revealed a normal matrix in decellularized ovaries. LDH assay showed a lack of cytotoxicity for CCs after 7-days compared to the control group. Immunohistochemistry staining showed both CD31- and estrogen-positive cells in CCs + HUVECs compared to the CCs group. CD31 cells appeared with flattened morphology aligned with matrix fibers. The existence of estrogen and CD31 positive cells showed the efficiency of decellularized ovarian tissue to restore cellular function and activity.

Interaction of alginate with nano-hydroxyapatite-collagen using strontium provides suitable osteogenic platform.

BACKGROUND: 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, Ca2+, Ba2+, and Sr2+, 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. RESULTS: Physicochemical properties of hydrogels illustrated that microspheres cross-linked with Sr2+ had reduced swelling, enhanced stability, and mechanical strength, as compared to the other groups. Human MG-63 osteoblasts inside Sr2+ 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 Sr2+ cross-linked microspheres appropriately induced various signaling transduction pathways in human osteoblasts leading to osteogenic activity and dynamic growth. Transplantation of Sr2+ 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. CONCLUSION: Sr2+ cross-linked Alg-nHA-Col hydrogel can promote functionality and dynamic growth of osteoblasts.

PCL/Col I-based magnetic nanocomposite scaffold provides an osteoinductive environment for ADSCs in osteogenic cues-free media conditions.

BACKGROUND: The bone tissue engineering (BTE) approach has been introduced as an alternative to conventional treatments for large non-healing bone defects. Magnetism promotes stem cells' adherence to biocompatible scaffolds toward osteoblast differentiation. Furthermore, osteogenic differentiation media are expensive and any changes in its composition affect stem cells differentiation. Moreover, media growth factors possess a short half-life resulting in the rapid loss of their functions in vivo. With the above in mind, we fabricated a multilayered nanocomposite scaffold containing the wild type of Type I collagen (Col I) with endogenous magnetic property to promote osteogenesis in rat ADSCs with the minimum requirement of osteogenic differentiation medium. METHODS: Fe3O4 NPs were synthesized by co-precipitation method and characterized using SEM, VSM, and FTIR. Then, a PCL/Col I nanocomposite scaffold entrapping Fe3O4 NPs was fabricated by electrospinning and characterized using SEM, TEM, AFM, VSM, Contact Angle, tensile stretching, and FTIR. ADSCs were isolated from rat adipose tissue and identified by flow cytometry. ADSCs were loaded onto PCL/Col I and PCL/Col I/Fe3O4-scaffolds for 1-3 weeks with/without osteogenic media conditions. The cell viability, cell adhesion, and osteogenic differentiation were evaluated using MTT assay, SEM, DAPI staining, ALP/ARS staining, RT-PCR, and western blotting, respectively. RESULTS: SEM, VSM, and FTIR results indicated that Fe3O4 was synthesized in nano-sized (15-30 nm) particles with spherical-shaped morphology and superparamagnetic properties with approved chemical structure as FTIR revealed. According to SEM images, the fabricated magnetic scaffolds consisted of nanofiber (500-700 nm). TEM images have shown the Fe3O4 NPs entrapped in the scaffold's fiber without bead formation. FTIR spectra analysis confirmed the maintenance of the natural structure of Col I, PCL, and Fe3O4 upon electrospinning. AFM data have shown that MNPs incorporation introduced stripe-like topography to nanofibers, while the depth of the grooves has decreased from 800 to 500 nm. Flow cytometry confirmed the phenotype of ADSCs according to their surface markers (i.e., CD29 and CD105). Additionally, Fe3O4 NP improved nanocomposite scaffold strength, wettability, porosity, biocompatibility and also facilitates the ALP activity, calcium-mineralization. Finally, magnetic nanocomposite scaffolds upregulated osteogenic-related genes or proteins' expression (e.g., Col I, Runx2, OCN, ON, BMP2) in seeded ADSCs with/without osteo-differentiation media conditions. CONCLUSIONS: Together, these results indicate that Fe3O4 NPs within the natural structure of Col I increase osteogenic differentiation in osteogenic cues-free media conditions. This effect could be translated in vivo toward bone defects healing. These findings support the use of natural ECM materials alongside magnetic particles as composite scaffolds to achieve their full therapeutic potential in BTE treatments.

Collagen and nano-hydroxyapatite interactions in alginate-based microcapsule provide an appropriate osteogenic microenvironment for modular bone tissue formation.

The addition of nano-hydroxyapatite (nHA) and collagen (Col) to the alginate (Alg) microcapsule hydrogel reduced swelling and degradation ratios while the compressive strength increased compared to Alg, Alg-Col, and Alg-nHA groups. MTT assay and Calcein-AM staining revealed an enhanced MG-63 osteoblasts viability in the Alg-nHA-Col hydrogel compared to the other groups. SEM showed the attachment of MG-63 osteoblasts inside Alg-Col hydrogels. Non-significant differences were found in antioxidant capacity of cells inside the Alg-nHA-Col hydrogel compared to the Alg group. Hematoxylin-Eosin staining showed the distribution of MG-63 osteoblasts inside microspheres. Calcium deposits, alkaline phosphatase (ALP) activity with the increase of intracellular calcium were found in Alg-nHA-Col group. Western blotting showed that levels of osteocalcin, ColA2, Sox-9, and ColA1 also significantly increased compared to the Alg, Alg-Col, Alg-nHA groups. The present study demonstrated that the addition of mineral nHA and protein (Col) into the Alg improves osteogenic potential and provides a 3D platform for modular bone tissue engineering.

Static and dynamic culture of human endothelial cells encapsulated inside alginate-gelatin microspheres.

This study aimed to explore the angiogenesis potential of human endothelial cells encapsulated inside alginate-gelatin microspheres under static and dynamic culture systems after 7 days. Human umbilical vein endothelial cells were encapsulated inside alginate (1%) and gelatin (1.2%) using an electrostatic encapsulation method. Cells were incubated for 7 days in vitro. The cell survival rate was measured using the MTT assay. The expression of VEGFR-2 and von Willebrand factor genes was studied by real-time PCR assay. Using western blot analysis, we monitored the protein contents of VEGFR-2, vWF, and Caspase 3. The levels of SOD and GPx enzymes were calculated using biochemical kits. Angiogenesis potential was assessed using in vitro Matrigel assay. Data showed an increased survival rate in encapsulated cells cultured under the static condition compared to the conventional 2D condition (p < 0.05). The culture of encapsulated cells under a dynamic bioreactor system did not alter cell viability. Compared to the dynamic culture system, the incubation of encapsulated cells in the static culture system swelled the microspheres (p < 0.05). Both dynamic and static culture models increased the expression of VEGFR-2 and von Willebrand factor in encapsulated cells compared to 2D culture (p < 0.05), showing enhanced functional maturation. Data showed a significant increase of vWF and reduction of apoptosis marker Caspase in the dynamic culture system (p < 0.05). The levels of SOD and GPx were significantly increased in dynamic and static culture models as compared to the control 2D group (p < 0.05). In vitro tubulogenesis assay showed significant induction of angiogenesis in dynamic encapsulated HUVECs indicated with a large number of vascular tubes and arborized ECs compared to the control and static encapsulated HUVECs (p < 0.05). The current study suggests a bioreactor dynamic system is a reliable approach, similar to a static condition, for the expansion of encapsulated human ECs in a 3D milieu.

Tissue Engineering Strategies to Increase Osteochondral Regeneration of Stem Cells; a Close Look at Different Modalities.

The homeostasis of osteochondral tissue is tightly controlled by articular cartilage chondrocytes and underlying subchondral bone osteoblasts via different internal and external clues. As a correlate, the osteochondral region is frequently exposed to physical forces and mechanical pressure. On this basis, distinct sets of substrates and physicochemical properties of the surrounding matrix affect the regeneration capacity of chondrocytes and osteoblasts. Stem cells are touted as an alternative cell source for the alleviation of osteochondral diseases. These cells appropriately respond to the physicochemical properties of different biomaterials. This review aimed to address some of the essential factors which participate in the chondrogenic and osteogenic capacity of stem cells. Elements consisted of biomechanical forces, electrical fields, and biochemical and physical properties of the extracellular matrix are the major determinant of stem cell differentiation capacity. It is suggested that an additional certain mechanism related to signal-transduction pathways could also mediate the chondro-osteogenic differentiation of stem cells. The discovery of these clues can enable us to modulate the regeneration capacity of stem cells in osteochondral injuries and lead to the improvement of more operative approaches using tissue engineering modalities.

Influence of gelatin and collagen incorporation on peroxidase-mediated injectable pectin-based hydrogel and bioactivity of fibroblasts.

Pectin has recently attracted increasing attention for biomedical and pharmaceutical applications. Due to the lack of adhesion molecules in polysaccharides, phenolic hydroxyl conjugated gelatin was added to enzymatically-gellable peroxidase-modified pectin derivative and compared with phenolic hydroxyl -pectin/collagen. Both pectin and gelatin were modified by tyramine hydrochloride in the presence of EDC/NHS. The phenolic hydroxyl -pectin/phenolic hydroxyl -gelatin, phenolic hydroxyl-pectin/collagen, and phenolic hydroxyl -pectin hydrogels were prepared using horseradish peroxidase and hydrogen peroxide. The hydrogels were characterized by gelation time analysis. Morphology, enzymatic biodegradation, mechanical and swelling properties as well as water vapor transmission rate were also evaluated. Fibroblasts were cultured for 7 days, and the survival rate was evaluated using conventional MTT assay. Hydrogels composed of Ph-pectin/Ph-gelatin showed decreased biodegradation rate, and WVTR and further improved mechanical performance in comparison with other groups. Both phenolic hydroxyl -pectin/collagen and phenolic hydroxyl -pectin/phenolic hydroxyl -gelatin hydrogels exhibited porous structures. The hydrogels composed of collagen promoted cell survival rate 1.4 and 3.5 times compared to phenolic hydroxyl -gelatin and phenolic hydroxyl -pectin based hydrogels at the end of 7 days, respectively (p < 0.001). The study demonstrated the potential of enzymatically-gellable pectin-based hydrogels as cost-effective frameworks for use in tissue engineering applications.