The dental follicle, also known as dental sac, is made up of mesenchymal cells and fibres surrounding the enamel organ and

dental papilla In embryology and prenatal development, the dental papilla is a condensation of ectomesenchymal cells called odontoblasts, seen in histologic sections of a developing tooth. It lies below a cellular aggregation known as the enamel organ. Th ...

of a developing tooth. It is a vascular fibrous sac containing the developing

tooth A tooth ( : teeth) is a hard, calcified structure found in the jaws (or mouths) of many vertebrates and used to break down food. Some animals, particularly carnivores and omnivores, also use teeth to help with capturing or wounding prey, tea ...

and its

odontogenic Tooth development or odontogenesis is the complex process by which teeth form from embryonic cells, grow, and erupt into the mouth. For human teeth to have a healthy oral environment, all parts of the tooth must develop during appropriate stage ...

organ. The dental follicle (DF) differentiates into the

periodontal ligament The periodontal ligament, commonly abbreviated as the PDL, is a group of specialized connective tissue fibers that essentially attach a tooth to the alveolar bone within which it sits. It inserts into root cementum one side and onto alveolar b ...

. In addition, it may be the precursor of other cells of the

periodontium The periodontium is the specialized tissues that both surround and support the teeth, maintaining them in the maxillary and mandibular bones. The word comes from the Greek terms περί ''peri''-, meaning "around" and -''odont'', meaning "tooth". ...

, including

osteoblast Osteoblasts (from the Greek combining forms for "bone", ὀστέο-, ''osteo-'' and βλαστάνω, ''blastanō'' "germinate") are cells with a single nucleus that synthesize bone. However, in the process of bone formation, osteoblasts functio ...

cementoblast A cementoblast is a biological cell that forms from the follicular cells around the root of a tooth, and whose biological function is cementogenesis, which is the formation of cementum (hard tissue that covers the tooth root). The mechanism of di ...

s and

fibroblast A fibroblast is a type of biological cell that synthesizes the extracellular matrix and collagen, produces the structural framework ( stroma) for animal tissues, and plays a critical role in wound healing. Fibroblasts are the most common cells o ...

s. They develop into the alveolar bone, the cementum with Sharpey's fibers and the periodontal ligament fibers respectively. Similar to

, the dental follicle provides nutrition to the

enamel organ The enamel organ, also known as the dental organ, is a cellular aggregation seen in a developing tooth and it lies above the dental papilla. The enamel organ which is differentiated from the primitive oral epithelium lining the stomodeum.The ename ...

and

and also have an extremely rich blood supply.

Role in tooth eruption

The formative role of the dental follicle starts when the crown of the tooth is fully developed and just before

tooth eruption Tooth eruption is a process in tooth development in which the teeth enter the mouth and become visible. It is currently believed that the periodontal ligament plays an important role in tooth eruption. The first human teeth to appear, the deciduo ...

into the

oral cavity In animal anatomy, the mouth, also known as the oral cavity, or in Latin cavum oris, is the opening through which many animals take in food and issue vocal sounds. It is also the cavity lying at the upper end of the alimentary canal, bounded on t ...

. Although

mechanisms have yet to be understood entirely, generally it can be agreed that many factors, together, affect the

process which is why it is very difficult to differentiate the causes and effects. There have been many theories proposed for

. Ideas such as remodelling of the

alveolar bone The alveolar process () or alveolar bone is the thickened ridge of bone that contains the tooth sockets on the jaw bones (in humans, the maxilla and the mandible). The structures are covered by gums as part of the oral cavity. The synonymous t ...

, root elongation and to a certain extent, the most probable reasoning for

in human beings is the formation of the

Bone remodelling

Bone remodelling of the jaws has been associated to

such that in the pre-eruptive phase of a tooth, the natural pattern of growth of the

maxilla The maxilla (plural: ''maxillae'' ) in vertebrates is the upper fixed (not fixed in Neopterygii) bone of the jaw formed from the fusion of two maxillary bones. In humans, the upper jaw includes the hard palate in the front of the mouth. The t ...

mandible In anatomy, the mandible, lower jaw or jawbone is the largest, strongest and lowest bone in the human facial skeleton. It forms the lower jaw and holds the lower tooth, teeth in place. The mandible sits beneath the maxilla. It is the only movabl ...

would theoretically move teeth by the selective deposition and reabsorption of bone in the adjacent surroundings of the tooth. A sequence of experiments in dogs does provide the most reliable substantiation to prove that bone remodelling is a cause of tooth movement. When an eruption is stopped by wiring the tooth germ on to the lower margin of the

or when the dental follicle remains undisturbed as the developing pre-molar is removed, osteoclasts enlarge the gubernacular canal while an eruptive pathway develops within the bone covering over the enucleated tooth. However, no eruptive pathway will develop if the dental follicle is removed. Furthermore, the replica will erupt with the development of an eruptive pathway as long as the dental follicle is preserved when an exact silicone or metal duplicate replaces the tooth germ. Such observations should be examined judiciously and in great detail. Firstly, eruptive pathways have unmistakably been demonstrated to develop in bone deprived of a budding or growing tooth. Secondly, they provided evidence to prove that the dental follicle is involved in the process. Therefore, it is only when concurrent bone deposition can be confirmed at the base of the crypt and inhibition of such bone deposition can be demonstrated to show interference with

, then the conclusion that an eruptive pathway forming within bone means that bony remodelling is the cause for tooth formation. In many studies, with the usage of tetracyclines as indicators of bone deposition have proven that bone resorption is principal activity in the fundus of an alveolus in a number of species, including human beings. For example, in human beings, the base of the crypt of the permanent first

molars The molars or molar teeth are large, flat teeth at the back of the mouth. They are more developed in mammals. They are used primarily to grind food during chewing. The name ''molar'' derives from Latin, ''molaris dens'', meaning "millstone to ...

and permanent third molars will repeatedly reabsorb as the eruption of these teeth occur, although, in the second molars and second premolars, there will be some bone deposition on the crypt floor. For the circumstance of a dormant duplicate’s demonstrated eruption, many would think that the bony remodelling would be the only reason. However, as per what will be discussed next, it can be concluded that follicular tissue is accountable for this movement as supported by pieces of evidence. Furthermore, in some recent research, it has been observed that alveolar bone growth at the base of the crypt is a prerequisite for molar

in rats. Undoubtedly, more attention needs to be given to the intraosseous

. Regardless of whether bone growth is a main moving force, it can widely be agreed that for

to happen, the dental follicle is required and that, as will be discussed later, the dental follicle regulates bone remodelling.

Dental follicle

Researches show a repeated series of cellular activities which involves the reduced dental epithelium and the follicle linked to

that assists bone absorption and connective tissue degradation. With the lack of colony-stimulating factor 1, a factor that encourages differentiation of osteoclasts, in osteopetrotic animals, no mechanism for bone removal is present and therefore eruption is prevented. The eruption will happen when differentiation of osteoclasts is allowed due to local administration of the colony-stimulating factor 1. Proteases, which is produced by the reduced enamel epithelium, results in a path of least resistance as it promotes the breakdown of connective tissue. When stimulating alveolar bone growth at the base of the crypt, expression of the bone morphogenetic protein-6 in the dental follicle may also be necessary. It is also thought that signalling between the dental follicle and the reduced enamel epithelium exists. This signalling could be a plausible reason for the noteworthy regularity of eruption timings because the enamel epithelium is most possibly programmed as part of its functional life cycle. Signalling would also aid in explaining why radicular follicle, that is not related to reduced enamel epithelium, is involved in the formation of the

but does not experience degeneration.

Periodontal ligament

Cells of dental follicle will differentiate into collagen forming

s and

s, that produces and secretes

cementum Cementum is a specialized calcified substance covering the root of a tooth. The cementum is the part of the periodontium that attaches the teeth to the alveolar bone by anchoring the periodontal ligament.Illustrated Dental Embryology, Histology, a ...

on the surface of the tooth roots. As the tooth roots break apart, some cells of the dental follicle will invade the roots of the tooth. Delicate fibres that appear along the developing roots near the cervical region of the crown will also be formed by some cells of the ligament. These are most likely the

stem cell In multicellular organisms, stem cells are undifferentiated or partially differentiated cells that can differentiate into various types of cells and proliferate indefinitely to produce more of the same stem cell. They are the earliest type of ...

s that form principle fibre groups that will surface as the roots lengthen. As fibres become implanted in the

of the root surface, the other end will be attached to the forming

. Rejuvenation and development of the

have been taken as a factor in the eruption of the tooth due to the traction power possessed by

s and because of experimental results relying on the unceasingly erupting rat incisor. The case is not the same where the existence of a

does not always correspond with resorption in teeth that have limited growth period. Cases do however happen in which rootless teeth erupt and when a

is present, and the tooth does not erupt. One significant difference in the formation of fibres exists between teeth that have predecessors and teeth that do not have predecessors. For the former group of teeth (such as the permanent incisors, the canines and the premolars), the principal fibres group will develop later than in the latter group of teeth (such as the deciduous teeth and the permanent molars). It can be observed that the coronal half of the

is made up of well composed, obliquely orientated principal collagen fibre bundles when an erupting permanent molar enters into the oral cavity. The opposite is true too. A majority of the

of an erupting permanent

premolar The premolars, also called premolar teeth, or bicuspids, are transitional teeth located between the canine and molar teeth. In humans, there are two premolars per quadrant in the permanent set of teeth, making eight premolars total in the mou ...

is deprived of a discernable number of organized principal collagen fibre bundles passing from tooth to

Molecular determinants of tooth eruption

Tooth eruption Tooth eruption is a process in tooth development in which the teeth enter the mouth and become visible. It is currently believed that the periodontal ligament plays an important role in tooth eruption. The first human teeth to appear, the deciduo ...

is a closely controlled process which involves the tooth organ, consisting of the dental follicle and the enamel organ, and adjacent alveolar tissues. The balance between tissue formation of bone,

and root, and tissue destruction of bone, connective tissue and epithelium causes tooth movement. Osteoclasts are gathered from circulating

monocyte Monocytes are a type of leukocyte or white blood cell. They are the largest type of leukocyte in blood and can differentiate into macrophages and conventional dendritic cells. As a part of the vertebrate innate immune system monocytes also infl ...

s which are chemically drawn to the location where bone resorption takes place during bone remodelling. A growth factor, colony-stimulating factor 1, produced by the dental follicle, encourages the differentiation of

s into

macrophage Macrophages (abbreviated as M φ, MΦ or MP) ( el, large eaters, from Greek ''μακρός'' (') = large, ''φαγεῖν'' (') = to eat) are a type of white blood cell of the immune system that engulfs and digests pathogens, such as cancer cell ...

s and osteoclasts. In addition, as a result of the epidermal

growth factor A growth factor is a naturally occurring substance capable of stimulating cell proliferation, wound healing, and occasionally cellular differentiation. Usually it is a secreted protein or a steroid hormone. Growth factors are important for re ...

, the enamel organ produces

interleukin-1 alpha Interleukin-1 alpha (IL-1 alpha) also known as hematopoietin 1 is a cytokine of the interleukin 1 family that in humans is encoded by the ''IL1A'' gene. In general, Interleukin 1 is responsible for the production of inflammation, as well as the p ...

, a promoter of bone resorption, that induces follicular cells to produce the colony-stimulating factor 1. In the process of

, monocyte chemotactic protein-1 may also be involved. Signalling via the receptor-activated Nuclear factor kB or receptor- activated Nuclear factor kB ligand or osteoprotegerin pathway controls osteoclastogenesis. In the apex of the dental follicle, osteoprotegerin prevents osteoclast formation, and its expression is down-regulated. Ultimately, accentuation of the differentiation of

s at the base of the alveolar crypt is accentuated. A high level of transcription factor Runt-related transcription factor- 2, that is involved in

differentiation and function, is indicated in the basal portion of the dental follicle. Down-regulation of the expression of the Runt-related transcription factor- 2 in the apex portion of the dental follicle, that supports bone removal along the surface which the tooth erupts, is due to the transforming growth factor b. Acceleration of

incisor Incisors (from Latin ''incidere'', "to cut") are the front teeth present in most mammals. They are located in the premaxilla above and on the mandible below. Humans have a total of eight (two on each side, top and bottom). Opossums have 18, wher ...

eruption in rodents has been proven to be affected by the epidermal growth factor which increases the level of expression for the transformation of growth factor b.

Role in development of Odontogenic Cysts and Tumours

Role in development of odontogenic cysts and tumour

The most common pathologies associated with dental follicle are dentigerous cyst, keratocystic odontogenic tumor and ameloblastoma. Carcinomas, such as primary intraosseous carcinoma and the other tumors including sarcoma and bromixoma can also be associated with dental follicle. Dentigerous (follicular) cyst The second most common odontogenic cyst is the follicular cyst. The cyst develops in normal dental follicule surrounding an unerupted tooth. It can also develop from break down of stellate reticulum or collection of fluid between the reduced enamel epithelium layers. Clinical features The dentigerous cyst is often found in areas where unerupted teeth are found. These areas, in decreasing order of frequency, are mandibular third molars, maxillary third molars and maxillary canines. The cyst may grow to a large size, replace the tooth with which they are associated, or hardly cause resorption of adjacent tooth roots. Diagnosis Clinical and radiographic assessments are required to diagnose dentigerous cysts. A cyst is present when the follicular space exceeds 5mm from the crown. However, it is possible that keratocysts and ameloblastomas mimic the radiographical appearance of follicular cysts. Aspiration can be used to differentiate the lesions. Treatment - Marsupialization This procedure is partial removal of associated tooth. The advantage of this procedure is that it maintains the vitality of teeth and is less invasive. The disadvantage is that it required substantial after care and heals very slowly. - Enucleation This procedure is complete removal of the associated tooth. The advantage of enucleation is that the cyst cavity heals eventually and the full cyst tissue is available for histological examination. The disadvantage is that if the cyst involves the apices of adjacent vital teeth, the surgery might deprive the teeth of their blood supply and kill the viral teeth.

Odontogenic Tumors

Odontogenic tumors may consist of odontogenic epithelium, odontogenic connective tissue or both. Odontogenic tumours consisting predominantly of epithelium arise from odontogenic epithelium. Odontogenic tumours composed of odontogenic connective tissue arise from the ectomesenchymal region of the tooth germ, either from the dental papilla or the dental follicle. Odontogenic tumours of mixed origin contain both ameloblastic epithelium and odontoblastic tissue while in active growth. After complete development, it principally consists of enamel, dentine and cementum.

Dental Follicle Stem Cells

The presence of stem cells in the dental is an integral part of the DF’s ability to differentiate into the

. Current knowledge about the stem cells present in DFs, is based on the extraction of DFs from immature roots of impacted teeth. In Comparison to the DF of a typically erupted tooth, the DF of an

impacted tooth An impacted tooth is one that fails to erupt into the dental arch within the expected developmental window. Because impacted teeth do not erupt, they are retained throughout the individual's lifetime unless extracted or exposed surgically. Tee ...

, e.g. third molar, does not surround the tooth and subsequently be divided into two parts. The periapical section: This surrounds the apex of the developing tooth root and mediates tooth eruption. The coronal section: This is attached to developing tooth root and mediates bone growth. Stem cells isolated from these two parts are summarised below.

Multipotentectomesenchymal Precursor Cells

Multipotentectomesenchymal precursor cells also referred to as DFCs are found in the coronal part of the DF of impacted human third molar teeth contains. DFCs are considered multipotent, and they are especially precursors for cells of the tooth attachment apparatus Cells of this tissue structure express typical markers for PDL fibroblast, alveolar osteoblasts, and cementoblasts. When cultivated, DFCs have a morphology that is comparable to that of a fibroblast and express markers such as Nestin and STRO-1 which are typical markers for dental stem cells. These cells have are highly proliferate and proliferate typically at a higher rate than that of a proliferating bone marrow- derived Mesenchymal stem cell.

Regulation of DFCs

The initiation of differentiation is controlled by a number of different extracellular factors, such as growth factors, cell-cell contacts, extracellular matrix, and mechanical loading. These factors coordinate to induce or modulate the process of differentiation into particular functional body cell type. There have been some studies done recently about the differentiation of cultivated DFCs into

biomineralising Biomineralization, also written biomineralisation, is the process by which living organisms produce minerals, often to harden or stiffen existing tissues. Such tissues are called mineralized tissues. It is an extremely widespread phenomenon; ...

cells. These studies revealed new ways in which the cell differentiation mechanisms work. Moreover, information about genome-wide expression profiles was provided by

proteomics Proteomics is the large-scale study of proteins. Proteins are vital parts of living organisms, with many functions such as the formation of structural fibers of muscle tissue, enzymatic digestion of food, or synthesis and replication of DNA. I ...

and

transcriptomics Transcriptomics technologies are the techniques used to study an organism's transcriptome, the sum of all of its RNA transcripts. The information content of an organism is recorded in the DNA of its genome and expressed through transcription. He ...

with DFCs. These help in showing more clearly the molecular mechanisms in cells. The extracellular signal regulated kinase ( ERK) pathway was also revealed during the osteogenic differentiation of DFCs by these investigations. The proteomics and transcriptomics identified regulated transcription factors such as SP1 and TP53. These

transcription factor In molecular biology, a transcription factor (TF) (or sequence-specific DNA-binding factor) is a protein that controls the rate of transcription of genetic information from DNA to messenger RNA, by binding to a specific DNA sequence. The fun ...

s were more precisely identified by bioinformatics after the analysis of the proteome. The role of these transcription factors regulate the cell proliferation and differentiation of DFCs. Human dental follicle cells are progenitor cells. Different studies suggested that osteogenic differentiation of DFCs is controlled by BMP2 and IGF2, which are growth factors. However, the influence of BMP2 and IGF2 on the differentiation of DFCs has not been analysed in too much depth. There was a study that examined DFCs after the induction of osteogenic differentiation with BMP2, IGF2 and a standard osteogenic differentiation medium (ODM) with

dexamethasone Dexamethasone is a glucocorticoid medication used to treat rheumatic problems, a number of skin diseases, severe allergies, asthma, chronic obstructive lung disease, croup, brain swelling, eye pain following eye surgery, superior vena cava s ...

. The alkaline

phosphatase In biochemistry, a phosphatase is an enzyme that uses water to cleave a phosphoric acid monoester into a phosphate ion and an alcohol. Because a phosphatase enzyme catalyzes the hydrolysis of its substrate, it is a subcategory of hydrolases. P ...

activity and the calcium accumulation showed osteogenic differentiation after all treatments, but with the most effective differentiation by ODM. Furthermore, markers of the process of osteoblast differentiation were much higher up-regulated in BMP2- or IGF2-treated cells than in ODM-treated cells. To find the reason between these differences, the genome-wide expression profiles were compared at an early differentiation stage. Chondroblast markers in BMP2-differentiated cells and general markers for cell differentiation/proliferation in IGF2-treated cells were significantly regulated. However, ODM-treated DFCs expressed late markers of osteogenic-differentiated DFCs such as the transcription factor ZBTB16 that is not expressed in BMP2- or IGF2-differentiated cells. Therefore, this study shows that osteogenic differentiation of DFCs can be triggered with all tested inducers. However to analyse this mechanism, the transcription factor ZBTB16 is a target for further investigations. DLX3, a

, which is related to the induced BMP2 pathway in osteogenic differentiated DFCs was able to trigger cell viability and the osteogenic differentiation of DFCs via the BMP2/Smad1 feedback loop ). DFCs control the proportional amount of all three periodontal tissues, which includes a good balance between the size of the periodontal ligament and the amount of the surrounding cementum and alveolar bone. A high level of

in the

supports the growth of the

and the

. This is why a soft extracellular matrix supports the osteogenic differentiation of DFCs.

The Migration Capacity of DFCs

The

migration Migration, migratory, or migrate may refer to: Human migration * Human migration, physical movement by humans from one region to another ** International migration, when peoples cross state boundaries and stay in the host state for some minimum le ...

capacity of DFCs can be histological examined, during this, DFCs demonstrate an extensive migratory capacity for dental

mesenchymal stem cell Mesenchymal stem cells (MSCs) also known as mesenchymal stromal cells or medicinal signaling cells are multipotent stromal cells that can differentiate into a variety of cell types, including osteoblasts (bone cells), chondrocytes (cartilage ce ...

s, during the initial stages of tooth root development. When compared with the migration capacity of stem cells from the

dental pulp The pulp is the connective tissue, nerves, blood vessels, and odontoblasts that comprise the innermost layer of a tooth. The pulp's activity and signalling processes regulate its behaviour. Anatomy The pulp is the neurovascular bundle centr ...

baby teeth Deciduous teeth or primary teeth, also informally known as baby teeth, milk teeth, or temporary teeth,Illustrated Dental Embryology, Histology, and Anatomy, Bath-Balogh and Fehrenbach, Elsevier, 2011, page 255 are the first set of teeth in the ...

and stem cells from the dental apical papilla (dental neural crest derived progenitor cells, dNC-PCs), DFCs are found to have the highest

cell migration Cell migration is a central process in the development and maintenance of multicellular organisms. Tissue formation during embryonic development, wound healing and immune responses all require the orchestrated movement of cells in particular direct ...

velocity. Furthermore, DFC migration can be accelerated during cultivation, with the use of growth factors found in the dental hard tissue matrix such as

TGF-β Transforming growth factor beta (TGF-β) is a multifunctional cytokine belonging to the transforming growth factor superfamily that includes three different mammalian isoforms (TGF-β 1 to 3, HGNC symbols TGFB1, TGFB2, TGFB3) and many other si ...

or BMP2 which have also been found to be involved in the differentiation of DFCs

Follicle-Derived Embryonic Neural Crest Stem Cells (FENCSCs)

FENCSCs are a sub-population of DFCs, however both differ in their in cell migration properties. FENSCs express high levels of embryonic stem cell markers (TRA1-60, TRA1-81, OCT-4) and mRNA transcripts for Nanog and Rex-1. They have the ability to differentiate into cells of all three germ layers. Some examples include, smooth and skeletal muscle, osteoblasts,

neuron A neuron, neurone, or nerve cell is an electrically excitable cell that communicates with other cells via specialized connections called synapses. The neuron is the main component of nervous tissue in all animals except sponges and placozoa. ...

glial cells Glia, also called glial cells (gliocytes) or neuroglia, are non-neuronal cells in the central nervous system (brain and spinal cord) and the peripheral nervous system that do not produce electrical impulses. They maintain homeostasis, form mye ...

, and

adipocyte Adipocytes, also known as lipocytes and fat cells, are the cells that primarily compose adipose tissue, specialized in storing energy as fat. Adipocytes are derived from mesenchymal stem cells which give rise to adipocytes through adipogenesis. ...

s and therefore display pluripotency. These cells also have high levels of

telomerase Telomerase, also called terminal transferase, is a ribonucleoprotein that adds a species-dependent telomere repeat sequence to the 3' end of telomeres. A telomere is a region of repetitive sequences at each end of the chromosomes of most euk ...

activity. A strategy that enables isolation of specific types of stem cells within the dental follicle such as FENCSCs, is known as Fluorescent Activated Cell Sorting. Cell culturing is also important to consider understanding cellular morphology. DFCs and FENCSCs spheroid cell clusters under serum – free cell culture conditions. The choice of adequate

cell culture Cell culture or tissue culture is the process by which cells are grown under controlled conditions, generally outside of their natural environment. The term "tissue culture" was coined by American pathologist Montrose Thomas Burrows. This t ...

conditions is of great importance for a specific feature of dental stem cells. For example, both DFCs and FENCSCs form spheroid-like cell clusters under serum-free cell culture conditions.

Periapical Follicle Stem Cells (PAFSCs)

Once the development of the tooth root is completed, the DF disappears thus meaning that all cells are considered to be part of the periodontium. Prior to this stage, a periapical part of the DF attaches to the apex of the developing tooth root and is referred to as periapical follicle. Therefore, undifferentiated cells within this tissue are known as Periapical Dental Follicle Stem cells (PAFSCs) and can be isolated from colony forming cells within periapical follicle cell cultures. Typical markers expressed within these cells include, CD44 and STRO1. Both Cell migration capacity and

cell proliferation Cell proliferation is the process by which ''a cell grows and divides to produce two daughter cells''. Cell proliferation leads to an exponential increase in cell number and is therefore a rapid mechanism of tissue growth. Cell proliferation re ...

capacity of these cells are greater than various types of dental mesenchymal stem cells. PFACs are targets of regenerative dentistry due to their high multipotent differentiation potential within all types of dental tissue. Despite the closely related developmental origin of PAFScs and DFScs, There is still much to learn about PAFSCs and how they compare to DFSc.

Role in regenerative dentistry

Human dental follicles can be separated from impacted wisdom teeth with undeveloped tooth roots. Therefore, undifferentiated ectomesenchymal cells can be isolated from both apical and coronal parts of the human dental follicle. The dental follicle contains various types of multipotent stem cells. They are progenitors for all types of periodontal cells providing a potential cell source for periodontal regeneration.