**3. Mesenchymal stem cells for oral and maxillofacial tissue reconstruction**

#### **3.1. Mesenchymal stem cells for bone engineering**

The bone marrow is not only the site where hematopoiesis occurs in postnatal life, it is also a reservoir of pluripotent stem cells for mesenchymal tissues [50]. Plated at low densities, single precursor cells derived from bone marrow, and referred to as colony-forming units, give rise to distinct and heterogeneous colonies. These colonies have been shown to undergo osteogenic, chondrogenic and adipogenic differentiation [51].

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

**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‐

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.

**3. Mesenchymal stem cells for oral and maxillofacial tissue reconstruction**

The bone marrow is not only the site where hematopoiesis occurs in postnatal life, it is also a reservoir of pluripotent stem cells for mesenchymal tissues [50]. Plated at low densities, single precursor cells derived from bone marrow, and referred to as colony-forming units, give rise

factors and tumor formation.

144 Regenerative Medicine and Tissue Engineering

cantly expanded the frontiers of reconstructive surgery.

**3.1. Mesenchymal stem cells for bone engineering**

Chang and colleagues showed that MSCs can produce ectopic bone generation in a mouse model [52]. A suspension of osteogenically induced MSCs was added to 2% alginate, which was then gelled by mixing with calcium sulfate. The gel was injected subcutaneously on the dorsal side of the experimental animals. Histological examination of the implants revealed signs of endochondrosis with woven bone deposition. The equilibrium modulus of the newly formed bone increased with time up to 678 kPa at 30 weeks, as determined by biomechanical analysis. This value is approximately 1.62% of native bovine cancellous bone. In another study [53] of large mandibular bone defect repair, dog MSCs cultured with ß-TCP to generate osteogenic cells were co-implanted with a titanium plate into a 30 mm segmental mandible defect. Biomechanical tests showed a significant difference between the experimental group (with cells) and the control group (without cells), highlighting the importance of the MSCs in bone formation. Pedicled bone flaps based on collagen I scaffolds, bone marrow stromal cells and a PTFE membrane have been successfully generated using the carotid artery and jugular vein or the saphenous bundle as a vascular axis in a mouse model [54]. The osteogenetic stimulus was supplied by the injection of mouse MSCs cultured in osteogenic medium inside the space delimited by the PTFE membrane. After only 4 weeks islands of bone tissue were present inside the membrane.

#### **3.2. Clinical trials for bone engineering with mesenchymal stem cells**

There is some clinical experience with bone reconstruction using expanded MSCs combined with scaffolds. Constructs of expanded autologous MSCs in macroporous hydroxyapatite were used in three patients with large segmental bone defects [55, 56].

Warnke and Terheyden have developed a two stage procedure for mandible reconstruction in humans [57]. This study used prefabrication in the latissimus dorsi muscle with the aim of reconstructing a 70 mm defect in the mandible of a man who underwent a tumor resection years previously. The entire construction of the mandible was built using blocks of Bio-Oss® and MSCs that had been cultured in the presence of BMP-7. The Bio-Oss® and MSCs were placed in a titanium cage, and implanted into the latissimus dorsi of the patient and maintained in situ for 7 weeks. Subsequently, this unit, together with the vascular bundle that supplied it, was removed and re-implanted in the mandible defect by fixation with titanium plates and microvascular sutures connecting the vasculature to the external carotid artery and the cephalic vein.
