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What is the periodontal ligament and what is its function?

1. Berkovitz BK. Periodontal ligament: structural and clinical correlates. Dent Update. 2004 Jan-Feb;31(1):46-50, 52, 54. Review.

2. Cho MI, Garant PR. Development and general structure of the periodontium. Periodontol 2000. 2000 Oct;24:9-27. Review.

3. Cohn SA: Transalveolar fibres in the human periodontium. Arch Oral Biol 20:257-259, 1975.

4. Selliseth NJ, Selvig KA. The vasculature of the periodontal ligament: A scanning electron microscopic study using corrosion casts in the rat. J Periodontol65:1079-1087, 1994.

What are the dimensions of the PDL? Do these change with age and/or function?

1. Oehmke MJ, Schramm CR, Knolle E, Frickey N, Bernhart T, Oehmke HJ. Age-dependent changes of the periodontal ligament in rats. Microsc Res Tech. 2004 Mar 1;63(4):198-202.

2. McCulloch, C.A., Lekic, P., McKee, M.D., Role of physical forces in regulating the form and function of the periodontal ligament. Periodontol 2000, 24: 56 – 72, 2000

3. Coolidge ED: The thickness of human periodontal membrane. JADA 24:1260-1270, 1937.

For all periodontal tissues, what are the various types of collagen and where they are found? How are these arranged and what are their functions?

1. Chavrier C, Couble MC, Maglorie H, Grimaud JA : Connective tissue organization of healthy human gingiva. Ultrastructural localization of collagen types I, II, III, and IV. J. Periodontal Res. 19:221-229, 1984.

2. Page RC, Ammons WF. Collagen turnover in gingiva and other mature connective tissues of the marmoset. Arch. Oral Biol. 19:651-658, 1974.

3. Zwarych PD, Quigley MB: The intermediate plexus of the periodontal ligament: History and further observations. J. Dent. Res., 44:383-391, 1965.

What cell type(s) is(are) responsible for collagen production in the periodontal tissues? What are some of the characteristics of these cells?

1. Giannopoulou C, Cimasoni G. Functional characteristics of gingival and periodontal ligament fibroblasts. J Dent Res 1996; 75: 895-902.

2. Mariotti AJ, Cochran DL : Characterization of fibroblasts derived from human periodontal ligament and gingiva. J. Periodontol. 61:103-111, 1990.

3. Palaiologou AA, Yukna RA, Moses R, Lallier TE. Gingival, dermal, and periodontal ligament fibroblasts express different extracellular matrix receptors. J Periodontol. 2001 Jun;72(6):798-807.

4. Kramer PR, Nares S, Kramer SF, Grogan D, Kaiser M. Mesenchymal stem cells acquire characteristics of cells in the periodontal ligament in vitro. J Dent Res. 2004 Jan;83(1):27-34

5. McCulloch C, Melcher A: Cell migration in the periodontal ligament of mice. J Periodontal Res.18:339-352, 1983

6. Lallier TE, Miner QW Jr, Sonnier J, Spencer A. A simple cell motility assay demonstrates differential motility of human periodontal ligament fibroblasts, gingival fibroblasts, and pre-osteoblasts. Cell Tissue Res. 2007 May;328(2):339-54. Epub 2007 Jan 31

What changes occur in collagen in periodontal disease? How is collagen degraded? What cells and agents are involved?

1. Chavrier C, et al. Immunohistochemical study of types I, II, III, and IV collagen in fibrosis of diseased gingiva during chronic periodontitis: A light and electron microscopic study. J. Periodontal Res. 22:29-36, 1987.

2. Buduneli N, Atilla G, Guner G, Oktay G. Biochemical analysis of total collagen content and collagen types I, III, IV, V and VI in gingiva of various periodontitis categories. J Int Acad Periodontol. 2001 Jan;3(1):1-6.

3. Christner P: Collagenase in the human periodontal ligament. J Periodontol 51:455-461, 1980

4. Bildt MM, Bloemen M, Kuijpers-Jagtman AM, Von den Hoff JW. Collagenolytic fragments and active gelatinase complexes in periodontitis. J Periodontol. 2008 Sep;79(9):1704-11.

5. Chang YC, Lai CC, Yang SF, Chan Y, Hsieh YS. Stimulation of matrix metalloproteinases by black-pigmented Bacteroides in human pulp and periodontal ligament cell cultures. J Endod. 2002 Feb;28(2):90-3.

How does smoking affect collagen and collagen production?

1. Zhou J, Olson BL, Windsor LJ. Nicotine increases the collagen-degrading ability of human gingival fibroblasts. J Periodontal Res. 2007 Jun;42(3):228-35.

2. Takeuchi H, Kubota S, Murakashi E, Zhou Y, Endo K, Ng PS, Takigawa M, Numabe Y. JNicotine-induced CCN2:from smoking to periodontal fibrosis. Dent Res. 2010 Jan;89(1):34-9. Epub .

What is the relationship between the periodontal ligament and periodontal regeneration?

1. MacNeil RL, Somerman MJ. Development and regeneration of the periodontium: parallels and contrasts. Perio 2000 19:8-20, 1999.

2. Sculean A, Donos N, et al. Presence of oxytalan fibers in human regenerated periodontal ligament. J Clin Periodontol 26:318-321, 1998.

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What is the periodontal ligament and what is its function?

Berkovitz 2004                              No Article

PURPOSE: To review certain structural aspects of the periodontal ligament (PDL) including: collagen, ground substance, cells, nerves, and blood vessels.

DISCUSSION:

Periodontal Ligament main functions:

• 1)tissue attachment between tooth and alveolar bone and is responsible for displacing forces

• 2)is responsible for the mechanisms whereby a tooth attains, and the maintains, its functional position

• 3)maintains and repairs alveolar bone and cementum

• 4)mechanoreceptors are involved in the neurological control of mastication

Extracellular matrix is made up of:

• 1)collagen fibers: mostly types I and III (3:1 ratio), but types V, VI, VII, and XII also exist. PDL collagen has a high rate of turnover but the significance of this has not been determined.  The nature of the collagen may change in periodontal disease (increase in type V collagen).

• 2)oxytalan fibers: pre-elastin type fibers make up about 3% of the PDL fibers, are attached to the cementum of the tooth and the function is unknown but may have some role in tooth support.

• 3)ground substance: turnover rate faster than collagen. Its functions are ion and water binding and exchange, control of collagen fibrillogenesis and fiber orientation. May play a role in tooth support, eruptive mechanisms and prevention of PDL mineralization.  Content changes in periodontal disease (dermatan sulphateàchondroitin sulphate).

Cells: PDL consists of heterogenous cell population including:connective tissue cells-fibroblasts (most numerous);  formative cells- cementoblasts, osteoblasts;resorbing cells- osteoclasts and odontoclasts/cementoclasts;  stem cells/precursors; defense cells and epithelial cells (rests of Malassez)

Fibroblasts:The typical fibroblast shows a well-developed rough endoplasmic reticulum, Golgi complex and many mitochondria and secretory vesicles. They synthesize and secrete collagen (and ground substance) as well as degrade collagen (through intracellular collagen vacuoles). Cellular activities of PDL fibroblasts can be modulated by bioactive molecules made by themselves, by local inflammatory cells, or be present within the extracellular matrix of the PDL or bone/cementum. PDL fibroblasts produce numerous growth factors and cytokines such as IGFI, BMPs, PDGF, IL-1, and TGFβ. PDL fibroblasts also release prostaglandins which may influence bone cell activity. They are also rich in alkaline phosphatase, cellular retinoic acid-binding protein, and in receptors to epidermal growth factor.

Cell Kinetics and Cell Phenotype:Cell formation and cell differentiation increases markedly with wounding or after the application of orthodontic loads, while different stimuli may recruit progenitors giving rise to different cell types. It is not clear whether periodontal fibroblasts, cementoblasts, and osteoblasts all arise from a common precursor, or whether each cell type has its own specific precursor cell.

Cementogenesis and Periodontal Regeneration:Cementogenesis is a key component of periodontal regeneration but there are still gaps in our knowledge concerning this process in the normal state. Recent studies have shown that enamel matrix protein (EMP) can be applied to a cleaned root surface and can result in periodontal regeneration. The underlying mechanism of this is unknown though it is postulated that EMP may interact with PL fibroblasts via integrins. Cementum attachment proteins (CAP) may play a role during cementogenesis and during periodontal regeneration.

Nerves:The sensory nerves of the PDL show endings of the Ruffini type that plan an important part in the reflex control of mastication. Sensory nerve endings also release neuropeptides, such as substance P, that can have widespread effects on both blood vessels and cells, though their exact role in PDL biology is yet unknown.

Vessels:Major blood vessels of the PDL lie between the principal fiber bundles, close to the wall of the alveolus. The majority of vessels appear to be postcapillary venules. The presence of fenestrated capillaries are related to the high metabolic requirements of the PDL. The number of fenestrations are not fixed and vary according to stage of eruption.

CONCLUSION/BLBasic knowledge concerning the structure and development of the PDL has relevance in understanding and achieving periodontal regeneration.

Cho 2000                             No Article

B: The periodontal tissues develop as the root forms, mostly arising from the dental follicle that is of neural crest origin. These tissues both develop and function as a unit.

P: Review of the development of periodontal tissues by emphasizing the origin and lineage of the cells responsible for formation of their structural components

D: Review of cell and tissue structure of periodontium using TEM, histochemistry, cytochemistry and radioautography to develop periodontal regeneration techniques.

I – Tooth bud formation

Tooth bud is capable of giving rise to all components of a mature tooth. 2 major components are the dental papilla (odontoblasts and dental pulp) and the dental follicle (cementum, PDL and alveolar bone)

The dental follicle develops as neural crest cells migrate to developing branchial arches then interact with early oral epithelium to form tooth primordial. These ectomesenchymal cells aggregate to form dental papilla and dental follicle.

II – Hertwig’s epithelial root sheath

Double layer (inner and outer enamel epithelial cells): separates dental papilla from dental follicle. It is continuous with the apical rim of the enamel organ, but continuity is lost at onset of root formation

The inner epithelial layer induces odontoblast differentiation. (the developing root gives rise to fibroblasts, preodontoblasts and precementoblasts). It also produces proteins such as bone sialoprotein, osteopontin and amelin along with components of the basement membrane. It has been hypothesized, but not proven, that these secreted elements cause differentiation of cementoblasts (stimulates cementogenesis), but this has not been proven

The outer layer of HERS breaks up at the onset of cementogenesis. More recent studies: possible that some of these epithelial cells undergo mesenchymal transition into fibroblasts (secrete acellular cementum) and cementoblasts (secrete cellular cementum), whereas the rest retain an epithelial phenotype and survive in the PDL as rests of Mallasez (traditional thinking)

III.- Cementum

Avascular mineralized tissue covering the entire root surface. Forms the interface between root dentin and PDL.

2 types: cellular cementum has cementocytes within, acellular does not. Further grouping is based on presence of collagen fibers; intrinsic has collagen fibers formed by cementoblasts and extrinsic has collagen fibers formed from fibroblasts.

Acellular afibrillar cemenum: over cervical enamel at CEJ, major component is glycosaminoglycans, its functional significance is unknown.

Cellular intrinsic fiber cememtum: contains cementocytes embedded in a collagenous matrix of intrinsic collagen fibers. Found in old resorption lacunae and root fracture sites

Cellular mixed stratified cementum: located primarily on the apical one third of the root and in the furcation area of multirooted teeth. It is composed of alternating layers of acellular extrinsic fiber cementum and cellular intrinsic fiber cementum/acellular intrinsic fiber cementum, and is covered by a thin layer of acellular extrinsic fiber cementum for attachment to the periodontal ligament. Serves to reshape the root surface in order to compensate for physiological drift and nonphysiological shifting of teeth in their alveolar sockets.

Acellular extrinsic fiber cementum covers 40% to 70% of the root sur- face and is comprised of collagen fibers and glycosa- minoglycans. It serves the exclusive function of an- choring the root to the periodontal ligament.

Cementogenesis of acellular extrinsic fiber cementum: In humans, cementoblast differentiation and cementogenesis are closely related with root formation. HERS detaches from dentin at the apical edge of the developing root. Fibroblasts of the dental follicle lay down “fringe fibers” which will be incorporated into the layer of acellular extrinsic fiber cementum once mineralization begins. These fringe fibers will become continuous with the principal fibers of the PDL once they develop.

When root development is ~ 2/3 complete, shifts to formation of the cellular varieties. Rapid deposition from multiple sites leads to entrapment of cementoblasts in the matrix as cementocytes.

IV. PDL

Develops prior to tooth eruption at the time of root formation. Perifollicular mesenchymal cells have increased cellular volume and synthetic activity, become elongated, then actively synthesize and deposit collagen fibrils and glycoproteins. These fiber bundles will eventually merge with the fringe fibers and be embedded in bone/cementum as Sharpey’s fibers.

Distinct groups of principle fibers histologically: dentogingival, alveolar crest, transseptal, interradicular, horizontal, oblique and apical fiber bundles.

Mature PDL has 3 distinct regions: 1. bone-related with lot of cells/blood vessels, 2. cementum-related with dense well organized collagen bundles, and 3. middle zone: fewer cells and thinner collagen

Contain undifferentitated stem cells that retain the potential to differentiate into fibroblasts, cementoblasts and osteoblasts (possible that damage to PDL causes differentiation to osteoblasts which then causes ankylosis)

Fibroblasts: most abundant cell in PDL. 1. needed to maintain normal width of PDL, 2. can give rise to osteoblasts and cementoblasts, 3. produce acellular extrinsic fiber cementum in mature PDL, 4. responsible for collagen fiber formation and removal, and 5. express numberous epidermal growth factor receptors

V. Gingiva

Comprised of gingival epithelium and CT. covers the tooth-bearing part of alveolar bone and cervical neck of tooth. Regional morphological variations ( oral GE, oral SE, JE)

At eruption: a thick reduced enamel epithelium overlying the enamel fuses with oral epithelium, transforms, then establishes dentogingival junction (JE directly attached to tooth). 10-20 cells wide at coronal end, 1-2 cells at apical end. In health, the apical aspect of JE is at CEJ.

CT attachment: densely packed collagen bundled anchored to the acellular extrinsic fiber cementum just below terminal part of JE. Stability of CT is key factor in limiting apical migration of JE. Gingival CT fibroblasts secrete collagen matrix organized into fiber bundles

Gingival supra-alveolar fiber apparatus: transseptal, circular, semicircular, transgingival & intergingival fibers: connect/link adjacent teeth in the arch. Secure against rotation and maintain linkage during drift.

VI. Alveolar Bone

Maxilla and mandible have 2 components: alveolar process (houses the tooth roots) and basal body.

Alveolar process consists of thin alveolar bone proper (socket wall), inner and outer cortical plates, and spongy bone in between. Size, shape, location and function of the teeth determines the morphology of alveolar process.

Begins formation during late bell stage of development, separating individual tooth germs. Changes occur as the root forms: osteoblasts differentiate from dental follicle and form alveolar bone proper.

Major function is to anchor roots of teeth and to absorb/distribute occlusal pressures. This is achieved by insertion of Sharpey’s fibers into alveolar bone proper.

Cohn 1975                             Article

BG: It has been established that numerous cemento-alveolar fibers in the mouse, marmoset, and macaque monkeys do not terminate in bone but pass without interruption through the wall of the alveolus. These unusual fibers were termed transalveolar fibers.

P:To examine the transalveolar fibers (the cemento-alveolar fibers that traverse the entire thickness of the alveolus) in humans.

M+M:Jaws of 16 adult cadavers and a few fresh specimens were fixed and cut into blocks of 2 adjacent teeth. Blocks then cut into 10um sections in a mesio-distal plane and stained by a modified Mallory (trichrome stain using aniline blue, acid fuchsin, and orange G reveals collagen of CT) method.

R:Many cemento-alveolar fibers traversed the entire thickness of the alveolus instead of being anchored in bone as conventional Sharpey’s fibers. Observed only in regions of lamellar bone, lacking Haversian systems. The fibers could join roots of adjacent teeth, the roots of the same tooth, the periosteum of the alveolar process, or to the lamina propria on gingival surfaces.

BL:The author proposes orientation and distribution of transalveolar fibers likely represents a functional adaptation to occlusal and muscular forces, permitting maximum support of the tooth in its alveolus.

CR:How many is many? This study does not specify.

Selliseth 1994                             No Article

P: To examine the 3-dimensional architecture of the microvascular system of the rat periodontal ligament (PDL).

M&M: 6 rats had liquid acrylic resin perfused through each of carotid arteries. Vascular corrosion casts were prepared and examined by SEM.

D: The results show that the microvasculature forms a highly organized system presumably related to the specialized functions of the periodontium. Cervically, arterioles and venules communicated with the profuse capillary network of the gingiva. The mid-root segment of the PDL contained arterioles and venules that coursed occluso-apically near the alveolar wall, as well as capillary loops located closer to the surface. Arterioles entered PDL through vascular canals from the bone marrow when proceeded coronally and branched into an interconnected capillary network. The capillaries formed hairpin loops pointing coronally. At the apical portion, capillary loops were larger in diameter, coursed apically, and anastomosed freely until entering a venule.

BL: Cervically, a dense capillary system may be required for antimicrobial defenses and rapid tissue turnover. Mid root vasculature supports the suspensory structures, while the apical region has a venous cap designed maybe for cushioning of masticatory forces. The large vessel diameter combined with an irregular lumina surface at the tip of capillary loops indicates reduced blood velocity and turbulence in the functional part of the PDL vasculature where exchange of metabolites mainly occur.

What are the dimensions of the PDL? Do these change with age and/or function?

Oehmke 2004                             Article

Purpose: To analyze the normal age-dependent changes and regional differences of the collagen renewal rate of the PDL in rats.

Materials and methods: Nine male rats were used and divided in 3 groups: Group A: 1 month old, Group B: 8 months old and Group C: 18 months old. Animals received an injection of 3H-proline for he labeling of newly formed collagen and were killed 8 hours after that. Parts from the mandible, muscles and blood were sampled. The mesial roof o the 1^stand 2^nd lower right molars of each animal were evaluated. Autoradiography was used to visualize the activity of fibroblasts as collagen-forming cells.

Results: Autoradiograms demonstrated age-related alterations and regional differences of the collagen renewal rate of the PDL. In the cervical third of the PDL of Group A, the density of silver grains (labeled molecules) was significantly higher than in the adjacent bone or dentine. In 8-month-old rats a lower density of silver grains was observed and was further diminished at 18-month-old specimen. In the middle root third, there was a marked decrease in the number of silver grains with increasing age and an irregular shaped PDL comparing to the cervical third. In the apical the situation in younger rats is similar to that in the cervical third. The density of silver grains was slightly reduced in the 8-month-old animals but was stull markedly higher in the apical zone. IN the 18-month-old rats there were even fewer silver grains. The labeling was reduced with age, it was always lowest in the middle root third and highest in the apical third, with the values of the cervical third in between.

Conclusion:In all age groups, the formation of collagen occurred mainly in the apical and cervical root thirds, presumably subject to functional demand.

McCulloch 2000                             Article

P: Review of how cytoskeletal proteins mediate protective responses to applied force which may enable the cells to survive in a mechanically active environment.

PDL: bundles of fibers arranged in a meshlike net stretching between the cementum and bone. It is the only ligament to span two distinct hard tissues (cementum and bone). It has a dynamic relationship to external forces, with specific metabolic requirements and architectural tissue design.

Maintenance and remodeling of collagen as well as calcification of the extremities to form Sharpey’s fibers require numerous cell types with multiple signaling mechanisms. The PDL fibroblast appears to be responsible for formation and remodeling of PDL fibers.

PDL fibroblast dispersed throughout the ligament, generally organized with their long axis parallel to the direction of the collagen fiber. There appear to be multiple organized contact points for cell signaling cascades to occur quickly in response to external stimuli. Other cells existing within the PDL including endothelial cells, epithelial rests of Malassez, sensory cells, osteogenic and osteoclastic cells and cementoblasts.

The ECM of the PDL appears to have a much higher turnover rate than other dental tissues. It is possible that only certain portions of the fibrils are broken down, and not the entire fiber, in response to certain stresses. This is important as portions of the fibers are embedded into bone which is remodeled under different circumstances than cementum.

B/c the PDL maintains a generally constant width throughout life, several mechanisms must maintain homeostasis. Cytokines and growth factors are important for acting locally, and there are several signaling signals in place to respond to mechanical forces in order to maintain width. The PDL secretes and expresses proteins for regulating PDL growth, but also appears to be able to influence bone metabolism as well.

If PDL cells are removed from the root or disturbed by medications, bone generally grows into the PDL space and ankylosis occurs.

PDL and alveolar bone cells are exposed to physical forces in vivo in response to mastication, parafunction, speech and ortho movement. The actual process of remodeling is, at this time, poorly understood. It also relatively unknown how force is specifically transferred from the ligament into the bone. Models being studied generally revolve around the stress-strain relationship.

BL: The fibroblasts and osteoblasts within the PDL have the necessary signaling and effector mechanisms to both sense forces and produce an applied response to maintain the PDL width and cell viability.

Coolidge 1937                             Article

P: To examine the thickness of the human periodontal ligament at different ages and in teeth showing different types of occlusion.

M&M:1145 measurements from 172 teeth of 15 human jaws were used. PDL thickness was measured at the alveolar crest, at mid-root, at the apex of the tooth, and at the bifurcation of multi-rooted teeth. Measurements were made at M/D, B/L, or all 4 surfaces. Plaster casts of the models were mounted on articulators to reproduce occlusal relationships and determine the amount of function: if there were fewer teeth in the arch they were labeled as heavy function while teeth w/o antagonists were labeled as no function. Unerupted or embedded teeth were labeled as a separate group.

R: The thickness of the PDL decreased with age (except around teeth in heavy function). PDL thickness generally increases with function, but can vary with the type of stress. PDL was thinner on the pressure side of drifting teeth, and thicker on the tension side. Drifting and malaposed teeth were found to have a relatively thick periodontal membrane. The following are averages of the findings:

11 – 16 years: 0.21mm heavy function: 0.18mm

32 – 50 years: 0.18mm no function: 0.13mm

51 – 67 years: 0.15mm embedded teeth: 0.08 mm

Malposed/drifted 0.19 mm

BL: The thickness of the PDL is a variable amount and is affected by age, intensity of functional forces, and the tooth position (drifting, malposition). Avg thickness is 0.10-0.20 mm.

For all periodontal tissues, what are the various types of collagen and where they are found? How are these arranged and what are their functions?

Chavrier 1984                             Article

BG: Type I and III collagen are the main collagenous components of the healthy human gingival connective tissue (99% of the total extractable collagen) with a predominance of type III collagen in the gingival papillae underlying gingival basement membrane, and around the blood vessel walls, while Type IV collagen was the main collagenous component of basement membrane (accounts for less than 1%.)

P: To examine the morphological pattern of organization of the gingival CT and its collagen

components, by using the indirect immunoperoxidase labeling procedure.

M&M: 7 healthy dental students had 6 mm³ of attached gingiva biopsied. 2 mm³ sample block

were subsequently cut and divided into two groups: a) Standard EM, and b) Indirect

Immunolabeling, c) controls.

R: Standard EM – 2 Patterns observed:

Dense tissue (Predominant), with large dense bundles of long, thick, striated collagen fibers (60-70nm). Often in close contact with mature fibroblasts

Loose CT, underlying gingival basement membrane or blood vessels walls. Short thin (40-60 nm) striated collagen fibers, mixed with non-striated material (mast and plasma cells).

Immunoperoxide labeling – 2 patterns observed:

Type I collagen arranged in thick bundles (60-70nm), with 64nm space between the fibers.

Mixed I and III collagen, with type III, to be the predominant and organized either in fibrous (short, thin, striated fibers) or fibrillar (wide-spread, thread-like material) form. Type IV, was limited to the lamina densa of the basement membrane.

D: Healthy gingival CT has heterogenicity of collagen that can be divided into 2 types of

organization and composition. One type of collagen predominates in each of the 2 patterns of

gingival CT. Fibroblasts in 1 region produce more of its respective collagen. Type 1 = stability

and Type III = early regeneration

BL: Collagen types are reflected in the tissue’s function. Immuno-typing is an advance in the attempt to distinguish different patterns of organization in healthy gingival connective tissue.

Page and Ammons 1974                             Article

P: To study the normal biologic properties of CT turnover in healthy tissues.

M&M:  12 marmosets were used in this study.  Each animal was given (U)¹⁴C-L-proline on 2 consecutive days.  24 hours after the second dose the animals were killed and blood and tissue was collected.  Several CT (tendons, skin, gingiva, palate) expected to exhibit varying rates of collagen turnover, were selected for analysis and comparison with the gingiva. Any areas that displayed gingival inflammation were excised and not included.   The extent of incorporation of  (U)¹⁴C-L-proline into collagen hydroxyproline and the subsequent loss of the label from the tissue was evaluated over 17 weeks.

R: Initially there was a very high incorporation of proline and conversion into hydroxyproline in all of the tissues.  During subsequent periods, significant numbers of counts remained only in the gingiva, but after 17 weeks activity began to approach the values seen in other CT.

D: The rate of conversion of ¹⁴C-proline into ¹⁴C-L-hydroxyproline by CT reflects the rate of collagen production, and the rate of loss of the labeled hydroxyproline from the tissue indicates the levels of collagen degradation. The gingiva differs from other CT in that a far greater portion of the newly synthesized molecules is required for incorporation into the insoluble collagen. The data shows that the turnover rate of mature insoluble collagen in normal gingiva is rapid (5X) when compared to other connective tissues.

In inflammation, the observed net loss of collagen may result from interference with collagen production and turnover, rather than from destruction of previously existing collagen.

Zwarych 1965                             Article

PURPOSE: To determine if an intermediate plexus exists in the mammalian periodontal ligament in teeth of limited growth.

METHODS: 8 adult white mice were sacrificed and decapitated.  Heads were fixed, bisected sagitally, and embedded in paraffin and sectioned so that mesio, distal, occlusal, and oblique plane sections could be studied histologically.

RESULTS: The sections of mouse molars support the concept of continuity of the principal fibers across the periodontal space.  No “intermediate plexus” was found.  Shortly after passing from the alveolar septum, the closely packed fibers became more loosely arranged and the individual fibers took separate courses joining a few adjacent fibers to become attached to the cementum.  A greater number of bundles containing fewer fibers were attached to the cementum in contrast to a lesser number of bundles containing more fibers attached to the alveolar septum. 

DISCUSSION:  The evidence indicates what the PDL fibers are continuous across the periodontal space.  More numerous bundles with fewer fibers are attached to cementum while fewer bundles with more fibers are attached to the alveolar bone of the socket, allowing displacement forces applied to a given area of cementum to be transmitted to a greater area of the alveolar septum.

CONCLUSION/BLThere is no “intermediate plexus” in mice molar teeth with limited eruption. Principle fibers are continuous from the cementum to alveolar bone, leading to enhanced compressibility and strength of the periodontal ligament in normal tooth movement.

What cell type(s) is(are) responsible for collagen production in the periodontal tissues? What are some of the characteristics of these cells?

Giannopoulou 1996                             Article

P:To study and compare the functional characteristics of gingival and PDL fibroblasts.

M&M:Gingival and PDL fibroblasts (GF & PDLF) from 5 healthy Caucasian males 25-30 years old were isolated and compared in vitro. Patients were undergoing extractions for orthodontic reasons. The CT cells were taken from PDL of premolar teeth and adjacent healthy gingiva or interdental papilla. The cells were prepared and observed under SEM. The effect of extracellular matrix components (ECM) on attachment, proliferation and protein synthesis were examined. The agents used were: collagen type I, IV, gelatin, fibronectin, laminin and vitronectin. Muscle differentiation markers and the effects of epithelial cells were also examined.

R:GF and PDLF appeared similar under SEM. They appeared rounded, with a spherical nucleus in the center and typical prolongations. Generally, in primary cultures, the proliferation of gingival fibroblasts was faster than that of PDL fibroblasts but the differences were not SS. All ECM components enhanced attachment; however, while collagen types I and IV were more effective in promoting the attachment of GF, gelatin, laminin, and vitronectin promoted attachment of PDLF. Both cell types demonstrated same degree of enhanced attachment with fibronectin. Most ECM components increased the proliferation rate of GF and the biosynthetic activity (protein synthesis) of PDLF.The biochemical markers were similarly distributed between the 2 cell types, except for alkaline phosphatase, which was detected only in the cellular extract of PDLF. Both GF & PDLF strongly expressed alpha-smooth-muscle actin, but only PDLF were positive for smooth-muscle myosin. Epithelial cells significantly stimulated the proliferation of both GF and PDLF but had no effect on their biosynthetic activity.

BL:This in vitro investigation confirmed that GF and PDLF have a similar morphology, but physiological and chemical differences may better explain their in vivo functional differences.

Mariotti 1990                             Article

P:To determine the characteristics of fibroblasts derived from human PDL and gingiva.

M+M: PDL fibroblasts (PDLF) were isolated from impacted 3^rdmolars (21-35year old) healthy adults. Human gingival fibroblast (hGF) were isolated from interproximal papilla of premolar or molar gingiva. PDLF and hGF were incubated then replanted on 500,000-cells/100mm-culture dish. Fibroblasts were used between the 3^rd and 5^th passages only.

R:

GF grew faster than PDLF, in a 500,000 cells / 100 mm culture; a total confluence occurred in 4 days for GF and 6 days for PDLF.

DNA content of growing cells was greater in GF than PDLF.

Total protein content in GF was slightly greater than PDLF at day 7 but NSSD.

Greater trend on non-collagen protein synthesis in GF, and more collagen synthesis in PDLF.

GF had greater amounts of hyaluronic acid and heparin and lesser amounts of chondroitin sulfates A and C.

The growth characteristics of PDLF and GF was similar but did exhibit specific differences in proliferate rates and macromolecular synthesis.

D: An explanation for GF cells reaching confluence earlier than PDLF may be because of the gingival cells being larger than PDLF cells. This is supported by the findings that the DNA and protein content of GF cultures are initially greater than PDLF.

BL: GF cells reached total confluence faster than PDLF cells and had different productions of macromolecules.

Palaiologou 2001                             Article

B: Fibroblasts are the main cell of the periodontal ligament and gingiva and play important roles in function/regeneration of periodontal tissues. Glycoproteins are important for cell-cell and cell-matrix interactions. Fibronectin is the main glycoprotein in connective tissue and serves to orient fibroblasts to collagen and provide protein attachment for cell-matrix adhesions linking collagen and fibrin to the cell surface and underlying actin cytoskeleton. Fibronectin contains Arg-Gly-Asp (RGD) sequence that is part of the cell binding site and plays a crutial role in migration of fibroblasts and maintaining structural integrity of connective tissue.

P: To evaluate any differences in binding of fibroblasts (human gingival (GF), periodontal ligament (PDLF, and dermal (DF))to various ECM proteins (fibronectin, laminin, vitronectin, other peptides) and collagen (Type I and IV).  Differences in integrin expression was also looked at for the different types of fibroblasts.

M&M: GF were taken from a 13-year-old boy that was systemically healthy and underwent surgery for hereditary gingival hyperplasia. Dermal fibroblasts were ordered and came from a 12 week old female embryo. Stock cultures of polyclonal human periodontal ligament fibroblasts were used. ECM proteins were commercially obtained. Cells were allowed to adhere to the substratum for 45 min to 2 hours. Non adherent cells were removed by the 3^rd wash of PBS and lysed by freezing. ECM proteins were prepared according to manufacturer’s instructions and added to each sample and incubated for 30 min. Adherent cells were quantified fluorometrically using a fluorescent die. Guanidine thiocyanate was used to extract the RNA from the 3 cell types. RT-PCR was used to assess transcript expression. The primers used, specific for the integrin subunits to quantify ECM receptor transcript expression, were derived from the published DNA sequence for human integrins.

R: GF and PDLF adhered to vitronectin and collagen type 1 and IV more than DF. PDLF adhered more to laminin than, whereas GF and DF did not. All adhered well to fibronectin and RGD peptide. There were found to be innate differences between the fibroblast types and the integrin transcripts they expressed.

BL: GF and PDLF are more similar in the ECM proteins that they adhere to and the integrins they express when compared to DF. Moving forward, experiments using dermal fibroblasts would not necessarily be useful in studies looking at oral tissues.

Cr: Could have tested several fibroblasts from other patients in addition to the 13 y/o boy with hereditary gingival hyperplasia to confirm results.

Kramer 2004                             Article                   mesenchymal stem cells

Purpose: To determine if mesenchymal cells differentiate into a specific cell type.

Materials and methods: Samples of extracted teeth were obtained from female subjects needing extractions. Proliferative cells having various morphologies were produced from the section in culture between 7 and 10 days. Cell with PDL morphology were diluted and cultured and processed for immunohistochemistry. Human male mesenchymal cells were obtained and cultured according to the manufacturer’s directions. Mesenchymal stem cells were mixed with periodontal cells isolated from the tooth explants at rations of 1:1, 2:1 and 10:1. Co-cultures of cells were isolated after 0, 3, 7, 14 and 21 days and processed for immunohistochemistry and in situ hybridization.

Results: Collagen III staining was restricted to PDL cells and not in the cementum and dentin layers. Osteopontin was present in the PDL, osteocalcin was heterogeneous in the PDL and observed in the bone, dentin and cementum and BMP-2/4 could be detected in the PDL, cementum and bone tissue. PDL did not stain for bone sialoprotein. These indicate that PDL can be differentiated from other periodontal tissues within the explant. The staining pattern for mesenchymal cells was different form that seen for PDL and have different morphology. Co-culture for 7 days led to an overall change in the mesenchymal stem cells structure, to a more fibroblast – like morphology. Osteocalcin and osteopontin expression was up-regulated in mesenchymal stem cells following co-culture for 7 and 21 days and expression of bone sialoprotein was reduced.

Conclusion: Data demonstrate mesenchymal stem cells’ potency to develop periodontal ligament characteristics and suggest that the cells may have the potential to form other periodontal tissues.

McCulloch 1983                             Article

Purpose: First to confirm that cells in a normally functioning PDL do migrate; second to analyze the rate at which the cells cycle; third to analyze the relationship between proliferating cells within the PDL in order to assess further their capacity for migration and for clonal proliferation within the PDL.

Materials and methods:

• 135 mice were injected ³H-Tdr dilated with PBS then sacrificed at 1hr, 1 day, 3days, 7days, 14days, 60days and three control mice were injected with PBS.

• Then mice mandibles were block sectioned in the molar area and analysis of radioautographs using labeling index and grain counts.

Results:

• Analysis of radioautographs using labeling index and grain counts demonstrated that the majority of labeled cells divided within 3days, but a measurable population of cells had not divided after 14days.

• The labeling index of cells adjacent to alveolar bone increased 8 times within 1 day, indicating that labeled cells had migrated to the bone surface.

• Migration of cells to the vicinity of the cementum was observed 3 days after labeling.

• The percentage of labeled cells located within 20 m of one another increase to 40% within 3 days, suggesting clonal proliferation of PDL cells.

BL: Cells of PDL migrate under physiological conditions. This conclusion is supported by first, the evidence that progeny of labeled cells can migrate from their paravascular location; second, that members of clones migrate from the site of their birth; and third, that cells migrate to the surfaces of bone and cementum.

Lallier 2007                             Article

P:To investigate the motility of the cells of the periodontium, since this may influence their ability to aid in tissue regeneration. Moreover, to determine whether different ECM proteins (collagen I, collagen III, collagen V,fibronectin, and laminin) can be used to promote differential cell (PDL and HGF and osteoblasts) motility.

M&M:Periodontal and gingival fibroblasts were established from patients who had healthy gingiva but who underwent oral surgery at the Louisiana State University School of Dentistry for the purpose of removing impacted wisdom teeth. Cell lines of pre-osteoblasts (ATCC-CRL-11372) were obtained from the American Type Culture Collection. Mature osteoblasts were obtained by growing pre-osteoblasts at 37°C for 5 days prior to use in subsequent assays. Cell motility assay, cell movement evaluation, cell adhesion assay and cell proliferation assay were carried out for different cell types and different ECM proteins. ELIZA was done to detect integrin β1, α1, and α2 subunit expression, using different Integrin subunit antibodies.

R:Gingival fibroblasts are twice as motile as PDL fibroblasts, whereas osteoblasts are essentially non-motile. Collagens promote the greatest motility of gingival fibroblasts in the following order: collagen III>collagen V>collagen I. Differences in motility do not correlate with cell proliferation or integrin expression. Osteoblasts display greater attachment to collagens than does either fibroblast population, but lower motility. Gingival fibroblast motility on collagen I is generally mediated by α2 integrins, whereas motility on collagen III involves α1 integrins.

BL:ECM proteins, differentially promote the cell motility of periodontal cells. Because of their greater motility, gingival fibroblasts have more of a potential to invade periodontal wound sites, which may explain the formation of disorganized connective tissue masses rather than the occurrence of the true regeneration of the periodontium.

What changes occur in collagen in periodontal disease? How is collagen degraded? What cells and agents are involved?

Chavrier 1987                             Article

P: To study the distribution, ultrastructure and organization of type I, III, IV collagen in fibrotic gingival CT of pts with long standing cases of chronic periodontitis.

M&M: 5 pts with progressive, long standing periodontitis provided tissue samples that were evaluated by immunofluorescence (IF), Standard electrom microscope (SEM) , and immunoperoxidase electron labeling (IPEL).

R: IF: The diseased CT was made up of both type I and type III collagen. Type I collagen was strongly fluorescent and appeared to be the main gingival collagenous comp