Green Hydrogels Composed of Sodium Mannuronate/Guluronate, Gelatin and Biointeractive Calcium Silicates/Dicalcium Phosphate Dihydrate Designed for Oral Bone Defects Regeneration.

Innovative green, eco-friendly, and biologically derived hydrogels for non-load bearing bone sites were conceived and produced. Natural polysaccharides (copolymers of sodium D-mannuronate and L-guluronate) with natural polypeptides (gelatin) and bioactive mineral fillers (calcium silicates CaSi and dicalcium phosphate dihydrate DCPD) were used to obtain eco-sustainable biomaterials for oral bone defects. Three PP-x:y formulations were prepared (PP-16:16, PP-33:22, and PP-31:31), where PP represents the polysaccharide/polypeptide matrix and x and y represent the weight % of CaSi and DCPD, respectively. Hydrogels were tested for their chemical-physical properties (calcium release and alkalizing activity in deionized water, porosity, solubility, water sorption, radiopacity), surface microchemistry and micromorphology, apatite nucleation in HBSS by ESEM-EDX, FT-Raman, and micro-Raman spectroscopies. The expression of vascular (CD31) and osteogenic (alkaline phosphatase ALP and osteocalcin OCN) markers by mesenchymal stem cells (MSCs) derived from human vascular walls, cultured in direct contact with hydrogels or with 10% of extracts was analysed. All mineral-filled hydrogels, in particular PP-31:31 and PP-33:22, released Calcium ions and alkalized the soaking water for three days. Calcium ion leakage was high at all the endpoints (3 h-28 d), while pH values were high at 3 h-3 d and then significantly decreased after seven days (p < 0.05). Porosity, solubility, and water sorption were higher for PP-31:31 (p < 0.05). The ESEM of fresh samples showed a compact structure with a few pores containing small mineral granules agglomerated in some areas (size 5-20 microns). PP-CTRL degraded after 1-2 weeks in HBSS. EDX spectroscopy revealed constitutional compounds and elements of the hydrogel (C, O, N, and S) and of the mineral powders (Ca, Si and P). After 28 days in HBSS, the mineral-filled hydrogels revealed a more porous structure, partially covered with a thicker mineral layer on PP-31:31. EDX analyses of the mineral coating showed Ca and P, and Raman revealed the presence of B-type carbonated apatite and calcite. MSCs cultured in contact with mineral-filled hydrogels revealed the expression of genes related to vascular (CD31) and osteogenic (mainly OCN) differentiation. Lower gene expression was found when cells were cultured with extracts added to the culture medium. The incorporation of biointeractive mineral powders in a green bio-derived algae-based matrix allowed to produce bioactive porous hydrogels able to release biologically relevant ions and create a suitable micro-environment for stem cells, resulting in interesting materials for bone regeneration and healing in oral bone defects.

Mineral-Doped Poly(L-lactide) Acid Scaffolds Enriched with Exosomes Improve Osteogenic Commitment of Human Adipose-Derived Mesenchymal Stem Cells.

Exosomes derived from mesenchymal stem cells are extracellular vesicles released to facilitate cell communication and function. Recently, polylactic acid (PLA), calcium silicates (CaSi), and dicalcium phosphate dihydrate (DCPD) have been used to produce bioresorbable functional mineral-doped porous scaffolds-through thermally induced phase separation technique, as materials for bone regeneration. The aim of this study was to investigate the effect of mineral-doped PLA-based porous scaffolds enriched with exosome vesicles (EVs) on osteogenic commitment of human adipose mesenchymal stem cells (hAD-MSCs). Two different mineral-doped scaffolds were produced: PLA-10CaSi-10DCPD and PLA-5CaSi-5DCPD. Scaffolds surface micromorphology was investigated by ESEM-EDX before and after 28 days immersion in simulated body fluid (HBSS). Exosomes were deposited on the surface of the scaffolds and the effect of exosome-enriched scaffolds on osteogenic commitment of hAD-MSCs cultured in proximity of the scaffolds has been evaluated by real time PCR. In addition, the biocompatibility was evaluated by direct-contact seeding hAD-MSCs on scaffolds surface-using MTT viability test. In both formulations, ESEM showed pores similar in shape (circular and elliptic) and size (from 10-30 µm diameter). The porosity of the scaffolds decreased after 28 days immersion in simulated body fluid. Mineral-doped scaffolds showed a dynamic surface and created a suitable bone-forming microenvironment. The presence of the mineral fillers increased the osteogenic commitment of hAD-MSCs. Exosomes were easily entrapped on the surface of the scaffolds and their presence improved gene expression of major markers of osteogenesis such as collagen type I, osteopontin, osteonectin, osteocalcin. The experimental scaffolds enriched with exosomes, in particular PLA-10CaSi-10DCPD, increased the osteogenic commitment of MSCs. In conclusion, the enrichment of bioresorbable functional scaffolds with exosomes is confirmed as a potential strategy to improve bone regeneration procedures.

Vascular Wall-Mesenchymal Stem Cells Differentiation on 3D Biodegradable Highly Porous CaSi-DCPD Doped Poly (α-hydroxy) Acids Scaffolds for Bone Regeneration.

Vascularization is a crucial factor when approaching any engineered tissue. Vascular wall-mesenchymal stem cells are an excellent in vitro model to study vascular remodeling due to their strong angiogenic attitude. This study aimed to demonstrate the angiogenic potential of experimental highly porous scaffolds based on polylactic acid (PLA) or poly-e-caprolactone (PCL) doped with calcium silicates (CaSi) and dicalcium phosphate dihydrate (DCPD), namely PLA-10CaSi-10DCPD and PCL-10CaSi-10DCPD, designed for the regeneration of bone defects. Vascular wall-mesenchymal stem cells (VW-MSCs) derived from pig thoracic aorta were seeded on the scaffolds and the expression of angiogenic markers, i.e. CD90 (mesenchymal stem/stromal cell surface marker), pericyte genes α-SMA (alpha smooth muscle actin), PDGFR-ß (platelet-derived growth factor receptor-ß), and NG2 (neuron-glial antigen 2) was evaluated. Pure PLA and pure PCL scaffolds and cell culture plastic were used as controls (3D in vitro model vs. 2D in vitro model). The results clearly demonstrated that the vascular wall mesenchymal cells colonized the scaffolds and were metabolically active. Cells, grown in these 3D systems, showed the typical gene expression profile they have in control 2D culture, although with some main quantitative differences. DNA staining and immunofluorescence assay for alpha-tubulin confirmed a cellular presence on both scaffolds. However, VW-MSCs cultured on PLA-10CaSi-10DCPD showed an individual cells growth, whilst on PCL-10CaSi-10DCPD scaffolds VW-MSCs grew in spherical clusters. In conclusion, vascular wall mesenchymal stem cells demonstrated the ability to colonize PLA and PCL scaffolds doped with CaSi-DCPD for new vessels formation and a potential for tissue regeneration.