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.

Controlled synthesis of dicalcium phosphate dihydrate (DCPD) from metastable solutions: insights into pathogenic calcification

Calcium phosphate (CaP) compounds may occur in the body as abnormal pathogenic phases in addition to their normal occurrence as bones and teeth. Dicalcium phosphate dihydrate (DCPD; CaPO4·2H2O), along with other significant CaP phases, have been observed in pathogenic calcifications such as dental calculi, kidney stones and urinary stones. While other studies have shown that polar amino acids can inhibit the growth of CaPs, these studies have mainly focused on hydroxyapatite (HAp; Ca10(PO4)6(OH)2) formation from highly supersaturated solutions, while their effects on DCPD nucleation and growth from metastable solutions have been less thoroughly explored. By further elucidating the mechanisms of DCPD formation and the influence of amino acids on those mechanisms, insights may be gained into ways that amino acids could be used in treatment and prevention of unwanted calcifications. The current study involved seeded growth of DCPD from metastable solutions at constant pH in the presence of neutral, acidic and phosphorylated amino acid side chains. As a comparison, solutions were also seeded with calcium pyrophosphate (CPP; Ca2P2O7), a known calcium phosphate inhibitor. The results show that polar amino acids inhibit DCPD growth; this likely occurs due to electrostatic interactions between amino acid side groups and charged DCPD surfaces. Phosphoserine had the greatest inhibitory ability of the amino acids tested, with an effect equal to that of CPP. Clustering of DCPD crystals giving rise to a “chrysanthemum-like” morphology was noted with glutamic acid. This study concludes that molecules containing an increased number of polar side groups will enhance the inhibition of DCPD seeded growth from metastable solutions.

Synthesis of Brushite from Phophogypsum Industrial Waste

Dicalcium phosphate dihydrate (DCPD) nanoparticles, also known as brushite, are considered an important bioceramic compound. In this study, brushite was prepared from Moroccan phosphogypsum (PG) using a new sol-gel method. A two-step technique undergoes the synthesis of brushite, the preparation of anhydrite from PG followed by adding phosphoric acid in the presence of sodium hydroxide. The morphology, the chemical composition, and the crystallites size were obtained using Scanning Electron Microscopy (SEM-EDAX), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR), respectively. According to the Debye-Scherrer equation, these characterization methods indicated that the synthesized brushite was highly pure according to the Ca/P ratio of 1.14 and an average crystallites size estimated at 66 nm. These results proved that the brushite was successfully synthesized from Moroccan phosphogypsum.

Effects of pullulan on the biomechanical and anti-collapse properties of dicalcium phosphate dihydrate bone cement

In this work, a modified dicalcium phosphate dihydrate (DCPD) bone cement with unique biodegradable ability in a calcium phosphate cement system was prepared by the hydration reaction of monocalcium phosphate monohydrate and calcium oxide and integration with pullulan (Pul), a non-toxic, biocompatible, viscous, and water-soluble polysaccharide that has been successfully used to improve defects in DCPD bone cement, especially its rapid solidification, fragile mechanical properties, and easy collapse. The effect of different contents of Pul on the structure and properties of DCPD were also studied in detail. The modified cement was characterised by X-ray diffraction, Fourier-transform infrared spectroscopy, scanning electron microscopy, ultraviolet–visible absorption, X-ray photoelectron spectroscopy analysis, and rheological property measurements. The results indicated that Pul promoted the hydration formation of DCPD, and interface bonding occurred between Pul and DCPD. With increasing content of Pul, the setting time of the DCPD bone cement increased from 2.6 min to 42.3 min, the compressive strength increased from 0 MPa to 20.4 MPa, and the anti-collapse ability also improved owing to the strong interface bonding, implying that the DCPD bone cement improved by Pul has better potential for application in the field of non-loading bone regenerative medicine compared to unmodified DCPD bone cement.