Bone is a mineralized tissue that confers multiple mechanical and metabolic functions to the skeleton. Bone is composed by cells and an extracellular matrix which becomes
mineralized by the deposition of calcium hydroxyapatite, giving the bone rigidity and strength. Bone has three distinct cell types: the osteoblasts, or bone-forming cells, the osteoclasts, or bone-resorbing cells, whose functions are intimately linked, and the osteocytes, which are osteoblasts entrapped within lacunae. In order to balance bone formation and resorption in healthy individuals, osteoblasts secrete factors that regulate the differentiation of osteoclasts and osteocytes secrete factors regulating the activity of both osteoblasts and osteoclasts. Bone is constantly being resorbed by osteoclasts and then replaced by osteoblasts in a process called bone remodelling, which is tightly synchronized by local and endocrine factors (42). It is apparent that any imbalance in this process will lead to problems. In endosseous implant therapy the clinical success is related to the formation and maintenance of bone at the bone implant interface (77). The formation of bone at the implant surface is associated with the osteoblast metabolic and secretory activities (39). Nevertheless, the maintenance of bone at the interface is related to the long term balance between bone resorption and reformation.
Osteoblasts are mononuclear, not terminally differentiated, specialized cells that form bone. They arise from osteoprogenitor cells of mesenchymal origin and terminally
differentiate to osteocytes. These cells have a set of distinctive characteristics that include the capability of forming a collagen rich matrix (osteoid) and mineralizing it, which results in the formation of calcified bone. When they are active they have a large Golgi apparatus and an abundant rough endoplasmic reticulum. In addition, they form tight junctions with adjacent osteoblasts and have regions of plasma membrane specialized in vesicular trafficking and secretion (23). Moreover, osteoblasts are autocrine regulatory cells that synthesize and deposit growth factors into the bone matrix and respond to these factors when they are
released during bone resorption phases of repair and remodelling. Moreover, osteoblast commitment, differentiation, and function are governed by several transcription factors, resulting in expression of phenotypic genes and acquisition of osteoblast phenotype (29, 73). Several ways can be considered to control osteoblastic activity at implants, such as; osteoblast recruitment, attachment, proliferation, and differentiation. These aspects of osteoblast physiology are interrelated represent potential targets for clinical improvement of bone formation at implant surface (29).
1.4.1. Osteoblast cell culture systems
Osteoblast cell culture model is widely used in osseointegration research. As mentioned earlier this model provides an invaluable opportunity to investigate aspects of bone formation and cell biology in the laboratory. Several cell lines and culture systems have been developed and utilized in investigating the osteoblast activity and interaction at implant surface. Cooper and his collaborates categorized these cells according to their origin into
1. Primary cultures such as bone marrow stromal cells, intramembranous bone, or trabecular long bone.
2. Nontransformed clonal cell lines such as the mouse osteoblast like cells MC3T3-E1. 3. Osteosarcoma cell lines such as, the human osteosarcoma MG63 cells and the rat
osteosarcoma ROS 17/2.8 cells.
4. Intentionally immortalized cell lines such as the rat calvarial cell line RCT-1(31). This classification reflects the origin of cell lines, but not the characteristics of each model. These different cell lines represent different states of phenotypic maturation in the osteoblast lineage and have substantially different levels of homogeneity (17). Nevertheless, in cell culture studies the significance and validity of each system depends on the question to be
addressed, and the biochemical and molecular properties of the system (31).The osteoblast cell lines most commonly used to study the interaction of cells with implant surface include; the human osteosarcoma MG63 cells, the mouse osteoblast like MC3T3-E1 cells, the rat osteosarcoma osteoblast like ROS 17/2.8 cells, and the fetal rat calvaria FRC cells (17). In the present investigation the mouse osteoblast like MC3Ts-E1 cells were used. These cells were originally cultured from newborn mouse calvaria. They have unique features that make them a good model to study the mechanisms of biological calcification, osteoblast differentiation, and matrix mineralization. The cells have a low alkaline phosphatase (ALP) activity in the growing state; however, the enzyme activity will increase in the confluent state. This osteogenic cell line has the capacity to differentiate into osteoblasts, osteocytes, and deposit minerals in vitro. The cells are capable of showing different stages of growth and development under dell culture conditions (79). The pattern of osteogenesis that this cell showed in vitro is similar to intramembranous ossification pattern in vivo. The cell
morphology in the growing state resembles a fibroblast. Nevertheless, when the cells grow they start to resemble the osteoblasts in morphology and they can grow in layers (117).In intramembranous ossification the process is triggered by the aggregation of undifferentiated mesenchymal cells into layers or membranes. These cells synthesize and secrete a loose organic matrix that regularly contains blood vessels, fibroblasts, and osteoprogenitor cells. The osteoprogenitor cells differentiate into osteoblasts and commence the assembly of osteoid (uncalcified bone matrix). This matrix will then mineralize side by side with vascular ingrowth to form woven bone that matures into lamellar bone. Similar observations were noted by Sennerby and his collaborates around titanium implants placed in rat cortical bone (104). Another important feature of the non-transformed osteoblast like cells is that they
demonstrate a well-defined inverse relationship between cell differentiation and proliferation (114). These unique characteristics make this osteogenic cell line an excellent model to study the interaction between the osteoblastic cell and the extracellular matrix (ECM) in vitro.