Inside Bone: The Hidden Architects and Their Cellular Symphony

1. Inside Bone: The Hidden Architects and Their Cellular Symphony Bone is a specialized connective tissue, contains calcium and specialized bone cells. Most bone also contains bone marrow, where blood cells form. Bone plays a crucial role in mechanically supporting of soft tissues, levers for muscle action, protection of the central nervous system, release calcium and other ions to maintain extracellular fluid, support hemopoiesis etc. Types of Bone cells Bone is composed of four types of cells: osteoblasts, osteoclasts, osteocytes, and osteoprogenitor. All these bone cells have unique functions and found in different locations in bones. Osteoprogenitors (Osteogenic Cells): Osteoprogenitors (osteogenic cells) are only proliferative cells in bone. Immature osteoprogenitors are located in deep periosteum and marrow. FIGURE 1: Bone Structure, types of bone cells and their location. Image resource: Ch. 1 Introduction – Anatomy and Physiology | Open Stax

2. Osteoblasts: When osteoprogenitors mature, they become osteoblast. Osteoblast is located in the growing portions of bone, including the periosteum and endosteum. Osteoblast secrete collagen matrix and calcium salts. As the secreted matrix around osteoblast calcifies, it gets trapped in the matrix and changes its morphology, and now called as osteocytes. Osteocytes: Osteocytes are non-proliferative, mature cells; it synthesizes and secrete the collagen matrix and calcium deposits; located in lacunae. Osteocytes make ~90% of cellular component of bone. Bone is the most dynamic tissue which undergo extensive wear and tear. When the new cells form; old, injured or unnecessary cells need to be reabsorbed. Osteoclasts: Osteoclasts are responsible for the reabsorption of these old osteoblasts, located on bone surface. Osteoclasts are multinucleated cells, originating from macrophages and monocytes, rather the osteoprogenitors. Bone homeostasis is a controlled dynamic equilibrium of three key bone cells, osteoclasts, osteoblasts and osteocytes. Primary Bone cells Human primary bone cells are directly isolated from bone tissue by mechanical, enzymatic (collagenase, trypsin, dispose etc.) or explant culture methods. The primary bone cells include osteoblasts, osteoclasts and osteocytes. Generally, osteocytes and osteoblasts are isolated from bone. Osteoclasts are typically differentiated from monocyte progenitors found in bone marrow or peripheral blood. FIGURE 2: Schematic representation of isolation of primary osteoblast from human bone. Image Resource: PMID: 36359752 Human Stem Cell-Derived Bone Cells Mesenchymal stem cells (MSC) and Induced Pluripotent Stem Cells(iPSCs) have potential to differentiate towards various bone cell types, providing renewal resource for bone cells. These cells are used for disease modelling, drug screening, bone tissue engineering etc.

3. FIGURE 3: Schematic representation of osteogenic differentiation of Mesenchymal Stem Cells (MSCs). Image resource: PMID: 24179602 Human Bone Cell lines Human bone cell lines are immortalized cells derived from bone tissue that can proliferate indefinitely in vitro, providing a consistent and renewable resource of cells. Osteoblast Cell Lines: Saos-2: Saos-2 cell line was derived from a female osteosarcoma patient. Saos-2 cells express bone-specific proteins such as osteocalcin and collagen type 1. These cells are useful in studying bone cancer, drug discovery etc. MG-63: MG-63 cell line was derived from a male osteosarcoma patient. MG-63 express alkaline phosphatase and osteocalcin. These cell lines are useful in studying bone biology, osteoblast etc. hFOB 1.19: hFOB 1.19 was derived by transfecting of foetal limb tissue with the temperature sensitive expression vector pUCSVtsA58 and the neomycin resistance expression vector pSV2-neo. This cell line is useful in studying osteoblast differentiation, effect of hormones and growth factors on bone etc. HOS (Human Osteosarcoma Cells): HOS was derived from female osteosarcoma patients. It exhibits high level of alkaline phosphatase activity. This cell line is useful in studying drug discovery, bone cancer etc. FIGURE 4: Schematic representation of osteogenic differentiation of iPSCs. Image resource: PMID: 37936199

4. Osteoclast Cell lines: U-937: U-937 cell line was derived from malignant cells obtained from the pleural effusion of a patient with histiocytic lymphoma. U-937 cells can be differentiated towards macrophage-like cells which further induced towards osteoclast-like cells. U-937 cells are useful in studying osteoclast biology, bone resorption, osteoclast-related diseases etc. Osteocyte Cell lines: MLO-Y4: MLO-Y4 cell line was derived from a transgenic mouse in which the immortalizing T-antigen was expressed under control of the osteocalcin promoter. MLO- Y4 exhibit dendritic morphology. Osteocalcin, Connexin 43 and antigen E11 are highly expressed by MLO-Y4. Characterization of Bone Cells Bone cells i.e. osteoblasts, osteoclasts, osteocytes, and osteoprogenitor are characterized by morphology, molecular markers and functional assessments. Morphological Analysis: Osteoblast exhibit cuboidal morphology. Osteoclast display dendritic shape and osteocytes possess large multinucleated structure. Light microscope is commonly used to observe the cell confluence, shape and structure of bone cells. Electron microscope is used to observe ultrastructure of bone cells. Protein Expression Analysis: Alkaline phosphatase, osteocalcin and collagen type 1 proteins are typically expressed by osteoblast. Sclerostin and dentin matrix protein 1 (DMP1) are expressed by osteocytes. Osteoclasts are identified by tartrate-resistant acid phosphatase (TRAP), cathepsin K, and the calcitonin receptor. Protein expression is generally assessed by western blot, flow cytometer and immunocytochemistry. Gene Expression Analysis: Gene expression analysis are performed by real-time PCR, Semi-quantitative PCR or RNA sequencing. COL1A1, ALPL, RUNX2, SPP1, BGLAP etc. genes are specific for osteoblasts. CTSK, ACP5, TNFSF11 (RANKL), CALCR, TCIRG1 etc. genes are specific for osteoclasts. SOST, DMP1, MEPE, FGF23, PHEX etc genes are specific for osteocytes. Functional Assays: Alizarin or Von Kossa staining: Calcium deposition capability of osteoblast and osteocytes are assessed by Alizarin or Von Kossa stain. Alkaline Phosphatase Activity Assay: Alkaline phosphatase is an early marker of osteoblast. Collagen Synthesis Assay: It assesses the synthesis of collagen, a major component of bone matrix. Tartrate-Resistant Acid Phosphatase (TRAP) Staining: Identifies osteoclasts based on their TRAP activity. Bone Resorption Assay (Pit Formation Assay): Measures the restoratives activity of osteoclasts. Actin Ring Formation Assay: The actin rings composed of podosomes, a cohesive superstructure. Formation of actin ring is one of the major features of osteoclast bone resorption.

5. Lacunar-Canalicular Network Formation: Osteocytes are terminally differentiated osteoblasts, its located in the bone matrix called as lacunae. The capability to form an interconnected network by osteocytes are assessed. Sclerostin Expression Assay: Sclerostin is a mature marker of osteocytes. Mechanical Strain Assay: Mechanical strain capability of osteocytes is evaluated. Applications of Bone Cells: Bone Tissue Engineering: Mesenchymal stem cells or osteoblasts combined with biodegradable scaffolds are commonly used for bone repair and regeneration. Bioprinting: Bone cells mixed with Bioink are commonly used to create precise bone architecture. Disease Modelling: Bone cells are commonly used to understand the mechanism of bone diseases such as osteoporosis, osteosarcoma, osteopetrosis, bone metastasis etc. Its also useful to develop and test therapeutic agents for bone disorders. High throughput Drug Screening: Bone cells are commonly used for potential drug screening for bone formation, resorption, disorders etc. Drug safety and efficacy profiling is performed on bone cells. Regenerative Medicine: Stem cell-derived osteoblast is commonly transplanted in patients suffering from bone disorders. Conclusion: At Kosheeka, we are committed to advance drug discovery and development paradigm through our advanced in vitro cellular models, as well as frontiers of cellular therapies for bone disorders. We are the best cell culture research company in India provide bone cells i.e. osteoprogenitors, osteoblast and osteocytes for in vitro applications, which is amenable for high throughput screenings, drug safety and toxicity profiling. CONTACT US +91-9654321400 www.kosheeka.com info@kosheeka.com