**3. Original biocompatible scaffolds validated for hASCs differentiation in the context of tissue engineering applications**

Advanced research in tissue engineering promotes the use of bioconstructs as an effective solution to perform regeneration of damaged tissues. Using 3D microenvironments brings benefits for tissue engineering applications, since 3D microenvironment recreates better *in vivo* conditions and mimics closely the natural tissue. A number of cell-scaffold bioconstructs with structure and properties adapted to the nature of the tissue in need of reconstruction have been shown to be useful in the production of functional *de novo* tissue. These bioconstructs can function as 3D (i) transport systems ("shuttle") to deliver the cells to the injured site, encouraging the self-healing ability of the tissue and (ii) biocompatible and biodegradable bioconstructs, supporting the cellular component during tissue reconstruction.

In this context, a group of researchers from the Department of Biochemistry and Molecular Biology, University of Bucharest, partially in collaboration with the Department of Histology from Western University Vasile Goldis of Arad, investigated hASCs potential to differentiate in contact with several original recipes of materials designed for adipose, cartilage and bone tissue engineering.

For adipose tissue engineering (ATE), the optimal conditions required for successful differentiation of hASCs in case of implantations at a wound healing site were studied. Cells should first proliferate and then receive the optimal amount of pro-adipogenic signals to induce the differentiation process with a certain rate. Therefore, a modulation of the adipogenic conditions would be required, aiming to ensure the long-term proliferation of the precursor cells and to control the kinetics of the differentiation process [87].

One novel scaffold validated for ATE by *in vitro* studies was a spongious 60% collagen and 40% sericin hydrogel preceded with hASCs (CollSS) [12]. In this case, the novel CollSS composition was compared in terms of biocompatibility and ability to support adipogenic differentiation of hASCs to a pure collagen hydrogel (Coll). The addition of the sticky protein sericin in the composition of a classical collagen sponge enhanced the adhesion and also the proliferation rate of hASCs. CollSS proved to be more biocompatible than pure Coll. hASCs-CollSS bioconstruct proved to efficiently support the adipogenic differentiation process, as confirmed by the expression levels of PPARγ2, fatty acid synthase (FAS), adipocyte protein 2 (aP2) and perilipin adipogenic markers [12].

hASCs ability to differentiate towards mature adipocytes in 3D environment was also confirmed when using a gelatin- alginate- polyacril amide (GAPAA) scaffold. This type of material proved to be at least as efficient as the gelatin-alginate biomaterial (GA) for adipogenic differentiation, hASCs generating mature adipocytes in this system in 21 days of induced differentiation [88].

**4. Most recent advances and future perspectives**

assess their utility for tissue engineering purposes.

positive chondrogenic differentiation.

for the repair of large bone tissue defects.

still have a good proliferation.

eration processes.

The optimization of biomaterials composition, structure and properties to mimic as well as possibilities in the *in vivo* architecture has led to the development of the advanced technology of bioprinting. Recently, highly advanced bioprinted 3D constructs comprising of material, stem cells and additives have been reported to successfully support differentiation and regen-

Human Adipose-Derived Stem Cells for Tissue Engineering Approaches: Current Challenges and Perspectives

http://dx.doi.org/10.5772/intechopen.68712

305

In order to combine as efficiently as possible this modern technology with hASCs properties, several groups have conducted studies to observe hASCs behaviour in these systems and to

One of these studies explored the behaviour of hASCs when printed in a free-scalable 3D grid pattern by means of laser-assisted bioprinting (LaBP). Cells in this system managed to differentiate towards the adipogenic lineage, thus proving that such 3D bioprints could be used

In order to be efficient, the bioprinted constructs should resemble the native tissue microenvironment as close as possible. For example, muscular soft tissue constructs can benefit from bioinks that mimic their nanofibrous matrix constitution [90]. Thus, it is critical to maximize the biocompatibility between cells and the type of fibre materials used to create the constructs. Narayanan et al. have used hASCs with a fibrous bioink composed of alginate hydrogel and polylactic acid nanofibers to obtain bioprinted constructs as replacement for knee meniscus. Results indicated favourable behaviour of hASCs, high levels of cell proliferation as well as

For bone reconstruction purposes, hASCs were included in a mixture together with nanohydroxyapatite and bioprinted in a 3D construct, further evaluated for cell proliferation and osteogenesis ability [91]. In this case, it was concluded that nano-hydroxyapatite could increase osteogenic differentiation of the hASCs mixture after bioprinting, in which the cells

Similarly, Wang et al. [92] aimed to investigate osteogenic differentiation of hASCs in 3D bioprinted tissue constructs, both *in vitro* and *in vivo*. After performing tests to assess osteogenic markers expression both at gene and protein levels, researchers reported significantly increased expression levels of RUNX2, OSX and OCN after 7 and 14 days of osteogenesis. *In vivo* studies demonstrated obvious bone matrix formation in the 3D bioprinted constructs. Based on this study, it was concluded that hASCs could be used in 3D bioprinted constructs

In cardiac regeneration, a great interest is directed to obtaining 3D cocultures of stem cells and endothelial cells to closely mimic the native tissue conditions. In this context, a team of researchers [93] used laser-induced-forward-transfer (LIFT) cell printing technique to prepare a polyester urethane urea (PEUU) cardiac patch seeded with human umbilical vein endothelial cells (HUVEC) and human MSCs. Results showed an increased production of blood ves-

sels, which reflected an improvement of functionality in infarcted tissue.

successfully in tissue regeneration or the biofabrication of living grafts [89].

With regard to bone tissue engineering (BTE) direction, original research validated a composite material based on chitosan (CHT) and graphene oxides (GO) (hASCs/CHT/GO 3 wt.%) as most suitable for hASCs osteogenesis [paper under review]. The study of the osteogenic differentiation potential of hASCs in contact with chitosan-based scaffolds enriched with 0.5–3 wt.% GO showed the highest rate of differentiation in cells cultured in the presence of 3 wt.% GO, suggesting that hASCs/CHT/GO 3 wt.% may be a candidate for future bone regeneration applications and BTE. Currently, the potential of graphene and its derivatives is exploited increasingly in tissue engineering because of their positive influence on cellular interaction with the material and on the bioactivity of the material. Graphene oxide (GO) in particular was shown to improve the biological properties of materials and to promote adhesion, proliferation and osteogenic differentiation of mesenchymal stem cells. Experiments demonstrated good biocompatibility of CHT/GO materials, where the degree of biocompatibility depends on the GO content. Similar to previous studies, GO favoured hASCs contact with the materials and influenced the proliferation rate.

For cartilage tissue engineering (CTE), two distinct bioconstructs were studied: (i) a three component inter-polymeric network (IPN) hydrogel based on gelatin, alginate and polyacrylamide (GAPAA) was evaluated for potential to support hASCs chondrogenesis (hASCs/ GAPAA) and (ii) a hydrogel based on collagen (Coll), improved with sericin (SS), hyaluronic acid (HA) and chondroitin sulphate (CS) (hASCs/CollSSHACS), was tested for hASCs proliferation, adhesion and chondrogenic differentiation [11, 13].

Chondrogenic differentiation studies conducted in GAPAA IPN have provided important information about the essential characteristics of a 3D scaffold to be used effectively in cartilage reconstruction. In this case, the adequate porosity of the system and the control over scaffold's 3D architecture for hASCs chondrogenic differentiation were achieved by adding the synthetic component polyacrylamide in the composition. Normally, acrylamide (AA) would exert a toxic effect on cells, but the polymerized form of PAA has a lower toxicity when used in very low concentration in the composition of GAPAA to control pore size [11].

Similarly, in hASCs/CollSSHACS bioconstruct exposed to pro-chondrogenic differentiation conditions, the results indicated a more efficient chondrogenesis of hASCs in the collagen hydrogel enriched with hyaluronic acid and chondroitin sulphate (CollSSHACS), two cartilage markers present in the extracellular matrix, as compared to the results obtained for the reference system (hASCs/Coll). The simultaneous presence of both molecules in HA and CS biomaterial composition favoured the initiation and controlled hASC chondrogenesis for 28 days *in vitro* [paper under review]. Therefore, hASCs/CollSSHACS bioconstruct would be a good candidate with high potential for use in CTE applications.

### **4. Most recent advances and future perspectives**

hASCs ability to differentiate towards mature adipocytes in 3D environment was also confirmed when using a gelatin- alginate- polyacril amide (GAPAA) scaffold. This type of material proved to be at least as efficient as the gelatin-alginate biomaterial (GA) for adipogenic differentiation, hASCs generating mature adipocytes in this system in 21 days of induced

With regard to bone tissue engineering (BTE) direction, original research validated a composite material based on chitosan (CHT) and graphene oxides (GO) (hASCs/CHT/GO 3 wt.%) as most suitable for hASCs osteogenesis [paper under review]. The study of the osteogenic differentiation potential of hASCs in contact with chitosan-based scaffolds enriched with 0.5–3 wt.% GO showed the highest rate of differentiation in cells cultured in the presence of 3 wt.% GO, suggesting that hASCs/CHT/GO 3 wt.% may be a candidate for future bone regeneration applications and BTE. Currently, the potential of graphene and its derivatives is exploited increasingly in tissue engineering because of their positive influence on cellular interaction with the material and on the bioactivity of the material. Graphene oxide (GO) in particular was shown to improve the biological properties of materials and to promote adhesion, proliferation and osteogenic differentiation of mesenchymal stem cells. Experiments demonstrated good biocompatibility of CHT/GO materials, where the degree of biocompatibility depends on the GO content. Similar to previous studies, GO favoured hASCs contact with the materials and influenced the prolif-

For cartilage tissue engineering (CTE), two distinct bioconstructs were studied: (i) a three component inter-polymeric network (IPN) hydrogel based on gelatin, alginate and polyacrylamide (GAPAA) was evaluated for potential to support hASCs chondrogenesis (hASCs/ GAPAA) and (ii) a hydrogel based on collagen (Coll), improved with sericin (SS), hyaluronic acid (HA) and chondroitin sulphate (CS) (hASCs/CollSSHACS), was tested for hASCs prolif-

Chondrogenic differentiation studies conducted in GAPAA IPN have provided important information about the essential characteristics of a 3D scaffold to be used effectively in cartilage reconstruction. In this case, the adequate porosity of the system and the control over scaffold's 3D architecture for hASCs chondrogenic differentiation were achieved by adding the synthetic component polyacrylamide in the composition. Normally, acrylamide (AA) would exert a toxic effect on cells, but the polymerized form of PAA has a lower toxicity when used in very low concentration in the composition of GAPAA to control

Similarly, in hASCs/CollSSHACS bioconstruct exposed to pro-chondrogenic differentiation conditions, the results indicated a more efficient chondrogenesis of hASCs in the collagen hydrogel enriched with hyaluronic acid and chondroitin sulphate (CollSSHACS), two cartilage markers present in the extracellular matrix, as compared to the results obtained for the reference system (hASCs/Coll). The simultaneous presence of both molecules in HA and CS biomaterial composition favoured the initiation and controlled hASC chondrogenesis for 28 days *in vitro* [paper under review]. Therefore, hASCs/CollSSHACS bioconstruct would be a

eration, adhesion and chondrogenic differentiation [11, 13].

good candidate with high potential for use in CTE applications.

differentiation [88].

304 Stem Cells in Clinical Practice and Tissue Engineering

eration rate.

pore size [11].

The optimization of biomaterials composition, structure and properties to mimic as well as possibilities in the *in vivo* architecture has led to the development of the advanced technology of bioprinting. Recently, highly advanced bioprinted 3D constructs comprising of material, stem cells and additives have been reported to successfully support differentiation and regeneration processes.

In order to combine as efficiently as possible this modern technology with hASCs properties, several groups have conducted studies to observe hASCs behaviour in these systems and to assess their utility for tissue engineering purposes.

One of these studies explored the behaviour of hASCs when printed in a free-scalable 3D grid pattern by means of laser-assisted bioprinting (LaBP). Cells in this system managed to differentiate towards the adipogenic lineage, thus proving that such 3D bioprints could be used successfully in tissue regeneration or the biofabrication of living grafts [89].

In order to be efficient, the bioprinted constructs should resemble the native tissue microenvironment as close as possible. For example, muscular soft tissue constructs can benefit from bioinks that mimic their nanofibrous matrix constitution [90]. Thus, it is critical to maximize the biocompatibility between cells and the type of fibre materials used to create the constructs. Narayanan et al. have used hASCs with a fibrous bioink composed of alginate hydrogel and polylactic acid nanofibers to obtain bioprinted constructs as replacement for knee meniscus. Results indicated favourable behaviour of hASCs, high levels of cell proliferation as well as positive chondrogenic differentiation.

For bone reconstruction purposes, hASCs were included in a mixture together with nanohydroxyapatite and bioprinted in a 3D construct, further evaluated for cell proliferation and osteogenesis ability [91]. In this case, it was concluded that nano-hydroxyapatite could increase osteogenic differentiation of the hASCs mixture after bioprinting, in which the cells still have a good proliferation.

Similarly, Wang et al. [92] aimed to investigate osteogenic differentiation of hASCs in 3D bioprinted tissue constructs, both *in vitro* and *in vivo*. After performing tests to assess osteogenic markers expression both at gene and protein levels, researchers reported significantly increased expression levels of RUNX2, OSX and OCN after 7 and 14 days of osteogenesis. *In vivo* studies demonstrated obvious bone matrix formation in the 3D bioprinted constructs. Based on this study, it was concluded that hASCs could be used in 3D bioprinted constructs for the repair of large bone tissue defects.

In cardiac regeneration, a great interest is directed to obtaining 3D cocultures of stem cells and endothelial cells to closely mimic the native tissue conditions. In this context, a team of researchers [93] used laser-induced-forward-transfer (LIFT) cell printing technique to prepare a polyester urethane urea (PEUU) cardiac patch seeded with human umbilical vein endothelial cells (HUVEC) and human MSCs. Results showed an increased production of blood vessels, which reflected an improvement of functionality in infarcted tissue.

#### **4.1. Risks associated with hASCs use for tissue engineering and regenerative medicine**

**Authors' contribution**

**Author details**

Sorina Dinescu1

Romania

**References**

reviewing the literature and writing the chapter.

, Anca Hermenean2

\*Address all correspondence to: anca.hermenean@gmail.com

therapies. Tissue Engineering. 2001;**7**:211-228

perspectives. Discovery Medicine. 2011;**11**(57):160-170

Thrombosis and Vascular Biology. 2009;**29**:503-510

Circulation Research. 2007;**100**(9):1249-1260

2011;**317**:2950-2957

Sorina Dinescu, Anca Hermenean and Marieta Costache contributed to designing the chapter,

Human Adipose-Derived Stem Cells for Tissue Engineering Approaches: Current Challenges and Perspectives

http://dx.doi.org/10.5772/intechopen.68712

307

\* and Marieta Costache1

1 Department of Biochemistry and Molecular Biology, University of Bucharest, Romania

2 Department of Histology, Faculty of Medicine, Vasile Goldis Western University of Arad,

[1] Zuk PA, Zhu M, Mizuno H, Huang JI, Futrell WJ, Katz AJ, Benhaim P, Lorenz HP, Hedrick, MH. Multilineage cells from human adipose tissue: Implications for cell-based

[2] Tobita M, Orbay H, Mizuno H. Adipose-derived stem cells: Current findings and future

[3] Dai R, Wang Z, Samanipour R, Koo KI, Kim K. Adipose-derived stem cells for tissue engineering and regenerative medicine applications. Stem Cells International. 2016;**2016**:6737345

[4] Ebrahimian TG, Pouzoulet F, Squiban C, Buard V, André M, Cousin B, Gourmelon P, Benderitter M, Casteilla L, Tamarat R. Cell therapy based on adipose tissue-derived stromal cells promotes physiological and pathological wound healing. Arteriosclerosis

[5] Gimble JM, Kate AJ, Bunnell BA. Adipose-derived stem cells for regenerative medicine.

[6] Rubio D, Garcia-Castro J, Martin MC, de la Fuente R, Cigudosa JC, Lloyd AC. Spontaneous human adult stem cell transformation. Cancer Research. 2005;**65**:3035-3039 [7] Rosland GV, Svendsen A, Torsvik A, Sobala E, McCormack E, Immervoll H. Long-term cultures of bone marrow-derived human mesenchymal stem cells frequently undergo

[8] Ren Z, Wang J, Zhu W, Guan Y, Zou C, Chen Z. Spontaneous transformation of adult mesenchymal stem cells from cynomolgus macaques *in vitro*. Experimental Cell Research.

spontaneous malignant transformation. Cancer Research. 2009;**69**:5331-5339

According to good manufacturing practices (GMP) rules, mesenchymal stem cells including hASCs are considered as advanced therapy medical products and are validated safe for medical practice. Thus, these cells are widely used in tissue engineering and regenerative medicine applications based on the fact that they are adult stem cells and that they have a relatively limited potential for proliferation, differentiation and an extremely low risk of transformation.

Most clinical applications involving hASCs, however, require the use of a large number of cells at the implant site for regenerative therapy success and therefore most often, the *in vitro* expansion of these cells is necessary. Risks associated with hASCs use in regenerative medicine are mainly associated with the *in vitro* handling of cells and, thus, most studies are directed towards assessing changes that may occur in the genome of these cells during *ex vivo* cultivation.

Furthermore, a series of contradictory studies discussed the transformation potential of hASCs in different circumstances since the implantation of these cells in the body for regenerative purposes can have long-term consequences if they have tumorigenic potential. Although the accumulation of chromosomal aberrations and mutations was repeatedly reported in the genome of hASCs during *in vitro* culture and expansion, most of these mutations are proven to have a transient nature and can be normally removed by DNA repair systems. Although no case of malignant transformation of hASCs implanted in humans was reported so far, caution is necessary. The interaction of hASCs with the *in vivo* existing microenvironment is specific to each organism and could have risks. In this context, hASCs genomic instability when in contact with tumour microenvironment should be further studied, considering the implications for tissue engineering.

### **5. Conclusions**

Human adipose-derived stem cells are a versatile source of mesenchymal*-like* stem cells, with enormous potential for tissue engineering and regenerative medicine. Regardless of the application and tissue that require restoration, hASCs proved to be efficient in a great number of cases. However, the stem-ness status of these cells and their unexplored risks of transformation should not be forgotten and thus more cautiousness should be taken before recommending therapy with hASCs. If correctly controlled, the potential of these cells could become a very powerful tool to increase the quality of life in patients with tissue defects.

### **Acknowledgements**

The authors acknowledge the sources of funding that supported this research in the field of stem cells: national grant PCCE248/2010, national project PNII-PCCA140/2012, national project Bridge Grant PNIII-P2-2.1-BG-2016-0458 (123BG/2016) as well as COST Action 15205. Additionally, the authors acknowledge the contribution made by UNESCO-L'Oreal foundation through Sorina Dinescu's UNESCO-L'Oreal Fellowship for Women in Science 2016, winner of Life Science Section.

### **Authors' contribution**

Sorina Dinescu, Anca Hermenean and Marieta Costache contributed to designing the chapter, reviewing the literature and writing the chapter.

### **Author details**

**4.1. Risks associated with hASCs use for tissue engineering and regenerative medicine**

According to good manufacturing practices (GMP) rules, mesenchymal stem cells including hASCs are considered as advanced therapy medical products and are validated safe for medical practice. Thus, these cells are widely used in tissue engineering and regenerative medicine applications based on the fact that they are adult stem cells and that they have a relatively limited potential for proliferation, differentiation and an extremely low risk of transformation.

Most clinical applications involving hASCs, however, require the use of a large number of cells at the implant site for regenerative therapy success and therefore most often, the *in vitro* expansion of these cells is necessary. Risks associated with hASCs use in regenerative medicine are mainly associated with the *in vitro* handling of cells and, thus, most studies are directed towards assessing changes that may occur in the genome of these cells during *ex vivo* cultivation.

Furthermore, a series of contradictory studies discussed the transformation potential of hASCs in different circumstances since the implantation of these cells in the body for regenerative purposes can have long-term consequences if they have tumorigenic potential. Although the accumulation of chromosomal aberrations and mutations was repeatedly reported in the genome of hASCs during *in vitro* culture and expansion, most of these mutations are proven to have a transient nature and can be normally removed by DNA repair systems. Although no case of malignant transformation of hASCs implanted in humans was reported so far, caution is necessary. The interaction of hASCs with the *in vivo* existing microenvironment is specific to each organism and could have risks. In this context, hASCs genomic instability when in contact with tumour microenvironment should be further studied, considering the implications for tissue engineering.

Human adipose-derived stem cells are a versatile source of mesenchymal*-like* stem cells, with enormous potential for tissue engineering and regenerative medicine. Regardless of the application and tissue that require restoration, hASCs proved to be efficient in a great number of cases. However, the stem-ness status of these cells and their unexplored risks of transformation should not be forgotten and thus more cautiousness should be taken before recommending therapy with hASCs. If correctly controlled, the potential of these cells could become a

The authors acknowledge the sources of funding that supported this research in the field of stem cells: national grant PCCE248/2010, national project PNII-PCCA140/2012, national project Bridge Grant PNIII-P2-2.1-BG-2016-0458 (123BG/2016) as well as COST Action 15205. Additionally, the authors acknowledge the contribution made by UNESCO-L'Oreal foundation through Sorina Dinescu's UNESCO-L'Oreal Fellowship for Women in Science 2016, winner of Life Science Section.

very powerful tool to increase the quality of life in patients with tissue defects.

**5. Conclusions**

306 Stem Cells in Clinical Practice and Tissue Engineering

**Acknowledgements**

Sorina Dinescu1 , Anca Hermenean2 \* and Marieta Costache1

\*Address all correspondence to: anca.hermenean@gmail.com

1 Department of Biochemistry and Molecular Biology, University of Bucharest, Romania

2 Department of Histology, Faculty of Medicine, Vasile Goldis Western University of Arad, Romania

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**Chapter 15**

**Provisional chapter**

**Tissue Engineering for Skin Replacement Methods**

The skin is the biggest structure of the body, and it plays a significant role in maintaining the unity of the body environment. The skin is important for the endurance of the organism as an outer coat for the thermal regulation and hydration preservation. With the intention of helping these significant utilities, the skin continually experiences regeneration and holds the capability to overhaul wound by repair and regeneration of several kinds of skin stem cells. Noteworthy, development has been accomplished throughout the recent times in the generation of engineered skin alternates which imitate human skin cells in vitro for replacement or modeling. Conversely, existing new skin alternatives do not reinstate completely the healthy skin anatomy and suffer from deficiency of natural supplements in skin covering, sebaceous glands, hair follicles, and sweat glands. Improvements in stem cell biology and skin morphogenesis show significant potentials to evidently advance the engineering of skin replacements which would preferably be vague from normal skin. This chapter reviews these developments in the in vivo and in

vitro techniques of engineered and manufactured skin scaffold biomaterials.

in vivo skin models, biomaterials, skin regeneration, skin renewal

**Keywords:** skin tissue engineering, scaffolds, skin, skin alternates, in vitro skin models,

Tissue engineering is growing as a novel area in biomedical engineering which purposes to redevelop newfangled material for substituting problematic or injured tissues. In order to accomplish this, not only is a basis of cells necessary but also a simulated extracellular matrix (ECM) that the cells which may be reinforced should exist. Human skin signifies about one-tenth of the body form, and injuries like physical distress, infection, burn, disease, or operation to a portion of this main organ carry intense penalties. Tissue engineering

**Tissue Engineering for Skin Replacement Methods**

DOI: 10.5772/intechopen.69905

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

Özge Sezin Somuncu, Ceren Karahan, Salih Somuncu and Fikrettin Şahin

Özge Sezin Somuncu, Ceren Karahan, Salih Somuncu and Fikrettin Şahin

http://dx.doi.org/10.5772/intechopen.69905

**Abstract**

**1. Introduction**

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter


**Provisional chapter**
