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- Discuss the complex tissue structure found in animals
As multicellular organisms, animals differ from plants and fungi because their cells don’t have cell walls, their cells may be embedded in an extracellular matrix (such as bone, skin, or connective tissue), and their cells have unique structures for intercellular communication (such as gap junctions). Epithelial tissues include the epidermis of the integument, the lining of the digestive tract and trachea, and make up the ducts of the liver and glands of advanced animals.
The animal kingdom is divided into Parazoa (sponges) and Eumetazoa (all other animals). As very simple animals, the organisms in group Parazoa (“beside animal”) do not contain true specialized tissues; although they do possess specialized cells that perform different functions, those cells are not organized into tissues. These organisms are considered animals since they lack the ability to make their own food. Animals with true tissues are in the group Eumetazoa (“true animals”). When we think of animals, we usually think of Eumetazoans, since most animals fall into this category.
The different types of tissues in true animals are responsible for carrying out specific functions for the organism. This differentiation and specialization of tissues is part of what allows for such incredible animal diversity. For example, the evolution of nerve tissues and muscle tissues has resulted in animals’ unique ability to rapidly sense and respond to changes in their environment. This allows animals to survive in environments where they must compete with other species to meet their nutritional demands.
Watch a presentation by biologist E.O. Wilson on the importance of diversity.
A YouTube element has been excluded from this version of the text. You can view it online here: pb.libretexts.org/bionm2/?p=360
Tissue Definition and Examples in Biology
In biology, a tissue is a group of cells and their extracellular matrix that share the same embryonic origin and perform a similar function. Multiple tissues then form organs. The study of animal tissues is called histology, or histopathology when it is concerned with diseases. The study of plant tissues is called plant anatomy. The word "tissue" comes from the French word "tissu," which means "woven." French anatomist and pathologist Marie François Xavier Bichat introduced the term in 1801, stating that body functions could be understood better if they were studied at the level of tissues rather than organs.
Key Takeaways: Tissue Definition in Biology
- A tissue is a group of cells with the same origin that serve a similar function.
- Tissues are found in animals and plants.
- The four main types of animal tissues are connective, nervous, muscle, and epithelial tissues.
- The three main tissue systems in plants are the epidermis, ground tissue, and vascular tissue.
Epithelial tissue is a highly cellular tissue that overlies body surfaces, lines cavities, and forms glands. In addition, specialized epithelial cells function as receptors for special senses (smell, taste, hearing, and vision). Epithelial cells are numerous, exist in close apposition to each other, and form specialized junctions to create a barrier between connective tissues and free surfaces. Free surfaces of the body include the outer surface of internal organs, lining of body cavities, exterior surface of the body, tubes and ducts. The extracellular matrix of epithelial tissue is minimal and lacks additional structures. Although epithelial tissue is avascular, it is innervated.
The cells of epithelial tissue have three types of surfaces differentiated by their location and functional specializations: basal, apical, and lateral.
The basal surface is nearest to the basement membrane. The basement membrane itself creates a thin barrier between connective tissues and the most basal layer of epithelial cells. Specialized junctions called hemidesmosomes secure the epithelial cells on the basement membrane.
The apical surface of an epithelial cell is nearest to the lumen or free space. Apical cell surfaces may display specialized extensions. Microvilli are small processes projecting from the apical surface to increase surface area. They are heavily involved in diffusion in the proximal convoluted tubule of the nephron and in the lumen of the small intestines.
Cilia are small processes found in the respiratory tract and female reproductive tract. Their complex structure facilitates movement that brushes small structures through the lumen of either the trachea or Fallopian tubes. Stereocilia are similar to cilia in size and shape, however they are immotile and more frequently found in the epithelium of the male reproductive tract, specifically in the ductus deferens and the epididymis.
The lateral surfaces of epithelial cells are located between adjacent cells. The most notable lateral surface structures are junctions. Adhering junctions link the cytoskeleton of neighboring cells to produce strength in the tissue. Desmosomes can be thought of as spot-welding for epithelial tissues. They are usually located deep to adhering junctions and are found in locations subject to stresses. For example in the stratified epithelium of the skin.
Tight junctions form a solid barrier to prevent movement of molecules between adjacent epithelial cells. Tight junctions are found in the simple columnar epithelium of the gut tube to regulate absorption of nutrients. Finally, gap junctions perform the opposite function. Gap junctions allow small molecules and structures to pass freely between cells. For example, gap junctions in cardiac muscle tissue allow for coordinated contraction of the heart.
|Characteristics||Highly cellular, function as receptors, form a barrier, minimal extracellular matrix, avascular, innervated,|
|Basal surface||Basement membrane, hemidesmosomes|
|Apical surface||Microvilli, cilia, stereocilia|
|Lateral surface||Adhering junctions, desmosomes, tight junctions, gap junctions|
Two major characteristics of epithelial tissue divide it into subclasses: the shape of the cells and the presence of layers.
- Squamous – cells are flattened, can be keratinized or nonkeratinized, involved in protection and diffusion, found in capillary walls and skin
- Cuboidal – cells are cube-shaped, can be found forming tubes in the nephrons of the kidney, involved in secretion and absorption
- Columnar – cells are rectangular, cilia are often present, involved in absorption, secretion, protection, and lubrication, form the inner lining of the gut tube
- Simple – one layer of cells
- Stratified – two or more layers of cells
- Pseudostratified – simple epithelia that appear to be stratified when viewed in cross-section though they are only one layer of cells
Specialized epithelial tissue
- Transitional epithelium – distends tissues of urinary tract
- Keratinized stratified squamous epithelium – makes up the epidermis of skin
- Nonkeratinized stratified squamous epithelium – found in regions subject to abrasion, for example oral mucosa and vaginal lining
- Pseudostratified ciliated columnar epithelium – lines the inner surface of the trachea
- Endothelium - lines the inner surface of blood vessels
- Ependymal cells - present in the nervous system
The plant tissue in which the process of growth has stopped are called the permanent tissue and it originates from both primary and secondary meristematic tissue. These cells have a definite shape and configuration but they do not have the power of division. The permanent tissues can be classified into three major types on the basis of its constituent cells. Those are :
(a) Simple tissue including parenchyma, collenchyma and sclerenchyma.
(b)Complex tissue including the xylem and phloem.
(c) Special tissue including the external and internal secretory tissue.
Here we shall discuss about the simple tissue of plants.
What is simple tissue?
The homogeneous group of non-meristematic or permanent cells having similar structure, function and origin is collectively known as simple tissue.
Classification of simple tissue:
There are three major types of simple tissues viz., parenchyma, collenchyma and sclerenchyma.
The Parenchyma is walled living cells. It is very soft in nature because of the presence of thin walled cells.
Origin : It usually develops from ground meristems. The parenchyma of vascular bundle develops from procambium but that of secondary vascular tissues develop from inter-fascicular cambium. The parenchyma of secondary epidermis is formed from phellogen or cork cambium.
Structures of Parenchyma :
The simple tissue, parenchyma shows the following characteristics: -
(1) They are living cells with thin cellulose wall, though secondary xylem. Parenchyma tissue are lignified as observed in the endosperm of date-palm.
(2) They are with equal diameter, more or less spherical or star shaped in Scirpus, and usually with intercellular spaces.
(3) The cells contain large nucleus and large vacuoles.
(4) They are either photosynthetic or non-photosynthetic, containing leucoplasts.
Occurrence of Parenchyma:
It occurs in the simple tissue of epidermis of root, stem and leaf, hypodermis, peric pith, medullary rays, mesophyll cells, mesocarp of fleshy fruits, embryo and endosp
Functions of parenchyma :
(1) The parenchyma of the epidermis protects the plant organs.
(2) It carries out functions like regeneration, repairing of tissues and reproduc
(3) It is the major organ taking part in diffusion and osmosis.
(4) It is the storage tissue for food and water.
(5) It helps in the transport of food matter.
(6) Photosynthesis occurs in chlorophyllous parenchyma.
There are mainly three types of parenchyma found in plant tissue. Those are : -
(1) Chlorenchyma:The chlorophyllous parenchyma is called chlorenchyma
(2) Aerenchymaparenchyma contai air cavities. e.g. chyma of the leaf and of plants giving buoy
*(3) Idioblast: specialized parenchyma with variable size storing oils, tannins and mineral crystals e.g. leaf of banyan.
The permanent simple tissue consisting of unevenly thick walled living cells are called collenchymas. The uneven thickening of these cell walls makes it partially hard giving mechanical support. It is derived mostly from the elongated cells of the ground meristem and sometimes from the procambium.
Structure of collenchyma:
This type of simple tissue shows the following characteristics:
(1) The living cells with unevenly thickened walls composed of hemicellulose and pectin.
(2) The cells are elongated containing scanty vacuolated rotoplasm, appear polygonal in cross section.
(3) They are constituted by short and long fibre like cells, the short cells remain ong the long axis but the long cells are interlocked with overlapping tapering end.
(4) The primary pit fields are present.
(5) The intercellular spaces may or may not be present.
(6) They may contain chloroplast, helping in photosynthesis.
Occurrence and function of collenchymas :
The collenchymas usually remain in the hypodermis of stem and also in the base petiole and pedicel. The functions are as follows:
(1) It gives rigidity to plant body.
(2) It has extensible and plastic cell walls, which gives effective mechanical strength.
(3) They may contain chlorophyll and help in photosynthesis.
Complex loading affects intervertebral disc mechanics and biology
Complex loading develops in multiple spinal motions and in the case of hyperflexion is known to cause intervertebral disc (IVD) injury. Few studies have examined the interacting biologic and structural alterations associated with potentially injurious complex loading, which may be an important contributor to chronic progressive degeneration.
This study tested the hypothesis that low magnitudes of axial compression loading applied asymmetrically can induce IVD injury affecting cellular and structural responses in a large animal IVD ex-vivo model.
Bovine caudal IVDs were assigned to either a control or wedge group (15°) and placed in organ culture for 7 days under static 0.2 MPa load. IVD tissue and cellular responses were assessed through confined compression, qRT-PCR, histology and structural and compositional measurements, including Western blot for aggrecan degradation products.
Complex loading via asymmetric compression induced cell death, an increase in caspase-3 staining (apoptosis), a loss of aggrecan and an increase in aggregate modulus in the concave annulus fibrosis. While an up-regulation of MMP-1, ADAMTS4, IL-1β, and IL-6 mRNA, and a reduced aggregate modulus were induced in the convex annulus.
Asymmetric compression had direct deleterious effects on both tissue and cells, suggesting an injurious loading regime that could lead to a degenerative cascade, including cell death, the production of inflammatory mediators, and a shift towards catabolism. This explant model is useful to assess how injurious mechanical loading affects the cellular response which may contribute to the progression of degenerative changes in large animal IVDs, and results suggest that interventions should address inflammation, apoptosis, and lamellar integrity.
Major Histocompatibility Complex (MHC) | Immune System | Immunology
In this article we will discuss about:- 1. Introduction to Major Histocompatibility Complex (MHC) 2. Structure of Major Histocompatibility Complex (MHC) 3. Nomenclature and Inheritance 4. Expression of MHC Molecules 5. Classification of MHC Molecules.
- Introduction to Major Histocompatibility Complex (MHC)
- Structure of Major Histocompatibility Complex (MHC)
- Nomenclature and Inheritance of Major Histocompatibility Complex (MHC)
- Expression of MHC Molecules
- Classification of MHC Molecules
1. Introduction to Major Histocompatibility Complex (MHC):
MHC complex is a large genomic region or group of genes found in most vertebrates on a single chromosome that codes the MHC molecules which plays a vital role in immune system. Major histocompatibility antigens (also called transplantation antigens) mediate rejection of grafts between two genetically different individuals. HLA (human leukocyte antigens) were first detected on leukocytes and so they are called MHC antigens of humans. H-2 antigens are their equivalent MHC antigens of mouse. A set of MHC alleles present on each chromosome is called an MHC haplo-type.
Monozygotic human twins have the same histocompatibility molecules on their cells, and they can accept transplants of tissue from each other. Histocompatibility molecules of one indi­vidual act as antigens when introduced into a different individual. George Snell, Jean Dausset and Baruj Benacerraf received the Nobel Prize in 1980 for their contributions to the discovery and understanding of the MHC in mice and humans MHC gene products were identified as responsible for graft rejection.
MHC gene products that control immune responses are called the immune response genes. Immune response genes influence responses to infections. The essential role of the HLA antigens lies in the induction and regulation of the immune response and defence against microorganisms. The physiologic function of MHC molecules is the presentation of peptide antigen to T lymphocytes.
There are two general classes of MHC molecules – Class I and Class II. Class I MHC molecules are found on all nucleated cells and present peptides to cytotoxic T cells. Class II MHC molecules are found on certain immune cells themselves, chiefly macrophages, B cells and dendritic cells, collectively known as professional antigen-presenting cells (APCs). These APCs specialize in the uptake of pathogens and subsequent processing into peptide fragments within phagosomes. The Class II MHC molecules on APCs present these fragments to helper T cells, which stimulate an immune reaction from other cells.
2. Structure of Major Histocompatibility Complex (MHC):
The MHC complex resides in the short arm of chromosome 6 and overall size of the MHC is approximately 3.5 million base pairs. The complete three-dimensional structure for both class I and class II MHC molecules has been determined by x-ray crystallography. The class I gene complex contains three loci A, B and C, each of which codes of α chain polypeptides.
The class II gene complex also contains at least three loci, DP, DQ and DR each of these loci codes for one α and a variable number of β chain polypeptides. Class III region is not actually a part of the HLA complex, but is located within the HLA region, because its components are either related to the functions of HLA antigens or are under similar control mechanisms to the HLA genes. Class III antigens are associated with proteins in serum and other body fluids (e.g., C4, C2, factor B, TNF) and have no role in graft rejection.
3. Nomenclature and Inheritance of Major Histocompatibility Complex (MHC):
HLA specificities are identified by a letter for locus and a number (A1, B5, etc.), and the haplotypes are identified by individual specificities (e.g., A1, B7, Cw4, DP5, DQ10, DR8). Specificities which are defined by genomic analysis (PCR), are named with a letter for the locus and a four digit number (e.g., A0101, B0701, C0401, etc.).
Inheritance of Major Histocompatibility Complex (MHC):
Histocompatibility genes are inherited as a group (haplotype), one from each parent. Thus, MHC genes are co-dominantly expressed in each individual. A heterozygous human inherits one paternal and one maternal haplotype, each containing three Class-I (B, C and A) and three Class II (DP, DQ and DR) loci. Each individual inherits a maximum of two alleles for each locus.
The maximum number of class I MHC gene products expressed in an individual is six that for class II MHC products can exceed six but is also limited. Thus, as each chromosome is found twice (diploid) in each individual, a normal tissue type of an individual will involve 12 HLA antigens. Haplotypes, normally, are inherited intact and hence antigens encoded by different loci are inherited together.
However, on occasions, there is crossing over between two parental chromosomes, thereby resulting in new recombinant haplotypes. There is no somatic DNA recombination that occurs for antibodies and for the TCR, so the MHC genes lack re-combinational mechanisms for generating diversity. Many alleles of each locus permit thousands of possible assortments. There are at least 1000 officially recognized HLA alleles.
4. Expression of MHC Molecules:
MHC class I molecules are widely expressed, though the level varies between different cell types. MHC class II molecules are constitutively expressed only by certain cells involved in immune responses, though they can be induced on a wider variety of cells.
5. Classification of MHC Molecules:
1. MHC Class I Molecule:
MHC Class I is a membrane spanning molecule composed of two proteins. The membrane spanning protein is approximately 350 amino acids in length, with about 75 amino acids at the carboxylic end comprising the trans-membrane and cytoplasmic portions. The remaining 270 amino acids, as shown in the diagram, are divided into three globular domains Alpha-1, Alpha-2 and Alpha-3 prime, with alpha-1 being closest to the amino terminus and alpha-3 closest to the membrane.
The second portion of the molecule is a small globular protein called Beta-2 Micro-globulin. It associates primarily with the alpha-3 prime domain and is necessary for MHC stability. The bound peptide sits within the groove. The MHC molecules ability to present a wide range of antigenic peptides for T cell recognition requires a compromise between broad specificity and high affinity.
The peptide main chain is tightly bound whilst peptide side chains show less restrictive interactions. It is primarily the peptide side-chain contacts and conformational variability that ensures that the peptide-MHC complex presents an antigenically unique surface to T cell receptors.
2. MHC Class II Molecule:
Although similar to Class I, the MHC Class II molecule is composed of two membrane spanning proteins. Each chain is approximately 30 kilodaltons in size, and made of two globular domains as shown in the diagram. The domains are named Alpha-1, Alpha-2, Beta-1 and Beta-2. The two regions farthest from the membrane are alpha-1 and beta-1. The two chains associate without covalent bonds.
The bound peptide is within the groove. The MHC molecules ability to present a wide range of antigenic peptides for T cell recognition requires a compromise between broad specificity and high affinity. The peptide main chain is tightly bound whilst peptide side chains show less restrictive interactions. It is primarily the peptide side-chain contacts and conformational variability that ensures that the peptide-MHC complex presents an antigenically unique surface to T cell receptors. Class II molecules are dimers consisting of an alpha and beta polypeptide chain.
Each chain contains an immunoglobulin like region, next to the cell membrane. The antigen binding cleft, composed of two alpha-helices above a beta-pleated sheet, specifically binds short peptides, about 15 to 24 residues long. The amino acid sequence around the binding site, which specifies the antigen binding properties, is the most variable site in the MHC molecule.
Differences between Class I and Class II structures can explain the different length requirements for the bound peptide. The ends of the antigen binding cleft of Class I molecules taper and are blocked by bulky tyrosine that bind the N terminus of the peptide. These conserved residues are not found in Class II molecules where smaller residues (glycine or valine) replace the larger tyrosine.
3. MHC Class III Molecule:
This class includes genes coding several secreted proteins with immune functions-components of the complement system (such as C2, C4 and B factor) and molecules related with inflammation (cytokines such as TNF-α, LTA, LTB) or heat shock proteins (hsp). Class-III molecules do not share the same function as class-I and II molecules, but they are located between them in the short arm of human chromosome 6. For this reason they are frequently described together.
HLA antigens are recognized on almost all of the tissues of the body (with few exceptions), the identification of HLA antigens is also described as “Tissue Typing”. HLA matching between donor and recipient is desirable for allogenic (distinct) transplantation. Class I typing methods include test such as microcytoxicity (for typing A, B, C loci) and cellular techniques such as CML (for HLA-DPw typing). Class II typing involves cellular techniques such as MLR/MLC (for DR typing) and molecular techniques such as PCR and direct sequencing (for DR, DQ typing).
Significance of HLA Typing:
The fact that HLA types vary very widely among different ethnic populations has allowed anthropologists to establish or confirm relationship among populations and migration pattern. HLA-A34, which is present in 78% of Australian Aborigines, has a frequency of less than 1% in both Australian Caucasoids and Chinese.
2. Paternity Testing:
If a man and child share a HLA haplotype, then there is possibility that the man may be the father but not proven. However, if they don’t match or share a haplotype then it is agreed that he is not the father.
Because HLA plays such a dominant role in transplant immunity, pre-transplant histocompatibility testing is very important for organ transplantation. Results with closely related living donors matched with the recipient for one more both haplotypes are superior than obtained with unrelated cadaveric donors.
A number of diseases have been found to occur at a higher frequency in individuals with certain MHC haplotypes. Most prominent among these are ankylosing spondylitis (B27), celiac disease (DR3), Reiter’s syndrome (B27).
I. Disease Associations with Class I HLA:
Ankylosing spondylitis (B27), Reiter’s disease (B27), Acute anterior Uvietis (B27), Psoriasis vulgaris (Cw6).
II. Disease Associations with Class II HLA:
Hashimoto’s disease (DR5), Primary myxedema (DR3), Graves thyrotoxicosis (DR3), Insulin-dependent diabetes (DQ2/8), Addison’s disease (adrenal) (DR3), Good pasture’s syndrome (DR2), Rheumatoid arthritis (DR4), Juvenile rheumatoid arthritis (DR8), Sjogren’s syndrome (DR3), Chronic active hepatitis (DR3), Multiple sclerosis (DR2, DR6), Celiac disease (DR3), Dermatitis herpetiformis (DR3). No definite reason is known for this association.
However, several hypotheses have been proposed-antigenic similarity between pathogens and MHC, antigenic hypo- and hyper-responsiveness controlled by the class II genes are included among them.
Possible explanation for these associations is that the HLA antigen itself plays a role in disease, by a method similar to one of the following models:
(a) By being a poor presenter of a certain viral or bacterial antigen.
(b) By providing a binding site on the surface of the cell for a disease provoking virus or bacterium.
(c) By providing a transport piece for the virus to allow it to enter the cell by having a such a close molecular similarity to the pathogen that the immune system fails to recognize the pathogen as foreign and so fails to mount an immune response against it.
Serologic techniques provide one of the simplest and fastest methods for histocompatibility testing. These methods use sera that contain specific antibodies to HLA antigens. Tissue typing sera for the HLA were obtained in the past, from multiparous women who were exposed to the child’s paternal antigens during the parturition and subsequently developed antibodies to these antigens. More recently they are being produced by the monoclonal antibody technology.
This is done by exposing the unknown lymphocyte to a battery of antisera of known HLA specificities. Lymphocytes are isolated from the peripheral blood (or from lymph node or spleen in cadavers) and separated from other cells by buoyant density gradient centrifugation. For HLA I antigens, T lymphocytes are chosen while for HLA II antigens, B lymphocytes are chosen.
An array of anti-HLA sera covering full range of HLA types is chosen. Individual serum is dispensed into microtitre wells. Approximately 2000 lymphocytes are dispensed per well and incubated. Complement is then added to each well and incubated. The duration of incubation is different for T and B lymphocytes.
If the antibodies bind to lymphocytes, complement gets activated and results in lysis of that lymphocyte. The damaged cells are not completely lysed but suffer sufficient membrane damage to allow uptake of vital stains such as eosin Y, Trypan Blue or fluorescent stains such as ethidium bromide. Live cells don’t stain but the dead cells take up the stain.
This is used to detect the presence of HLA antibodies in the potential transplant recipients. A highly sensitive solid phase ELISA is used to detect antibodies in recipient’s serum. Purified preparations of HLA antigens are adsorbed on the solid phase of plastic plates. Recipient’s serum is then added to different HLA antigen coated wells.
After the removal of unbound antibodies by washing the wells are treated with enzyme-labeled anti-gamma globulin. The wells are washed and treated with color generating substrate. If the recipient is positive for the HLA type of the donor (that means recipient has antibodies directed against donor’s antigens), then transplantation is not possible.
Lymphocytes from one donor, when cultured with lymphocytes from an unrelated donor, are stimulated to proliferate or become cytotoxic. This proliferation is due to a disparity in the class IIMHC (DR) antigens.
Mixed Leukocyte Reaction (MLR) or Mixed Leukocyte Culture (MLC):
T cells of one individual interact with allogeneic class-II MHC antigen bearing cells (e.g., B cells) of unrelated individual. When lymphocytes from individuals of different class II haplotypes are cultured together, blast cell transformation and mitosis occurs. The irradiated or mitomycin-C treated stimulator cells of recipient (usually containing B cells, macrophages, and dendritic cells) are mixed with CD4 cells of responder (donor).
The donor cells respond to different class II antigens on stimulator cells and undergo transformation (DNA synthesis and enlargement) and proliferation (mitogenesis). These changes were recorded by the addition of radioactive (tritiated, 3H) thymidine into the culture and monitoring its incorporation into DNA.
Cell Mediated Lympholysis (CML):
The responder cells not only undergo blast transformation and proliferation on contact with different MHC II molecules, they also give rise to cytotoxic cells. These cytotoxic cells in turn identify the HLA I antigen on the stimulator cells and kill them.
These methods involve detection of the genes coding for the antigens rather than detecting the antigen itself. These are Sequence-specific PCR, Restriction fragment length polymorphism and sequence specific oligonucleotide probe etc.
2. Bone Cells
Osteoblasts are cuboidal cells that are located along the bone surface comprising 4𠄶% of the total resident bone cells and are largely known for their bone forming function . These cells show morphological characteristics of protein synthesizing cells, including abundant rough endoplasmic reticulum and prominent Golgi apparatus, as well as various secretory vesicles [22, 23]. As polarized cells, the osteoblasts secrete the osteoid toward the bone matrix  (Figures 1(a) , 1(b) , and 2(a) ).
(a)–(d) Light micrographs of portions of alveolar bone of rats. (a) HE-stained section showing a portion of a bony trabecula (B). Polarized osteoblasts (Ob) and giant multinucleated osteoclasts (Oc) are observed in the bone surface osteocyte (Ot) surrounding bone matrix is also observed. (b) Section subjected to immunohistochemistry for osteocalcin detection and counterstained with hematoxylin. Note osteocalcin-positive osteoblasts (arrows) on the surface of a bony trabecula (B). BV: blood vessel. (c) Undecalcified section subjected to the Gomori method for the detection of alkaline phosphatase, evidencing a portion of bone matrix (B) positive to the alkaline phosphatase (in brown/black). Ob: osteoblasts. (d) Undecalcified section subjected to the von Kossa method for calcium detection (brown/dark color). von Kossa-positive bone matrix (B) is observed some positive granules (arrow) can also be observed on the surface of the bone trabeculae. Scale bar: 15 μm.
Electron micrographs of portions of alveolar bone of rats. (a) Oteoblasts exhibiting abundant rough endoplasmic reticulum are observed adjacent to the bone (B) surface. A layer of bundles of collagen fibrils situated between osteoblasts (Ob) and calcified bone surface (B) constitutes the osteoid (Otd). Scale bar: 2.7 μm. (b) Bone lining cells (BLC) exhibiting scarce cytoplasm are situated on the osteoid surface (Otd). Bone lining cells (BLC) extend some thin cytoplasmic projections (arrows) towards the osteoid (Otd). Scale bar: 2 µm. N: nucleus.
Osteoblasts are derived from mesenchymal stem cells (MSC). The commitment of MSC towards the osteoprogenitor lineage requires the expression of specific genes, following timely programmed steps, including the synthesis of bone morphogenetic proteins (BMPs) and members of the Wingless (Wnt) pathways . The expressions of Runt-related transcription factors 2, Distal-less homeobox 5 (Dlx5), and osterix (Osx) are crucial for osteoblast differentiation [22, 26]. Additionally, Runx2 is a master gene of osteoblast differentiation, as demonstrated by the fact that Runx2-null mice are devoid of osteoblasts [26, 27]. Runx2 has demonstrated to upregulate osteoblast-related genes such as ColIA1, ALP, BSP, BGLAP, and OCN .
Once a pool of osteoblast progenitors expressing Runx2 and ColIA1 has been established during osteoblast differentiation, there is a proliferation phase. In this phase, osteoblast progenitors show alkaline phosphatase (ALP) activity, and are considered preosteoblasts . The transition of preosteoblasts to mature osteoblasts is characterized by an increase in the expression of Osx and in the secretion of bone matrix proteins such as osteocalcin (OCN), bone sialoprotein (BSP) I/II, and collagen type I. Moreover, the osteoblasts undergo morphological changes, becoming large and cuboidal cells [26, 29].
There is evidence that other factors such as fibroblast growth factor (FGF), microRNAs, and connexin 43 play important roles in the osteoblast differentiation . FGF-2 knockout mice showed a decreased bone mass coupled to increase of adipocytes in the bone marrow, indicating the participation of FGFs in the osteoblast differentiation . It has also been demonstrated that FGF-18 upregulates osteoblast differentiation in an autocrine mechanism . MicroRNAs are involved in the regulation of gene expression in many cell types, including osteoblasts, in which some microRNAs stimulate and others inhibit osteoblast differentiation [37, 38]. Connexin 43 is known to be the main connexin in bone . The mutation in the gene encoding connexin 43 impairs osteoblast differentiation and causes skeletal malformation in mouse .
The synthesis of bone matrix by osteoblasts occurs in two main steps: deposition of organic matrix and its subsequent mineralization (Figures 1(b) – 1(d) ). In the first step, the osteoblasts secrete collagen proteins, mainly type I collagen, noncollagen proteins (OCN, osteonectin, BSP II, and osteopontin), and proteoglycan including decorin and biglycan, which form the organic matrix. Thereafter, mineralization of bone matrix takes place into two phases: the vesicular and the fibrillar phases [40, 41]. The vesicular phase occurs when portions with a variable diameter ranging from 30 to 200 nm, called matrix vesicles, are released from the apical membrane domain of the osteoblasts into the newly formed bone matrix in which they bind to proteoglycans and other organic components. Because of its negative charge, the sulphated proteoglycans immobilize calcium ions that are stored within the matrix vesicles [41, 42]. When osteoblasts secrete enzymes that degrade the proteoglycans, the calcium ions are released from the proteoglycans and cross the calcium channels presented in the matrix vesicles membrane. These channels are formed by proteins called annexins .
On the other hand, phosphate-containing compounds are degraded by the ALP secreted by osteoblasts, releasing phosphate ions inside the matrix vesicles. Then, the phosphate and calcium ions inside the vesicles nucleate, forming the hydroxyapatite crystals . The fibrillar phase occurs when the supersaturation of calcium and phosphate ions inside the matrix vesicles leads to the rupture of these structures and the hydroxyapatite crystals spread to the surrounding matrix [44, 45].
Mature osteoblasts appear as a single layer of cuboidal cells containing abundant rough endoplasmic reticulum and large Golgi complex (Figures 2(a) and 3(a) ). Some of these osteoblasts show cytoplasmic processes towards the bone matrix and reach the osteocyte processes . At this stage, the mature osteoblasts can undergo apoptosis or become osteocytes or bone lining cells [47, 48]. Interestingly, round/ovoid structures containing dense bodies and TUNEL-positive structures have been observed inside osteoblast vacuoles. These findings suggest that besides professional phagocytes, osteoblasts are also able to engulf and degrade apoptotic bodies during alveolar bone formation .
Light (a and b) and electron micrographs of portions of alveolar bone rats. (a) a semithin section stained with toluidine blue showing a portion of a bony trabecula (B). Osteoblasts (Ob) and bone lining cells (BLC) are present on bone surface while osteocytes (Ot) are observed entrapped in the bone matrix. BV: blood vessels. Scale bar: 15 μm. (b) Section subjected to the silver impregnation method. Note the cytoplasmic processes (arrows) of the osteocytes (Ot) connecting them with each other. Scale bar: 15 μm. (c) Scanning electron micrograph showing two osteocytes (Ot) surrounded by bone matrix (B). Note that the cytoplasmic processes (arrows) are observed between the osteocytes (Ot) forming an interconnected network. Scale bar: 2 μm. (d) Transmission electron micrograph showing a typical osteocyte (Ot) inside a lacuna (La) in the bone matrix (B), with its cytoplasmic processes (arrows) inside the canaliculi (Ca). Scale bar: 2 μm. N: nucleus.
2.2. Bone Lining Cells
Bone lining cells are quiescent flat-shaped osteoblasts that cover the bone surfaces, where neither bone resorption nor bone formation occurs . These cells exhibit a thin and flat nuclear profile its cytoplasm extends along the bone surface and displays few cytoplasmic organelles such as profiles of rough endoplasmic reticulum and Golgi apparatus  ( Figure 2(b) ). Some of these cells show processes extending into canaliculi, and gap junctions are also observed between adjacent bone lining cells and between these cells and osteocytes [50, 51].
The secretory activity of bone lining cells depends on the bone physiological status, whereby these cells can reacquire their secretory activity, enhancing their size and adopting a cuboidal appearance . Bone lining cells functions are not completely understood, but it has been shown that these cells prevent the direct interaction between osteoclasts and bone matrix, when bone resorption should not occur, and also participate in osteoclast differentiation, producing osteoprotegerin (OPG) and the receptor activator of nuclear factor kappa-B ligand (RANKL) [14, 53]. Moreover, the bone lining cells, together with other bone cells, are an important component of the BMU, an anatomical structure that is present during the bone remodeling cycle .
Osteocytes, which comprise 90% of the total bone cells, are the most abundant and long-lived cells, with a lifespan of up to 25 years . Different from osteoblasts and osteoclasts, which have been defined by their respective functions during bone formation and bone resorption, osteocytes were earlier defined by their morphology and location. For decades, due to difficulties in isolating osteocytes from bone matrix led to the erroneous notion that these cells would be passive cells, and their functions were misinterpreted . The development of new technologies such as the identification of osteocyte-specific markers, new animal models, development of techniques for bone cell isolation and culture, and the establishment of phenotypically stable cell lines led to the improvement of the understanding of osteocyte biology. In fact, it has been recognized that these cells play numerous important functions in bone .
The osteocytes are located within lacunae surrounded by mineralized bone matrix, wherein they show a dendritic morphology [15, 55, 56] (Figures 3(a) – 3(d) ). The morphology of embedded osteocytes differs depending on the bone type. For instance, osteocytes from trabecular bone are more rounded than osteocytes from cortical bone, which display an elongated morphology .
Osteocytes are derived from MSCs lineage through osteoblast differentiation. In this process, four recognizable stages have been proposed: osteoid-osteocyte, preosteocyte, young osteocyte, and mature osteocyte . At the end of a bone formation cycle, a subpopulation of osteoblasts becomes osteocytes incorporated into the bone matrix. This process is accompanied by conspicuous morphological and ultrastructural changes, including the reduction of the round osteoblast size. The number of organelles such as rough endoplasmic reticulum and Golgi apparatus decreases, and the nucleus-to-cytoplasm ratio increases, which correspond to a decrease in the protein synthesis and secretion .
During osteoblast/osteocyte transition, cytoplasmic process starts to emerge before the osteocytes have been encased into the bone matrix . The mechanisms involved in the development of osteocyte cytoplasmic processes are not well understood. However, the protein E11/gp38, also called podoplanin may have an important role. E11/gp38 is highly expressed in embedding or recently embedded osteocytes, similarly to other cell types with dendritic morphology such as podocytes, type II lung alveolar cells, and cells of the choroid plexus . It has been suggested that E11/gp38 uses energy from GTPase activity to interact with cytoskeletal components and molecules involved in cell motility, whereby regulate actin cytoskeleton dynamics [60, 61]. Accordingly, inhibition of E11/gp38 expression in osteocyte-like MLO-Y4 cells has been shown to block dendrite elongation, suggesting that E11/gp38 is implicated in dendrite formation in osteocytes .
Once the stage of mature osteocyte totally entrapped within mineralized bone matrix is accomplished, several of the previously expressed osteoblast markers such as OCN, BSPII, collagen type I, and ALP are downregulated. On the other hand, osteocyte markers including dentine matrix protein 1 (DMP1) and sclerostin are highly expressed [8, 62].
Whereas the osteocyte cell body is located inside the lacuna, its cytoplasmic processes (up to 50 per each cell) cross tiny tunnels that originate from the lacuna space called canaliculi, forming the osteocyte lacunocanalicular system  (Figures 3(b) – 3(d) ). These cytoplasmic processes are connected to other neighboring osteocytes processes by gap junctions, as well as to cytoplasmic processes of osteoblasts and bone lining cells on the bone surface, facilitating the intercellular transport of small signaling molecules such as prostaglandins and nitric oxide among these cells . In addition, the osteocyte lacunocanalicular system is in close proximity to the vascular supply, whereby oxygen and nutrients achieve osteocytes .
It has been estimated that osteocyte surface is 400-fold larger than that of the all Haversian and Volkmann systems and more than 100-fold larger than the trabecular bone surface [67, 68]. The cell-cell communication is also achieved by interstitial fluid that flows between the osteocytes processes and canaliculi . By the lacunocanalicular system ( Figure 3(b) ), the osteocytes act as mechanosensors as their interconnected network has the capacity to detect mechanical pressures and loads, thereby helping the adaptation of bone to daily mechanical forces . By this way, the osteocytes seem to act as orchestrators of bone remodeling, through regulation of osteoblast and osteoclast activities [15, 69]. Moreover, osteocyte apoptosis has been recognized as a chemotactic signal to osteoclastic bone resorption . In agreement, it has been shown that during bone resorption, apoptotic osteocytes are engulfed by osteoclasts .
The mechanosensitive function of osteocytes is accomplished due to the strategic location of these cells within bone matrix. Thus, the shape and spatial arrangement of the osteocytes are in agreement with their sensing and signal transport functions, promoting the translation of mechanical stimuli into biochemical signals, a phenomenon that is called piezoelectric effect . The mechanisms and components by which osteocytes convert mechanical stimuli to biochemical signals are not well known. However, two mechanisms have been proposed. One of them is that there is a protein complex formed by a cilium and its associated proteins PolyCystins 1 and 2, which has been suggested to be crucial for osteocyte mechanosensing and for osteoblast/osteocyte-mediated bone formation . The second mechanism involves osteocyte cytoskeleton components, including focal adhesion protein complex and its multiple actin-associated proteins such as paxillin, vinculin, talin, and zyxin . Upon mechanical stimulation, osteocytes produce several secondary messengers, for example, ATP, nitric oxide (NO), Ca 2+ , and prostaglandins (PGE2 and PGI2,) which influence bone physiology [8, 80]. Independently of the mechanism involved, it is important to mention that the mechanosensitive function of osteocytes is possible due to the intricate canalicular network, which allows the communication among bone cells.
Osteoclasts are terminally differentiated multinucleated cells (Figures 4(a) – 4(d) ), which originate from mononuclear cells of the hematopoietic stem cell lineage, under the influence of several factors. Among these factors the macrophage colony-stimulating factor (M-CSF), secreted by osteoprogenitor mesenchymal cells and osteoblasts , and RANK ligand, secreted by osteoblasts, osteocytes, and stromal cells, are included . Together, these factors promote the activation of transcription factors [81, 82] and gene expression in osteoclasts [83, 84].
Light (a and c) and electron micrographs (b and d) of portions of alveolar bone of rats. In (a) tartrate-resistant acid phosphatase (TRAP) activity (in red color) is observed in the cytoplasm of osteoclasts (OC) adjacent to the alveolar bone (B) surface. Note that in the opposite side of the bony trabecula B is covered by large and polarized osteoblasts (Ob). Ot, osteocytes (Ot) BV: blood vessel. Bar: 40 μm. (b) Multinucleated osteoclast (OC) shows evident ruffled border (RB) adjacent to the excavated bone surface (arrows). Several vacuoles (V) are observed in the cytoplasm adjacent to ruffled border (RB). N: nucleus. Bar: 4 μm. (c) Portions of TRAP-positive osteoclasts (Oc and Oc1) are observed in a resorbing bone lacuna. A round cell (Ap) with condensed irregular blocks of chromatin, typical apoptotic cell, is observed inside a large vacuole of the Oc1. B: bone matrix Ot: osteocyte. Bar: 15 μm. (d) An osteoclast (Oc) showing ruffled border (RB) and clear zone (CZ) is in close juxtaposition to the excavation of the bone surface (arrows), that is, Howship lacuna. Vacuoles (V) with varied size are present next to the ruffled border (RB) one of them contains a round cell with masses of condensed chromatin (Ap), typical of cell undergoing apoptosis. B: bone matrix N: nucleus. Bar: 3 μm.
M-CSF binds to its receptor (cFMS) present in osteoclast precursors, which stimulates their proliferation and inhibits their apoptosis [82, 85]. RANKL is a crucial factor for osteoclastogenesis and is expressed by osteoblasts, osteocytes, and stromal cells. When it binds to its receptor RANK in osteoclast precursors, osteoclast formation is induced . On the other hand, another factor called osteoprotegerin (OPG), which is produced by a wide range of cells including osteoblasts, stromal cells, and gingival and periodontal fibroblasts , binds to RANKL, preventing the RANK/RANKL interaction and, consequently, inhibiting the osteoclastogenesis  ( Figure 5 ). Thus, the RANKL/RANK/OPG system is a key mediator of osteoclastogenesis [19, 86, 89].
Schematic summary of bone tissue showing bone cells and the relationships among them and with bone matrix (B). Osteoclast (Oc) activation occurs after binding of RANKL to its receptor RANK, present in the membrane of osteoclast precursors. Then, osteoclast becomes polarized through its cytoskeleton reorganization the ruffled border (RB) and clear zone (CZ) are membrane specializations observed in the portion of the osteoclast juxtaposed to the bone resorption surface, Howship lacuna (HL). Dissolution of hydroxyapatite occurs in the bone surface adjacent to the ruffled border (RF) upon its acidification due to pumping of hydrogen ions (H + ) to the HL. H + and ions bicarbonate (HCO3 − ) originate from the cleavage of carbonic acid (H2CO3) under the action of carbonic anhydrase II (CAII). After dissolution of mineral phase, osteoclast (Oc) releases cathepsin (Cp), matrix metalloproteinase-9 (MMP-9), and tartrate-resistant acid phosphatase (TRAP) that degrade the organic matrix. EphrinB2 (Eph2) present in osteoclast membrane binds to ephrinB4 (Eph4) in osteoblast (Ob) membrane, promoting its differentiation, whereas the reverse signaling (ephrinB4/ephrinB2) inhibits osteoclastogenesis. Sema4D produced by osteoclasts inhibits osteoblasts, while Sema3A secreted by osteoblasts inhibits osteoclasts. Osteoblasts (Ob) also produce receptor activator of nuclear factor KB (RANKL) and osteoprotegerin (OPG), which increase and decrease osteoclastogenesis, respectively. Osteoblasts (Ob) secrete collagenous (Col1) and noncollagenous proteins such as osteocalcin (OCN), osteopontin (OSP), osteonectin (OSN), bone sialoprotein (BSP), and bone morphogenetic proteins (BMP). Osteocytes (Ot) are located within lacunae surrounded by mineralized bone matrix (B). Its cytoplasmic processes cross canaliculi to make connection with other neighboring osteocytes processes by gap junctions, mainly composed by connexin 43 (Cx3), as well as to cytoplasmic processes of osteoblasts (Ob) and bone lining cells (BLC) on bone surface. RANKL secreted by osteocytes stimulates osteoclastogenesis, while prostaglandin E2 (PGE2), nitric oxide (NO), and insulin-like growth factor (IGF) stimulate osteoblast activity. Conversely, osteocytes produce OPG that inhibits osteoclastogenesis moreover, osteocytes produce sclerostin and dickkopf WNT signaling pathway inhibitor (DKK-1) that decrease osteoblast activity.
The RANKL/RANK interaction also promotes the expression of other osteoclastogenic factors such as NFATc1 and DC-STAMP. By interacting with the transcription factors PU.1, cFos, and MITF, NFATc1 regulates osteoclast-specific genes including TRAP and cathepsin K, which are crucial for osteoclast activity . Under the influence of the RANKL/RANK interaction, NFATc1 also induces the expression of DC-STAMP, which is crucial for the fusion of osteoclast precursors [91, 92].
Despite these osteoclastogenic factors having been well defined, it has recently been demonstrated that the osteoclastogenic potential may differ depending on the bone site considered. It has been reported that osteoclasts from long bone marrow are formed faster than in the jaw. This different dynamic of osteoclastogenesis possibly could be, due to the cellular composition of the bone-site specific marrow .
During bone remodeling osteoclasts polarize then, four types of osteoclast membrane domains can be observed: the sealing zone and ruffled border that are in contact with the bone matrix (Figures 4(b) and 4(d) ), as well as the basolateral and functional secretory domains, which are not in contact with the bone matrix [94, 95]. Polarization of osteoclasts during bone resorption involves rearrangement of the actin cytoskeleton, in which an F-actin ring that comprises a dense continuous zone of highly dynamic podosome is formed and consequently an area of membrane that develop into the ruffled border is isolated. It is important to mention that these domains are only formed when osteoclasts are in contact with extracellular mineralized matrix, in a process which α v β 3-integrin, as well as the CD44, mediates the attachment of the osteoclast podosomes to the bone surface . Ultrastructurally, the ruffled border is a membrane domain formed by microvilli, which is isolated from the surrounded tissue by the clear zone, also known as sealing zone. The clear zone is an area devoid of organelles located in the periphery of the osteoclast adjacent to the bone matrix . This sealing zone is formed by an actin ring and several other proteins, including actin, talin, vinculin, paxillin, tensin, and actin-associated proteins such as α-actinin, fimbrin, gelsolin, and dynamin . The α v β 3-integrin binds to noncollagenous bone matrix containing-RGD sequence such as bone sialoprotein, osteopontin, and vitronectin, establishing a peripheric sealing that delimits the central region, where the ruffled border is located  (Figures 4(b) – 4(d) ).
The maintenance of the ruffled border is also essential for osteoclast activity this structure is formed due to intense trafficking of lysosomal and endosomal components. In the ruffled border, there is a vacuolar-type H + -ATPase (V-ATPase), which helps to acidify the resorption lacuna and hence to enable dissolution of hydroxyapatite crystals [20, 100, 101]. In this region, protons and enzymes, such as tartrate-resistant acid phosphatase (TRAP), cathepsin K, and matrix metalloproteinase-9 (MMP-9) are transported into a compartment called Howship lacuna leading to bone degradation [94, 101] ( Figure 5 ). The products of this degradation are then endocytosed across the ruffled border and transcytosed to the functional secretory domain at the plasma membrane [7, 95].
Abnormal increase in osteoclast formation and activity leads to some bone diseases such as osteoporosis, where resorption exceeds formation causing decreased bone density and increased bone fractures . In some pathologic conditions including bone metastases and inflammatory arthritis, abnormal osteoclast activation results in periarticular erosions and painful osteolytic lesions, respectively [83, 105, 106]. In periodontitis, a disease of the periodontium caused by bacterial proliferation [107, 108] induces the migration of inflammatory cells. These cells produce chemical mediators such as IL-6 and RANKL that stimulate the migration of osteoclasts [89, 109, 110]. As a result, an abnormal increased bone resorption occurs in the alveolar bone, contributing to the loss of the insertions of the teeth and to the progression of periodontitis [89, 111].
On the other hand, in osteopetrosis, which is a rare bone disease, genetic mutations that affect formation and resorption functions in osteoclasts lead to decreased bone resorption, resulting in a disproportionate accumulation of bone mass . These diseases demonstrate the importance of the normal bone remodeling process for the maintenance of bone homeostasis.
Furthermore, there is evidence that osteoclasts display several other functions. For example, it has been shown that osteoclasts produce factors called clastokines that control osteoblast during the bone remodeling cycle, which will be discussed below. Other recent evidence is that osteoclasts may also directly regulate the hematopoietic stem cell niche . These findings indicate that osteoclasts are not only bone resorbing cells, but also a source of cytokines that influence the activity of other cells.
2.5. Extracellular Bone Matrix
Bone is composed by inorganic salts and organic matrix . The organic matrix contains collagenous proteins (90%), predominantly type I collagen, and noncollagenous proteins including osteocalcin, osteonectin, osteopontin, fibronectin and bone sialoprotein II, bone morphogenetic proteins (BMPs), and growth factors . There are also small leucine-rich proteoglycans including decorin, biglycan, lumican, osteoaderin, and seric proteins .
The inorganic material of bone consists predominantly of phosphate and calcium ions however, significant amounts of bicarbonate, sodium, potassium, citrate, magnesium, carbonate, fluorite, zinc, barium, and strontium are also present [1, 2]. Calcium and phosphate ions nucleate to form the hydroxyapatite crystals, which are represented by the chemical formula Ca10(PO4)6(OH)2. Together with collagen, the noncollagenous matrix proteins form a scaffold for hydroxyapatite deposition and such association is responsible for the typical stiffness and resistance of bone tissue .
Bone matrix constitutes a complex and organized framework that provides mechanical support and exerts essential role in the bone homeostasis. The bone matrix can release several molecules that interfere in the bone cells activity and, consequently, has a participation in the bone remodeling . Once loss of bone mass alone is insufficient to cause bone fractures , it is suggested that other factors, including changes in the bone matrix proteins and their modifications, are of crucial importance to the understanding and prediction of bone fractures . In fact, it is known that collagen plays a critical role in the structure and function of bone tissue .
Accordingly, it has been demonstrated that there is a variation in the concentration of bone matrix proteins with age, nutrition, disease, and antiosteoporotic treatments [119, 121, 122] which may contribute to postyield deformation and fracture of bone . For instance, in vivo and in vitro studies have reported that the increase in hyaluronic acid synthesis after parathyroid hormone (PTH) treatment was related to a subsequent bone resorption  suggesting a possible relationship between hyaluronic acid synthesis and the increase in osteoclast activity.
2.6. Interactions between Bone Cells and Bone Matrix
As previously discussed, bone matrix does not only provides support for bone cells, but also has a key role in regulating the activity of bone cells through several adhesion molecules [117, 128]. Integrins are the most common adhesion molecules involved in the interaction between bone cells and bone matrix . Osteoblasts make interactions with bone matrix by integrins, which recognize and bind to RGD and other sequences present in bone matrix proteins including osteopontin, fibronectin, collagen, osteopontin, and bone sialoprotein [130, 131]. The most common integrins present in osteoblasts are α 1 β 1, α 2 β 1, and α 5 β 1 . These proteins also play an important role in osteoblast organization on the bone surface during osteoid synthesis .
On the other hand, the interaction between osteoclasts and bone matrix is essential for osteoclast function, since as previously mentioned, bone resorption occurs only when osteoclasts bind to mineralized bone surface . Thus, during bone resorption osteoclasts express α v β 3 and α 2 β 1 integrins to interact with the extracellular matrix, in which the former bind to bone-enriched RGD-containing proteins, such as bone sialoprotein and osteopontin, whereas β 1 integrins bind to collagen fibrils [133, 134]. Despite these bindings, osteoclasts are highly motile even active resorption and, as migrating cells, osteoclasts do not express cadherins. However, it has been demonstrated that cadherins provide intimate contact between osteoclast precursors and stromal cells, which express crucial growth factors for osteoclast differentiation .
Integrins play a mediating role in osteocyte-bone matrix interactions. These interactions are essential for the mechanosensitive function of these cells, whereby signals induced by tissue deformation are generated and amplified . It is still not clear which integrins are involved, but it has been suggested that β 3 and β 1 integrins are involved in osteocyte-bone matrix interaction [137, 138]. These interactions occur between osteocyte body and the bone matrix of the lacuna wall as well as between canalicular wall with the osteocyte processes .
Only a narrow pericellular space filled by a fluid separates the osteocyte cell body and processes from a mineralized bone matrix . The space between osteocyte cell body and the lacunar wall is approximately 0.5𠄱.0 μm wide, whereas the distance between the membranes of osteocyte processes and the canalicular wall varies from 50 to 100 nm . The chemical composition of the pericellular fluid has not been precisely defined. However, a diverse array of macromolecules produced by osteocytes such as osteopontin, osteocalcin, dentin matrix protein, proteoglycans, and hyaluronic acid is present [136, 140, 141].
The osteocyte and their processes are surrounded by a nonorganized pericellular matrix delicate fibrous connections were observed within the canalicular network, termed “tethers” . It has been suggested that perlecan is a possible compound of these tethers . Osteocyte processes can also attach directly by the “hillocks,” which are protruding structures originating from the canalicular walls. These structures form close contacts, possibly by means of β 3-integrins, with the membrane of osteocyte processes [137, 142]. Thus, these structures seem to play a key role in the mechanosensitive function of osteocytes, by sensing the fluid flux movements along with the pericellular space, provoked by mechanical load forces . In addition, the fluid flux movement is also essential for the bidirectional solute transport in the pericellular space, which influences osteocyte signaling pathways and communication among bone cells [144, 145].
2.7. Local and Systemic Factor That Regulate Bone Homeostasis
Bone remodeling is a highly complex cycle that is achieved by the concerted actions of osteoblasts, osteocytes, osteoclasts, and bone lining cells . The formation, proliferation, differentiation, and activity of these cells are controlled by local and systemic factors [18, 19]. The local factors include autocrine and paracrine molecules such as growth factors, cytokines, and prostaglandins produced by the bone cells besides factors of the bone matrix that are released during bone resorption [46, 146]. The systemic factors which are important to the maintenance of bone homeostasis include parathyroid hormone (PTH), calcitonin, 1,25-dihydroxyvitamin D3 (calcitriol), glucocorticoids, androgens, and estrogens [16, 147]. Similar to PTH, PTH related protein (PTHrP), which also binds to PTH receptor, has also been reported to influence bone remodeling .
Estrogen plays crucial roles for bone tissue homeostasis the decrease in estrogen level at menopause is the main cause of bone loss and osteoporosis . The mechanisms by which estrogen act on bone tissue are not completely understood. Nevertheless, several studies have shown that estrogen maintains bone homeostasis by inhibiting osteoblast and osteocyte apoptosis  and preventing excessive bone resorption. The estrogen suppresses the osteoclast formation and activity as well as induces osteoclast apoptosis [16, 76, 104, 154]. It has been suggested that estrogen decreases osteoclast formation by inhibiting the synthesis of the osteoclastogenic cytokine RANKL by osteoblasts and osteocytes. Moreover, estrogen stimulates these bone cells to produce osteoprotegerin (OPG), a decoy receptor of RANK in osteoclast, thus inhibiting osteoclastogenesis [19, 155]. In addition, estrogen inhibits osteoclast formation by reducing the levels of other osteoclastogenic cytokines such as IL-1, IL-6, IL-11, TNF-α, TNF-β, and M-CSF [160, 161].
Estrogen acts directly on bone cells by its estrogen receptors α and β present on these cells . Moreover, it has been shown that osteoclast is a direct target for estrogen [163, 164]. Accordingly, immunoexpression of estrogen receptor β has been demonstrated in alveolar bone cells of estradiol-treated female rats. Moreover, the enhanced immunoexpression observed in TUNEL-positive osteoclasts indicates that estrogen participates in the control of osteoclast life span directly by estrogen receptors . These findings demonstrate the importance of estrogen for the maintenance of bone homeostasis.
2.8. Bone Remodeling Process
The bone remodeling cycle takes place within bone cavities that need to be remodeled . In these cavities, there is the formation of temporary anatomical structures called basic multicellular units (BMUs), which are comprised of a group of osteoclasts ahead forming the cutting cone and a group of osteoblasts behind forming the closing cone, associated with blood vessels and the peripheral innervation [11, 166]. It has been suggested that BMU is covered by a canopy of cells (possibly bone lining cells) that form the bone remodeling compartment (BRC) . The BRC seems to be connected to bone lining cells on bone surface, which in turn are in communication with osteocytes enclosed within the bone matrix [13, 14].
The bone remodeling cycle begins with an initiation phase, which consists of bone resorption by osteoclasts, followed by a phase of bone formation by osteoblasts but between these two phases, there is a transition (or reversal) phase. The cycle is completed by coordinated actions of osteocytes and bone lining cells [10, 11]. In the initiation phase, under the action of osteoclastogenic factors including RANKL and M-CSF, hematopoietic stem cells are recruited to specific bone surface areas and differentiate into mature osteoclasts that initiate bone resorption [167, 168].
It is known that during bone remodeling cycle, there are direct and indirect communications among bone cells in a process called coupling mechanism, which include soluble coupling factors stored in bone matrix that would be released after osteoclast bone resorption . For instance, factors such as insulin-like growth factors (IGFs), transforming growth factor β (TGF-β), BMPs, FGF, and platelet-derived growth factor (PDGF) seem to act as coupling factors, since they are stored in bone matrix and released during bone resorption . This idea is supported by genetic studies in humans and mice as well as by pharmacological studies [105, 171].
Recently, it has been suggested that another category of molecules called semaphorins is involved in the bone cell communication during bone remodeling . During the initial phase, osteoblast differentiation and activity must be inhibited, in order to completely remove the damaged or aged bone. The osteoclasts express a factor called semaphorin4D (Sema4D) that inhibits bone formation during bone resorption . Semaphorins comprise a large family of glycoproteins which are not only membrane-bound but also exist as soluble forms that are found in a wide range of tissues and shown to be involved in diverse biological processes such as immune response, organogenesis, cardiovascular development, and tumor progression [172, 173]. In bone, it has been suggested that semaphorins are also involved in cell-cell communication between osteoclasts and osteoblasts during the bone remodeling cycle .
Sema4D expressed in osteoclasts binds to its receptor (Plexin-B1) present in osteoblasts and inhibits IGF-1 pathway, essential for osteoblast differentiation , suggesting that osteoclasts suppress bone formation by expressing Sema4D. Conversely, another member of semaphorin family (Sema3A) has been found in osteoblasts and is considered an inhibitor of osteoclastogenesis . Thus, during the bone remodeling cycle, osteoclasts inhibit bone formation by expressing Sema4D, in order to initiate bone resorption, whereas osteoblasts express Sema3A that suppresses bone resorption, prior to bone formation  ( Figure 5 ).
Recent studies also suggest the existence of other factors involved in the coupling mechanism during the bone remodeling cycle. One of these factors is ephrinB2, a membrane-bound molecule expressed in mature osteoclasts, which bind to ephrinB4, found in the plasma membrane of osteoblasts. The ephrinB2/ephrinB4 binding transduces bidirectional signals, which promote osteoblast differentiation, whereas the reverse signaling (ephrinB4/ephrinB2) inhibits osteoclastogenesis  ( Figure 5 ). These findings suggest that ephrinB2/ephrinB4 pathway may be involved in the ending of bone resorption and induces osteoblast differentiation in the transition phase .
In addition, it has been shown that ephrinB2 is also expressed in osteoblasts . Furthermore, mature osteoclasts secrete a number of factors that stimulate osteoblast differentiation such as the secreted signaling molecules Wnt10b, BMP6, and the signaling sphingolipid, sphingosine-1-phosphate . These findings suggest a highly complex mechanism of ephrins and the involvement of other factors in osteoclast/osteoblast communication during the bone remodeling cycle. On the other hand, despite the studies reporting the involvement of semaphorins and ephrins on osteoclast/osteoblast communication, the direct contact between mature osteoblasts and osteoclasts has not been demonstrated in vivo and it is still controversial.
Besides osteoclasts and osteoblasts, it has been demonstrated that osteocytes play key roles during the bone remodeling cycle . In fact, under the influence of several factors, the osteocytes act as orchestrators of the bone remodeling process, producing factors that influence osteoblast and osteoclast activities  ( Figure 5 ). For example, mechanical loading stimulates osteocyte to produce factors that exert anabolic action on bone such as PGE2, prostacyclin (PGI2), NO, and IGF-1 . On the other hand, mechanical unloading downregulates anabolic factors and stimulates osteocytes to produce sclerostin and DKK-1, which are inhibitors of osteoblast activity , as well as specific factors that stimulate local osteoclastogenesis . Sclerostin is a product of the SOST gene and is known to be a negative regulator of bone formation, by antagonizing in osteoblasts the actions of Lrp5, a key receptor of the Wnt/β-catenin signaling pathway .
Osteocyte apoptosis has been shown to act as a chemotactic signal for local osteoclast recruitment [70, 150, 152, 190, 191]. Accordingly, it has been reported that osteoclasts engulf apoptotic osteocytes [74, 75, 192], suggesting that osteoclasts are able to remove dying osteocytes and/or osteoblasts from a remodeling site (Figures 4(c) and 4(d) ). Moreover, it is reported that the osteoclastogenic factors is also produced by viable osteocytes nearby the dying osteocytes . There is evidence that osteocytes act as the main source of RANKL to promote osteoclastogenesis [167, 168], although this factor has also been demonstrated to be produced by other cell types such as stromal cells , osteoblasts, and fibroblasts [88, 89].
Thus, there are still uncertainties about the precise osteoclastogenesis-stimulating factors produced by osteocytes. Recent reviews have focused on some molecules that may be candidates for signaling between osteocyte apoptosis and osteoclastogenesis [72, 73]. For instance, in bones subjected to fatigue loading, viable osteocytes near the apoptotic ones express, besides high RANKL/OPG ratio, increased levels of vascular endothelial growth factor (VEGF) and monocyte chemoattractant protein-1 (CCL2) promoting an increase in local osteoclastogenesis [194, 195]. It has been suggested that osteocytes act as the main source of RANKL to promote osteoclastogenesis [166, 167]. In addition, an increase in RANKL/OPG ratio expressed by osteocytes was also observed in connexin43-deficient rats, suggesting that a disruption in cell-to-cell communication between osteocytes may induce the release of local proosteoclastogenic cytokines [33, 196, 197]. High mobility group box protein 1 (HMGB1)  and M-CSF  have also been suggested to be produced by osteocytes that stimulate osteoclast recruitment during bone remodeling [72, 73]. Thus, future studies are required to address this issue.
2.9. Endocrine Functions of Bone Tissue
The classical functions of bone tissue, besides locomotion, include support and protection of soft tissues, calcium, and phosphate storage and harboring of bone marrow. Additionally, recent studies have focused on the bone endocrine functions which are able to affect other organs . For instance, osteocalcin produced by osteoblasts has been shown to act in other organs . Osteocalcin can be found in two different forms: carboxylated and undercarboxylated. The carboxylated form has high affinity to the hydroxyapatite crystals, remaining into bone matrix during its mineralization. The undercarboxylated form shows lower affinity to minerals, due to acidification of bone matrix during osteoclast bone resorption, and then it is ferried by the bloodstream, reaching other organs [204, 205]. It has been shown that the undercarboxylated osteocalcin has some effects in pancreas, adipose tissue, testis, and the nervous system. In the pancreas, osteocalcin acts as a positive regulator of pancreatic insulin secretion and sensitivity as well as for the proliferation of pancreatic β-cells . In the adipose tissue, osteocalcin stimulates adiponectin gene expression that in turn enhances insulin sensitivity . In the testis, osteocalcin can bind to a specific receptor in Leydig cells and enhances testosterone synthesis and, consequently, increases fertility . Osteocalcin also stimulates the synthesis of monoamine neurotransmitters in the hippocampus and inhibits gamma-aminobutyric acid (GABA) synthesis, improving learning and memory skills .
Another endocrine function of bone tissue is promoted by osteocytes. These cells are able to regulate phosphate metabolism by the production of FGF23, which acts on other organs including parathyroid gland and kidneys to reduce the circulating levels of phosphates [208, 209]. Osteocytes also act on the immune system by modifying the microenvironment in primary lymphoid organs and thereby influencing lymphopoiesis . Not only osteocyte but also osteoblast and osteoclast activities are known to influence the immune system, mainly upon bone inflammatory destruction. Indeed, the discovery of communication interplay between skeletal and immune systems led to a new field of study called osteoimmunology .
Golgi Apparatus Structure
The image below shows the structure of the Golgi apparatus. The cis face of the organelle is closest to the endoplasmic reticulum. The trans face is the side furthest from the nucleus, which secretes vesicles to various parts of the cell. Further, there are a number of lumens and cisternae through which products flow. These appear as a series of flattened sacs stack on each other, much like the endoplasmic reticulum.
What is xylem?
The xylem is one of the conductive tissues in plants. It is a complex tissue composed of many types of cells. The term xylem was proposed by Nageli (1858) and he derived the word from a Greek word ‘xylos’ meaning wood. The main function of xylem is to conduct water and minerals from roots to leaves. The secondary xylem also provides mechanical support due to the presence of thick lignified cell wall.
What are the components or elements of xylem?
The xylem composed of four types of cells. Among these cells, some cells are living and some are dead.
The four elements of xylem are:
(3). Xylem Fibres
(4). Xylem Parenchyma
Tracheids are the fundamental cell type in the xylem. They are elongated tube like cells with tapering ends and chisel like in appearance. The cells are non-living at their maturity and the mature cells are empty without protoplast. They have highly lignified secondary cell wall and the cells angular and polygonal in cross section. The average length of tracheid is 5 – 6 mm.
Major portions of the cell wall of tracheids are perforated with pits. They also possess pit pairs between two adjacent tracheids at their common walls. Pits may be simple circular pits or advanced bordered pits.
Tracheids are the only xylem element in Pteridophytes. In Gymnosperms, major portion of the secondary xylem composed of tracheids. In Angiosperms, tracheids occur with other xylem elements. In some primitive Angiosperms such as Drimys, Trochodendron, Tetracentron, the xylem composed only of tracheids (vessels absent).
Patterns of secondary thickening in tracheids:
The secondary cell wall materials are laid down on the lateral walls of the tracheids in specific patterns. The most common patterns are the following types:
(a). Annular thickening: Secondary wall thickening occurs as rings arranged one above the other. Annular thickening is considered as the most primitive type of wall thickening.
(b). Spiral thickening (helical thickening): Here the secondary wall materials is deposited in the form of spirals along the inner wall of the tracheids.
(c). Scalariform thickening (ladder like thickening): The wall materials are deposited as transverse bands along the wall. The bands are with few interconnections.
(d). Reticulate thickening (net-like thickening): Here the wall thickening pattern is net-like (reticulate).
(e). Pitted thickening: It is the most advanced type of secondary wall thickening in tracheids. Here, the secondary wall materials are evenly distributed over the inner portion of the cell and the cell wall looks more or less uniform in their thickness. Many pits are distributed over the cell wall. Nature and arrangement the pits vary in different plant groups. The pits may be circular or elongate bordered type. Scalariform pitted thickening is a highly advanced type of pitting pattern where elongated bordered pits are arranged in a ladder like (scalariform) pattern.
Structural advancement of tracheids in relation to their functions:
Tracheids are specially adapted to do its function such as the conduction of water and mineral and providing mechanical support in plants. The structural advancements of tracheids which best suits to do these functions are given below:
Ø Tracheid cells are elongated with tapering ends
Ø Cells are devoid of any protoplasts at their maturity (ensure easy flow of water)
Ø Thick lignified secondary cell wall (provide mechanical support)
Ø Lateral walls and end walls are provided with pit pairs (facilitate lateral conduction of water)
Ø Cells are placed end to end to the long axis of the organ in which they occur.
Ø Water and mineral passage takes place through pit membrane
Ø Torus of pit act as valves which can regulate the passage of water
Vessels (also called as trachea) are the second category of xylem elements composed of short and tube like cells. Vessels are arranged as a series in an end to end fashion to the long axis of the organ in which they occur. Components of the vessel are called vessel segments or vessel element. Each vessel elements are shorter than tracheids in their length however, the diameter of the vessel lumen is much larger than that of tracheids. The cells are non-living and they are devoid of protoplast at their maturity. Numerous pits are present in the lateral walls of the vessels for communication.
Distribution of vessels among plants:
Vessels occur mainly in the xylem of Angiosperms. Usually, vessels are absent in Pteridophytes and Gymnosperms. The wood of Gnetum, an advanced Gymnosperm, contains plenty of vessels. The presence of vessels in the secondary wood of Gnetum is considered as one of the strongest evidence for the Gymnospermic origin of Angiosperms and thus Gnetum acts as a connecting link between Gymnosperms & Angiosperms.
Very rarely vessels are also present in some Pteridophytes such as Pteridium, Selaginella and Equisetum. In some primitive Angiosperms, such as Trochodendron, Tetracentron and Drimys, the vessels are absent. Aquatic plants usually do not have vessels in their poorly developed xylem. Xylem in aquatic plants will be ill developed, since these plants do not require a well specialized water conducting system.
Some parasitic plants and few succulent plants also do not show vessels in their xylem. In some monocots like Dracaena and Yucca, vessels are completely absent. The absence of vessels in these plants is due to the evolutionary reduction.
Structure of Vessels in relations to its functions:
Vessel system is made up of a series of cells placed end to end as a long tube like structure. Each cell is called vessel member or vessel element. Each vessel member has perforations (large openings) at their end walls for the easy passage of water and minerals between the cells. Usually, vessels members are shorter than tracheids. However, the diameter of vessels is much larger than tracheids. This facilitates a rapid and efficient flow of water through the vessel lumen.
In highly advanced forms, the vessel cells are with shorter length and wider diameter and they appear as drum shaped structures (as in Quercus alba). The end wall of each vessel members is oblique or transverse. Vessels with oblique end are considered as primitive, whereas those with transverse ends are treated as highly advanced. In some plants, such as Malus, tail like tip occurs beyond the end wall. The openings or pores in each vessel end wall are known as perforations (Perforation plate: the region of the vessel with perforation occurs). Usually perforations occur at the end wall, sometimes lateral perforations also occur on the walls.
Different types of perforation plates seen in vessels are
1. Simple perforation plate: a plate with single perforation (advanced type)
2. Multiple perforation plate: many perforations
3. Scalariform perforation plate: a multiple perforation plate with perforations arranged in parallel series. The wall region of pores in scalariform perforation plate is called as perforation bar
4. Reticulate perforation plate: pores arranged in reticulate fashion
5. Forminate type perforation: many pores arranged more or in a less circular pattern. This type is also called Ephedoid perforation plate.
Secondary wall of Vessels
The secondary wall thickening of vessels is similar to that of tracheids. Different types of thickenings pattern seen in vessels are Annular thickening, Spiral thickening (helical), Scalariform thickening, Reticulate thickening and Pitted thickening.
The pitted thickening is a characteristic of the vessels of meta-xylem and secondary xylem. The pits on the xylem are commonly bordered type. The distribution pattern of pits varies greatly in different plant groups. The pits are distributed in three basic patterns, they are:
1. Scalariform pitting: elongated with pits in ladder like arrangement
2. Opposite pitting: pits arranged in horizontal rows in pairs
3. Alternate pitting: pits arranged in diagonal rows
Differentiation of vessels
The primary xylem vessels are formed from the longitudinal cells of the pro-cambia. The secondary xylem vessels are formed from cells of vascular cambium. Initials of vessels in both cambia are called primordial vessel members. Primordial vessels members have dense cytoplasm with prominent nucleus. Vessel’s secondary cell wall is laid down by the content of primordial cell. Secondary wall layers are deposited in a pattern characteristic of the given type of vessel element.
The perforation areas are not thickened by deposition of wall materials. After the secondary thickening is complete, the protoplasm of the primordial cell disintegrates. The vacuole secretes many hydrolytic enzymes which degrade the primary cell wall region which is not covered by lignified secondary wall. The non-cellulosic components in the perforation plate are degraded, leaving cellulose micro-fibrils intact. Protoplast completely disappears once wall deposition is completed. Dead cytoplasm forms a layer over the inner side of the lumen called Warty layer.
Structure of vessels in relation to its functions:
The main function of vessels is conduction of water and nutrients. Apart from this, vessels also provide mechanical support. The structure of vessel is best suited to do these two functions. The vessel elements are arranged end-to-end to form long tube like channels. This is suitable for uninterrupted passage of water along with minerals. Thick lignified cell wall provides mechanical support.
Evolutionary origin of vessels in Angiosperms:
The vessels are believed to be originated from the tracheids. In Angiosperms, the vessels originated from tracheids with pitted, reticulate or helical secondary thickening. The formation of the perforation plate of vessels at the end wall of each vessel element is considered as the most important event in stelar evolution.
(3). Xylem Fibres
Xylem fibres are the third components of xylem and it is also called as xylary fibres. Similar to tracheids and vessels, they are also dead cells and they do not contain protoplast at their maturity. Cells are with very thick lignified secondary cell wall. The main function is to provide mechanical support. There are two types of xylary fibres, they are:
Fibre tracheids are longer than tracheids and they have apical intrusive growth. Fibre tracheids have less developed bordered pits.
Libriform fibres are highly specialized fibres. They have simple pits on their walls. Gelatinous fibres are special category of xylem fibre found in the tension wood (a reaction wood in Angiosperms). The secondary cell wall of gelaginous fibres do not have lignin but have cellulosic cell wall. Thus this part of cell wall appears as gelatinous in cross section. Gelatinous fibres are highly hygroscopic and they can absorb and store plenty of water.
(4). Xylem Parenchyma
Xylem parenchyma is the fourth component of xylem. It is the only living component in the xylem. The cells are with plenty of cytoplasm and prominent nucleus. They have thin cellulosic cell wall. Lignified secondary cell wall is absent in xylem parenchyma. Very rarely parenchyma cells in the secondary xylem undergo secondary growth. Parenchyma in the xylem can store starch, oil and other ergastic substances.
Classification of Xylem Parenchyma:
Two types of xylem parenchyma occurs in the xylem
(a). Axial parenchyma
(b). Ray parenchyma
(a). Axial parenchyma
Axial parenchyma is originated from the elongated fusiform initials of the cambial cells. They are arranged parallel to the long axis of the organ in which they occur.
(b). Ray parenchyma
Ray parenchyma originated from the ray initials of the cambium. There are two types of ray parenchyma in the xylem.
(A). Procumbent ray cells: long axis of the cell are radially elongated
(B). Upright ray cells: long axis of the cell vertically elongated
Based on the composition of cell types, two types of rays occur in the xylem:
Ø Homocellular ray: composed of single type of ray cells (either procumbent or upright).
Ø Heterocellular ray: composed of both types of ray cells (procumbent and upright)
Tyloses are the outgrowth of parenchymatous cells to the lumen of tracheids or vessels of the secondary xylem through pit openings. Tyloses accumulate resins and other secondary materials in their protoplasm. They are responsible for the characteristic odor of wood. They also prevent the degradation of wood by termites and mites.
Classification of xylem:
Based on origin, xylem classified into two groups
Primary xylem is formed during the primary growth of the plant. It is derived from procambium (a meristem) and consists of two parts namely Protoxylem and Metaxylem. Protoxylem is the first formed xylem and it contains fewer amounts of tracheary elements and more amount of parenchyma. Usually proto-xylem gets destroyed during the maturation of the plant. Metaxylem is derived or differentiated after protoxylem in the vascular bundles. Metaxylem usually contains more tracheary elements than parenchyma. Plants without secondary thickening, metaxylem are functional xylem part throughout the life cycle of the plant. Those plants with secondary thickening the metaxylem are replaced by the secondary xylem.
Secondary xylem is the xylem formed during the secondary growth of the plant. It is developed from the vascular cambium (a lateral meristem). The main function is the conduction of water and mineral in the secondary plant body. They also provide mechanical support.
Function of Xylem:
Ø Conduction of water from roots to leaves
Ø Conduction of minerals and nutrients from roots to leaves
Ø Provide mechanical support
Ø Ray parenchyma forms tyloses which store ergastic substances
Ø These ergastic substances give the wood a characteristic colour and odour
Ø Ergastic substances present in the tyloses also protect the wood from termites and mites.