Skin Tissue Engineering Research Articles

Cancer Spheroids Embedded in Tissue-Engineered Skin Substitutes: A New Method to Study Tumorigenicity In Vivo.

Tumorigenic assays are used during a clinical translation to detect the transformation potential of cell-based therapies. One of these in vivo assays is based on the separate injection of each cell type to be used in the clinical trial. However, the injection method requires many animals and several months to obtain useful results. In previous studies, we showed the potential of tissue-engineered skin substitutes (TESs) as a model for normal skin in which cancer cells can be included in vitro. Herein, we showed a new method to study tumorigenicity, using cancer spheroids that were embedded in TESs (cTES) and grafted onto athymic mice, and compared it with the commonly used cell injection assay. Tumors developed in both models, cancer cell injection and cTES grafting, but metastases were not detected at the time of sacrifice. Interestingly, the rate of tumor development was faster in cTESs than with the injection method. In conclusion, grafting TESs is a sensitive method to detect tumor cell growth with and could be developed as an alternative test for tumorigenicity.

Bulk Modification with Inorganic Particles and Immobilization of Extracellular Vesicles onto PDO Composite for Facial Rejuvenation.

The skin, a vital organ protecting against microorganisms and dehydration, undergoes structural decline with aging, leading to visible issues such as wrinkles and sagging. Reduced blood vessels exacerbate vulnerability, hindering optimal cellular function and compromising skin health. Polydioxanone (PDO) biomaterials address aging concerns but produce acidic byproducts, causing inflammation. Inorganic particles and nitric oxide (NO) play crucial roles in inhibiting inflammation and promoting skin regeneration. Stem cell-derived extracellular vesicles (EVs) contribute to intercellular communication, offering the potential to enhance cell functions. The study proposes a method to enhance PDO-based medical devices by incorporating inorganic particles and immobilizing EVs, focusing on facial rejuvenation, anti-inflammatory response, collagen formation, and angiogenesis. PDO composites with inorganic particles such as magnesium hydroxide (MH) and zinc oxide (ZO) were prepared and followed by EV immobilization. Comprehensive characterization included biocompatibility, anti-inflammation, collagen formation ability, and angiogenesis ability. Bulk-modified PDO composites demonstrated even dispersion of inorganic particles, pH neutralization, and enhanced biocompatibility. EVs immobilized on the composite surface exhibited spherical morphology. Inflammation-related gene expressions decreased, emphasizing anti-inflammatory effects. Collagen-related gene and protein expressions increased, showcasing collagen formation ability. In addition, angiogenic capabilities were notably improved, indicating potential for skin rejuvenation. The study successfully developed and characterized PDO composites with inorganic particles and EVs, demonstrating promising attributes for medical applications. These composites exhibit biocompatibility, anti-inflammatory properties, collagen formation ability, and angiogenic potential, suggesting their utility in skin rejuvenation and tissue engineering. Further research and clinical validation are essential.

Recent advances in fabrication of dECM-based composite materials for skin tissue engineering.

Chronic wound management is an intractable medical and social problem, affecting the health of millions worldwide. Decellularized extracellular matrix (dECM)-based materials possess remarkable biological properties for tissue regeneration, which have been used as commercial products for skin regeneration in clinics. However, the complex external environment and the longer chronic wound-healing process hinder the application of pure dECM materials. dECM-based composite materials are constructed to promote the healing process of different wounds, showing noteworthy functions, such as anti-microbial activity and suitable degradability. Moreover, fabrication technologies for designing wound dressings with various forms have expanded the application of dECM-based composite materials. This review provides a summary of the recent fabrication technologies for building dECM-based composite materials, highlighting advances in dECM-based molded hydrogels, electrospun fibers, and bio-printed scaffolds in managing wounds. The associated challenges and prospects in the clinical application of dECM-based composite materials for wound healing are finally discussed.

Bioactive self-assembling silk fibroin–sericin films for skin tissue engineering

The quest for an ideal wound dressing material has been a strong motivation for researchers to explore novel biomaterials for this purpose. Such explorations have led to the extensive use of silk fibroin (SF) as a suitable polymer for several applications over the years. Unfortunately, another major silk protein—sericin has not received its due attention yet in spite of having favorable biological properties. In this study, we report an approach of blending SF and silk sericin (SS) without the usage of chemical crosslinkers is made possible by the usage of formic acid which evaporates to induce β-sheets formation to form cytocompatible films. Raman spectroscopy confirms the presence of SF/SS components in blend and formation of β-sheet in films. In situ, gelation kinetics studies were conducted to understand the change in gelation properties with addition of sericin into SF. Methyl thiazolyl tetrazolium and live/dead assays were performed to study cellular attachment, viability and proliferation on SF/SS films. The antibacterial properties of SF/SS films were tested using Gram-negative and Gram-positive bacteria. The re-structured SF/SS films were stable, transparent, show good mechanical properties, antibacterial activity and cytocompatibility, therefore can serve as suitable biomaterial candidates for skin regeneration applications.

Rapid innervation and physiological epidermal regeneration by bioengineered dermis implanted in mouse

Tissue-engineered skin substitutes are promising tools to cover large and deep skin defects. However, the lack of a synergic and fast regeneration of the vascular network, nerves, and skin appendages limits complete skin healing and impairs functional recovery. It has been highlighted that an ideal skin substitute should mimic the structure of the native tissue to enhance clinical effectiveness. Here, we produced a pre-vascularized dermis (PVD) comprised of fibroblasts embedded in their own extracellular matrix (ECM) and a capillary-like network. Upon implantation in a mouse full-thickness skin defect model, we observed a very early innervation of the graft in 2 weeks. In addition, mouse capillaries and complete epithelialization were detectable as early as 1 week after implantation and, skin appendages developed in 2 weeks. These anatomical features underlie the interaction with the skin nerves, thus providing a further cue for reinnervation guidance. Further, the graft displays mechanical properties, collagen density, and assembly features very similar to the host tissue. Taken together our data show that the pre-existing ECM components of the PVD, physiologically organized and assembled similarly to the native tissue, support a rapid regeneration of dermal tissue. Therefore, our results suggest a promising potential for PVD in skin regeneration.

Preparation and characterization of a jelly fig (Ficus awkeotsang Makino) polysaccharide-based bioactive 3D scaffold for improved vascularization and skin tissue engineering applications

Jelly fig polysaccharides (JFP) were extracted from Ficus awkeotsang Makino achenes. The yield of JFP was approximately 10–15 %. FT-IR spectrum of the extracted JFP confirmed that it was made of low methoxyl pectin (LMP). 3D scaffolds of JFP (JFP scaffold) were fabricated using ionic crosslinking of 2 % (w/v) JFP solution with Ca2+ ions and freeze-drying. The JFP scaffold showed 73.46 ± 1.97 % porosity and a 12-fold swelling capacity. The porous morphology was also observed in SEM micrographs. JFP scaffolds were completely degraded in 14 days when incubated in 1 mg/mL lysozyme solution, compared to the 50 % degradation observed in PBS alone. The antioxidant activity of the JFP and JFP scaffold was approximately 40 %. The hemolytic assay of the JFP scaffold showed <5 % (3.0 ± 0.4) RBC lysis. The cytocompatibility of the JFP scaffold was evaluated using L929 mouse fibroblasts and human dermal fibroblasts (HDF). The in vitro studies using L929 cells showed that the JFP scaffold is cytocompatible. HDF cells cultured in the presence of JFP scaffolds show a higher fold cell viability, proliferation, and migration. Collagen expression and deposition were also studied, and no significant changes occurred with JFP scaffold treatment. In vivo CAM assay showed an increase in the number and thickness of blood vessels by 1.185-fold and 1.19-fold, respectively. These results confirm the angiogenic property of the JFP scaffold. These biocompatible and bioactive properties of the JFP scaffold could be beneficial for tissue engineering and regenerative medicine applications.

Preparation, design, and characterization of an electrospun polyurethane/calcium chloride nanocomposite scaffold with improved properties for skin tissue regeneration

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.

Biocompatible recombinant type III collagen enhancing skin repair and anti-wrinkle effects.

Treating sunburn and other UV-induced skin damage issues remains a significant challenge in the field of dermatology. In this study, we synthesized a highly bioactive recombinant type III collagen (rCol III) to accelerate the healing of UV-damaged skin. The high-purity rCol III demonstrated excellent biocompatibility and bioactivity, significantly promoting the adhesion, proliferation, and migration of HFF-1 cells. In a mouse UV-damage model, Combo evaluations demonstrated that rCol III contributed to restore transepidermal water loss (TEWL) values of UV-damaged skin to normal levels. Histological analysis further confirmed that rCol III substantially accelerated skin repair by enhancing collagen regeneration. Additionally, rCol III facilitated the regeneration of zebrafish tail fin tissue and alleviated shrinkage caused by excessive UV exposure. The biocompatible and bioactive rCol III offers a novel strategy for treating UV-induced skin damage, holding immense potential for applications in skin tissue engineering.

Development of citric acid-based biomaterials for biomedical applications.

The development of bioactive materials with controllable preparation is of great significance for biomedical engineering. Citric acid-based biomaterials are one of the few bioactive materials with many advantages such as simple synthesis, controllable structure, biocompatibility, biomimetic viscoelastic mechanical behavior, controllable biodegradability, and further functionalization. In this paper, we review the development of multifunctional citrate-based biomaterials for biomedical applications, and summarize their multifunctional properties in terms of physical, chemical, and biological aspects, and finally the applications of citrate-based biomaterials in biomedical engineering, including bone tissue engineering, skin tissue engineering, drug/cell delivery, vascular and neural tissue engineering, and bioimaging.

A sustainable and self-healable silk fibroin nanocomposite with antibacterial and drug eluting properties for 3D printed wound dressings.

The development of self-healable and 3D printable hydrogels with decent biocompatibility, mechanical durability, adhesiveness to tissues, and antibacterial activity is of great importance for wound healing applications. In this study, we present a sustainable and environmentally friendly composite hydrogel consisting of silk fibroin (SF), oxidized salep (OS), and kappa carrageenan nanoparticles (NPs) for efficient wound care. The injectable nanocomposite hydrogel is highly stretchable and exhibits strong tissue adhesiveness and self-healing response through Schiff-base cross-linking between OS and SF. The tunable shear-thinning viscoelastic properties of the hydrogel facilitate 3D bioprinting with excellent shape adaptability (97.7 ± 1.1% recovery), enabling the fabrication of complex-shaped constructs. In vitro release kinetics of tetracycline (TC) encapsulated in kappa carrageenan NPs indicate a distinctive Korsmeyer-Peppas profile, including an initial burst release followed by a triphasic pattern controlled by the embedded NPs within the hydrogel matrix. The composite hydrogel shows a remarkable broad-spectrum antibacterial activity with substantial zones of inhibition against S. aureus (34.00 ± 1.00 mm) and E. coli (27.60 ± 2.08 mm) after 24 h of incubation at 37 °C. The addition of TC further enhances the zones of inhibition by approximately 45% for S. aureus and 27% for E. coli. The control group without kappa NP incorporation shows no zone of inhibition, underscoring the critical role of the nanoparticles in imparting antibacterial activity to the hydrogel. Cytocompatibility assays show the high viability of fibroblast (L929) cells (>90%) in vitro. In vivo biocompatibility studies through subcutaneous implantation also do not show malignancy, infection, abscess, necrosis, epidermal or dermal modifications, or inflammation of the wounds after 14 days post-injection. H&E staining shows that the biodegradation of the developed hydrogel facilitates the growth of non-inflammatory cells, leading to the substitution of the injected hydrogel with autologous tissue. The detailed analyses affirm that the multifunctional injectable hydrogel with self-healing and antibacterial properties has high potential for wound healing and skin tissue engineering.

Nanocellulose-based hydrogels as versatile materials with interesting functional properties for tissue engineering applications.

Tissue engineering has emerged as a remarkable field aiming to restore or replace damaged tissues through the use of biomimetic constructs. Among the diverse materials investigated for this purpose, nanocellulose-based hydrogels have garnered attention due to their intriguing biocompatibility, tunable mechanical properties, and sustainability. Over the past few years, numerous research works have been published focusing on the successful use of nanocellulose-based hydrogels as artificial extracellular matrices for regenerating various types of tissues. The review emphasizes the importance of tissue engineering, highlighting hydrogels as biomimetic scaffolds, and specifically focuses on the role of nanocellulose in composites that mimic the structures, properties, and functions of the native extracellular matrix for regenerating damaged tissues. It also summarizes the types of nanocellulose, as well as their structural, mechanical, and biological properties, and their contributions to enhancing the properties and characteristics of functional hydrogels for tissue engineering of skin, bone, cartilage, heart, nerves and blood vessels. Additionally, recent advancements in the application of nanocellulose-based hydrogels for tissue engineering have been evaluated and documented. The review also addresses the challenges encountered in their fabrication while exploring the potential future prospects of these hydrogel matrices for biomedical applications.

Development of Machine Learning Regression Models for Predicting the Performance of Nanofibrous Scaffolds for Skin Tissue Engineering

Development of Machine Learning Regression Models for Predicting the Performance of Nanofibrous Scaffolds for Skin Tissue Engineering

FRESH 3D Bioprinting of Alginate – Cellulose – Gelatin Constructs for Soft Tissue Biofabrication

The fabrication of three-dimensional (3D) bioprinted free-standing, low viscous, cell-laden hydrogels with good resolution, low cytotoxicity, and mechanical properties, comparable to native soft tissues, is a current challenge in tissue engineering. Recently, a new syringe extrusion approach, called Freeform Reversible Embedding of Suspended Hydrogels (FRESH), has been introduced to enhance 3D-bioprinting of soft hydrogels. Printing is conducted with the material embedded in a thermo-reversible gelatin bath, which acts as supporting material and can also initiate in-situ crosslinking when proper crosslinker agents are added. This work is the first to develop a 3D FRESH printable, low-cost, polymeric hydrogel composed of sodium alginate (SA), carboxymethylcellulose (CMC), and gelatin. A factorial design is investigated to identify best printing parameters. Printability Pr and CAD fidelity indexes are defined as responses of interest against scaffold resolution and shape fidelity. Results show a free-standing scaffold with a printability index, Pr ~ 1, a compression Young’s Modulus of about 4 kPa, and a cellular ATP content of stromal cells up to 50% compared to a 2D cell culture after 14 days. These promising results demonstrate the potential of such hydrogel to print soft constructs, applicable for neural or skin tissue engineering applications.

Three-dimensional structured PLCL/ADM bioactive aerogel for rapid repair of full-thickness skin defects.

The failure to treat deep skin wounds can result in significant complications, and the limitations of current clinical treatments highlight the pressing need for the development of new deep wound healing materials. In this study, a series of three-dimensional structured PLCL/ADM composite aerogels were fabricated by electrospinning and subsequently characterized for their microstructure, compression mechanics, exudate absorption, and hemostatic properties. Additionally, the growth of HSFs and HUVECs, which are involved in wound repair, was observed in the aerogels. The composite aerogel was subsequently employed in wound repair experiments on rat full-thickness skin with the objective of observing the wound healing rate and examining histological utilizing H&E, Masson, CD31, and COL-I staining. The findings indicated that the PLCL/ADM composite aerogel with a 10% concentration exhibited uniform pore size distribution, a good three-dimensional structure, and compression properties comparable to those of human skin, which could effectively absorb exudate and exert hemostatic effect. In vivo experiment results demonstrated that the aerogel exhibited superior efficacy to conventional oil-gauze overlay therapy and ADM aerogel in promoting wound healing and could facilitate rapid, high-quality in situ repair of deep wounds, thereby offering a novel approach for skin tissue engineering and clinical wound treatment.

Haloarchaeal poly[(3-hydroxybutyrate)-co-(3-hydroxyvalerate)] composite films reinforced with graphene nanoplatelets as a biomaterial for skin tissue engineering.

Halophilic archaea are an untapped source for a wide range of applications. This study explores the potential of a copolymer poly[(3-hydroxybutyrate)-co-(3-hydroxyvalerate) (PHBV), naturally synthesized by the halophilic archaeon, Halogeometricum borinquense E3, as a potential candidate for a tissue engineering biomaterial. Composites and blends from natural PHBV were fabricated with poly(l-lactic acid) (PLLA), poly(ε-caprolactone) (PCL) and graphene nanoplatelets (GNP) to enhance the properties of the material. This significantly improved the tensile strength of the blend to 4.729 MPa (359%). The reinforcement with 0.3% w/v of GNP further increased the tensile strength to 13.268 MPa (981%). Characterization of the films was done using ATR-FTIR, XRD, TGA, and SEM. The haloarchaeal PHBV exhibited the highest porosity ergo the highest swelling percentage while the PHBV/GNP showed the least. All the films showed good biocompatibility compared to tissue culture plastic (TCP). The viability of HaCaT cells and L929 fibroblast cells was maximum on the PHBV/PLLA/PCL blend albeit no significant change in the cell viability was observed in the graphene-reinforced nanocomposite. The films were also highly hemocompatible (<5% hemolysis).

A supervised machine learning approach to predict performance and aid decision making of biomaterials design for skin tissue engineering applications

A supervised machine learning approach to predict performance and aid decision making of biomaterials design for skin tissue engineering applications

Three-dimensional model of normal human dermal tissue using serial tissue sections.

Background: This study aims to construct a three-dimensional model of skin dermis utilizing continuous tissue sections, with the primary objective of obtaining anatomical structure data for normal human dermal tissues. Methods: Normal skin tissue specimens were acquired, paraffin-embedded, and subjected to HE staining. Panoramic images of skin sections were captured using a microscope. Tissue section images were aligned using the SIFT and StackReg image alignment methods, with analysis conducted using the OpenCV module. Mimics17 software facilitated the reconstruction of the skin dermal 3D model, enabling the calculation of dermal porosity and the void diameter. Results: Panoramic skin slices exhibited high-resolution differentiation of dermal fibers and cellular structures. Both SIFT and StackReg image alignment methods yielded similar results, although the SIFT method demonstrated greater robustness. Successful reconstruction of the three-dimensional dermal structure was achieved. Quantitative analysis revealed a dermal porosity of 18.96 ± 4.41% and an average pore diameter of 219.29 ± 34.27 μm. Interestingly, the porosity of the dermis exhibited a gradual increase from the papillary layer to the fourth layer, followed by a transient decrease and then a gradual increase. The distribution of the mean pore diameter mirrored the pattern observed in porosity distribution. Conclusion: Utilizing the continuous skin tissue slice reconstruction technique, this study successfully reconstructed a high-precision three-dimensional tissue structure of the skin. The quantitative analysis of dermal tissue porosity and average pore diameter provides a standardized dataset for the development of biomimetic tissue-engineered skin.

Coaxial electrospun PGS/PCL and PGS/PGS-PCL nanofibrous membrane containing platelet-rich plasma for skin tissue engineering

Wound healing will be enhanced using structures with therapeutic effects. This study fabricated a novel nanofibrous scaffold for skin tissue regeneration using a coaxial structure polyglycerol sebacate (PGS)/platelet-rich plasma (PRP) was embedded in the core and two different compositions were selected for the shell; in one group, polycaprolactone (PCL), and in the other group, PGS/PCL blend was used. The physical, mechanical behavior, drug delivery patterns, and cell response of scaffolds were evaluated. Results revealed that by adding PRP to the core and PGS to the shell, fiber diameters decreased to 260.8 ± 31.3 nm. It also decreased the water contact angle from 66° to 32°, that is ideal candidate for cell attachment. The drug release showed a burst release pattern in the first 30 min, followed by a continuous and slow release during the first day. Adding PGS to the shell decreased the elastic modulus, and its value reached about 500 kPa, which is near the skin elastic modulus and will lead to greater mechanical compatibility for cell proliferation. Particularly, the addition of PRP to the fiber structure enhanced the cell viability and cell adhesion with a suitable morphology. Based on the results, nanofibrous PGS-PRP/PGS-PCL dressing can enhance skin tissue regeneration.

Innervation of an Ultrasound-Mediated PVDF-TrFE Scaffold for Skin-Tissue Engineering.

In this work, electrospun polyvinylidene-trifluoroethylene (PVDF-TrFE) was utilized for its biocompatibility, mechanics, and piezoelectric properties to promote Schwann cell (SC) elongation and sensory neuron (SN) extension. PVDF-TrFE electrospun scaffolds were characterized over a variety of electrospinning parameters (1, 2, and 3 h aligned and unaligned electrospun fibers) to determine ideal thickness, porosity, and tensile strength for use as an engineered skin tissue. PVDF-TrFE was electrically activated through mechanical deformation using low-intensity pulsed ultrasound (LIPUS) waves as a non-invasive means to trigger piezoelectric properties of the scaffold and deliver electric potential to cells. Using this therapeutic modality, neurite integration in tissue-engineered skin substitutes (TESSs) was quantified including neurite alignment, elongation, and vertical perforation into PVDF-TrFE scaffolds. Results show LIPUS stimulation promoted cell alignment on aligned scaffolds. Further, stimulation significantly increased SC elongation and SN extension separately and in coculture on aligned scaffolds but significantly decreased elongation and extension on unaligned scaffolds. This was also seen in cell perforation depth analysis into scaffolds which indicated LIPUS enhanced perforation of SCs, SNs, and cocultures on scaffolds. Taken together, this work demonstrates the immense potential for non-invasive electric stimulation of an in vitro tissue-engineered-skin model.

Advanced Applications of Silk-Based Hydrogels for Tissue Engineering: A Short Review.

Silk has been consistently popular throughout human history due to its enigmatic properties. Today, it continues to be widely utilized as a polymer, having first been introduced to the textile industry. Furthermore, the health sector has also integrated silk. The Bombyx mori silk fibroin (SF) holds the record for being the most sustainable, functional, biocompatible, and easily produced type among all available SF sources. SF is a biopolymer approved by the FDA due to its high biocompatibility. It is versatile and can be used in various fields, as it is non-toxic and has no allergenic effects. Additionally, it enhances cell adhesion, adaptation, and proliferation. The use of SF has increased due to the rapid advancement in tissue engineering. This review comprises an introduction to SF and an assessment of the relevant literature using various methods and techniques to enhance the tissue engineering of SF-based hydrogels. Consequently, the function of SF in skin tissue engineering, wound repair, bone tissue engineering, cartilage tissue engineering, and drug delivery systems is therefore analysed. The potential future applications of this functional biopolymer for biomedical engineering are also explored.