Marine Collagen from European Sea Bass (Dicentrarchus labrax) Waste for the Development of Chitosan/Collagen Scaffolds in Skin Tissue Engineering

Gelatin–GAG electrospun nanofibrous scaffold for skin tissue engineering: Fabrication and modeling of process parameters

Tissue engineering is a novel regenerative approach in the medicinal field that promises the regeneration of damaged tissues. Moreover, tissue engineering involves synthetic and natural biomaterials that facilitate tissue or organ growth outside the body. Not surprisingly, the demand for polymer-based therapeutical approaches in skin tissue defects has increased at an effective rate, despite the pressing clinical need. Among the 3D scaffolds for tissue engineering and regeneration approaches, hydrogel scaffolds have shown significant importance for their use as 3D cross-linked scaffolds in skin tissue regeneration due to their ideal moisture retention property and porosity biocompatibility, biodegradable, and biomimetic characteristics. In this review, we demonstrated the choice of ideal biomaterials to fabricate the novel hydrogel scaffolds for skin tissue engineering. After a short introduction to the bioactive and drug-loaded polymeric hydrogels, the discussion turns to fabrication and characterisation techniques of the polymeric hydrogel scaffolds. In conclusion, we discuss the excellent wound healing potential of stem cell-loaded hydrogels and Nano-based approaches to designing hydrogel scaffolds for skin tissue engineering.

Biomedical applications of herbals and their derivatives for skin tissue engineering have become increasingly popular in recent years. Herbals and their derivatives can be used to create scaffolds for skin tissue engineering and wound healing, as they provide a biocompatible and biodegradable structure that can be used to promote skin tissue regeneration. In the literature search for relevant papers, sixty-eight studies have been included from January 2000 to September 2023 from PubMed and Google Scholar database sources. This review provided an overview of current knowledge on biomedical applications of herbal medicine such as Aloe Vera, Calendula Officinalis, Hypericum perforatum, Lawsonia inermis, Nicotine, Propolis, Honey, Perovskia abrotanoides Karel, Oak fruit inner, Lithospermum officinale, and their derivatives in skin tissue engineering. We showed that tissue engineering is a prominent therapeutic strategy that can be cost-effective in treatment of skin wounds by applying different natural herbal products, nanotechnology, material science, and regenerative medicine to suit the current wound care demands such as tissue repair, restoration of lost tissue integrity and scarless healing. So the design, synthesis, modification, evaluation, and characterization of herbal products are needed to target skin tissue engineering and wound healing. New strategies based on the drug delivery systems and nanocarriers such as nanofibers, nanoparticles, and vesicular structures, based on materials such as gelatin, PCL, collagen, chitosan, PLGA, PEG, PEO, PVA, natural gums, and PU can also be developed to facilitate the wound healing process. The homeostasis, re-epithelialization, regeneration, and immunocytes can be investigated by inducing fibroblasts proliferation and/or collagen production. Nevertheless, further clinical studies are needed before introducing commercial products in the market.

Keratin-based nanofibers were fabricated using the electrospinning technique, and their potential as scaffolds for tissue engineering was investigated. Keratin, extracted from the human hair, was blended with poly(vinyl alcohol) (PVA) in an aqueous medium. Morphological characterizations of the fabricated PVA-keratin nanofiber (PK-NF) random and aligned scaffolds performed using a scanning electron microscope (SEM) revealed the formation of uniform and randomly oriented nanofibers with an interconnected three-dimensional network structure. The mean diameter of the nanofibers ranged from 100 to 250nm. Functional groups and structural studies were done by infrared spectroscopy (FTIR) and X-ray diffraction (XRD) analysis. FTIR study suggested that PVA interacted with keratin by hydrogen bonding. Moreover, the in vitro cell culture study could suggest that PK-NF scaffolds were non-cytotoxic by supporting the growth of murine embryonic stem cells (ESCs), human keratinocytes (HaCaT), and dermal fibroblast (NHDF) cell lines. Further, the immunocytochemical characterization revealed the successful infiltration, adhesion, and growth of ESCs, HaCaT, and NHDF cells seeded on PK-NF scaffolds. However, there was no noteworthy difference observed concerning cell growth and viability irrespective of the random and aligned internal fibril arrangement of the PK-NF scaffolds. The infiltration and growth pattern of HaCaT and NHDF cells adjacent to each other in a 3D co-culture study mimicked that of epidermal and dermal skin cells and indeed underscored the potential of PK-NFs as a scaffold for skin tissue engineering.

Skin tissue engineering has emerged as a promising field for developing wound dressings and skin substitutes. Recently, cryogel based scaffolds have gained significant attention due to their biocompatibility, tunable properties and porous structure resembling the native extracellular matrix. Polysaccharides like sodium alginate, chitosan, dextran, and agarose are widely explored for fabricating cryogels due to their inherent biocompatibility and bioactivity. The review begins by highlighting the significance of skin tissue engineering in treating different dermatological conditions and injuries. It then explores the fundamental properties of polysaccharide based cryogel scaffolds, focusing on their biocompatibility, biodegradability, fabrication methods and biomedical applications of polysaccharides based cryogel scaffolds in skin tissue engineering. In addition, it explores the potential of integrating 3D and 4D printing technologies to enhance the functionality of these scaffolds, leading to their widespread adoption in the clinical settings for wound healing and for personalized medicine by offering tailored solutions for tissue repair and regeneration. Overall, this review emphasizes the immense potential of polysaccharide based cryogel scaffolds in advancing the field of skin tissue engineering, offering novel solutions for wound healing, and personalized medicine.

Fabrication and characterization of PCL/zein/gum arabic electrospun nanocomposite scaffold for skin tissue engineering

In the search for a novel and scalable skin scaffold for wound healing and tissue regeneration, we fabricated a class of fibrin/polyvinyl alcohol (PVA) scaffolds using an emulsion templating method. The fibrin/PVA scaffolds were formed by enzymatic coagulation of fibrinogen with thrombin in the presence of PVA as a bulking agent and an emulsion phase as the porogen, with glutaraldehyde as the cross-linking agent. After freeze drying, the scaffolds were characterized and evaluated for biocompatibility and efficacy of dermal reconstruction. SEM analysis showed that the formed scaffolds had interconnected porous structures (average pore size e was around 330 µm) and preserved the nano-scale fibrous architecture of the fibrin. Mechanical testing showed that the scaffolds' ultimate tensile strength was around 0.12 MPa with an elongation of around 50%. The proteolytic degradation of scaffolds could be controlled over a wide range by varying the type or degree of cross-linking and by fibrin/PVA composition. Assessment of cytocompatibility by human mesenchymal stem cell (MSC) proliferation assays shows that MSC can attach, penetrate, and proliferate into the fibrin/PVA scaffolds with an elongated and stretched morphology. The efficacy of scaffolds for tissue reconstruction was evaluated in a murine full-thickness skin excision defect model. The scaffolds were integrated and resorbed without inflammatory infiltration and, compared to control wounds, promoted deeper neodermal formation, greater collagen fiber deposition, facilitated angiogenesis, and significantly accelerated wound healing and epithelial closure. The experimental data showed that the fabricated fibrin/PVA scaffolds are promising for skin repair and skin tissue engineering.

Skin tissue engineering (STE) is widely regarded as an effective approach for skin regeneration. Several synthetic biomaterials utilized for STE have demonstrated favorable fibrillar characteristics, facilitating the regeneration of skin tissue at the site of injury, yet they have exhibited a lack of in situ degradation. Various types of skin regenerative materials, such as hydrogels, nanofiber scaffolds, and 3D-printing composite scaffolds, have recently emerged for use in STE. Electrospun nanofiber scaffolds possess distinct advantages, such as their wide availability, similarity to natural structures, and notable tissue regenerative capabilities, which have garnered the attention of researchers. Hence, electrospun nanofiber scaffolds may serve as innovative biological materials possessing the necessary characteristics and potential for use in tissue engineering. Recent research has demonstrated the potential of electrospun nanofiber scaffolds to facilitate regeneration of skin tissues. Nevertheless, there is a need to enhance the rapid degradation and limited mechanical properties of electrospun nanofiber scaffolds in order to strengthen their effectiveness in soft tissue engineering applications in clinical settings. This Review centers on advanced research into electrospun nanofiber scaffolds, encompassing preparation methods, materials, fundamental research, and preclinical applications in the field of science, technology, and engineering. The existing challenges and prospects of electrospun nanofiber scaffolds in STE are also addressed.

Polymeric nanofibrous scaffolds have the potential to interact and regulate specific regenerative events at molecular level to restore the damaged tissues. Nanofibers can be fabricated using various techniques like electrospinning, phase separation, self–assembly, etc. Electrospinning is the most widely employed method to fabricate nanofibers for applications in tissue engineering. The electrospun ultrafine fibers can be tuned to exhibit desired pore distribution, high surface area–to–volume ratio, cell adhesion and proliferation due to their structural resemblance to the native extracellular matrix. Electrospun polymeric nanofibers possess various advantages as skin substitutes because they can prevent fluid and proteins loss from the wound area, help in the removal of exudates, inhibit microbial infection, exhibit excellent anti–adhesion properties and guide endogenous cells to proliferate and remodel. Nanofibrous scaffolds are currently being fabricated in combination with growth factors and / or cells to accelerate orchestrated wound healing. The use of electrospun nanofibrous scaffolds for skin tissue engineering is detailed in this review. Further, recent advances in fabricating biocomposite nanofibrous scaffolds, electrospinning-coupled electrospraying of cells and stem cells for skin tissue engineering are also highlighted.

Chapter 17 - Nanofibrous scaffolds for skin tissue engineering and wound healing applications

Background: Skin is a 3-dimensional (3-D) tissue that mainly consists 2 layers, comprising the epidermis and dermis. Skin tissue engineering scaffolds are used commonly as 3-D analogs of the extracellular matrix (ECM) of the skin. Bacterial cellulose (BC) has great importance in skin tissue engineering because of its resemblance to ECM and its biocompatibility. The lack of 3-D microporosity and limited biodegradation capacity has restricted its application as a scaffold for skin tissue engineering. Controlled 3-D microporosity of BC via surface modification techniques are required for potential tissue engineering applications.Methods: Freeze-drying is an ex-situ surface modification technique for making macroporous BC scaffolds (MBCSs). This study proposed a new approach to the freeze-drying method for the arrangement of the pore size of MBCSs specifically for the human keratinocyte cell line (KER-CT). Different concentrations of MBCS (0.25%, 0.50%, and 0.75%) were prepared and the KER-CT cell viability was detected via 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay.Result: The results of this study indicated that the KER-CT cells were able to proliferate all of the concentrations of MBCS, and the best cell viability value was observed with 0.75% MBCS. The results were supported by FESEM and light microscopic observations.Conclusion: These findings suggested that 0.75% MBCS might be of use in epidermal tissue engineering applications.

The present research paper explores the potential of electrospun nanofibers in the promising field of skin tissue engineering. Specifically, we propose an advanced preparation and characterization of an electrospun Polyurethane/Calcium Chloride (PU/CaCl2) nanocomposite scaffold, devised to boost the scaffold’s physicochemical and biological properties for skin tissue regeneration. By incorporating CaCl2 into the PU matrix using an electrospinning process, we were able to fabricate a novel nanocomposite scaffold. The morphological examination through Field Emission Scanning Electron Microscope (FESEM) revealed that the fiber diameter of the PU/CaCl2 (563 ± 147 nm) scaffold was notably smaller compared to the control (784 ± 149 nm). The presence of CaCl2 in the PU matrix was corroborated by Fourier-Transform Infrared Spectroscopy (FTIR) and Thermogravimetric Analysis (TGA). Furthermore, the PU/CaCl2 scaffold exhibited superior tensile strength (10.81 MPa) over pristine PU (Tensile −6.16 MPa, Contact angle - 109° ± 1° and Roughness - 854 ± 32 nm) and revealed enhanced wettability (72° ± 2°) and reduced surface roughness (274 ± 104 nm), as verified by Contact angle and Atomic Force Microscopy. The developed scaffold demonstrated improved anticoagulant properties, indicating its potential for successful integration within a biological environment. The improved properties of the PU/CaCl2 nanocomposite scaffold present a significant advancement in electrospun polymer nanofibers, offering a potential breakthrough in skin tissue engineering. However, additional studies are required to thoroughly evaluate the scaffold’s effectiveness in promoting cell adhesion, proliferation, and differentiation. We aim to catalyze significant advancements in the field by revealing the creation of a potent skin scaffold leveraging electrospun nanofibers. Encouraging deeper exploration into this innovative electrospun composite scaffold for skin tissue engineering, the PU/CaCl2 scaffold stands as a promising foundation for pioneering more innovative, efficient, and sustainable solutions in biomedical applications.

Creating scaffolds for skin tissue engineering remain challenging in terms of their mechanical and biological properties. In this paper, we present a study on the nanocomposite polyurethane (PU)/polycaprolactone (PCL) scaffolds with graphene oxide (GO), which were fabricated by using electrospinning method, for potential skin tissue engineering. For this, homogenous and soft PU nanofibers containing varying percent of polycaprolactone (12% and 15%) and nano GO (0.5-4%) were electrospun, respectively, and then characterized by different techniques/assays in vitro. For the scaffold characterization, scanning electron microscopy (SEM) and Fourier-transform infrared spectroscopy (FTIR) were used. The SEM results show the spun scaffolds have 3D porous structure (90%) with the fiber diameter increased with the GO concentration, while the FTIR results confirmed the presence of PU, PCL, and Go in the scaffolds. Also, the biocompatibility, via the cytotoxicity, of the scaffolds was examined by MTT assay with the human skin fibroblast cells, along with their wettability in terms of contact angle. Our results show that the scaffolds are biocompatible to the skin fibroblast cell, illustrating their potential use in skin tissue engineering. Also, our results illustrate that the addition of GO to the PU/PCL composite can increase the wettability (or hydrophilicity) and biocompatibility of scaffolds. Combined together, the nanocomposite PU/PCL scaffolds with GO are promising as biocompatible constructs for skin tissue engineering.

After decades of research, fully functional skin regeneration is still a challenge. Skin is a multilayered complex organ exhibiting a cascading healing process affected by various mechanisms. Specifically, nutrients, oxygen, and biochemical signals can lead to specific cell behavior, ultimately conducive to the formation of high-quality tissue. This biomolecular exchange can be tuned through scaffold engineering, one of the leading fields in skin substitutes and equivalents. The principal objective of this investigation was the design, fabrication, and evaluation of a new class of three-dimensional fibrous scaffolds consisting of poly(ε-caprolactone) (PCL)/calcium alginate (CA), with the goal to induce keratinocyte differentiation through the action of calcium leaching. Scaffolds fabricated by electrospinning using a PCL/sodium alginate solution were treated by immersion in a calcium chloride solution to replace alginate-linked sodium ions by calcium ions. This treatment not only provided ion replacement, but also induced fiber crosslinking. The scaffold morphology was examined by scanning electron microscopy and systematically assessed by measurements of the pore size and the diameter, alignment, and crosslinking of the fibers. The hydrophilicity of the scaffolds was quantified by contact angle measurements and was correlated to the augmentation of cell attachment in the presence of CA. The in vitro performance of the scaffolds was investigated by seeding and staining fibroblasts and keratinocytes and using differentiation markers to detect the evolution of basal, spinous, and granular keratinocytes. The results of this study illuminate the potential of the PCL/CA scaffolds for tissue engineering and suggest that calcium leaching out from the scaffolds might have contributed to the development of a desirable biological environment for the attachment, proliferation, and differentiation of the main skin cells (i.e., fibroblasts and keratinocytes).

This study aims to fabricate PYA/PEG/Chitosan nanofiber scaffolds for skin tissue engineering. The scaffolds in the nanofiber structure closely match with the ECM structure of the skin tissue. PYA/PEG/Chitosan nanofibers were prepared by electrospinning technique with various compositions (v/v) % (95:5, 90:10, 85:15, 80:20). The scaffold surface morphology was characterized by scanning electron microscopy (SEM), and the functional groups were characterized by Fourier transform infrared (FTIR). The biodegradation test was carried out by immersing the sample in a simulated body fluid (SBF) solution with time variations of 1, 2, 3, and 4 weeks. The scaffolds have diameters in the range of 110-520 nm, which correspond to the skin ECM, and porosity in the range of 63-71%, which facilitates cell adhesion, and proliferation. The rate of biodegradation is faster in the composition (95:5). The surface morphology with random fiber diameter, porosity, and biodegradation are suitable for skin tissue scaffolding.