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One of the main hurdles to improving scaffolds for regenerative medicine is the development of non-invasive methods to monitor cell proliferation within three-dimensional environments. Recently, an electrical impedance-based approach has been identified as promising for three-dimensional proliferation assays. A low-cost impedance-based solution, easily integrable with multi-well plates, is here presented. Sensors were developed using biocompatible carbon-based ink on foldable polyimide substrates by means of a novel aerosol jet printing technique. The setup was tested to monitor the proliferation of human mesenchymal stromal cells into previously validated gelatin-chitosan hybrid hydrogel scaffolds. Reliability of the methodology was assessed comparing variations of the electrical impedance parameters with the outcomes of enzymatic proliferation assay. Results obtained showed a magnitude increase and a phase angle decrease at 4 kHz (maximum of 2.5 kΩ and −9 degrees) and an exponential increase of the modeled resistance and capacitance components due to the cell proliferation (maximum of 1.5 kΩ and 200 nF). A statistically significant relationship with enzymatic assay outcomes could be detected for both phase angle and electric model parameters. Overall, these findings support the potentiality of this non-invasive approach for continuous monitoring of scaffold-based cultures, being also promising in the perspective of optimizing the scaffold-culture system. [ABSTRACT FROM AUTHOR]

Purpose: Radiotherapy (RT) plays a crucial role in head and neck (HN) cancer treatment. Nevertheless, it can lead to serious and challenging adverse events such as osteoradionecrosis (ORN). A preclinical rabbit model of irradiated bone and ORN is herein proposed, with the aim to develop a viable model to be exploited for investigating new therapeutic approaches., Methods: Nine New Zealand white rabbits were irradiated using a single beam positioned to the left of the mandible and directed perpendicular to the left mandible. A 10 × 10 mm 2 region of interest (ROI) located below the first molar tooth on the left side was identified and irradiated with 7 Gy each fraction, once every 2 days, for five fractions. Dose distributions demonstrated that the corresponding ROI on the contralateral (right) mandibular side received approximately 5 Gy each fraction, thus bilateral irradiation of the mandible was achieved. ROIs were categorized as ROI H on the left side receiving the high dose and ROI L on the right side receiving the low dose. Rabbits were followed up clinically and imaged monthly. After 4 months, the irradiated bone was excised, and histological examination of ROIs was performed., Results: Radiological signs suggestive for ORN were detected in the entire population (100%) 16 weeks after irradiation on ROI H , which consisted of cortical erosion and loss of trabeculae. ROI L did not show any radiological evidence of bone damage. Histologically, both sides showed comparable signs of injury, with marked reduction in osteocyte count and increase in empty lacunae count., Conclusions: A preclinical double model was successfully developed. The side receiving the higher dose showed radiological and histological signs of bone damage, resulting in an ORN model. Whereas the contralateral side, receiving the lower dose, presented with histological damage only and a normal radiological appearance. This work describes the creation of a double model, an ORN and irradiated bone model, for further study using this animal species., (Head & Neck© 2024 The Author(s). Head & Neck published by Wiley Periodicals LLC.)

Human mesenchymal stromal cells (hMSCs), whether used alone or together with three-dimensional scaffolds, are the best-studied postnatal stem cells in regenerative medicine. In this study, innovative composite scaffolds consisting of a core-shell architecture were seeded with bone-marrow-derived hMSCs (BM-hMSCs) and tested for their biocompatibility and remarkable capacity to promote and support bone regeneration and mineralization. The scaffolds were prepared by grafting three different amounts of gelatin-chitosan (CH) hydrogel into a 3D-printed polylactic acid (PLA) core (PLA-CH), and the mechanical and degradation properties were analyzed. The BM-hMSCs were cultured in the scaffolds with the presence of growth medium (GM) or osteogenic medium (OM) with differentiation stimuli in combination with fetal bovine serum (FBS) or human platelet lysate (hPL). The primary objective was to determine the viability, proliferation, morphology, and spreading capacity of BM-hMSCs within the scaffolds, thereby confirming their biocompatibility. Secondly, the BM-hMSCs were shown to differentiate into osteoblasts and to facilitate scaffold mineralization. This was evinced by a positive Von Kossa result, the modulation of differentiation markers (osteocalcin and osteopontin), an expression of a marker of extracellular matrix remodeling (bone morphogenetic protein-2), and collagen I. The results of the energy-dispersive X-ray analysis (EDS) clearly demonstrate the presence of calcium and phosphorus in the samples that were incubated in OM, in the presence of FBS and hPL, but not in GM. The chemical distribution maps of calcium and phosphorus indicate that these elements are co-localized in the same areas of the sections, demonstrating the formation of hydroxyapatite. In conclusion, our findings show that the combination of BM-hMSCs and PLA-CH, regardless of the amount of hydrogel content, in the presence of differentiation stimuli, can provide a construct with enhanced osteogenicity for clinically relevant bone regeneration., Competing Interests: The authors declare no conflicts of interest.

Background: Reconstruction of mandibular bone defects is a surgical challenge, and microvascular reconstruction is the current gold standard. The field of tissue bioengineering has been providing an increasing number of alternative strategies for bone reconstruction. Methods: In this preclinical study, the performance of two bioengineered scaffolds, a hydrogel made of polyethylene glycol-chitosan (HyCh) and a hybrid core-shell combination of poly (L-lactic acid)/poly ( ε -caprolactone) and HyCh (PLA-PCL-HyCh), seeded with different concentrations of human mesenchymal stromal cells (hMSCs), has been explored in non-critical size mandibular defects in a rabbit model. The bone regenerative properties of the bioengineered scaffolds were analyzed by in vivo radiological examinations and ex vivo radiological, histomorphological, and immunohistochemical analyses. Results: The relative density increase (RDI) was significantly more pronounced in defects where a scaffold was placed, particularly if seeded with hMSCs. The immunohistochemical profile showed significantly higher expression of both VEGF-A and osteopontin in defects reconstructed with scaffolds. Native microarchitectural characteristics were not demonstrated in any experimental group. Conclusion: Herein, we demonstrate that bone regeneration can be boosted by scaffold- and seeded scaffold-reconstruction, achieving, respectively, 50% and 70% restoration of presurgical bone density in 120 days, compared to 40% restoration seen in spontaneous regeneration. Although optimization of the regenerative performance is needed, these results will help to establish a baseline reference for future experiments., Competing Interests: Some of the following authors (LS, DR, SP, PN, MF, RG, and JI) declare the present patents, but declare no other financial or non-financial competing interests: LS, DR., SP, PN, MF, RG, JI “Integrated core-shell bioactive structure for the regeneration of bone and osteochondral tissues” licensed to PCT: WO2022009126 (2022); priority IT20200016579 (2020). LS, DR, PG, KD, Salmeron-Sanchez M., Borsani E. “Tridimensional bioactive porous body for bone tissue regeneration and process for its preparation “licensed to ”PCT: WO2022009125 (2022); priority: IT20200016576 (2020). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Ferrari, Taboni, Chan, Townson, Gualtieri, Franz, Ruaro, Mathews, Daly, Douglas, Eu, Sahovaler, Muhanna, Ventura, Dey, Pandini, Pasini, Re, Bernardi, Bosio, Mattavelli, Doglietto, Joshi, Gilbert, Nicolai, Viswanathan, Sartore, Russo and Irish.)

Motivated by the enormous potential of hydrogels in regenerative medicine, new biocompatible gelatin-based hybrid hydrogels were developed through a green process using poly(ethylene glycol) diglycidyl ether as a cross-linking agent, adding carrageenan and chitosan polysaccharides to the network to better mimic the hybrid composition of native extracellular matrix. Overall, the hydrogels show suitable structural stability, high porosity and pore interconnectivity, good swellability, and finally, biocompatibility. Their mechanical behavior, investigated by tensile and compression tests, appears to be characterized by nonlinear elasticity with high compliance values, fast stress-relaxation, and good strain reversibility with no sign of mechanical failure for compressive loading-unloading cycles at relatively high deformation levels of 50%. Degradation tests confirm the hydrogel bioresorbability by gradual hydrolysis, during which the structural integrity of both materials is maintained, while their mechanical behavior becomes more and more compliant. Human Umbilical Cord-derived Mesenchymal Stem Cells (hUC-MSCs) were used to test the hydrogels as potential carriers for cell delivery in tissue engineering. hUC-MSCs cultured inside the hydrogels show a homogenous distribution and maintain their growth and viability for at least 21 days of culture, with an increasing proliferation trend. Hence, this study contributes to a further understanding of the potential use of hybrid hydrogels and hUC-MSCs for a wide range of biomedical applications, particularly in soft tissue engineering.

Scaffolds for tissue engineering are expected to respond to a challenging combination of physical and mechanical requirements, guiding the research towards the development of novel hybrid materials. This study introduces innovative three-dimensional bioresorbable scaffolds, in which a stiff poly(lactic acid) lattice structure is meant to ensure temporary mechanical support, while a bioactive gelatin-chitosan hydrogel is incorporated to provide a better environment for cell adhesion and proliferation. The scaffolds present a core-shell structure, in which the lattice core is realized by additive manufacturing, while the shell is nested throughout the core by grafting and crosslinking a hydrogel forming solution. After subsequent freeze-drying, the hydrogel network forms a highly interconnected porous structure that completely envelops the poly(lactic acid) core. Thanks to this strategy, it is easy to tailor the scaffold properties for a specific target application by properly designing the lattice geometry and the core/shell ratio, which are found to significantly affect the scaffold mechanical performance and its bioresorption. Scaffolds with a higher core/shell ratio exhibit higher mechanical properties, whereas reducing the core/shell ratio results in higher values of bioactive hydrogel content. Hydrogel contents up to 25 wt% could be achieved while maintaining high compression stiffness (>200 MPa) and strength (>5 MPa), overall, within the range of values displayed by human bone tissue. In addition, mechanical properties remain stable after prolonged immersion in water at body temperature for several weeks. On the other hand, the hydrogel undergoes gradual and homogeneous degradation over time, but the core-shell integrity and structural stability are nevertheless maintained during at least 7-week hydrolytic degradation tests. In vitro experiments with human mesenchymal stromal cells reveal that the core-shell scaffolds are biocompatible, and their physical-mechanical properties and architecture are suitable to support cell growth and osteogenic differentiation, as demonstrated by hydroxyapatite formation. These results suggest that the bioresorbable core-shell scaffolds can be considered and further studied, in view of clinically relevant endpoints in bone regenerative medicine.

Conductive nanocomposites play a significant role in tissue engineering by providing a platform to support cell growth, tissue regeneration, and electrical stimulation. In the present study, a set of electroconductive nanocomposite hydrogels based on gelatin (G), chitosan (CH), and conductive carbon black (CB) was synthesized with the aim of developing novel biomaterials for tissue regeneration application. The incorporation of conductive carbon black (10, 15 and 20 wt.%) significantly improved electrical conductivity and enhanced mechanical properties with the increased CB content. We employed an oversimplified unidirectional freezing technique to impart anisotropic morphology with interconnected porous architecture. An investigation into whether any anisotropic morphology affects the mechanical properties of hydrogel was conducted by performing compression and cyclic compression tests in each direction parallel and perpendicular to macroporous channels. Interestingly, the nanocomposite with 10% CB produced both anisotropic morphology and mechanical properties, whereas anisotropic pore morphology diminished at higher CB concentrations (15 and 20%), imparting a denser texture. Collectively, the nanocomposite hydrogels showed great structural stability as well as good mechanical stability and reversibility. Under repeated compressive cyclic at 50% deformation, the nanocomposite hydrogels showed preconditioning, characteristic hysteresis, nonlinear elasticity, and toughness. Overall, the collective mechanical behavior resembled the mechanics of soft tissues. The electrical impedance associated with the hydrogels was studied in terms of the magnitude and phase angle in dry and wet conditions. The electrical properties of the nanocomposite hydrogels conducted in wet conditions, which is more physiologically relevant, showed a decreasing magnitude with increased CB concentrations, with a resistive-like behavior in the range 1 kHz-1 MHz and a capacitive-like behavior for frequencies <1 kHz and >1 MHz. Overall, the impedance of the nanocomposite hydrogels decreased with increased CB concentrations. Together, these nanocomposite hydrogels are compositionally, morphologically, mechanically, and electrically similar to native ECMs of many tissues. These gelatin-chitosan-carbon black nanocomposite hydrogels show great promise for use as conducting substrates for the growth of electro-responsive cells in tissue engineering.

Bone regenerative medicine is a clinical approach combining live osteoblast progenitors, such as mesenchymal stromal cells (MSCs), with a biocompatible scaffold that can integrate into host bone tissue and restore its structural integrity. Over the last few years, many tissue engineering strategies have been developed and thoroughly investigated; however, limited approaches have been translated to clinical application. Consequently, the development and clinical validation of regenerative approaches remain a centerpiece of investigational efforts towards the clinical translation of advanced bioengineered scaffolds. The aim of this review was to identify the latest clinical trials related to the use of scaffolds with or without MSCs to regenerate bone defects. A revision of the literature was performed in PubMed, Embase, and Clinicaltrials.gov from 2018 up to 2023. Nine clinical trials were analyzed according to the inclusion criteria: six presented in the literature and three reported in Clinicaltrials.gov. Data were extracted covering background trial information. Six of the clinical trials added cells to scaffolds, while three used scaffolds alone. The majority of scaffolds were composed of calcium phosphate ceramic alone, such as β-tricalcium phosphate (TCP) (two clinical trials), biphasic calcium phosphate bioceramic granules (three clinical trials), and anorganic bovine bone (two clinical trials), while bone marrow was the primary source of the MSCs (five clinical trials). The MSC expansion was performed in GMP facilities, using human platelet lysate (PL) as a supplement without osteogenic factors. Only one trial reported minor adverse events. Overall, these findings highlight the importance and efficacy of cell-scaffold constructs in regenerative medicine under different conditions. Despite the encouraging clinical results obtained, further studies are needed to assess their clinical efficacy in treating bone diseases to optimize their application.

A great promise for tissue engineering is represented by scaffolds that host stem cells during proliferation and differentiation and simultaneously replace damaged tissue while maintaining the main vital functions. In this paper, a novel process was adopted to develop composite scaffolds with a core-shell structure for bone tissue regeneration, in which the core has the main function of temporary mechanical support, and the shell enhances biocompatibility and provides bioactive properties. An interconnected porous core was safely obtained, avoiding solvents or other chemical issues, by blending poly(lactic acid), poly(ε-caprolactone) and leachable superabsorbent polymer particles. After particle leaching in water, the core was grafted with a gelatin/chitosan hydrogel shell to create a cell-friendly bioactive environment within its pores. The physicochemical, morphological, and mechanical characterization of the hybrid structure and of its component materials was carried out by means of infrared spectroscopy, thermogravimetric analysis, scanning electron microscopy, and mechanical testing under different loading conditions. These hybrid polymer devices were found to closely mimic both the morphology and the stiffness of bones. In addition, in vitro studies showed that the core-shell scaffolds are efficiently seeded by human mesenchymal stromal cells, which remain viable, proliferate, and are capable of differentiating towards the osteogenic phenotype if adequately stimulated.

The mechanical milieu of the extracellular matrix (ECM) plays a key role in modulating the cellular responses. The native ECM exhibits viscoelasticity with stress relaxation behavior. Here, we reported the preparation of degradation-mediated stress relaxing semi-interpenetrating (semi-IPN) polymeric networks of hydroxyethyl cellulose in the crosslinked gelatin-polyethylene glycol (PEG) architecture, leveraging a newly developed synthesis protocol which successively includes one-pot gelation under physiological conditions, freeze-drying and a post-curing process. Fourier transform infrared (FTIR) confirmed the formation of the semi-IPN blend mixture. A surface morphology analysis revealed an open pore porous structure with a compact skin on the surface. The hydrogel showed a high water-absorption ability (720.00 ± 32.0%) indicating the ability of retaining a hydrophilic nature even after covalent crosslinking with functionalized PEG. Detailed mechanical properties such as tensile, compressive, cyclic compression and stress relaxation tests were conducted at different intervals over 28 days of hydrolytic degradation. Overall, the collective mechanical properties of the hydrogel resembled the mechanics of cartilage tissue. The rate of stress relaxation gradually increased with an increasing swelling ratio. Hydrolytic degradation led to a marked increase in the percentage dissipation energy and stress relaxation response, indicating the degradation-dependent viscoelasticity of the hydrogel. Strikingly, the hydrogel maintained the structural stability even after degrading two-thirds of its initial mass after a month-long hydrolytic degradation. This study demonstrates that this semi-IPN G-PEG-HEC hydrogel possesses bright prospects as a potential scaffolding material in tissue engineering.

Background: Endoscopic transnasal transclival intradural surgery is limited by a high postoperative cerebrospinal fluid leak rate. The aim of this study was to investigate the role of three-dimensional printing to create a personalized, rigid scaffold for clival reconstruction., Methods: Two different types of clivectomy were performed in 5 specimens with the aid of neuronavigation, and 11 clival reconstructions were simulated. They were repaired with polylactide, three-dimensional-printed scaffolds that were manually designed in a computer-aided environment based either on the real or on the predicted defect. Scaffolds were printed with a fused filament fabrication technique and different offsets. They were positioned and fixed either following the gasket seal technique or with screws. Postdissection radiological evaluation of scaffold position was performed in all cases. In 3 specimens, the cerebrospinal fluid leak pressure point was measured immediately after reconstruction., Results: The production process took approximately 30 hours. The designed scaffolds were satisfactory when no offset was added. Wings were added during the design to allow for screw positioning, but broke in 30% of cases. Radiological assessment documented maximal accuracy of scaffold positioning when the scaffold was created on the real defect; accuracy was satisfactory when the predicted clivectomy was performed under neuronavigation guidance. The cerebrospinal fluid leak pressure point was significantly higher when the scaffold was fixed with screws compared with the gasket technique., Conclusions: In this preclinical setting, additive manufacturing allows the creation of customized scaffolds that are effective in reconstructing even large and geometrically complex clival defects., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Gelatin-dextran hydrogel scaffolds (G-PEG-Dx) were evaluated for their ability to activate the bone marrow human mesenchymal stromal cells (BM-hMSCs) towards mineralization. G-PEG-Dx1 and G-PEG-Dx2, with identical composition but different architecture, were seeded with BM-hMSCs in presence of fetal bovine serum or human platelet lysate (hPL) with or without osteogenic medium. G-PEG-Dx1, characterized by a lower degree of crosslinking and larger pores, was able to induce a better cell colonization than G-PEG-Dx2. At day 28, G-PEG-Dx2, with hPL and osteogenic factors, was more efficient than G-PEG-Dx1 in inducing mineralization. Scanning electron microscopy (SEM) and Raman spectroscopy showed that extracellular matrix produced by BM-hMSCs and calcium-positive mineralization were present along the backbone of the G-PEG-Dx2, even though it was colonized to a lesser degree by hMSCs than G-PEG-Dx1. These findings were confirmed by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), detecting distinct lipidomic signatures that were associated with the different degree of scaffold mineralization. Our data show that the architecture and morphology of G-PEG-Dx2 is determinant and better than that of G-PEG-Dx1 in promoting a faster mineralization, suggesting a more favorable and active role for improving bone repair.

Selection of feasible hybrid-hydrogels for best chondrogenic differentiation of human mesenchymal stromal cells (hMSCs) represents an important challenge in cartilage regeneration. In this study, three-dimensional hybrid hydrogels obtained by chemical crosslinking of poly (ethylene glycol) diglycidyl ether (PEGDGE), gelatin (G) without or with chitosan (Ch) or dextran (Dx) polysaccharides were developed. The hydrogels, namely G-PEG, G-PEG-Ch and G-PEG-Dx, were prepared with an innovative, versatile and cell-friendly technique that involves two preparation steps specifically chosen to increase the degree of crosslinking and the physical-mechanical stability of the product: a first homogeneous phase reaction followed by directional freezing, freeze-drying and post-curing. Chondrogenic differentiation of human bone marrow mesenchymal stromal cells (hBM-MSC) was tested on these hydrogels to ascertain whether the presence of different polysaccharides could favor the formation of the native cartilage structure. We demonstrated that the hydrogels exhibited an open pore porous morphology with high interconnectivity and the incorporation of Ch and Dx into the G-PEG common backbone determined a slightly reduced stiffness compared to that of G-PEG hydrogels. We demonstrated that G-PEG-Dx showed a significant increase of its anisotropic characteristic and G-PEG-Ch exhibited higher and faster stress relaxation behavior than the other hydrogels. These characteristics were associated to absence of chondrogenic differentiation on G-PEG-Dx scaffold and good chondrogenic differentiation on G-PEG and G-PEG-Ch. Furthermore, G-PEG-Ch induced the minor collagen proteins and the formation of collagen fibrils with a diameter like native cartilage. This study demonstrated that both anisotropic and stress relaxation characteristics of the hybrid hydrogels were important features directly influencing the chondrogenic differentiation potentiality of hBM-MSC., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

In mammals, mechanics at multiple stages-nucleus to cell to ECM-underlie multiple physiological and pathological functions from its development to reproduction to death. Under this inspiration, substantial research has established the role of multiple aspects of mechanics in regulating fundamental cellular processes, including spreading, migration, growth, proliferation, and differentiation. However, our understanding of how these mechanical mechanisms are orchestrated or tuned at different stages to maintain or restore the healthy environment at the tissue or organ level remains largely a mystery. Over the past few decades, research in the mechanical manipulation of the surrounding environment-known as substrate or matrix or scaffold on which, or within which, cells are seeded-has been exceptionally enriched in the field of tissue engineering and regenerative medicine. To do so, traditional tissue engineering aims at recapitulating key mechanical milestones of native ECM into a substrate for guiding the cell fate and functions towards specific tissue regeneration. Despite tremendous progress, a big puzzle that remains is how the cells compute a host of mechanical cues, such as stiffness (elasticity), viscoelasticity, plasticity, non-linear elasticity, anisotropy, mechanical forces, and mechanical memory, into many biological functions in a cooperative, controlled, and safe manner. High throughput understanding of key cellular decisions as well as associated mechanosensitive downstream signaling pathway(s) for executing these decisions in response to mechanical cues, solo or combined, is essential to address this issue. While many reports have been made towards the progress and understanding of mechanical cues-particularly, substrate bulk stiffness and viscoelasticity-in regulating the cellular responses, a complete picture of mechanical cues is lacking. This review highlights a comprehensive view on the mechanical cues that are linked to modulate many cellular functions and consequent tissue functionality. For a very basic understanding, a brief discussion of the key mechanical players of ECM and the principle of mechanotransduction process is outlined. In addition, this review gathers together the most important data on the stiffness of various cells and ECM components as well as various tissues/organs and proposes an associated link from the mechanical perspective that is not yet reported. Finally, beyond addressing the challenges involved in tuning the interplaying mechanical cues in an independent manner, emerging advances in designing biomaterials for tissue engineering are also explored.

Regenerative medicine aims to restore damaged tissues and mainly takes advantage of human mesenchymal stromal cells (hMSCs), either alone or combined with three-dimensional scaffolds. The scaffold is generally considered a support, and its contribution to hMSC proliferation and differentiation is unknown or poorly investigated. The aim of this study was to evaluate the capability of an innovative three-dimensional gelatin-chitosan hybrid hydrogel scaffold (HC) to activate the osteogenic differentiation process in hMSCs. We seeded hMSCs from adipose tissue (AT-hMSCs) and bone marrow (BM-hMSCs) in highly performing HC of varying chitosan content in the presence of growing medium (GM) or osteogenic medium (OM) combined with Fetal Bovine Serum (FBS) or human platelet lysate (hPL). We primarily evaluated the viability and the proliferation of AT-hMSCs and BM-hMSCs under different conditions. Then, in order to analyse the activation of osteogenic differentiation, the osteopontin ( OPN ) transcript was absolutely quantified at day 21 by digital PCR. OPN was expressed under all conditions, in both BM-hMSCs and AT-hMSCs. Cells seeded in HC cultured with OM+hPL presented the highest OPN transcript levels, as expected. Interestingly, both BM-hMSCs and AT-hMSCs cultured with GM+FBS expressed OPN . In particular, BM-hMSCs cultured with GM+FBS expressed more OPN than those cultured with GM+hPL and OM+FBS; AT-hMSCs cultured with GM+FBS presented a lower expression of OPN when compared with those cultured with GM+hPL, but no significant difference was detected when compared with AT-hMSCs cultured with OM+FBS. No OPN expression was detected in negative controls. These results show the capability of HC to primarily and independently activate osteogenic differentiation pathways in hMCSs. Therefore, these scaffolds may be considered no more as a simple support, rather than active players in the differentiative and regenerative process.

Cartilage tissue engineering remains problematic because no systems are able to induce signals that contribute to native cartilage structure formation. Therefore, we tested the potentiality of gelatin-polyethylene glycol scaffolds containing three different concentrations of chitosan (CH; 0%, 8%, and 16%) on chondrogenic differentiation of human platelet lysate-expanded human bone marrow mesenchymal stromal cells (hBM-MSCs). Typical chondrogenic (SOX9, collagen type 2, and aggrecan), hypertrophic (collagen type 10), and fibrotic (collagen type 1) markers were evaluated at gene and protein level at Days 1, 28, and 48. We demonstrated that 16% CH scaffold had the highest percentage of relaxation with the fastest relaxation rate. In particular, 16% CH scaffold, combined with chondrogenic factor TGFβ3, was more efficient in inducing hBM-MSCs chondrogenic differentiation compared with 0% or 8% scaffolds. Collagen type 2, SOX9, and aggrecan showed the same expression in all scaffolds, whereas collagen types 10 and 1 markers were efficiently down-modulated only in 16% CH. We demonstrated that using human platelet lysate chronically during hBM-MSCs chondrogenic differentiation, the chondrogenic, hypertrophic, and fibrotic markers were significantly decreased. Our data demonstrate that only a high concentration of CH, combined with TGFβ3, creates an environment capable of guiding in vitro hBM-MSCs towards a phenotypically stable chondrogenesis., (© 2019 John Wiley & Sons, Ltd.)

Rational design and development of tailorable simple synthesis process remains a centerpiece of investigational efforts toward engineering advanced hydrogels. In this study, a green and scalable synthesis approach is developed to formulate a set of gelatin-based macroporous hybrid hydrogels. This approach consists of four sequential steps starting from liquid-phase pre-crosslinking/grafting, unidirectional freezing, freeze-drying, and finally post-curing process. The chemical crosslinking mainly involves between epoxy groups of functionalized polyethylene glycol and functional groups of gelatin both in liquid and solid state. Importantly, this approach allows to accommodate different polymers, chitosan or hydroxyethyl cellulose, under identical benign condition. Structural and mechanical anisotropy can be tuned by the selection of polymer constituents. Overall, all hydrogels show suitable structural stability, good swellability, high porosity and pore interconnectivity, and maintenance of mechanical integrity during 3-week-long hydrolytic degradation. Under compression, hydrogels exhibit robust mechanical properties with nonlinear elasticity and stress-relaxation behavior and show no sign of mechanical failure under repeated compression at 50% deformation. Biological experiment with human bone marrow mesenchymal stromal cells (hMSCs) reveals that hydrogels are biocompatible, and their physicomechanical properties are suitable to support cells growth, and osteogenic/chondrogenic differentiation, demonstrating their potential application for bone and cartilage regenerative medicine toward clinically relevant endpoints., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Bone marrow and adipose tissue human mesenchymal stem cells were seeded in highly performing 3D gelatin-chitosan hybrid hydrogels of varying chitosan content in the presence of human platelet lysate and evaluated for their proliferation and osteogenic differentiation. Both bone marrow and adipose tissue human mesenchymal stem cells in gelatin-chitosan hybrid hydrogel 1 (chitosan content 8.1%) or gelatin-chitosan hybrid hydrogel 2 (chitosan 14.9%) showed high levels of viability (80%-90%), and their proliferation and osteogenic differentiation was significantly higher with human platelet lysate compared to fetal bovine serum, particularly in gelatin-chitosan hybrid hydrogel 1. Mineralization was detected early, after 21 days of culture, when human platelet lysate was used in the presence of osteogenic stimuli. Proteomic characterization of human platelet lysate highlighted 59 proteins mainly involved in functions related to cell adhesion, cellular repairing mechanisms, and regulation of cell differentiation. In conclusion, the combination of our gelatin-chitosan hybrid hydrogels with hPL represents a promising strategy for bone regenerative medicine using human mesenchymal stem cells., Competing Interests: Declaration of conflicting interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Tissue engineers have explored a set of materials cues that can allow control of cell viability and guide cell fate and functions. Although the effect of substrate stiffness on cell fate has been extensively studied and established, the role of substrate stress relaxation, the ability of a substrate to dissipate cell-induced forces, is only emerging. Recently, several studies have demonstrated that substrate stress relaxation is an important mechanical cue for cell spreading, proliferation and differentiation in vitro. In this mini-review, we highlight the influence of substrate stress relaxation on cell behavior and function as well as provide future perspectives. Firstly, we describe the methods used for characterizing the stress relaxation/creep responses of hydrogels along with the molecular origin of viscoelastic properties. Then, we highlight the most recent studies elucidating the stress relaxation effect on cellular behavior using physically cross-linked hydrogels. Finally, we report on an emerging alternative design of tunable viscoelastic hydrogels: chemically cross-linked (reversible linkages) adaptable hydrogels that have been used as stable 3D cell culture platforms for a few years in the era of hydrogel systems.

Polymeric protein-based biocomposites were used in this work as water dispersions to generate, in situ, biobased mulching coatings by spray technique, as alternative to low density polyethylene films for soil mulching. At the end of their lifetime, these biodegradable coatings degrade in soil thank to the microbial community that mineralizes them. Protein hydrolysates (PH) were derived from waste products of the leather industry, while poly(ethylene glycol) diglycidyl ether (PEG) and epoxidized soybean oil (ESO) were used to make the biodegradable spray coatings. A study under greenhouse condition was carried out using seedling test plots in order to investigate the performance of the spray coatings and their possible influence on some aspects of leaf growth, functionality and nutritional quality of lettuce (Lactuca sativa L., Mortarella selection Romanella variety Duende) and on soil properties. The biodegradable coatings showed the same good agronomic performances comparable with the ones of a commercial low density polyethylene mulching film, maintaining the mulching effect for the requested cultivation period and ensuring at the same time a similar rate of plant growth and dry matter accumulation. The research showed that 2 months after the tillage carried out at the end of the cultivation the amount of coating residues present in the soil was <5% of the initial weight of the biodegradable coatings. At the end of the field test, the soil mulched with the polyethylene film recorded an electrical conductivity value lower with respect to the soil mulched with the sprayed coatings, which release nutrients in the soil during their decomposition., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A flow type quartz crystal microbalance (QCM) chemical sensor was developed for monitoring of heavy metal ions in aqueous solutions (that is suitable for environmental monitoring). The sensor is based upon surface chelation of the metal ions at multifunctional polymer modified gold electrodes on 9 MHz AT-cut quartz resonators, functioning as a QCM. New processes have been developed which enable to obtain surface-modified gold electrodes with high heavy metal ions complexing ability. These polymer grafted QCM sensors can selectively adsorb heavy metal ions, such as copper lead chrome and cadmium, from solution over a wide range from 0.01 to 1000 ppm concentration by complexation with functional groups in the polymers. Cations typically present in natural water did not interfere with the detection of heavy metals. X-Ray Reflectivity (XRR) and Total Reflection X-ray Fluorescence (TXRF) were carried out to characterise the unmodified and modified gold surfaces as well as to verify the possibility to selectively bond and remove metal ions.