Granulation tissue forms after hematoma facilitating vascular ingrowth and angiogenesis. Within the granulation tissue, endochondral formation occurs in between the fracture ends. Following the differentiation of MSCs into chondrocytes and osteoblasts, the cartilaginous tissue forms a soft callus. Periosteum, bone marrow, and endosteum are three sources of osteoblasts and osteocytes whereas chondrocytes within the fracture callus are primarily derived from the periosteum. Types I and II collagen are produced in order to form a matrix that restores stability to the bone ends. This corresponds roughly to the time when the fragments are no longer moving freely, approximately 2–3 weeks after the initial fracture occurs. TGF-β, FGF-1 and 2, PDF and IGF-1 are all expressed in the soft callus and regulate chondrocyte differentiation. FGF-2 regulates expression of the transcription factor Sox-9, which activates the expression of type I, IX, and collagen genes and the gene for aggrecan protein [61].

The hard callus appears with the conversion of cartilage to a calcified cartilage matrix with terminal differentiation of the chondrocytes. Macrophages produce matrix metalloproteinases (MMPs) to degrade the cartilage matrix. These MMPs have a central role in soft-to-hard callus switch [11, 62–65]. Other immune cells also reappear during the mineralization of cartilaginous callus. This includes T- and B-lymphocytes that were found to be located in a close contact with osteoblasts and osteoclasts. These lymphocytes produce TNF-α which trigger the death of mature chondrocytes aiding the transition from cartilage into bone [28, 42, 66]. Monoclonal antibodies to BMP-2, 4, and 7 showed an increasing intensity of staining for the BMPs in periosteal mesenchymal cells in the region of hard callus formation, in the proliferating mesenchymal cells of the early soft callus and chondroblasts differentiated from these cells [61]. IL-17 is another cytokine that can affect the conversion of soft callus into the hard callus. Also, IL-17 enhances the MSC differentiation into osteoblasts [67]. Given the reduction in the number of chondrocytes, the dominant cell types during the hard callus phase are the osteoblast and osteoclast. Several growth factors including PDGF, TGF-β, IGF, fibroblast growth factor-1 (FGF-1), and BMPs involved in proliferation and the chondrogenic differentiation of MSCs as well as deposition of collagen [38, 68, 69]. Overall, the conversion of soft callus into hard callus is highly controlled by macrophages, T- and B-lymphocytes, and various cytokines and growth factors [25].

Fracture healing is completed during the remodeling phase in which the healing bone is restored nearer to its original shape, structure, and mechanical strength. Cutting cones are formed by osteoclasts that first remove the disorganized woven bone. Then, osteoblasts follow and lay down lamellar bone in an organized pattern around a central blood vessel. During remodeling, the canalicular architecture of bone is reestablished, and the Haversian system with its osteocytes is restored. This process starts in concert with bone consolidation and continues for months or years after a solid osseous union has been achieved. The remodeling of the fractured bone is mainly related to the balance between osteoblast and osteoclast functions under both systemic and local control pathways. The osteoblast/osteoclast function is controlled by MSCs, macrophages, and cytokines such as TNF-α and IL-17 [25]. The TNF family molecule RANKL and its receptor RANK are key regulators of bone remodeling [70]. Osteoprotegerin (OPG), a soluble decoy receptor for receptor RANKL, inhibits both differentiation and function of osteoclasts and decreasing bone resorption [71]. At the same time, IL-17 enhances the expression of RANKL on MSCs enhancing the osteoclastogenesis when co-cultured with peripheral blood mononuclear cells (PBMCs) [63, 72]. Another cytokine, TNF-α produced by MSCs and osteoblasts during the late phase of bone healing [37] can also influence both osteoblast and osteoclast functions demonstrating its vital role in the remodeling phase [73, 74]. Also, several systemic hormonal pathways are involved in bone remodeling. PTH, PTHrP, 1,25-20H vitamin D3, and leptin increase bone resorption by increasing osteoclast differentiation. Conversely, sex steroids inhibit osteoclast differentiation, while intermittent PTH administration and high doses of vitamin D3 stimulate osteoprogenitor cells to differentiate into osteoblasts.

The biological sequence of fracture healing is an overlapping process with many cytokines and growth hormones involved in different phases. The inflammatory phase is characterized by pro-inflammatory cytokines and inflammatory cells dominancy. MSC migration to the fracture site and licensing of these cells controlled by a chemokine cocktail. After the suppression of the initial inflammatory response repair phase starts and MSCs differentiate into osteoblasts and chondroblasts which form callus. Fracture healing is completed by remodeling which is regulated between osteoblast and osteoclast functions both systemic and local control pathways involved. Remodeling of the bone occurs slowly over months to several years. For successful bone remodeling, adequate blood supply and mechanical stability is essential. Moreover, with the current knowledge the final destination of the fracture is seemed to be controlled at the beginning with the early secretion of growth factors and cytokines further function in the later stages. With the recent advances, understanding of the biologic events occurs during healing phases, it will be possible to control healing process of a fracture at the beginning before any adverse outcomes occur (Table 34.1).