Histology, Osteocytes

Structure

Osteocyte resides within the lacunae located between the lamellae of the matrix. Each lacuna consists of one osteocyte. Osteocytes are flat, almond-shaped in the structure; they have a depth of about 7 micrometers and length of about 15 micrometers. The cell body diameter ranges from 5 to 20 micrometers and may obtain 40 to 60 processes per cell within a cellular distance of 20 to 30 micrometers. A mature osteocyte holds a single nucleus, which has a membrane and one or two nucleoli. The nucleus usually exists toward the vascular side of the surface. These cells exhibit a predominantly reduced Golgi complex and rough endoplasmic reticulum. These specialized structures also exhibit a more condensed nuclear chromatin. Osteocytes compose an extensive connecting syncytial network with small dendritic, or cytoplasmic, processes taking place in canaliculi. Osteocytes have expanded longevity and are long-living by nature.[2][3]

Function

Nutrient exchange involving osteocytes may nourish a chain of approximately 15 cells. Processes involving adjacent cells allow contact with gap junctions; molecules pass intracellularly via these structures. The small amount of extracellular substance between the bone matrix and osteocytes may instigate a few molecular exchanges between blood vessels and osteocytes. Osteocytes are actively involved in the perseverance of the bony matrix; osteocytic necrosis leads to resorption of the bony matrix. Despite their inert qualities, osteocytes perform molecular modification and synthesis along with signal transmission over long distances. After a bone fracture, for instance, osteocytic glutamate transporters compose nerve growth factors, proving their ability to sense and transfer information. Matured osteocytes are the most common cells in bone tissues, and the majority of receptor activities involved with bone function are available in these cells. Osteocytic necrosis also leads to trabecular bone decay, decreased bone formation, and malfunctioning loading.

Osteocytes also control osteoblastic and osteoclastic activities within a basic multicellular unit (BMU), a transient anatomic structure for insulating bone remodeling. Osteocytic proteins like sclerostin contribute to mineral metabolism aside from other molecules such as  MEPE, FGF-23, PHEX, and DMP-1, which help to regulate phosphate and biomineralization. Osteocytes also regulate bone mass and act as an essential endocrine regulator of phosphate metabolism.[4][5][6]

Tissue Preparation

Medical scientists typically use trabecular bone samples from the anterior iliac crest, as this sample can be collected from healthy patients as surgical waste material from procedures like the sinus floor elevation surgery. Creating human osteocyte culture is a tedious process: firstly, researchers collect the sample in sterile phosphate-based saline (PBS) with antimicrobial agents. They wash the biopsy materials three times using phosphate-buffered saline and then allocate the biopsy samples to a petri dish. They use a 23 scalpel to fragment the trabecular bone from the original sample in a PBS-treated petri dish.

Afterward, they use a 10 blade to divide the trabecular bone pieces into 1 to 2 mm square samples. In serum-free DMEM, they make 2 mg ml^−1 collagenase II solution and filter the solution with a 0.2 um filter unit. They place the bone fragments inside a 50 ml centrifuge tube, which is premixed with collagenase II and mix properly. They incubate those fragments using collagenase II for two hours at 37 degrees C inside a rocking water bath to clean any existing adhering cells on top of the bone surfaces. They then wash those samples with DMEM consisting of 10% serum made off fetal Clone I.

A subdivision phase follows: they submerge the bone samples into another petri dish; they then divide them into equal sections weighing about 100 mg. They transfer those portions into wells of a culture plate (12 wells suggested); the samples are treated with cCM. After incubating the samples at 37 degrees C with 5% CO2 overnight, they wash the samples with PBS 4 or 5 times the next day, followed by a repetition of the overnight treatments. The medium is replenished twice a week, and the process continues for seven days. At the preparation period, the scientists continue to add cytokines, hormones, chemicals, etc. into the osteocytic culture; osteocytic signaling factors are also measured in this modified culture medium. The additional analysis derives from the collection of total RNA from the samples using triazole.[7]

Histochemistry and Cytochemistry

Internal osteocytic histochemical design is still under active research; scientists generated a few hypotheses. The first theory suggests that osteocytic enzymatic reactions occur due to the artifactual diffusion of TRAP’s (Type 5 tartrate-resistant acid phosphatase) histochemical reaction yields from the actual reaction site (bone-resorbing surface). A second theory suggests that the enzyme reactions are not histochemical diffusion artifacts but true reactions that display TRAP proteins in osteocytes; by this hypothesis, the scientists proposed that the proteins diffuse from the bone-resorbing surface via the bone canaliculi under physiological treatments in vivo.

A final hypothesis indicates that osteocytic enzymatic reactions directly show the TRAP enzyme proteins produced by the respective osteocytes. While all of the proposed hypotheses are under study, one investigation strongly suggests that osteocytes in proximal areas to bone resorption surface create TRAP proteins and display histochemical TRAP reactions taking place within cytoplasmic granular structures. The probable TRAP protein diffusion deriving from osteoclasts via bone canaliculi to the TRAP-positive osteocytes is significant.[8][9]

Microscopy, Light

The visualization of sclerostin confirms the presence of osteocytes in the bone pieces. On day 7, researchers fix bone samples in cold 4% phosphate-buffered formaldehyde. They dehydrate the samples in graded ethanol by repeating the incubation process in 100% ethanol at least twice; methylmethacrylate mixture emission follows the incubation procedure. They cut the samples with Leica/Reichert-Jung Polycut S (SM2500) microtome into 5 um pieces. The researchers rehydrate the sections and douse the endogenous peroxidase with about 3% H2O2 in 40% methanol in PBS. They proceed to incubate the samples with 1% trypsin for 15 min at 37 °C to retrieve antigen. They incubate the bones with a 3-amino-9-ethylcarbazole reagent to develop a color visualization and counterstain the samples with hematoxylin. They visualize the sclerostin staining under preferred light microscopes, such as a Zeiss Apotome 2 microscope.[7]

Pathophysiology

Degraded or malfunctioning osteocytes may lead to various pathological conditions, such as hypophosphatemic rickets, sclerosteosis, and necrotic bone. Autosomal recessive hypophosphatemic rickets (ARHR) type 1 happens because of a lack of function mutations in dentin matrix protein 1, a type of noncollagenous bone matrix protein found in osteocytes and pre-osteocytic osteoblasts. This protein contributes to osteocyte proliferation and FGF23 downregulation. ARHR 2 happens because of a lack of function mutations in ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1); ENPP1 produces inorganic pyrophosphate (PPi), a crucial calcification inhibitor, and is related to ectopic calcification disorders in some cases. 

SOST codes for a specific protein known as the 190-residue glycoprotein sclerostin, which is mainly released by osteocytes. Sclerostin is an osteogenesis inhibitor that suppresses the canonical Wnt signaling pathway within osteoblast lineage cells. Sclerostin articulates to Wnt-signaling coreceptors LRP5 and LRP6, blockading Wnt particle articulation to those receptors. Sclerosteosis, caused by a decreased sclerostin expression, results in less restrained osteogenesis, leading to progressive hyperostosis. Sclerostin, therefore, relies on its coreceptor, LRP4.

Osteonecrosis refers to the classic pattern of cell death and complex osteogenesis and bone resorption processes. Osteocyte necrosis (ON) initiates with hematopoietic and adipocytic cellular necrosis along with interstitial marrow edema. ON happens after about 2 to 3 hours of anoxia; histological signs of osteocytic necrosis do not display until about 24 to 72 hours after hypoxia. ON is first characterized by pyknosis of nuclei, followed by hollow osteocyte lacunae. Capillary revascularization and reactive hyperemia slightly take place at the periphery of the necrosis site, followed by a repair process combining both bone resorption and production that incompletely changes dead with living bone. Nouveau bone overlays onto dead trabeculae along with fragmentary resorption of dead bone. Bone resorption outperforms formation resulting in a net removal of bone, deformed structural integrity of the subchondral trabeculae, joint incongruity, and subchondral fracture.[10][11][12][13]

Clinical Significance

Contemporary data suggests that osteocytic functions have led to various therapeutic measures to alter their physiology. Osteocytes, the prevalent and advanced-most bone cells, and osteocyte-derived proteins (i.e., sclerostin) lead to the development of newer strategies to treat various orthopedic conditions.

Researchers recognize that sclerostin as a vital molecule governing bone health. Osteocytes, source of sclerostin, are the main integrators of mechanical and chemical signals that lead to significant changes within the human body. The deep reach of the sophisticated vascular system passing via the canalicular fluid and osteocytic network composes a model delivery system for various small molecules.

Further applications of sclerostin action are under active research, as seen in chondrocyte expression of sclerostin in osteoarthritis. Maintaining sclerostin levels is crucial to protecting the intricate balance among the osteoblast, the osteocytes, and the osteoclast.[14][15]