**2.1 Adipose-derived stem cells**

*Clinical Implementation of Bone Regeneration and Maintenance*

**2. Sources of cells for tissue-engineered grafts**

interest for application of ADSCs in cell-based BTE.

respectively [11, 16].

better graft incorporation, osteoconductivity, osteoinductivity, and osteogenicity [5, 9–11]. Combining tissue scaffolds, growth factors, cells (especially stem cells), and gene therapies, along with three-dimensional printing and other new technological products, makes BTE a promising option. BTE has progressed over time, producing grafts with increasing ability to regenerate and repair bone [5–7, 9–12]. Cell-based treatment is emerging as a more promising approach in regenerative medicine. Cells (e.g., osteogenic or mesenchymal stem cells and others) are used to create new bone alone or are seeded onto a support matrix or scaffold to form bone tissue in vitro [6, 7, 9, 10, 13]. On these principles, engineered vascularized bone grafts can be created with some similarities to autografts [9, 10, 14]. Of particular interest in BTE is the application of mesenchymal stem cells because of their multipotency and the presence of osteogenic potential [6, 9, 10, 13, 15]. Cells can also be used as a vehicle for osteoinductive genes [6, 10]. Recent developments include ex vivo bioreactors capable at the very automated and controlled way to imitate in vivo environment producing bone with appropriate biomechanical properties before implantation [7, 9, 10, 12, 13]. To test the new features and products of BTE, as well as preclinical testing, many in vitro and in vivo methods and models have been developed with various advantages and disadvantages

Nowadays, adult stem cells can be purified from different adult tissues and used

in BTE as potential progenitors of osteogenic cells. It is reported that stem cells intended for BTE can be derived from many different tissues such as bone marrow, umbilical cord, dental pulp, as well as adipose tissue [17]. For instance, mesenchymal stem cells (MSCs) derived from bone marrow can be successfully differentiated into cells of various connective tissues [18] as well as in bone cells which give opportunity for its implementation in cell-based BTE [10, 19]. Despite its frequent use in BTE, bone marrow MSCs are reported to have some disadvantages, especially low cell quantity at isolation [20]. This is also an unfavorable characteristic for many other adult stem cells which imply in vitro cell expansion after their isolation in order to obtain sufficient quantity of cells which is mandatory for its further implementation in cell-based BTE. Morbidity and pain are also reported as accompanying side effects that are consequences of bone marrow MSC harvesting [20]. Nevertheless, MSCs are easier to isolate from accessible adipose tissue, which can be harvested with minimally harmful [21] and less painful methods. These multipotent cells derived from adipose tissue were reported by Zuk et al. [22] and suggested as a possible alternative to bone marrow MSCs [22]. Others also reported

that stromal cells derived from adipose tissue rapidly proliferate and can be

obtained in sufficient quantity, eliminating the need for in vitro expansion [23, 24] which is a tremendous advantage of adipose-derived stem cells (ADSCs) over other tissues that are potential sources of adult stem cells. The more recent study showed that bone morphogenic protein 2-transduced human adipose-derived MSCs had higher capacity for osteogenic differentiation than bone morphogenic protein 2-transduced MSCs from bone marrow [25]. With all this advantages, MSCs from adipose tissue are also reported to have similar potential to differentiate toward osteoblast and form bone as bone marrow MSCs [26]. Adipose tissue therefore represents a reliable source of adult stem cells intended for therapeutic purposes in BTE. In addition to this, in the past decade, we are witnessing an increase of

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The capacity to differentiate toward a broad spectrum of specialized cells such as bone cells, cartilage cells, muscle cells, endothelial cells, liver cells, neural cells, and others [17, 21, 27] makes ADSCs suitable generally for cell-based tissue engineering as well as for BTE. ADSCs represent a form of MSCs [28] that could be simply isolated from both subcutaneous and visceral adipose tissue that are usually sufficiently abundant in almost every individual. Nevertheless, it is reported that ADSCs derived from visceral fat have higher osteogenic potential than ADSCs from subcutaneous fat in rabbits [29] which should be taken into consideration when selecting the type of adipose tissue as starting source for ADSC isolation. Adipose tissue in humans could be obtained by liposuction or resection method that are both reported to yield similar quantity and good quality of MSCs [30]. More precisely, a direct source of ADSCs is stromal vascular fraction (SVF) which is obtained by enzymatic digestion of previously isolated fat tissue and its subsequent centrifugation [15]. There are well-described markers intended for ADSC characterization. Particularly, ADSCs are reported to have recognizable fibroblast-like, spindleshaped morphology [22, 26], good longevity and plastic adhesion properties in in vitro condition [31], and capacity for rapid proliferation in cell culture [23, 32]. ADSCs also have characteristic expression pattern of CD cell surface markers. In general, there is an accordance in literature about the expression pattern of CD markers which should be used for ADSC characterization. For instance, Cai et al. [27] summarized that some characteristic CDs, particularly CD166, CD105, CD90, CD73, CD44, CD29, and CD13, have high expression in adipose-derived stem cells [27] which is in accordance with other reviews [33]. Also, according to Bajek et al. (2016) high expressions of CD73, CD44, CD105 and CD90 are indications mainly used for adipose-derived stem cell identification [34]. Therefore, before further steps in their application and analyses, it is very important to examine and determine expression of CDs and other markers like cell morphology in order to characterize ADSCs.
