**4. Adipose-derived stem cells for oral and maxillofacial tissue engineering**

#### **4.1. Characterization of adipose-derived stem cells**

There is a general consensus that SVF cells are a heterogeneous population, and no specific ranges for each subpopulation have been agreed upon formally [21]. In contrast, the Interna‐

tional Society for Cell Therapy has provided guidelines for the definition of MSCs, as follows. (1) MSCs must be plastic-adherent when maintained in standard culture conditions. (2) MSCs must express CD105, CD73 and CD90, and lack expression of CD45, CD34, CD14 or CD11b, CD79α or CD19 and HLA-DR surface antigens. (3) MSCs must differentiate into osteoblasts, adipocytes and chondroblasts in vitro [58].

SVF cells include preadipocytes, fibroblasts, vascular smooth muscle cells, endothelial cells (ECs), resident monocytes/macrophages, lymphocytes and ASCs [59]. Although the criteria to define SVF cells remain in contention, the heterogeneous SVF cell population includes putative ASCs (CD31-, CD34+/-, CD45-, CD90+, CD105-, CD146-), endothelial (progenitor) cells (CD31+, CD34+, CD45-, CD90+, CD105-, CD146+), vascular smooth muscle cells or pericytes (CD31-, CD34+/-, CD45-, CD90+, CD105-, CD146+), and hematopoietic cells (CD45+) in uncultured conditions [60]. Cultured ASCs show an extensive proliferative ability in an uncommitted state while retaining their multilineage differentiation potential. ASCs express the mesenchymal stem cell markers CD10, CD13, CD29, CD34, CD44, CD54, CD71, CD90, CD105, CD106, CD117, CD166 and STRO-1. They are negative for the hematopoietic lineage markers CD45, CD14, CD16, CD56, CD61, CD62E, CD104, and CD106 and for the EC markers CD31, CD144, and von Willebrand factor [20, 61, 62]. Morphologically, cultured ASCs are fibroblast-like and preserve their shape after expansion in vitro [20, 63]. The ASC specific surface markers CD29, CD90, and CD166 increase during culture [64]. In later passages, ASC cultures are homogeneous and exhibit fibroblastoid morphology. The composition of the subpopulations, therefore, may change during expansion [65, 66]. Therefore ASCs match the standard criteria for MSCs.

#### **4.2. Differentiation potential of osteogenic cells in vitro and in vivo**

Numerous studies have presented results that clearly show that ASCs can differentiate into osteoblasts [20, 59, 63, 67, 68]. ASCs exhibit a time-dependent expression of genes and proteins associated with the osteoblast phenotype, including ALP, Type I Collagen, OPN, ON, RUNX2, BMP-2, BMP-4 and BMP receptors I and II [20, 67, 69, 70]. Additionally, between 2 and 4 weeks of culture, mineralization of the extracellular matrix begins and proceeds via the activity of ALP, an enzyme that hydrolyzes phosphate esters making available inorganic phosphate to form hydroxyapatite [19, 20, 71].

Furthermore, recent reports have shown that ASCs co-cultured with ECs exhibit enhanced osteogenesis [72, 73]. ASCs exhibited increased secretion of alkaline phosphatase and osteo‐ calcin, and an overall increase in osteogenesis in the co-cultured situation compare with other experimental groups. These interactions may be important to regenerate bone in large bone defects since angiogenesis plays a key role in regeneration of large amounts of tissue.

#### **4.3. Fabricated bone engineering with adipose-derived stem cells**

To make a functional prefabricated bone, three elements are required: scaffolds to provide a three-dimensional support, growth factors to stimulate neovascularization, and MSCs to give an osteoinductive stimulus. Okuda et al. have reported prefabrication of tissue engineered bone grafts using ASCs in a rat model [74]. ASCs and porous β-TCP as scaffold material were implanted into the superficial inferior epigastric artery flap. After prefabrication for eight weeks, the prefabricated flaps were elevated and the pedicles were clamped for 4 h; prefabri‐ cated tissue was harvested two weeks later. The osteogenic capacity of the prefabricated graft was not significantly different from non-prefabricated grafts examined after two weeks in a rat model. Furthermore, an analysis of angiogenesis suggested that the prefabricated model possessed significantly greater capillary density than the non-prefabricated model.

tional Society for Cell Therapy has provided guidelines for the definition of MSCs, as follows. (1) MSCs must be plastic-adherent when maintained in standard culture conditions. (2) MSCs must express CD105, CD73 and CD90, and lack expression of CD45, CD34, CD14 or CD11b, CD79α or CD19 and HLA-DR surface antigens. (3) MSCs must differentiate into osteoblasts,

SVF cells include preadipocytes, fibroblasts, vascular smooth muscle cells, endothelial cells (ECs), resident monocytes/macrophages, lymphocytes and ASCs [59]. Although the criteria to define SVF cells remain in contention, the heterogeneous SVF cell population includes putative ASCs (CD31-, CD34+/-, CD45-, CD90+, CD105-, CD146-), endothelial (progenitor) cells (CD31+, CD34+, CD45-, CD90+, CD105-, CD146+), vascular smooth muscle cells or pericytes (CD31-, CD34+/-, CD45-, CD90+, CD105-, CD146+), and hematopoietic cells (CD45+) in uncultured conditions [60]. Cultured ASCs show an extensive proliferative ability in an uncommitted state while retaining their multilineage differentiation potential. ASCs express the mesenchymal stem cell markers CD10, CD13, CD29, CD34, CD44, CD54, CD71, CD90, CD105, CD106, CD117, CD166 and STRO-1. They are negative for the hematopoietic lineage markers CD45, CD14, CD16, CD56, CD61, CD62E, CD104, and CD106 and for the EC markers CD31, CD144, and von Willebrand factor [20, 61, 62]. Morphologically, cultured ASCs are fibroblast-like and preserve their shape after expansion in vitro [20, 63]. The ASC specific surface markers CD29, CD90, and CD166 increase during culture [64]. In later passages, ASC cultures are homogeneous and exhibit fibroblastoid morphology. The composition of the subpopulations, therefore, may change during expansion [65, 66]. Therefore ASCs match the standard criteria for MSCs.

Numerous studies have presented results that clearly show that ASCs can differentiate into osteoblasts [20, 59, 63, 67, 68]. ASCs exhibit a time-dependent expression of genes and proteins associated with the osteoblast phenotype, including ALP, Type I Collagen, OPN, ON, RUNX2, BMP-2, BMP-4 and BMP receptors I and II [20, 67, 69, 70]. Additionally, between 2 and 4 weeks of culture, mineralization of the extracellular matrix begins and proceeds via the activity of ALP, an enzyme that hydrolyzes phosphate esters making available inorganic phosphate to

Furthermore, recent reports have shown that ASCs co-cultured with ECs exhibit enhanced osteogenesis [72, 73]. ASCs exhibited increased secretion of alkaline phosphatase and osteo‐ calcin, and an overall increase in osteogenesis in the co-cultured situation compare with other experimental groups. These interactions may be important to regenerate bone in large bone

To make a functional prefabricated bone, three elements are required: scaffolds to provide a three-dimensional support, growth factors to stimulate neovascularization, and MSCs to give an osteoinductive stimulus. Okuda et al. have reported prefabrication of tissue engineered bone grafts using ASCs in a rat model [74]. ASCs and porous β-TCP as scaffold material were

defects since angiogenesis plays a key role in regeneration of large amounts of tissue.

**4.3. Fabricated bone engineering with adipose-derived stem cells**

**4.2. Differentiation potential of osteogenic cells in vitro and in vivo**

adipocytes and chondroblasts in vitro [58].

146 Regenerative Medicine and Tissue Engineering

form hydroxyapatite [19, 20, 71].

Recently, repair of a large bony defect using ASCs was clinically reported [75-77] (Table. 1). Mesima°ki and colleagues published a clinical case report of prefabricated bone tissue engineering [77]. The large bony defect was reconstructed with a microvascular flap using autologous ASCs, β-TCP and BMP-2, 36 months after a hemimaxillectomy due to a large keratocyst. After expansion of ASCs and cultivation with β-TCP and BMP-2 in vitro, a titanium cage filled with ASCs and β-TCP was inserted through a vertical incision into a pouch prepared in the patient's left rectus abdominis muscle. The rectus abdominis free flap was raised. Before severing the vascular supply to the muscle, the muscle pouch was carefully opened and the titanium cage was opened. After severing the vessels, the flap was placed in the maxillary defect; the inferior epigastric artery was anastomosed end-to-end to the facial artery and the vein end-to-end to the facial vein.


Abbreviations: SVF; Stromal Vascular Fraction, ASCs; Adipose-derived Stem Cells, β-TCP; Beta-tricalcium phosphate, MSCs; mesenchymal stem cells, BMP; bone morphogenetic protein

(\*Identifier on Clinical trials website: \*http://clinicaltrials.gov/ct2/results?term=adipose+derived+cells+bone).

**Table 1.** Clinical reports/trials for large bony defect using adipose-derived stem cells

### **5. Future perspective**

In the past decade, basic research characterizing ASCs shows that these cells have the potential to regenerate tissue defects such as large bone defects, and clinical studies have examined the potential use of ASCs to reconstruct oral and maxillofacial tissue. Although clinical studies have only just begun, the use of ASCs in the clinical setting is extremely promising because ASCs are a readily available, multipotent, and abundant cell type with the capability to undergo robust osteogenesis. However, further studies, including research to determine the mechanism of osteogenic differentiation and studies to evaluate the safety of ASC usage, will be necessary to realize the potential of ASCs in clinical regenerative medicine of the future.
