Design and Fabrication of Human Skin by Three-Dimensional Bioprinting
Vivian LeeGurtej SinghJohn P. TrasattiChris S. BjornssonXiawei XuThanh Nga TranSeung‐Schik YooGuohao DaiPankaj Karande
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Three-dimensional (3D) bioprinting, a flexible automated on-demand platform for the free-form fabrication of complex living architectures, is a novel approach for the design and engineering of human organs and tissues. Here, we demonstrate the potential of 3D bioprinting for tissue engineering using human skin as a prototypical example. Keratinocytes and fibroblasts were used as constituent cells to represent the epidermis and dermis, and collagen was used to represent the dermal matrix of the skin. Preliminary studies were conducted to optimize printing parameters for maximum cell viability as well as for the optimization of cell densities in the epidermis and dermis to mimic physiologically relevant attributes of human skin. Printed 3D constructs were cultured in submerged media conditions followed by exposure of the epidermal layer to the air-liquid interface to promote maturation and stratification. Histology and immunofluorescence characterization demonstrated that 3D printed skin tissue was morphologically and biologically representative of in vivo human skin tissue. In comparison with traditional methods for skin engineering, 3D bioprinting offers several advantages in terms of shape- and form retention, flexibility, reproducibility, and high culture throughput. It has a broad range of applications in transdermal and topical formulation discovery, dermal toxicity studies, and in designing autologous grafts for wound healing. The proof-of-concept studies presented here can be further extended for enhancing the complexity of the skin model via the incorporation of secondary and adnexal structures or the inclusion of diseased cells to serve as a model for studying the pathophysiology of skin diseases.Keywords:
3D bioprinting
Human skin
Skeletal muscle tissue engineering (TE) and adipose tissue engineering have undergone significant progress in recent years. This review focuses on the key findings in these areas, particularly highlighting the integration of 3D bioprinting techniques to overcome challenges and enhance tissue regeneration. In skeletal muscle TE, 3D bioprinting enables the precise replication of muscle architecture. This addresses the need for the parallel alignment of cells and proper innervation. Satellite cells (SCs) and mesenchymal stem cells (MSCs) have been utilized, along with co-cultivation strategies for vascularization and innervation. Therefore, various printing methods and materials, including decellularized extracellular matrix (dECM), have been explored. Similarly, in adipose tissue engineering, 3D bioprinting has been employed to overcome the challenge of vascularization; addressing this challenge is vital for graft survival. Decellularized adipose tissue and biomimetic scaffolds have been used as biological inks, along with adipose-derived stem cells (ADSCs), to enhance graft survival. The integration of dECM and alginate bioinks has demonstrated improved adipocyte maturation and differentiation. These findings highlight the potential of 3D bioprinting techniques in skeletal muscle and adipose tissue engineering. By integrating specific cell types, biomaterials, and printing methods, significant progress has been made in tissue regeneration. However, challenges such as fabricating larger constructs, translating findings to human models, and obtaining regulatory approvals for cellular therapies remain to be addressed. Nonetheless, these advancements underscore the transformative impact of 3D bioprinting in tissue engineering research and its potential for future clinical applications.
Decellularization
3D bioprinting
Regenerative Medicine
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Abstract Cardiac tissue engineering strategies are based on the development of functional models of heart muscle in vitro . Our research is focused on evaluating the feasibility of different tissue engineering platforms to support the formation of heart muscle. Our previous work was focused on developing three‐dimensional (3D) models of heart muscle using self‐organization strategies and biodegradable hydrogels. To build on this work, our current study describes a third tissue engineering platform using polymer‐based scaffolding technology to engineer functional heart muscle in vitro . Porous scaffolds were fabricated by solubilizing chitosan in dilute glacial acetic acid, transferring the solution to a mold, freezing the mold at −80°C followed by overnight lyophilization. The scaffolds were rehydrated in sodium hydroxide to neutralize the pH, sterilized in 70% ethanol and cellularized using primary cardiac myocytes. Several variables were studied: effect of polymer concentration and chitosan solution volume (i.e., scaffold thickness) on scaffold fabrication, effect of cell number and time in culture on active force generated by cardiomyocyte‐seeded scaffolds and the effect of lysozyme on scaffold degradation. Histology (hematoxylin and eosin) and contractility (active, baseline and specific force, electrical pacing) were evaluated for the cellularized constructs under different conditions. We found that a polymer concentration in the range 1.0–2.5% (w/v) was most suitable for scaffold fabrication while a scaffold thickness of 200 μm was optimal for cardiac cell functionality. Direct injection of the cells on the scaffold did not result in contractile constructs due to low cell retention. Fibrin gel was required to retain the cells within the constructs and resulted in the formation of contractile constructs. We found that lower cell seeding densities, in the range of 1–2 million cells, resulted in the formation of contractile heart muscle, termed s mart m aterial i ntegrated h eart m uscle (SMIHMs). Chitosan concentration of 1–2% (w/v) did not have a significant effect on the active twitch force of SMIHMs. We found that scaffold thickness was an important variable and only the thinnest scaffolds evaluated (200 μm) generated any measurable active twitch force upon electrical stimulation. The maximum active force for SMIHMs was found to be 439.5 μN while the maximum baseline force was found to be 2850 μN, obtained after 11 days in culture. Histological evaluation showed a fairly uniform cell distribution throughout the thickness of the scaffold. We found that lysozyme concentration had a profound effect on scaffold degradation with complete scaffold degradation being achieved in 2 h using a lysozyme concentration of 1 mg/mL. Slower degradation times (in the order of weeks) were achieved by decreasing the lysozyme concentration to 0.01 mg/mL. In this study, we provide a detailed description for the formation of contractile 3D heart muscle utilizing scaffold‐based methods. We demonstrate the effect of several variables on the formation and culture of SMIHMs. © 2007 Wiley Periodicals, Inc. J Biomed Mater Res, 2008
Contractility
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In this study, small intestinal submucosa (SIS) was prepared and engineered cardiac tissue was constructed using the SIS as scaffold. SIS was decellularized by mechanical and chemical treatment. Its mechanical capability and biocompatibility were evaluated, and then neonatal rat cardiomyocytes were seeded on SIS, thus the engineered cardiac tissue sheets were constructed in vitro. The results showed that the SIS was decellularized completely; its mechanical capability and biocompatibility were both satisfactory. The engineered cardiac tissue could beat spontaneously for a long time; it was consisted of layers of cardiomyocytes. In conclusion, the SIS with good capability was prepared successfully, and engineered cardiac tissue was constructed successfully based on the SIS scaffold.
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Biocompatibility
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Human skin phantoms are essential to enable fast, label-free, and reliable testing of pharmaceutical and cosmetic products. We report the characterisation of polyvinyl alcohol-based hydrogel phantoms along with in-vivo skin measurements of three volunteers from 0.2 to 1 THz. The results indicate that frequency-dependent properties of hydrogel phantoms are similar to human skin and show promising prospects of being utilised as a skin equivalent.
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Human skin
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The clinical need for alternatives to autologous vein and artery grafts for small-diameter vascular reconstruction have led researches to a tissue-engineering approach. Bioengineered vascular grafts provide a mechanically robust conduit for blood flow while implanted autologous cells remodel the construct to form a fully functional vessel [1]. A typical tissue-engineering approach involves fabricating a vascular scaffold from natural or synthetic materials, seeding the lumen of a vessel with endothelial cells (EC) and the vessel wall with smooth muscle cells or fibroblasts to mimic the functional properties of a native vessel. The cell-seeded vascular scaffold is then preconditioned in vitro using a pulsatile bioreactor to mimic in vivo conditions to enhance vessel maturation before implantation (Fig. 1).
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Engineering new tissues using cell transplantation may provide a valuable tool for reconstructive surgery applications. Chondrocyte transplantation in particular has been successfully used to engineer new tissue masses due to the low metabolic requirements of these cells. However, the engineered cartilaginous tissue is too rigid for many soft tissue applications. We propose that hybrid tissue engineered from chondrocytes and smooth muscle cells could reflect mechanical properties intermediate between these two cell types. In this study, rat aortic smooth muscle cells and pig auricular chondrocytes were co-cultured on polyglycolic acid fiber-based matrices to address this hypothesis. Mixed cell suspensions were seeded by agitating the polymer matrices and a cell suspension with an orbital shaker. After seeding, cell-polymer constructs were cultured in stirred bioreactors for 8 weeks. The cell density and extracellular matrix (collagen, elastin, and glycosaminoglycan) content of the engineered tissues were determined biochemically. After 8 weeks in culture, the hybrid tissue had a high cell density (5.8 × 108 cells/cm3), and elastin (519 μg/g wet tissue sample), collagen (272 μg/g wet tissue sample), and glycosaminoglycan (GAG; 10 μg/g wet tissue sample) content. Mechanical testing indicated the compressive modulus of the hybrid tissues after 8 weeks to be 40.8 ± 4.1 kPa and the equilibrium compressive modulus to be 8.4 ± 0.8 kPa. Thus, these hybrid tissues exhibited intermediate stiffness; they were less stiff than native cartilage but stiffer than native smooth muscle tissue. This tissue engineering approach may be useful to engineer tissues for a variety of reconstructive surgery applications.
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Muscle tissue
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Injectable composites combined with tissue-printing technology for improved bioengineered devices. The invisible engineering problem, the one often ignored, is the design of a readily implantable, precisely assembled cellular construct. Previous studies have consistently shown that composite tissue-engineered devises are readily implanted via minimally invasive means and, in the systems tested, produce minimal inflammation and fibrous encapsulation. Gels of optimal viscosity are able to maintain separation between the cellular scaffold and allow tissue growth. Studies with the cell/substrate printing system have shown that it is possible to define, in a controlled manner, spatial arrangement of cells within a gel substrate.
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Objective To explore the cell adhesion and growth on the scaffold under a certain magnitude of electric field,and to compare with the cells incubated using conventional static culture techniques.Methods A synthetic polycaprolactone (PCL) nanofiberous scaffold for incubation of the endothelial progenitor cellswas constructed and was sewn on the electrical stimulator.This entailed the allocation of the cells to four groups according to the voltage (0 V,1 V,2 V and 4 V).Haematoxylin-eosin staining,transmission electron microscopy and MTF technique were applied to determine the cell adhesion and viability.Results The electric fields improved cell adhesion on the PCL scaffolds,with the optimal condition being the stimulation of 50 Hz and 2 V/cm.Conclusion The pulsatile electric field may be a promising approach for vascular tissue engineering.
Key words:
Tissue engineering; Blood vessels ; Nanocomposites; Endothelial progenitor cell
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In vitro application of pulsatile electrical stimulation to neonatal rat cardiomyocytes cultured on polymer scaffolds has been shown to improve the functional assembly of cells into contractile engineered cardiac tissues. However, to date, the conditions of electrical stimulation have not been optimized. We have systematically varied the electrode material, amplitude and frequency of stimulation to determine the conditions that are optimal for cardiac tissue engineering. Carbon electrodes, exhibiting the highest charge-injection capacity and producing cardiac tissues with the best structural and contractile properties, were thus used in tissue engineering studies. Engineered cardiac tissues stimulated at 3 V/cm amplitude and 3 Hz frequency had the highest tissue density, the highest concentrations of cardiac troponin-I and connexin-43 and the best-developed contractile behaviour. These findings contribute to defining bioreactor design specifications and electrical stimulation regime for cardiac tissue engineering.
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