Skin Regeneration Applications Research Articles

Effects of Gelatin Methacrylate Bio-ink Concentration on Mechano-Physical Properties and Human Dermal Fibroblast Behavior.

Gelatin-methacryloyl (GelMa) is a very versatile biomaterial widely used in various biomedical applications. The addition of methacryloyl makes it possible to have hydrogels with varying mechanical properties due to its photocuring characteristics. In addition, gelatin is obtained and derived from natural material; thus, it retains various cell-friendly motifs, such as arginine-glycine-aspartic acid, which then provides implanted cells with a friendly environment for proliferation and differentiation. In this study, we fabricated human dermal fibroblast cell (hDF)-laden photocurable GelMa hydrogels with varying physical properties (5%, 10%, and 15%) and assessed them for cellular responses and behavior, including cell spreading, proliferation, and the degree of extracellular matrix remodeling. Under similar photocuring conditions, lower concentrations of GelMa hydrogels had lower mechanical properties than higher concentrations. Furthermore, other properties, such as swelling and degradation, were compared in this study. In addition, our findings revealed that there were increased remodeling and proliferation markers in the 5% GelMa group, which had lower mechanical properties. However, it was important to note that cellular viabilities were not affected by the stiffness of the hydrogels. With this result in mind, we attempted to fabricate 5–15% GelMa scaffolds (20 × 20 × 3 mm3) to assess their feasibility for use in skin regeneration applications. The results showed that both 10% and 15% GelMa scaffolds could be fabricated easily at room temperature by adjusting several parameters, such as printing speed and extrusion pressure. However, since the sol-gel temperature of 5% GelMa was noted to be lower than its counterparts, 5% GelMa scaffolds had to be printed at low temperatures. In conclusion, GelMa once again was shown to be an ideal biomaterial for various tissue engineering applications due to its versatile mechanical and biological properties. This study showed the feasibility of GelMa in skin tissue engineering and its potential as an alternative for skin transplants.

A review on wound management with special reference to nanotechnology

Management of wound care mainly depends upon the advancement of innovative and effective wound care product to achieve speedy recovery and minimising scar formation. Wound healing management has been always an interesting field of research till date due to serious need for new wound treatment. Appropriate wound care is a significant challenge because of the complications associated with wounds as well as low permeability through the skin. The interconnected events of wound healing occur simultaneously to restore the tissue integrity and functions of the cells. Wound healing gets hampered by numerous factors. These physiological events occur easily in normal human being, but in some situations these molecular events are affected, resulting in hard to heal/ chronic wounds. In current years nanotechnology has emerged as an exciting field with several applications in skin regeneration. These nanoscale delivery systems have numerous benefits in the healing process such as decrease in drug’s cytotoxicity, administration of poorly water-soluble drugs, better skin permeation, controlled release behaviour, antimicrobial activity, as well as stimulation of fibroblast proliferation and reduced inflammation. Thus, emerged as an effective strategy and approach for the treatment of chronic wounds. This review briefly discusses about the wounds, characteristics of an ideal wound dressings along with a special mention regarding the several strategies of wound healing by nanotechnology with their functions and advantages.

In vitro and in vivo evaluation of chitosan-alginate/gentamicin wound dressing nanofibrous with high antibacterial performance

Wound dressings based on nanofiber polymer scaffolds with good antimicrobial performance and skin reconstruction ability are promising options to thwart wound infection and accelerate wound healing. This paper reports on the synthesis via electrospinning of chitosan-alginate (CS-Alg) nanofiber dressings with various amounts of gentamicin (Gn; 0–10 wt%) as a drug delivery system. Smooth and continuous nanofibers with no obvious beads were created, with increases in the amount of Gn resulting in reduced fiber diameter. Antimicrobial tests showed the Gn-loaded nanofibers had good antibacterial performance as indicated by the inhibition of bacterial growth. CS-Alg nanofibers loaded with higher Gn concentrations exhibited greater antibacterial performance than those with lower Gn concentrations. In vitro cell culture studies demonstrated that CS-Alg wound dressings with 1–3% Gn improved L929 cell attachment and proliferation more than wound dressings with higher Gn concentrations. In vivo experiments revealed that Cs-Alg nanofibers loaded with 3% Gn significantly enhanced skin regeneration in a Balb/C mice model by stimulating the formation of a thicker dermis, increasing collagen deposition, and increasing the formation of new blood vessels and hair follicles. Collectively, Gn-loaded CS-Alg wound dressings can be considered a good candidate for drug delivery systems and skin regeneration applications.

Electrospun Polyurethane–Gelatin Composite: A New Tissue-Engineered Scaffold for Application in Skin Regeneration and Repair of Complex Wounds

Wound healing is vital for patients with complex wounds including burns. While the gold standard of skin transplantation ensures a surgical treatment to heal wounds, it has its limitations, for example, insufficient donor sites for patients with large burn wounds and creation of wounds and pain when harvesting the donor skin. Therefore, tissue-engineered skin is of paramount importance. The aim of this study is to investigate and characterize an elastomeric acellular scaffold that would demonstrate the ability to promote skin regeneration. A hybrid gelatin-based electrospun scaffold is fabricated via the use of biodegradable polycarbonate polyurethane (PU). It is hypothesized that the addition of PU would enable a tailored degradation rate and an enhanced mechanical strength of electrospun gelatin. Introducing 20% PU to gelatin scaffolds (Gel80-PU20) results in a significant increase in the degradation resistance, yield strength, and elongation of these scaffolds without altering the cell viability. In vivo studies using a mouse excisional wound biopsy grafted with the scaffolds reveals that the Gel80-PU20 scaffold enables greater cell infiltration than clinically established matrices, for example, Integra (dermal regeneration matrix, DRM), a benchmark scaffold. Immunostaining shows fewer macrophages and myofibroblastic cells on the Gel80-PU20 scaffold when compared with the DRM. The findings show that electrospun Gel80-PU20 scaffolds hold potential for generating tissue substitutes and overcoming some limitations of conventional wound care matrices.

Chitosan-laponite nanocomposite scaffolds for wound dressing application.

The pivotal issue of skin regeneration research is the development of effective biomaterials that exhibit biological activities as fungicide and bactericide, combining simple and low cost manufacturing technologies. In this context, nanocomposite scaffolds based on chitosan (Ch)/Laponite (Lap) were produced by using different concentrations of Lap via freeze-drying process for potential application in skin regeneration. The influence of Lap concentration on the scaffold properties was evaluated. The prepared scaffolds were characterized by x-ray diffraction (XRD), scanning electron microscopy (SEM), porosity, swelling capacity, and mechanical analyses. The results revealed that the scaffolds exhibited a porous architecture, besides the increase in the clay content, leads to an increase in the porosity, an improvement of mechanical strength, and a decrease of swelling capacity. In vitro tests were also carried out to evaluate the biocompatibility of the materials, such as bioadhesion, antibacterial activity, viability, and cell adhesion. Viability and cell adhesion demonstrated that all scaffolds were not cytotoxic and the fibroblast cells readily attached on the surface of the scaffolds. Thereby, the results suggested that the nanocomposite scaffolds are biomaterials potentially useful as wound dressings.

Biomimetic nanocomposite scaffolds based on surface modified PCL-nanofibers containing curcumin embedded in chitosan/gelatin for skin regeneration

Recently, nanofibrous-hydrogel composites are luring attention for tissue regeneration applications as they mimic soft-tissues' microstructure. Mostly, the electrospun nanofibers based on synthetic polymers such as Polycaprolactone (PCL) are placed in a crosslinked hydrogels. Due to hydrophobic nature of PCL, integration of these nanofibers with the hydrophilic hydrogels of matrix is not sufficient. In this study, we applied Poly(ethylene glycol) methyl ether methacrylate (PEGMA)-surface-modified PCL nanofibers within chitosan-gelatin hydrogels for skin regeneration applications. In addition, curcumin was loaded into PCL nanofibers due to its great impact on skin regeneration process. Fabricated nanofibrous-hydrogel scaffolds were characterized using scanning electron microscopy (SEM), porosimetery, Fourier transform infrared spectroscopy (FTIR), mechanical compression test, and water uptake studies. Curcumin release was investigated using UV/Vis spectrophotometry. In order to study biocompatibility of scaffolds MTT assay and cell culture was performed using L929 cells. FTIR spectra confirmed PEGMA modification of PCL nanofibers. Results of mechanical test determined that surface modified PCL nanofibers improved mechanical strength and modulus of scaffolds. Porosimetery studies showed proper porosity of scaffolds for skin regeneration from 90.43 to 71.48% and pore size of 101–256 μm. Biological test confirmed proper biocompatibility and good cell attachment to the scaffolds. Taken together, the chitosan/gelatin hydrogel incorporating PEGMA modified PCL nanofibers containing curcumin shows great potential for skin regeneration.

Low molecular weight ε-caprolactone-p-coumaric acid copolymers as potential biomaterials for skin regeneration applications.

ε-caprolactone-p-coumaric acid copolymers at different mole ratios (ε-caprolactone:p-coumaric acid 1:0, 10:1, 8:1, 6:1, 4:1, and 2:1) were synthesized by melt-polycondensation and using 4-dodecylbenzene sulfonic acid as catalyst. Chemical analysis by NMR and GPC showed that copolyesters were formed with decreasing molecular weight as p-coumaric acid content was increased. Physical characteristics, such as thermal and mechanical properties, as well as water uptake and water permeability, depended on the mole fraction of p-coumaric acid. The p-coumarate repetitive units increased the antioxidant capacity of the copolymers, showing antibacterial activity against the common pathogen Escherichia coli. In addition, all the synthesized copolyesters, except the one with the highest concentration of the phenolic acid, were cytocompatible and hemocompatible, thus becoming potentially useful for skin regeneration applications.

Characterization and Evaluation of Carboxymethyl Cellulose-Based Films for Healing of Full-Thickness Wounds in Normal and Diabetic Rats.

Artificial skin substitute made of polymeric films are of great demand in the field of skin tissue engineering. We report here the fabrication of carboxymethyl cellulose (CMC) and poly(ethylene glycol) (PEG) blend films by solution casting method for wound healing applications. The physicochemical characteristics and the thermal stability of the films were analyzed. The surface morphology shows crystalline structures with large hexagonal-like platelet crystals of CMC on the surface of the films. Pure CMC films exhibited higher tensile strength than the CMC/PEG blend films. The swelling ratio (SR) of the films was influenced by the pH of Tris–HCL buffer (2.0, 5.0, and 7.0), which increased with increase in pH. The hemocompatibility assay and cytotoxicity test using NIH 3T3 fibroblast cells showed that the films were biocompatible. To evaluate the wound healing efficacy, the films were applied in full-thickness wounds created in normal and diabetic Wistar albino rats. The wounds healed faster with pure CMC film compared to blend films in both normal and diabetic rats, evidenced by intensive collagen formation in histopathological analysis. Thus, the films have potential application in skin regeneration, thereby to restore the structural and functional characteristics of the skin.

Preparation and structural characterization of surface modified microporous bacterial cellulose scaffolds: A potential material for skin regeneration applications in vitro and in vivo

This study reports the fabrication of porogen-induced, surface-modified, 3-dimensionally microporous regenerated bacterial cellulose (rBC)/gelatin (3DMP rBC/G) scaffolds for skin regeneration applications. Round shaped gelatin microspheres (GMS), fabricated using a water-in-oil emulsion (WOE) method, were utilized as the porogen. The dissolution of GMS from the solution casted BC scaffolds led to surface-modified microporous rBC. The scaffolds were characterized using field emission scanning electron microscopy (FE-SEM) and elemental analysis. FE-SEM analysis confirmed the regular microporosity of the 3DMP rBC/G scaffolds, while elemental analysis confirmed the successful surface modification of cellulose with gelatin. In vitro tests showed good adhesion and proliferation of human keratinocytes (HaCaT) on the 3DMP rBC/G scaffolds during 7 days of incubation. Confocal microscopy showed penetration of HaCaT cells into the scaffolds, up to 300 μm in depth. In vivo wound healing and skin regeneration experiments, in experimental mice, showed complete skin regeneration within 2 weeks. The wound closure efficacy of the 3DMP rBC/G scaffolds was much higher (93%) than that of the control (47%) and pure BC-treated (63%) wounds. These results indicated that our 3DMP rBC/G scaffolds represent future candidate materials for skin regeneration applications.

Nano-/Microfibrous Cotton-Wool-Like 3D Scaffold with Core-Shell Architecture by Emulsion Electrospinning for Skin Tissue Regeneration.

Electrospun nanofibrous scaffold has long been studied as skin substitutes for their structural resemblance to the dermal extracellular matrix. However, packed fibrous architecture with small pore size restricts cellular infiltration into nanofibrous mat. In this article, we report highly porous, nano-/microfibrous 3D structure using polycaprolactone-chitosan emulsion and its application in skin regeneration. Under the influence of electric field, the emulsion containing encapsulated charged chitosan droplets enhances charge of the spinning solution and residual charge in the core of the deposited fiber, thereby creating core-shell, cotton-like fluffy structure with average pore size 62 μm, fiber diameter ∼1.62 μm, contact angle of 72° and 80% water uptake capacity of the scaffold. Further, differential stirring period of the specific emulsion developed compact nanofibrous membrane with nanometer ranged pore size emphasizing the role played by emulsion droplet size and the charge carried thereafter. Presence of nanofibers with high-interconnected porosity promoted efficient cellular infiltration and proliferation from initial days of cell seeding. The scaffold supported extracellular matrix protein expression and stratified epithelialization in vitro. Effective integration and attachment of scaffold with margins of a full-thickness excision wound created in a rat model with accelerated healing within 3 weeks proved the efficiency of the scaffold as skin substitute. Additionally, gradual and prolong release of acidic chitosan from the core section benefitted wound healing by lowering the pH of wound environment. Simple technique with inexpensive raw materials endorsed the scaffold as a promising off-the-shelf matrix for skin tissue regeneration.

Nanotechnology: A Promising Tool Towards Wound Healing.

Nanotechnology is an exciting emerging field with multiple applications in skin regeneration. Nanofibers have gained special attention in skin regeneration based on their structural similarity to the extracellular matrix. A wide variety of polymeric nanofibers with distinct properties have been developed and tested as scaffolds for skin regeneration. Besides providing support for tissue repair, nanofibrous materials can act as delivery systems for drugs, proteins, growth factors, and other molecules. Moreover, the morphology, biodegradability, and other functionalities of nanofibrous materials can be controlled towards specific conditions of wound healing. Other nanostructured drug delivery systems, such as nanoparticles, micelles, nanoemulsions, and liposomes, have been used to improve wound healing at different stages. These nanoscale delivery systems have demonstrated several benefits for the wound healing process, including reduced cytotoxicity of drugs, administration of poorly water-soluble drugs, improved skin penetration, controlled release properties, antimicrobial activity, and protection of drugs against light, temperature, enzymes or pH degradation, as well as stimulation of fibroblast proliferation and reduced inflammation.

Characterization of A Three-Dimensional Organotypic Co-Culture Skin Model for Epidermal Differentiation of Rat Adipose-Derived Stem Cells.

The organotypic co-culture is a well-known technique to examine cellular interactions and their roles in stem cell proliferation and differentiation. This study aims to evaluate the effects of dermal fibroblasts (DFs) on epidermal differentiation of adipose-derived stem cells (ASCs) using a three-dimensional (3D) organotypic co- culture technique. In this experimental research study, rat DFs and ASCs were isolated and cultured separately on electrospun polycaprolactone (PCL) matrices. The PCL matrices seeded by ASCs were superimposed on to the matrices seeded by DFs in order to create a 3D organotypic co-culture. In the control groups, PCL matrices seeded by ASCs were placed on matrices devoid of DFs. After 10 days, we assessed the expressions of keratinocyte-related genes by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and expression of pan-cytokeratin protein by immunofluorescence in the differentiated keratinocyte-like cells from co- culture and control groups. Keratinocyte-like cell morphologies were also observed by scanning electron microscopy (SEM). The early, intermediate, and terminal differentiation keratinocyte markers-Cytokeratin14, Filaggrin, and Involucrin significantly expressed in the co-culture groups com- pared to the control ones (P<0.05). We observed pan-cytokeratin in keratinocyte-like cells of both groups by immunofluorescence. SEM observation of the co-culture groups showed that the differentiated keratinocyte-like cells developed a polygonal cobblestone shape, considered characteristic of keratinocytes. The 3D organotypic co-culture bilayered construct that consisted of DFs and ASCs was an effective technique for epidermal differentiation of ASCs. This co-culture might be useful for epidermal differentiation of stem cells for future applications in skin regeneration.

Microstructure and invitro cellular response to novel soy protein-based porous structures for tissue regeneration applications.

Interest in the development of new bioresorbable structures for various tissue engineering applications is on the rise. In the current study, we developed and studied novel soy protein-based porous blends as potential new scaffolds for such applications. Soy protein has several advantages over the various types of natural proteins employed for biomedical applications due to its low price, non-animal origin and relatively long storage time and stability. In the present study, blends of soy protein with other polymers (gelatin, pectin and alginate) were added and chemically cross-linked using the cross-linking agents carbodiimide or glyoxal, and the porous structure was obtained through lyophilization. The resulting blend porous structures were characterized using environmental scanning microscopy, and the cytotoxicity of these scaffolds was examined invitro. The biocompatibility of the scaffolds was also evaluated invitro by seeding and culturing human fibroblasts on these scaffolds. Cell growth morphology and adhesion were examined histologically. The results show that these blends can be assembled into porous three-dimensional structures by combining chemical cross-linking with freeze-drying. The achieved blend structures combine suitable porosity with a large pore size (100-300 µm). The pore structure in the soy-alginate scaffolds possesses adequate interconnectivity compared to that of the soy-gelatin scaffolds. However, porous structure was not observed for the soy-pectin blend, which presented a different structure with significantly lower porosities than all other groups. The invitro evaluation of these porous soy blends demonstrated that soy-alginate blends are advantageous over soy-gelatin blends and exhibited adequate cytocompatibility along with better cell infiltration and stability. These soy protein scaffolds may be potentially useful as a cellular/acellular platform for skin regeneration applications.

Novel porous soy protein-based blend structures for biomedical applications: Microstructure, mechanical, and physical properties.

Use of naturally derived materials for biomedical applications is steadily increasing. Soy protein has advantages over various types of natural proteins employed for biomedical applications due to its low price, nonanimal origin, and relatively long storage time and stability. In the current study, blends of soy protein with other polymers (gelatin, alginate, pectin, polyvinyl alcohol, and polyethylene glycol) were developed and studied. The mechanical tensile properties of dense films were studied in order to select the best secondary polymer for porous three-dimensional structures. The porous soy-gelatin and soy-alginate structures were then studied for physical properties, degradation behavior, and microstructure. The results show that these blends can be assembled into porous three-dimensional structures by combining chemical crosslinking with freeze-drying. The soy-alginate blends are advantageous over soy-gelatin blends, demonstrated better stability, and degradation time along with controlled swelling behavior due to more effective crosslinking and higher water uptake than soy-gelatin blends. Water vapor transmission rate experiments showed that all porous blend structures were in the desired range for burn treatment [2000-2500 g/(m(2) d)] and can be controlled by the crosslinking process. We conclude that these novel porous three-dimensional structures have a high potential for use as scaffolds for tissue engineering, especially for skin regeneration applications. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1109-1120, 2016.

Electrospinning of poly (3-hydroxybutyric acid) and gelatin blended thin films: fabrication, characterization, and application in skin regeneration

A tissue engineering scaffold should mimic the structure and biological function of native extracellular matrix proteins. Electrospinning is a simple and versatile method to produce ultrathin fibers for tissue engineering. Blended submicron fibers of poly (3-hydroxybutyric acid) and gelatin were electrospun using 1,1,1,3,3,3 hexafluoro-2-propanol as solvent. Cross linking of fibers was achieved using glutaraldehyde, and the resultant fibers were tested and analyzed using scanning electron microscopy (SEM), differential scanning calorimetry, thermogravimetric analysis, X-ray diffraction, and Fourier transformed infrared spectroscopy (FTIR).The fibers were found to exhibit good tensile strength. Degradation studies were performed and analyzed using SEM and FTIR and proved the stability of fibers for tissue engineering applications. The fibrous scaffold supported the growth and rapid proliferation of human dermal fibroblasts and keratinocytes with normal morphology, thus proving its reliability in using it as a potential scaffold for skin regeneration.

Stem cell differentiation to epidermal lineages on electrospun nanofibrous substrates for skin tissue engineering

Bone marrow (BM) mesenchymal stem cells (MSC) capable of differentiating along the epidermal lineage on engineered nanofibrous scaffolds have great potential for bionanomaterial–cell transplantation therapy of skin wounds. MSC have been the focus of many tissue engineering studies, mainly because of their multipotential properties. We investigated the potential of human BM-derived MSC for epidermal cell differentiation in vitro on electrospun collagen/poly(l-lactic acid)–co-poly(3-caprolactone) (Coll/PLLCL) nanofibrous scaffolds. PLLCL and Coll/PLLCL nanofibrous scaffolds were fabricated by an electrospinning process and their chemical and mechanical characterization carried out by scanning electron microscopy (SEM), water contact angle determination, Fourier transform infrared spectroscopy, and tensile testing. The differentiation of MSC was carried out using epidermis inducing factors, including epidermal growth factor (EGF) and 1,25-dihydroxyvitamin D3, in culture medium. The proliferation of MSC evaluated by cell proliferation assay showed that the number of cells grown on Coll/PLLCL nanofibrous scaffolds was significantly higher than those on PLLCL scaffolds. The SEM results showed that MSC differentiated on Coll/PLLCL nanofibrous scaffolds showed a round keratinocyte morphology and expressed keratin 10, filaggrin and partial involucrin protein by immunofluorescent microscopic studies. The interaction of MSC and nanofibers was studied and we concluded that the electrospun Coll/PLLCL nanofibers could mimic the native skin extracellular matrix environment and are promising substrates for advanced skin tissue engineering. Our studies on the differentiation of MSC along the epidermal lineage on nanofibrous scaffolds suggest their potential application in skin regeneration without regional differentiation.

Fabrication and characterization of chitosan‐gelatin blend nanofibers for skin tissue engineering

Tissue engineering scaffolds produced by electrospinning feature a structural similarity to the natural extracellular matrix. Polymer blending is one of the effective methods to provide new and desirable biocomposites for tissue engineering applications. In this study chitosan was blended with gelatin and the effect of processing parameters of electrospinning and the solution properties of the polymer on the morphology of the fibers obtained were investigated. The morphology of the electrospun chitosan, gelatin and the chitosan-gelatin blend were characterized using a scanning electron microscope (SEM). The miscibility of the blend was determined using a SEM, and differential scanning calorimetry (DSC) Fourier transform Infrared spectrometer (FTIR). Further the tensile properties of the blend nanofibers were studied and compared with chitosan and gelatin fibers. In this study we have been able to electrospin defect-free chitosan, gelatin and chitosan-gelatin blend nanofibers with smooth morphology and diameter ranging from 120 to 200 nm, 100 to 150 nm, and 120-220 nm, respectively by optimizing the process and solution parameters. Chitosan and gelatin formed completely miscible blends as evidenced from DSC and FTIR measurements. The tensile strength of the chitosan-gelatin blend nanofibers (37.91 +/- 4.42 MPa) was significantly higher than the gelatin nanofibers (7.23 +/- 1.15 MPa) (p < 0.05) and comparable with that of normal human skin. Thus the novel chitosan-gelatin blend nanofiber system has potential application in skin regeneration.