Electrospun Collagen Nanofibers Research Articles

Electrospun Collagen Nanofibers Reduce Inflammation, Inhibit Fibrosis, and Promote Wound Healing on the Ocular Surface

Electrospun Collagen Nanofibers Reduce Inflammation, Inhibit Fibrosis, and Promote Wound Healing on the Ocular Surface

Comparative Study of the Dehydrothermal Crosslinking of Electrospun Collagen Nanofibers: The Effects of Vacuum Conditions and Subsequent Chemical Crosslinking.

Collagen nanofibrous materials have become integral to tissue engineering due to their exceptional properties and biocompatibility. Dehydrothermal crosslinking (DHT) enhances stability and maintains structural integrity without the formation of toxic residues. The study involved the crosslinking of electrospun collagen, applying DHT with access to air and under vacuum conditions. Various DHT exposure times of up to 72 h were applied to examine the time dependance of the DHT process. The DHT crosslinked collagen was subsequently chemically crosslinked using carbodiimides. The material crosslinked in this way evinced elevated Young's modulus values and ultimate tensile strength values, a lower swelling rate and lower shrinkage ratio during crosslinking, and a higher degree of resistance to degradation than the material crosslinked solely with DHT or carbodiimides. It was shown that the crosslinking mechanism using DHT occupies different binding sites than those using chemical crosslinking. Access to air for 12 h or less did not exert a significant impact on the material properties compared to DHT under vacuum conditions. However, concerning longer exposure times, it was determined that access to air results in the deterioration of the properties of the material and that reactions take place that occupy the free bonding sites, which subsequently reduces the effectiveness of chemical crosslinking using carbodiimides.

Preparation of Short Collagen Nanofibers for Injectable Hydrogels: Comparative Assessment of Fragmentation Methods, Physicomechanical Properties, and Biocompatibility

AbstractCollagen nanofibers can be employed in hydrogels to create injectable nanocomposite hydrogels, mimicking the fibrous architecture of the natural extracellular matrix (ECM). As long continuous electrospun collagen nanofibers are not applicable, fragmentation is inevitable to obtain injectable hydrogels with a fine viscosity. Here, four methods: hand grinding (HG), homogenizer (HM), mixer milling (MM), and ultrasonication (UH) are used to disintegrate and shorten collagen nanofiber mats before incorporation into an injectable hyaluronic acid hydrogel as a matrix. The Length‐to‐diameter (L/d) ratio and morphology of fragmented collagen are compared by SEM. The injection force, mechanical properties, and cell viability of the selected collagen‐incorporated hydrogels are also evaluated. UH emerges as the most effective method, yielding the highest L/d ratio of 46 and a notable compressive modulus of 8.7 ± 0.92 kPa. Assessment of the in vitro cell viability of the encapsulated chondrocytes in the collagen‐incorporated hydrogels demonstrates good biocompatibility, and hydrogels containing UH short nanofiber, in particular, show an increase in cell proliferation. This work indicates how collagen mats can be effectively broken down and combined with injectable hydrogels to enhance both their mechanical behavior and biocompatibility.

Green Electrospun Nanofibers for Biomedicine and Biotechnology

Green electrospinning harnesses the potential of renewable biomaterials to craft biodegradable nanofiber structures, expanding their utility across a spectrum of applications. In this comprehensive review, we summarize the production, characterization and application of electrospun cellulose, collagen, gelatin and other biopolymer nanofibers in tissue engineering, drug delivery, biosensing, environmental remediation, agriculture and synthetic biology. These applications span diverse fields, including tissue engineering, drug delivery, biosensing, environmental remediation, agriculture, and synthetic biology. In the realm of tissue engineering, nanofibers emerge as key players, adept at mimicking the intricacies of the extracellular matrix. These fibers serve as scaffolds and vascular grafts, showcasing their potential to regenerate and repair tissues. Moreover, they facilitate controlled drug and gene delivery, ensuring sustained therapeutic levels essential for optimized wound healing and cancer treatment. Biosensing platforms, another prominent arena, leverage nanofibers by immobilizing enzymes and antibodies onto their surfaces. This enables precise glucose monitoring, pathogen detection, and immunodiagnostics. In the environmental sector, these fibers prove invaluable, purifying water through efficient adsorption and filtration, while also serving as potent air filtration agents against pollutants and pathogens. Agricultural applications see the deployment of nanofibers in controlled release fertilizers and pesticides, enhancing crop management, and extending antimicrobial food packaging coatings to prolong shelf life. In the realm of synthetic biology, these fibers play a pivotal role by encapsulating cells and facilitating bacteria-mediated prodrug activation strategies. Across this multifaceted landscape, nanofibers offer tunable topographies and surface functionalities that tightly regulate cellular behavior and molecular interactions. Importantly, their biodegradable nature aligns with sustainability goals, positioning them as promising alternatives to synthetic polymer-based technologies. As research and development continue to refine and expand the capabilities of green electrospun nanofibers, their versatility promises to advance numerous applications in the realms of biomedicine and biotechnology, contributing to a more sustainable and environmentally conscious future.

Electrospinning of collagen: enzymatic and spectroscopic analyses reveal solvent-independent disruption of the triple-helical structure.

Electrospinning has become a well-established method for creating nanofibrous meshes for tissue-engineering applications. The incorporation of natural extracellular components, such as electrospun pure collagen nanofibers, has proven to be particularly challenging, as electrospun collagen nanofibers do not constitute native collagen fibers anymore. In this study, we show that this detrimental effect is not only limited to fluorinated solvents, as previously thought. Rat tail collagen was dissolved in acetic acid and ethanol and electrospun at various temperatures. Electrospun collagen mats were analyzed using circular dichroism, enzymatic digestion, SDS-PAGE, western blotting, and Raman spectroscopy and compared to heat-denaturated and electrospun collagen in HFIP. Our data suggest that even non-fluorinated electrospinning solvents, such as acid-based solvents, do not yield structurally intact fibers. Interestingly, neither epithelial cells nor fibroblasts displayed a different cellular response to electrospun collagen compared to collagen-coated polyurethane scaffolds in F-actin staining and metabolic analysis using fluorescent lifetime imaging. The disruption of the structural integrity of collagen might therefore be underestimated based on the cell-material interactions alone. These observations expose the larger than anticipated vulnerability of collagen in the electrospinning process. Based on these findings, the influence of the electrospinning process on the distinct biochemical properties of collagen should always be considered, when ECM-mimicking fibrous constructs are manufactured.

A bilayer vascular scaffold with spatially controlled release of growth factors to enhance in situ rapid endothelialization and smooth muscle regeneration

When transplanting a scaffold to repair defected vascular tissues, there is a critical need to achieve in-situ rapid endothelialization together with the circumferential alignment and ingrowth of smooth muscle cells. To this end, we hereby design and fabricate a bilayer vascular scaffold with spatially controlled release of growth factors. The lumen of such a scaffold was made of electrospun poly(l-lactide-co-caprolactone) and collagen (PLCL/COL) nanofibers loaded with uniform heparin and vascular endothelial growth factors (VEGF), while its outer layer was composed of circumferentially aligned, PLCL/COL nanofiber yarns loaded with graded platelet derived growth factors (PDGF) increasing from the outermost toward the interior of the scaffold. With the controlled release of VEGF and PDGF from the scaffold, we showed a continuous greater release percentage (ca. 15%) of VEGF relative to PDGF over a duration of almost one month in vitro. At two-month post implantation in a rat abdominal aorta defect model, we observed rapid endothelialization at the luminal surface and orientated smooth muscles infiltrating inside the vascular wall. In particular, loose connective tissues rich in collagen fibers were produced at the outermost layer of the vascular scaffold, indicating the capability of such kind of vascular scaffold for in-situ vascular repair or regeneration.

Synchrotron Fourier transform infrared microspectroscopy (sFTIRM) analysis of unfolding behavior of electrospun collagen nanofibers

Collagen nanofibers are popular extracellular matrix (ECM) materials in regenerative medicine. Electrospinning of collagen dissolved in organic solvents is widely used for fabricating anisotropic collagen nanofibers; however, such fibers are water-soluble and require cross-linking before use as scaffolds for cell culture. Herein, in-situ crosslinking during electrospinning process is suggested by using different chemical agents, namely genipin and glutaraldehyde, and physical crosslinking method (UV light). sFTIRM; Synchrotron Fourier-Transform Infrared Microspectroscopy is a powerful tool that sheds light on the molecular structure of collagen nanofibers. Applied extraction methods caused shifts on protein band positions. Electrospinning process prevents self-assembly of collagen molecules and obtained electrospun collagen nanofibers have lower band positions. Crosslinkers have effect on the secondary structure of collagen molecules. Among different crosslinkers, genipin in-situ crosslinking process perform better in preserving the native structure of electrospun collagen nanofibers than the physical crosslinking method (UV).

Antimicrobial quaternary ammonium organosilane cross-linked nanofibrous collagen scaffolds for tissue engineering.

IntroductionIn search for cross-linkers with multifunctional characteristics, the present work investigated the utility of quaternary ammonium organosilane (QOS) as a potential cross-linker for electrospun collagen nanofibers. We hypothesized that the quaternary ammonium ions improve the electrospinnability by reducing the surface tension and confer antimicrobial properties, while the formation of siloxane after alkaline hydrolysis could cross-link collagen and stimulate cell proliferation.Materials and methodsQOS collagen nanofibers were electrospun by incorporating various concentrations of QOS (0.1%–10% w/w) and were cross-linked in situ after exposure to ammonium carbonate. The QOS cross-linked scaffolds were characterized and their biological properties were evaluated in terms of their biocompatibility, cellular adhesion and metabolic activity for primary human dermal fibroblasts and human fetal osteoblasts.Results and discussionThe study revealed that 1) QOS cross-linking increased the flexibility of otherwise rigid collagen nanofibers and improved the thermal stability; 2) QOS cross-linked mats displayed potent antibacterial activity and 3) the biocompatibility of the composite mats depended on the amount of QOS present in dope solution – at low QOS concentrations (0.1% w/w), the mats promoted mammalian cell proliferation and growth, whereas at higher QOS concentrations, cytotoxic effect was observed.ConclusionThis study demonstrates that QOS cross-linked mats possess anti-infective properties and confer niches for cellular growth and proliferation, thus offering a useful approach, which is important for hard and soft tissue engineering and regenerative medicine.

Study on structure, mechanical property and cell cytocompatibility of electrospun collagen nanofibers crosslinked by common agents

Collagen electrospun scaffolds properly reproduce the framework of the extracellular matrix (ECM) of tissues that are natural with the fibrous morphology of the protein by coupling large biomimetism of the biological material. However, traditional solvents employed for collagen electrospinning lead to poor mechanical attributes and bad hydro-stability. In this work, by N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride with N-hydroxysulfosuccinimide (EDC-NHS), glutaraldehyde (GTA) and genipin (GP) respectively, electrospun collagen fibers cross-linked, effectively stabilized the fiber morphology over 2months and improved the mechanical properties in both dry and wet state, especially EDC-NHS with large ultimate tensile stress and εb. The secondary structure of collagen structure still remained and had no obvious difference among various crosslinked samples according to FTIR. On the cell assessment, electrospun collagen fibers crosslinked by EDC-NHS, GTA and GP, were found to support cell adhesion, spreading and proliferation of MC3T3-E1. By contrast, GTA was more effective in preserving explicit fibrous morphology with a relatively lower cell viability both in FBS and BSA soaked mats. Interestingly, GP also had the similar cytocompatibility of MC3T3-E1 as EDC-NHS did. The study proved the feasibility of chemical crosslinker to electrospun collagen for biomedical application.

Electrospinning of in situ crosslinked recombinant human collagen peptide/chitosan nanofibers for wound healing.

Electrospun collagen nanofibers are effective for wound healing; however, many problems, such as the tedious preparation process, weak strength and poor structure integration, limit further applications. In this study, recombinant human collagen (RHC) peptides and a simple one-step crosslinking strategy were used to prepare RHC/chitosan nanofibers. With the nonpathogenic, water-soluble RHC and a mild electrospinning solvent, in situ crosslinked nanofibers (S-CN) not only simplified the preparation procedure but also maintained a more integrated morphology. Compared with the immersed crosslinked nanofibers (I-CN), S-CN showed better performance in moisture retention, degradation and mechanical strength tests. In vitro cell proliferation, morphology and RT-PCR studies confirmed that fibroblasts presented better activities on nanofibers crosslinked in situ. Importantly, after treating with the nanofibers, rapid epidermidalization and angiogenesis were observed in an SD rat scalding model. All these data suggest that electrospun RHC/chitosan nanofibers crosslinked in situ are an ideal candidate that can be used for wound healing applications.

Antimicrobial electrospun collagen nanofibers loaded with Silver nanoparticles and Vancomycin

Antimicrobial electrospun collagen nanofibers loaded with Silver nanoparticles and Vancomycin

Observation of triple helix motif on electrospun collagen nanofibers and its effect on the physical and structural properties

Collagen is a very popular natural biomaterial due to its high biocompatibility and bioactivity. Electrospinning is currently the only technique that allows the fabrication of continuous fibers with diameters down to a few nanometers. In order to regenerate collagen in the forms of nanofibers, it is necessary to dissolve it in suitable solvents. The solvents and electrospinning process cause unfolding of collagen nanofibers. It is proposed that acidic solvents preserve better the natural structure of collagen fibers. In this paper, the structures of collagen nanofibers were examined by using circular dichroism (CD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), Raman spectroscopy, differential scanning calorimetry (DSC) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) methods in order to test this hypothesis. The increase in PP-II fraction, representing the triple helix structure in collagen, that was observed in CD analysis of HAc derived collagen nanofibers, for the first time was successfully confirmed and illustrated by using SEM and TEM methods. Furthermore, CD revealed the mostly detrimental effect of stabilization conditions such as heat, vacuum and UV treatment on the secondary structure of the collagen nanofibers.

Electrospun Collagen Nanofibers and Their Applications in Skin Tissue Engineering.

Electrospinning is a simple and versatile technique to fabricate continuous fibers with diameter ranging from micrometers to a few nanometers. To date, the number of polymers that have been electrospun has exceeded 200. In recent years, electrospinning has become one of the most popular scaffold fabrication techniques to prepare nanofiber mesh for tissue engineering applications. Collagen, the most abundant extracellular matrix protein in the human body, has been electrospun to fabricate biomimetic scaffolds that imitate the architecture of native human tissues. As collagen nanofibers are mechanically weak in nature, it is commonly cross-linked or blended with synthetic polymers to improve the mechanical strength without compromising the biological activity. Electrospun collagen nanofiber mesh has high surface area to volume ratio, tunable diameter and porosity, and excellent biological activity to regulate cell function and tissue formation. Due to these advantages, collagen nanofibers have been tested for the regeneration of a myriad of tissues and organs. In this review, we gave an overview of electrospinning, encompassing the history, the instrument settings, the spinning process and the parameters that affect fiber formation, with emphasis given to collagen nanofibers' fabrication and application, especially the use of collagen nanofibers in skin tissue engineering.

Solvent and temperature effects on folding of electrospun collagen nanofibers

Electrospinning is a well-known method to obtain nanofibers and collagen is a very attractive material for biological applications due to its bioactive nature. However, the preservation of the natural structure of collagen at the end products due to the different production parameters is an open question. In this study the importance of the preparation temperature for CD measurements and the solvent effect that employed in the electrospinning process was discussed. The decrease of the preparation temperature by 10° increased the measured PP-II (folded) fraction ratio from 37% to 52.5% and the nanofibers that were obtained from acidic solvent scored 59% of PP-II fraction ratio.

Resemblance of Electrospun Collagen Nanofibers to Their Native Structure

Electrospinning is a promising method to mimic the native structure of the extracellular matrix. Collagen is the material of choice, since it is a natural fibrous structural protein. It is an open question how much the spinning process preserves or alters the native structure of collagen. There are conflicting results in the literature, mainly due to the different solvent systems in use and due to the fact that gelatin is employed as a reference state for the completely unfolded state of collagen in calculations. Here we used circular dichroism (CD) and Fourier-transform infrared spectroscopy (FTIR) to investigate the structure of regenerated collagen samples and scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to illuminate the electrospun nanofibers. Collagen is mostly composed of folded and unfolded structures with different ratios, depending on the applied temperature. Therefore, CD spectra were acquired as a temperature series during thermal denaturation of native calf skin collagen type I and used as a reference basis to extract the degree of collagen folding in the regenerated electrospun samples. We discussed three different approaches to determine the folded fraction of collagen, based on CD spectra of collagen from 185 to 260 nm, since it would not be sufficient to obtain simply the fraction of folded structure θ from the ellipticity at a single wavelength of 221.5 nm. We demonstrated that collagen almost completely unfolded in fluorinated solvents and partially preserved its folded structure θ in HAc/EtOH. However, during the spinning process it refolded and the PP-II fraction increased. Nevertheless, it did not exceed 42% as deduced from the different secondary structure evaluation methods, discussed here. PP-II fractions in electrospun collagen nanofibers were almost same, being independent from the initial solvent systems which were used to solubilize the collagen for electrospinning process.

Nanofibrous Collagen Nerve Conduits for Spinal Cord Repair

Nerve regeneration in an injured spinal cord is often restricted, contributing to the devastating outcome of neurologic impairment below the site of injury. Although implantation of tissue-engineered scaffolds has evolved as a potential treatment method, the outcomes remain sub-optimal. One possible reason may be the lack of topographical signals from these constructs to provide contact guidance to invading cells or regrowing axons. Nanofibers mimic the natural extracellular matrix architecturally and may therefore promote physiologically relevant cellular phenotypes. In this study, the potential application of electrospun collagen nanofibers (diameter=208.2±90.4 nm) for spinal cord injury (SCI) treatment was evaluated in vitro and in vivo. Primary rat astrocytes and dorsal root ganglias (DRGs) were seeded on collagen-coated glass cover slips (two-dimensional [2D] substrate controls), and randomly oriented or aligned collagen fibers to evaluate scaffold topographical effects on astrocyte behavior and neurite outgrowth, respectively. When cultured on collagen nanofibers, astrocyte proliferation and expression of glial fibrillary acidic protein (GFAP) were suppressed as compared to cells on 2D controls at days 3 (p<0.05) and 7 (p<0.01). Aligned fibers resulted in elongated astrocytes (elongation factor >4, p<0.01) and directed the orientation of neurite outgrowth from DRGs along fiber axes. In the contrast, neurites emanated radially on randomly oriented collagen fibers. By forming collagen scaffolds into spiral tubular structures, we demonstrated the feasibility of using electrospun nanofibers for the treatment of acute SCI using a rat hemi-section model. At days 10 and 30 postimplantation, extensive cellular penetration into the constructs was observed regardless of fiber orientation. However, scaffolds with aligned fibers appeared more structurally intact at day 30. ED1 immunofluorescent staining revealed macrophage invasion by day 10, which decreased significantly by day 30. Neural fiber sprouting as evaluated by neurofilament staining was observed as early as day 10. In addition, GFAP immunostained astrocytes were found only at the boundary of the lesion site, and no astrocyte accumulation was observed in the implantation area at any time point. These findings indicate the feasibility of fabricating 3D spiral constructs using electrospun collagen fibers and demonstrated the potential of these scaffolds for SCI repair.

Sustained release of neurotrophin‐3 and chondroitinase ABC from electrospun collagen nanofiber scaffold for spinal cord injury repair

Nerve regeneration after spinal cord injuries (SCI) remains suboptimal despite recent advances in the field. One major hurdle is the rapid clearance of drugs from the injury site, which greatly limits therapeutic outcomes. Nanofiber scaffolds represent a potential class of materials for enhancing nerve regeneration because of its biomimicking architecture. In this study, we investigated the feasibility of incorporating neurotrophin-3 (NT-3) and chondroitinase ABC (ChABC) onto electrospun collagen nanofibers for SCI treatment. By using microbial transglutaminase (mTG) mediated crosslinking, proteins were loaded onto electrospun collagen nanofibers at an efficiency of ∼45-48%. By combining NT-3 with heparin during the protein incorporation process, a sustained release of NT-3 was obtained (∼96% by day 28). As indicated by dorsal root ganglion outgrowth assay, NT-3 incorporated collagen scaffolds supported neuronal culture and neurite outgrowth for a longer time period than bolus delivery of NT-3. The presence of heparin also protected ChABC from degradation. Specifically, as evaluated by dimethylmethylene blue assay, bioactive ChABC was detected from collagen scaffolds for at least 32 days in vitro in the presence of heparin (∼32% of bioactivity retained). In contrast, ChABC bioactivity was only ∼1.9% by day 22 in the absence of heparin. Taken together, these results clearly demonstrated the feasibility of incorporating NT-3 and ChABC via mTG immobilization to produce protein-incorporated collagen nanofibers. Such biofunctional nanofiber constructs may find useful applications in SCI treatment by providing topographical signals and multiple biochemical cues that can promote nerve regeneration while antagonizing axonal growth inhibition for CNS regeneration.

Three-Dimensional Hierarchical Composite Scaffolds Consisting of Polycaprolactone, β-Tricalcium Phosphate, and Collagen Nanofibers: Fabrication, Physical Properties, and In Vitro Cell Activity for Bone Tissue Regeneration

β-Tricalcium phosphate (β-TCP) and collagen have been widely used to regenerate various hard tissues, but although Bioceramics and collagen have various biological advantages with respect to cellular activity, their usage has been limited due to β-TCP's inherent brittleness and low mechanical properties, along with the low shape-ability of the three-dimensional collagen. To overcome these material deficiencies, we fabricated a new hierarchical scaffold that consisted of a melt-plotted polycaprolactone (PCL)/β-TCP composite and embedded collagen nanofibers. The fabrication process was combined with general melt-plotting methods and electrospinning. To evaluate the capability of this hierarchical scaffold to act as a biomaterial for bone tissue regeneration, physical and biological assessments were performed. Scanning electron microscope (SEM) micrographs of the fabricated scaffolds indicated that the β-TCP particles were uniformly embedded in PCL struts and that electrospun collagen nanofibers (diameter = 160 nm) were well layered between the composite struts. By accommodating the β-TCP and collagen nanofibers, the hierarchical composite scaffolds showed dramatic water-absorption ability (100% increase), increased hydrophilic properties (20%), and good mechanical properties similar to PCL/β-TCP composite. MTT assay and SEM images of cell-seeded scaffolds showed that the initial attachment of osteoblast-like cells (MG63) in the hierarchical scaffold was 2.2 times higher than that on the PCL/β-TCP composite scaffold. Additionally, the proliferation rate of the cells was about two times higher than that of the composite scaffold after 7 days of cell culture. Based on these results, we conclude that the collagen nanofibers and β-TCP particles in the scaffold provide good synergistic effects for cell activity.

Photochemical crosslinked electrospun collagen nanofibers: Synthesis, characterization and neural stem cell interactions

Currently available crosslinking methods for electrospun collagen nanofibers do not preserve the fibrous architecture over prolonged periods of time. In addition, electrospinning of collagen often involves solvents that lead to extensive protein denaturation. In this study, we demonstrate the advantage of acetic acid over 1,1,1,3,3,3 hexafluoroisopropanol (HFP) in preventing collagen denaturation. A novel photochemical crosslinking method using rose bengal as the photoinitiator is also introduced. Using circular dichorism analyses, we demonstrate the fraction of collagen helical structure to be significantly greater in acetic acid-spun fibers than HFP-spun fibers (28.9 +/- 5.9% vs. 12.5 +/- 2.0%, p < 0.05). By introducing 0.1% (w/v) rose bengal into collagen fibers and subjecting these scaffolds to laser irradiation at a wavelength of 514 nm for 100 sec, biodegradable crosslinked scaffolds were obtained. Scaffold degradation as evaluated by soaking crosslinked collagen scaffolds in PBS at 37 degrees C, indicated a mass loss of 47.7 +/- 7.4% and 68.9 +/- 24.7% at day 7 and day 15, respectively. However, these scaffolds retained fibrous architecture for at least 21 days under physiological conditions. Neural stem cell line, C17.2, cultured on crosslinked collagen scaffolds proliferated after 7 days by forming a confluent layer of cells with extensive cellular projections that were indicative of neurite outgrowth. Taken together, these findings support the potential of acetic acid-electrospun photochemical crosslinked collagen nanofibers for neural tissue engineering.

Comparative Performance of Electrospun Collagen Nanofibers Cross-linked by Means of Different Methods

Collagen, as the major structural protein of the extracellular matrix in animals, is a versatile biomaterial of great interest in various engineering applications. Electrospun nanofibers of collagen are regarded as very promising materials for tissue engineering applications because they can reproduce the morphology of the natural bone but have as a drawback a poor structural consistency in wet conditions. In this paper, a comparative study between the performance of different cross-linking methods such as a milder enzymatic treatment procedure using transglutaminase, the use of N-[3-(dimethylamino)propyl]-N'-ethylcarbodiimide hydrochloride/N-hydroxysuccinimide, and genipin, and the use of a physical method based on exposure to ultraviolet light was carried out. The chemical and enzymatic treatments provided, in this order, excellent consistency, morphology, cross-linking degree, and osteoblast viability for the collagen nanofibers. Interestingly, the enzymatically cross-linked collagen mats, which are considered to be a more biological treatment, promoted adequate cell adhesion, making the biomaterial biocompatible and with an adequate degree of porosity for cell seeding and in-growth.