Dermal Constructs Research Articles

Bioinspired and Photo-Clickable Thiol-Ene Bioinks for the Extrusion Bioprinting of Mechanically Tunable 3D Skin Models.

Bioinks play a fundamental role in skin bioprinting, dictating the printing fidelity, cell response, and function of bioprinted 3D constructs. However, the range of bioinks that support skin cells' function and aid in the bioprinting of 3D skin equivalents with tailorable properties and customized shapes is still limited. In this study, we describe a bioinspired design strategy for bioengineering double crosslinked pectin-based bioinks that recapitulate the mechanical properties and the presentation of cell-adhesive ligands and protease-sensitive domains of the dermal extracellular matrix, supporting the bioprinting of bilayer 3D skin models. Methacrylate-modified pectin was used as a base biomaterial enabling hydrogel formation via either chain-growth or step-growth photopolymerization and providing independent control over bioink rheology, as well as the mechanical and biochemical cues of cell environment. By tuning the concentrations of crosslinker and polymer in bioink formulation, dermal constructs were bioprinted with a physiologically relevant range of stiffnesses that resulted in strikingly site-specific differences in the morphology and spreading of dermal fibroblasts. We also demonstrated that the developed thiol-ene photo-clickable bioinks allow for the bioprinting of skin models of varying shapes that support dermis and epidermis reconstruction. Overall, the engineered bioinks expand the range of printable biomaterials for the extrusion bioprinting of 3D cell-laden hydrogels and provide a versatile platform to study the impact of material cues on cell fate, offering potential for in vitro skin modeling.

A heparin-functionalized bioink with sustained delivery of vascular endothelial growth factor for 3D bioprinting of prevascularized dermal constructs

Skin tissue engineering faces challenges due to the absence of vascular architecture, impeding the development of permanent skin replacements. To address this, a heparin-functionalized 3D-printed bioink (GH/HepMA) was formulated to enable sustained delivery of vascular endothelial growth factor (VEGF), comprising 0.3 % (w/v) hyaluronic acid (HA), 10 % (w/v) gelatin methacrylate (GelMA), and 0.5 % (w/v) heparin methacrylate (HepMA). The bioink was then used to print dermal constructs with angiogenic functions, including fibroblast networks and human umbilical vein endothelial cell (HUVEC) networks. GH/HepMA, with its covalently cross-linked structure, exhibits enhanced mechanical properties and heparin stability, allowing for a 21-day sustained delivery of VEGF. Cytocompatibility experiments showed that the GH/HepMA bioink supported fibroblast proliferation and promoted collagen I production. With VEGF present, the GH/HepMA bioink promoted HUVEC proliferation, migration, as well as the formation of a richer capillary-like network. Furthermore, HA within the GH/HepMA bioink enhanced rheological properties and printability. Additionally, 3D-bioprinted dermal constructs showed significant deposition of collagen I and III and mature stable capillary-like structures along the axial direction. In summary, this study offers a promising approach for constructing biomimetic multicellular skin substitutes with angiogenesis-induced functions.

Investigation of the application of a Taylor-Couette bioreactor in the post-processing of bioprinted human dermal tissue

Over the past decade, 3D printing has been explored as a means of producing human skin mimics for applications such as cosmetics testing, drug screening and wound healing. When it comes to the post-processing step of bioprinting skin, choices in recent times have mainly been the static culture of constructs or the use of perfusion and rotatory bioreactors. In this study, we investigated the feasibility of the use of a Taylor-Couette bioreactor in the maturation of bioprinted dermal constructs. Here, a set of bioprinted dermal constructs were cultured in a Taylor-Couette bioreactor up to a period of 7 days and was simultaneously compared with statically cultured constructs. Evaluation of cellular viability using MTS Assay and LIVE/DEAD Assay showed the capability of the Taylor-Couette bioreactor for improving maturation of fibroblasts in the dermal constructs. Histology and immunostaining revealed no adverse effect of the dynamic culture environment on fibroblasts in the constructs. This study thus demonstrates the possibility of utilizing the Taylor-Couette bioreactor as an alternative means for the maturation of the dermal layer in the skin bioprinting pipeline.

Bioengineering the microanatomy of human skin.

Recreating the structure of human tissues in the laboratory is valuable for fundamental research, testing interventions, and reducing the use of animals. Critical to the use of such technology is the ability to produce tissue models that accurately reproduce the microanatomy of the native tissue. Current artificial cell‐based skin systems lack thorough characterisation, are not representative of human skin, and can show variation. In this study, we have developed a novel full thickness model of human skin comprised of epidermal and dermal compartments. Using an inert porous scaffold, we created a dermal construct using human fibroblasts that secrete their own extracellular matrix proteins, which avoids the use of animal‐derived materials. The dermal construct acts as a foundation upon which epidermal keratinocytes were seeded and differentiated into a stratified keratinised epithelium. In‐depth morphological analyses of the model demonstrated very close similarities with native human skin. Extensive immunostaining and electron microscopy analysis revealed ultrastructural details such as keratohyalin granules and lamellar bodies within the stratum granulosum, specialised junctional complexes, and the presence of a basal lamina. These features reflect the functional characteristics and barrier properties of the skin equivalent. Robustness and reproducibility of in vitro models are important attributes in experimental practice, and we demonstrate the consistency of the skin construct between different users. In summary, a new model of full thickness human skin has been developed that possesses microanatomical features reminiscent of native tissue. This skin model platform will be of significant interest to scientists researching the structure and function of human skin.

Validation of in vitro assays in three-dimensional human dermal constructs.

Three-dimensional cell culture systems are urgently needed for cytocompatibility testing of biomaterials. This work aimed at the development of three-dimensional in vitro dermal skin models and their optimization for cytocompatibility evaluation. Initially “murine in vitro dermal construct” based on L929 cells was generated, leading to the development of “human in vitro dermal construct” consisting of normal human dermal fibroblasts in rat tail tendon collagen type I. To assess the viability of the cells, different assays CellTiter-Blue®, RealTime-Glo™ MT, and CellTiter-Glo® (Promega) were evaluated to optimize the best-suited assay to the respective cell type and three-dimensional system. Z-stack imaging (Live/Dead and Phalloidin/DAPI-Promokine) was performed to visualize normal human dermal fibroblasts inside matrix revealing filopodia-like morphology and a uniform distribution of normal human dermal fibroblasts in matrix. CellTiter-Glo was found to be the optimal cell viability assay among those analyzed. CellTiter-Blue reagent affected the cell morphology of normal human dermal fibroblasts (unlike L929), suggesting an interference with cell biological activity, resulting in less reliable viability data. On the other hand, RealTime-Glo provided a linear signal only with a very low cell density, which made this assay unsuitable for this system. CellTiter-Glo adapted to three-dimensional dermal construct by optimizing the “shaking time” to enhance the reagent penetration and maximum adenosine triphosphate release, indicating 2.4 times higher viability value by shaking for 60 min than for 5 min. In addition, viability results showed that cells were viable inside the matrix. This model would be further advanced with more layers of skin to make a full thickness model.

Anti-proliferative and anti-migratory effects of hyperforin in 2D and 3D artificial constructs of human dermal fibroblasts – A new option for hypertrophic scar treatment?

Hyperforin (HYP), one of the main bioactive compounds in extracts of Hypericum perforatum, is a potential drug candidate for the treatment of skin diseases. Since extracts have proven to support wound healing, in the present study effects of HYP on human dermal fibroblasts (HDF) were evaluated in 2D and 3D in vitro dermal constructs. Viability and cytotoxicity assays as well as a live-dead cell staining were performed to test at which concentration HYP reduces viability and/or shows cytotoxicity. Furthermore a differentiation between cytotoxic, anti-proliferative and anti-migratory effects was done. For the latter purpose a 2D migration assay was performed. HDF-induced contraction of a 3D artificial dermal (AD) construct was determined at given HYP concentration. Induction of apoptosis was examined by determination of caspase 3/7 activities. HYP reduced viability of HDF down to 70% at concentrations of 5–10µM. This decrease was not due to cytotoxicity but to a reduction in proliferation as shown from both the proliferation assay and the cytotoxicity assay as well as from live-dead cell staining. The 2D migration assay showed that HYP reduced migration activity of HDF cells at a concentration of 10µM. At this concentration HYP also reduced the HDF-induced contraction of collagen gels as 3D AD constructs. Apoptotic effects of HYP were excluded performing a caspase 3/7 activity detecting assay. The results show for the first time that HYP may be rather a potential candidate for treatment of hypertrophic scars than promoting effects which are understood as important in wound healing.

Wound healing of different molecular weight of hyaluronan; in-vivo study

Recruitment of cells and mediators is altered during impaired wound healing, thereby delaying this process. To overcome this problem, the correlation of wound healing in older rats, and the impact of different molecular weight of hyaluronan without silver nanoparticles; (low-HA1), (High-HA2), (Medium- HA3) and with silver nanoparticles (High-HA4) is investigated. The superior HA were selected to be further investigated onto diabetic wounds. Our results pointed to a marked deficiency in wounds granulation in older rats, which was accompanied with impairment of healing process. In older rats group treated with HA2 or HA4, granulation and dermal construction were improved. Furthermore, the number of pathogenic bacteria on wounds was declined throughout the first 24h by HA2 and HA4. The wound size in HA4-treated older rats was significantly smaller than that in other HA1, HA2 or HA3-treated older ones. Also, diabetes impaired the level of inflammatory cytokine, in diabetic model. On contrary, HA4 was found to normalize the level of inflammatory cytokine, in the diabetic model. Furthermore, HA4 was found to recover all oxidative and toxicity markers in diabetic models. This data confirms the critical role of HA4 to improve granulation and inflammatory mediators in impaired older and diabetic rat wound healing.

Plasma-activated air mediates plasmid DNA delivery in vivo

Plasma-activated air (PAA) provides a noncontact DNA transfer platform. In the current study, PAA was used for the delivery of plasmid DNA in a 3D human skin model, as well as in vivo. Delivery of plasmid DNA encoding luciferase to recellularized dermal constructs was enhanced, resulting in a fourfold increase in luciferase expression over 120 hours compared to injection only (P < 0.05). Delivery of plasmid DNA encoding green fluorescent protein (GFP) was confirmed in the epidermal layers of the construct. In vivo experiments were performed in BALB/c mice, with skin as the delivery target. PAA exposure significantly enhanced luciferase expression levels 460-fold in exposed sites compared to levels obtained from the injection of plasmid DNA alone (P < 0.001). Expression levels were enhanced when the plasma reactor was positioned more distant from the injection site. Delivery of plasmid DNA encoding GFP to mouse skin was confirmed by immunostaining, where a 3-minute exposure at a 10 mm distance displayed delivery distribution deep within the dermal layers compared to an exposure at 3 mm where GFP expression was localized within the epidermis. Our findings suggest PAA-mediated delivery warrants further exploration as an alternative approach for DNA transfer for skin targets.

Biologic Augmentation of the Ulnar Collateral Ligament in the Elbow of a Professional Baseball Pitcher.

Tears of the ulnar collateral ligament (UCL) of the elbow are common injuries in overhead athletes. Although surgical reconstruction of the UCL has improved outcomes, not all athletes return to their previous level of competition and when this goal is achieved, the time required averages one to two years. Therefore, additional techniques are needed to further improve return to play and the rate of return to play in overhead athletes. A construct comprising a dermal allograft, platelet rich plasma (PRP), and mesenchymal stem cells (MSCs) has been shown to successfully improve healing in the rotator cuff. Given the promising provisional findings, we postulated that this construct could also improve healing if applied to the UCL. Therefore, the purpose of the present report was to examine the feasibility of utilizing a dermal allograft, PRP, and MSC construct to augment UCL reconstruction in a professional baseball pitcher. No complications were encountered. Although limited to minimal follow-up, the patient has demonstrated excellent progress and has returned to activity.

Experimental tissue engineering of fetal skin.

In some human fetuses undergoing prenatal spina bifida repair, the skin defect is too large for primary closure. The aim of this study was to engineer an autologous fetal skin analogue suitable for in utero skin reconstruction during spina bifida repair. Keratinocytes (KC) and fibroblasts (FB) isolated from skin biopsies of 90-day-old sheep fetuses were cultured. Thereafter, plastically compressed collagen hydrogels and fibrin gels containing FB were prepared. KC were seeded onto these dermal constructs and allowed to proliferate using different culture media. Constructs were analyzed histologically and by immunohistochemistry and compared to normal ovine fetal skin. Development of a stratified epidermis covering the entire surface of the collagen gel was observed. The number of KC layers and degree of organization was dependent on the cell culture media used. The collagen hydrogels exhibited a strong tendency to shrink after eight to ten days of culture in vitro. On fibrin gels, we did not observe the formation of a physiologically organized epidermis. Collagen-gel-based ovine fetal cell-derived skin analogues with near normal anatomy can be engineered in vitro and may be suitable for autologous fetal transplantation.

Feasibility of Dermal Substitute Construction on Hybrid Scaffold Made of Poly(&lt;I&gt;ε&lt;/I&gt;-caprolactone) and Bio Mimetic Fibrin Composite

Feasibility of Dermal Substitute Construction on Hybrid Scaffold Made of Poly(&lt;I&gt;ε&lt;/I&gt;-caprolactone) and Bio Mimetic Fibrin Composite

All-trans retinoic acid is an effective inhibitor of hyaluronate synthesis in a human dermal equivalent

All-trans retinoic acid (ATRA) is known to have beneficial effects on skin. It has been used extensively for the treatment of photodamaged skin. To assess the effects of all-trans retinoic acid on the dermis, specifically its effect on hyaluronate (hyaluronic acid, HA) synthesis and inhibition, tissue-engineered human dermal equivalents were prepared in the presence of varying concentrations of ATRA using the method of "self-assembly". A substantial extracellular matrix was formed at the end of the culture period. The extracellular matrices of these dermal constructs were characterized and compared to the construct prepared in the absence of ATRA (Normal). Inhibition of hyaluronate was observed in constructs prepared in the presence of varying concentrations of all-trans retinoic acid. Chondroitin sulfate synthesis was unaffected up to 1μM ATRA. Collagen synthesis was enhanced at lower concentrations of ATRA (250 and 500nM) and inhibited at higher concentrations of ATRA. Differential gene array experiments were performed comparing the construct grown in the presence of 500nM ATRA to one grown in the absence of ATRA, to obtain preliminary information regarding the gene(s) involved in HA inhibition using a GLYCOv4 gene chip. These preliminary experiments demonstrated the differential expression of 127 genes and suggest that down-regulation of five key enzymes in the HA biosynthetic pathway may be involved in this inhibitory process.

Sensory neurons accelerate reepithelialization through Substance P release in an innervated tissue‐engineered model of skin wound healing.

The keratinocytes are responsible for the restoration of the epidermal barrier through reepithelialization during the wound healing process in humans. The influence of sensory neurons on this mechanism is poorly understood. We tested whether sensory neurons influence reepithelialization via a secretion of the neuropeptide substance P (SP). We developed a new tissueengineered model of skin wound healing. It consists of a perforated tissue‐engineered skin made of keratinocytes expressing GFP and that was stacked on a dermis to reconstitute the wound bed. The dermal construct is endothelialized by human endothelial cells and innervated or not by mouse sensory neurons. The migration of keratinocytes was imaged and quantified with GFP fluorescence. Sensory neurons produce SP in the construct as shown with ELISA and immunofluorescence analysis. Keratinocytes were found to express the NK1 cell receptor for SP in the reconstructed epidermis. The rate of reephithelialization was increased in presence of sensory neurons and SP was shown to contribute to this effect using antagonist to NK1 and recombinant SP instead of neurons. We conclude that this new model is promising for the study of the influence of the peripheral nervous system on reepithelialization.Grant Funding Source: N/A

A tissue‐engineered human dermal construct utilizing fibroblasts and transforming growth factor β1 to promote elastogenesis

Numerous studies have shown that extracellular matrix (ECM)-based scaffolds are suitable for dermal constructs for the differentiation of various cell types in vitro and for constructive tissue remodeling after implantation in vivo. However, a shortcoming of these ECM materials is its limited elastogenesis. Elastic fibers constitute an essential component of mammalian connective tissue and the presence of elastic fibers is crucial for the proper function of the cardiovascular, pulmonary, and intestinal systems. Since it is still largely unknown how cells coordinate the molecular events of elastic-fiber assembly, understanding the ability to regenerate elastic fibers in tissues remains a significant challenge. For this reason, human neonatal dermal fibroblasts (HDFneo) were analyzed for their potential to serve as a cell culture model for elastic fiber assembly. Using optical technologies such as multiphoton laser-scanning microscopy (MPSLM) we demonstrate that HDFneo stimulated with transforming growth factor β1 (TGF-β1) are able to produce a distinct and complex elastic fiber system in vitro. As shown by the desmosine and isodesmosine content, crosslinked elastic fibers were formed within the 3D ECM-based scaffold. This tissue-engineered dermal construct may prove to be an effective template for the development of medicinal approaches in regenerative soft skin tissue reconstruction through TGF-β1 induction.

Morphogenesis of chimeric hair follicles in engineered skin substitutes with human keratinocytes and murine dermal papilla cells

Engineered skin substitutes (ESS) have been used successfully to treat life-threatening burns, but lack cutaneous appendages. To address this deficiency, dermal constructs were prepared using collagen-glycosaminoglycan scaffolds populated with murine dermal papilla cells expressing green fluorescent protein (mDPC-GFP), human dermal papilla cells (hDPC) and/or human fibroblasts (hF). Subsequently, human epidermal keratinocytes (hK) or hK genetically modified to overexpress stabilized β-catenin (hK') were used to prepare ESS epithelium. After 10days incubation at air-liquid interface, ESS were grafted to athymic mice and were evaluated for 6weeks. Neofollicles were observed in ESS containing mDPC-GFP, but not hDPC or hF, independent of whether or not the hK were genetically modified. Based on detection of GFP fluorescence, mDPC were localized to the dermal papillae of the well-defined follicular structures of grafted ESS. In addition, statistically significant increases in LEF1, WNT10A and WNT10B were found in ESS with neofollicles. These results demonstrate a model for generation of chimeric hair in ESS.

Tissue-engineered fetal dermal matrices

In the early to mid-gestation fetus, skin wounds heal with no scar formation and perfect restoration of dermal architecture. This phenomenon is intrinsic to fetal skin. The intrinsic phenotypic properties of the fetal fibroblast are believed to be "the effector of scarless repair". We sought to prepare dermal matrices with high similarity to the mid-gestation fetal dermis using the technology of "self-assembly" with fetal dermal cells of 18, 20, and 22wk gestation. Comparison of these dermal constructs to those prepared with neonatal dermal cells, adult skin, neonatal foreskin, and mid-gestation fetal skin demonstrates that these fetal dermal matrices bear marked morphological and biochemical resemblance to the mid-gestation fetal dermis. In order to shed further light on the genes involved in scarless wound healing, we conducted a differential gene array analysis of the neonatal and fetal dermal matrices. Using a gene chip (GLYCOv4 gene chip) of approximately 1,260 human genes, we observed differential expression of 67 genes. A number of fibrotic genes were observed to be downregulated and anti-fibrotic genes upregulated.

Characterization of chitosan-gelatin scaffolds for dermal tissue engineering

Porous scaffolds for dermal tissue engineering were fabricated by freeze-drying a mixture of chitosan and gelatin (CG) solutions. Different crosslinking agents including glutaraldehyde, 1-(3-dimethylaminopropyl)-3-ethyl-carbodimide hydrochloride (EDC), and genipin were used to crosslink the scaffolds and improve their biostability. The porous structure and mechanical properties were determined for the scaffolds. The proliferation of human fibroblasts in the scaffolds was analyzed. It was found that EDC crosslinked scaffolds had the greatest amount of cells after four days. EDC crosslinked CG scaffolds had tensile modulus in a dry state and compressive modulus in a wet state similar to commercial collagen wound dressing. They also showed appropriate pore size, high water absorption, and good dimensional stability during cell culture. When human fibroblasts were seeded on acellular porcine dermis (APD), acellular human dermis (AHD), and CG scaffolds for 3D cell culture, they were well-distributed in the centre of the CG scaffolds but stayed only on the superficial layer of APD or AHD after seven days. A gelatin-based bioglue was applied to the CG scaffolds where the keratinocytes were seeded to mimic epidermal structure. After 14 days, the bioglue degraded and keratinocytes grew to form monolayers on the scaffolds. This study showed that CG scaffolds crosslinked by EDC and seeded with human fibroblasts could serve as dermal constructs, while the bioglue coating seeded with keratinocytes could serve as an epidermal construct. Such a combination could help regenerate skin with integrated dermal and epidermal layers and a have potential use in tissue-engineered skin.

A preliminary investigation of the reinnervation and return of sensory function in burn patients treated with INTEGRA ®

Background Loss of sensory function in scar after burn is common, although the basis for this loss is not clear. Additionally, little is known about the effects of different treatment modalities on sensory function and neuroanatomical outcomes in burn patients. Here, we investigated the effects of the use of the INTEGRA ® dermal scaffold on neuroanatomy and sensory function in acute burn patients. Hypothesis and objectives We hypothesized that the use of artificial dermal templates would inhibit or reduce reinnervation after excision, since regrowth of nerves requires complex molecular interactions. Therefore the primary objective of this study was to identify whether there is regrowth of nerve fibres in the INTEGRA ® dermal scaffold. The secondary objective was to identify whether the INTEGRA ® dermal scaffold reduced nerve regrowth or limited sensory function outcomes in acute burn patients. Methods Five patients treated with INTEGRA ®, cultured epithelial autograft spray (prepared using ReCell ® (CEA)) and split skin graft (SSG) were assessed for sensory function in scar and uninjured contralateral control skin. Neuroanatomy of scar and control sites was assessed using immunohistochemistry for PGP9.5, CGRP and substance P neuronal markers. Nerve density and sensory function was also assessed in a comparative group ( n = 8) treated with CEA and SSG only. Results Neuroanatomy was not significantly different in the INTEGRA ® patients when compared to the CEA/SSG group only. The patients treated with INTEGRA ® had worse sensory function than those with CEA/SSG only. Conclusions Peripheral nerves do reinnervate the INTEGRA ® dermal scaffold. There is no statistically significant reduction in reinnervation observed when compared to a control group. It is possible that the use of artificial dermal constructs, while permissive for nerve regrowth, limit functionality when compared to nerves that regrow through dermal tissue. Further research to understand the causes of this, and into enhancing reinnervation in dermal scaffolds may improve sensory outcome in the most severely burned patients.

Hair Follicles Guide Nerve Migration In Vitro and In Vivo in Tissue-Engineered Skin

Hair Follicles Guide Nerve Migration In Vitro and In Vivo in Tissue-Engineered Skin

Tissue-Engineered Skin Preserving the Potential of Epithelial Cells to Differentiate into Hair After Grafting

The aim of this study was to evaluate whether tissue-engineered skin produced in vitro was able to sustain growth of hair follicles in vitro and after grafting. Different tissues were designed. Dissociated newborn mouse keratinocytes or newborn mouse hair buds (HBs) were added onto dermal constructs consisting of a tissue-engineered cell-derived matrix elaborated from either newborn mouse or adult human fibroblasts cultured with ascorbic acid. After 7-21 days of maturation at the air-liquid interface, no hair was noticed in vitro. Epidermal differentiation was observed in all tissue-engineered skin. However, human fibroblast-derived tissue-engineered dermis (hD) promoted a thicker epidermis than mouse fibroblast-derived tissue-engineered dermis (mD). In association with mD, HBs developed epithelial cyst-like inclusions presenting outer root sheath-like attributes. In contrast, epidermoid cyst-like inclusions lined by a stratified squamous epithelium were present in tissues composed of HBs and hD. After grafting, pilo-sebaceous units formed and hair grew in skin elaborated from HBs cultured 10-26 days submerged in culture medium in association with mD. However, the number of normal hair follicles decreased with longer culture time. This hair-forming capacity after grafting was not observed in tissues composed of hD overlaid with HBs. These results demonstrate that epithelial stem cells can be kept in vitro in a permissive tissue-engineered dermal environment without losing their potential to induce hair growth after grafting.