Bioprinting of human dermal microtissues precursors as building blocks for endogenousin vitroconnective tissue manufacturing.

The advent of 3D bioprinting technologies in tissue engineering has unlocked the potential to fabricatein vitrotissue models, overcoming the constraints associated with the shape limitations of preformed scaffolds. However, achieving an accurate mimicry of complex tissue microenvironments, encompassing cellular and biochemical components, and orchestrating their supramolecular assembly to form hierarchical structures while maintaining control over tissue formation, is crucial for gaining deeper insights into tissue repair and regeneration. Building upon our expertise in developing competent three-dimensional tissue equivalents (e.g. skin, gut, cervix), we established a two-step bottom-up approach involving the dynamic assembly of microtissue precursors (µTPs) to generate macroscopic functional tissue composed of cell-secreted extracellular matrix (ECM). To enhance precision and scalability, we integrated extrusion-based bioprinting technology into our established paradigm to automate, control and guide the coherent assembly ofµTPs into predefined shapes. Compared to cell-aggregated bioink, ourµTPs represent a functional unit where cells are embedded in their specific ECM.µTPs were derived from human dermal fibroblasts dynamically seeded onto gelatin-based microbeads. After 9 days,µTPs were suspended (50% v/v) in Pluronic-F127 (30% w/v) (µTP:P30), and the obtained formulation was loaded as bioink into the syringe of the Dr.INVIVO-4D6 extrusion based bioprinter.µTP:P30 bioink showed shear-thinning behavior and temperature-dependent viscosity (gel atT> 30 °C), ensuringµTPs homogenous dispersion within the gel and optimal printability. The bioprinting involved extruding several geometries (line, circle, and square) into Pluronic-F127 (40% w/v) (P40) support bath, leveraging its shear-recovery property. P40 effectively held the bioink throughout and after the bioprinting procedure, untilµTPs fused into a continuous connective tissue.µTPs fusion dynamics was studied over 8 days of culture, while the resulting endogenous construct underwent 28 days culture. Histological, immunofluorescence analysis, and second harmonic generation reconstruction revealed an increase in endogenous collagen and fibronectin production within the bioprinted construct, closely resembling the composition of the native connective tissues.

Functional and Molecular Analysis of Human Osteoarthritic Chondrocytes Treated with Bone Marrow-Derived MSC-EVs.

Osteoarthritis (OA) is a degenerative joint disease, causing impaired mobility. There are currently no effective therapies other than palliative treatment. Mesenchymal stromal cells (MSCs) and their secreted extracellular vesicles (MSC-EVs) have shown promise in attenuating OA progression, promoting chondral regeneration, and modulating joint inflammation. However, the precise molecular mechanism of action driving their beneficial effects has not been fully elucidated. In this study, we analyzed MSC-EV-treated human OA chondrocytes (OACs) to assess viability, proliferation, migration, cytokine and catabolic protein expression, and microRNA and mRNA profiles. We observed that MSC-EV-treated OACs displayed increased metabolic activity, proliferation, and migration compared to the controls. They produced decreased proinflammatory (Il-8 and IFN-γ) and increased anti-inflammatory (IL-13) cytokines, and lower levels of MMP13 protein coupled with reduced expression of MMP13 mRNA, as well as negative microRNA regulators of chondrogenesis (miR-145-5p and miR-21-5p). In 3D models, MSC-EV-treated OACs exhibited enhanced chondrogenesis-promoting features (elevated sGAG, ACAN, and aggrecan). MSC-EV treatment also reversed the pathological impact of IL-1ß on chondrogenic gene expression and extracellular matrix component (ECM) production. Finally, MSC-EV-treated OACs demonstrated the enhanced expression of genes associated with cartilage function, collagen biosynthesis, and ECM organization and exhibited a signature of 24 differentially expressed microRNAs, associated with chondrogenesis-associated pathways and ECM interactions. In conclusion, our data provide new insights on the potential mechanism of action of MSC-EVs as a treatment option for early-stage OA, including transcriptomic analysis of MSC-EV-treated OA, which may pave the way for more targeted novel therapeutics.

A cytokine-induced spheroid-based in vitro model for studying osteoarthritis pathogenesis.

Given the lack of in vitro models faithfully reproducing the osteoarthritis (OA) disease on-set, this work aimed at manufacturing a reliable and predictive in vitro cytokine-based Articular Cartilage (AC) model to study OA progression. Cell spheroids of primary human fetal chondrocytes (FCs) and h-TERT mesenchymal stem cells differentiated chondrocytes (Y201-C) were analysed in terms of growth kinetics, cells proliferation and apoptosis over 10 days of culture, in healthy condition or in presence of cytokines (interleukin-1ß, -6 and TNF-α). Then, the spheroids were assembled into chondrospheres using a bottom-up strategy, to obtain an in vitro cytokines-induced OA model. The resulting chondrospheres were evaluated for gene expression and anabolic ECM proteins. Compared to the healthy environment, the simulated OA environment induced chondrocyte hyperproliferation and apoptotic pathway, decreased expression of anabolic ECM proteins, and diminished biosynthetic activity, resembling features of early-stage OA. These characteristics were observed for both Y201-C and HC at high and low concentrations of cytokines. Both HC and Y201-C demonstrated the suitability for the manufacturing of a scaffold-free in vitro OA model to facilitate studies into OA pathogenesis and therapeutic strategies. Our approach provides a faithful reproduction of early-stage osteoarthritis, demonstrating the ability of obtaining different disease severity by tuning the concentration of OA-related cytokines. Given the advantages in easy access and more reproducible performance, Y201-C may represent a more favourable source of chondrocytes for establishing more standardized protocols to obtain OA models.

An In Vitro Engineered Osteochondral Model as Tool to Study Osteoarthritis Environment.

Osteoarthritis (OA) is a joint degenerative pathology characterized by mechanical and inflammatory damages affecting synovium, articular cartilage (AC), and subchondral bone (SB). Several in vitro, in vivo, and ex vivo models are developed to study OA, but to date the identification of specific pharmacological targets seems to be hindered by the lack of models with predictive capabilities. This study reports the development of a biomimetic in vitro model of AC and SB interface. Gellan gum methacrylated and chondroitin sulfate/dopamine hydrogels are used for the AC portion, whereas polylactic acid functionalized with gelatin and nanohydroxyapatite for the SB. The physiological behavior of immortalized stem cells (Y201s) and Y201s differentiated in chondrocytes (Y201-Cs), respectively, for the SB and AC, is demonstrated over 21 days of culture in vitro in healthy and pathological conditions, whilst modeling the onset of cytokines-induced OA. The key metrics are: lower glycosaminoglycans production and increased calcification given by a higher Collagen X content, in the AC deep layer; higher expression of pro-angiogenic factor (vegf) and decreased expression of osteogenic markers (coll1, spp1, runx2) in the SB. This novel approach provides a new tool for studying the development and progression of OA.

Adhesive Bioinspired Coating for Enhancing Glass-Ceramics Scaffolds Bioactivity.

Bioceramic scaffolds, composed of a biphasic composite containing bioactive glass and hydroxyapatite, were prepared in this work to overcome the intrinsic limits of the two components taken separately (in particular, their specific reactivities and dissolution rates, which should be tunable as a function of the given clinical requirements). To mimic the biological environment and tune the different stages of cellular response, a coating with gelatin and chondroitin sulphate via Layer-by-Layer (LbL) assembly was presented and discussed. The resulting functionalized scaffolds were affected by the coating in terms of microstructure and porosity. In addition, the LbL coating significantly enhanced the seeded cell behaviour, with high adhesion, proliferation and osteogenic activity, as revealed by the alkaline phosphatase activity and overexpression of osteopontin and osteocalcin.

A bioprintable gellan gum/lignin hydrogel: a smart and sustainable route for cartilage regeneration.

In this work a hydrogel, based on a blend of two gellan gums with different acyl content embedding lignin (up to 0.4%w/v) and crosslinked with magnesium ions, was developed for cartilage regeneration. The physico-chemical characterizations established that no chemical interaction between lignin and polysaccharides was detected. Lignin achieved up to 80 % of ascorbic acid's radical scavenging activity in vitro on DPPH and ABTS radicals. Viability of hMSC onto hydrogel containing lignin resulted comparable to the lignin-free one (>70 % viable cells, p > 0.05). The presence of lignin improved the hMSC 3D-constructs chondrogenesis, bringing to a significant (p < 0.05) up-regulation of the collagen type II, aggrecan and SOX 9 chondrogenic genes, and conferred bacteriostatic properties to the hydrogel, reducing the proliferation of S. aureus and S. epidermidis. Finally, cellularized 3D-constructs were manufactured via 3D-bioprinting confirming the processability of the formulation as a bioink and its unique biological features for creating a physiological milieu for cell growth.

Valuable effect of Manuka Honey in increasing the printability and chondrogenic potential of a naturally derived bioink.

Hydrogel-based bioinks are the main formulations used for Articular Cartilage (AC) regeneration due to their similarity to chondral tissue in terms of morphological and mechanical properties. However, the main challenge is to design and formulate bioinks able to allow reproducible additive manufacturing and fulfil the biological needs for the required tissue. In our work, we investigated an innovative Manuka honey (MH)-loaded photocurable gellan gum methacrylated (GGMA) bioink, encapsulating mesenchymal stem cells differentiated in chondrocytes (MSCs-C), to generate 3D bioprinted construct for AC studies. We demonstrated the beneficial effect of MH incorporation on the bioink printability, leading to the obtainment of a more homogenous filament extrusion and therefore a better printing resolution. Also, GGMA-MH formulation showed higher viscoelastic properties, presenting complex modulus G∗ values of ∼1042 â€‹Pa, compared to ∼730 â€‹Pa of GGMA. Finally, MH-enriched bioink induced a higher expression of chondrogenic markers col2a1 (14-fold), sox9 (3-fold) and acan (4-fold) and AC ECM main element production (proteoglycans and collagen).

A Chondrosphere-Based Scaffold Free Approach to Manufacture an In Vitro Articular Cartilage Model.

In vitro engineering of human articular cartilage (AC) is a regenerative medicine challenge. The main objective of this study was the development of a repeatable scaffold-free in vitro model of chondrocyte spheroid-based treatments of cartilage defects, to allow for systematic study and further optimization of this type of treatment. Human articular chondrocytes (HC) and immortalized mesenchymal cells differentiated in chondrocytes (Y201-Cs) were cultured in round-bottom 96-well plates to produce multicellular spheroids and their growth kinetics, and viability was evaluated over 7 days of culture. Then, the spheroids were assembled and cultured for 21 days on a gelatin-coated poly(lactic-co-glycolic acid) electrospun membrane (10 spheroids/cm2), following a protocol in line with the clinically approved Chondrosphere® (CO.DON AG) technique. Both HC and Y201-C cells formed compact and viable spheroids after 7 days of culture with a reduction of diameter over the 7 days from 1300 ± 150 µm to 600 ± 90 µm and from 1250 ± 60 µm to 800 ± 20 µm for HC and Y201-C, respectively. When the spheroids were transferred onto the support membrane, these adhered on the membrane itself and fused themselves, producing collagen type II (COL2A1) and aggrecan (ACAN), according to gene expression and glycosaminoglycans quantification analyses. We detected higher expression of COL2A1 in HC cells, while the Y201-C constructs were characterized by an increased ACAN expression. The approach we presented allows a standardizable production of spheroids with predictable geometry and the creation of a reproducible scaffold-free in vitro AC-like construct showing high expression of chondrogenic markers, using both HC and Y201-C. In addition, the bankable Y201-C cells provide an effective base model for experimentation with the spheroid approach to further enhance the process. Impact statement This is first work on the development of a repeatable scaffold-free in vitro model based on an optimized protocol in line with a recent clinically approved Chondrosphere® (CO.DON AG) technique. In addition, we demonstrated that a bankable cell type (Y201-C) could produce an engineered cartilage-like construct, giving a repeatable model as a key tool for experimentation of therapeutic treatment ahead of studies with heterogeneous cell populations.

Insights into oxidative stress in bone tissue and novel challenges for biomaterials.

The presence of Reactive Oxygen Species (ROS) in bone can influence resident cells behaviour as well as the extra-cellular matrix composition and the tissue architecture. Aging, in addition to excessive overloads, unbalanced diet, smoking, predisposing genetic factors, lead to an increase of ROS and, if it is accompanied with an inappropriate production of scavengers, promotes the generation of oxidative stress that encourages bone catabolism. Furthermore, bone injuries can be triggered by numerous events such as road and sports accidents or tumour resection. Although bone tissue possesses a well-known repair and regeneration capacity, these mechanisms are inefficient in repairing large size defects and bone grafts are often necessary. ROS play a fundamental role in response after the implant introduction and can influence its success. This review provides insights on the mechanisms of oxidative stress generated by an implant in vivo and suitable ways for its modulation. The local delivery of active molecules, such as polyphenols, enhanced bone biomaterial integration evidencing that the management of the oxidative stress is a target for the effectiveness of an implant. Polyphenols have been widely used in medicine for cardiovascular, neurodegenerative, bone disorders and cancer, thanks to their antioxidant and anti-inflammatory properties. In addition, the perspective of new smart biomaterials and molecular medicine for the oxidative stress modulation in a programmable way, by the use of ROS responsive materials or by the targeting of selective molecular pathways involved in ROS generation, will be analysed and discussed critically.

PLGA Membranes Functionalized with Gelatin through Biomimetic Mussel-Inspired Strategy.

Electrospun membranes have been widely used as scaffolds for soft tissue engineering due to their extracellular matrix-like structure. A mussel-inspired coating approach based on 3,4-dihydroxy-DL-phenylalanine (DOPA) polymerization was proposed to graft gelatin (G) onto poly(lactic-co-glycolic) acid (PLGA) electrospun membranes. PolyDOPA coating allowed grafting of gelatin to PLGA fibers without affecting their bulk characteristics, such as molecular weight and thermal properties. PLGA electrospun membranes were dipped in a DOPA solution (2 mg/mL, Tris/HCl 10 mM, pH 8.5) for 7 h and then incubated in G solution (2 mg/mL, Tris/HCl 10 mM, pH 8.5) for 16 h. PLGA fibers had an average diameter of 1.37 ± 0.23 µm. Quartz crystal microbalance with dissipation technique (QCM-D) analysis was performed to monitor DOPA polymerization over time: after 7 h the amount of deposited polyDOPA was 71 ng/cm2. After polyDOPA surface functionalization, which was, also revealed by Raman spectroscopy, PLGA membranes maintained their fibrous morphology, however the fiber size and junction number increased. Successful functionalization with G was demonstrated by FTIR-ATR spectra, which showed the presence of G adsorption bands at 1653 cm-1 (Amide I) and 1544 cm-1 (Amide II) after G grafting, and by the Kaiser Test, which revealed a higher amount of amino groups for G functionalized membranes. Finally, the biocompatibility of the developed substrates and their ability to induce cell growth was assessed using Neonatal Normal Human Dermal Fibroblasts.

Development of Natural-Based Bone Cement for a Controlled Doxorubicin-Drug Release.

Osteosarcoma (OS) accounts for 60% of all global bone cancer diagnoses. Intravenous administration of Doxorubicin Hydrochloride (DOXO) is the current form of OS treatment, however, systemic delivery has been linked to the onset of DOXO induced cardiomyopathy. Biomaterials including calcium phosphate cements (CPCs) and nanoparticles (NPs) have been tested as localized drug delivery scaffolds for OS cells. However, the tumor microenvironment is critical in cancer progression, with mesenchymal stem cells (MSCs) thought to promote OS metastasis and drug resistance. The extent of MSC assisted survival of OS cells in response to DOXO delivered by CPCs is unknown. In this study, we aimed at investigating the effect of DOXO release from a new formulation of calcium phosphate-based bone cement on the viability of OS cells cocultured with hMSC in vitro. NPs made of PLGA were loaded with DOXO and incorporated in the formulated bone cement to achieve local drug release. The inclusion of PLGA-DOXO NPs into CPCs was also proven to increase the levels of cytotoxicity of U2OS cells in mono- and coculture after 24 and 72 h. Our results demonstrate that a more effective localized DOXO delivery can be achieved via the use of CPCs loaded with PLGA-DOXO NPs compared to CPCs loaded with DOXO, by an observed reduction in metabolic activity of U2OS cells in indirect coculture with hMSCs. The presence of hMSCs offer a degree of DOXO resistance in U2OS cells cultured on PLGA-DOXO NP bone cements. The consideration of the tumor microenvironment via the indirect inclusion of hMSCs in this study can act as a starting point for future direct coculture and in vivo investigations.

pH-Triggered Adhesiveness and Cohesiveness of Chondroitin Sulfate-Catechol Biopolymer for Biomedical Applications.

Nature provides biomaterials that tend to be effective to control both their adhesive and cohesive properties. A catecholamine motif found in the marine mussels, the mytilus edulis foot protein, can play adhesiveness and cohesiveness. Particularly, acidic pH drives catechol (Cat) to have adhesive function, resulting in surface coating, while basic pH allows to enhance its cohesive properties, resulting in the formation of hydrogels. In this work, we demonstrated the usefulness of Cat-conjugated chondroitin sulfate (CS) as a platform for mesenchymal stem cell culture, utilizing the adhesive property of CS-Cat as coating for different substrates and the cohesive properties as hydrogel for cells encapsulation. To prepare the CS-Cat biopolymer, dopamine (DP) was coupled to the CS by carbodiimide coupling reaction and the Cat content was determined by UV-Vis spectroscopy (4.8 ± 0.6%). To demonstrate the adhesive properties of the biopolymer, PLA, PCL, TiO2, and SiO2 substrates were immersed in CS-Cat solution (pH < 2). Following the coating, the surfaces became highly hydrophilic, exhibiting a contact angle less than 35°. Also, in the presence of an oxidizing agent at pH 8, CS-Cat solution immediately became a hydrogel, as shown by inverted-vial test. Finally, immortalized TERT human mesenchymal stem cells (Y201) confirmed the high cytocompatibility of the biopolymer. The CS-Cat coating significantly enabled the Y201 adhesion onto PLA substrates, while the prepared hydrogel demonstrated to be a suitable environment for the encapsulation of cells as suitable bioink for further bioprinting applications.

From the sea to the bee: Gellan gum-honey-diatom composite to deliver resveratrol for cartilage regeneration under oxidative stress conditions.

Carbohydrate-based porous scaffolds are promising biomaterials to support cartilage regeneration. In this respect, their composition could be designed to face clinical challenges, i.e., articular load bearing, infections and oxidative stress. Herein, an innovative scaffold has been developed, combining raw materials belonging to different kingdoms of life. Indeed, gellan gum, a bacterial-derived carbohydrate, was blended with a beehive product (Manuka honey) with prominent antibacterial features. Moreover, resveratrol, a phytoalexin with powerful antioxidant activity, was loaded into the silica shells of diatoms, unicellular microalgae with cytocompatible features. The developed composite porous scaffolds demonstrated mechanical properties suitable for cartilage regeneration. Furthermore, they allowed the controlled release of resveratrol, hindering bacterial proliferation and oxidative stress damage, while supporting stem cell colonization and chondrogenic differentiation.

Data on the influence of inorganic clays to improve mechanical and healing properties of antibacterial Gellan gum-Manuka honey hydrogels.

This work contains original data supporting our research paper "Advances in cartilage repair: the influence of inorganic clays to improve mechanical and healing properties of antibacterial Gellan gum-Manuka honey hydrogels", by Maria A. Bonifacio, Andrea Cochis, Stefania Cometa, Annachiara Scalzone, Piergiorgio Gentile, Giuseppe Procino, Serena Milano, Alessandro C. Scalia, Lia Rimondini, Elvira De Giglio [1]. The main paper describes how four different clays (i.e., mesoporous silica, bentonite and halloysite nanotubes, coded as MS, BE and HNT) as cheap, abundant and versatile feed materials can be used for the preparation of highly performant hydrogels as cartilage substitutes, based on Gellan Gum (GG) and Manuka Honey (MH). Here the composites were further examined by means of Thermogravimetric Analysis (TGA), histological analysis (Alcian blue and Safranin-O) and static compression tests. This set of data strengthens the evidence that these hydrogels possess biological and physicochemical characteristics suitable for their application as reinforcing inorganic fillers in composite materials designed for cartilage regeneration.

Advances in cartilage repair: The influence of inorganic clays to improve mechanical and healing properties of antibacterial Gellan gum-Manuka honey hydrogels.

Effective treatment of cartilage defects represents a challenging problem, mainly due to the tissue's limited intrinsic self-repair capacity; the use of polymeric scaffolds as tissue substitute is rapidly increasing, but it is still limited by poor mechanical properties. Moreover, the onset of an infection can irreversibly affect the healing process. Accordingly, in this work we describe, for the first time, the preparation of composite scaffolds based on gellan gum, antibacterial Manuka honey and an inorganic clay (mesoporous silica, sodium­calcium bentonite or halloysite nanotubes). The surface composition, morphology, mechanical and biological features of such composites are herein assessed, aiming to optimize the composition of a superior scaffold for cartilage repair. Results demonstrated that after 45 days of in vitro incubation with human mesenchymal stem cells, the mesoporous silica-composite hydrogels exhibited significant changes in peak elastic and dynamic moduli over time thus demonstrating superior mechanical properties. Moreover, mesoporous silica provided the best performances in terms of in vitro cytocompatibility and antibacterial preventive activity in protection of cells in a co-culture model. Therefore, this selected composition was exploited for subcutaneous implantation in mice to investigate materials biocompatibility and infection prevention. Results demonstrated that composites did not cause severe immune response as well as they were able to restrain the infection. Accordingly, GG-MH-MS composites represent a very promising tool for cartilage tissue engineering.

The interplay between chondrocyte spheroids and mesenchymal stem cells boosts cartilage regeneration within a 3D natural-based hydrogel.

Articular cartilage (AC) lacks the ability to self-repair and cell-based approaches, primarily based on using chondrocytes and mesenchymal stem cells (MSCs), are emerging as effective technology to restore cartilage functionality, because cells synergic functionality may support the maintenance of chondrogenic phenotype and promote extracellular matrix regeneration. This work aims to develop a more physiologically representative co-culture system to investigate the influence of MSCs on the activity of chondrocytes. A thermo-sensitive chitosan-based hydrogel, ionically crosslinked with ß-glycerophosphate, is optimised to obtain sol/gel transition at physiological conditions within 5 minutes, high porosity with pores diameter <30 µm, and in vitro mechanical integrity with compressive and equilibrium Young's moduli of 37 kPa and 17 kPa, respectively. Live/dead staining showed that after 1 and 3 days in culture, the encapsulated MSCs into the hydrogels are viable and characterised by round-like morphology. Furthermore chondrocyte spheroids, seeded on top of gels that contained either MSCs or no cells, show that the encapsulated MSCs stimulate chondrocyte activity within a gel co-culture, both in terms of maintaining the coherence of chondrocyte spheroids, leading to a larger quantity of CD44 (by immunofluorescence) and a higher production of collagen and glycosaminoglycans (by histology) compared with the mono-culture.