**3. Bone substitutes and regulatory factors**

As it is mentioned in the previous text, apart from cells, there are two more components in cell-based BTE, and those are bone substitute materials and regulatory factors. These three components together, combined in appropriate manner, makes specific construct which is an adequate alternative to bone grafts for the treatment of bone tissue defects. Bone substitutes are used as appropriate scaffolds for osteogenic cells. Chao et al. [12] described scaffold as logistic templates for guided formation of tissue [12]. Three-dimensional scaffolds that support osteogenic differentiation of the stem cells are seen as crucial components for in vitro engineering of bone construct which can be clinically usable [9]. Even more, to respond to special requirements at the defect site, engineering of the customized bone grafts was also proposed [35]. Bone substitute materials provide appropriate microenvironment for differentiation and proliferation of bone cells [36], and porosity, particle size, and material composition also play important roles [37]. At the first place, bone substitutes should be biocompatible which, among the other things, implies that they are non-toxic and non-genotoxic [38]. Biomaterials are also used to fill defects and compensate lost part of bone tissue at the defect site. Bone substitutes that belong to a group of bioactive biomaterials are capable for interaction with the biological environment and can provide conditions for cellular actions [39]

which is of benefit for bone regeneration. Nowadays, many different synthetic and natural bone substitutes are reported to be in use [10, 39, 40]. Particularly, materials based on hydroxyapatite and β-tricalcium phosphate are suitable for BTE [41]. In addition, materials based on bone mineral matrix are frequently used [42–45]. Also, regulatory factors are not less important components in BTE, and at the first place, they should induce and support osteogenic differentiation, adhesion, and proliferation of implanted cells. Because of its significant properties, here the focus will be on platelet-rich plasma as a source of regulatory factors for cell-based BTE. PRP is one of the well-known natural sources of different stimulative regulatory factors [46–48]. Last decade, PRPs are constantly drawing attention of many researchers in fields of regenerative biology as well as in BTE. That is reasonable because regulatory factors from PRP is reported to enhance adhesion, differentiation, and proliferation of the cells and also enhance angiogenesis [46, 49] which may support regeneration and reparation of bone tissue. In addition, it was reported that regulatory factors from PRP can enhance osteogenic process by inducing proliferation and differentiation of MSCs [50, 51]. Nevertheless, the question about adequate and stimulating concentration of platelets in prepared PRP is of great importance, and it is still topical. The opinions about adequate concentration of platelets in PRP intended for BTE are different. There are reports that higher platelet concentrations might have an inhibitory effect [52], and on the other hand, concentrations that are lower than the physiological level is reported to be useful for bone regeneration [53]. Finally, there is a recommendation that optimal concentration of platelets in PRP intended for bone treatment should be from four to eight folds higher than the normal physiological level of platelets in the blood [46]. Another advantage of activated PRP is that it can form fibrin fibers which can couple ADSCs with bone substitute material and improve retention of all construct components [54, 55] similar to reports where fibrin was used for this purpose [56, 57]. Finally, there is a growing interest about synergistic effects of ADSCs and PRP for bone regeneration. Many studies reported promising osteogenic potential of ADSCs and PRP combination [45, 54, 55, 58] and stimulative potential of PRP that improves osteogenesis in combination with cells [50, 59]. In addition, Fernandes and Yang [48] reviewed and summarized some recently published data which implies that adipose-derived stem cells obtained from human, mouse, rat and rabbit in combination with PRP in in vitro and/or in vivo conditions are related with outcomes which are of benefit for BTE [48].
