**Abstract**

Despite excellent self-regeneration capacity of bone tissue, there are some large bone defects that cannot be healed spontaneously. Numerous literature data in the field of cell-based bone tissue engineering showed that adipose-derived stem cells (ADSCs) after isolation could be subsequently applied in a one-step approach for treatment of bone defect, without previous in vitro expansion and osteoinduction. However, standard approaches usually involve in vitro expansion and osteoinduction of ADSCs as an additional preparation step before its final application. Bioreactors are also used for the preparation of ADSC-based graft prior application. The commonly used approaches are reviewed, and their outcomes, advantages, disadvantages, as well as their potential for successful application in the treatment of bone defects are discussed. Difficulty in spontaneous healing of bone defects is very often due to poor vascularization. To overcome this problem, numerous methods in bone tissue engineering (BTE) were developed. We focused on freshly isolated stromal vascular fraction (SVF) cells and ADSCs in vitro induced into endothelial cells (ECs) as cells with vasculogenic capacity for the further application in bone defect treatment. We have reviewed orthotopic and ectopic models in BTE that include the application of SVFs or ADSCs in vitro induced into ECs, with special reference to co-cultivation.

**Keywords:** stromal vascular fraction, adipose-derived stem cells, endothelial cells, in vitro-induced differentiation, bone tissue engineering, vascularization, osteogenic process stromal vascular fraction, adipose-derived stem cells, endothelial cells, in vitro-induced differentiation, bone tissue engineering, vascularization, osteogenic process

### **1. Introduction**

Structure of the bone tissue is very dynamic due to environmental influence but also because of many factors that act inside the body [1]. The bone can regenerate and repair itself, but large fractures and bone defects fail to heal and repair successfully. In addition to other adverse factors, this results in delayed unions, malunions, or nonunions. To aid bone healing and repair in such situations, build bone-deficient areas, or replace missing bone as well as in purposes of joint reconstruction, bone grafting is used [2–5].

Bone grafting is one of the most common options for the treatment of major bone defects, and the use of bone grafts is among the most common procedures in orthopedic surgery and the second most frequent transplantation of tissue after blood. Over 2 million bone grafting procedures are performed annually in surgery [5, 6]. Bone graft material, alone or in combination with other materials, can perform bone healing function by having at least one of the features among osteogenicity, osteoinduction, and osteoconduction and therefore usually have one or more components—scaffold, as an osteoconductive matrix that supports bone growth, osteoinductive proteins and factors, and osteogenic cells [5, 7]. The main types of bone grafting materials are autografts, allografts, xenografts, synthetic and biological tissue engineering biomaterials, and combinations of these materials [2–6]. Bone grafts and graft substitutes may differ by material type, source, and origin and may also be categorized as osteogenic, osteoinductive, and osteoconductive agents [7]. The choice of bone graft depends on the condition of the bone tissue, defect size, surgical feasibility of the procedure, possible health complications, graft structure, its biological and mechanical characteristics, size and shape, cost, and ethical issues [5].

Each of the bone graft material and its substituents has its advantages and disadvantages, of which there is considerable agreement in surgical practice. Autografts are the best clinical solution among grafts because they have all the necessary properties to stimulate bone repair and regeneration—osteogenicity, osteoinductivity, and osteoconductivity—as they provide osteogenic cells, osteoinductive factors, and osteoconductive scaffold for bone growth. So far, autografts have proven superior in quality and time to bone healing. That is why in orthopedic practice, for the purpose of reconstruction of small defects and replacement of lost bone, autografts are the "gold standard" [3, 5–7]. Autografts can be of different types such as vascularized grafts, bone tissue, or bone marrow. They are usually obtained from the iliac crest, as well as from mandibular bone in dentistry, and by osteectomy and osteoplasty in various procedures. The vascularized fibula is used for the treatment of congenital bone damage, replacement of the bone segment after trauma, and in the case of a malignant tumor so that the periosteum and nutrient artery allow the graft to live and grow in the transplanted site [4, 8, 9]. The use of autografts often involves additional surgery and additional pain, and there is a frequent occurrence of morbidity at the donor site, which with limited availability is a limitation to their use. Morbidity, although low, is significant and ranges from minor to major complications, and there is a risk of injury to large blood vessels and visceral organs while taking the graft. The type of complications and their severity depends on the donor site as well as whether the graft material is taken at the same incision on the primary surgical site [2, 3, 5–8].

Allografts are much more available for orthopedic purposes. They have an osteoconductivity property, they can be well remodeled, but they are poorly osteoinductive, which can be present if they have an organic matrix. All of these properties depend on the size of the graft and the grafting site [3–6, 8]. Allografts are obtained from a bone bank containing cadaveric bones and can be found in various forms. Compared to autografts, allografts lack osteogenic properties and thus have an overall weaker bone regenerative potential, and their integration into the recipient bone over the long term may be insufficient. Allografts can induce immune response and rejection and requires caution because of the possibility of transmission of pathogens from the donor organism, and their disadvantages are also high costs and lack of availability of donors [3, 5–8]. To reduce some disadvantages and limitations by allografts, cells and proteins that can elicit an immune response can be removed, thereby also reducing the possibility of transmission of viral infection by donors. Usually, the sterilization and deactivation process of the proteins is performed before the use of the allograft, but then they lack osteoinductive factors, which in turn remain if the demineralized bone matrix is prepared [4, 8].

**181**

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

as is the case with the PRP and DBM, is still an open question [6, 10].

The shortcomings in the outcomes of bone healing were the impetus to seek new types of grafts and bone substitutes. On these motives, a bone tissue engineering (BTE) as a new field began to develop in the last decades of the last century, integrating multiple disciplines such as cell biology, developmental and molecular biology, biomechanics, biomaterials science, immunology, and others. With the progressive introduction of innovations and new technologies, BTE has been offering more and more solutions to reduce the disadvantages of traditional bone grafts and improve the process of healing fractures and defects by achieving

Xenografts are obtained from species other than humans, e.g., bovine bone, which is much used in dentistry. There are also nonbone xenograft materials such as sclera, collagen membranes, and coral-derived materials. Compared to autografts, xenografts as allografts only have osteoconductive and osteoinductive but lack osteogenic features. There is a risk of zoonotic transmission in xenografts treatment, and rejection is more likely and stronger than in the case of an allograft. Xenografts are cheaper, but the results of their application are inconsistent [4–6]. In addition to bone grafts (autograft, allograft, xenograft), newer bone substitutes are the ceramic types (calcium compounds: hydroxyapatite, tricalcium phosphate (TCP), calcium sulfate) and biological factors such as growth factors and others (bone morphogenetic proteins (BMPs), platelet-rich plasma (PRP), demineralized bone matrix (DBM)). Bone substitutes have been used for decades and have been defined as synthetic, inorganic, or organic, as well as biological origin materials, or a combination of those used to treat bone defect instead of bone [3, 4, 6]. Bone substitutes are especially used in traumatology and oncologic, spine, and prosthetic surgeries. Suitable bone substitutes should be biocompatible, not provoking an adverse inflammatory response, osteoinductive, osteoconductive, resorbable, easily molded into the bone defect, nonconductive, sterilizable, available, traceable in vivo, and at a reasonable cost [6]. The integration of bone substitutes over the long term may not be sufficient. Bone substitute materials of a synthetic nature such as calcium phosphate (CaP)-based biomaterials often behave osteoconductively only and can be remodeled. Osteoinductivity is possessed by biological factors such as bone matrix proteins or BMP-type growth factors that can be added to other bone substitutes [3, 8]. Ceramic-based bone graft substitutes include hydroxyapatite (HA), TCP, calcium sulfate, and bioglass used alone or in combination. Ceramics can be used in the form of granules, blocks, or moldable paste shape, and the occurrence of injectable cements was particularly significant because it enabled a mininvasive application [6, 7, 10]. Calcium phosphate-based bone substitutes have wide clinical use, since they are generally therapeutically effective, although they have poor mechanical properties, are less strong than bone tissue, and can be completely resorbed. The more advanced variants of HAs have biomimetic properties, since they include ions (carbonates, Mg, fluoride, Sr), so that natural HA is imitated [6]. Polymer-based bone substitutes can be degradable and nondegradable polymers and are applied alone, as co-polymers, or in combination with other materials [7, 10]. Various marine biomaterials are also used as bone substitutes, including chitosan, corals, and sponge skeleton [7]. Although biological factors generally influence good bone formation, the clinical application is not widespread due to high prices and possible adverse side effects [3]. Growth factors as bone substitutes, such as BMPs, transforming growth factor-beta (TGF-beta), platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF), can be natural and recombinant and can be used alone or in combination with other materials [7]. BMPs are bone growth factors that are widely used in spine surgery and for the treatment of tibial nonunion. The efficiency of some biological factors,

*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*

Xenografts are obtained from species other than humans, e.g., bovine bone, which is much used in dentistry. There are also nonbone xenograft materials such as sclera, collagen membranes, and coral-derived materials. Compared to autografts, xenografts as allografts only have osteoconductive and osteoinductive but lack osteogenic features. There is a risk of zoonotic transmission in xenografts treatment, and rejection is more likely and stronger than in the case of an allograft. Xenografts are cheaper, but the results of their application are inconsistent [4–6].

In addition to bone grafts (autograft, allograft, xenograft), newer bone substitutes are the ceramic types (calcium compounds: hydroxyapatite, tricalcium phosphate (TCP), calcium sulfate) and biological factors such as growth factors and others (bone morphogenetic proteins (BMPs), platelet-rich plasma (PRP), demineralized bone matrix (DBM)). Bone substitutes have been used for decades and have been defined as synthetic, inorganic, or organic, as well as biological origin materials, or a combination of those used to treat bone defect instead of bone [3, 4, 6]. Bone substitutes are especially used in traumatology and oncologic, spine, and prosthetic surgeries. Suitable bone substitutes should be biocompatible, not provoking an adverse inflammatory response, osteoinductive, osteoconductive, resorbable, easily molded into the bone defect, nonconductive, sterilizable, available, traceable in vivo, and at a reasonable cost [6]. The integration of bone substitutes over the long term may not be sufficient. Bone substitute materials of a synthetic nature such as calcium phosphate (CaP)-based biomaterials often behave osteoconductively only and can be remodeled. Osteoinductivity is possessed by biological factors such as bone matrix proteins or BMP-type growth factors that can be added to other bone substitutes [3, 8]. Ceramic-based bone graft substitutes include hydroxyapatite (HA), TCP, calcium sulfate, and bioglass used alone or in combination. Ceramics can be used in the form of granules, blocks, or moldable paste shape, and the occurrence of injectable cements was particularly significant because it enabled a mininvasive application [6, 7, 10]. Calcium phosphate-based bone substitutes have wide clinical use, since they are generally therapeutically effective, although they have poor mechanical properties, are less strong than bone tissue, and can be completely resorbed. The more advanced variants of HAs have biomimetic properties, since they include ions (carbonates, Mg, fluoride, Sr), so that natural HA is imitated [6]. Polymer-based bone substitutes can be degradable and nondegradable polymers and are applied alone, as co-polymers, or in combination with other materials [7, 10]. Various marine biomaterials are also used as bone substitutes, including chitosan, corals, and sponge skeleton [7]. Although biological factors generally influence good bone formation, the clinical application is not widespread due to high prices and possible adverse side effects [3]. Growth factors as bone substitutes, such as BMPs, transforming growth factor-beta (TGF-beta), platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF), can be natural and recombinant and can be used alone or in combination with other materials [7]. BMPs are bone growth factors that are widely used in spine surgery and for the treatment of tibial nonunion. The efficiency of some biological factors, as is the case with the PRP and DBM, is still an open question [6, 10].

The shortcomings in the outcomes of bone healing were the impetus to seek new types of grafts and bone substitutes. On these motives, a bone tissue engineering (BTE) as a new field began to develop in the last decades of the last century, integrating multiple disciplines such as cell biology, developmental and molecular biology, biomechanics, biomaterials science, immunology, and others. With the progressive introduction of innovations and new technologies, BTE has been offering more and more solutions to reduce the disadvantages of traditional bone grafts and improve the process of healing fractures and defects by achieving

*Clinical Implementation of Bone Regeneration and Maintenance*

ethical issues [5].

in orthopedic surgery and the second most frequent transplantation of tissue after blood. Over 2 million bone grafting procedures are performed annually in surgery [5, 6]. Bone graft material, alone or in combination with other materials, can perform bone healing function by having at least one of the features among osteogenicity, osteoinduction, and osteoconduction and therefore usually have one or more components—scaffold, as an osteoconductive matrix that supports bone growth, osteoinductive proteins and factors, and osteogenic cells [5, 7]. The main types of bone grafting materials are autografts, allografts, xenografts, synthetic and biological tissue engineering biomaterials, and combinations of these materials [2–6]. Bone grafts and graft substitutes may differ by material type, source, and origin and may also be categorized as osteogenic, osteoinductive, and osteoconductive agents [7]. The choice of bone graft depends on the condition of the bone tissue, defect size, surgical feasibility of the procedure, possible health complications, graft structure, its biological and mechanical characteristics, size and shape, cost, and

Each of the bone graft material and its substituents has its advantages and disadvantages, of which there is considerable agreement in surgical practice. Autografts are the best clinical solution among grafts because they have all the necessary properties to stimulate bone repair and regeneration—osteogenicity, osteoinductivity, and osteoconductivity—as they provide osteogenic cells, osteoinductive factors, and osteoconductive scaffold for bone growth. So far, autografts have proven superior in quality and time to bone healing. That is why in orthopedic practice, for the purpose of reconstruction of small defects and replacement of lost bone, autografts are the "gold standard" [3, 5–7]. Autografts can be of different types such as vascularized grafts, bone tissue, or bone marrow. They are usually obtained from the iliac crest, as well as from mandibular bone in dentistry, and by osteectomy and osteoplasty in various procedures. The vascularized fibula is used for the treatment of congenital bone damage, replacement of the bone segment after trauma, and in the case of a malignant tumor so that the periosteum and nutrient artery allow the graft to live and grow in the transplanted site [4, 8, 9]. The use of autografts often involves additional surgery and additional pain, and there is a frequent occurrence of morbidity at the donor site, which with limited availability is a limitation to their use. Morbidity, although low, is significant and ranges from minor to major complications, and there is a risk of injury to large blood vessels and visceral organs while taking the graft. The type of complications and their severity depends on the donor site as well as whether the graft material is taken at the same incision on the primary surgical site [2, 3, 5–8]. Allografts are much more available for orthopedic purposes. They have an osteoconductivity property, they can be well remodeled, but they are poorly osteoinductive, which can be present if they have an organic matrix. All of these properties depend on the size of the graft and the grafting site [3–6, 8]. Allografts are obtained from a bone bank containing cadaveric bones and can be found in various forms. Compared to autografts, allografts lack osteogenic properties and thus have an overall weaker bone regenerative potential, and their integration into the recipient bone over the long term may be insufficient. Allografts can induce immune response and rejection and requires caution because of the possibility of transmission of pathogens from the donor organism, and their disadvantages are also high costs and lack of availability of donors [3, 5–8]. To reduce some disadvantages and limitations by allografts, cells and proteins that can elicit an immune response can be removed, thereby also reducing the possibility of transmission of viral infection by donors. Usually, the sterilization and deactivation process of the proteins is performed before the use of the allograft, but then they lack osteoinductive factors,

which in turn remain if the demineralized bone matrix is prepared [4, 8].

**180**

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 respectively [11, 16].
