Using Three-Dimensional Bioprinting to Generate Realistic Models of Wound Healing

3D Bioprinting strategies

The skin is a complex organ with three distinct layers—epidermis, dermis, and hypodermis—each with specific functions. ³¹ The epidermis, the outermost layer, acts as a protective barrier and continuously renews itself through keratinocytes. ³² Below, the dermis comprises fibroblasts and fibrous proteins such as elastin and collagen, providing the skin with strength. The hypodermis, the deepest layer, contains adipocytes that support the ECM, secrete cytokines, and facilitate wound repair. ²⁷ Therefore, 3D bioprinting technologies, utilizing layer-by-layer deposition of biocompatible bioinks loaded with biologics such as skin cells, growth factors, and live biotherapeutic products (LBPs), have the potential to replicate the complex structure of native skin.

Extrusion-based bioprinting is a widely utilized technique, where bioinks are extruded through a nozzle to form continuous filaments that create the desired structure. ³³ The extrusion process is driven by pneumatic, mechanical (piston or screw), or hydrodynamic systems, making it suitable for high-viscosity bioinks. These bioinks maintain structural integrity, prevent cell sedimentation, and ensure uniform cell distribution. ³⁴ However, higher-viscosity bioinks require greater pressure during printing, increasing shear stress at the nozzle tip, which can reduce cell viability and affect protein signaling and cell phenotype maturation. ³⁵ Optimizing factors such as bioink viscosity, nozzle diameter, and extrusion pressure is essential to balance these effects. Approaches to predict cell viability based on shear stress have been explored. Zhang et al. summarized the main mathematical models that can be used to quantify the cell resistance to shear stress for optimizing bioprinting systems. ³⁶ In addition, shear stress was also demonstrated to affect printability; excessive stress can cause nozzle clogging and uneven extrusion, while insufficient stress can lead to poor flow properties and a compromised structure. ³⁷ To mitigate these effects, researchers can adjust printing parameters, such as extrusion speed and nozzle size, or modify bioink rheology with thickeners, crosslinking agents, or the use of sacrificial and support bioinks to enhance structural integrity without compromising cell health. ^{38 –40} According to Tian et al., some of the most important factors for achieving good cell viability in extrusion-based bioprinting are porosity, permeability, stiffness, and diffusivity. ⁴¹ However, because these parameters can inversely and simultaneously affect both the biological and mechanical performance of bioprinted tissues, it is crucial to strike a balance to achieve optimal biological functionality and structural integrity. ⁴¹ Bebiano et al. demonstrated that by adjusting crosslinker and polymer concentrations in the bioink, the stiffness of bioprinted skin constructs could be tailored, leading to site-specific differences in fibroblast morphology and spreading. ²⁹ The versatility of extrusion-based bioprinting also allows for the creation of complex, multilayered skin grafts. For instance, implantable skin grafts have been generated using multiple bioinks: one containing fibroblasts and endothelial cells to form the dermis, followed by another containing keratinocytes to form the epidermis. When implanted in immunodeficient mice, these grafts became perfused through both graft and host microvessels, indicating successful integration with the host tissue. ⁴²

Jetting-based bioprinting technology is classified into inkjet printing, microvalve printing, acoustic printing, laser-assisted printing, electrospinning, and electrohydrodynamic jet printing. These methods provide advantages such as high precision, rapid throughput, single-cell printing, and droplet-continuous capabilities for fabricating tissue constructs. ⁴³ The technique’s ability to eject singular bioink droplets ensures accuracy and facilitates the simultaneous deposition of multiple cell types and bioactive molecules. However, the heat- or piezoelectric-based energy required for droplet formation may cause cell damage or lysis, and the reliance on low-viscosity bioinks limits the diversity of biomaterials that can be utilized. ⁴⁴ Therefore, careful optimization is required to ensure cell viability and functionality. Ng et al. identified two primary factors influencing the quality of jetting-based bioprinting: droplet impact velocity and droplet volume. ⁴⁵ Their study, utilizing high-speed imaging, demonstrated that slower droplet impact velocities reduce splashing, thereby improving printing accuracy and enhancing cell viability, while higher velocities can induce mechanical stress on cells. For thermal inkjet bioprinters, the group emphasized that controlling droplet volume is critical to maintaining cell viability, particularly when printing duration must be limited to prevent excessive evaporation. ⁴⁵ The same group also investigated the influence of bioink properties on printing performance and cell health by playing with the concentration of polyvinylpyrrolidone. It was observed that higher bioink viscoelasticity was found to stabilize droplet filaments and help maintain the integrity of printed droplets. For accuracy and printability, the group observed that the increase in bioink viscosity was shown to facilitate droplet deposition on the substrate surface without splashing. ⁴⁶ In the context of skin models, Albanna et al. developed a mobile inkjet bioprinter capable of depositing autologous fibroblasts and keratinocytes in situ. This device featured integrated imaging technology that allowed for real-time management of wound sizes and topologies, enabling researchers to print directly over the skin wounds of mice and adjust the thickness to achieve uniform wound closure. This approach not only replicated the layers of skin but also demonstrated quicker healing and faster growth of normal skin due to its precise control. ⁴⁷ To further illustrate the effectiveness of inkjet-based bioprinting, Yanes et al. successfully deposited endothelial cells in specific locations to build capillary-like networks within a collagen scaffold containing fibroblasts and keratinocytes. This bilayer skin graft with printed microvasculature was implanted into mice, resulting in improved wound healing and the formation of neoskin closely resembling normal skin. ⁴⁸

Light-based bioprinting, also known as stereolithography or vat photopolymerization, utilizes light to cure or solidify photopolymerizable bioinks layer by layer, enabling the creation of highly detailed tissue structures. ⁴⁹ This technique excels in achieving extremely high resolution, making it ideal for fabricating intricate tissue architectures. However, the requirement for photopolymerizable bioinks restricts the range of usable materials. Moreover, exposure to light and photo initiators can negatively affect cell viability, necessitating careful selection and optimization of bioinks and printing parameters. ⁵⁰ As highlighted by Chartrain et al. and Ng et al., the success of vat photopolymerization depends on critical factors such as the type and wavelength of the light source, which must be compatible with the photo initiator to ensure efficient polymerization. The wavelength is particularly important as it influences the depth of light penetration and the resolution of the printed layers. Additionally, key printing parameters such as layer thickness, exposure time, and printing speed must be finely tuned to optimize the resolution, surface finish, and mechanical properties of the printed scaffold. ^30,51 To address the challenges associated with light-based bioprinting, Hafa et al. developed a system that simultaneously prints and images the constructs in real time. This innovation allows for continuous monitoring and adjustment during the printing process, significantly improving the speed, resolution, and viability of printed cells such as fibroblasts and keratinocytes. ⁵²

To harness the advantages of different bioprinting techniques, combining multiple approaches is becoming increasingly popular. In a recent study, Lee et al. demonstrated this by integrating extrusion and inkjet-based bioprinting to create a skin model composed of fibroblasts and keratinocytes. The process began with a pneumatic microextrusion technique to produce a stable dermal layer of collagen and primary human dermal fibroblasts. This was followed by piezoelectric inkjet printing of primary human epidermal keratinocytes to form an epidermal layer with high viability and uniformity. ⁵³ This approach underscores the importance of integrating multiple cell types, biomaterials, and technologies to accurately replicate the complex, layered architecture of human skin.

Current skin substitutes, such as skin grafts, fail to fully replicate the functionality of native skin. They are mechanically fragile, prone to contamination, expensive, and exhibit low engraftment rates. ⁵⁴ Additionally, many substitutes are acellular or lack diverse cell types, providing only temporary coverage. The absence of key fibrous proteins such as collagen and elastin, responsible for tensile strength, contributes to mechanical fragility. ⁸ Furthermore, the alkaline pH of the wound microenvironment promotes pathogen growth, impairs fibroblast activity, and delays wound remodeling and healing. ^16,18 These limitations can be addressed with 3D bioprinting, as this technology allows precise positioning of cells in specific patterns and orientations within layered constructs. Kim et al. demonstrated that densely populated fibroblasts align along ridges and elongate more compared with less dense regions. Their study highlighted that cell migration and morphology are influenced by the density and orientation of patterns, underscoring the importance of topography in cell–matrix interactions. ⁵⁵ This is particularly crucial in wound healing, where fibroblasts naturally migrate to open wounds. ⁵⁶ Therefore, 3D bioprinting holds the potential to create customized skin constructs with enhanced functionality and compatibility. By precisely placing bioactive molecules and cells at wound sites, this approach could promote efficient hemostasis recovery, limit the development of pathogenic bacteria, and enhance the wound-healing process. This would be achieved by facilitating cell growth, differentiation, migration, and proliferation around the area where the construct is located.

Biomaterials in wound healing

The appropriate selection of biomaterials directly influences the functionality and success of bioinks encapsulating living cells or bioactive factors. ⁵⁷ When selecting a biomaterial, several key factors must be considered, including mechanical strength, rheological properties for printability, and biological compatibility with skin tissue. Bioinks must also support cellular growth and proliferation without adversely affecting cell phenotypes. ²⁶ For skin bioprinting, a suitable bioink should mimic the natural ECM found in the skin, providing an optimal tissue microenvironment that enhances cellular processes such as migration, nutrient exchange, and tissue viability maintenance. ⁵⁸ Biomaterials can be designed not only as structural frameworks but also as delivery systems for bioactive molecules. ⁵⁹ They can be derived from natural sources such as collagen, fibrin, and hyaluronic acid (HA), which form hydrogels with inherent biocompatibility and biodegradability, making them ideal for in vivo applications. ⁶⁰ Alternatively, synthetic materials, engineered to mimic the mechanical properties of natural tissues, can offer greater customization. ⁶¹ Combining natural and synthetic materials allows bioinks to fulfill the three essential properties required for effective bioprinting: (i) biocompatibility with the host to maintain cell viability and reduce the risk of immune rejection, (ii) rheological properties for smooth deposition, and (iii) rapid crosslinking to retain 3D structure postbioprinting. ^62,63 This approach enables the creation of customizable platforms that provide an ideal microenvironment for skin cells, promoting their proliferation, migration, and differentiation while maintaining the mechanical strength, rigidity, and shape needed for wound healing. ⁶⁴

The importance of in vivo wound healing highlights the need for developing vascularized skin substitutes. Vascularization is critical for supplying oxygen and nutrients, removing metabolic waste, and facilitating angiogenesis—the formation of new blood vessels from existing ones—which is essential for the survival and integration of bioprinted skin. ⁶⁵ Biomaterials that support the incorporation of cells and growth factors to promote proper vascularization are, therefore, crucial. Dai et al. utilized biodegradable polyurethane–gelatin (4:1) to extrude human fibroblasts, endothelial progenitor cells (EPCs), and keratinocytes, creating bilayered dermoepidermal constructs. Incorporating EPCs into the dermal layer enhanced vascularization, as demonstrated by improved wound healing and microvessel formation when grafted onto skin wounds in immunodeficient mice. ⁶⁶

As we discussed for appropriate bioprinting process, the biomaterials that composed the bioink should be low viscous to protect the cells from shear stress and prevent clogging and solidify rapidly to maintain shape. Hydrogel-based bioinks are commonly used for skin regeneration and wound healing, with key features including their remarkable capacity to retain significant amounts of water, without losing structural integrity, due to hydrophilic components in their polymer structure. ^67,68 This unique attribute of hydrogels highlights their suitability for replicating ECM properties and promoting tissue regeneration.

The commonly used biomaterials for skin bioprinting are HA, chitosan, alginate, collagen, gelatin, and fibrin. ^26,69 HA derives from the ECM, offering inherent biocompatibility and supporting cell adhesion and tissue regeneration. ⁷⁰ HA oligomers (∼6 − 20 monomers) alleviate the inflammatory response, enhance re-epithelialization, and facilitate angiogenesis by stimulating growth factors such as transforming growth factor beta, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF), which recruit and activate keratinocytes, endothelial cells, and fibroblasts. ⁷¹ However, HA has poor mechanical strength and limited viscoelasticity, ⁷² requiring combination with other biomaterials to enhance its performance. For instance, Maiz-Fernandez et al. improved HA’s mechanical stability by integrating it with chitosan, forming a robust, self-stabilizing hydrogel. ⁷³

Chitosan, a linear polysaccharide derived from the deacetylation of chitin, is biocompatible, biodegradable, and antibacterial, making it effective against pathogens such as S. aureus while promoting hemostasis recovery of the rest of the microbiome. ^44,74 However, chitosan’s weak mechanical integrity, rapid dissociation under physiological conditions, and lack of cell-binding domains limit its utility in tissue engineering. ⁷⁵ Ng et al. addressed these issues by formulating a chitosan-gelatin polyelectrolyte hydrogel, which improved cell viability. ⁷⁶

Alginate, a polysaccharide-based hydrogel, is widely utilized due to its biocompatibility, low toxicity, and excellent printability. Moreover, alginate is utilized as a shear-thinning solution, wherein its viscosity decreases as the shear rate increases. This property protects cells from excessive shear stress during bioprinting. Once deposited, the bioink rapidly regains rigidity, allowing the scaffold to maintain its structural integrity. ³⁶

Collagen is a fundamental component of the skin ECM, offering structural integrity and mechanical support. ⁷⁷ Its porous structure and permeability enable efficient nutrient exchange and waste removal when used as a scaffold. ⁷⁸ However, as a standalone biomaterial ink, collagen lacks stability and is challenging to pattern. ³³ To address this, chemical crosslinking is employed, but it compromises biocompatibility and increases antigenicity. Using high-purity collagen, as demonstrated by Osidak et al., enhances mechanical properties in bioprinted structures. ⁷⁹

Gelatin, a biodegradable polymer derived from the partial hydrolysis of collagen, demonstrates versatility in forming hydrogels via a sol-gel transition at low temperatures, which supports the maintenance of scaffolds at specific printing temperatures. ^33,80 However, optimizing printing conditions, particularly temperature and viscosity, presents challenges when gelatin is used as a standalone bioink due to its inherent limitations. ⁸¹

Fibrin plays a pivotal role in promoting cell proliferation, differentiation, and vascularization. ⁸² Fibrinogen polymerizes into fibrin gel upon activation by thrombin. However, the weak mechanical properties of fibrin gel limit its ability to sustain 3D structures, requiring its combination with other biomaterials to address its mechanical deficiencies and enhance structural stability. ⁸³

Understanding the advantages and limitations of each biomaterial is crucial for developing hydrogel-based bioinks. Recognizing that some materials enhance mechanical properties, while others are vital for supporting cell growth, differentiation, and vascularization, simplifies the selection of complementary biomaterials. This approach enables the creation of an optimized skin scaffold that can be effectively bioprinted, offering structural strength, biocompatibility, and a supportive microenvironment. These scaffolds foster cell viability, proliferation, maturation, and differentiation, enabling vascularization, ECM production, and cell migration to wounds, ultimately improving the healing process and tissue repair. However, chronic wounds are often characterized by persistent inflammation, pH alkalinization, and oxygen limitation, which disrupt the homeostasis between the host and microbiome, prolonging the injury and making it more susceptible to pathogen colonization. Therefore, refining current biomaterials by incorporating bioactive molecules such as LBPs or growth factors can help protect the wound environment, preventing or delaying enzymatic, pH-mediated degradation, and supporting oxygenation. ⁸⁴ This approach would make them “smart hydrogels” that respond to stimuli from the wound microenvironment, triggering the release of bioactive molecules to develop an advanced therapeutic system. ⁸⁵

Smart hydrogels

The terminology of “smart hydrogels” refers to materials that can recognize natural or pathological cues in the tissue microenvironment and using those as passive triggers such as pH or enzymes. ⁸⁶ These hydrogels can monitor wounds in real time, deliver drugs tailored to different healing stages, and adjust their degradation rate for optimal bioactive molecule release ⁸⁷ (Fig. 4). In the following sections, we review several approaches for using intelligent hydrogels to enhance both wound healing and the effectiveness of skin microbiome management.

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Figure 4.

Smart hydrogels for wound-healing applications. Smart hydrogels are materials designed to adapt to external conditions by responding to natural or pathological signals, such as changes in pH or the presence of enzymes. These cues serve as passive triggers, allowing the hydrogels to adjust their properties, promote vascularization, or interact with surrounding enzymes. For instance, during the inflammatory phase of the healing process, hydrogels can react with matrix metalloproteinase-1 (MMP-1) to release bioactive molecules such as antibiotics, while, in the granulation phase, they respond to MMP-7 to release growth factors trapped within the biomaterials. Adapted from Olivia et al., 2020. Created in BioRender. Willerth, S. (2024) https://BioRender.com/k12h568.

Bioactive molecules

Approximately 1 million bacteria reside per square centimeter, amounting to over 10¹⁰ bacterial cells on human skin across the 1.8 m² surface of the skin. ¹⁰⁷ The skin’s surface comprises diverse microenvironments with distinct pH, moisture, temperature, and topography that regulate the delicate balance between the host and its resident microbes. Through immune tolerance, the skin can distinguish between harmless commensal microorganisms and harmful pathogens. TLRs may become desensitized by prolonged exposure to commensal microorganisms, either through reduced TLR expression on the cell surface or activation of inhibitors such as IL-1 receptor-associated kinase 3 and suppressor of cytokine signaling 1. ¹⁰⁸ Additionally, commensals can create an environment hostile to pathogens that invade, as seen with antagonism against S. aureus, by modulating the local pH. ¹⁰⁹

Among commensals, S. epidermidis has been shown to modulate the host’s innate immune response. Phenol-soluble modules produced by S. epidermidis selectively inhibit pathogens such as S. aureus and Group A Streptococcus while cooperating with host AMPs to enhance pathogen-killing. ¹⁹ Recent studies highlight that commensal-induced TLR signaling is essential for cell survival and repair during infections. ²⁰

LBPs are emerging as an innovative class of therapeutics, incorporating engineered living microorganisms such as yeast. The integration of LBPs into smart hydrogels represents a major advancement in wound-healing technologies. These microorganisms act as natural probiotics with anti-inflammatory and immunomodulatory properties, degrade toxins, and enhance the overall healing process. ¹¹⁰

TAKE HOME MESSAGES

Inappropriate wound management often causes acute injuries to progress into chronic wounds, which may require weeks or months for recovery.

Wound healing involves four phases: homeostasis, inflammation, proliferation, and remodeling. Chronic wounds remain “stuck” in the inflammatory phase, delaying recovery.

The microenvironment of a chronic wound is characterized by high levels of pro-inflammatory macrophages, overexpression of inflammatory mediators, and increased matrix metalloproteinase (MMP) activity, which degrades the extracellular matrix essential for cell attachment, communication, migration, and proliferation.

The skin’s microbiome, such as S. epidermidis, coexists in a mutualistic symbiotic relationship with the host. It suppresses excessive inflammation and promotes the release of antimicrobial peptides by keratinocytes, enhancing the skin’s defense against opportunistic pathogens like S. aureus.

Scaffolds created using 3D bioprinting technology must exhibit key biomaterial properties: mechanical strength mimicking the extracellular matrix, biodegradability, non-toxicity, immune compatibility, and good rheological properties to ensure smooth printing without causing shear stress on cells or deformation of the construct.

“Smart hydrogels” incorporate bioactive molecules within biomaterials that respond to stimuli from the wound microenvironment, such as pH or enzymes. These stimuli act as passive triggers to release drugs and adjust the hydrogel’s degradation rate for optimal bioactive molecule delivery across different healing stages.

Angiogenesis, the formation of new blood vessels from existing ones, is crucial for promoting tissue vascularization.

The skin hosts approximately 1 million bacteria per square centimeter. Through immune tolerance, it distinguishes between harmless commensal microorganisms and harmful pathogens.

LBPs (Live Biotherapeutic Products) are bioactive molecules with probiotic, anti-inflammatory, and immunomodulatory properties that degrade toxins, reducing inflammation when incorporated into smart hydrogels for chronic wound healing.

Restoring the balance between the microbiome and host homeostasis is crucial for regulating microbial invasion, controlling bacterial proliferation, and preventing excessive inflammation.

Etter et al. demonstrated the potential of bioprinting by encapsulating yeast within a GelMA hydrogel patch to assess drug delivery parameters and nutrient availability, allowing microbial growth and sustained protein secretion. ¹¹¹ Similarly, Schaffner et al. bioprinted hydrogels immobilizing P. putida and A. xylinum, which degraded pollutants and produced medically relevant bacterial cellulose. ¹¹² These studies suggest the possibility of incorporating microbiota into hydrogels to restore homeostasis, thereby reducing the likelihood of dysbiosis, inefficient skin cell proliferation, and opportunistic infections during the wound-healing process.

This highlights the critical importance of the synergistic interaction between the microbiome and the host in restoring homeostasis. Such balance is essential to regulate microbial invasion and bacterial proliferation, preventing excessive inflammatory responses. By creating an environment that naturally regulates bacterial behavior—similar to healthy skin—this approach paves the way for biotherapeutic treatments with smart hydrogels that constitute the 3D-bioprinted dermal substitutes (Fig. 5).

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Figure 5.

Smart bioinks with live biotherapeutic products. The skin’s diverse microenvironments host over 10¹⁰ bacterial cells, maintaining a delicate balance between commensal microorganisms and pathogens through immune tolerance mechanisms. Commensals like Staphylococcus epidermidis modulate the host’s immune response by producing phenol-soluble modulins that inhibit pathogens such as Staphylococcus aureus while cooperating with antimicrobial peptides to enhance pathogen elimination. The integration of live biotherapeutic products into smart hydrogels and bioinks offers innovative wound-healing strategies, enabling immune modulation, toxin degradation, and enhanced healing. Bioprinted hydrogels incorporating engineered microorganisms further advance this approach by restoring homeostasis, promoting efficient skin cell regeneration, and preventing infections. Created in BioRender. Willerth, S. (2024) https://BioRender.com/k12h568.