**2. Current therapy for large bone reconstruction**

Bone tissue is composed of heterogeneous cell types embedded in a three-dimensional mineralized extracellular matrix. The scaffolds for repair of large bone defects, including autogenous bone grafts or biomaterials, must provide the necessary support for cells to proliferate while maintaining their potential to differentiate, and must possess an architecture suitable for matching the final shape of the newly formed bone [28].

#### **2.1. Autologous bone reconstruction**

The current standard of care for repair of critical large bone defects consists of autoge‐ nous bone grafting using bone from the rib or iliac crest of the patient. An autologous bone graft is still the ideal material for the repair of craniofacial defects; however, the availability of autologous bone is limited and harvesting can be associated with complica‐ tions [29]. Vascularized and avascular autogenous bone has a greater osteogenic capacity than any other bone replacement material, as revascularization attracts mesenchymal differentiation into osteogenic, chondrogenic and other cell types. Autogenous bone transplants possess an inherent biocompatibility and are therefore more easily incorporat‐ ed without immunogenic responses [30]. However, the clinical use of autologous bone transplants is limited by considerable donor site morbidity, which increases with the amount of harvested bone. Bleeding, hematomas, infections, and chronic pain are com‐ mon complications of autologous bone graft harvests [31, 32].

#### **2.2. Allogenic/Xenogenic bone reconstruction**

The concept of prefabricated bone engineering with MSCs for large bone defects may play a pivotal role in future therapies. However, bone marrow-derived MSCs have been reported to require selective sera lots and growth factor supplements for culture expansion [11]. Further‐ more, traditional bone marrow procurement, particularly in volumes larger than a few

Bone marrow tissue provides the most universal and attractive source of MSCs; however, other tissues such as periosteal [15], muscle [16], synovial membrane [17] and adipose [18-20] tissues also appear to possess MSCs. Particularly, adipose tissue is an important source of stem cells because subcutaneous adipose tissue is an abundant and accessible source of both uncultured stromal vascular fraction (SVF) cells and cultured homogeneous adipose-derived stem cells (ASCs) (21). ASCs obtained from lipoaspirates have multilineage potential and will differen‐ tiate into adipogenic, chondrogenic, myogenic, osteogenic, and neurogenic cells [19, 22, 23]. Thus, ASCs have great potential for clinical applications such as the repair of damaged tissues and angiogenic therapy. Injection of human ASCs was recently shown to improve neovascu‐ larization in an ischemic hindlimb mouse model and osteoid matrix formation in immunoto‐ lerant mice [24-26]. Further, ASCs have been shown to increase the functional capacity of damaged skeletal muscle in vivo [27]. Therefore, these reports suggest that ASCs may also have the potential for use in large bone tissue engineering techniques such as prefabrication. Recently, prefabricated bone engineered with ASCs was reported both with in vivo studies in rat and a clinical human case. Thus, the use of ASCs in maxillofacial tissue reconstruction should be viewed favorably and these novel approaches may have advantages for tissue

In this chapter, the current approaches and the biomaterials used for repair of large bone defects are presented, and the novel approach of prefabricated bone engineering with MSCs

Bone tissue is composed of heterogeneous cell types embedded in a three-dimensional mineralized extracellular matrix. The scaffolds for repair of large bone defects, including autogenous bone grafts or biomaterials, must provide the necessary support for cells to proliferate while maintaining their potential to differentiate, and must possess an architecture

The current standard of care for repair of critical large bone defects consists of autoge‐ nous bone grafting using bone from the rib or iliac crest of the patient. An autologous bone graft is still the ideal material for the repair of craniofacial defects; however, the availability of autologous bone is limited and harvesting can be associated with complica‐ tions [29]. Vascularized and avascular autogenous bone has a greater osteogenic capacity than any other bone replacement material, as revascularization attracts mesenchymal

**2. Current therapy for large bone reconstruction**

suitable for matching the final shape of the newly formed bone [28].

milliliters may be painful, frequently requiring general or spinal anesthesia [12-14].

reconstruction.

and ASCs is introduced.

142 Regenerative Medicine and Tissue Engineering

**2.1. Autologous bone reconstruction**

Demineralized bone matrix (DMB) is the de-cellularized and organic component of bone, and is a commercially available osteoinductive and osteoconductive biomaterial. DMB represents a concentrated source of bone morphogenetic proteins (BMPs) and has been used in numerous animals systems since its initial description in 1965 [33]. The widespread use of DMB in humans still remains restricted since the immunologic properties of donor DMB are unknown [34].

With the disadvantages of host morbidity and the limits in suitable harvesting sites and material for autologous grafts, the use of xenografts might be considered for large bone reconstructions, although the histocompatibility issues between the human recipient and animal donor preclude the use of bone xenografts [34]. However, bovine-derived DMB is currently used in oral and maxillofacial surgery [35].

#### **2.3. Synthetic scaffolds for bone reconstruction**

A wide variety of synthetic (alloplastic) scaffolds such as ceramics and polymers are used clinically for bone grafting [30]. Ceramics are crystalline, inorganic, nonmetallic minerals that are held together by ionic bonds and usually densified by sintering [36]. Ceramics such as hydroxyapatite and β-tricalcium phosphate (TCP) are currently in use clinically for bone tissue regeneration of large bone defects.

Various synthetic polymer scaffolds exhibit different structural, mechanical and degradation properties that make then suitable for bone tissue engineering [36]. Blending polymers of different molecular weights can achieve both optimal degradation rates and mechanical properties [37]. Some synthetic polymer scaffolds such as polycaprolactone (PCL) scaffold, polylactic acid (PLLA), polyglycolic acid (PGA) and polylactic-co-glycolic acid (PLGA) materials have been approved by the FDA for craniofacial applications or as absorbable sutures and bone pins/screws [36].

#### **2.4. Gene therapy for bone reconstruction**

The use of exogenous cytokines and growth factors, which are essential for bone regeneration, promotes cell adhesion, proliferation, migration and osteogenic differentiation [28]. Growth factors such as BMPs, fibroblast growth factors (FGFs), insulin-like growth factors (IGF), vascular endothelial growth factors (VEGF) and platelet-derived growth factors (PDGF) have been used in bone regeneration [28, 36].

Recently the use of combinations of growth factors, such as BMP-2 and NEL-like molecule-1 (NELL-1), was tested in rapid distraction osteogenesis in a rabbit model. The combined treatment produced significantly greater bone healing compared to single growth factor treatments after four weeks of treatment [38]. However, some reports have cautioned that the clinical use of BMPs and VEGF is in its infancy, and some risks may accompany their use. VEGF is commonly upregulated in various types of tumors to enhance their vascularization, and subcutaneous sarcomas were found in some rats administered recombinant human BMP-7 [39, 40], although no clinical relationship has been established between the use of these growth factors and tumor formation.

#### **2.5. Prefabricated bone engineering for oral and maxillofacial tissue reconstruction**

Prefabrication is an interesting area of oral and maxillofacial surgery and plastic and recon‐ structive surgery, because it represents a bridge between conventional reconstructive surgery and tissue engineering [41, 42]. The purpose of prefabrication is to build a tissue (muscle, bone, skin, or composite) with characteristics as similar as possible to those of the defect that is to be repaired [43]. Conventional osteomyocutaneous flaps do not always meet the requirements for repairing a composite defect. A prefabricated composite flap can be created according to the complex geometry of the defect. Prefabrication of multi-component flaps is a well estab‐ lished procedure in plastic and reconstructive surgery [41]. This concept is based on the revascularization phenomenon directly related to host tissue vascularity [44] and has signifi‐ cantly expanded the frontiers of reconstructive surgery.

Hirase et al. were the first to report the use of prefabricated myocutaneous and osteomyocu‐ taneous tissue in a rat model [45]. Flap prefabrication using conventional bone grafts allows for generation of new types of flaps independent of the vascular anatomy of the bone trans‐ plant. However, the donor site morbidity after harvesting of bone for grafting is still a problem. Recently, biomaterials, osteogenic cells and osteoinductive growth factors have been used for generation of vascularized bone tissues in combination with a vascular axis or vascularized flaps. An inflammatory wound healing response as a reaction to the surgical implantation induces vascularization of the scaffolds [31]. Induction of axial vascularization protected the porous biomaterials from bacterial infection and transfer of this vascularized hard tissue as a free flap has been demonstrated [46]. Prefabricated vascularized bone grafts have been used in a clinical setting for mandibular reconstruction following thorough in vivo evaluation in a pig model [47-49]. In these studies, granules of xenogenic bone minerals soaked with recombi‐ nant Osteogenic protein-1 were implanted into the latissimus dorsi muscle and the neo-tissue was subsequently transferred to sites of mandibular defects using microsurgical techniques.
