Question

In a bicycle accident, a patient suffered a full-thickness skin wound of the forearm with an area of about 7 cm x 3 cm. Briefly describe the experimental strategy to engineer a 3-dimensional replacement tissue, considering (a)cell type, (b) biomaterial, (c) bioengineering technique, and (d) bioreactor

Answer 1

The in vitro tissue engineering has four steps.

(1) A biomaterial scaffold may be a flat sheet to mimic skin.

(2) The construct is bioactivated with primary or stem cells (using either endogenous or allogeneic cell lines (keratinocytes and fibroblasts)).

(3) After seeding the cells, the construct is cultured in a bioreactor, simulating at least one aspect of the in vivo environment (eg, chemistry, mechanical stresses).

(4) The final step is implantation.

Introduction

The human skin comprises three layers: the epidermis (outermost), dermis (middle), and hypodermis (deeper). The epidermis is a 0.2 mm thick, packed sheath of cells consisting of keratinocytes, which are in several stages of differentiation, alongside melanocytes and epidermal stem cells confined to the basal proliferative layer. Furthermore, there are 4 layers within the epidermis, namely, the corneum (dead cornified layer with 15-30 sheets of corneocytes), stratum (3-5 sheets of flattened keratinocytes with arrested division), stratum spinosum (possessing 8-10 layers of keratinocytes with restricted cell division), and stratum germinativum (proliferative layer). The “bricks-and-mortar” array kind of organization of corneocytes within the epidermis acts as a barrier separating the inside body environment from the external in conjunction with regulating fluid loss. The dermis, comprising of thick connective tissue, is sandwiched in the middle of the epidermis and the hypodermis. It is constituted of a bed of glycosaminoglycans (GAGs), elastin, and collagen extracellular matrix (ECM) with embedded fibroblasts. It also possesses numerous skin appendages like sebaceous and sweat glands, mechanoreceptors, hair follicles, vasculature, and nerve endings. The dermis imparts sensory and mechanical properties to the skin.

Types of Skin Substitutes

Depending upon the depth of the tissue, skin substitutes are often categorized into four distinct types.

• Epidermal Skin Constructs

The epidermal skin constructs comprise of keratinocytes cultured on a layer of irradiated feeder cells of murine fibroblasts. The autologous keratinocytes isolated from the patient usually take 2-3 weeks in expansion media to develop cell sheets of cultured epithelial autografts (CEAs). CEAs are typically 2 to 8 layers thick.

• Dermal Skin Constructs

Dermis comprises of ECM with fibroblasts which are further divided into an upper papillary and lower reticular region. Most of the commercial dermal skin replacements are cell-free and act as an initial framework for facilitating the infiltration of cells and blood vessels from the host tissue.

• Epidermal-Dermal Skin Constructs

Currently, the closest and the most sophisticated skin biomimetic available in the market is an epidermal-dermal skin substitute comprising of both of the upper layers of the skin.

• Trilayered Skin Construct

Trilayered skin constructs include the hypodermal fat alongside the dermis and therefore the epidermis. It is often considered because of the closest mimic to the native human skin for full-thickness wounds. The hypodermal layer consists of fatty connective tissue with predominantly collagen VI ECM and a multicellular organization (preadipocytes, adipocytes, vascular endothelial cells, and adipose macrophages).

A Trilayered Skin Construct can be considered in this case.

Biomaterials

• Silk

Silk has long been used as a dressing for wounds thanks to its beneficial properties, like good biodegradability, simple chemical alteration, good oxygen permeability, and therefore the ability of moisture retention. The relative molecular mass of SF has been found to affect wound healing. SF with a narrow range of molecular-weight distribution accelerates healing with better reepithelization, reduced scarring, lower infections, and immunogenic responses as compared to SF with a wider molecular-weight range.

• Hyaluronic Acid (HA)

HA is a common biological constituent of connective tissues of the cardiac valves, skin, bone, neuronal tissue, and umbilical cord. Being a crucial element of the vertebrate ECM, HA is nonimmunogenic and provides a congenial environment for cellular growth.

• Fibrin Glue

Fibrinogen is a glycoprotein in blood. After its isolation via precipitation by ammonium sulfate, PEG, or ethanol, the extracted fibrinogen is converted to fibrin glue (FG) via cross-linking by thrombin. The structural and mechanical properties of FG are often controlled by changing the extent of cross-linking.

• Collagen

Collagens are a bunch of fibrous proteins having a triple-helical structure comprising of α-chains. They form the elemental components of the ECM of just about all the tissues and play an important role by aiding within the regulation of tissue remodeling at the time of tissue repair. Collagen possesses target motifs for integrin receptors of cells thus regulating various properties associated with adhesion, migration, proliferation, and differentiation. Apart from this, easy isolation and purification; reduced toxic levels; and proven chemical, physical, and immunological properties mark its suitability for skin tissue engineering.

• Decellularized Extracellular Matrix (ECM)

Decellularized ECM scaffolds are widely utilized in the fabrication of several tissue substitutes during which the donor tissue undergoes removal of cellular components without disturbing the ECM comprising of collagen, GAGs, elastins, and growth factors. Decellularization of the skin allows for complete removal of the resident cell populations (hence reducing immune responses) while retaining the collagen framework, which acts because of the reservoir for growth factors and protein components within this ECM network.

Optimal Cell Source

• Embryonic stem cells have been isolated and differentiated into keratinocytes and fibroblasts.
• Cell lines: keratinocyte cell line (for example, HaCaT, immortalized adult skin keratinocyte) and the fibroblast cell line (HFF, human foreskin fibroblast) is also used.
• Induced pluripotent stem cells
• Differentiated primary cells
• Adult stem cells

1. Bone marrow-derived mesenchymal stem cells
2. Adipose-derived stem cells
3. Skin stem cells

Bioengineering technique

Current strategies include,

(a) nano-functionalized materials for triggering specific responses using nanotechnology

(b) automated and robotic fabrication of engineered tissues to extend efficacy, reduce costs, and cater to individual patient needs using 3D bioprinting

(c) regenerative therapy using stem cell technology for targeting pigmentation, wound closure, angiogenesis, and cutaneous sensation.

Bio-Reactors

Bioreactors have played a vital role in tissue engineering as they are capable of cultivating mammalian cells under a controlled environment up to an industrial scale. Several operational conditions can be modified and controlled including pH, temperature, oxygen tension, and perfusion of the cells as well as external stimuli such as mechanical forces, etc. Bioreactors can be used to aid in the in vitro development of new tissues. They provide the biochemical and physical regulatory signals required for cells to proliferate, differentiate, and to supply ECM.

• Expanding Bioreactors for skin grafts:

The bioreactor system applies continuous physio-mechanical stress load in culture with the hope of harvesting a larger area of products. It contained an actuator mounted on the tissue container which secures the culturing skin or scaffold, a linear motor-driven device, and an incubator. It is programmed to uniaxially expand the fixed tissue or scaffold to 20% of its initial length per day, so it will double the surface area in 5 days approximately.

Reference :

Kaur, A., Midha, S., Giri, S., & Mohanty, S. (2019). Functional Skin Grafts: Where Biomaterials Meet Stem Cells. Stem Cells International, 2019, 1–20. doi:10.1155/2019/1286054