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Type I collagen physiological scaffold for tissue regeneration is considered one of the widely used biomaterials for tissue engineering and medical applications. It is hierarchically organized: five laterally staggered molecules are packed within fibrils, arranged into fascicles and bundles. The structural organization is correlated to the direction and intensity of the forces which can be loaded onto the tissue. For a tissue-specific regeneration, the required macro- and microstructure of a suitable biomaterial has been largely investigated. Conversely, the function of multiscale structural integrity has been much less explored but is crucial for scaffold design and application. In this work, collagen was extracted from different animal sources with protocols that alter its structure. Collagen of tendon shreds excised from cattle, horse, sheep and pig was structurally investigated by wide- and small-angle X-ray scattering techniques, at both molecular and supramolecular scales, and thermo-mechanically with thermal and load-bearing tests. Tendons were selected because of their resistance to chemical degradation and mechanical stresses. The multiscale structural integrity of tendons’ collagen was studied in relation to the animal source, anatomic location and source for collagen extraction.

Filter masks are disposable devices intended to be worn in order to reduce exposure to potentially harmful foreign agents of 0.1–10.0 microns. However, to perform their function correctly, these devices should be replaced after a few hours of use. Because of this, billions of non-biodegradable face masks are globally discarded every month (3 million/minute). The frequent renewal of masks, together with the strong environmental impact of non-biodegradable plastic-based mask materials, highlights the need to find a solution to this emerging ecological problem. One way to reduce the environmental impact of masks, decrease their turnover, and, at the same time, increase their safety level is to make them able to inhibit pathogen proliferation and vitality by adding antibacterial materials such as silver, copper, zinc, and graphene. Among these, silver and copper are the most widely used. In this study, with the aim of improving commercial devices’ efficacy and eco-sustainability, Ag-based and Cu-based antibacterial treatments were performed and characterized from morphological, compositional, chemical–physical, and microbiological points of view over time and compared with the antibacterial treatments of selected commercial products. The results demonstrated the good distribution of silver and copper particles onto the surface of the masks, along with almost 100% antibacterial capabilities of the coatings against both Gram-positive and Gram-negative bacteria, which were still confirmed even after several washing cycles, thus indicating the good potential of the developed prototypes for mask application.

Type I collagen is the most abundant protein of the human body. Due to its favourable properties, collagen extracted from animal tissues is adopted to manufacture a wide range of devices for biomedical applications. Compared to bovine and porcine collagens, which are the most largely used, equine collagen is free from the risk of zoonosis, has no reported immune reactions, and has not religious constraints. In this work, a recently available type I collagen extracted from horse tendon was evaluated and compared with a commercially available collagen isoform derived from the same species and tissue. Detailed physical, chemical and biological investigations were performed, in agreement with the requirements of the current standard for the characterization of type I collagen to be used for the manufacture of Tissue Engineering Medical Products. To the best of our knowledge, this is the first report on the complete primary structure of the investigated collagen.

Soft tissues diseases significantly affect patients quality of life and usually require targeted, costly and sometimes constant interventions. With the average lifetime increase, a proportional increase of age-related soft tissues diseases has been witnessed. Due to this, the last two decades have seen a tremendous demand for minimally invasive one-step resolutive procedures. Intensive scientific and industrial research has led to the recognition of injectable formulations as a new advantageous approach in the management of complex diseases that are challenging to treat with conventional strategies. Among them, collagen-based products are revealed to be one of the most promising among bioactive biomaterials-based formulations. Collagen is the most abundant structural protein of vertebrate connective tissues and, because of its structural and non-structural role, is one of the most widely used multifunctional biomaterials in the health-related sectors, including medical care and cosmetics. Indeed, collagen-based formulations are historically considered as the “gold standard” and from 1981 have been paving the way for the development of a new generation of fillers. A huge number of collagen-based injectable products have been approved worldwide for clinical use and have routinely been introduced in many clinical settings for both aesthetic and regenerative surgery. In this context, this review article aims to be an update on the clinical outcomes of approved collagen-based injectables for both aesthetic and regenerative medicine of the last 20 years with an in-depth focus on their safety and effectiveness for the treatment of diseases of the integumental, gastrointestinal, musculoskeletal, and urogenital apparatus.

Fish collagen garnered significant academic and commercial focus in the last decades featuring prospective applications in a variety of health-related industries, including food, medicine, pharmaceutics, and cosmetics. Due to its distinct advantages over mammalian-based collagen, including the reduced zoonosis transmission risk, the absence of cultural-religious limitations, the cost-effectiveness of manufacturing process, and its superior bioavailability, the use of collagen derived from fish wastes (i.e., skin, scales) quickly expanded. Moreover, by-products are low cost and the need to minimize fish industry waste’s environmental impact paved the way for the use of discards in the development of collagen-based products with remarkable added value. This review summarizes the recent advances in the valorization of fish industry wastes for the extraction of collagen used in several applications. Issues related to processing and characterization of collagen were presented. Moreover, an overview of the most relevant applications in food industry, nutraceutical, cosmetics, tissue engineering, and food packaging of the last three years was introduced. Lastly, the fish-collagen market and the open technological challenges to a reliable recovery and exploitation of this biopolymer were discussed.

In the last two decades, fisheries and fish industries by-products have started to be recovered for the extraction of type I collagen because of issues related to the extraction of traditional mammalian tissues. In this work, special attention has been paid to by-products from fish bred in aquaponic plants. The valorization of aquaponic fish wastes as sources of biopolymers would make the derived materials eco-friendlier and attractive in terms of profitability and cost effectiveness. Among fish species, Nile Tilapia is the second-most farmed species in the world and its skin is commonly chosen as a collagen extraction source. However, to the best of our knowledge, no studies have been carried out to investigate, in depth, the age-related differences in fish skin with the final aim of selecting the most advantageous fish size for collagen extraction. In this work, the impact of age on the structural and compositional properties of Tilapia skin was evaluated with the aim of selecting the condition that best lends itself to the extraction of type I collagen for biomedical applications, based on the known fact that the properties of the original tissue have a significant impact on those of the final product. Performed analysis showed statistically significant age-related differences. In particular, an increase in skin thickness (+110 µm) and of wavy-like collagen fiber bundle diameter (+3 µm) besides their organization variation was observed with age. Additionally, a preferred collagen molecule orientation along two specific directions was revealed, with a higher fiber orientation degree according to age. Thermal analysis registered a shift of the endothermic peak (+1.7 °C) and an increase in the enthalpy (+3.3 J/g), while mechanical properties were found to be anisotropic, with an age-dependent brittle behavior. Water (+13%) and ash (+0.6%) contents were found to be directly proportional with age, as opposed to protein (−8%) and lipid (−10%) contents. The amino acid composition revealed a decrease in the valine, leucine, isoleucine, and threonine content and an increase in proline and hydroxyproline. Lastly, fatty acids C14:0, C15:0, C16:1, C18:2n6c, C18:3n6, C18:0, C20:3n3, and C23:0 were revealed to be upregulated, while C18:1n9c was downregulated with age.

Urethral stenosis is a pathological condition that consists in the narrowing of the urethral lumen because of the formation of scar tissue. Unfortunately, none of the current surgical approaches represent an optimal solution because of the high stricture recurrence rate. In this context, we preliminarily explored the potential of an insoluble type-I collagen from horse tendon as scaffolding material for the development of innovative devices for the regeneration of injured urethral tracts. Non-porous collagen-based substrates were produced and optimized, in terms of crosslinking density of the macromolecular structure, to either provide mechanical properties compliant with the urinary tract physiological stress and better sustain tissue regeneration. The effect of the adopted crosslinking strategy on the protein integrity and on the substrate physical–chemical, mechanical and biological properties was investigated in comparison with a decellularized matrix from porcine small intestinal submucosa (SIS patch), an extensively used xenograft licensed for clinical use in urology. The optimized production protocols allowed the preservation of the type I collagen native structure and the realization of a substrate with appealing end-use properties. The biological response, preliminarily investigated by immunofluorescence experiments on human adult renal stem/progenitor cells until 28 days, showed the formation of a stem-cell monolayer within 14 days and the onset of spheroids within 28 days. These results suggested the great potential of the collagen-based material for the development of scaffolds for urethral plate regeneration and for in vitro cellular studies.

Three-dimensional (3D) cell culture systems mimic the structural complexity of the tissue microenvironment that includes the extracellular matrix (ECM) in addition to the cellular components Thus, 3D culture systems are increasingly important as they resemble the ECM-cell and cell-cell physical interactions occurring in vivo. So far, several scaffold-based culture systems and techniques have been proposed as valuable approaches for large-scale production of spheroids, but often suffering of poor reproducible conditions or high costs of production. In this work we present a reliable 3D culture system based on collagen I-blended agarose hydrogels and show how the variation of the agarose weight percentage affects the physical and mechanical properties of the resulting hydrogel, being that with a lower amount of agarose more permeable, softer and more prone to degradation compared to hydrogels with higher agarose concentrations. We have also evaluated the effect of the different physical and mechanical properties of the agarose hydrogels on the growth, size, morphology and cell motility of spheroids obtained by culturing three different breast cancer cell lines (MCF-7, MDA-MB-361and MDA-MB-231). As proof of concept, the cisplatin penetration and its cytotoxic effect on the tumor spheroids was evaluated as function of the hydrogel stiffness. Noteworthy, the possibility to recover the spheroids from the hydrogels for further processing and other biological studies has been also considered.

In the present work, we investigated the potential of novel semi-interpenetrating polymer network (semi-IPN) cryogels, obtained through ultraviolet exposure of aqueous mixtures of poly(ethylene glycol) diacrylate and type I collagen, as tunable off-the-shelf platforms for 3D cancer cell research. We synthesized semi-IPN cryogels with variable collagen amounts (0.1% and 1% w/v) and assessed the effect of collagen on key cryogel properties for cell culture, for example, porosity, degradation rate and mechanical stiffness. Then, we investigated the ability of the cryogels to sustain the long-term growth of two pancreatic ductal adenocarcinoma (PDAC) cell populations, the parenchymal Panc1 cells and their derived cancer stem cells. Results revealed that both cell lines efficiently infiltrated, attached and expanded in the cryogels over a period of 14 days. However, only when grown in the cryogels with the highest collagen concentration, both cell lines reproduced their characteristic growth pattern previously observed in collagen-enriched organotypic cultures, biomimetic of the highly fibrotic PDAC stroma. Cellular preembedding in Matrigel, that is, the classical approach to develop/grow organoids, interfered with an efficient intra-scaffold migration and growth. Although preliminary, these findings highlight the potential of the proposed cryogels as reproducible and tunable cancer cell research platforms.

Type I collagen has always aroused great interest in the field of life-science and bioengineering, thanks to its favorable structural properties and bioactivity. For this reason, in the last five decades it has been widely studied and employed as biomaterial for the manufacture of implantable medical devices. Commonly used sources of collagen are represented by bovine and swine but their applications are limited because of the zoonosis transmission risks, the immune response and the religious constrains. Thus, type-I collagen isolated from horse tendon has recently gained increasing interest as an attractive alternative, so that, although bovine and porcine derived collagens still remain the most common ones, more and more companies started to bring to market a various range of equine collagen-based products. In this context, this work aims to overview the properties of equine collagen making it particularly appealing in medicine, cosmetics and pharmaceuticals, as well as its main biomedical applications and the currently approved equine collagen-based medical devices, focusing on experimental studies and clinical trials of the last 15 years. To the best of our knowledge, this is the first review focusing on the use of equine collagen, as well as on equine collagen-based marketed products for healthcare.

In the last two decades, marine collagen has attracted great scientific and industrial interest as a 'blue resource', with potential for use in various health-related sectors, such as food, medicine, pharmaceutics and cosmetics. In particular, the large availability of polluting by-products from the fish processing industry has been the key factor driving the research towards the conversion of these low cost by-products (e.g. fish skin and scales) into collagen-based products with high added value and low environmental impact. After addressing the extraction of collagen from aquatic sources and its physicochemical properties, this review focuses on the use of marine collagen and its derivatives (e.g. gelatin and peptides) in different healthcare sectors. Particular attention is given to the bioactive properties of marine collagen that are being explored in preclinical and clinical studies, and pave the way to an increased demand for this biomaterial in the next future. In this context, in addition to the use of native collagen for the development of tissue engineering or wound healing devices, particularly relevant is the use of gelatin and peptides for the development of dietary supplements and nutraceuticals, specifically directed to weight management and glycemic control. The marine collagen market is also briefly discussed to highlight the opportunities and the most profitable areas of interest.

Peripheral nerve injuries may lead to a significant function loss, which deeply affect patient’s quality of life. In this context, tissue engineered collagen-based nerve guides are a promising alternative to autografts. To enhance the regeneration of the injured nerve tract, several bioactive molecules can be adopted as further components of the collagen-based conduits. Herein, sericin (Ser), a waste product of textile industry, was combined with type I collagen (Col), for the development of a bioactive substrate potentially able to support and enhance the regeneration of the peripheral nervous system. In particular, in order to identify an optimal substrate composition in terms of physicochemical and biological properties, thin Ser-Col films with different Ser:Col ratios were produced by air-drying and subsequently crosslinked through two different crosslinking methods. Then, Ser:Col films were characterized from a physicochemical point of view by examining secondary protein structure modifications via FTIR spectroscopy, swelling ratios, degradation rates and Ser release kinetics. Moreover, Ser:Col films ability to promote adhesion and proliferation of Schwann cells was evaluated in vitro. The results obtained in this study, although preliminary, suggest that Ser:Col blends could represent a promising strategy to enhance the repair of damaged peripheral nerves, by providing a sustained release of Ser from collagen-based nerve substrates.

The aim of this paper is to model and to compare the results of the mechanical characterization, carried out on numerical models and real specimens, of uniform P-scaffolds with different porosity values. The analysis includes the morphological characterization of 3D printed specimens and the implementation of a FEM shell model to reproduce a compressive test suitable for mechanical properties evaluation of PLA scaffolds. Young modulus and yield strength were also obtained, in order to verify the numerical model accuracy, by experimental tests on 3D printed PLA scaffolds. Numerical results showed that the shell model was able to reproduce, more efficiently compared to a solid model proposed in a previous work, both elastic and plastic behavior of the scaffolds, providing elastic modulus values very close to the experimental ones. On the other hand, the not very high quality of the 3D printing, detected by MicroCT analysis, caused a significant dispersion in the yield strength numerical values respect to the real data. Anyway, an inverse correlation between mechanical properties and porosity was found as expected. The elastic modulus values were similar to the typical values of the trabecular bone for whose regeneration this kind of scaffolds is usually employed.

The aim of this work is to evaluate the effects of different extraction and material processing protocols on the collagen structure and hierarchical organization of equine tendons. Wide and Small Angle X-ray Scattering investigations on raw powders and thin films revealed that not only the extraction and purification treatments, but also the processing conditions may affect the extent of the protein crystalline domain and induce a nanoscale “shield effect.” This is due to the supramolecular fiber organization, which protects the atomic scale structure from the modifications that occur during fabrication protocols. Moreover, X-ray analyses and Fourier Transform Infrared spectroscopy performed on the biomaterial sheds light on the relationship between processing conditions, triple helical content and the organization in atomic and nanoscale domains. It was found that the mechanical homogenization of the slurry in acidic solution is a treatment that ensures a high content of super-organization of collagen into triple helices and a lower crystalline domain in the material. Finally, mechanical tensile tests were carried out, proving that the acidic solution is the condition which most enhances both mechanical stiffness and supramolecular fiber organization of the films.

Hyaluronic acid (HA), also termed hyaluronan, is a key extracellular matrix glycosaminoglycan that not only provides tissue hydration, but also shows intrinsic bioactivity towards cellular proliferation, differentiation and inflammatory response. These properties make HA attractive both as a potential target for the treatment of several diseases and as an effective biomaterial to control cell behavior. This concise review aims at emphasizing the increasing role of HA as a promising biomaterial for advanced medical applications that include, among others, the development of targeted therapies for cancer treatment and the development of scaffolds for regenerative medicine. Although the bioactive mechanisms of HA are not fully understood, its molecular weight and purity are well known to affect the cellular and immunological responses to HA-based products. Furthermore, in spite of the complex translation of novel HA-based medical devices from the lab bench to the clinic, a rapid growth of the HA market is foreseen in the next future, also due to the growing aging of the population and the increasing demand for anti-aging products. In this perspective, the industrial HA production is briefly addressed, with reference to the technological issues that are currently undergoing extensive research to improve the HA yield and quality, in an attempt to meet the quickly rising HA demand.

The optimization of collagen-based scaffolds for tissue engineering goes through the careful selection of the crosslinking method(s), which should impart the prerequisite mechanical and degradation properties without impairing the cell/tissue response. Here, we investigated the chemically effective (ρxch) and the elastically effective (ρxel) crosslink density of collagen-based scaffolds, induced by various crosslinking methods. The aim was to get a deeper insight into the influence of intramolecular and intermolecular crosslinks on several scaffold properties. Freeze-dried collagen matrices were crosslinked via a dehydrothermal treatment (DHT), and then treated with different chemical agents, including carbodiimide (EDC), glutaraldehyde (GTA), formaldehyde (FA), genipin (GP) and dimethyl suberimidate (DMS). Quantification of primary amines and stress-relaxation compressive tests were performed to evaluate ρxch and ρxel, respectively. Scaffolds were then assessed for their water uptake, thermal stability and in vitro resistance to enzymatic degradation. Interestingly, for the various crosslinking treatments ρxch was found to increase in the order DHT

Chitosan (Cs) mats obtained by electrospinning are potentially ideal scaffolds for tissue engineering. This technique allows obtaining nanometric fibrous structures with preferred orientation, which in turn enable cells to align themselves and produce extracellular matrix along desired orientations. In this study, we fabricated aligned Cs electrospun nanofiber mats and investigated the role of the amino acid l -Arginine (L-Arg) as stabilizing agent. Morphological, chemical, mechanical and biological characterizations were performed on untreated and L-Arg treated nanofibrous mats showing the role of L-Arg as biomimetic stabilizer. L-Arg acts as chemical stabilizer of nanofibrous mats, providing improved wettability behavior, mechanical properties and stability even after 60 days in aqueous medium in comparison to untreated mats. Moreover, preliminary biological tests demonstrated favorable cell-material interactions implying physiological responses in terms of viability and proliferation. The proposed L-Arg-treated Cs mats can be considered as potential scaffolds for highly oriented tissue patterning.

Collagen represents one of the most widely used biomaterial for scaffolds fabrication in tissue engineering as it represents the mechanical support of natural tissues. It also provides physical scaffolding for cells and it influences their attachment, growth, and tissue regeneration. Among all fibrillary collagens, type I is considered one of the gold standard for scaffolds fabrication, thanks to its high biocompatibility, biodegradability, and hemostatic properties. It can be extracted by chemical and enzymatic protocols from several collagen-rich tissues, such as tendon and skin, of different animal species. Both the extraction processes and the manufacturing protocols for scaffolds fabrication provide structural and mechanical changes that can be tuned in order to deeply impact the properties of the final biomaterial. The aim of this review is to discuss the role of X-rays to study structural changes of type I collagen from fresh collagen-rich tissues (bovine, equine, fish) to the final scaffolds, with the aim to screen across available collagen sources and scaffolds fabrication protocols to be used in tissue regeneration.

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.