**5. Solving vascularization problems in bone tissue engineering by SVF and ADSCs in vitro induced into endothelial cells**

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

Bone tissue is a self-renewable tissue with an excellent regeneration capacity [80]. 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 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 selected randomly [102].

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,

*Clinical Implementation of Bone Regeneration and Maintenance*

osteogenic factors needed to support osteogenic process.

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

was demonstrated [63].

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

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

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

**188**

leukocytes, pre-adipocytes, mastocytes, and adipose-derived mesenchymal stem [104, 105]. SVF with heterogeneous cell populations can be obtained upon simple enzyme-based adipose tissue isolation procedure that includes enzymatic digestion, filtration, and centrifugation [76, 106–109]. After this procedure, SVF can be used as a source of ADSCs directly [108] or after expansion in cell culture through few passages [104] and subjecting to differentiation toward certain cell line [66, 76, 105, 109–111].

ADSCs secrete numerous angiogenesis-related mediators including vascular endothelial growth factor (VEGF), bone morphogenic proteins, placental growth factor (PGF), angiopoietin-1, hepatocyte growth factor (HGF), transforming growth factor-β, and fibroblast growth factor 2 (FGF-2) [112]. Secretion of these angiogenic factors makes ADSCs convenient for regenerative cell therapy [106]. Among the abovementioned growth factors, VEGF and BMP2 are considered to be the main factors during bone regeneration, VEGF on the vascular and BMP2 on the osteogenic side [113]. Besides soluble growth factors, ADSCs release plasma membrane-derived vesicles (MVs) that can contain some pro-angiogenic and osteogenic molecules [114] with an influence on adjacent cells. In addition, growth factors and other molecules contained within MVs (cytokines, RNAs, microRNAs) can be transported to some more distant target cells all over the body. By taking up the content of MVs, target cells use these molecules and perform certain biological activity including angiogenesis of target cells which is of crucial importance in bone tissue regeneration [115].

In order to apply SVF as a source of cells with vasculogenic capacity in bone tissue engineering, several methods have been described. These methods include application of SVFs immediately after isolation from adipose tissue as well as after in vitro induction of endothelial differentiation.

### **5.2 Potential of stromal vascular fraction as a source of cells with vasculogenic capacity for application in bone tissue engineering**

Freshly isolated SVF contains both endothelial and skeletal progenitor cells [116]. This property was widely used to construct osteogenic and vasculogenic grafts. It has been found that three-dimensional (3D) cultures of ECs and osteoblasts (OBs) as well as osteogenic-vasculogenic constructs could be achieved by using perfusion-based bioreactor system of single cell source human SVFs in ceramic scaffolds [116]. Namely, SVFs were seeded on 3D porous ceramic scaffolds and cultivated during 5 days using bioreactor system. Eight weeks after implantation of resulting scaffolds in nude mice, formation of functional vascular network that was connected with the host's vasculature as well as ectopic bone formation was observed. Nude mice model was also used by Todorov and his colleagues [117]. This team isolated SVFs from human abdominal lipoaspirates and obtained hypertrophic cartilage (HC) pellets from bone marrow-derived stromal cells. Devitalized HC was embedded in fibrin gel and implanted with and without SVFs ectopically and in calvarial defects. Twelve weeks after implantations, vascularization and bone formation of grafts enriched with SVFs were enhanced in ectopic, subcutaneous, and in orthotopic experimental model.

The use of human SVF-derived vascular progenitor cells can speed up the engraftment of critical-sized osteogenic constructs which improves in vivo formation of the bone tissue [118]. Human SVFs have been isolated, seeded, and cultured into hydroxyapatite scaffolds. Perfusion bioreactor system was used in order to preserve the cells of CD34<sup>+</sup> /CD31+ endothelial lineage from the SVF. As a result of in vitro cultivation, 5 days after seeding, endothelial and mesenchymal progenitors from SVF constituted capillary networks that were able to anastomose with the host vasculature 7 days after implantation on an ectopic nude rat model [118].

**191**

tion procedure.

stages of bone healing.

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

With an aim to simulate an intraoperative procedure on an ectopic bone forming model, autologous SVFs isolated from epididymal adipose tissue of Balb/c mice and platelet-rich plasma were added to the bone mineral matrix (BMM) [108]. BMM served as a carrier for cells and growth factors. The constructs were implanted subcutaneously, and osteogenic capacity of this combination was examined. Eight weeks after implantations, these implants had significantly higher percentage of infiltrated tissue and percentage of vascularization than the control (BMM-only implants). According to osteogenesis-related gene expression analysis, implants with SVFs induced rapid onset of osteogenesis process. One of the reasons for such results probably lies in the fact that freshly isolated SVFs had upregulated expression of endothelial- and osteogenic-related genes which was proved by using

Non-induced ADSCs cultivated in vitro up to the third passage were mixed with allogeneic PRP taken from three healthy Wistar rats [119]. ADSCs-PRP constructs were implanted into the jaws of rats that had bisphosphonate-related osteonecrosis of the jaw. Eight weeks after the treatment, the incidence of osteonecrosis was lower, while the degree of bone turnover and number of osteoclasts were higher than the experimental groups that were not treated with ADSCs [119]. These data could be explained by the fact that PRP release numerous growth factors that can fasten both in vitro and in vivo ADSC differentiation without previous addition of any other factors inductive toward certain cell line [109]. One of the possible mechanisms of synergistic effect of ADSCs and PRP could be ascribed to the release of platelet-derived growth factor from PRP. PDGF has an impact on selectable expansion and recruitment of non-induced MSCs, as well as on proliferation and migration of progenitors of blood vessel wall cells [120]. It also triggers differentiation of MSCs into blood vessel cells [107] and enhances release of extracellular vesicles (EVs)—MVs and exosomes from ADSCs which further influence both

On an ectopic model, Man and associates [109] pointed out to the effect of ADSCs that is similar to the one shown in the abovementioned orthotopic model. ADSCs isolated from inguinal rabbit fat pads and mixed with PRP were loaded onto alginate microspheres. Well-developed blood vessel network and good bone mineralization were observed 3 months after subcutaneous implantations. Opposite results were obtained in another research where non-induced ADSCs were seeded on poly (D, L-lactide) scaffolds and implanted in the large rat palatal bone defect [122]. Bone formation did not occur even 12 weeks after implantations, and the defects were filled with dense fibrous tissue. The differences between the outcomes of the different studies could be attributed to the fact that adipose tissue was taken from the different localizations of the donors' body. Also, the implant preparation was done in a different manner, and different models were chosen for the implanta-

Adipose tissue can also be used as a source of microvascular fragments (MVF) that can be further applied as vascularization units [123]. MVF isolated from CD-1 mice were incorporated into thermoresponsive hydrogel (TRH), cultivated, and used for filling the osteotomy gaps in the femurs of CD-1 mice. Bone healing was assessed 14 and 35 days after induction of osteotomy, while non-incorporated MVF as well as no material group served as control groups. It was found that TRH is a suitable carrier for MVF since vascularization in MVF-loaded TRH was improved in comparison to the control groups. In contrast to this finding, bone formation in this group was impaired, probably due to low levels of VEGF expression during the early

Bone substitute biomaterials based on calcium phosphate, including hydroxyapatite (HA), β-tricalcium phosphate (β-TCP), and HA/β-TCP combination, are

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

in vitro and in vivo angiogenic process [121].

real-time PCR analysis.

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

With an aim to simulate an intraoperative procedure on an ectopic bone forming model, autologous SVFs isolated from epididymal adipose tissue of Balb/c mice and platelet-rich plasma were added to the bone mineral matrix (BMM) [108]. BMM served as a carrier for cells and growth factors. The constructs were implanted subcutaneously, and osteogenic capacity of this combination was examined. Eight weeks after implantations, these implants had significantly higher percentage of infiltrated tissue and percentage of vascularization than the control (BMM-only implants). According to osteogenesis-related gene expression analysis, implants with SVFs induced rapid onset of osteogenesis process. One of the reasons for such results probably lies in the fact that freshly isolated SVFs had upregulated expression of endothelial- and osteogenic-related genes which was proved by using real-time PCR analysis.

Non-induced ADSCs cultivated in vitro up to the third passage were mixed with allogeneic PRP taken from three healthy Wistar rats [119]. ADSCs-PRP constructs were implanted into the jaws of rats that had bisphosphonate-related osteonecrosis of the jaw. Eight weeks after the treatment, the incidence of osteonecrosis was lower, while the degree of bone turnover and number of osteoclasts were higher than the experimental groups that were not treated with ADSCs [119]. These data could be explained by the fact that PRP release numerous growth factors that can fasten both in vitro and in vivo ADSC differentiation without previous addition of any other factors inductive toward certain cell line [109]. One of the possible mechanisms of synergistic effect of ADSCs and PRP could be ascribed to the release of platelet-derived growth factor from PRP. PDGF has an impact on selectable expansion and recruitment of non-induced MSCs, as well as on proliferation and migration of progenitors of blood vessel wall cells [120]. It also triggers differentiation of MSCs into blood vessel cells [107] and enhances release of extracellular vesicles (EVs)—MVs and exosomes from ADSCs which further influence both in vitro and in vivo angiogenic process [121].

On an ectopic model, Man and associates [109] pointed out to the effect of ADSCs that is similar to the one shown in the abovementioned orthotopic model. ADSCs isolated from inguinal rabbit fat pads and mixed with PRP were loaded onto alginate microspheres. Well-developed blood vessel network and good bone mineralization were observed 3 months after subcutaneous implantations. Opposite results were obtained in another research where non-induced ADSCs were seeded on poly (D, L-lactide) scaffolds and implanted in the large rat palatal bone defect [122]. Bone formation did not occur even 12 weeks after implantations, and the defects were filled with dense fibrous tissue. The differences between the outcomes of the different studies could be attributed to the fact that adipose tissue was taken from the different localizations of the donors' body. Also, the implant preparation was done in a different manner, and different models were chosen for the implantation procedure.

Adipose tissue can also be used as a source of microvascular fragments (MVF) that can be further applied as vascularization units [123]. MVF isolated from CD-1 mice were incorporated into thermoresponsive hydrogel (TRH), cultivated, and used for filling the osteotomy gaps in the femurs of CD-1 mice. Bone healing was assessed 14 and 35 days after induction of osteotomy, while non-incorporated MVF as well as no material group served as control groups. It was found that TRH is a suitable carrier for MVF since vascularization in MVF-loaded TRH was improved in comparison to the control groups. In contrast to this finding, bone formation in this group was impaired, probably due to low levels of VEGF expression during the early stages of bone healing.

Bone substitute biomaterials based on calcium phosphate, including hydroxyapatite (HA), β-tricalcium phosphate (β-TCP), and HA/β-TCP combination, are

*Clinical Implementation of Bone Regeneration and Maintenance*

105, 109–111].

tissue regeneration [115].

in vitro induction of endothelial differentiation.

**capacity for application in bone tissue engineering**

/CD31+

in vitro cultivation, 5 days after seeding, endothelial and mesenchymal progenitors from SVF constituted capillary networks that were able to anastomose with the host

vasculature 7 days after implantation on an ectopic nude rat model [118].

leukocytes, pre-adipocytes, mastocytes, and adipose-derived mesenchymal stem [104, 105]. SVF with heterogeneous cell populations can be obtained upon simple enzyme-based adipose tissue isolation procedure that includes enzymatic digestion, filtration, and centrifugation [76, 106–109]. After this procedure, SVF can be used as a source of ADSCs directly [108] or after expansion in cell culture through few passages [104] and subjecting to differentiation toward certain cell line [66, 76,

ADSCs secrete numerous angiogenesis-related mediators including vascular endothelial growth factor (VEGF), bone morphogenic proteins, placental growth factor (PGF), angiopoietin-1, hepatocyte growth factor (HGF), transforming growth factor-β, and fibroblast growth factor 2 (FGF-2) [112]. Secretion of these angiogenic factors makes ADSCs convenient for regenerative cell therapy [106]. Among the abovementioned growth factors, VEGF and BMP2 are considered to be the main factors during bone regeneration, VEGF on the vascular and BMP2 on the osteogenic side [113]. Besides soluble growth factors, ADSCs release plasma membrane-derived vesicles (MVs) that can contain some pro-angiogenic and osteogenic molecules [114] with an influence on adjacent cells. In addition, growth factors and other molecules contained within MVs (cytokines, RNAs, microRNAs) can be transported to some more distant target cells all over the body. By taking up the content of MVs, target cells use these molecules and perform certain biological activity including angiogenesis of target cells which is of crucial importance in bone

In order to apply SVF as a source of cells with vasculogenic capacity in bone tissue engineering, several methods have been described. These methods include application of SVFs immediately after isolation from adipose tissue as well as after

**5.2 Potential of stromal vascular fraction as a source of cells with vasculogenic** 

Freshly isolated SVF contains both endothelial and skeletal progenitor cells [116]. This property was widely used to construct osteogenic and vasculogenic grafts. It has been found that three-dimensional (3D) cultures of ECs and osteoblasts (OBs) as well as osteogenic-vasculogenic constructs could be achieved by using perfusion-based bioreactor system of single cell source human SVFs in ceramic scaffolds [116]. Namely, SVFs were seeded on 3D porous ceramic scaffolds and cultivated during 5 days using bioreactor system. Eight weeks after implantation of resulting scaffolds in nude mice, formation of functional vascular network that was connected with the host's vasculature as well as ectopic bone formation was observed. Nude mice model was also used by Todorov and his colleagues [117]. This team isolated SVFs from human abdominal lipoaspirates and obtained hypertrophic cartilage (HC) pellets from bone marrow-derived stromal cells. Devitalized HC was embedded in fibrin gel and implanted with and without SVFs ectopically and in calvarial defects. Twelve weeks after implantations, vascularization and bone formation of grafts enriched with SVFs were enhanced in ectopic, subcutaneous, and in orthotopic experimental model. The use of human SVF-derived vascular progenitor cells can speed up the engraftment of critical-sized osteogenic constructs which improves in vivo formation of the bone tissue [118]. Human SVFs have been isolated, seeded, and cultured into hydroxyapatite scaffolds. Perfusion bioreactor system was used in order to

endothelial lineage from the SVF. As a result of

**190**

preserve the cells of CD34<sup>+</sup>

often used in BTE due to their good biocompatibility and the absence of toxicity of their chemical compounds [124]. These materials also have such 3D features that allow immediate colonization by MSCs and extensive revascularization [125]. For those reasons, Farré-Guasch and his associates used ADSCs-containing SVF seeded on calcium-based biomaterials to treat the patients subjected to maxillary sinus floor elevation (MSFE)—a surgical procedure that in some patients must precede dental implant placement [126]. Autologous SVFs taken from patients were seeded on two types of carriers for two different groups of patients: group 1 had SVFs seeded on β-tricalcium phosphate, and group 2 had SVFs seeded on biphasic calcium phosphate, while the control group had only ceramics, without cells. Histomorphometrical analysis and immunohistochemical staining for blood vessel markers such as CD34 and alpha-smooth muscle actin revealed higher number of blood vessels and immunoexpression of blood vessel markers in both experimental than a control group. These results point out pro-angiogenic influence of SVF.

One of the recently published papers regarding pre-vascularization of various engineered tissues compares the use of ADSCs as a potential source of cells with vasculogenic capacity in combination with different types of gel-based scaffolds [127]. ADSCs were isolated from human fat tissue, cultivated, and, after second passage, molded in fibrin as well as agarose-collagen gels. After 14 days of incubation have passed, the gels were analyzed by two-photon laser scanning microscopy. Vascularization was achieved in both types of gels which were detected as branched networks of tubular vascular structures in both hydrogels. Nevertheless, volume, area, and length of vascular structures supported by ADSCs in agarose-collagen hydrogels were comparable to human dermal fibroblast control.

## **5.3 Potential of adipose-derived stem cells in vitro induced into ECs for application in bone tissue engineering**

#### *5.3.1 Orthotopic model*

In order to increase vascularization, ADSCs induced into ECs were applied in BTE by using several models, among which orthotopic model is one of the most commonly used. Rat ADSCs in vitro induced into ECs during 8 days was used for the construction of allografts [128]. The cells were combined with sterilized and decellularized banked allografts made of calvaria from earlier sacrificed Lewis rats. Allografts were implanted into critical-sized calvarial defects in rats, and 8 weeks after the implantation, blood vessel density was increased. As a consequence, bone volume was also increased. In parallel, two other types of allografts were constructed—allografts seeded with ADSCs induced into OBs and allografts seeded with the combination of ADSCs induced into ECs and ADSCs induced into OBs. These implants had weaker vascularization and lower bone volume 8 weeks after the implantation than the ECs-only allografts.

ADSCs in vitro induced into ECs were also seeded onto poly(D, L-lactide) scaffolds and implanted into critical-sized calvarial defects of Lewis rats [110]. Scaffolds prevascularized in this manner did not caused an increase in bone formation by itself, but according to the conclusion of this team, they could possibly be used as a source of cells for accomplishing better vascularization and function of the existing OBs. On the other hand, the constructs that were constructed out of undifferentiated ADSCs or ADSCs induced into OBs had statistically greater bone volume than the implants containing ADSCs induced into ECs.

In another study, also performed on critical-sized calvarial defect model of Lewis rats, hydroxyapatite/poly(lactide-co-glycolide) [HA-PLG] was used as a biomaterial carrier, while adipose tissue was extracted from inguinal fat pads [129].

**193**

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

found differences had statistical significance (*P* > 0.05).

ADSCs were induced into ECs as well as into OBs. Vascularization and osteogenic process were evaluated in the following groups: (I) HA-PLG scaffolds without cells, (II) HA-PLG scaffolds seeded with non-induced ADSCs, (III) HA-PLG scaffolds seeded with ADSCs induced into ECs, and (IV) HA-PLG scaffolds seeded with ADSCs induced into OBs. The highest bone mineral density, bone regeneration, and vascular density in regenerated bone were found in group IV, although none of the

In order to repair critical-sized bone defects in rat femur, ADSCs induced into ECs were used for prevascularization of the modified hierarchical mesoporous bioactive glass (MBG) scaffold with an enhanced compressive strength and then combined with ADSCs subjected to osteogenic differentiation [108]. Prevascularized MBG carrying ADSCs induced into OBs had more advanced angiogenesis both on the surface and in the interior than the non-vascularized MBG carrying ADSCs induced into OBs and MBG scaffolds that were not seeded with cells. Moreover, the group with prevascularized MBG scaffolds had the highest mineral deposition rate postoperatively. These results indicate that time-phase sequential utilization of ADSCs on MBG scaffolds is a good strategy for reparation

Ectopic models are important in bone tissue engineering since they provide reducing external influences and side effects, thus concentrating on intrinsic potential of the applied implant components [53] and their interactions [42]. With an aim to overcome the problem of inadequate blood vessels development and consequent inability of bone tissue regeneration, the influence of ADSCs in vitro induced into ECs on vascularization and osteogenic process in ectopic osteogenic implants was examined [109]. The implants composed of ADSCs, BMM, and PRP and the ones composed of BMM and PRP were subcutaneously implanted into BALB/c mice. Endothelial-related gene expression, high percentage of vascularization, and VEGFR-2 immunoexpression show that implants enriched with ECs have increased vascularization compared to the cell-free implants. This was followed by more pronounced signs of osteogenic process in ADSCs-BMM-PRP implants than in BMM-PRP implants. By examining endothelial-related gene expression, vascular cell adhesion molecule-1 (VCAM-1) and osteopontin immunoexpression, it was also shown that the composition of implants based on biological triad (ADSCs induced into ECs, PRP and BMM) is more favorable for improving vascularization

in the ectopic bone-forming model than in the BMM-only implants [76].

implantations, at each single observation point.

Uninduced ADSCs cultivated up to the 12th day after the third passage, combined with PRP and BMM and subcutaneously implanted into BALB/c mice, also have vasculogenic potential [109]. However, their vasculogenic potential is lower than that of ADSCs in vitro induced into osteogenic cells and implanted in combination with BMM and PRP. Specifically, relative gene expression analysis of endothelial gene markers *Vwf*, *Egr1*, *Flt1*, and *Vcam1* was significantly higher *(p < 0.05)* in the group that contained osteoinduced ADSCs than in the group with uninduced ADSCs for each single gene and with the exception of *Vwf* at 1 and 4 weeks after

Endothelial differentiated ADSCs and osteogenic differentiated ADSCs can be simultaneously applied for the construction of ectopic osteogenic implants. A method of in vitro prevascularization applied by Zhang and his team included construction of double cell sheets (DCS) out of rabbit ADSCs previously in vitro induced into ECs and into osteogenic cells [113]. DCS were combined with coral hydroxyapatite (CHA) in four different manners. Twelve weeks after ectopic

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

of massive bone defects.

*5.3.2 Ectopic model*

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

ADSCs were induced into ECs as well as into OBs. Vascularization and osteogenic process were evaluated in the following groups: (I) HA-PLG scaffolds without cells, (II) HA-PLG scaffolds seeded with non-induced ADSCs, (III) HA-PLG scaffolds seeded with ADSCs induced into ECs, and (IV) HA-PLG scaffolds seeded with ADSCs induced into OBs. The highest bone mineral density, bone regeneration, and vascular density in regenerated bone were found in group IV, although none of the found differences had statistical significance (*P* > 0.05).

In order to repair critical-sized bone defects in rat femur, ADSCs induced into ECs were used for prevascularization of the modified hierarchical mesoporous bioactive glass (MBG) scaffold with an enhanced compressive strength and then combined with ADSCs subjected to osteogenic differentiation [108]. Prevascularized MBG carrying ADSCs induced into OBs had more advanced angiogenesis both on the surface and in the interior than the non-vascularized MBG carrying ADSCs induced into OBs and MBG scaffolds that were not seeded with cells. Moreover, the group with prevascularized MBG scaffolds had the highest mineral deposition rate postoperatively. These results indicate that time-phase sequential utilization of ADSCs on MBG scaffolds is a good strategy for reparation of massive bone defects.

#### *5.3.2 Ectopic model*

*Clinical Implementation of Bone Regeneration and Maintenance*

hydrogels were comparable to human dermal fibroblast control.

**application in bone tissue engineering**

the implantation than the ECs-only allografts.

volume than the implants containing ADSCs induced into ECs.

*5.3.1 Orthotopic model*

**5.3 Potential of adipose-derived stem cells in vitro induced into ECs for** 

In order to increase vascularization, ADSCs induced into ECs were applied in BTE by using several models, among which orthotopic model is one of the most commonly used. Rat ADSCs in vitro induced into ECs during 8 days was used for the construction of allografts [128]. The cells were combined with sterilized and decellularized banked allografts made of calvaria from earlier sacrificed Lewis rats. Allografts were implanted into critical-sized calvarial defects in rats, and 8 weeks after the implantation, blood vessel density was increased. As a consequence, bone volume was also increased. In parallel, two other types of allografts were constructed—allografts seeded with ADSCs induced into OBs and allografts seeded with the combination of ADSCs induced into ECs and ADSCs induced into OBs. These implants had weaker vascularization and lower bone volume 8 weeks after

ADSCs in vitro induced into ECs were also seeded onto poly(D, L-lactide) scaffolds and implanted into critical-sized calvarial defects of Lewis rats [110]. Scaffolds prevascularized in this manner did not caused an increase in bone formation by itself, but according to the conclusion of this team, they could possibly be used as a source of cells for accomplishing better vascularization and function of the existing OBs. On the other hand, the constructs that were constructed out of undifferentiated ADSCs or ADSCs induced into OBs had statistically greater bone

In another study, also performed on critical-sized calvarial defect model of Lewis rats, hydroxyapatite/poly(lactide-co-glycolide) [HA-PLG] was used as a biomaterial carrier, while adipose tissue was extracted from inguinal fat pads [129].

often used in BTE due to their good biocompatibility and the absence of toxicity of their chemical compounds [124]. These materials also have such 3D features that allow immediate colonization by MSCs and extensive revascularization [125]. For those reasons, Farré-Guasch and his associates used ADSCs-containing SVF seeded on calcium-based biomaterials to treat the patients subjected to maxillary sinus floor elevation (MSFE)—a surgical procedure that in some patients must precede dental implant placement [126]. Autologous SVFs taken from patients were seeded on two types of carriers for two different groups of patients: group 1 had SVFs seeded on β-tricalcium phosphate, and group 2 had SVFs seeded on biphasic calcium phosphate, while the control group had only ceramics, without cells. Histomorphometrical analysis and immunohistochemical staining for blood vessel markers such as CD34 and alpha-smooth muscle actin revealed higher number of blood vessels and immunoexpression of blood vessel markers in both experimental than a control group. These results point out pro-angiogenic influence of SVF. One of the recently published papers regarding pre-vascularization of various engineered tissues compares the use of ADSCs as a potential source of cells with vasculogenic capacity in combination with different types of gel-based scaffolds [127]. ADSCs were isolated from human fat tissue, cultivated, and, after second passage, molded in fibrin as well as agarose-collagen gels. After 14 days of incubation have passed, the gels were analyzed by two-photon laser scanning microscopy. Vascularization was achieved in both types of gels which were detected as branched networks of tubular vascular structures in both hydrogels. Nevertheless, volume, area, and length of vascular structures supported by ADSCs in agarose-collagen

**192**

Ectopic models are important in bone tissue engineering since they provide reducing external influences and side effects, thus concentrating on intrinsic potential of the applied implant components [53] and their interactions [42]. With an aim to overcome the problem of inadequate blood vessels development and consequent inability of bone tissue regeneration, the influence of ADSCs in vitro induced into ECs on vascularization and osteogenic process in ectopic osteogenic implants was examined [109]. The implants composed of ADSCs, BMM, and PRP and the ones composed of BMM and PRP were subcutaneously implanted into BALB/c mice. Endothelial-related gene expression, high percentage of vascularization, and VEGFR-2 immunoexpression show that implants enriched with ECs have increased vascularization compared to the cell-free implants. This was followed by more pronounced signs of osteogenic process in ADSCs-BMM-PRP implants than in BMM-PRP implants. By examining endothelial-related gene expression, vascular cell adhesion molecule-1 (VCAM-1) and osteopontin immunoexpression, it was also shown that the composition of implants based on biological triad (ADSCs induced into ECs, PRP and BMM) is more favorable for improving vascularization in the ectopic bone-forming model than in the BMM-only implants [76].

Uninduced ADSCs cultivated up to the 12th day after the third passage, combined with PRP and BMM and subcutaneously implanted into BALB/c mice, also have vasculogenic potential [109]. However, their vasculogenic potential is lower than that of ADSCs in vitro induced into osteogenic cells and implanted in combination with BMM and PRP. Specifically, relative gene expression analysis of endothelial gene markers *Vwf*, *Egr1*, *Flt1*, and *Vcam1* was significantly higher *(p < 0.05)* in the group that contained osteoinduced ADSCs than in the group with uninduced ADSCs for each single gene and with the exception of *Vwf* at 1 and 4 weeks after implantations, at each single observation point.

Endothelial differentiated ADSCs and osteogenic differentiated ADSCs can be simultaneously applied for the construction of ectopic osteogenic implants. A method of in vitro prevascularization applied by Zhang and his team included construction of double cell sheets (DCS) out of rabbit ADSCs previously in vitro induced into ECs and into osteogenic cells [113]. DCS were combined with coral hydroxyapatite (CHA) in four different manners. Twelve weeks after ectopic

implantation into nude mice, a group that contained CHA covered with DCS such that endothelial cell sheet was inside and osteogenic cell sheet outside exhibited the most favorable results regarding vascularization and bone maturation of the graft.

#### *5.3.3 Co-cultivation*

Co-cultivation of ECs with other cell types is one of the approaches for resolving the problem of inadequate vascularization in BTE [130]. ECs were co-cultivated with different types of cells before the implantation procedure, but the most important for BTE is co-cultivation of ECs and OBs since numerous interactions between these two cell types exist during normal bone regeneration process [131, 132]. One of the mechanisms of those interactions was found by Kaigler and associates [133]. They have shown that in vitro, ECs improve osteogenic capacity of bone mesenchymal stem cells (BMSCs) during co-cultivation, in direct contact or near each other. In part, this role of ECs could be attributed to release of BMP-2. In vivo, transplanted ECs enhanced capability of transplanted BMSCs to form the bones.

The positive effect of combination of ECs and OBs was estimated in ectopically implanted HA/bTCP scaffolds. The 3D porous ceramic scaffolds were seeded with in vitro co-cultivated ADSCs induced into OBs, ADSCs induced into ECs, and CD14+ osteoclast progenitors derived from human peripheral blood osteoclasts [134]. This three-dimensional organotypic culture model based on human cells was cultivated during 21 days in the perfusion bioreactor system. After cultivation, the system was implanted subcutaneously into dorsal pockets of nude mice. Eight weeks after implantation, blood vessels and bone-like tissue were formed.

In another study, co-cultivation of ADSCs induced into ECs and ADSCs induced into OBs did not have such positive effect on vascularization and osteogenic process [130]. Co-cultivation increased proliferation of this two cell types in vitro in comparison with monocultures or undifferentiated ADSCs. Nevertheless, co-cultures in a ratio 1:1 = OBs:ECs seeded on polylactic acid gas-plasma-treated scaffolds have not induced increased vascularization and signs of osteogenic process compared to the implants constituted out of non-induced ADSCs and polymer scaffolds. Unlike the co-culture group, ECs seeded on polymer scaffolds have increased vascularization, and OBs seeded on polymer scaffolds have improved osteogenesis more than a group with undifferentiated ADSCs [130].

Unlike the results of the above discussed study, ADSCs in vitro induced into OBs and ADSCs in vitro induced into ECs and seeded into self-assembling peptide RADA16-I scaffolds as co-cultures (1:1) had better osteogeneration and vascularization than the scaffolds seeded with monocultures of this type of cells or noninduced ADSCs [135]. Before seeding the cells into RADA16-I scaffolds, it has been shown that the best interaction of ADSCs induced into OBs and ADSCs induced into ECs in co-cultures was achieved when the ratio of the cells was 1:1.

The results obtained by some of the abovementioned research teams, which were worse than expected regarding a group of implants that contained previously co-cultured ECs and OBs, could lie in the ratio of the applied cell types. It has been confirmed that application of too many ECs decreases graft neovascularization probably due to increase in metabolic load and enhanced competition for nutrients [136]. The other reason for those differences can be the differences in the applied biomaterials used for the implant construction [130].

#### *5.3.4 Arteriovenous vascular bundle*

Formation of arteriovenous vascular bundle (AVB) belongs to in vivo prevascularization approaches. Incorporation of AVB into scaffolds represents a model

**195**

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

of scaffold that have artery and vein inserted into its central part. In this way, stem cells as well as cytokines, oxygen, and nutrients can be transported, while waste products can be removed from the scaffold. Altogether, that kind of construction leads to excellent vascularization and osteogenesis within the scaffold [137]. Scaffolds with AVB were combined with rat ADSCs previously subjected to in vitro endothelial differentiation. Obtained ECs were incorporated into porous nano-hydroxyapatite-polyamide 66 (nHA-PA 66) scaffolds in vitro [111]. After that, AV bundle was inserted into ECs-based nHA-PA 66 in vivo. Also, AV bundle was inserted into nHA-PA 66 scaffolds seeded with non-induced ADSCs and into empty nHA-PA 66 scaffolds, while one experimental group had nHA-PA 66 scaffolds without inserted AV bundle and without cells. Two and four weeks after the implantation procedure, the implants were extracted from the animals and analyzed. Density of blood vessels was significantly higher, and the diameter of blood vessels was larger in ECs-based nHA-PA 66 scaffolds than that in all other groups of

Combining ex vivo gene therapy with cell transplantation techniques that include endothelial cell line is another approach for overcoming the problem of insufficient vascularization in bone. The benefit of this method was assessed by using 3D poly (lactide-co-glycolide) sintered microsphere scaffolds in a BTE approach [138]. ADSCs were isolated from human infrapatellar fat tissue, and the cells were transfected with adenovirus that encodes cDNA of VEGF and combined with endothelial ones. As a result, genetically modified ADSCs combined with ECs caused prominent growth within 3D poly (lactide-co-glycolide) scaffolds, which indicates the potential for ADSCs application in improving vascularization in BTE. Another study where gene therapy and ADSCs were combined was conducted by Peterson and associates [139]. First, ADSCs were transfected with the BMP-2 gene, then loaded on the collagen-ceramic carrier, and finally implanted in criticalsized femoral defect of nude mice. Eight weeks after implantations, histologic, radiographic, and biomechanical analyses showed that the collagen-ceramic carrier combined with ADSCs previously transfected with the BMP-2 gene caused bone

The study was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (project III41017).

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

implants.

*5.3.5 Gene therapy*

formation within the defect.

**Acknowledgements**

**Conflict of interest**

The authors declare no conflict of interest.

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

of scaffold that have artery and vein inserted into its central part. In this way, stem cells as well as cytokines, oxygen, and nutrients can be transported, while waste products can be removed from the scaffold. Altogether, that kind of construction leads to excellent vascularization and osteogenesis within the scaffold [137].

Scaffolds with AVB were combined with rat ADSCs previously subjected to in vitro endothelial differentiation. Obtained ECs were incorporated into porous nano-hydroxyapatite-polyamide 66 (nHA-PA 66) scaffolds in vitro [111]. After that, AV bundle was inserted into ECs-based nHA-PA 66 in vivo. Also, AV bundle was inserted into nHA-PA 66 scaffolds seeded with non-induced ADSCs and into empty nHA-PA 66 scaffolds, while one experimental group had nHA-PA 66 scaffolds without inserted AV bundle and without cells. Two and four weeks after the implantation procedure, the implants were extracted from the animals and analyzed. Density of blood vessels was significantly higher, and the diameter of blood vessels was larger in ECs-based nHA-PA 66 scaffolds than that in all other groups of implants.

### *5.3.5 Gene therapy*

*Clinical Implementation of Bone Regeneration and Maintenance*

*5.3.3 Co-cultivation*

CD14+

implantation into nude mice, a group that contained CHA covered with DCS such that endothelial cell sheet was inside and osteogenic cell sheet outside exhibited the most favorable results regarding vascularization and bone maturation of the graft.

Co-cultivation of ECs with other cell types is one of the approaches for resolving the problem of inadequate vascularization in BTE [130]. ECs were co-cultivated with different types of cells before the implantation procedure, but the most important for BTE is co-cultivation of ECs and OBs since numerous interactions between these two cell types exist during normal bone regeneration process [131, 132]. One of the mechanisms of those interactions was found by Kaigler and associates [133]. They have shown that in vitro, ECs improve osteogenic capacity of bone mesenchymal stem cells (BMSCs) during co-cultivation, in direct contact or near each other. In part, this role of ECs could be attributed to release of BMP-2. In vivo, transplanted

The positive effect of combination of ECs and OBs was estimated in ectopically implanted HA/bTCP scaffolds. The 3D porous ceramic scaffolds were seeded with in vitro co-cultivated ADSCs induced into OBs, ADSCs induced into ECs, and

 osteoclast progenitors derived from human peripheral blood osteoclasts [134]. This three-dimensional organotypic culture model based on human cells was cultivated during 21 days in the perfusion bioreactor system. After cultivation, the system was implanted subcutaneously into dorsal pockets of nude mice. Eight

In another study, co-cultivation of ADSCs induced into ECs and ADSCs induced into OBs did not have such positive effect on vascularization and osteogenic process [130]. Co-cultivation increased proliferation of this two cell types in vitro in comparison with monocultures or undifferentiated ADSCs. Nevertheless, co-cultures in a ratio 1:1 = OBs:ECs seeded on polylactic acid gas-plasma-treated scaffolds have not induced increased vascularization and signs of osteogenic process compared to the implants constituted out of non-induced ADSCs and polymer scaffolds. Unlike the co-culture group, ECs seeded on polymer scaffolds have increased vascularization, and OBs seeded on polymer scaffolds have improved osteogenesis more than a

Unlike the results of the above discussed study, ADSCs in vitro induced into OBs and ADSCs in vitro induced into ECs and seeded into self-assembling peptide RADA16-I scaffolds as co-cultures (1:1) had better osteogeneration and vascularization than the scaffolds seeded with monocultures of this type of cells or noninduced ADSCs [135]. Before seeding the cells into RADA16-I scaffolds, it has been shown that the best interaction of ADSCs induced into OBs and ADSCs induced

The results obtained by some of the abovementioned research teams, which were worse than expected regarding a group of implants that contained previously co-cultured ECs and OBs, could lie in the ratio of the applied cell types. It has been confirmed that application of too many ECs decreases graft neovascularization probably due to increase in metabolic load and enhanced competition for nutrients [136]. The other reason for those differences can be the differences in the applied

Formation of arteriovenous vascular bundle (AVB) belongs to in vivo prevascularization approaches. Incorporation of AVB into scaffolds represents a model

into ECs in co-cultures was achieved when the ratio of the cells was 1:1.

biomaterials used for the implant construction [130].

*5.3.4 Arteriovenous vascular bundle*

ECs enhanced capability of transplanted BMSCs to form the bones.

weeks after implantation, blood vessels and bone-like tissue were formed.

group with undifferentiated ADSCs [130].

**194**

Combining ex vivo gene therapy with cell transplantation techniques that include endothelial cell line is another approach for overcoming the problem of insufficient vascularization in bone. The benefit of this method was assessed by using 3D poly (lactide-co-glycolide) sintered microsphere scaffolds in a BTE approach [138]. ADSCs were isolated from human infrapatellar fat tissue, and the cells were transfected with adenovirus that encodes cDNA of VEGF and combined with endothelial ones. As a result, genetically modified ADSCs combined with ECs caused prominent growth within 3D poly (lactide-co-glycolide) scaffolds, which indicates the potential for ADSCs application in improving vascularization in BTE. Another study where gene therapy and ADSCs were combined was conducted by Peterson and associates [139]. First, ADSCs were transfected with the BMP-2 gene, then loaded on the collagen-ceramic carrier, and finally implanted in criticalsized femoral defect of nude mice. Eight weeks after implantations, histologic, radiographic, and biomechanical analyses showed that the collagen-ceramic carrier combined with ADSCs previously transfected with the BMP-2 gene caused bone formation within the defect.
