Craniofacial ApplicationsBone has important functions as an endocrine organ, regulating global levels of calcium, insulin, seratonin, and other hormones [1-3]. However, bone treatments primarily emphasize restoring structural function. As a result, anatomically shaped inorganic materials have sufficed to treat cranial defects or to recapitulate maxillofacial structures. Alternatively, bone tissue engineering (BTE) uses biological components. The major difference is the new tissue’s ability to integrate with host tissues and remodel in response to environmental cues. The traditional BTE approach combines osteoprogenitor cells with scaffolds and growth factors. Potential cell sources include mesenchymal stem cells derived from various sites within the body including bone marrow and adipose tissues. These cells are readily available with limited morbidity, and can be easily differentiated into bone-forming cells through exposure to growth factors or other biophysical stimuli. Several objective parameters are assessed to determine the choice of scaffold biomaterial including its mechanical properties, osteoconductivity, and osteoinductivity. While the classical BTE approach employs a combination of cells with biomaterials to induce tissue regeneration,
the economic and regulatory hurdles associated with cell-based therapies have also led to the proliferation of cell-free biomaterial approaches, which stimulate the activity of endogenous stem cells. For large craniomaxillofacial defects, the damage to physical appearance has negative psychosocial effects on the patients and the cosmetic outcome has become a primary consideration.In this chapter we describe in detail the application of BTE principles to the facial skeleton with special emphasis on cells, scaffolds, growth factors, and bioreactor technologies used to obtain bone substitutes. We focus on the relative success of these approaches in animal studies and human trials. Additionally, we report on the impact of recent advances in the fields of micro-and nanotechnology on scaffold properties, modulation of cell-scaffold interactions, and growth factor delivery. The clinical impact and limitations of these approaches are also discussed. 17.2 Principles of Craniofacial Graft Design
The traditional strategy for BTE-mediated repair of nonhealing defects is to isolate cells (most commonly mesenchymal stem cells or progenitor cells), expand them in vitro, and seed them onto biocompatible and biodegradable scaffolds that meet the structural and mechanical requirements of the defect site. Appropriate growth factors are added to accelerate healing, initiate proliferation, and differentiation of local osteoprogenitor cells and improve bone formation. After in vitro maturation and mineralization of this cell-seeded construct, preferably in bioreactors, it is implanted at the target site. In time, the biodegradable scaffold is resorbed while the cells produce their own ECM and replace the implant. In this section, these components-cells, biomaterials, growth-factors, and bioreactors-are described in detail in the particular context of craniofacial graft design. 17.2.1 Cell Sources for Craniofacial Bone Tissue
EngineeringIt is widely assumed that exogenous cells are required to stimulate functional bone regeneration. Implantation of viable cells within appropriate scaffolds is indeed beneficial for BTE, since soft callus formation after surgery often depends on bone formation by
the transplanted osteoblasts. Possible sources of cells for tissue engineering include autogenic, allogenic and xenogenic cells. The use of allogenic cells is restricted due to possible immunogenic responses of the host , while the use of xenogenic cells is limited to research purposes and application in humans is rare . In tissue engineering, an appropriate source is autologous cells taken from a healthy region of the patient’s damaged tissue. However, due to limited availability of the healthy tissue as well as difficulty in tissue harvesting for mature cell isolation (especially in the case of bone), other cell sources have been considered.Stem cells are widely used in the engineering of bone substitutes. The use of pluripotent embryonic stem cells (ESCs) in BTE is promising  as they are capable of indefinite undifferentiated proliferation in vitro and can provide an unlimited supply of cells . However, there are technical and ethical issues that have to be addressed before they can be used in clinical applications. Ethical issues may be circumvented by the use of induced pluripotent stem (iPS) cells, which have similar characteristics to ESCs [8,9]. Although this technique has remarkable potential in tissue engineering and stem cell therapy, current limitations such as low efficiency and risks associated with tumor formation and viral transgenes should be eliminated before use in a clinical setting .Multipotent mesenchymal stem cells (MSCs) are of great interest to tissue engineers. In the current clinical practice, MSCs can be differentiated into the osteogenic lineage by culturing in the presence of osteogenic supplements . MSCs and bone repair cells  from bone marrow have been shown to enhance craniofacial bone regeneration in both orthotopic and ectopic models (Table 17.1). Bone marrow MSCs isolated from beagle dogs were labeled with green fluorescent protein (GFP) and were implanted into periodontal defects within collagen matrices . At 4 weeks, histological evaluation revealed that the defect was fully regenerated by GFPpositive cementoblasts, osteoblasts, osteocytes, and fibroblasts indicating the extent of differentiation of MSCs in orthotopic defect sites. In another recent orthotopic implantation study, bone marrow MSCs were again harvested from beagle dogs, encapsulated within HAp particles, and this time implanted within jaw cleft defects . At 6 months’ follow-up, complete regeneration of the defect was reported by histological and radiological analysis. The potential of bone marrow-derived MSCs was also verified in ectopic models.