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AUTHOR
Title
Utilization of patterned bioprinting for heterogeneous and physiologically representative reconstructed epidermal skin models
[Abstract]
Year
2021
Journal/Proceedings
Scientific Reports
Reftype
Madiedo-Podvrsan2021
DOI/URL
DOI
Groups
AbstractOrganotypic skin tissue models have decades of use for basic research applications, the treatment of burns, and for efficacy/safety evaluation studies. The complex and heterogeneous nature of native human skin however creates difficulties for the construction of physiologically comparable organotypic models. Within the present study, we utilized bioprinting technology for the controlled deposition of separate keratinocyte subpopulations to create a reconstructed epidermis with two distinct halves in a single insert, each comprised of a different keratinocyte sub-population, in order to better model heterogonous skin and reduce inter-sample variability. As an initial proof-of-concept, we created a patterned epidermal skin model using GPF positive and negative keratinocyte subpopulations, both printed into 2 halves of a reconstructed skin insert, demonstrating the feasibility of this approach. We then demonstrated the physiological relevance of this bioprinting technique by generating a heterogeneous model comprised of dual keratinocyte population with either normal or low filaggrin expression. The resultant model exhibited a well-organized epidermal structure with each half possessing the phenotypic characteristics of its constituent cells, indicative of a successful and stable tissue reconstruction. This patterned skin model aims to mimic the edge of lesions as seen in atopic dermatitis or ichthyosis vulgaris, while the use of two populations within a single insert allows for paired statistics in evaluation studies, likely increasing study statistical power and reducing the number of models required per study. This is the first report of human patterned epidermal model using a predefined bioprinted designs, and demonstrates the relevance of bioprinting to faithfully reproduce human skin microanatomy.
AUTHOR
Title
A 3D biofabricated cutaneous squamous cell carcinoma tissue model with multi-channel confocal microscopy imaging biomarkers to quantify antitumor effects of chemotherapeutics in tissue
[Abstract]
Year
2020
Journal/Proceedings
Oncotarget; Vol 11, No 27
Reftype
DOI/URL
URL
Groups
Abstract// James R. Browning 1 , Paige Derr 2 , Kristy Derr 2 , Nicole Doudican 3 , Sam Michael 2 , Samantha R. Lish 1 , Nicholas A. Taylor 3 , James G. Krueger 1 , Marc Ferrer 2 , John A. Carucci 3 and Daniel S. Gareau 1 1 Laboratory for Investigative Dermatology, The Rockefeller University, New York, New York, USA 2 National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland, USA 3 The Ronald O. Perelman Department of Dermatology, New York University School of Medicine, New York, New York, USA Correspondence to: Daniel S. Gareau, email: dgareau@rockefeller.edu Keywords: squamous cell carcinoma; screening; 3D printing; in vitro model; confocal microscopy Received: January 05, 2020 Accepted: April 03, 2020 Published: July 07, 2020 ABSTRACT Cutaneous squamous cell carcinoma (cSCC) causes approximately 10,000 deaths annually in the U. S. Current therapies are largely ineffective against metastatic and locally advanced cSCC. There is a need to identify novel, effective, and less toxic small molecule cSCC therapeutics. We developed a 3-dimensional bioprinted skin (3DBPS) model of cSCC tumors together with a microscopy assay to test chemotherapeutic effects in tissue. The full thickness SCC tissue model was validated using hematoxylin and eosin (H&E) and immunohistochemical histological staining, confocal microscopy, and cDNA microarray analysis. A nondestructive, 3D fluorescence confocal imaging assay with tdTomato-labeled A431 SCC and ZsGreen-labeled keratinocytes was developed to test efficacy and general toxicity of chemotherapeutics. Fluorescence-derived imaging biomarkers indicated that 50% of cancer cells were killed in the tissue after 1?M 5-Fluorouracil 48-hour treatment, compared to a baseline of 12% for untreated controls. The imaging biomarkers also showed that normal keratinocytes were less affected by treatment (11% killed) than the untreated tissue, which had no significant killing effect. Data showed that 5-Fluorouracil selectively killed cSCC cells more than keratinocytes. Our 3DBPS assay platform provides cellular-level measurement of cell viability and can be adapted to achieve nondestructive high-throughput screening (HTS) in bio-fabricated tissues.
AUTHOR
Title
Fully 3D Bioprinted Skin Equivalent Constructs with Validated Morphology and Barrier Function
[Abstract]
Year
2019
Journal/Proceedings
Tissue Engineering Part C: Methods
Reftype
DOI/URL
DOI
Groups
AbstractDevelopment of high throughput, reproducible, three-dimensional bioprinted skin equivalents that are morphologically and functionally comparable to native skin tissue is advancing research in skin diseases, and providing a physiologically relevant platform for the development of therapeutics, transplants for regenerative medicine, and testing of skin products like cosmetics. Current protocols for the production of engineered skin rafts are limited in their ability to control three dimensional geometry of the structure and contraction leading to variability of skin function between constructs. Here we describe a method for the biofabrication of skin equivalents that are fully bioprinted using an open market bioprinter, made with commercially available primary cells and natural hydrogels. The unique hydrogel formulation allows for the production of a human-like skin equivalent with minimal lateral tissue contraction in a multiwell plate format, thus making them suitable for high throughput bioprinting in a single print with fast print and relatively short incubation times. The morphology and barrier function of the fully three-dimensional bioprinted skin equivalents are validated by immunohistochemistry staining, optical coherence tomography, and permeation assays.
AUTHOR
Year
2018
Journal/Proceedings
Biofabrication
Reftype
DOI/URL
DOI
Groups
AbstractThree-dimensional (3D) pigmented human skin constructs have been fabricated using a 3D bioprinting approach. The 3D pigmented human skin constructs are obtained from using three different types of skin cells (keratinocytes, melanocytes and fibroblasts from three different skin donors) and they exhibit similar constitutive pigmentation (pale pigmentation) as the skin donors. A two-step drop-on-demand bioprinting strategy facilitates the deposition of cell droplets to emulate the epidermal melanin units (pre-defined patterning of keratinocytes and melanocytes at the desired positions) and manipulation of the microenvironment to fabricate 3D biomimetic hierarchical porous structures found in native skin tissue. The 3D bioprinted pigmented skin constructs are compared to the pigmented skin constructs fabricated by conventional a manual-casting approach; in-depth characterization of both the 3D pigmented skin constructs has indicated that the 3D bioprinted skin constructs have a higher degree of resemblance to native skin tissue in term of the presence of well-developed stratified epidermal layers and the presence of a continuous layer of basement membrane proteins as compared to the manually-cast samples. The 3D bioprinting approach facilitates the development of 3D in vitro pigmented human skin constructs for potential toxicology testing and fundamental cell biology research.
AUTHOR
Title
Combining bioengineered human skin with bioprinted cartilage for ear reconstruction
[Abstract]
Year
2023
Journal/Proceedings
Science Advances
Reftype
DOI/URL
DOI
Groups
AbstractMicrotia is a congenital disorder that manifests as a malformation of the external ear leading to psychosocial problems in affected children. Here, we present a tissue-engineered treatment approach based on a bioprinted autologous auricular cartilage construct (EarCartilage) combined with a bioengineered human pigmented and prevascularized dermo-epidermal skin substitute (EarSkin) tested in immunocompromised rats. We confirmed that human-engineered blood capillaries of EarSkin connected to the recipient’s vasculature within 1 week, enabling rapid blood perfusion and epidermal maturation. Bioengineered EarSkin displayed a stratified epidermis containing mature keratinocytes and melanocytes. The latter resided within the basal layer of the epidermis and efficiently restored the skin color. Further, in vivo tests demonstrated favorable mechanical stability of EarCartilage along with enhanced extracellular matrix deposition. In conclusion, EarCartilage combined with EarSkin represents a novel approach for the treatment of microtia with the potential to circumvent existing limitations and improve the aesthetic outcome of microtia reconstruction. A therapy for microtia patients utilizes bioprinted auricular cartilage combined with a tissue-engineered skin graft.
AUTHOR
Title
3D-Printed and Recombinant Spider Silk Particle Reinforced Collagen Composite Scaffolds for Soft Tissue Engineering
[Abstract]
Year
2024
Journal/Proceedings
Advanced Functional Materials
Reftype
DOI/URL
DOI
Groups
AbstractAbstract Collagen is one main component of the extracellular matrix (ECM) in natural tissues and is, therefore, well suited as a biomaterial for tissue engineering. In this study, a method is presented to 3D-bioprint collagen into a precipitation bath comprising recombinantly produced spider silk protein eADF4(C16) yielding a composite with excellent mechanical properties. The spider silk precipitation bath induced assembly of the collagen into fibrils, and subsequent addition of potassium phosphate buffer lead to the formation of silk particles and stabilization of the collagen fibrils. The produced collagen-silk composite scaffolds show an internal structure of homogeneously distributed and interacting collagen fibrils and spider silk particles with significantly better mechanical properties compared to plain collagen scaffolds. Further, enzymatic degradation assays of the scaffolds over a 7-day period show higher stability of the collagen-silk scaffolds compared to plain collagen scaffolds in the presence of wound proteases. Using the spider silk variant eADF4(C16-RGD) further increases compressive stress and elastic modulus compared to that of the unmodified variant. Finally, it is shown that the unique collagen-spider silk composite scaffolds comprising the cell-binding domains of collagen and the RGD sequence in the spider silk variant represent a promising material for soft tissue regeneration.
AUTHOR
Title
Integration of Melt Electrowritten Polymeric Scaffolds and Bioprinting for Epithelial Healing via Localized Periostin Delivery
[Abstract]
Year
2024
Journal/Proceedings
Reftype
DOI/URL
DOI
Groups
AbstractACS Macro Lett. 0.0:959-965
AUTHOR
Title
Bioprinting and plastic compression of large pigmented and vascularized human dermo-epidermal skin substitutes by means of a new robotic platform
[Abstract]
Year
2022
Journal/Proceedings
Journal of Tissue Engineering
Reftype
Groups
AbstractExtensive availability of engineered autologous dermo-epidermal skin substitutes (DESS) with functional and structural properties of normal human skin represents a goal for the treatment of large skin defects such as severe burns. Recently, a clinical phase I trial with this type of DESS was successfully completed, which included patients own keratinocytes and fibroblasts. Yet, two important features of natural skin were missing: pigmentation and vascularization. The first has important physiological and psychological implications for the patient, the second impacts survival and quality of the graft. Additionally, accurate reproduction of large amounts of patient’s skin in an automated way is essential for upscaling DESS production. Therefore, in the present study, we implemented a new robotic unit (called SkinFactory) for 3D bioprinting of pigmented and pre-vascularized DESS using normal human skin derived fibroblasts, blood- and lymphatic endothelial cells, keratinocytes, and melanocytes. We show the feasibility of our approach by demonstrating the viability of all the cells after printing in vitro, the integrity of the reconstituted capillary network in vivo after transplantation to immunodeficient rats and the anastomosis to the vascular plexus of the host. Our work has to be considered as a proof of concept in view of the implementation of an extended platform, which fully automatize the process of skin substitution: this would be a considerable improvement of the treatment of burn victims and patients with severe skin lesions based on patients own skin derived cells.
AUTHOR
Title
Exploring the Potential of Alginate-Gelatin-Diethylaminoethyl Cellulose-Fibrinogen based Bioink for 3D Bioprinting of Skin Tissue Constructs
[Abstract]
Year
2022
Journal/Proceedings
Carbohydrate Polymer Technologies and Applications
Reftype
Groups
AbstractDesigning printable bioinks for 3D bioprinting capable of supporting cellular viability with post-printing functionality remains challenging. Native ECM offers several physical, chemical, and biological cues that are difficult to restore using only a single component. Herein, we have optimized a multicomponent-based bioink formulation comprising alginate (ALG), gelatin (GEL), diethylaminoethyl cellulose (DCEL) and fibrinogen (FIB), termed as ALG-GEL-DCEL-FIB bioink for potential application in bioprinting and biofabrication of skin tissue equivalents. The designed formulation was extensively studied for its printability, physico-chemical, rheological, and biocompatibility properties. Excellent printability, shape fidelity and cell-laden tissue equivalent printing were established using the RegenHu 3D Discovery Bioprinter. The human primary fibroblast and keratinocyte-laden bioprinted constructs exhibited good cell viability. Long term culture of 4 weeks comprising 5 days of air-liquid-interphase followed by 21 days of submerged culture produced biomimetic tissue histology in the ALG-GEL-DCEL-FIB bioink printed constructs. Specific epidermal-dermal marker expressions proving functionality were evident in immunohistochemical, biochemical and gene expression analysis. The ALG-GEL-DCEL-FIB bioink may be explored further for potential biofabrication and therapeutic applications.
AUTHOR
Title
Impact of microstructure on cell behavior and tissue mechanics in collagen and dermal decellularized extra-cellular matrices
[Abstract]
Year
2022
Journal/Proceedings
Acta Biomaterialia
Reftype
Groups
AbstractSkin models are used for many applications such as research and development or grafting. Unfortunately, most lack a proper microenvironment producing poor mechanical properties and inaccurate extra-cellular matrix composition and organization. In this report we focused on mechanical properties, extra-cellular matrix organization and cell interactions in human skin samples reconstructed with pure collagen or dermal decellularized extra-cellular matrices (S-dECM) and compared them to native human skin. We found that Full-thickness S-dECM samples presented stiffness two times higher than collagen gel and similar to ex vivo human skin, and proved for the first time that keratinocytes also impact dermal mechanical properties. This was correlated with larger fibers in S-dECM matrices compared to collagen samples and with a differential expression of F-actin, vinculin and tenascin C between S-dECM and collagen samples. This is clear proof of the microenvironment's impact on cell behaviors and mechanical properties. Statement of significance In vitro skin models have been used for a long time for clinical applications or in vitro knowledge and evaluation studies. However, most lack a proper microenvironment producing a poor combination of mechanical properties and appropriate biological outcomes, partly due to inaccurate extra-cellular matrix (ECM) composition and organization. This can lead to limited predictivity and weakness of skin substitutes after grafting. This study shows, for the first time, the importance of a complex and rich microenvironment on cell behaviors, matrix macro- and micro-organization and mechanical properties. The increased composition and organization complexity of dermal skin decellularized extra-cellular matrix populated with differentiated cells produces in vitro skin models closer to native human skin physiology.