Bone cell response to 3D bioinspired scaffolds was tested on osteoblast culture supernatants and by means of quantitative polymerase chain reaction (qPCR). Foaming and freeze-drying method was optimized in order to obtain three-dimensional interconnected porous scaffolds of gelatin at different contents of nanocrystalline hydroxyapatite (HA). Addition of a non toxic crosslinking agent during foaming stabilized the scaffolds, as confirmed by the slow and relatively low gelatin release in phosphate buffer up to 28 days. Micro-computed tomography reconstructed images showed porous interconnected structures, with interconnected pores displaying average diameter ranging from about 158 to about 71 μm as the inorganic phase content increases from 0 to 50 wt %. The high values of connectivity (>99%), porosity (> 60%), and percentage of pores with a size in the range 100-300 μm (>50%) were maintained up to 30 wt % HA, whereas higher content provoked a reduction of these parameters, as well as of the average pore size, and a significant increase of the compressive modulus and collapse strength up to 8 ± 1 and 0.9 ± 0.2 MPa, respectively. Osteoblast cultured on the scaffolds showed good adhesion, proliferation and differentiation. The presence of HA promoted ALP activity, TGF-β1, and osteocalcin production, in agreement with the observed upregulation of ALP, OC, Runx2, and TGF-β1 gene in qPCR analysis, indicating that the composite scaffolds enhanced osteoblast activation and extra-cellular matrix mineralization processes.
Sterilization through γ-irradiation has been reported to affect collagen mechanical properties, but its possible effects on gelatin based materials have not been investigated up to now. Herein we report the results of a mechanical, chemical and thermal study performed on gelatin films before and after γ-irradiation. The investigation was performed on uncrosslinked films as well as on crosslinked films. To this aim, two common crosslinking agents, glutaraldehyde and genipin, at different concentration (0.15, 0.30 and 0.67%) were used. The results indicate that sterilization significantly affects the mechanical properties of uncrosslinked films, whereas it displays a modest effect on gelatin swelling, release in solution, thermal stability and molecular structure. Both glutaraldehyde and genipin enhance the mechanical properties and stability in solution of the gelatin films. In particular, the values of Young modulus increase as a function of crosslinker concentration up to about 10 and 18 MPa for genipin and glutaraldehyde treated samples respectively. The results of in vitro study demonstrate that the films crosslinked with genipin do not display any cytotoxic reaction, whereas glutaraldehyde crosslinking provokes an acute and dose dependent cytotoxic effect.
3D highly porous (93% total porosity) gelatin scaffolds were prepared according to a novel, simple method, which implies gelatin foaming, gelification, soaking into ethanol and successive freeze-drying. Reinforcement of the as-prepared scaffolds (GEL) was performed through immersion in aqueous solutions at different gelatin concentrations. Reinforcement solutions with and without genipin addition allowed to prepare two series of samples:cross-linked and uncross-linked samples, respectively. The amount of gelatin adsorbed onto the reinforced samples increases as a function of gelatin concentration in solution and provokes a drastic improvement of the compressive modulus and collapse strength up to values of about 30 and 4 MPa, respectively. The open and interconnected porosity, although slightly reduced, is still of the order of 80% in the samples reinforced with the highest concentration of gelatin. Water uptake ability evaluated after immersion in PBS for 20 s decreases with gelatin reinforcement. The presence of genipin in cross-linked samples reduces gelatin release and stabilizes the scaffolds in solution. Chondrocytes from human articular cartilage adhere, proliferate, and penetrate into the scaffolds. The evaluation of differentiation markers both on the supernatants of cell culture and by means of quantitative polymerase chain reaction (qPCR) indicates a dose-dependent promotion of cell differentiation.
A promising strategy for osteochondral interface regeneration consists in the development of hybrid scaffolds, composed of distinct but integrated layers able to mimic the different regions of cartilage and bone. We developed multi-layered scaffolds by assembling a gelatin layer with layers containing different amounts of gelatin and hydroxyapatite nanocrystals, and using a gelatin solution (as a glue) to stick layers together. The scaffolds exhibit a high, interconnected porosity and mechanical properties varying with composition along the thickness of the scaffolds up to values of compressive stress and modulus of about 1 and 14 MPa, respectively. In vitro tests demonstrate that the different layers of the scaffolds promote chondrogenic and osteogenic differentiation of Human Mesenchimal Stem Cells (hMSC).
The systemic administration of bisphosphonates (BPs) for the treatment of metabolic diseases characterized by abnormal bone loss suffers from several adverse side effects, which can be reduced by implementation of alternative modes of administration. In this work, glutaraldehyde cross-linked gelatin scaffolds are proposed as delivery systems of calcium alendronate monohydrate (CaAL•H O). The 3D highly porous scaffolds display a relevant interconnected porosity (>94%), independently from CaAL•H O content (0, 3, and 6 wt%). At variance, pore size varies with composition. The relative increase of the number of smaller pores on increasing BP content is in agreement with the parallel significant increase of the compressive modulus and collapse strength. The scaffolds exhibit a sustained CaAL•H O release profile, and a significant amount of the drug is retained in the scaffolds even after 14 d. In vitro tests are carried out using cocultures of osteoblast (OB) and osteoclast (OC). The evaluation of differentiation markers is performed both on the supernatants of cell culture and by means of quantitative polymerase chain reaction. The results indicate that BP containing scaffolds support osteoblast proliferation and differentiation, whereas they inhibit osteoclast viability and activity, displaying a promising beneficial role on bone repair processes.
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