**Author details**

**7. Conclusion**

several experimentations [84].

284 Stem Cells in Clinical Practice and Tissue Engineering

**Acknowledgements**

**Conflict of interest**

of this research.

More clinical trials are needed to remove the obstacles [86].

Unfortunately, limiting factors still existed; most of them could be the differences of lab environment and human body such as unknown exact dose of BMP and applicate high dose of this material to creation of bony scaffold [83]. On the other hand, unpredictable effects of BMP and complications about application of BMP together with oncogenesis. Based on these evidences, Food and Drug Administration (FDA) restricted application of BMP to sinus alveolar process augmentation in the United States; other substitute materials in maxillofacial region should be considered moral issues even if these materials reveal high level of evidence in

Another problem like tissue transfer decreases the chance of high-quality and priced experimentations [85], and also Ripamonti et al. study revealed that growth factor and signaling systems in animal and human are totally different and huge variation between them is observed. Unfortunately, there are few clinical trials in the maxillofacial region, but the question is what the obstacles are? The main obstacles are how to predict regenerate cells to not become oncogenesis and produce our wanted cells, how to manage signaling factors to facilitate the procedure of regeneration, and how to create the scaffold that permits cell growth in the best way.

Tissue engineering is the field that is surrounded by other fields like histology, medical engineering, and pathology that every progress in these fields could change principles of tissue engineering. Our goals are simple which are to know how to regenerate human tissues from host cells and somehow that these regenerates have desirable function and esthetics. To reach to this goal, we have long way, but today engineers progressed biocompatible scaffolds, increase the flexibility to 3D tissue constructs, and designed complex tissue for the different facial areas. The latest progress guarantees that tissue engineering is the trustworthy choice for the treatment of maxillofacial defects. In future, the role of tissue engineering will increase and become routine in surgeries.

Conducting this review will not be possible without team work and cooperation. We have to thank people for their work in helping us through this way, authors of all referenced articles which are used for construction of this review, Dr. Tannaz Tafakori, Eng. MT Amirzade-

Authors declare that there is no conflict of interest that may damage the integrity and validity

Iranaq, and Mr. MS Amirzade-Iranaq with their search and companionship.

Seied Omid Keyhan1,2,3,4, Hamidreza Fallahi<sup>5</sup> , Alireza Jahangirnia6 , Seyed Mohammad Reza Masoumi<sup>7</sup> , Mohammad Hossein Khosravi7,8 and Mohammad Hosein Amirzade-Iranaq7,8,9,10\*

\*Address all correspondence to: h.amirzade@gmail.com

1 Department of Oral & Maxillofacial Surgery, Faculty of Dentistry, Birjand University of Medical Science, Birjand, Iran

2 Vice Presidential Organization of Technology of the Islamic Republic of Iran, Iran

3 Stem Cell & Regenerative Medicine Network, Shahid Beheshti University of Medical Sciences, Tehran, Iran

4 Cranio Maxillofacial Research Center, Tehran Dental Branch, Islamic Azad University, Tehran, Iran

5 Oral and Maxillofacial Surgery, Jundishapur University of Medical Sciences, Ahvaz, Iran

6 Private Practice, Tehran, Iran

7 Student Research Committee (SRC), Baqiyatallah University of Medical Sciences, Tehran, Iran

8 International Otorhinolaryngology Research Association (IORA), Universal Scientific Education and Research Network (USERN), Tehran, Iran

9 Student Research Committee, Shahid Sadoughi University of Medical Sciences, Yazd, Iran

10 Universal Network of Interdisciplinary Research in Oral and Maxillofacial Surgery (UNIROMS), Universal Scientific Education and Research Network (USERN), Tehran, Iran

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290 Stem Cells in Clinical Practice and Tissue Engineering


**Chapter 14**

**Provisional chapter**

**Human Adipose-Derived Stem Cells for Tissue**

**Human Adipose-Derived Stem Cells for Tissue** 

**Perspectives**

**Perspectives**

Marieta Costache

**Abstract**

**1. Introduction**

Marieta Costache

Sorina Dinescu, Anca Hermenean and

Sorina Dinescu, Anca Hermenean and

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

Additional information is available at the end of the chapter

the quality of life in patients with tissue defects.

medicine, stem cell differentiation

differentiation potential of stem cells.

Additional information is available at the end of the chapter

**Engineering Approaches: Current Challenges and**

Human adipose-derived stem cells (hASCs) currently represent a viable source of mesenchymal*-like* stem cells, with similar properties and differentiation potential to bonemarrow-derived mesenchymal stem cells (BM-MSCs) but with a different and more accessible source—the adipose tissue. hASCs are able to produce almost all of the factors that contribute to normal wound healing, and therefore, they are preferred for all types of tissue engineering (TE) and regenerative medical applications. This chapter will review hASCs regeneration potential and the most modern approaches in TE for bone, cartilage and adipose tissue regeneration using hASCs. Furthermore, an overview of novel and original hASCs-scaffold constructs studied in our group completes an up-to-date presentation of hASCs multiple uses. Additionally, this chapter will highlight the relevance of ultimate advances in regenerative medicine and the need for this evolution to increase

**Keywords:** human adipose-derived stem cells, tissue engineering, regenerative

**Engineering Approaches: Current Challenges and** 

DOI: 10.5772/intechopen.68712

© 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.

Current tissue engineering (TE) approaches registered an evolution over the past few years in the area of regenerative medicine. If the last decade was dedicated to repair and reconstructive procedures involving the implantation of inert materials to solve tissue defects, the modern trend uses regenerative strategies frequently based on advanced biomaterials and the

**Provisional chapter**

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

**Human Adipose-Derived Stem Cells for Tissue** 

DOI: 10.5772/intechopen.68712

Sorina Dinescu, Anca Hermenean and Marieta Costache Marieta Costache Additional information is available at the end of the chapter

Sorina Dinescu, Anca Hermenean and

Additional information is available at the end of the chapter

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

#### **Abstract**

Human adipose-derived stem cells (hASCs) currently represent a viable source of mesenchymal*-like* stem cells, with similar properties and differentiation potential to bonemarrow-derived mesenchymal stem cells (BM-MSCs) but with a different and more accessible source—the adipose tissue. hASCs are able to produce almost all of the factors that contribute to normal wound healing, and therefore, they are preferred for all types of tissue engineering (TE) and regenerative medical applications. This chapter will review hASCs regeneration potential and the most modern approaches in TE for bone, cartilage and adipose tissue regeneration using hASCs. Furthermore, an overview of novel and original hASCs-scaffold constructs studied in our group completes an up-to-date presentation of hASCs multiple uses. Additionally, this chapter will highlight the relevance of ultimate advances in regenerative medicine and the need for this evolution to increase the quality of life in patients with tissue defects.

**Keywords:** human adipose-derived stem cells, tissue engineering, regenerative medicine, stem cell differentiation

### **1. Introduction**

Current tissue engineering (TE) approaches registered an evolution over the past few years in the area of regenerative medicine. If the last decade was dedicated to repair and reconstructive procedures involving the implantation of inert materials to solve tissue defects, the modern trend uses regenerative strategies frequently based on advanced biomaterials and the differentiation potential of stem cells.

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. © 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.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

The basic principle underlying this modern tissue engineering concept is that an equilibrium should be established simultaneously between the elements required to obtain a functional engineered tissue—suitable cells, appropriate scaffolds and the adequate signalling molecules. The goal of tissue engineering is to recreate a microenvironment as similar as possible to the *in vivo* natural tissue.

such as bioprinting, patterning, self-assembling and organ-on-a-chip [10]. According to Dai et al., most of these approaches have been employed to encapsulate hASCs in 3D structures

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

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

295

Related to *in vivo* experiments using hASCs, it was both experimentally and clinically shown that the topical administration of hASCs to full-thickness radiated wounds increases the healing rate of the wound [4]. It was also shown that hASCs stimulate fibroblast proliferation and migration and type I collagen secretion in an *in vivo* wound model. These findings suggest

hASCs are currently recognized as an attractive and efficient adult stem cell type for regenerative medicine. Still, there are problems that need to be clarified including the mechanisms of the interactions among hASCs and their long-term safety. Only a small number of clinical

The majority of clinical trials involving hASCs or hASCs-enriched fat grafts are incipient phase clinical trials (phase I or II), while only one trial reached phase IV in human subjects (NCT00616135). Several directions were approached by clinicians in order to test the efficiency of hASCs in tissue regeneration. Breast reconstruction is one of the major applications approached by clinicians, not only for breast augmentation purposes (NCT01771913) but also for breast reconstruction after tumour resection (NCT00616135). Special care should be given for implantation of hASCs in a former tumour microenvironment, since little is known about hASCs stability at genomic level. Both clinical trials resulted in a favourable primary outcome. However, this direction is considered to have limitations since the implanted fat graft suffered resorption over time and thus the implant shape and dimension altered after 6–12 months. Additional successful applications where hASCs were involved are liver tissue reconstruction (NCT01062750), cardiovascular disease (NCT01449032; NCT00442806; NCT00426868; NCT01216995), osteoarthritis (NCT01585857) or brain injury (NCT01649700). All results showed hASCs display high therapeutic potential and generally display safety for

Considering all the abovementioned, the chapter will further present a few biomaterial compositions that we used in conjunction with hASCs and stimulating factors. In particular, we have studied the potential of hASCs differentiation towards the adipose, cartilage and bone lineages during *in vitro* studies in different 3D original scaffolds. For adipose tissue engineering (ATE), we have identified novel biomaterials based on gelatin-alginate-polyacrylamide (GAPAA) [11] and collagen-sericin (CollSS) [12] which proved to be efficient for soft tissue reconstruction. In parallel, the addition of hyaluronic acid and chondroitin sulphate proved to increase the quality of the CollSS hydrogel and to transform it in a scaffold designed for cartilage tissue engineering (CTE) [13]. We have also tested and validated different scaffolds based on chitosan, polyvinyl alcohol, polysulphone, etc. each improved by the addition of graphene oxide (GO), giving stability to the structure. An appropriate concentration of GO in scaffolds composition resulted in significantly better cell differentiation towards bone tissue. Both chitosan/GO biomaterials and improved collagen scaffolds for cartilage repair were tested for response during *in vivo* studies on mouse models. Results supported the conclusions

(resulting in 3D culture systems) specifically designed for a TE application.

that hASCs may promote *in vivo* wound healing.

trials have been performed by now [3].

*in vivo* implantation.

Human adipose-derived stem cells (hASCs) represent a viable source of mesenchymal*like* stem cells, with similar properties to bone-marrow-derived mesenchymal stem cells (BM-MSCs) but with a different and more accessible source—the adipose tissue. A particular population of stem cells with self-renewal properties and multilineage differentiation potential, isolated from the adipose stromal-vascular compartment, was first reported by Zuk et al. [1]. In further studies, it has been proved that these cells display (i) a hypoimmunogenic profile [2], (ii) can be easily harvested from subcutaneous adipose tissue during liposuction procedures [3] and (iii) since they can be isolated from autologous fat, there is no ethical issue involved in case of transplantation for regenerative purposes. All these properties that hASCs display make them a more viable solution for regenerative medicine approaches then MSCs or other adult stem cells.

Apart from the fact they can be more easily harvested than MSCs, hASCs are able to produce almost all of the factors that contribute to normal wound healing [4]. Consequently, at the injury site, implanted cells that undergo differentiation generate not only an inert filling tissue but also are able to stimulate cell recruitment from stem cell niches in order to aesthetically restore the site of injury in a paracrine manner (by secretion of growth factors and cytokines) [5]. These observations suggest that hASCs could be better candidates for TE applications than other traditional cell sources.

*hASCs' clinical and TE applications:* Modern regenerative therapies use hASCs, based on their abundance, distribution and multilineage differentiation ability. There are certain studies that put, under a question mark, the safety issues related to hASCs use for regeneration purposes, since several genetic abnormalities after their *in vitro* expansion or differentiation have been reported by several groups [6–8]. However, no interdiction of hASCs in clinical practice for TE has been yet announced.

Regarding the scaffolds appropriate for TE applications, there has been an evolution in the composition and properties of biomaterials in the last decade. Nowadays, biomaterials tend to be made of natural and biodegradable compounds, thus favouring their biocompatibility. In addition, a 3D scaffold displays a significantly increase in capacity to closely mimic *in vivo* cellular microenvironments [9, 10].

Different biomaterials have been used in studies to favour hASCs growth in 3D scaffolds. hASCs displayed potential to attach, proliferate and differentiate in contact with favourable biomaterial compositions. Ideally, biofabricated scaffolds should offer hASCs proper environments to facilitate their proliferation and maintain their differentiation potentials [3]. Besides the biocompatibility condition, these materials should also be synthesized to have highly porous structures with interconnected architecture to mimic the native tissue niche [3]. Science in the field of scaffold engineering has evolved towards biofabrication using modern approaches such as bioprinting, patterning, self-assembling and organ-on-a-chip [10]. According to Dai et al., most of these approaches have been employed to encapsulate hASCs in 3D structures (resulting in 3D culture systems) specifically designed for a TE application.

The basic principle underlying this modern tissue engineering concept is that an equilibrium should be established simultaneously between the elements required to obtain a functional engineered tissue—suitable cells, appropriate scaffolds and the adequate signalling molecules. The goal of tissue engineering is to recreate a microenvironment as similar as possible

Human adipose-derived stem cells (hASCs) represent a viable source of mesenchymal*like* stem cells, with similar properties to bone-marrow-derived mesenchymal stem cells (BM-MSCs) but with a different and more accessible source—the adipose tissue. A particular population of stem cells with self-renewal properties and multilineage differentiation potential, isolated from the adipose stromal-vascular compartment, was first reported by Zuk et al. [1]. In further studies, it has been proved that these cells display (i) a hypoimmunogenic profile [2], (ii) can be easily harvested from subcutaneous adipose tissue during liposuction procedures [3] and (iii) since they can be isolated from autologous fat, there is no ethical issue involved in case of transplantation for regenerative purposes. All these properties that hASCs display make them a more viable solution for regenerative medicine approaches then MSCs

Apart from the fact they can be more easily harvested than MSCs, hASCs are able to produce almost all of the factors that contribute to normal wound healing [4]. Consequently, at the injury site, implanted cells that undergo differentiation generate not only an inert filling tissue but also are able to stimulate cell recruitment from stem cell niches in order to aesthetically restore the site of injury in a paracrine manner (by secretion of growth factors and cytokines) [5]. These observations suggest that hASCs could be better candidates for TE applications

*hASCs' clinical and TE applications:* Modern regenerative therapies use hASCs, based on their abundance, distribution and multilineage differentiation ability. There are certain studies that put, under a question mark, the safety issues related to hASCs use for regeneration purposes, since several genetic abnormalities after their *in vitro* expansion or differentiation have been reported by several groups [6–8]. However, no interdiction of hASCs in clinical practice for

Regarding the scaffolds appropriate for TE applications, there has been an evolution in the composition and properties of biomaterials in the last decade. Nowadays, biomaterials tend to be made of natural and biodegradable compounds, thus favouring their biocompatibility. In addition, a 3D scaffold displays a significantly increase in capacity to closely mimic *in vivo*

Different biomaterials have been used in studies to favour hASCs growth in 3D scaffolds. hASCs displayed potential to attach, proliferate and differentiate in contact with favourable biomaterial compositions. Ideally, biofabricated scaffolds should offer hASCs proper environments to facilitate their proliferation and maintain their differentiation potentials [3]. Besides the biocompatibility condition, these materials should also be synthesized to have highly porous structures with interconnected architecture to mimic the native tissue niche [3]. Science in the field of scaffold engineering has evolved towards biofabrication using modern approaches

to the *in vivo* natural tissue.

294 Stem Cells in Clinical Practice and Tissue Engineering

or other adult stem cells.

than other traditional cell sources.

TE has been yet announced.

cellular microenvironments [9, 10].

Related to *in vivo* experiments using hASCs, it was both experimentally and clinically shown that the topical administration of hASCs to full-thickness radiated wounds increases the healing rate of the wound [4]. It was also shown that hASCs stimulate fibroblast proliferation and migration and type I collagen secretion in an *in vivo* wound model. These findings suggest that hASCs may promote *in vivo* wound healing.

hASCs are currently recognized as an attractive and efficient adult stem cell type for regenerative medicine. Still, there are problems that need to be clarified including the mechanisms of the interactions among hASCs and their long-term safety. Only a small number of clinical trials have been performed by now [3].

The majority of clinical trials involving hASCs or hASCs-enriched fat grafts are incipient phase clinical trials (phase I or II), while only one trial reached phase IV in human subjects (NCT00616135). Several directions were approached by clinicians in order to test the efficiency of hASCs in tissue regeneration. Breast reconstruction is one of the major applications approached by clinicians, not only for breast augmentation purposes (NCT01771913) but also for breast reconstruction after tumour resection (NCT00616135). Special care should be given for implantation of hASCs in a former tumour microenvironment, since little is known about hASCs stability at genomic level. Both clinical trials resulted in a favourable primary outcome. However, this direction is considered to have limitations since the implanted fat graft suffered resorption over time and thus the implant shape and dimension altered after 6–12 months. Additional successful applications where hASCs were involved are liver tissue reconstruction (NCT01062750), cardiovascular disease (NCT01449032; NCT00442806; NCT00426868; NCT01216995), osteoarthritis (NCT01585857) or brain injury (NCT01649700). All results showed hASCs display high therapeutic potential and generally display safety for *in vivo* implantation.

Considering all the abovementioned, the chapter will further present a few biomaterial compositions that we used in conjunction with hASCs and stimulating factors. In particular, we have studied the potential of hASCs differentiation towards the adipose, cartilage and bone lineages during *in vitro* studies in different 3D original scaffolds. For adipose tissue engineering (ATE), we have identified novel biomaterials based on gelatin-alginate-polyacrylamide (GAPAA) [11] and collagen-sericin (CollSS) [12] which proved to be efficient for soft tissue reconstruction. In parallel, the addition of hyaluronic acid and chondroitin sulphate proved to increase the quality of the CollSS hydrogel and to transform it in a scaffold designed for cartilage tissue engineering (CTE) [13]. We have also tested and validated different scaffolds based on chitosan, polyvinyl alcohol, polysulphone, etc. each improved by the addition of graphene oxide (GO), giving stability to the structure. An appropriate concentration of GO in scaffolds composition resulted in significantly better cell differentiation towards bone tissue.

Both chitosan/GO biomaterials and improved collagen scaffolds for cartilage repair were tested for response during *in vivo* studies on mouse models. Results supported the conclusions obtained *in vitro* and confirmed the efficiency of these differentiation-specific cell-scaffold systems. However, only a small number of studies have addressed *in vivo* applications involving ASCs. More studies need to be developed in this direction in order to evaluate the good and the bad potential of hASCs.

tissues [20]. In many experiments, hASCs administered at a lesion site have stimulated the development of granulation tissue and increased cell density and traumatic wound re-epithelialization [20, 21]. Recent research developments have shown that hASCs produce a volume of cytokines and growth factors superior to BM-MSCs and to human dermal fibroblasts, thus

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

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

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Since fat tissue actively intervenes in the endocrine processes, paracrine action of transplanted hASCs can negatively influence peripheral tissues. For example, the secretion of active hormones such as leptin, certain cytokines such as IL-6 and TNF-α or of certain growth factors

Most organs display "reservoirs" of adult stem cells that are activated in case of trauma, infection or disease [24]. In many of these cases, the endogenous stem cell populations are insufficient to cope with compromised tissue regeneration process, and therefore, modern strategies in the field of regenerative medicine involve the use of exogenous stem cells. Restoration of damaged structures, as well as the resumption of the restored tissue functionality, was associated with stem cells ability to adopt a specific phenotype through differentiation and to

Numerous studies showed that hASCs have the potential to differentiate into bone, cartilage and muscle, as well as adipose and neural tissue [15, 26, 27]. This ability to differentiate towards different mesenchymal lineages has stimulated interest in their clinical use. hASCs have also been used for breast augmentation and to treat congenital deformities and other defects as well as for reconstruction after mastectomy [28]. Immunomodulatory molecules, growth factors, angiogenic and antifibrotic factors released by hASCs, matrix metalloproteinases and collagen stimulate the regeneration and remodelling of altered structures [29], whereas secreted anti-apoptotic molecules and antioxidants protect cells in proximity [30]. The most important differentiation pathways reported for hASCs will be further described:

Adipogenic differentiation is induced *in vitro* by treatment with a cocktail based on dexamethasone, indomethacin, isobutyl-methyl-xanthine [31] and insulin [32]. The mixture stimulates expression of the receptor peroxisome proliferation-activated receptor ɣ2 (PPAR-ɣ2), key inducer of adipogenic differentiation, which, once activated, triggers the transcription of a set of genes involved in differentiation of terminal adipocytes [33]. These include genes encoding the synthase fatty acid (FAS), the protein binding of fatty acids P2 (aP2), perilipin marker of adipogenic differentiation, lipoprotein lipase (LPL), the carrier protein fatty acid-1 (FATP-1) and adipocytokines (adiponectin, leptin and resistin). All of these events stimulate the lipid

Paracrine action of MSCs plays a key role in the modulation of adipogenic differentiation. Thus, BMP, with cytokine function, stimulates *in vivo* adipogenesis. Bone morphogenetic

*2.2.1. Adipogenic differentiation and adipose tissue engineering involving hASCs*

metabolism, leading in the end to the formation of intracellular lipid vesicles [34].

can lead to a disturbance of the peripheral metabolic pathways [23].

being ideal tools in tissue regeneration [22].

**2.2. hASCs regenerative potential**

paracrine actions of stem cells [25].
