**3. Tissue engineering history and techniques**

As medical treatments and expanded lifespan expectancies in both males and females have improved, the number of individuals waiting for organ transplants or blood vessel bypasses is constantly increasing but the availability of organs does not often match the demand. To circumvent this shortage in organ and tissue supplies, many efforts in cell culture methods were deployed to engineer tissues that could be used as an alternative therapeutical option.

#### **3.1. Self-assembly technique**

The self-assembly technique is based on the ability of MSCs to secrete and organize their own ECM to produce sheets. This tissue engineering method allows the production of autologous living tissues, free of exogenous biomaterials [35–37]. The self-assembly technique has exploited the inherent characteristics of MSCs to produce ECM. For instance, it was well documented that ascorbic acid, a vitamin C derivate, promotes collagen protein synthesis and deposition of sulfated glycosaminoglycans in human skin substitutes [38]. Once fibroblasts supplemented with ascorbic acid are cultured for 21–35 days, they form sheets of matrix where stromal cells are embedded within [35]. These sheets can be peeled from the culture dish and superimposed. The superimposed layers are maintained for an additional week for further cell-matrix reorganization and layer fusion (**Figure 1A**). Holes can be made in the multilayer dermal equivalent and hair follicles can be added to mimic the presence of native skin component. An additional culture time is required before seeding keratinocytes. Thereafter, the skin equivalent is maintained for 21 days at an air–liquid interface to induce the cornifi‐ cation of the epidermis [36, 39].

**Figure 1. Schematic representation of self-assembled tissues**. **A**) The classical self-assembly technique requires stack‐ ing of three fibroblast (Fb) cell sheets that are cultured for 4 weeks in the presence of ascorbic acid. Sheet fusion lasts 4 to 7 days. **B**) After 4 weeks of Fb cultures, the stroma is seeded with endothelial cells (ECs) and then a fusion set is carried on to generate endothelialized tissues. **C**) Modification to the self-assembly technique consists of an initial Fb culture for 2 weeks followed by an additional Fb reseeding. The culture is maintained for an additional 2 weeks before using the stroma without stacking. **D**) Fb and EC are co-seeded in order to generate endothelialized tissues with a 3D capillary-like network.

#### *3.1.1. Engineered human skin substitutes*

*2.1.2. Myofibroblasts: functions and origins*

344 Composition and Function of the Extracellular Matrix in the Human Body

**2.2. Adipose tissue–derived stem cells**

**3.1. Self-assembly technique**

Myofibroblasts, or activated fibroblasts, are contractile, resistant to apoptosis and have an upregulated rate of matrix deposition. They also express different cytokine and chemokine receptors that enable fiber regulation and wound contraction at injury sites [21–24]. In this sense, activated fibroblasts not only favor wound healing, but can also cause injuries when their activation is uncontrolled, producing a pathological fibrotic response [25, 26]. The precursor of myofibroblast is not precisely known, but many cells can differentiate into myofibroblasts through different signaling pathways or gene regulation. Some of these precursors include epithelial cells, ECs, pericytes, multipotent monocytes, and fibroblasts.

Fat is an abundant and accessible source of stem cells. Adipose tissue–derived stem cells (ASCs) include preadipocytes, and a subpopulation of stromal cells able to differentiate into multilineages, including neuronal cells, chondrocytes, and osteoblasts [27, 28]. Moreover, these MSCs are able to secrete cytokines and growth factors promoting regenerative processes because they can influence cell recruitment, proliferation rates, or inhibit apoptosis [29, 30]. ASCs extracted from liposuctions can be expanded in culture and used as building blocks for tissue engineering. Both connective and adipose tissues were engineered in vitro using ASCs [31–33]. Both allogeneic and xenogeneic ASCs can be transplanted in patients regardless of their immunocompatibility and without the need of immunosuppression therapy, making

As medical treatments and expanded lifespan expectancies in both males and females have improved, the number of individuals waiting for organ transplants or blood vessel bypasses is constantly increasing but the availability of organs does not often match the demand. To circumvent this shortage in organ and tissue supplies, many efforts in cell culture methods were deployed to engineer tissues that could be used as an alternative therapeutical option.

The self-assembly technique is based on the ability of MSCs to secrete and organize their own ECM to produce sheets. This tissue engineering method allows the production of autologous living tissues, free of exogenous biomaterials [35–37]. The self-assembly technique has exploited the inherent characteristics of MSCs to produce ECM. For instance, it was well documented that ascorbic acid, a vitamin C derivate, promotes collagen protein synthesis and deposition of sulfated glycosaminoglycans in human skin substitutes [38]. Once fibroblasts supplemented with ascorbic acid are cultured for 21–35 days, they form sheets of matrix where stromal cells are embedded within [35]. These sheets can be peeled from the culture dish and superimposed. The superimposed layers are maintained for an additional week for further

them an unlimited source for regenerative medicine applications [34].

**3. Tissue engineering history and techniques**

One of the great breakthroughs in medicine was achieved by engineering human skin substitutes for grafting purposes. Self-assembled skin substitutes were generated by extracting the patient's own cells, thus avoiding immunological incompatibility problems upon grafting. These skin substitutes were characterized and showed a fully differentiated epidermis, structural and morphological resemblance to native human skin. Moreover, these in vitro engineered tissues were able to deliver cytokines, chemokines, and growth factors at the grafted site, improving the wound closure [36, 40–43]. Because of all these characteristics, selfassembled human skin is clinically used for wound healing and burn treatments [44, 45]. Selfassembled skin substitutes possess a near-to-native architecture and maintain their cell growth potential and matrix deposition. Therefore, these equivalents are free of exogenous material, cytotoxicity, and have clinically reduced morbidity in burnt patients (reviewed in Refs. [46, 47].

Over the years, self-assembled skin substitutes were also produced from extracted cells of patients having psoriasis [48]. The reconstructed tissues from psoriatic donors represent an ideal model to study one of the most common human skin diseases. In fact, this model outlines the excessive growth and aberrant differentiation of keratinocytes. It offers a reliable in vitro mean to measure the efficacy of appropriate treatments, perform tests directly on human primary cells, and avoid animal use [48–52]. Furthermore, self-assembled skin substitutes were used to extensively characterize cellular and molecular players involved in the pathogenesis of hypertrophic scars and scleroderma [53–55]. More recently, a skin substitute derived from patients diagnosed with amyotrophic lateral sclerosis (ALS) was similarly generated. As one of the early perturbations in ALS patients is skin alterations that often precede the neurological symptoms, this human skin model is designed to better identify disease-specific biomarkers and early diagnostic tools to monitor disease progression [56].

#### *3.1.2. Cardiovascular tissues*

The self-assembly technique was customized to engineer human blood vessel that displayed excellent physiological and mechanical properties without the need for any exogenous scaffold [35, 57]. Blood vessels are constituted of a functional endothelium seeded onto an internal membrane of human skin fibroblasts. In order to mimic the shape of a blood vessel, a smooth muscle cell (SMC) sheet is first rolled, followed by the fibroblast sheet around a cylindrical

**Figure 2. Vascular self-assembled tissue**. **A**) A sheet of smooth muscle cells (SMC) (first) and a sheet of fibroblasts (Fb) (second) are rolled around a cylindrical support after 4 weeks of culture. The construct is allowed to fuse and ma‐ ture in a bioreactor. **B**) Modification to the technique shown in (**A**) consists of co-seeding SMCc and Fb each at an ex‐ tremity of the same sheet, which is rolled around a cylindrical support after 4 weeks of culture and then allowed to fuse and mature in a bioreactor.

support, and cultured until fusion (**Figure 2A**). Analyses of these in vitro engineered vessels confirmed the presence of numerous ECM proteins (collagen types I, III, IV, laminin, fibro‐ nectin, and chondroitin sulfates) and a functional endothelium [35, 58, 59].

Progress in developing self-assembled valves was reported over the years [60, 61]. Valve leaflets made of self-assembled tissue sheets can organize into a characteristic three-layer structure featuring appropriate dynamic fluidics [60]. This tissue remains to be grafted into living recipients in order to assess in vivo survival and behavior of the transplanted valve. Nevertheless, this stentless bioprosthetic offers a great alternative to artificial valves for cardiovascular surgeries [60, 61].

### *3.1.3. Cornea*

potential and matrix deposition. Therefore, these equivalents are free of exogenous material, cytotoxicity, and have clinically reduced morbidity in burnt patients (reviewed in Refs. [46, 47]. Over the years, self-assembled skin substitutes were also produced from extracted cells of patients having psoriasis [48]. The reconstructed tissues from psoriatic donors represent an ideal model to study one of the most common human skin diseases. In fact, this model outlines the excessive growth and aberrant differentiation of keratinocytes. It offers a reliable in vitro mean to measure the efficacy of appropriate treatments, perform tests directly on human primary cells, and avoid animal use [48–52]. Furthermore, self-assembled skin substitutes were used to extensively characterize cellular and molecular players involved in the pathogenesis of hypertrophic scars and scleroderma [53–55]. More recently, a skin substitute derived from patients diagnosed with amyotrophic lateral sclerosis (ALS) was similarly generated. As one of the early perturbations in ALS patients is skin alterations that often precede the neurological symptoms, this human skin model is designed to better identify disease-specific biomarkers

The self-assembly technique was customized to engineer human blood vessel that displayed excellent physiological and mechanical properties without the need for any exogenous scaffold [35, 57]. Blood vessels are constituted of a functional endothelium seeded onto an internal membrane of human skin fibroblasts. In order to mimic the shape of a blood vessel, a smooth muscle cell (SMC) sheet is first rolled, followed by the fibroblast sheet around a cylindrical

**Figure 2. Vascular self-assembled tissue**. **A**) A sheet of smooth muscle cells (SMC) (first) and a sheet of fibroblasts (Fb) (second) are rolled around a cylindrical support after 4 weeks of culture. The construct is allowed to fuse and ma‐ ture in a bioreactor. **B**) Modification to the technique shown in (**A**) consists of co-seeding SMCc and Fb each at an ex‐ tremity of the same sheet, which is rolled around a cylindrical support after 4 weeks of culture and then allowed to

and early diagnostic tools to monitor disease progression [56].

346 Composition and Function of the Extracellular Matrix in the Human Body

*3.1.2. Cardiovascular tissues*

fuse and mature in a bioreactor.

Corneal tissue engineering was developed in an attempt to cure corneal opacity by replacing the damaged area with a clear substitute. Proulx *et al*. [62] generated a self-assembled threelayer equivalent of human cornea. These substitutes presented near-to-native stromal, endothelial, and epithelial morphology with an intact basement membrane filled with laminin V and collagen VII proteins. The differentiated epithelial layer had defined basal and wing cells that expressed Na+ /K+ ATPase α1 protein, keratin 3/12, and basic keratins. This human cornea model was also used to study the pattern of MMP genes expression during corneal wound healing [63].
