3D BioPrinting Skin: Guide

Human skin appears simple, flat, non-vascularized, superficial. But in fact, skin is the largest organ in our body and perhaps no less complex than our brain. Billions of dollars each year are spent on skincare, ranging from life-threatening conditions such as severe burn injuries, full-thickness skin wounds, diabetic ulcers, skin cancer, to annoying diseases like atopic dermatitis, psoriasis. Not to mention the much larger consumer-facing cosmetic skin products, which is projected to amount to 20 billion total revenue based on one report. In this article, we will explore the past, current status, and future of skin bioprinting based on our recent virtual event on the subject manner. 

Why do we need artificial skin? 

Bioprinting human skin could end animal testing – Regulatory force

In addition to the technological advancements in skin tissue engineering, 3D bioprinting, and our fundamental understanding of biology, a huge driving force behind developing bioprinted skin constructs or products is a trend towards eliminating animal testing.

In 2013, after more than two decades of efforts, the European Commission officially banned animal testing for cosmetic products. In the pharmaceutical industry, while currently, most drugs require pre-clinical animal testing, there is a growing sentiment against such practice not just due to concerns for animal cruelty, but also the efficacy of such treatment. Ninety percent of all drugs that passed pre-clinical phase animal testing cannot pass clinical testing in humans. Additionally, because of the genetic and biological differences between humans and animal models, some drugs that are toxic and ineffective in animal models may be so in humans. Parallel with the cosmetic industry, a similar trend is observed in the pharmaceutical industry in terms of developing animal testing alternatives.

3D tissue construct is one of these alternatives, offering a promising approach. 

Scientific and technological force

While 3D bioprinting (or 3D printing technology in general) is still in its infancy as an industry,

“It is a misconception that bioprinting is still only in the research lab, but in fact, bioprinting is at a stage where it can achieve scalable, reproducible, controllable printed products”, said Prof. Colin McGuckin’s during his presentation on CTIBIOTECH’s work in the space at our recent virtual event.  

Large addressable market – Economic force

In addition to the potentially formidable growing market of alternative testing tools replacing animal testing for the cosmetic and pharmaceutical industries, advanced wound care is also a sizable growing market driven by increasing spending on acute, chronic, surgical wounds, more patients affected by diseases and conditions affecting wound healing capabilities including diabetes, peripheral vascular disease, and a growing aging population in general. The global advanced wound care market is projected to reach USD 12.8 billion by 2026 from USD 9.4 billion in 2021, at a CAGR of 6.2% based on one market report. 

The 3D structures of the skin

Skin, while appearing flat to human eyes macroscopically, has complex 3D structures. Here is a quick overview of skin anatomy at a scale that matters to skin tissue engineering before we dig deeper into how to recreate it. 

Mammalian skin is composed of two primary layers: epidermis and dermis. Hypodermis or subcutaneous soft tissues, while not part of the skin technically, is also important to understand. 

Epidermis

The epidermis is the outermost layer of the skin, acting as a protective waterproof and pathogen-proof barrier over the body’s surface. The epidermal layer is a stratified squamous epithelium composed of proliferating basal and differentiated suprabasal epidermal keratinocytes. Human keratinocytes account for 95% of all cells of the epidermis. Other 5% of cells include melanocytes (cells that are responsible for skin color), Langerhan cells (part of the immune system), Merkle cells (cells responsible for touch). [Ref] Additionally, the epidermis contains no blood vessels, and the cells in the deeper layers are nourished by diffusion from blood capillaries extending to the upper layers of the dermis.

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Caption: A simplified illustration of skin anatomy (Source: Wikipedia)

Basement Membrane

The basement membrane is a thin fibrous layer that separates the epidermis from the dermis, controlling the traffic of cells and molecules between the two layers. It also acts as a reservoir for controlled release of a variety of cytokines and growth factors during remodeling or repair processes, [Ref]  

Dermis

The dermal layer is the thicker lower layer that contains skin appendages, acting as a cushion from stress and strain. The dermal structure provides tensile strength and elasticity to the skin through an extracellular matrix composed of collagen fibrils, microfibrils, and elastic fibers in hyaluronan and proteoglycan. Depending on where the skin is located, it also contains nerve endings that allow us to feel touch, heat, sweat glands, sebaceous glands, hair follicles, apocrine glands, lymphatic vessels, and blood vessels. It also contains dermal fibroblasts, which will regenerate connective tissues in case of injury. 

 Hypodermis (Subcutaneous tissue)

While not part of the skin, it is immediately below the dermis, attaching the skin to the underlying bone and muscles as well as supplying blood vessels and nerves. It also consists of loose connective tissue, elastin, human fibroblasts, macrophages, adipocytes (fat cells), which is 50% of body fat.

The history of wound healing

For those who are history buffs, this article provides a succinct recount of the history of wound care since five millennia ago. The first five minutes of Dr. Abbas Shafiee’s lecture during our previous event also give us a more visual presentation of this interesting history.  While the principles of cleaning the wound, dressing the wound, and bandaging the wound were documented since 2200 B.C., most of the wound care technological advancements in wound care happened in the past 100 years. Currently, we have 5000 different dressings on the market, and more than 1000 wound care centers in the United States alone. [ Ref ]

In full-thickness wounds, the dermis, epidermis, and hypodermis are all destroyed and do not heal well, exposing patients to opportunistic infection and dehydration. For large full-thickness wounds, autografting is one preferred method, but still with less to be desired. In particular, in severe burn patients who have no healthy skin left, finding a donor site from the same patient will be challenging. 

What are the advantages of 3D bioprinted skin? 

Some of the well-known benefits of 3D printing technology include automation, the ability to create reproducible complex structures previously thought impossible, personalization, decentralized manufacturing process, and many other intrinsic advantages of being part of a controllable digital manufacturing process. The complex but layered organization of skin is perhaps another reason scientists chose 3D printing in research in skin tissue engineering. Of all the organs the scientific community set out to regenerate, the skin remains the least technically challenging organ to reproduce or bioprinting,

For those who are more driven, a good opinion piece from CellPress by a group from the National University of Singapore comparing bioprinted skin tissue and conventional skin tissue engineering can be found here. In particular, the article points out that current tissue engineering (TE) constructs through traditional TE methods cannot fulfill the requirements of a complex more native-like skin replacement that has appropriate vascularization, pigmentation, and hair follicles. 

Concurrent with traditional bioprinting systems, there is also a growing trend in creating in situ skin bioprinting system, which was described by professor Axel Guenther during our previous event. We will elaborate more in the latter part of this blog. That is, newer delivery methods of tissue engineering constructs are simultaneously invented along with the bioprinted construct itself.

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Photo credit: Poietis

Which bioprinting process has been used to create bioprinted skin constructs? 

Similar to 3D printing technology, bioprinting uses computer-aided design (CAD) and aims to precisely deposit cells and biomaterials, either separately or simultaneously to create a 3D construct that hopefully will have similar characteristics and functions to native tissues after maturation.

Several bioprinting approaches have been investigated for suitability for bioprinting skin, including extrusion-based bioprinting (Cellink/BICO), melt electrowriting (Dr. Abbas Shafiee’s presentation), laser-assisted bioprinting (i.e. Poietis), drop on demand bioprinting (which includes microvalve based, inkjet, and laser-assisted bioprinting) (i.e. Inventia Life Sciences), among others. [Ref] In addition to adapting one promising approach at the time, some innovations combine several approaches creatively to achieve end products. For example, Poietis, a France-based company, combines laser-assisted bioprinting, microvalve printing, and extrusion bioprinting. In another example of creatively combining techniques, a Canadian research group (led by Professor Axel Guenther) invented a handheld in situ bioprinting skin device combining microfluidics and extrusion-based bioprinting.

Extrusion based bioprinting

This is also known as “continuous printing”. The most common and well-known process is FDM (Fused Deposition Modeling). A commercial example is Allevi (previously Biobots), which is now part of 3D Systems. 

Laser-assisted bioprinting

This is one type of DOD (Drop-On-Demand) bioprinting technique. A typical laser-based bioprinting setup includes a pulsed laser beam, a focusing system, a donor slide

coated with a layer of energy-absorbing layer and cell-encapsulated hydrogel, and a collector slide facing the ribbon.  It is a nozzle-free printing technique that prevents clogging issues and facilitates deposition at a high cellular density. The energy-absorbing layer first absorbs a large amount of energy from a moving pulsed laser beam, and then the local evaporation of the energy-absorbing layer creates a high gas pressure, which propels droplets of cell-encapsulated hydrogel toward the collector slide. [Ref] One commercial example includes Poietis, a French startup. 

Melt Electrowriting (MEW)

MEW is an adjacent technology to electrospinning that “draws fibers using electrical instabilities and is researched extensively to make filters, textiles, and tissue engineering scaffolds. In general, electrospinning has NOT been considered an additive manufacturing technique…” (Dalton Lab). However, melt electrowriting combines the principles behind electrospinning and additive manufacturing, and is generally considered an additive manufacturing technique. “In a typical MEW system, a molten polymer is extruded through a spinneret connected to a high voltage supply and deposited at the grounded collector, which is moving according to a predefined pattern.” [Ref] MEW is capable of depositing predefined micron-sized fibers, which has been shown to be useful in tissue engineering and the creation of smart or bioactive dressing. Recently, Dr. Shafiee et al. has published methods for 3D-printing medical-grade polycaprolactone (mPCL) wound dressing with biomimetically designed architecture wfor accelerating wound closure.[Ref] “The hierarchical design of fabricated MEW mPCL, assimilated the J-shaped strain stiffening, anisotropic behavior of natural skin tissue, and created a cellular environment similar to native stem cell niche that enhanced human gingival tissue multipotent MSC (hGMSC) attachment and proliferation.” [Ref]

Microvalved based bioprinting

This is a type of DOD (Drop-On-Demand) bioprinting technique (see below). “The microvalve-based system typically contains an array of microvalves and a three-axis movable robotic stage. Each microvalve is connected to an individual gas regulator that provides the pneumatic pressure and the valve opening time is controlled by a pulse generator. Microvalve-based bioprinting could be utilized for the direct deposition of cell-laden hydrogels or the simultaneous deposition of cell droplets and matrix materials. The main advantage of microvalve-based bioprinting is the simultaneous deposition of both cellular and material components from different printing cartridges and high throughput printing.” [Ref]

Drop-on-demand bioprinting

In this bioprinting approach, the bioprinter deposits cells and matrix components onto the surface of a well plate in droplets, and builds these components layer-by-layer to form the desired 3D structure. Microvalved based bioprinting (one of the more commonly used methods for bioprinting skin in the past), inkjet, and laser-assisted bioprinting are all considered a type of DOD.  Compared to extrusion-based bioprinting, this technique achieves potentially higher cell viability by reducing shear stresses on cells, as the pressure required to eject a droplet from the printhead is lower than that used in extrusion printing. Additionally, one advantage of DOD over continuous printing (i.e. extrusion-based bioprinting) is the ability to create a truly controlled heterogeneous 3D construct. A good commercial example is the Australia-based startup Inventia Life Sciences. A good article comparing the advantages and disadvantages of different DOD technologies can be found in this article.

What biological materials have been used for bioprinted construct?

Both synthetic and native 3D printing biomaterials have been investigated for skin regeneration and wound healing, with natural biopolymer accounting for about 90% of bioinks used in bioprinting according to one meta-analysis. “Natural-based biopolymers have different advantages over synthetic biopolymers, owing to their high similarity with human ECM composition which mimics cells’ native microenvironment to facilitate cell attachment, proliferation, migration, and differentiation.”[Ref]

An ideal bioink needs to be biocompatible to facilitate cell growth, be mechanically stable, and have high shape fidelity post-printing. Parameters such as cell-laden parameters (i.e., cell type, cell density, and incubation period), physicochemical properties (i.e., shear-thinning, viscosity, crosslinking degree, and gelation time), and printing parameters (i.e., nozzle temperature and diameter, feed rate, and printing duration) are also crucial to functional bioink.

Some of the more common natural-biopolymer used for bioprinting skin constructs include collagen and gelatin, but alginate and decellularized extracellular matrix have also been used. [Ref] Interested readers can find a more in-depth comparison of these materials in this article.[Ref]

What are the potential applications of bioprinted skin constructs?

There are three main categories of bioprinted 3D construct in general, regardless of organ system: 

  • Tissue regeneration – For example, in full-thickness skin wounds like burn patients, skin substitute will be life-saving especially when an autologous skin donor site is not sufficient. 
  • Better drug discovery, toxicity, the screening process
  • In vitro model to understand disease process better 

More specifically for skin, because of its self-regenerating ability, additional applications of a 3D construct are also made to accelerate the innate healing process. This includes the various smart dressings discussed by Dr. Shafiee and the point of care delivery of in situ handheld bioprinting device by Dr. Guenther.

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Which companies are working on commercializing 3D bioprinted functional skin? 

As we have alluded to in the body of this blog, there are a few companies that are actively commercializing bioprinted skin construct, skin bioprinting services. Most notably, they are:

  • Poietis – Poietis is a Bordeaux, France-based startup that has been active in bioprinting skin space for about a decade, and it offers both hardware products, service, and 3D construct (Poieskin®).
    Poieskin® is a bioprinted human full-thickness skin model that consists of a dermal layer of primary human fibroblasts embedded in a collagen I matrix overlaid by a stratified epidermis derived from primary human keratinocytes. It aims to start Phase I clinical trial with Poieskin® in 2022 according to CEO/founder Fabien Guillemot during our past webinar, which will be considered a milestone not just for bioprinted skin model, but perhaps for the whole industry. For those who are interested, we have done an interview with Fabien recently.
  • Inventia Life Sciences – is an Australia-based startup that focuses on DOD (drop-on-demand) bioprinting techniques to create tissue models and organoids for a variety of organ systems including the skin. Currently, the company offers bioprinter, materials, and workflow, aiming at drug discovery, screening, and biomedical research. 
  • Cellink/BICO – With an incredibly diverse and large portfolio of tissue engineering technologies, the company offers a variety of products including a variety of bioprinters (from extrusion to laser-assisted bioprinting), materials, and workflow specifically for in-vitro tissue models and analogous skin production. 

While not yet fully commercialized, we are looking forward to seeing handheld in situ bioprinting devices and smart 3D printed wound dressing could come to the market and benefit mankind soon. 

Which research labs are active in 3D bioprinting skin?

  • Advanced wound care – The need for better wound care will only increase. Various challenging wounds ranging from full thickness injury in burn victims to chronic wounds in diabetes are not being well addressed with current strategies and are often life-threatening. The other challenge of current wound care strategies is scar formation, which is not just a cosmetic problem but can cause severe disaiblity. A better skin substitute or “smart dressing” is needed to address this cohort of patients. 
  • 4D Bioprinting? While I am not a huge fan of adding numbers to bioprinting technique, however, I am a big fan of automation in biology. Unmanned factory and simultaneous 24/7 continuous manufacturing process is any capitalist’s dream. Poietis along with a handful of other bioprinting startups (such as BioAssemblyBots) have created a streamlined fully automated, robotics based, reproducing workflow to provide a quality-controlled end product that can be used at point of care. According to CEO Fabien Guillemot, two of Poietis bioprinters have been installed at the University of Marseille.
  • Variety of skin models – Prof. McGuckin at our virtual event shared that CTIBiotech is aiming to develop a variety of skin models focusing on skin color, lymphatic systems, and models with skin appendages that will serve to answer critical and specific clinical questions. I anticipate that these models will be more complex and bio-similar as the bioprinting technology improves over time, and that seems to be accelerating. 
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References: 

About the Author:

Hui Jenny Chen, M.D.

jenny chen

Jenny Chen, MD, is the Founder and CEO of 3DHEALS, a company focusing on educating, connecting, and discovering innovators and entrepreneurs in the space of bioprinting, regenerative medicine, healthcare applications using 3D printing. With a focus on healthcare technology, Dr. Chen serves as a startup mentor and advisor to 3D technology startups as well as Kyto Life Sciences and Technologies. She created the Pitch3D program that connects early-stage startups to various fundraising strategies and directly to 35+ institutional investors in the space of healthcare 3D printing and bioprinting. Her interests lie in automated biology, patient-specific medicine, biofabrication, and has a vision of a decentralized and personalized healthcare delivery system for our near future.

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