In vivo assessment of marine vs bovine origin collagen-based composite scaffolds promoting bone regeneration in a New Zealand rabbit model.

The ability of human tissues to self-repair is limited, which motivates the scientific community to explore new and better therapeutic approaches to tissue regeneration. The present manuscript provides a comparative study between a marine-based composite biomaterial, and another composed of well-established counterparts for bone tissue regeneration. Blue shark skin collagen was combined with bioapatite obtained from blue shark's teeth (mColl:BAp), while bovine collagen was combined with synthetic hydroxyapatite (bColl:Ap) to produce 3D composite scaffolds by freeze-drying. Collagens showed similar profiles, while apatite particles differed in their composition, being the marine bioapatite a fluoride-enriched ceramic. The marine-sourced biomaterials presented higher porosities, improved mechanical properties, and slower degradation rates when compared to synthetic apatite-reinforced bovine collagen. The in vivo performance regarding bone tissue regeneration was evaluated in defects created in femoral condyles in New Zealand rabbits twelve weeks post-surgery. Micro-CT results showed that mColl:BAp implanted condyles had a slower degradation and an higher tissue formation (17.9 ± 6.9 %) when compared with bColl:Ap implanted ones (12.9 ± 7.6 %). The histomorphometry analysis provided supporting evidence, confirming the observed trend by quantifying 13.1 ± 7.9 % of new tissue formation for mColl:BAp composites and 10.4 ± 3.2 % for bColl:Ap composites, suggesting the potential use of marine biomaterials for bone regeneration.

From Its Nature to Its Function: Marine-Collagen-Based-Biomaterials for Hard Tissue Applications.

Rapidly growing demand for collagen-based therapeutic applications requires a great amount of collagen stock. Commercial collagen is mainly confined to mammalian sources, which have concerns about zoonotic disease transfer and, additionally, the problem of terrestrial animals' overexploitation, which, even so, does not meet the crescent demand for collagen. The extraction of collagen from marine organisms, including the wastes of vertebrates and invertebrates, has both economic and environmental benefits. Marine collagen (MC) is easy to extract, has excellent biocompatibility and good absorption properties, is low in zoonotic and immunological risks for patients, and has fewer religious and regulatory restrictions. This review discusses the research done using MC on biomaterials for bone, cartilage, and osteochondral tissue regenerative applications and the underlying technologies that enable their development. The main challenges on processing MC associated with specific features, such as the low denaturation temperature and weak mechanical properties, are also addressed. A combination of blends and physical or chemical crosslinking treatments with conventional processing methodologies is still traditionally used to prepare MC biomaterials. However, the growing role of MC in the health care-related field, particularly in the treatment of musculoskeletal defects, has been pushing the scientific community to explore advanced techniques to design and develop safe, yet functional materials to better meet tissues' functionality.

Cell-Laden Marine Gelatin Methacryloyl Hydrogels Enriched with Ascorbic Acid for Corneal Stroma Regeneration.

Corneal pathologies from infectious or noninfectious origin have a significant impact on the daily lives of millions of people worldwide. Despite the risk of organ rejection or infection, corneal transplantation is currently the only effective treatment. Finding safe and innovative strategies is the main goal of tissue-engineering-based approaches. In this study, the potential of gelatin methacryloyl (GelMA) hydrogels produced from marine-derived gelatin and loaded with ascorbic acid (as an enhancer of the biological activity of cells) was evaluated for corneal stromal applications. Marine GelMA was synthesized with a methacrylation degree of 75%, enabling effective photocrosslinking, and hydrogels with or without ascorbic acid were produced, encompassing human keratocytes. All the produced formulations exhibited excellent optical and swelling properties with easy handling as well as structural stability and adequate degradation rates that may allow proper extracellular matrix remodeling by corneal stromal cells. Formulations loaded with 0.5 mg/mL of ascorbic acid enhanced the biological performance of keratocytes and induced collagen production. These results suggest that, in addition to marine-derived gelatin being suitable for the synthesis of GelMA, the hydrogels produced are promising biomaterials for corneal regeneration applications.

3D Biocomposites Comprising Marine Collagen and Silica-Based Materials Inspired on the Composition of Marine Sponge Skeletons Envisaging Bone Tissue Regeneration.

Ocean resources are a priceless repository of unique species and bioactive compounds with denouement properties that can be used in the fabrication of advanced biomaterials as new templates for supporting the cell culture envisaging tissue engineering approaches. The collagen of marine origin can be sustainably isolated from the underrated fish processing industry by-products, while silica and related materials can be found in the spicules of marine sponges and diatoms frustules. Aiming to address the potential of biomaterials composed from marine collagen and silica-based materials in the context of bone regeneration, four different 3D porous structure formulations (COL, COL:BG, COL:D.E, and COL:BS) were fabricated by freeze-drying. The skins of Atlantic cod (Gadus morhua) were used as raw materials for the collagen (COL) isolation, which was successfully characterized by SDS-PAGE, FTIR, CD, and amino acid analyses, and identified as a type I collagen, produced with a 1.5% yield and a preserved characteristic triple helix conformation. Bioactive glass 45S5 bioglass® (BG), diatomaceous earth (D.E.) powder, and biosilica (BS) isolated from the Axinella infundibuliformis sponge were chosen as silica-based materials, which were obtained as microparticles and characterized by distinct morphological features. The biomaterials revealed microporous structures, showing a porosity higher than 85%, a mean pore size range of 138-315 µm depending on their composition, with 70% interconnectivity which can be favorable for cell migration and ensure the needed nutrient supply. In vitro, biological assays were conducted by culturing L929 fibroblast-like cells, which confirmed not only the non-toxic nature of the developed biomaterials but also their capability to support cell adhesion and proliferation, particularly the COL:BS biomaterials, as observed by calcein-AM staining upon seven days of culture. Moreover, phalloidin and DAPI staining revealed well-spread cells, populating the entire construct. This study established marine collagen/silica biocomposites as potential scaffolds for tissue engineering, setting the basis for future studies, particularly envisaging the regeneration of non-load-bearing bone tissues.

Growth Factor-Free Vascularization of Marine-Origin Collagen Sponges Using Cryopreserved Stromal Vascular Fractions from Human Adipose Tissue.

The successful integration of transplanted three-dimensional tissue engineering (TE) constructs depends greatly on their rapid vascularization. Therefore, it is essential to address this vascularization issue in the initial design of constructs for perfused tissues. Two of the most important variables in this regard are scaffold composition and cell sourcing. Collagens with marine origins overcome some issues associated with mammal-derived collagen while maintaining their advantages in terms of biocompatibility. Concurrently, the freshly isolated stromal vascular fraction (SVF) of adipose tissue has been proposed as an advantageous cell fraction for vascularization purposes due to its highly angiogenic properties, allowing extrinsic angiogenic growth factor-free vascularization strategies for TE applications. In this study, we aimed at understanding whether marine collagen 3D matrices could support cryopreserved human SVF in maintaining intrinsic angiogenic properties observed for fresh SVF. For this, cryopreserved human SVF was seeded on blue shark collagen sponges and cultured up to 7 days in a basal medium. The secretome profile of several angiogenesis-related factors was studied throughout culture times and correlated with the expression pattern of CD31 and CD146, which showed the formation of a prevascular network. Upon in ovo implantation, increased vessel recruitment was observed in prevascularized sponges when compared with sponges without SVF cells. Immunohistochemistry for CD31 demonstrated the improved integration of prevascularized sponges within chick chorioalantoic membrane (CAM) tissues, while in situ hybridization showed human cells lining blood vessels. These results demonstrate the potential of using cryopreserved SVF combined with marine collagen as a streamlined approach to improve the vascularization of TE constructs.

Mineralized collagen as a bioactive ink to support encapsulation of human adipose stem cells: A step towards the future of bone regeneration.

Bioprinting - printing with incorporated living cells - has earned special attention on tissue engineering approaches, aiming to closer reproduce the 3D microenvironment of the target tissue. However, it raises extra complexity related to the need to use cell-friendly printing conditions that still comply with material printing fidelity. Inspired by the composite nano structural organization of mineralized tissues, this work reports the efficiency of the chemical approach followed to in situ mineralize blue shark skin collagen, at a nano scale level, to ultimately produce stable inks. The influence of initial cellular density was evaluated by assessing three different concentrations (2.5, 5 and 7.5 × 106 cells·ml-1) of human adipose stem cells (hASC), with the higher density of encapsulated cells presenting improved viability in a long culture term. Immunodetection of osteogenic-related markers, like RUNX2 and osteopontin, 21 days after cell culture in basal conditions confirmed the potential of the ink to be applied for osteogenic purposes, which may be associated with the success of the cell-to-ink interaction and the Ca2+ ions released from the co-precipitated hydroxyapatite. A combination of mineralized shark collagen, alginate and hASC is thus proposed as a bioactive bioink with potential properties for regeneration of bone tissue.

Engineering 3D printed bioactive composite scaffolds based on the combination of aliphatic polyester and calcium phosphates for bone tissue regeneration.

In this study, polylactic acid (PLA) filled with hydroxyapatite (HA) or beta-tricalcium phosphate (TCP) in 5 wt% and 10 wt% of concentration were produced employing twin-screw extrusion followed by fused filament fabrication in two different architectures, varying the orientation of fibers of adjacent layers. The extruded 3D filaments presented suitable rheological and thermal properties to manufacture of 3D scaffolds envisaging bone tissue engineering. The produced scaffolds exhibited a high level of printing accuracy related to the 3D model; confirmed by micro-CT and electron microscopy analysis. The developed architectures presented mechanical properties compatible with human bone replacement. The addition of HA and TCP made the filaments bioactive, and the deposition of new calcium phosphates was observed upon 7 days of incubation in simulated body fluid, exemplifying a microenvironment suitable for cell attachment and proliferation. After 7 days of cell culture, the constructs with a higher percentage of HA and TCP demonstrated a significantly superior amount of DNA when compared to neat PLA, indicating that higher concentrations of HA and TCP could guide a good cellular response and increasing cell cytocompatibility. Differentiation tests were performed, and the biocomposites of PLA/HA and PLA/TCP exhibited earlier markers of cell differentiation as confirmed by alkaline phosphatase and alizarin red assays. The 3D printed composite scaffolds, manufactured with bioactive materials and adequate porous size, supported cell attachment, proliferation, and differentiation, which together with their scalability, promise a high potential for bone tissue engineering applications.

Prionace glauca skin collagen bioengineered constructs as a promising approach to trigger cartilage regeneration.

Representing a strategy of marine by-products valorization, based on isolation of biocompounds and assessment of biomedical applicability, the potential of blue shark (Prionace glauca (PG)) skin collagen to induce chondrogenic differentiation of human adipose stem cells (hASC) was investigated, with and without exogenous stimulation. For that, a cryogelation method was applied to produce highly interconnected porous 3-dimensional (3D) constructs made of collagen and collagen:hyaluronic acid (20:1). In vitro studies reveal that hASC adhere abundantly to the constructs which then suggests the early chondrogenic differentiation of those cells. These findings are supported by the mRNA expression encoding chondrogenic-related markers like Coll II and Sox-9 that are markedly upregulated at an early stage for both conditions, with and without exogenous stimulation. The introduction of hyaluronic acid (Hya) seems to play a crucial role at later time points, as shown by the evident immunodetection of aggrecan (ACAN), even without exogenous stimulation. It is hypothesized that the PG collagen itself can support chondrogenic differentiation at early time points, but exogenous stimulation is required to ensure phenotype maintenance. The present work highlights the relevance of using blue shark collagen biopolymer as a building block to produce highly effective temporary matrices for cartilage applications.

Cell-Laden Biomimetically Mineralized Shark-Skin-Collagen-Based 3D Printed Hydrogels for the Engineering of Hard Tissues.

Mineralization processes based on coprecipitation methods have been applied as a promising alternative to the most commonly used methods of polymer-ceramic combination, direct mixing, and incubation in simulated body fluid (SBF) or modified SBF. In the present study, for the first time, the in situ mineralization (ideally hydroxyapatite formation) of blue shark (Prionace glauca (PG)) collagen to fabricate 3D printable cell-laden hydrogels is proposed. In the first part, several parameters for collagen mineralization were tested until optimization. The hydroxyapatite formation was confirmed by FT-IR, XRD, and TEM techniques. In the second part, stable bioinks combining the biomimetically mineralized collagen with alginate (AG) (1:1, 1:2, 1:3, and AG) solution were used for 3D printing of hydrogels. The addition of Ca2+ ions into the system did present a synergistic effect: by one side, the in situ mineralization of the collagen occurred, and at same time, they were also useful to ionically cross-link the blends with alginate, avoiding the addition of any cytotoxic chemical cross-linking agent. Mouse fibroblast cell line survival during and after printing was favored by the presence of PG collagen as exhibited by the biological performance of the hydrogels. Inspired in a concept of marine byproduct valorization, 3D bioprinting of in situ mineralized blue shark collagen is thus proposed as a promising approach, envisioning the engineering of mineralized tissues.

Altered bone microarchitecture in a type 1 diabetes mouse model Ins2Akita.

Type 1 diabetes mellitus (T1DM) has been associated to several cartilage and bone alterations including growth retardation, increased fracture risk, and bone loss. To determine the effect of long term diabetes on bone we used adult and aging Ins2 Akita mice that developed T1DM around 3-4 weeks after birth. Both Ins2 Akita and wild-type (WT) mice were analyzed at 4, 6, and 12 months to assess bone parameters such as femur length, growth plate thickness and number of mature and preapoptotic chondrocytes. In addition, bone microarchitecture of the cortical and trabecular regions was measured by microcomputed tomography and gene expression of Adamst-5, Col2, Igf1, Runx2, Acp5, and Oc was quantified by quantitative real-time polymerase chain reaction. Ins2 Akita mice showed a decreased longitudinal growth of the femur that was related to decreased growth plate thickness, lower number of chondrocytes and to a higher number of preapoptotic cells. These changes were associated with higher expression of Adamst-5, suggesting higher cartilage degradation, and with low expression levels of Igf1 and Col2 that reflect the decreased growth ability of diabetic mice. Ins2 Akita bone morphology was characterized by low cortical bone area (Ct.Ar) but higher trabecular bone volume (BV/TV) and expression analysis showed a downregulation of bone markers Acp5, Oc, and Runx2. Serum levels of insulin and leptin were found to be reduced at all-time points Ins2 Akita . We suggest that Ins2 Akita mice bone phenotype is caused by lower bone formation and even lower bone resorption due to insulin deficiency and to a possible relation with low leptin signaling.

Marine Collagen/Apatite Composite Scaffolds Envisaging Hard Tissue Applications.

The high prevalence of bone defects has become a worldwide problem. Despite the significant amount of research on the subject, the available therapeutic solutions lack efficiency. Autografts, the most commonly used approaches to treat bone defects, have limitations such as donor site morbidity, pain and lack of donor site. Marine resources emerge as an attractive alternative to extract bioactive compounds for further use in bone tissue-engineering approaches. On one hand they can be isolated from by-products, at low cost, creating value from products that are considered waste for the fish transformation industry. One the other hand, religious constraints will be avoided. We isolated two marine origin materials, collagen from shark skin (Prionace glauca) and calcium phosphates from the teeth of two different shark species (Prionace glauca and Isurus oxyrinchus), and further proposed to mix them to produce 3D composite structures for hard tissue applications. Two crosslinking agents, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride/N-Hydroxysuccinimide (EDC/NHS) and hexamethylene diisocyanate (HMDI), were tested to enhance the scaffolds' properties, with EDC/NHS resulting in better properties. The characterization of the structures showed that the developed composites could support attachment and proliferation of osteoblast-like cells. A promising scaffold for the engineering of bone tissue is thus proposed, based on a strategy of marine by-products valorisation.

Extraction and characterization of collagen from Antarctic and Sub-Antarctic squid and its potential application in hybrid scaffolds for tissue engineering.

Collagen is the most abundant protein found in mammals and it exhibits a low immunogenicity, high biocompatibility and biodegradability when compared with others natural polymers. For this reason, it has been explored for the development of biologically instructive biomaterials with applications for tissue substitution and regeneration. Marine origin collagen has been pursued as an alternative to the more common bovine and porcine origins. This study focused on squid (Teuthoidea: Cephalopoda), particularly the Antarctic squid Kondakovia longimana and the Sub-Antarctic squid Illex argentinus as potential collagen sources. In this study, collagen has been isolated from the skins of the squids using acid-based and pepsin-based protocols, with the higher yield being obtained from I. argentinus in the presence of pepsin. The produced collagen has been characterized in terms of physicochemical properties, evidencing an amino acid profile similar to the one of calf collagen, but exhibiting a less preserved structure, with hydrolyzed portions and a lower melting temperature. Pepsin-soluble collagen isolated from I. argentinus was selected for further evaluation of biomedical potential, exploring its incorporation on poly-ε-caprolactone (PCL) 3D printed scaffolds for the development of hybrid scaffolds for tissue engineering, exhibiting hierarchical features.