# Nanomaterials in Bone Regeneration
## 2 Bone Biology
### Bone Development  
Bone formation (ossification) is a complex process by which other types of connective tissue are converted into bone. Different parts of the mammalian skeleton are made up of different cell lines. An example is a difference in the formation of the skull bones. Neural crest cells migrate from the dorsal edges of the closing neural tube to the anterior region of the skull and contribute to the craniofacial skeleton. These cells produce dental dentin, connective tissue, and some bones at the front of the skull. On the other hand, prechordal mesodermal cells form the cartilage and bones of the back of the skull. The paraxial mesoderm (somites) forms the axial skeleton, while the mesodermal cells of the lateral plate form the appendicular skeleton [57]. Two different types of bone formation can be distinguished, intramembranous and endochondral ossification. The process of intramembranous ossification is initiated by MSCs, which after replication and formation of special clusters within primitive connective tissue, differentiate directly into osteoblasts. Osteoblasts secrete bone matrix proteins able to mineralize by binding calcium salts. The intramembranous type of ossification is how flat bones are formed. Endochondral ossification is a multi-stage process of bone formation in vertebrates. It is a formation of the mineralized bone according to the cartilage pattern. Endochondral bone formation occurs in the skull base and the posterior part of the skull, the axial skeleton, and the appendicular skeleton [58]. Intramembranous ossification is based on osteoblast lineage comprising the following differentiation stages: osteoporosis, preosteoblasts, osteoblasts, bone lining cells, and osteocytes. The key elements regulating osteoblast differentiation and function are Wnt and Notch signaling and the transcription factors RUNX2 and osterix (Oxx). Osteoblasts are characterized by OC expression. Preosteoblasts are characterized by the expression of RUNX2, or in the more differentiated stage, RUNX2 and Oxx. Osteocytes play an important role as regulators of bone remodeling in response to mechanical and hormonal signals. This relationship is partly driven by sclerostin produced by osteocytes. Sclerostin inhibits Wnt signaling in osteoblasts, leading to osteoblast differentiation. Wnt/\(\beta\)-catenin signaling is a key signaling determining the differentiation of mesenchymal progenitors. Genetic inactivation of \(\beta\)-catenin causes ectopic chondrocyte formation at the expense of osteoblast differentiation during intramembranous and endochondral ossification [59]. Endochondral ossification is based on the differentiation of mesenchymal cells (progenitors) into chondrocytes and periodontal cells [60]. Endochondral bone formation is regulated by many morphogens and growth factors, such as Wnts, hedgehogs, Notch, vascular endothelial growth factor (VEGF), hypoxia-inducible factor 1-alpha (HIF1\(\alpha\)), fibroblast growth factor (FGF), insulin-like growth factor-1 (IGF-1), transforming growth factor-beta (TGF-\(\beta\)), paired box transcription factor (PAX), Homeobox transcription factor (HOX) and parathyroid hormone-like peptide (PTHrP) [61; 62]. In the beginning, there is always an aggregate of mesenchymal cells that form mesenchymal condensations, which predetermine the formation of future skeletal elements. After mesenchymal cell condensation, mesenchymal cells differentiate into chondrocytes. Chondrocytes then form a cartilage template for future bone. Fibroblast growth factor receptor 3 gene (FGFR3) is a unique negative regulator of chondrocyte proliferation [63]. FGFR3 acts on chondrocyte proliferation via the transcription factor STAT1 and the ligand FGF18. Proliferation is also controlled by growth hormones and IGF-1 [64]. Hypertrophy of chondrocytes occurs under the influence of Runx-2, Indian hedgehog, and BMP-6.