Towards Complex Tissues Replication: Multilayer Scaffold Integrating Biomimetic Nanohydroxyapatite/Chitosan Composites.

This study explores an approach to design and prepare a multilayer scaffold mimicking interstratified natural tissue. This multilayer construct, composed of chitosan matrices with graded nanohydroxyapatite concentrations, was achieved through an in situ biomineralization process applied to individual layers. Three distinct precursor concentrations were considered, resulting in 10, 20, and 30 wt% nanohydroxyapatite content in each layer. The resulting chitosan/nanohydroxyapatite (Cs/n-HAp) scaffolds, created via freeze-drying, exhibited nanohydroxyapatite nucleation, homogeneous distribution, improved mechanical properties, and good cytocompatibility. The cytocompatibility analysis revealed that the Cs/n-HAp layers presented cell proliferation similar to the control in pure Cs for the samples with 10% n-HAp, indicating good cytocompatibility at this concentration, while no induction of apoptotic death pathways was demonstrated up to a 20 wt% n-Hap concentration. Successful multilayer assembly of Cs and Cs/n-HAp layers highlighted that the proposed approach represents a promising strategy for mimicking multifaceted tissues, such as osteochondral ones.

pH-sensing hybrid hydrogels for non-invasive metabolism monitoring in tumor spheroids.

The constant increase in cancer incidence and mortality pushes biomedical research towards the development of in vitro 3D systems able to faithfully reproduce and effectively probe the tumor microenvironment. Cancer cells interact with this complex and dynamic architecture, leading to peculiar tumor-associated phenomena, such as acidic pH conditions, rigid extracellular matrix, altered vasculature, hypoxic condition. Acidification of extracellular pH, in particular, is a well-known feature of solid tumors, correlated to cancer initiation, progression, and resistance to therapies. Monitoring local pH variations, non-invasively, during cancer growth and in response to drug treatment becomes extremely important for understanding cancer mechanisms. Here, we describe a simple and reliable pH-sensing hybrid system, based on a thermoresponsive hydrogel embedding optical pH sensors, that we specifically apply for non-invasive and accurate metabolism monitoring in colorectal cancer (CRC) spheroids. First, the physico-chemical properties of the hybrid sensing platform, in terms of stability, rheological and mechanical properties, morphology and pH sensitivity, were fully characterized. Then, the proton gradient distribution in the spheroids proximity, in the presence or absence of drug treatment, was quantified over time by time lapse confocal light scanning microscopy and automated segmentation pipeline, highlighting the effects of the drug treatment in the extracellular pH. In particular, in the treated CRC spheroids the acidification of the microenvironment resulted faster and more pronounced over time. Moreover, a pH gradient distribution was detected in the untreated spheroids, with more acidic values in proximity of the spheroids, resembling the cell metabolic features observed in vivo in the tumor microenvironment. These findings promise to shed light on mechanisms of regulation of proton exchanges by cellular metabolism being essential for the study of solid tumors in 3D in vitro models and the development of personalized medicine approaches.

Genipin-crosslinked collagen scaffolds inducing chondrogenesis: a mechanical and biological characterization.

Articular cartilage degeneration is still an unsolved issue owing to its weak repairing capabilities, which usually result in fibrocartilage tissue formation. This fibrous tissue lacks of structural and bio-mechanical properties, degrading over time. Currently, arthroscopic techniques and autologous transplantation are the most used clinical procedures. However, rather than restoring cartilage integrity, these methods only postpone further cartilage deterioration. Therefore, tissue engineering strategies aimed at selecting scaffolds that remarkably support the chondrogenic differentiation of human mesenchymal stem cells (hMSCs) could represent a promising solution, but they are still challenging for researchers. In this study, the influence of two different genipin (Gp) crosslinking routes on collagen (Coll)-based scaffolds in terms of hMSCs chondrogenic differentiation and biomechanical performances was investigated. Three-dimensional (3D) porous Coll scaffolds were fabricated by freeze-drying techniques and were crosslinked with Gp following a "two-step" and an in "bulk" procedure, in order to increase the physico-mechanical stability of the structure. Chondrogenic differentiation efficacy of hMSCs and biomechanical behavior under compression forces through unconfined stress-strain tests were assessed. Coll/Gp scaffolds revealed an isotropic and highly homogeneous pore distribution along with an increase in the stiffness, also supported by the increase in the Coll denaturation temperature (Td  = 57-63°C) and a significant amount of Coll and GAG deposition during the 3 weeks of chondrogenic culture. In particular, the presence of Gp in "bulk" led to a more uniform and homogenous chondral-like matrix deposition by hMSCs if compared to the results obtained from the Gp "two-step" functionalization procedure.

Valorization of porcine by-products: a combined process for protein hydrolysates and hydroxyapatite production.

The meat industry generates large amounts of by-products that are costly to be treated and discarded ecologically; moreover, they could be used to extract high added-value compounds. In this work, we present an innovative combined process which allowed the parallel extraction of both organic and mineral compounds; more specifically protein hydrolysates and single-phase hydroxyapatite were obtained. The protein hydrolysates, extracted through an enzymatic hydrolysis with alcalase, showed a degree of hydrolysis of 53.3 ± 5.1%; moreover, they had a high protein content with peptides with molecular weight lower than 1.2 kDa. Their antioxidant activities, measured with ABTS and ORAC tests, were 21.1 ± 0.5 mg ascorbic acid equivalent/g of dry extract and 87.7 ± 6.3 mg Trolox equivalent/g of dry extract, respectively. Single-phase hydroxyapatite, obtained with a simple calcination at 700 °C on the residues of the hydrolysis process, showed a Ca/P ratio close to the stoichiometric one (1.65 vs. 1.67) and presented a nanometric structure. This study reports a simple and feasible process for the valorization of porcine by-products in a large-scale up generating products with potential applications for environment remediation, biomedicine, nutrition and catalysis/bioenergy.

Preparation and Characterization of Salt-Mediated Injectable Thermosensitive Chitosan/Pectin Hydrogels for Cell Embedding and Culturing.

In recent years, growing attention has been directed to the development of 3D in vitro tissue models for the study of the physiopathological mechanisms behind organ functioning and diseases. Hydrogels, acting as 3D supporting architectures, allow cells to organize spatially more closely to what they physiologically experience in vivo. In this scenario, natural polymer hybrid hydrogels display marked biocompatibility and versatility, representing valid biomaterials for 3D in vitro studies. Here, thermosensitive injectable hydrogels constituted by chitosan and pectin were designed. We exploited the feature of chitosan to thermally undergo sol-gel transition upon the addition of salts, forming a compound that incorporates pectin into a semi-interpenetrating polymer network (semi-IPN). Three salt solutions were tested, namely, beta-glycerophosphate (ßGP), phosphate buffer (PB) and sodium hydrogen carbonate (SHC). The hydrogel formulations (i) were injectable at room temperature, (ii) gelled at 37 °C and (iii) presented a physiological pH, suitable for cell encapsulation. Hydrogels were stable in culture conditions, were able to retain a high water amount and displayed an open and highly interconnected porosity and suitable mechanical properties, with Young's modulus values in the range of soft biological tissues. The developed chitosan/pectin system can be successfully used as a 3D in vitro platform for studying tissue physiopathology.

Sintering of magnesium-strontium doped hydroxyapatite nanocrystals: Towards the production of 3D biomimetic bone scaffolds.

The development of biomimetic scaffolds is a challenging aim in the field of bone repair for fabrication of osteoconductive and osteoinductive scaffolds. Biogenic hydroxyapatite (HA) is not stoichiometric but is substituted by several ions. An approach to improve synthetic scaffolds biomimetism can be the doping with osteoinductive ions. To this aim, herein thermally stable magnesium-strontium hydroxyapatite (HAMgSr) nanocrystals were synthesized and used for the fabrication of sintered highly porous scaffolds. The chemical and physical properties of the obtained scaffolds were analyzed by X-ray diffraction, scanning electron microscopy, mechanical testing. Three different substituting ions percentage were analyzed and among these, the copresence of Mg and Sr at 0.5 wt% has shown the best results in terms of thermal stability and mechanical properties. The potential utilization of these materials for bone regeneration purposes was preliminarily evaluated in vitro, by assaying proliferation of viable osteoblast-like cells; the experimental evidences suggest that the scaffolds can be exploited as bone-mimicking substrates suitable to support cell growth and proliferation. These observations underline the importance of the presence of Mg and Sr in scaffolds for bone remodeling as well as the good potential of the newly developed scaffolds for clinical use in tissue engineering.

Evaluation of in Vivo Response of Three Biphasic Scaffolds for Osteochondral Tissue Regeneration in a Sheep Model.

Osteochondral defects are a common problem in both human medicine and veterinary practice although with important limits concerning the cartilaginous tissue regeneration. Interest in the subchondral bone has grown, as it is now considered a key element in the osteochondral defect healing. The aim of this work was to generate and to evaluate the architecture of three cell-free scaffolds made of collagen, magnesium/hydroxyapatite and collagen hydroxyapatite/wollastonite to be implanted in a sheep animal model. Scaffolds were designed in a bilayer configuration and a novel "Honey" configuration, where columns of hydroxyapatite were inserted within the collagen matrix. The use of different types of scaffolds allowed us to identify the best scaffold in terms of integration and tissue regeneration. The animals included were divided into four groups: three were treated using different types of scaffold while one was left untreated and represented the control group. Evaluations were made at 3 months through CT analysis. The novel "Honey" configuration of the scaffold with hydroxyapatite seems to allow for a better reparative process, although we are still far from obtaining a complete restoration of the defect at this time point of follow-up.

Influence of Nanofiber Orientation on Morphological and Mechanical Properties of Electrospun Chitosan Mats.

This work explored the use of chitosan (Cs) and poly(ethylene oxide) (PEO) blends for the fabrication of electrospun fiber-orientated meshes potentially suitable for engineering fiber-reinforced soft tissues such as tendons, ligaments, or meniscus. To mimic the fiber alignment present in native tissue, the CS/PEO blend solution was electrospun using a traditional static plate, a rotating drum collector, and a rotating disk collector to get, respectively, random, parallel, circumferential-oriented fibers. The effects of the different orientations (parallel or circumferential) and high-speed rotating collector influenced fiber morphology, leading to a reduction in nanofiber diameters and an improvement in mechanical properties.

Biomechanical evaluation of hMSCs-based engineered cartilage for chondral tissue regeneration.

Articular cartilage regeneration is still an open challenge in the field of tissue engineering. Although autologous chondrocytes seeded on collagen scaffolds (CSs) have already showed interesting results in the long-term repair of chondral lesions, they are not exempt from disadvantages that could be overcome using mesenchymal stem cells (MSCs). The ability of polymeric scaffolds to support MSCs proliferation and differentiation has been widely documented. However, few studies assessed their mechanical performances and additionally performing a single mechanical test, i.e. stress-strain or stress-relaxation in compression. Articular cartilage, though, possesses unique and multifaceted mechanical properties that can be exhaustively described only implementing a complete set of mechanical tests. Hence, the final aim of this study was to in depth assess the mechanical properties of human MSCs-cultured collagen scaffolds applying unconfined stress-strain, stress-relaxation and dynamic compression tests and identify key mechanical parameters. Firstly, plain CSs were fabricated and cultured under chondrogenic conditions with human MSCs (hMSCs). CSs displayed a high-interconnected porosity permitting uniform hMSCs distribution along the scaffold depth. Within CSs, hMSCs differentiated in chondroblasts, characterized by the presence of the lacunae and by a pericellular matrix positive for GAGs and for type 2 collagen deposition. The deep implemented mechanical characterization highlighted that the engineered constructs display (i) higher resistance to compression, (ii) more marked viscoelastic behavior over time and (iii) increased dynamic properties compared to naked CSs. In particular, stress-strain testes showed significant increase in the engineered constructs' stiffness that can be related to the proteoglycan deposition, observed by histology at the end of culture. Stress-relaxation and dynamic tests pointed out a substantial increase of peak and equilibrium stresses, relaxation time and dynamic modulus in the engineered constructs compared to empty CSs, suggesting a considerable decrease in scaffold permeability due to a strong chondral matrix deposition. Overall, the obtained results indicate a significant improvement of cell/CS mechanical performance toward a cartilage-like mechanical behavior.

Osteochondral repair by a novel interconnecting collagen-hydroxyapatite substitute: a large-animal study.

A novel three-dimensional bicomponent substitute made of collagen type I and hydroxyapatite was tested for the repair of osteochondral lesions in a swine model. This scaffold was assembled by a newly developed method that guarantees the strict integration between the organic and the inorganic parts, mimicking the biological tissue between the chondral and the osseous phase. Thirty-six osteochondral lesions were created in the trochlea of six pigs; in each pig, two lesions were treated with scaffolds seeded with autologous chondrocytes (cell+group), two lesions were treated with unseeded scaffolds (cell- group), and the two remaining lesions were left untreated (untreated group). After 3 months, the animals were sacrificed and the newly formed tissue was analyzed to evaluate the degree of maturation. The International Cartilage Repair Society (ICRS) macroscopic assessment showed significantly higher scores in the cell- and untreated groups when compared with the cell+ group. Histological evaluation showed the presence of repaired tissue, with fibroblast-like and hyaline-like areas in all groups; however, with respect to the other groups, the cell- group showed significantly higher values in the ICRS II histological scores for "cell morphology" and for the "surface/superficial assessment." While the scaffold seeded with autologous chondrocytes promoted the formation of a reparative tissue with high cellularity but low glycosaminoglycans (GAG) production, on the contrary, the reparative tissue observed with the unseeded scaffold presented lower cellularity but higher and uniform GAG distribution. Finally, in the lesions treated with scaffolds, the immunohistochemical analysis showed the presence of collagen type II in the peripheral part of the defect, indicating tissue maturation due to the migration of local cells from the surroundings. This study showed that the novel osteochondral scaffold was easy to handle for surgical implantation and was stable within the site of lesion; at the end of the experimental time, all implants were well integrated with the surrounding tissue and no signs of synovitis were observed. The quality of the reparative tissue seemed to be superior for the lesions treated with the unseeded scaffolds, indicating the promising potential of this novel biomaterial for use in a one-stage procedure for osteochondral repair.