Bioactive glass ions for in vitro osteogenesis and microvascularization in gellan gum-collagen hydrogels.

Lack of bone grafts appeals for bone augmentation solutions. We aimed at osteogenic differentiation of human adipose stem cells (hASCs) and microvascularization in coculture with human umbilical vein endothelial cells (HUVECs) embedded in three-dimensional (3D) gellan gum (GG) and collagen type I (COL) hydrogel mixture. We compared endothelial growth medium-2 (EGM-2) and bioactive glass extract-based endothelial and osteogenic medium (BaG EM-OM) for vascularized bone-like graft development in vitro. Cell viability, cell number, and osteogenic and endothelial gene expression were analyzed. Mineralized hydroxyapatite residues, immunocytochemical staining of endothelial marker CD31 production and late osteogenic marker osteocalcin were imaged. With both media, good cell viability was observed within 3D hydrogel. EGM-2 condition induced significantly higher cell number compared to BaG EM-OM condition at both 7 and 14 days. Interestingly, both media supported osteogenic as well as endothelial marker gene expression. Moreover, formation of reticulated cellular structures was observed in both EGM-2 and BaG EM-OM conditions. However, hydroxyapatite mineralization and strong osteocalcin staining were detected only in BaG EM-OM condition. Importantly, strong production of CD31 and elongated tube-like structures were apparent in EGM-2 culture alone. In conclusion, we demonstrated efficient hASC osteogenic differentiation and microvessel-like network formation in coculture with HUVECs.

Design of modular gellan gum hydrogel functionalized with avidin and biotinylated adhesive ligands for cell culture applications.

This article proposes the coupling of the recombinant protein avidin to the polysaccharide gellan gum to create a modular hydrogel substrate for 3D cell culture and tissue engineering. Avidin is capable of binding biotin, and thus biotinylated compounds can be tethered to the polymer network to improve cell response. The avidin is successfully conjugated to gellan gum and remains functional as shown with fluorescence titration and electrophoresis (SDS-PAGE). Self-standing hydrogels were formed using bioamines and calcium chloride, yielding long-term stability and adequate stiffness for 3D cell culture, as confirmed with compression testing. Human fibroblasts were successfully cultured within the hydrogel treated with biotinylated RGD or biotinylated fibronectin. Moreover, human bone marrow stromal cells were cultured with hydrogel treated with biotinylated RGD over 3 weeks. We demonstrate a modular and inexpensive hydrogel scaffold for cell encapsulation that can be equipped with any desired biotinylated cell ligand to accommodate a wide range of cell types.

Bioactive glass ions induce efficient osteogenic differentiation of human adipose stem cells encapsulated in gellan gum and collagen type I hydrogels.

BACKGROUND: Due to unmet need for bone augmentation, our aim was to promote osteogenic differentiation of human adipose stem cells (hASCs) encapsulated in gellan gum (GG) or collagen type I (COL) hydrogels with bioactive glass (experimental glass 2-06 of composition [wt-%]: Na2O 12.1, K2O 14.0, CaO 19.8, P2O5 2.5, B2O3 1.6, SiO2 50.0) extract based osteogenic medium (BaG OM) for bone construct development. GG hydrogels were crosslinked with spermidine (GG-SPD) or BaG extract (GG-BaG). METHODS: Mechanical properties of cell-free GG-SPD, GG-BaG, and COL hydrogels were tested in osteogenic medium (OM) or BaG OM at 0, 14, and 21 d. Hydrogel embedded hASCs were cultured in OM or BaG OM for 3, 14, and 21 d, and analyzed for viability, cell number, osteogenic gene expression, osteocalcin production, and mineralization. Hydroxyapatite-stained GG-SPD samples were imaged with Optical Projection Tomography (OPT) and Selective Plane Illumination Microscopy (SPIM) in OM and BaG OM at 21 d. Furthermore, Raman spectroscopy was used to study the calcium phosphate (CaP) content of hASC-secreted ECM in GG-SPD, GG-BaG, and COL at 21 d in BaG OM. RESULTS: The results showed viable rounded cells in GG whereas hASCs were elongated in COL. Importantly, BaG OM induced significantly higher cell number and higher osteogenic gene expression in COL. In both hydrogels, BaG OM induced strong mineralization confirmed as CaP by Raman spectroscopy and significantly improved mechanical properties. GG-BaG hydrogels rescued hASC mineralization in OM. OPT and SPIM showed homogeneous 3D cell distribution with strong mineralization in BaG OM. Also, strong osteocalcin production was visible in COL. CONCLUSIONS: Overall, we showed efficacious osteogenesis of hASCs in 3D hydrogels with BaG OM with potential for bone-like grafts.

Human Adipose Stem Cells Differentiated on Braided Polylactide Scaffolds Is a Potential Approach for Tendon Tissue Engineering.

Growing number of musculoskeletal defects increases the demand for engineered tendon. Our aim was to find an efficient strategy to produce tendon-like matrix in vitro. To allow efficient differentiation of human adipose stem cells (hASCs) toward tendon tissue, we tested different medium compositions, biomaterials, and scaffold structures in preliminary tests. This is the first study to report that medium supplementation with 50 ng/mL of growth and differentiation factor-5 (GDF-5) and 280 µM l-ascorbic acid are essential for tenogenic differentiation of hASCs. Tenogenic medium (TM) was shown to significantly enhance tendon-like matrix production of hASCs compared to other tested media groups. Cell adhesion, proliferation, and tenogenic differentiation of hASCs were supported on braided poly(l/d)lactide (PLA) 96l/4d copolymer filament scaffolds in TM condition compared to foamed poly(l-lactide-co-ɛ-caprolactone) (PLCL) 70L/30CL scaffolds. A uniform cell layer formed on braided PLA 96/4 scaffolds when hASCs were cultured in TM compared to maintenance medium (MM) condition after 14 days of culture. Furthermore, total collagen content and gene expression of tenogenic marker genes were significantly higher in TM condition after 2 weeks of culture. The elastic modulus of PLA 96/4 scaffold was more similar to the elastic modulus reported for native Achilles tendon. Our study showed that the optimized TM is needed for efficient and rapid in vitro tenogenic extracellular matrix production of hASCs. PLA 96/4 scaffolds together with TM significantly stimulated hASCs, thus demonstrating the potential clinical relevance of this novel and emerging approach to tendon injury treatments in the future.