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Martina Trentini,1,* Ugo D’Amora,2,* Alfredo Ronca,2 Luca Lovatti,2 José Luis Calvo-Guirado,3 Danilo Licastro,4 Simeone Dal Monego,4 Lucia Gemma Delogu,5 Mariusz R Wieckowski,6 Shlomo Barak,7 Oleg Dolkart,7 Barbara Zavan1 1Translational Medicine Department, University of Ferrara, Ferrara, 44121, Italy; 2Institute of Polymers, Composites and Biomaterials - National Research Council (IPCB-CNR), Naples, 80125, Italy; 3Faculty of Health Sciences, Universidad Autonoma de Chile, Santiago de Chile, 7500912, Chile; 4AREA Science Park, Trieste, 34149, Italy; 5Biomedical Science Department University of Padova, Padova, Italy; 6Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland; 7Magdent B.S.R. 3, Bnei Brak, Tel Aviv, Israel*These authors contributed equally to this workCorrespondence: Alfredo Ronca; Barbara Zavan, Email alfredo.ronca@cnr.it; barbara.zavan@unife.itIntroduction: In the process of bone regeneration, a prominent role is played by macrophages involved in both the initial inflammation and the regeneration/vascularization phases, due to their M2 anti-inflammatory phenotype. Together with osteoclasts, they participate in the degradation of the bone matrix if the inflammatory process does not end. In this complex scenario, recently, much attention has been paid to extracellular communication mediated by nanometer-sized vesicles, with high information content, called exosomes (EVs). Considering these considerations, the purpose of the present work is to demonstrate how the presence of a pulsed electromagnetic field (PEMF) can positively affect communication through EVs.Methods: To this aim, macrophages and osteoclasts were treated in vitro with PEMF and analyzed through molecular biology analysis and by electron microscopy. Moreover, EVs produced by macrophages were characterized and used to verify their activity onto osteoclasts.Results: The results confirmed that PEMF not only reduces the inflammatory activity of macrophages and the degradative activity of osteoclasts but that the EVS produced by macrophages, obtained from PEMF treatment, positively affect osteoclasts by reducing their activity.Discussion: The co-treatment of PEMF with M2 macrophage-derived EVs (M2-EVs) decreased osteoclastogenesis to a greater degree than separate treatments. Keywords: PEMF, exosomes, macrophages, bone, osteoclast

Background: In the case of a degenerated intervertebral disc (IVD), even though spinal fusion has provided good short-term clinical results, an alteration of the spine stability has been demonstrated by long-term studies. In this context, different designs of IVD prostheses have been proposed as alternative to spinal fusion. However, over the past few years, much of the recent research has been devoted to IVD tissue engineering, even if several limitations related to the complex structure of IVD are still presented.Purpose/Aim: Accordingly, the aim of the current paper was to develop a strategy in designing customised multiphasic nucleus/annulus scaffolds for IVD tissue engineering, benefiting from the great potential of reverse engineering, additive manufacturing and gels technology.Materials and Methods: The device consisted of a customised additive-manufactured poly(ε-caprolactone) scaffold with tailored architectural features as annulus and a cell-laden collagen-low molecular weight hyaluronic acid-based material as nucleus with specific rheological and functional properties. To this aim, injectability and viscoelastic properties of the hydrogel were analyzed. Furthermore, a mechanical and biological characterization of cell-laden multiphasic nucleus/annulus scaffold was performed.Results and Conclusions: Analyses on the developed devices demonstrated appropriate viscoelastic and mechanical properties. As evidenced by rheological tests, the hydrogel showed a shear-thinning behaviour, supporting the possibility to inject the material. The mechanical characterization highlighted a compressive modulus which falls in the range of lumbar discs, with the typical initial J-shaped stress–strain curve of natural IVDs. Furthermore, preliminary biological tests showed that human mesenchymal stem cells were viable over the culture period. [ABSTRACT FROM AUTHOR]

The main driving idea of the present study was the comparison between two different chemical modifications of hyaluronic acid (HA) followed by the development of nanocomposite hydrogels directly in situ by biomineralization of photocrosslinkable HA polymers through sol-gel synthesis. In this way, it has been possible to overcome some limitations due to classical approaches based on the physical blending of inorganic fillers into polymer matrix. To this aim, methacrylated and maleated HA, synthesized with similar degree of substitution (DS) were compared in terms of mechanical and physico-chemical properties. The success of in situ biomineralization was highlighted by reflect Fourier transform infrared spectroscopy and thermogravimetric analysis. Furthermore, mechanical characterization demonstrated the reinforcing effect of inorganic fillers evidencing a strong correlation with DS. The swelling behavior resulted to be correlated with filler concentration. Finally, the cytotoxicity tests revealed the absence of toxic components and an increase of cell proliferation over culture time was observed, highlighting these bio-nanocomposite hyaluronan derivatives as biocompatible hydrogel with tunable properties. [ABSTRACT FROM AUTHOR]

To improve wound healing, advanced biofabrication techniques are proposed here to develop customized wound patches to release bioactive agents targeting cell function in a controlled manner. Three-dimensional (3D) bioprinted "smart" patches, based on methacrylated gellan gum (GGMA), loaded with tannic acid (TA) or L-ascorbic acid (AA) have been manufactured. To improve stability and degradation time, gellan gum (GG) was chemically modified by grafting methacrylic moieties on the polysaccharide backbone. GGMA patches were characterized through physicochemical, morphological and mechanical evaluation. Kinetics release and antioxidant potential of TA and AA as well as antimicrobial activity against common pathogens Pseudomonas aeruginosa , Staphylococcus aureus and Escherichia coli in accordance with ISO 22196:2011 are reported. The cytocompatibility of the patches was demonstrated by direct and indirect tests on human dermal fibroblasts (HDF) as per ISO 10993. The positive effect of GGMA patches on cell migration was assessed through a wound healing assay. The results highlighted that the patches are cytocompatible, speed up wound healing and can swell upon contact with the hydration medium and release TA and AA in a controlled way. Overall, the TA- and AA-loaded GGMA patches demonstrated suitable mechanical features; no cytotoxicity; and antioxidant, antimicrobial and wound healing properties, showing satisfactory potential for wound dressing applications.

Although the multidisciplinary area of liver tissue engineering is in continuous progress, research in this field is still focused on developing an ideal liver tissue template. Innovative strategies are required to improve membrane stability and bioactivity. In our study, sustainable biomimetic membranes were developed by blending methacrylated chitosan (CSMA) with jellyfish collagen (jCol) for liver tissue engineering applications. The in vitro biological behaviour demonstrated the capability of the developed membranes to create a suitable milieu to enable hepatocyte growth and differentiation. The functionalization of chitosan together with the biocompatibility of marine collagen and the intrinsic membrane properties offered the ideal biochemical, topographical, and mechanical cues to the cells. Thanks to the enhanced CSMA/jCol membranes' characteristics, hepatocytes on such biomaterials exhibited improved growth, viability, and active liver-specific functions when compared to the cell fate achieved on CSMA membranes. Our study provides new insights about the influence of membrane properties on liver cells behaviour for the design of novel instructive biomaterials. The enrichment of functionalized chitosan with marine collagen represents a promising and innovative approach for the development of an appropriate platform for hepatic tissue engineering., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Chronic wounds (CWs) treatment still represents a demanding medical challenge. Several intrinsic physiological signals (i.e., pH) help to stimulate and support wound healing. CWs, in fact, are characterized by a predominantly alkaline pH of the exudate, which acidifies as the wound heals. Therefore, pH-responsive wound dressings hold great potential owing to their capability of tuning their functions according to the wound conditions. Herein, porous chitosan (CS)-based scaffolds loaded with cellulose nanocrystals (CNCs) and graphene oxide (GO) were successfully fabricated using a freeze-drying method. CNCs were extracted from bagasse pulps fibers through acid hydrolysis. GO was synthesised by Hummer's method. The scaffolds were then ionically cross-linked using the amino acid L-Arginine (Arg), as a bioactive agent, and tested as potential pH-responsive wound dressing. Notably, the effect of CNCs and GO singly and simultaneously loaded within the CS-Arg scaffolds was investigated. The modulation of CNCs and GO content within CS-Arg scaffolds facilitated the development of scaffolds with an optimal pH-dependent swelling ratio capability and extended degradation time. Furthermore, CS/CNC/GO-Arg scaffolds exhibited tuned biological features, in terms of antimicrobial activity, cellular proliferation/migration ability, and the expression of extracellular matrix specific markers (i.e., elastin and collagen I) related to wound healing in human dermal fibroblasts., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Stefania Scialla reports financial support was provided by National Research Council. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Wound healing is a dynamic process that requires an optimal extracellular environment, as well as an accurate synchronization between various cell types. Over the past few years, great efforts have been devoted to developing novel approaches for treating and managing burn injuries, sepsis, and chronic or accidental skin injuries. Multifunctional smart-polymer-based dressings represent a promising approach to support natural healing and address several problems plaguing partially healed injuries, including severe inflammation, scarring, and wound infection. Naturally derived compounds offer unique advantages such as minimal toxicity, cost-effectiveness, and outstanding biocompatibility along with potential anti-inflammatory and antimicrobial activity. Herein, the main driving idea of the work was the design and development of konjac glucomannan d-glucono-1,5-lactone (KG) films bioactivated by tannic acid and d-glucono-1,5-lactone (GL) addition. Our analysis, using attenuated total reflectance-Fourier transform infrared, atomic force microscopy, and surface energy measurements demonstrated that tannic acid (TA) clearly interacted with the KG matrix, acting as its cross-linker, whereas GL was embedded within the polymer structure. All developed films maintained a moist environment, which represents a pivotal property for wound dressing. Hemocompatibility experiments showed that all tested films exhibited no hemolytic impact on human erythrocytes. Moreover, the presence of TA and GL enhanced the metabolic and energetic activity in human dermal fibroblasts, as indicated by the MTT assay, showing results exceeding 150%. Finally, all films demonstrated high antibacterial properties as they significantly reduced the multiplication rate of both Staphylococcus aureus and Escherichia coli in bacterial broth and created the inhibition zones for S. aureus in agar plates. These remarkable outcomes make the KG/TA/GL film promising candidates for wound healing applications.

The brain consists of an interconnected network of neurons tightly packed in the extracellular matrix (ECM) to form complex and heterogeneous composite tissue. According to recent biomimicry approaches that consider biological features as active components of biomaterials, designing a highly reproducible microenvironment for brain cells can represent a key tool for tissue repair and regeneration. Indeed, this is crucial to support cell growth, mitigate inflammation phenomena and provide adequate structural properties needed to support the damaged tissue, corroborating the activity of the vascular network and ultimately the functionality of neurons. In this context, electro-fluid dynamic techniques (EFDTs), i.e., electrospinning, electrospraying and related techniques, offer the opportunity to engineer a wide variety of composite substrates by integrating fibers, particles, and hydrogels at different scales-from several hundred microns down to tens of nanometers-for the generation of countless patterns of physical and biochemical cues suitable for influencing the in vitro response of coexistent brain cell populations mediated by the surrounding microenvironment. In this review, an overview of the different technological approaches-based on EFDTs-for engineering fibrous and/or particle-loaded composite substrates will be proposed. The second section of this review will primarily focus on describing current and future approaches to the use of composites for brain applications, ranging from therapeutic to diagnostic/theranostic use and from repair to regeneration, with the ultimate goal of providing insightful information to guide future research efforts toward the development of more efficient and reliable solutions.

Aims: The first aim of this study was to characterize the surface topography of a novel 3D-printed dental implant at the micro- and macro-level. Its second aim was to evaluate the osteogenic, angiogenic, and immunogenic responses of human oral osteoblasts (hOBs), gingival fibroblasts (hGFs), mesenchymal stem cells (hAD-MSCs), and monocytes to this novel implant surface., Methods: A 3D-printed Ti-6Al-4 V implant was produced by selective laser melting and subjected to organic acid etching (TEST). It was then compared to a machined surface (CTRL). Its biological properties were evaluated via cell proliferation assays, morphological observations, gene expression analyses, mineralization assessments, and collagen quantifications., Results: Scanning electron microscopy analysis showed that the TEST group was characterized by a highly interconnected porous architecture and a roughed surface. The morphological observations showed good adhesion of cells cultured on the TEST surface, with a significant increase in hOB growth. Similarly, the gene expression analysis showed significantly higher levels of osseointegration biomarkers. Picrosirius staining showed a slight increase in collagen production in the TEST group compared to the CTRL group. hAD-MSCs showed an increase in endothelial and osteogenic commitment-related markers. Monocytes showed increased mRNA synthesis related to the M2 (anti-inflammatory) macrophagic phenotype., Conclusions: Considering the higher interaction with hOBs, hGFs, hAD-MSCs, and monocytes, the prepared 3D-printed implant could be used for future clinical applications., Clinical Relevance: This study demonstrated the excellent biological response of various cells to the porous surface of the novel 3D-printed implant., Competing Interests: Declaration of Competing Interest No conflict of interest is reported. No financial support was obtained for conducting the present investigation., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Over the past years, the development of innovative smart wound dressings is revolutionizing wound care management and research. Specifically, in the treatment of diabetic foot wounds, three-dimensional (3D) bioprinted patches may enable personalized medicine therapies. In the present work, a methacrylated hyaluronic acid (MeHA) bioink is employed to manufacture 3D printed patches to deliver small extracellular vesicles (sEVs) obtained from human mesenchymal stem cells (MSC-sEVs). The production of sEVs is maximized culturing MSCs in bioreactor. A series of in vitro analyses are carried out to demonstrate the influence of MSC-sEVs on functions of dermal fibroblasts and endothelial cells, which are the primary functional cells in skin repair process. Results demonstrate that both cell populations are able to internalize MSC-sEVs and that the exposure to sEVs stimulates proliferation and migration. In vivo experiments in a well-established diabetic mouse model of pressure ulcer confirm the regenerative properties of MSC-sEVs. The MeHA patch enhances the effectiveness of sEVs by enabling controlled release of MSC-sEVs over 7 days, which improve wound epithelialization, angiogenesis and innervation. The overall findings highlight that MSC-sEVs loading in 3D printed biomaterials represents a powerful technique, which can improve the translational potential of parental stem cell in terms of regulatory and economic impact., (© 2023. The Author(s).)

The most prevalent extracellular matrix (ECM) protein in the meniscus is collagen, which controls cell activity and aids in preserving the biological and structural integrity of the ECM. To create stable and high-precision 3D printed collagen scaffolds, ink formulations must possess good printability and cytocompatibility. This study aims to overlap the limitation in the 3D printing of pure collagen, and to develop a highly concentrated collagen ink for meniscus fabrication. The extrusion test revealed that 12.5 % collagen ink had the best combination of high collagen concentration and printability. The ink was specifically designed to have load-bearing capacity upon printing and characterized with respect to rheological and extrusion properties. Following printing of structures with different infill, a series of post-processing steps, including salt stabilization, pH shifting, washing, freeze-drying, crosslinking and sterilization were performed, and optimised to maintain the stability of the engineered construct. Mechanical testing highlighted a storage modulus of 70 kPa for the lower porous structure while swelling properties showed swelling ratio between 9 and 11 after 15 min of soaking. Moreover, human avascular and vascular meniscus cells cultured on the scaffolds deposited a meniscus-like matrix containing collagen I, II and glycosaminoglycans after 28 days of culture. Finally, as proof-of-concept, human size 3D printed meniscus scaffold were created., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Luigi Ambrosio reports financial support was provided by 10.13039/501100007601EU (European) Framework Programme for Research and Innovation Leadership in Enabling and Industrial Technologies. Carla Zihlmann reports a relationship with Geistlich Pharma AG that includes: employment. Niklaus Stiefel reports a relationship with Geistlich Pharma AG that includes: employment., (© 2023 The Authors.)

Critical-size skull defects caused by trauma, infection, and tumor resection raise great demands for efficient bone substitutes. Herein, a hybrid cross-linked hierarchical microporous hydrogel scaffold (PHCLS) was successfully assembled by a multistep procedure, which involved (i) the preparation of poly(lactic- co -glycolic)/nanohydroxyapatite (PLGA-HAP) porous microspheres, (ii) embedding the spheres in a solution of dopamine-modified hyaluronic acid and collagen I (Col I) and cross-linking via dopamine polyphenols binding to (i) Col I amino groups (via Michael addition) and (ii) PLGA-HAP (via calcium ion chelation). The introduction of PLGA-HAP not only improved the diversity of pore size and pore communication inside the matrix but also greatly enhanced the compressive strength (5.24-fold, 77.5 kPa) and degradation properties to construct a more stable mechanical structure. In particular, the PHCLS (200 mg, nHAP) promoted the proliferation, infiltration, and angiogenic differentiation of bone marrow mesenchymal stem cells in vitro, as well as significant ectopic angiogenesis and mineralization with a storage modulus enhancement of 2.5-fold after 30 days. Meanwhile, the appropriate matrix microenvironment initiated angiogenesis and early osteogenesis by accelerating endogenous stem cell recruitment in situ. Together, the PHCLS allowed substantial skull reconstruction in the rabbit cranial defect model, achieving 85.2% breaking load strength and 84.5% bone volume fractions in comparison to the natural cranium, 12 weeks after implantation. Overall, this study reveals that the hierarchical microporous hydrogel scaffold provides a promising strategy for skull defect treatment.

Pathological angiogenesis is a crucial attribute of several chronic diseases such as cancer, age-related macular degeneration, and osteoarthritis (OA). In the case of OA, pathological angiogenesis mediated by the vascular endothelial growth factor (VEGF), among other factors, contributes to cartilage degeneration and to implants rejection. In line with this, the use of the anti-VEGF bevacizumab (BVZ) has been shown to prevent OA progression and support cartilage regeneration. The aim of this work was to functionalize a medical grade collagen with poly (lactic-co-glycolic acid) (PLGA) microparticles containing BVZ via three-dimensional (3D) printing to target pathological angiogenesis. First, the effect of several formulation parameters on the encapsulation and release of BVZ from PLGA microparticles was studied. Then, the anti-angiogenic activity of released BVZ was tested in a 3D cell model. The 3D printability of the microparticle-loaded collagen ink was tested by evaluating the shape fidelity of 3D printed structures. Results showed that the release and the encapsulation efficiency of BVZ could be tuned as a function of several formulation parameters. In addition, the released BVZ was observed to reduce vascularization by human umbilical vein endothelial cells. Finally, the collagen ink with embedded BVZ microparticles was successfully printed, leading to shape-stable meniscus-, nose- and auricle-like structures. Taken altogether, we defined the conditions for the successful combination of BVZ-loaded microparticles with the 3D printing of a medical grade collagen to target pathological angiogenesis., Competing Interests: Declaration of Competing interest Niklaus Stiefel and Carla Zihlmann are employees of Geistlich Pharma AG. Geistlich Pharma AG provided the collagen material used in this study. Geistlich Pharma AG manufactures and commercializes collagen-based products for tissue regeneration. The other authors do not have any conflict of interests., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Gellan gum (GG) was chemically modified with methacrylic moieties to produce a photocrosslinkable biomaterial ink, hereinafter called methacrylated GG (GGMA), with improved physico-chemical properties, mechanical behavior and stability under physiological conditions. Afterwards, GGMA was functionalized by incorporating two different bioactive compounds, a naturally derived eumelanin extracted from the black soldier fly (BSF-Eumel), or hydroxyapatite nanoparticles (HAp), synthesized by the sol-gel method. Different ink formulations based on GGMA (2 and 4% ( w / v )), BSF-Eumel, at a selected concentration (0.3125 mg/mL), or HAp (10 and 30% w HAp /w GGMA ) were developed and processed by three-dimensional (3D) printing. All the functionalized GGMA-based ink formulations allowed obtaining 3D-printed GGMA-based scaffolds with a well-organized structure. For both bioactive signals, the scaffolds with the highest GGMA concentration (4% ( w / v )) and the highest percentage of infill (45%) showed the best performances in terms of morphological and mechanical properties. Indeed, these scaffolds showed a good structural integrity over 28 days. Given the presence of negatively charged groups along the eumelanin backbone, scaffolds consisting of GGMA/BSF-Eumel demonstrated a higher stability. From a mechanical point of view, GGMA/BSF-Eumel scaffolds exhibited values of storage modulus similar to those of GGMA ones, while the inclusion of HAp at 30% (w HAp /w GGMA ) led to a storage modulus of 32.5 kPa, 3.5-fold greater than neat GGMA. In vitro studies proved the capability of the bioactivated 3D-printed scaffolds to support 7F2 osteoblast cell growth and differentiation. BSF-Eumel and HAp triggered a different time-dependent physiological response in the osteoblasts. Specifically, while the ink with BSF-Eumel acted as a stimulus towards cell proliferation, reaching the highest value at 14 days, a higher expression of alkaline phosphatase activity was detected for scaffolds consisting of GGMA and HAp. The overall findings demonstrated the possible use of these biomaterial inks for 3D-printed bone tissue-engineered scaffolds.

Graphene oxide (GO) and its reduced form (rGO) have recently attracted a fascinating interest due to their physico-chemical properties, which have opened up new and interesting opportunities in a wide range of biomedical applications, such as wound healing. It is worth noting that GO and rGO may offer a convenient access to its ready dispersion within various polymeric matrices (such as cellulose and its derivative forms), owing to their large surface area, based on a carbon skeleton with many functional groups (i.e., hydroxyl, carboxyl, epoxy bridge, and carbonyl moieties). This results in new synergic properties due to the presence of both components (GO or rGO and polymers), acting at different length-scales. Furthermore, they have shown efficient antimicrobial and angiogenic properties, mostly related to the intracellular formation of reactive oxygen species (ROS), which are advantageous in wound care management. For this reason, GO or rGO integration in cellulose-based matrixes have allowed for designing highly advanced multifunctional hybrid nanocomposites with tailored properties. The current review aims to discuss a potential relationship between structural and physico-chemical properties (i.e., size, edge density, surface chemistry, hydrophilicity) of the nanocomposites with antimicrobials and angiogenic mechanisms that synergically influence the wound healing phenomenon, by paying particular attention to recent findings of GO or rGO/cellulose nanocomposites. Accordingly, after providing a general overview of cellulose and its derivatives, the production methods used for GO and rGO synthesis, the mechanisms that guide antimicrobial and angiogenic processes of tissue repair, as well as the most recent and remarkable outcomes on GO/cellulose scaffolds in wound healing applications, will be presented.

Hybrid bone substitute made up of a 3D printed polyetheretherketone (PEEK) scaffold coated with methacrylated hyaluronic acid (MeHA)-hydroxyapatite (HAp) hydrogel is the objective of the present work. Development and characterization of the scaffold and of the MeHA-HAp after its infiltration and UV photocrosslinking have been followed by analyses of its biological properties using human mesenchymal stem cells (MSCs). Interconnected porous PEEK matrices were produced by fused deposition modeling (FDM) characterized by a reticular pattern with 0°/90° raster orientation and square pores. In parallel, a MeHA-HAp slurry has been synthesized and infiltrated in the PEEK scaffolds. The mechanical properties of the coated and pure PEEK scaffold have been evaluated, showing that the inclusion of MeHA-HAp into the lattice geometry did not significantly change the strength of the PEEK structure with Young's modulus of 1034.9 ± 126.1 MPa and 1020.0 ± 63.7 MPa for PEEK and PEEK-MeHA-HAp scaffolds, respectively. Human MSCs were seeded on bare and coated scaffolds and cultured for up to 28 days to determine the adhesion, proliferation, migration and osteogenic differentiation. In vitro results showed that the MeHA-HAp coating promotes MSCs adhesion and proliferation and contributes to osteogenic differentiation and extracellular matrix mineralization. This study provides an efficient solution for the development of a scaffold combining the great mechanical performances of PEEK with the bioactive properties of MeHA and HAp, having high potential for translational clinical applications.

The aim of this review is to give an updated perspective about the methods for chemical modifications of hyaluronic acid (HA) toward the development of new applications in medical devices and material engineering. After a brief introduction on chemical, structural and biological features of this important natural polysaccharide, the most important methods for chemical and physical modifications are disclosed, discussing both on the formation of new covalent bonds and the interaction with other natural polysaccharides. These strategies are of paramount importance in the production of new medical devices and materials with improved properties. In particular, the use of HA in the development of new materials by means of additive manufacturing techniques as electro fluid dynamics, i.e., electrospinning for micro to nanofibres, and three-dimensional bioprinting is also discussed.

An optimized extraction protocol for eumelanins from black soldier flies (BSF-Eumel) allows an in-depth study of natural eumelanin pigments, which are a valuable tool for the design and fabrication of sustainable scaffolds. Here, water-soluble BSF-Eumel sub-micrometer colloidal particles were used as bioactive signals for developing a composite biomaterial ink for scaffold preparation. For this purpose, BSF-Eumel was characterized both chemically and morphologically; moreover, biological studies were carried out to investigate the dose-dependent cell viability and its influence on human mesenchymal stem cells (hMSCs), with the aim of validating suitable protocols and to find an optimal working concentration for eumelanin-based scaffold preparation. As proof of concept, 3D printed scaffolds based on methacrylated hyaluronic acid (MEHA) and BSF-Eumel were successfully produced. The scaffolds with and without BSF-Eumel were characterized in terms of their physico-chemical, mechanical and biological behaviours. The results showed that MEHA/BSF-Eumel scaffolds had similar storage modulus values to MEHA scaffolds. In terms of swelling ratio and stability, these scaffolds were able to retain their structure without significant changes over 21 days. Biological investigations demonstrated the ability of the bioactivated scaffolds to support the adhesion, proliferation and osteogenic differentiation of human mesenchymal stem cells.

In regenerative medicine and tissue engineering, the possibility to: (I) customize the shape and size of scaffolds, (II) develop highly mimicked tissues with a precise digital control, (III) manufacture complex structures and (IV) reduce the wastes related to the production process, are the main advantages of additive manufacturing technologies such as three-dimensional (3D) bioprinting. Specifically, this technique, which uses suitable hydrogel-based bioinks, enriched with cells and/or growth factors, has received significant consideration, especially in cartilage tissue engineering (CTE). In this field of interest, it may allow mimicking the complex native zonal hyaline cartilage organization by further enhancing its biological cues. However, there are still some limitations that need to be overcome before 3D bioprinting may be globally used for scaffolds' development and their clinical translation. One of them is represented by the poor availability of appropriate, biocompatible and eco-friendly biomaterials, which should present a series of specific requirements to be used and transformed into a proper bioink for CTE. In this scenario, considering that, nowadays, the environmental decline is of the highest concerns worldwide, exploring naturally-derived hydrogels has attracted outstanding attention throughout the scientific community. For this reason, a comprehensive review of the naturally-derived hydrogels, commonly employed as bioinks in CTE, was carried out. In particular, the current state of art regarding eco-friendly and natural bioinks' development for CTE was explored. Overall, this paper gives an overview of 3D bioprinting for CTE to guide future research towards the development of more reliable, customized, eco-friendly and innovative strategies for CTE.

Wound care management urgently needs the development of innovative smart wound dressings. The complexity of the wound often requires the use of personalized medication and the advent of three-dimensional (3D) bioprinting fits strongly with this need. In this view, in the present work a methacrylated hyaluronic acid (MeHA) bioink was tested for the fabrication of advanced smart patches as a delivery system of exosomes derived from human mesenchymal stem cells (hMSC-EXOs) suitable for wound healing purposes. MeHA patches were realized by 3D bioprinting technique and they were loaded with hMSC-EXOs. The 3D printed MeHA patches revealed improved mechanical performance, appropriate swelling ratio, extended degradation time, and suitable biocompatibility. Furthermore, MeHA patches loaded with hMSC-EXOs improved the proliferation, migration, angiogenic ability, and expression of specific markers related to wound healing process in human fibroblasts and human endothelial cells., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Articular cartilage is characterized by a poor self-healing capacity due to its aneural and avascular nature. Once injured, it undergoes a series of catabolic processes which lead to its progressive degeneration and the onset of a severe chronic disease called osteoarthritis (OA). In OA, important alterations of the morpho-functional organization occur in the cartilage extracellular matrix, involving all the nearby tissues, including the subchondral bone. Osteochondral engineering, based on a perfect combination of cells, biomaterials and biomolecules, is becoming increasingly successful for the regeneration of injured cartilage and underlying subchondral bone tissue. To this end, recently, several peptides have been explored as active molecules and enrichment motifs for the functionalization of biomaterials due to their ability to be easily chemically synthesized, as well as their tunable physico-chemical features, low immunogenicity issues and functional group modeling properties. In addition, they have shown a good aptitude to penetrate into the tissue due to their small size and stability at room temperature. In particular, growth-factor-derived peptides can play multiple functions in bone and cartilage repair, exhibiting chondrogenic/osteogenic differentiation properties. Among the most studied peptides, great attention has been paid to transforming growth factor-β and bone morphogenetic protein mimetic peptides, cell-penetrating peptides, cell-binding peptides, self-assembling peptides and extracellular matrix-derived peptides. Moreover, recently, phage display technology is emerging as a powerful selection technique for obtaining functional peptides on a large scale and at a low cost. In particular, these peptides have demonstrated advantages such as high biocompatibility; the ability to be immobilized directly on chondro- and osteoinductive nanomaterials; and improving the cell attachment, differentiation, development and regeneration of osteochondral tissue. In this context, the aim of the present review was to go through the recent literature underlining the importance of studying novel functional motifs related to growth factor mimetic peptides that could be a useful tool in osteochondral repair strategies. Moreover, the review summarizes the current knowledge of the use of phage display peptides in osteochondral tissue regeneration.

Over the past years, research attention has been focusing more on waste-derived, naturally derived, and renewable materials, in the view of a more sustainable economy. In this work, different topical formulations were obtained from the valorization of marine and agro-industrial by-products and the use of Carbopol 940 as gelling agent. In particular, the combination of extracts obtained from the marine snail, Rapanosa venosa, with Cladophora vagabunda and grape pomace extracts, was investigated for wound healing purposes. Rapana venosa has demonstrated wound healing properties and antioxidant activity. Similarly, grape pomace extracts have been shown to accelerate the healing process. However, their synergic use has not been explored yet. To this aim, four different formulations were produced. Three formulations differed for the presence of a different extract of Rapana venosa : marine collagen, marine gelatin, and collagen hydrolysate, while another formulation used mammalian gelatin as further control. Physico-chemical properties of the extracts as well as of the formulations were analyzed. Furthermore, thermal stability was evaluated by thermogravimetric analysis. Antioxidant capacity and biological behavior, in terms of cytocompatibility, wound healing, and antimicrobial potential, were assessed. The results highlighted for all the formulations (i) a good conservation and thermal stability in time, (ii) a neutralizing activity against free radicals, (iii) and high degree of cytocompatibility and tissue regeneration potential. In particular, collagen, gelatin, and collagen hydrolysate obtained from the Rapana venosa marine snail represent an important, valuable alternative to mammalian products.

Over the past few years, different in vitro and in vivo studies have been highlighting the great potentiality of hyaluronic acid (HA) as a biomaterial in wound healing treatment thanks to its good capability to induce mesenchymal and epithelial cell growth and differentiation, angiogenesis, and collagen deposition. However, the need to improve its mechanical properties as well as its residence time has led scientists to study new functionalization strategies. In this work, chemically modified HA-based hydrogels were obtained by methacrylic and maleic functionalization. Methacrylated (MEHA) and maleated HA (MAHA) hydrogels have shown important physico-chemical properties. The present study provides a deeper insight into the biocompatibility of both synthesized materials and their effects on tissue inflammation using in vitro and in vivo models. To this aim, different cell lines involved in wound healing, human dermal fibroblasts, human adipose-derived stem cells and human umbilical vein endothelial cells, were seeded on MEHA and MAHA hydrogels. Furthermore, an inflammation study was carried out on a murine macrophage cell line to assess the effects of both hydrogels on inflammatory and anti-inflammatory interleukin production. The results showed that both MAHA and MEHA supported cell proliferation with anti-inflammation ability as highlighted by the increased levels of IL-10 (57.92 ± 9.87 pg mL -1 and 68.08 ± 13.94 pg mL -1 , for MEHA and MAHA, respectively). To investigate the inflammatory response at tissue/implant interfaces, an in vivo study was also performed by subcutaneous implantation of the materials in BALB/c mice for up to 28 days. In these analyses, no significant chronic inflammation reaction was demonstrated in either MEHA or MAHA in the long-term implantation., Competing Interests: Authors declare no conflict of interest related to this study., (This journal is © The Royal Society of Chemistry.)

Injectable materials represent very attractive ready-to-use biomaterials for application in minimally invasive surgical procedures. It is shown that this approach to treat, for example, vertebral fracture, craniofacial defects, or tumor resection has significant clinical potential in the biomedical field. In the last four decades, calcium phosphate cements have been widely used as injectable materials for orthopedic surgery due to their excellent properties in terms of biocompatibility and osteoconductivity. However, few clinical studies have demonstrated certain weaknesses of these cements, which include high viscosity, long degradation time, and difficulties being manipulated. To overcome these limitations, the use of sol-gel technology has been investigated, which has shown good results for synthesis of injectable calcium phosphate-based materials. In the last few decades, injectable hydrogels have gained increasing attention owing to their structural similarities with the extracellular matrix, easy process conditions, and potential applications in minimally invasive surgery. However, the need to protect cells during injection leads to the development of double network injectable hydrogels that are capable of being cross-linked in situ. This review will provide the current state of the art and recent advances in the field of injectable biomaterials for minimally invasive surgery., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Over the past years, fundamentals of magnetism opened a wide research area of interest, in the field of tissue engineering and regenerative medicine. The integration of magnetic nanoarchitectures into synthetic/natural scaffold formulations allowed obtaining "on demand" responsive structures able to guide the regeneration process. The aim of this work was the design and characterization of three-dimensional (3D) chitosan-based scaffolds containing dextran-grafted maghemite nanoarchitectures (DM) and functionalized with l-arginine (l-Arg) amino acid as bioactive agent. A homogeneous pore distribution and a high degree of interconnection were obtained for all the structures with DMs, which resulted well distributed inside the polymer matrix. All the results suggest that the simultaneous presence of DMs and l-Arg conferred interesting mechano-structural and bioactive properties toward osteoblast-like and human mesenchymal stem cells, differentially stimulating their proliferation both in the absence and in the presence of a time-dependent magnetic field. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1244-1252, 2019., (© 2019 Wiley Periodicals, Inc.)

Osteoarthritis (OA) is a common degenerative disease which involves articular cartilage, and leads to total joint disability in the advanced stages. Due to its avascular and aneural nature, damaged cartilage cannot regenerate itself. Stem cell therapy and tissue engineering represent a promising route in OA therapy, in which cooperation of mesenchymal stem cells (MSCs) and three-dimensional (3D) scaffolds contribute to cartilage regeneration. However, this approach still presents some limits such as poor mechanical properties of the engineered cartilage. The natural dynamic environment of the tissue repair process involves a collaboration of several signals expressed in the biological system in response to injury. For this reason, tissue engineering involving exogenous "influencers" such as mechanostimulation and functional biomolecule delivery systems (BDS), represent a promising innovative approach to improve the regeneration process. BDS provide a controlled release of biomolecules able to interact between them and with the injured tissue. Nano-dimensional BDS is the future hope for the design of personalized scaffolds, able to overcome the delivery problems. MSC-derived extracellular vesicles (EVs) represent an attractive alternative to BDS, due to their innate targeting abilities, immunomodulatory potential and biocompatibility. Future advances in cartilage regeneration should focus on multidisciplinary strategies such as modular assembly strategies, EVs, nanotechnology, 3D biomaterials, BDS, mechanobiology aimed at constructing the functional scaffolds for actively targeted biomolecule delivery. The aim of this review is to run through the different approaches adopted for cartilage regeneration, with a special focus on biomaterials, BDS and EVs explored in terms of their delivery potential, healing capabilities and mechanical features., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

In this study, an innovative polymer blend, based on double network (DN) approach, has been developed by integrating a poly(ethylene glycol) diacrylate (PEGDA) network into a chemically modified hyaluronic acid sodium salt (HAs) hydrogel matrix. Here, the HAs was chemically functionalized with photocrosslinkable moieties by reacting with maleic anhydride (MAA) to obtain a maleated hyaluronic acid (MaHA). Furthermore, nanocomposite DN hydrogels were suitably prepared by physical blending of hydroxyapatite nanoparticles (HAp), obtained by sol-gel synthesis, within the hydrogel. Physico-chemical, thermal, morphological and mechanical analyses were performed. Results showed enhanced mechanical properties and a homogenous microstructure as highlighted by mechanical and morphological investigations. This suggests that nanocomposite DN hydrogels are promising candidates for biomedical applications.

Hyaluronic acid (HA)-based hydrogels are one of the most promising naturally derived biomaterials for tissue engineering applications, as they can play an important role in many key cellular processes. In this study, HA was chemically functionalized with photo-cross-linkable motifs by reacting with methacrylic anhydride (MA) to obtain methacrylated hyaluronic acid (MeHA). A range of MA/HA molar ratios was used to obtain different degrees of substitution (DS) ranging from 3.5% to 74.5%, as showed by nuclear magnetic resonance and attenuated total reflection spectroscopy. By fine tuning the DS, the chemical reaction parameters, and the polymer concentration, it was demonstrated the possibility to tailor their mechanical features. Double network (DN) hydrogels were prepared through the synergic use of MeHA and polyethylene glycole diacrylate (PEGDA). To improve the biological properties of DN hydrogels, bioactive solid signals such as hydroxyapatite nanoparticles (HAp) prepared by sol-gel approach were used in combination with DN hydrogels to obtain an advanced composite material with dual function in terms of mechanical and biological support for soft/hard tissue formation. The results highlighted that composite-DN hydrogels showed a 10-time increase of the storage modulus, if compared to neat MeHA, and an early alkaline phosphatase expression from human mesenchymal stem cells in basal medium. This work can be considered a first systematic approach for the designing of photo-cross-linkable hydrogels, based on a combination of natural/synthetic polymers and HAp, that could be applied in three-dimensional additive manufacturing techniques such as stereolithography. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 3079-3089, 2018., (© 2018 Wiley Periodicals, Inc.)

The ability to engineer scaffolds that resemble the transition between tissues would be beneficial to improve repair of complex organs, but has yet to be achieved. In order to mimic tissue organization, such constructs should present continuous gradients of geometry, stiffness and biochemical composition. Although the introduction of rapid prototyping or additive manufacturing techniques allows deposition of heterogeneous layers and shape control, the creation of surface chemical gradients has not been explored on three-dimensional (3D) scaffolds obtained through fused deposition modelling technique. Thus, the goal of this study was to introduce a gradient functionalization method in which a poly(ε-caprolactone) surface was first aminolysed and subsequently covered with collagen via carbodiimide reaction. The 2D constructs were characterized for their amine and collagen contents, wettability, surface topography and biofunctionality. Finally, chemical gradients were created in 3D printed scaffolds with controlled geometry and porosity. The combination of additive manufacturing and surface modification is a viable tool for the fabrication of 3D constructs with controlled structural and chemical gradients. These constructs can be employed for mimicking continuous tissue gradients for interface tissue engineering., (Copyright © 2017 John Wiley & Sons, Ltd.)

Aging is the most prominent risk factor triggering several degenerative diseases, such as osteoarthritis (OA). Due to its poor self-healing capacity, once injured cartilage needs to be reestablished. This process might be approached through resorting to cell-based therapies and/or tissue engineering. Human mesenchymal stem cells (MSCs) represent a promising approach due to their chondrogenic differentiation potential. Presently, in vitro chondrogenic differentiation of MSCs is limited by two main reasons as follows: aging of MSCs, which determines the loss of cell proliferative and differentiation capacity and MSC-derived chondrocyte hypertrophic differentiation, which limits the use of these cells in cartilage tissue regeneration approach. The effect of aging on MSCs is fundamental for stem cell-based therapy development, especially in older subjects. In the present review we focus on homeostasis alterations occurring in MSC-derived chondrocytes during in vitro aging. Moreover, we deal with potential cell aging regulation approaches, such as cell stimulation through telomerase activators, mechanical strain, and epigenetic regulation. Future investigations in this field might provide new insights into innovative strategies for cartilage regeneration and potentially inspire novel therapeutic approaches for OA treatment.

Magnetic nanocomposite scaffolds based on poly(ε-caprolactone) and poly(ethylene glycol) were fabricated by 3D fibre deposition modelling (FDM) and stereolithography techniques. In addition, hybrid coaxial and bilayer magnetic scaffolds were produced by combining such techniques. The aim of the current research was to analyse some structural and functional features of 3D magnetic scaffolds obtained by the 3D fibre deposition technique and by stereolithography as well as features of multimaterial scaffolds in the form of coaxial and bilayer structures obtained by the proper integration of such methods. The compressive mechanical behaviour of these scaffolds was investigated in a wet environment at 37 °C, and the morphological features were analysed through scanning electron microscopy (SEM) and X-ray micro-computed tomography. The capability of a magnetic scaffold to absorb magnetic nanoparticles (MNPs) in water solution was also assessed. confocal laser scanning microscopy was used to assess the in vitro biological behaviour of human mesenchymal stem cells (hMSCs) seeded on 3D structures. Results showed that a wide range of mechanical properties, covering those spanning hard and soft tissues, can be obtained by 3D FDM and stereolithography techniques. 3D virtual reconstruction and SEM showed the precision with which the scaffolds were fabricated, and a good-quality interface between poly(ε-caprolactone) and poly(ethylene glycol) based scaffolds was observed for bilayer and coaxial scaffolds. Magnetised scaffolds are capable of absorbing water solution of MNPs, and a preliminary information on cell adhesion and spreading of hMSCs was obtained without the application of an external magnetic field.

The design of hybrid poly-ε-caprolactone (PCL)-self-assembling peptides (SAPs) matrices represents a simple method for the surface functionalization of synthetic scaffolds, which is essential for cell compatibility. This study investigates the influence of increasing concentrations (2.5%, 5%, 10% and 15% w/w SAP compared to PCL) of three different SAPs on the physico-chemical/mechanical and biological properties of PCL fibers. We demonstrated that physico-chemical surface characteristics were slightly improved at increasing SAP concentrations: the fiber diameter increased; surface wettability increased with the first SAP addition (2.5%) and slightly less for the following ones; SAP-surface density increased but no change in the conformation was registered. These results could allow engineering matrices with structural characteristics and desired wettability according to the needs and the cell system used. The biological and mechanical characteristics of these scaffolds showed a particular trend at increasing SAP concentrations suggesting a prevailing correlation between cell behavior and mechanical features of the matrices. As compared with bare PCL, SAP enrichment increased the number of metabolic active h-osteoblast cells, fostered the expression of specific osteoblast-related mRNA transcripts, and guided calcium deposition, revealing the potential application of PCL-SAP scaffolds for the maintenance of osteoblast phenotype.

In the past few years, researchers have focused on the design and development of three-dimensional (3D) advanced scaffolds, which offer significant advantages in terms of cell performance. The introduction of magnetic features into scaffold technology could offer innovative opportunities to control cell populations within 3D microenvironments, with the potential to enhance their use in tissue regeneration or in cell-based analysis. In the present study, 3D fully biodegradable and magnetic nanocomposite scaffolds for bone tissue engineering, consisting of a poly(ε-caprolactone) (PCL) matrix reinforced with iron-doped hydroxyapatite (FeHA) nanoparticles, were designed and manufactured using a rapid prototyping technique. The performances of these novel 3D PCL/FeHA scaffolds were assessed through a combination of theoretical evaluation, experimental in vitro analyses and in vivo testing in a rabbit animal model. The results from mechanical com- pression tests were consistent with FEM simulations. The in vitro results showed that the cell growth in the magnetized scaffolds was 2.2-fold greater than that in non-magnetized ones. In vivo experiments further suggested that, after only 4 weeks, the PCL/FeHA scaffolds were completely filled with newly formed bone, proving a good level of histocompatibility. All of the results suggest that the introduction of magnetic features into biocompatible materials may confer significant advantages in terms of 3D cell assembly.

The grafting of galactose units onto poly(ε-caprolactone) (PCL) substrates by a wet chemistry two-step procedure is proposed. Even though a reduction of hardness from 0.58-0.31 GPa to 0.12-0.05 GPa is achieved, the chemical functionalization does not negatively affect the tensile modulus (332.2±31.3 MPa and 328.5±34.7 MPa for unmodified and surface-modified PCL, respectively) and strength (15.1±1.3 MPa and 14.8±1.5 MPa as assessed before and after the surface modification, respectively), as well as the mechanical behaviour evaluated through small punch test. XPS and enzyme-linked lectin assay (ELLA) demonstrate the presence, and also the correct exposition of the saccharidic epitope on PCL substrates. The introduction of carbohydrate moieties on the PCL surfaces clearly enhances the hydrophilicity of the substrate, as the water contact angle decreases from 82.1±5.8° to 62.1±4.2°. Furthermore, preliminary biological analysis shows human mesenchymal stem cell viability over time and an improvement of cell adhesion and spreading., (Copyright © 2014 Elsevier Ltd. All rights reserved.)