**4.1 One-step versus multistep approaches**

Numerous literature data in the past decade are related with extensive studies about regenerative potential of adipose-derived stem cells. It is well-known that ADSCs in combination with bone substitute materials and regulatory factors possess certain osteogenic potential that can initiate and boost osteogenic process both in orthotopic and ectopic conditions. Recent studies showed that good yield of ADSCs after isolation from adipose tissue provides opportunity for its subsequent application in one-step approach in cell-based bone tissue engineering without previous in vitro expansion and differentiation. Nevertheless, standard approach in cell-based BTE usually involves in vitro pretreatment of ADSCs as an additional step before its final application in order to induce differentiation of ADSCs into osteogenic cells. Literature data showed that both approaches are promising for implementation in treatment of bone tissue defects. Our experience about

**185**

preparation.

*Application of Adipose-Derived Stem Cells in Treatment of Bone Tissue Defects*

great importance for treatment of bone defects (**Figure 1**).

Also, it is reported that the entire procedure can be finished in a few hours [13, 56, 64] which reduces wait time for surgery [60] and is one of the major advantages of this approach. In addition, Jurgens et al. [61] showed that ADSCs can adhere promptly onto materials collagen type I/III and poly(L-lactide-cocaprolactone); particularly it is reported that nearly 10 minutes is sufficient time for ADSC adhesion [61] that significantly reduces time needed for construct

On the other hand, there are many well-described approaches which involve several steps for in vitro expansion and osteogenic differentiation of ADSCs before its final application [26, 45, 65]. In these approaches, by in vitro cultivation, ADSCs are additionally expanded and purified from heterogenous SVF [32] that are directly derived from adipose tissue (**Figure 1**). After that step, purified ADSCs are subjected to in vitro induction in osteogenic media to differentiate toward osteogenic cells before further application which follows in the next step. The characteristic components of osteogenic medium are dexamethasone, ascorbic acid, and β-glycerophosphate [31, 33, 66] which are frequently used for ADSC osteoinduction [67]. There are different data about the duration of in vitro osteogenic induction needed for ADSC differentiation toward osteogenic cells, but literature data mostly referred to 2 weeks [22, 33, 66, 68, 69] or between 2 and 3 weeks [21] (**Figure 2**). It was summarized that during osteogenic differentiation, ADSCs start expression of lineage-specific genes for osteogenesis such as osteocalcin, transcription factor osterix, transcription factor Runx2, bone sialoprotein, alkaline phosphatase, and others [32, 40] which might be detected by gene expression analysis [61] as confirmation of successful osteogenic differentiation of ADSCs. For instance,

osteogenic capacity of differently prepared ADSCs is mostly in accordance with other related studies. Based on our previously performed experimental studies and published results, these two approaches have quite different outcomes, and each approach has its advantages and potential to be successfully applied in treatment of bone tissue defects. Also, there is a growing number of researches that employed

There are well-described methods for ADSC preparation prior their application in BTE. Without purification, in vitro expansion, and osteoinduction, ADSCs contained in freshly isolated adipose-derived SVF could be prepared and applied in just one step which is described as intraoperative approach [56, 57, 60] or onestep procedure [61]. Intraoperative approach implies construct assembling during surgical procedure [13] by combining cells, bone substitutes, and regulatory factors together in one construct. One of the earlier reports by Aslan et al. [62] presented usage of noncultured human MSCs isolated from bone marrow [62]. More recently published articles demonstrated intraoperative application of freshly isolated SVF cells from human adipose tissue [56, 57] and freshly isolated adipose-derived SVF cells from mice epididymal adipose tissue in ectopic bone-forming model [55]. One-step surgical procedure is also used in oral and maxillofacial surgery for maxillary sinus floor elevation [37, 63]. All these studies suggest that previous in vitro pretreatment of ADSCs and their separation from SVF population prior their implementation in BTE is not necessary. Good yield of ADSCs enables bypassing of in vitro expansion step which goes in favor of the one-step concept in BTE. In addition, the autotransplantation of freshly isolated ADSCs for the treatment of bone tissue defects also enables avoiding problems with immune response [60] that might occur in allotransplantations and xenotransplantations. Adipose-derived SVF is reported to have great capacity for regeneration processes thanks to its heterogeneity [28] because it is composed of different cell types and growth factors [15], especially with cells that have osteogenic and angiogenic potential [37] which is of

*DOI: http://dx.doi.org/10.5772/intechopen.92897*

bioreactors for bone graft engineering.

#### *Application of Adipose-Derived Stem Cells in Treatment of Bone Tissue Defects DOI: http://dx.doi.org/10.5772/intechopen.92897*

osteogenic capacity of differently prepared ADSCs is mostly in accordance with other related studies. Based on our previously performed experimental studies and published results, these two approaches have quite different outcomes, and each approach has its advantages and potential to be successfully applied in treatment of bone tissue defects. Also, there is a growing number of researches that employed bioreactors for bone graft engineering.

There are well-described methods for ADSC preparation prior their application in BTE. Without purification, in vitro expansion, and osteoinduction, ADSCs contained in freshly isolated adipose-derived SVF could be prepared and applied in just one step which is described as intraoperative approach [56, 57, 60] or onestep procedure [61]. Intraoperative approach implies construct assembling during surgical procedure [13] by combining cells, bone substitutes, and regulatory factors together in one construct. One of the earlier reports by Aslan et al. [62] presented usage of noncultured human MSCs isolated from bone marrow [62]. More recently published articles demonstrated intraoperative application of freshly isolated SVF cells from human adipose tissue [56, 57] and freshly isolated adipose-derived SVF cells from mice epididymal adipose tissue in ectopic bone-forming model [55]. One-step surgical procedure is also used in oral and maxillofacial surgery for maxillary sinus floor elevation [37, 63]. All these studies suggest that previous in vitro pretreatment of ADSCs and their separation from SVF population prior their implementation in BTE is not necessary. Good yield of ADSCs enables bypassing of in vitro expansion step which goes in favor of the one-step concept in BTE. In addition, the autotransplantation of freshly isolated ADSCs for the treatment of bone tissue defects also enables avoiding problems with immune response [60] that might occur in allotransplantations and xenotransplantations. Adipose-derived SVF is reported to have great capacity for regeneration processes thanks to its heterogeneity [28] because it is composed of different cell types and growth factors [15], especially with cells that have osteogenic and angiogenic potential [37] which is of great importance for treatment of bone defects (**Figure 1**).

Also, it is reported that the entire procedure can be finished in a few hours [13, 56, 64] which reduces wait time for surgery [60] and is one of the major advantages of this approach. In addition, Jurgens et al. [61] showed that ADSCs can adhere promptly onto materials collagen type I/III and poly(L-lactide-cocaprolactone); particularly it is reported that nearly 10 minutes is sufficient time for ADSC adhesion [61] that significantly reduces time needed for construct preparation.

On the other hand, there are many well-described approaches which involve several steps for in vitro expansion and osteogenic differentiation of ADSCs before its final application [26, 45, 65]. In these approaches, by in vitro cultivation, ADSCs are additionally expanded and purified from heterogenous SVF [32] that are directly derived from adipose tissue (**Figure 1**). After that step, purified ADSCs are subjected to in vitro induction in osteogenic media to differentiate toward osteogenic cells before further application which follows in the next step. The characteristic components of osteogenic medium are dexamethasone, ascorbic acid, and β-glycerophosphate [31, 33, 66] which are frequently used for ADSC osteoinduction [67]. There are different data about the duration of in vitro osteogenic induction needed for ADSC differentiation toward osteogenic cells, but literature data mostly referred to 2 weeks [22, 33, 66, 68, 69] or between 2 and 3 weeks [21] (**Figure 2**). It was summarized that during osteogenic differentiation, ADSCs start expression of lineage-specific genes for osteogenesis such as osteocalcin, transcription factor osterix, transcription factor Runx2, bone sialoprotein, alkaline phosphatase, and others [32, 40] which might be detected by gene expression analysis [61] as confirmation of successful osteogenic differentiation of ADSCs. For instance,

*Clinical Implementation of Bone Regeneration and Maintenance*

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

**4. Approaches in application of ADSCs in cell-based BTE**

Numerous literature data in the past decade are related with extensive studies about regenerative potential of adipose-derived stem cells. It is well-known that ADSCs in combination with bone substitute materials and regulatory factors possess certain osteogenic potential that can initiate and boost osteogenic process both in orthotopic and ectopic conditions. Recent studies showed that good yield of ADSCs after isolation from adipose tissue provides opportunity for its subsequent application in one-step approach in cell-based bone tissue engineering without previous in vitro expansion and differentiation. Nevertheless, standard approach in cell-based BTE usually involves in vitro pretreatment of ADSCs as an additional step before its final application in order to induce differentiation of ADSCs into osteogenic cells. Literature data showed that both approaches are promising for implementation in treatment of bone tissue defects. Our experience about

**4.1 One-step versus multistep approaches**

**184**

for BTE [48].

#### **Figure 1.**

*Expansion and purification of ADSCs from adipose-derived SVF through in vitro cultivation. (a) Adiposederived SVF 24 h after isolation; (b) adipose-derived SVF 72 h after isolation; (c) adipose-derived SVF 7 days after isolation; and (d) ADSCs the first day after the first passage. Black arrows show ADSCs. Magnification 100×.*

#### **Figure 2.**

*In vitro osteoinduction of ADSCs. (a) ADSCs 3 days after cultivation in osteogenic media; (b) ADSCs 2 weeks after cultivation in osteogenic media. Black arrows show ADSCs. Magnification 100×.*

Cvetković et al. [45] recently published that bone-related genes osteocalcin, transcription factor osterix, alkaline phosphatase, and collagen I alpha1 chain had the highest expression in in vitro osteoinduced ADSCs at 15th day of osteoinduction [45]. Also, it is reported that changes in cell morphology during osteogenic induction to a more cuboidal shape could be observed in ADSC culture [69]. The presence of mineralization signs and proliferation of the ADSCs are also reported as markers of osteogenic differentiation during in vitro osteoinduction [65, 69, 70]. Particularly, after in vitro osteogenic differentiation, mineralization of the cell matrix could be evaluated using Von Kossa and alizarin red staining [31, 66]. In the next step, when osteoinduction

**187**

*Application of Adipose-Derived Stem Cells in Treatment of Bone Tissue Defects*

is confirmed, osteoinduced ADSCs are seeded on bone substitute material and supplemented with regulatory factors prior using in BTE. There are examples where prepared constructs, after ADSCs seeding on biomaterial (i.e. bone substitute material), are subjected to in vitro osteoinduction before implantation [26] which requires additional time for construct preparation and delays final application. Overall, in vitro osteoinduction is proven to be an effective method for preparation of the ADSCs, and that is confirmed in many different studies [26, 45, 65] which makes this method suitable for BTE. But methods that consider at least two steps usually few weeks for performing and additional material for preparation of ADSCs are timeand money-consuming and more complicated to perform than the one-step method. According to literature data, both in vitro osteoinduced ADSCs and untreated freshly isolated ADSCs are capable to induce osteogenesis to some level in orthotopic as well as in ectopic conditions. However, there are differences in their capacity to initiate and maintain osteogenic process. By comparing our two recently published studies, it could be concluded that ADSCs prepared and utilized in different manners induced different expression patterns of analyzed osteogenic markers in ectopic implants [45, 55]. In one of the studies, it was shown that untreated ADSCs contained in freshly isolated SVF are capable to quickly initiate osteogenic process, but between the 4th and 8th week of implantation, decreasing bone-related gene expression was detected [55]. On the other hand, Cvetković et al. [45] reported that in vitro osteoinduced ADSCs cause steady osteogenesis with peak at the 8th week [45]. In addition, there is a study which confirmed that osteogenically differentiated human ADSCs induced forming of bone tissue after 8 weeks in ectopic condition [26]; thus the application of osteoinduced ADSCs seems to have a favorable effect on osteogenesis. In other words, all these findings indicate that freshly isolated untreated ADSCs cannot maintain osteogenesis in ectopic condition to that extent as in vitro osteoinduced ADSCs can do. Previous osteoinduction triggered differentiation of ADSCs toward osteogenic cells which had sufficient potential to start and maintain osteogenesis for a longer period [45]. It was concluded that one of the reasons why untreated ADSCs within SVF failed to maintain osteogenic process for a longer period may be because of the lack of osteogenic factors in the ectopic environment which was used as model in this study [55]. Bone tissue normally had factors such as cytokines, mechanotransduction, and closeness of osteoprogenitor cells that are reported to act stimulative in bone formation, but they are reduced in ectopic bone-forming models [71]. Therefore, these factors are characteristic only for orthotopic models which allow evaluation of osteogenic potential of examined engineered construct in real natural milieu of the bone tissue. For that purpose the frequently used orthotopic models in BTE are criticalsized defects in calvaria bone [23] and long bones [44, 72, 73] where large rodents and other mammals are suitable. In addition, it was reported that the presence of sufficient doses of osteogenic factors are needed to support osteogenic differentia-

tion of implanted cells and bone formation in ectopic conditions [56, 64].

Deficiency of osteogenic factors in ectopic models may be bypassed to some extent by addition of stimulating factors to ADSCs such as activated PRP. The addition of activated PRP can make microenvironmental conditions similar to the natural ones that occur during bone trauma. But it is known that platelets release a major portion of regulatory factors immediately after injury [74], which might be sufficient to stimulate ADSCs immediately after its implantation but definitely not for a longer period. The manner and applied doses of PRP obviously was enough to support development and maintenance of osteogenesis guided by previously in vitro osteoinduced ADSCs [45] and was not sufficient to support maintenance of osteogenic process guided by untreated ADSCs within SVF [55]. Regarding the short period of efficient action of PRP, Fernandes and Yang [48] reported that

*DOI: http://dx.doi.org/10.5772/intechopen.92897*

#### *Application of Adipose-Derived Stem Cells in Treatment of Bone Tissue Defects DOI: http://dx.doi.org/10.5772/intechopen.92897*

is confirmed, osteoinduced ADSCs are seeded on bone substitute material and supplemented with regulatory factors prior using in BTE. There are examples where prepared constructs, after ADSCs seeding on biomaterial (i.e. bone substitute material), are subjected to in vitro osteoinduction before implantation [26] which requires additional time for construct preparation and delays final application. Overall, in vitro osteoinduction is proven to be an effective method for preparation of the ADSCs, and that is confirmed in many different studies [26, 45, 65] which makes this method suitable for BTE. But methods that consider at least two steps usually few weeks for performing and additional material for preparation of ADSCs are timeand money-consuming and more complicated to perform than the one-step method.

According to literature data, both in vitro osteoinduced ADSCs and untreated freshly isolated ADSCs are capable to induce osteogenesis to some level in orthotopic as well as in ectopic conditions. However, there are differences in their capacity to initiate and maintain osteogenic process. By comparing our two recently published studies, it could be concluded that ADSCs prepared and utilized in different manners induced different expression patterns of analyzed osteogenic markers in ectopic implants [45, 55]. In one of the studies, it was shown that untreated ADSCs contained in freshly isolated SVF are capable to quickly initiate osteogenic process, but between the 4th and 8th week of implantation, decreasing bone-related gene expression was detected [55]. On the other hand, Cvetković et al. [45] reported that in vitro osteoinduced ADSCs cause steady osteogenesis with peak at the 8th week [45]. In addition, there is a study which confirmed that osteogenically differentiated human ADSCs induced forming of bone tissue after 8 weeks in ectopic condition [26]; thus the application of osteoinduced ADSCs seems to have a favorable effect on osteogenesis. In other words, all these findings indicate that freshly isolated untreated ADSCs cannot maintain osteogenesis in ectopic condition to that extent as in vitro osteoinduced ADSCs can do. Previous osteoinduction triggered differentiation of ADSCs toward osteogenic cells which had sufficient potential to start and maintain osteogenesis for a longer period [45]. It was concluded that one of the reasons why untreated ADSCs within SVF failed to maintain osteogenic process for a longer period may be because of the lack of osteogenic factors in the ectopic environment which was used as model in this study [55]. Bone tissue normally had factors such as cytokines, mechanotransduction, and closeness of osteoprogenitor cells that are reported to act stimulative in bone formation, but they are reduced in ectopic bone-forming models [71]. Therefore, these factors are characteristic only for orthotopic models which allow evaluation of osteogenic potential of examined engineered construct in real natural milieu of the bone tissue. For that purpose the frequently used orthotopic models in BTE are criticalsized defects in calvaria bone [23] and long bones [44, 72, 73] where large rodents and other mammals are suitable. In addition, it was reported that the presence of sufficient doses of osteogenic factors are needed to support osteogenic differentiation of implanted cells and bone formation in ectopic conditions [56, 64].

Deficiency of osteogenic factors in ectopic models may be bypassed to some extent by addition of stimulating factors to ADSCs such as activated PRP. The addition of activated PRP can make microenvironmental conditions similar to the natural ones that occur during bone trauma. But it is known that platelets release a major portion of regulatory factors immediately after injury [74], which might be sufficient to stimulate ADSCs immediately after its implantation but definitely not for a longer period. The manner and applied doses of PRP obviously was enough to support development and maintenance of osteogenesis guided by previously in vitro osteoinduced ADSCs [45] and was not sufficient to support maintenance of osteogenic process guided by untreated ADSCs within SVF [55]. Regarding the short period of efficient action of PRP, Fernandes and Yang [48] reported that

*Clinical Implementation of Bone Regeneration and Maintenance*

Cvetković et al. [45] recently published that bone-related genes osteocalcin, transcription factor osterix, alkaline phosphatase, and collagen I alpha1 chain had the highest expression in in vitro osteoinduced ADSCs at 15th day of osteoinduction [45]. Also, it is reported that changes in cell morphology during osteogenic induction to a more cuboidal shape could be observed in ADSC culture [69]. The presence of mineralization signs and proliferation of the ADSCs are also reported as markers of osteogenic differentiation during in vitro osteoinduction [65, 69, 70]. Particularly, after in vitro osteogenic differentiation, mineralization of the cell matrix could be evaluated using Von Kossa and alizarin red staining [31, 66]. In the next step, when osteoinduction

*In vitro osteoinduction of ADSCs. (a) ADSCs 3 days after cultivation in osteogenic media; (b) ADSCs 2 weeks* 

*after cultivation in osteogenic media. Black arrows show ADSCs. Magnification 100×.*

*Expansion and purification of ADSCs from adipose-derived SVF through in vitro cultivation. (a) Adiposederived SVF 24 h after isolation; (b) adipose-derived SVF 72 h after isolation; (c) adipose-derived SVF 7 days after isolation; and (d) ADSCs the first day after the first passage. Black arrows show ADSCs. Magnification* 

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**Figure 1.**

*100×.*

**Figure 2.**

there is a need for carrier that would deliver PRP in the manner that it can act more efficiently and release growth factors sustainably [48]. This might be the key for enhancing osteogenesis guided by freshly isolated and untreated ADSCs within SVF. Despite disadvantages of ectopic models, there are many literature data where ectopic subcutaneous implantations were conducted [42, 43, 71, 75, 76] because they allow examining the real potential of the implanted cells [64] alone and without the impact of the factors that are normally present in bone tissue. Therefore, selection of adequate animal model should depend on experimental goal, and finally it is important for interpretation and extrapolation of obtained results [1]. Nevertheless, osteogenic capacity of previously discussed combinations of differently prepared ADSCs should be evaluated in orthotopic bone-forming models because they are closer to real situations where treatment of bone defect is needed. That is especially of great importance for intraoperative (one-step) method because it is still unclear if this method ready for clinical implementation. In addition, recently a safe and feasible one-step surgical procedure for maxillary sinus floor elevation with implants consisting of calcium phosphate and freshly isolated SVF was demonstrated [63].

Preparation and utilization of freshly isolated and untreated ADSCs at the first place provides tremendous acceleration of procedure performance which is very important especially in everyday clinical practice where intraoperative method is desirable. Despite much longer period which is needed for ADSC preparation and utilization, there is no doubt that osteoinduction is a reliable method for the purposes of BTE. Overall, according to studies published so far [45, 49] that are mostly in accordance with other related studies, it could be concluded that untreated ADSCs contained in freshly isolated SVF have different potential from in vitro osteoinduced ADSCs to start and maintain osteogenic process which leads to quite different outcomes, at least in ectopic condition. Surely, each of presented approaches has its own advantages and potential to be successfully applied in treatment of bone tissue defects. We believe that both approaches could be successfully utilized in the treatment of bone tissue defects, but additional research should be conducted especially in orthotopic models in order to determine required doses of osteogenic factors needed to support osteogenic process.

#### **4.2 ADSCs-based grafts prepared in bioreactors**

We previously discuss some methods for ex vivo engineering of the grafts in in vitro static condition using cells and bone substitutes as scaffold. The advanced method for graft engineering involves the use of bioreactors. Bioreactors are defined as devices for precise monitoring and controlling of conditions which are necessary for biological and biochemical processes [77]. The controlling and monitoring of the conditions are in favor of minimalizing variability of graft production as well as standardization of the process [13] for graft engineering. Amini et al. [10] reviewed several types of bioreactors which include perfusion bioreactors, rotating bioreactors, and spinner flask bioreactors. Perfusion-based bioreactors, which are marked as mostly used, significantly stimulate osteogenic cells due to fluid flow [10]. Thus, the dynamic conditions, which bioreactors provide, are more similar to in vivo conditions which allow proliferation and differentiation of seeded cells of three-dimensional scaffolds in a much appropriate way than static in vitro conditions. There is literature evidence where human ADSCs were successfully used for engineering bone grafts in stirrer flask bioreactors [78] as well as viable bone tissue construct in perfusion bioreactor [79]. However, the engineering of the ready-touse bone grafts using bioreactors could last for weeks, and it must be performed in several steps. Also, bioreactor devices for BTE purposes have high prizes, and

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selected randomly [102].

*Application of Adipose-Derived Stem Cells in Treatment of Bone Tissue Defects*

medium and other materials which reduces accessibility of this method.

**and ADSCs in vitro induced into endothelial cells**

these cells are desirable as one of the cell lines in co-culture [95, 96]*.*

An important question regarding the use of ECs, not only in BTE but in tissue engineering generally, is "which type of ECs should be used?" [97]. Sources of autologous ECs are limited [98], and mature ECs have limited proliferative capacity [99, 100]. In addition, the procedure of isolation is invasive, it is hard to collect a plentiful number of cells, and there is a possibility of contamination, infection, and change of phenotype and function of ECs during in vitro cultivation [94, 99, 101]. Also, ECs isolated from different organs manifest different phenotypes in vivo, which mean that EC types for the application in tissue engineering should not be

Bearing all this in mind, alternative methods have been developed in order to obtain more stable sources of ECs. These methods include in vitro induction of MSCs into ECs. An easily accessible tissue [103] that attracts a great attention in the last few decades is adipose tissue. Primarily cell components of adipose tissue are cells filled with lipids—adipocytes. Besides adipocytes, stromal vascular fraction of adipose tissue consists of microvascular ECs, pericytes, fibroblasts, macrophages,

the whole procedure is not economical because it requires a higher consumption of

**5. Solving vascularization problems in bone tissue engineering by SVF** 

Bone tissue is a self-renewable tissue with an excellent regeneration capacity [80].

**5.1 The problem of insufficient vascularization in bone tissue engineering**

Some bone fractures, tumors, or bone loss can cause such large bone deficiencies that cannot be healed spontaneously [80], since their size goes beyond bone selfrenewing capacity [81]. In those cases, classical surgical procedures or bone tissue engineering strategies must be applied [82]. The main problem that has not yet been completely overcome is insufficient vascularization in critical-sized bone defects. Therefore, a perfect bone substitute must have excellent angiogenic features [83]. Until now, various BTE methods for resolving insufficient vascularization were performed: changing the architecture and interconnectivity of pores of the applied biomaterial [84], co-cultivation of different types of cells [85], using mechanical stimulation [86], and adding one [87] or a couple of growth factors in the implants at the same time [88]. The implants were placed into highly vascularized areas or, before the implantation procedure, seeded with cells that secrete chemoattractants for attraction of the host's cells and blood vessels with small diameter [89]. Also, microvascular fragments of adipose tissue were incorporated [90], and tissue flap procedures [91] and scaffolds made of nano- or microfibers were applied [92]. One of the possible methods to facilitate anastomosing between the bioengineered vascular structures with the ones from the surrounding tissue is the construction of prevascularized scaffolds that contain endothelial cells (ECs) [93]. ECs constitute a continuous monolayer among interstitial fluid and blood and have crucial importance in vascularization and in controlling the function of blood vessels [94]. By producing, metabolizing, and releasing numerous humoral and hormonal agents, these cells create an active antithrombotic surface in order to ease transit of plasma and cell components through the blood vessels [94]. Nevertheless, not only that ECs are important for successful developing of the functional vascular system, other cells that constitute blood vessel wall, smooth muscle cells, and perycites are also of great importance [9]. Mural precursor cells participate in vascular remodeling and contribute to better vascularization in cell cultures which is the reason why

*DOI: http://dx.doi.org/10.5772/intechopen.92897*

the whole procedure is not economical because it requires a higher consumption of medium and other materials which reduces accessibility of this method.
