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The role of fibroblasts in the remodeling of periodontal ligament during physiologic tooth movement
The role of fibroblasts in the remodeling of periodontal ligament during physiologic tooth movement A. R. Ten Cate, B.D.S., B.Sc., Ph.D.,* and E. Freeman, D.D.S., M.Sc.D.
Toronto,
Ontario,
D. A. Deporter,
D.D.S.,
Ph.D.,
Cnnndn
1t is probably
fair comment to state that, to most orthodontists, tooth movement is usually considered as its translocation through bone and it is on this biologic basis that, until recently, most orthodontic therapy has been c0nceived.l It is easy to understand why this should be so. The resorption and deposition of bone-in other words, bony remodeling-can be readily demonstrated in tissue sections by conventional histologic methods and with the aid of various bone-seeking labels such as the tetracyclines. Thus, the responses of bone during tooth movement have been fairly easy to analyze. On the other hand, the remodeling of the soft connective tissue intervening between bone and tooth cannot be demonstrated by conventional histology and this phenomenon is, therefore, not properly understood. To explain how the periodontal ligament is remodeled during physiologic tooth movement, especially during tooth eruption, Siche? introduced the concept of an intermediate plexus within the ligament. In this plexus the dissolution and reformation of collagen fibers supposedly occurred, thereby permitting tooth movement. Although the concept of an intermediate plexus is attractive to explain movements such as tooth eruption and tooth rotation, it is not adequate to explain how the ligament remodels during movement of the tooth in, for example, a mesial or distal direction. Furthermore, there is considerable debate as to whether the intermediate plexus exists at all. Edwards3 has recently reviewed the literature on the intermediate plexus and concluded that there is no such structure. Instead, he offered From the Toronto.
Division
This study was
of
Biological
Sciences,
Faculty
of
Dentistry,
University
of
supported by the Medical Research Council of Canada.
*1975 Milo Hellman
Research Award
of the American
Association
of Orthodontists.
155
the following alternative mechanism to explain how remodeling of the pcriotion tal ligament might occur in response to tooth movement. He proposed ( 1) that progressive osteogenic activity (and cementogenic act,irity to a far lesser degree 1 played an active role in the shortening of extended fibers and in the rcattadment of new fibers developed during tooth movement ; (2) that the stretching of the wavy collagen fibers and reorientation of their directional morphology coultl permit a certain amount of tooth movement; and (3) that the existence of a type of intermediate plexus might allow an elongation of fiber bundles by “slippage” of the fibers over one anot,hcr and subsequent reorientation of the fibers in a new position. Edwards also made the point that the final return of the slozo-nzetnbol~zi?zn connective tissue fibers to their original and stable relationship to the tooth and each other depended on the remodeling of osseous tissue. Unfortunately, these alternate suggestions explaining how the ligament remodels are also largely speculative. A real problem is that collagen is generally considered to be a very stable protein and any explanation of ligament remodeling has to accommodate this supposed fact; hence such terms as “slippage” and “stretching.” But is collagen a stable protein? Certainly some collagen in the body, for example in tendon, is relatively inert. On the other hand, experiments using radioactive proline indicate that there is a high rate of collagen turnover in the periodontal ligament, probably higher than anywhere else in the body.’ Given the possibility that thcrc is a rapid rate of’ collagen remodeling in the periodontal ligament, a further problem is to explain how the tlissolution and synthesis of collagen takes p1ac.e in orderly and cont,rolled fashion during physiologic tooth movement. With bone there is no problem in explaining how it is remodeled. The occurrence of two cell types, one producing bone (the osteoblast) and the other destroying bone (the osteoclast), and acting in concert, is well documented and understood. There would also be no problem in explaining how connective tissue remodeling occurs if ttio cell types, one synthesizing collagen and the other degrading it, were recognized. The work coming from this laborator> +I” has shown that there is indeed a cellular mechanism involved in collagen remodeling and that, unlike the bone analogy where two distinct cell types are involved, this is achieved by a single cell, the fibroblast-in other words, that the fibroblast is capable of both synthesizing and degrading collagen at any one time. The purpose of this article is to review some of the work from this laboratory which has led to the concept of the fibroblast as a cell capable of synthesizing and tlegrading collagen, to introduce some new information in support of this concept, and to discuss tooth movement, be it physiologic or therapeutic, in the light of this concept. The
macrophage
As there is a parallel between macrophage function and fibroblast function, it is convenient to begin by giving a description of the events that occur when a macrophage ingests foreign material because this is a well-understood (and therefore undisputed) physiologic phenomenon. The sequence of events
Volunle 69 Number 2
Role of fibroblasts
Fig. 1. Diagram grades In B the of
foreign
lysosomes
foreign
illustrating
a foreign
body
body with takes
the
sequence
of
In A pseudopodia
body.
is now
the
intracellular
phagosome
to
events
of the and form
as
PDL
the
macrophage
macrophage
surround
is located a
in
within
phagolysosome.
remodeli?tg
ingests the
foreign
a phagosome
after
In C degradation
157
and
debody.
fusion of
the
place.
which occur during and after phagocytosis is illustrated in diagrammatic form in Fig. 1. The material to be ingested is engulfed by pseudopodal processes of the cell so that it eventually comes to lie within it in a vesicle lined by cell membrane. This vesicular structure is termed the phagosome. The Golgi apparatus of the macrophage then synthesizes lysosomes, vesicles which contain a whole battery of catabolic enzymes. The lpsosomes fuse with the phagosome, exposing its ingested material to the enzymes, and it is in this structure, now termed the phagolysosome, that the final degradation of the ingested material occurs. The phagolysomes then disappear or persist as electron-dense residual bodies. Furthermore, it has been clearly demonstrated that the macrophage can, on occasions, ingest and degrade collagen. The classical situation where this occurs is in uterine involution after pregnancy. Here macrophages achieve the rapid removal of collagen from the uterine wall by ingesting collagen and degrading it intracellularly. The question is, can macrophages act in a similar manner to bring about remodeling of the periodontal ligament? Unfortunately, when the periodontal ligament is examined with the electron microscope, no macrophages can be demonstrated within its cell population. The bulk of the cells in the normal functioning periodontal ligament are fibroblasts and this, therefore, prompts the question whether or not the fibroblasts may have a phagocytic function similar to that of the macrophage.
Fig. 2. Electron
micrograph
dioxide
intravenously
dense some
injected particules)
has
been
of
part
of
8 hours taken
up
a fibroblast previously. !by the
which The
fibroblast
has
thorium and
been dioxide
sequestered
exposed
to
(seen in
as a
thorium electron-
phagolyso-
(arrowed).
Evidence indicating
that the fibroblast
may act as a macrophage
The standard way of demonstrating the occurrence of phagocytosis at the fine structural level is to inject into the vascular system of an experimental animal an electron-dense material, such as thorium dioxide. This marker rapidly leaves the vascular system and enters the extracellular compartment, where it is usually picked up by scavenger cells in the immediate vicinity. Because thorium dioxide is electron dense, its location can be readily discerned with the electron microscope. Fig. 2 illustrates a fibroblast from such an experimental animal and shows the accumulation of thorium dioxide within a phagolysosome: clear evidence that phagocytosis has occurred. Thus there can be no doubt that the fibroblast is capable of ingesting foreign material. The key question, however, is whether or not the fibroblast can ingest its own product, collagen. Evidence indicating
that the fibroblast
can ingest and degrade
collagen
When the fine structure of the periodontal ligament is examined it is a fairly easy matter to assemble a series. of pictures which appear to indicate the phagocytosis of collagen by ligament fibroblasts (Fig. 3), an assumption strengthened by the demonstration of acid phosphatase activity within the collagen-containing vesicles or phagolysosomes. Convincing as this pictorial evidence may seem, however, unequivocal proof of phagocptosis is still required,
Fig.
3. A series
demonstrates illustrates the
collagen
development
which is also
of the
banded present
pictures actual within
indicating
the
phagocytosis
of
a phugosome
of a phagolysosome collagen fibrils in this cell.
are
phagocytosis collagen and
this
(C in Fig. still
discernible.
ot collagen and
corresponds l),
and A
by
corresponds
the to
A
to B in Fig.
3, d illustrates phagolysosome
fibroblast. in
Fig.
3, a 1. 3,
b
1. 3, c illustrates residual
containing
bodies collagen
in
for it is just possible that the morphologic appearance of collagen partly within and partly without the fibroblast, as seen in Fig. 3, n, could bc caused b) plane of section. Also Fig. 3, b and c, could be interpreted as indicating an overproduction of collagen and its subsequent degradation without the collagen ever leaving the fibroblast. We have attempted to provide unequivocal evidence two ways and our results are reported hcrr in detail for the first time. ElsewherelO we have suggested the following equation : synthesis amino acids , ’ collagen degradation so that if the rates of synthesis and degradation are equal the amount of collagen should remain steady. Now, if this equation holds good for the rapidly remodeling collagen in the periodontal ligament, it should be possible to predict an over-all loss of extracellular collagen if its synthesis is arrested without interfering with the fibroblasts’ phagocytic function. There are two ways whereby the synthesis of collagen by the fibroblast can be halted: by depriving the cell either of vitamin C or of the basic amino acids required to make collagen. Vitamin C (ascorbic acid) has a specific action on a particular sequence in the several steps of collagen synthesis. In the assembly of the collagen molecule the amino acid prolinc is first incorporated, with other amino acids, at a ribosomal site. The collagen precursor molecule formed here is then transferred from the ribosomes to the cisternae of the rough endoplasmic reticulum. On the unit membrane of the rough endoplasmic reticulum the proline is hydroxylated to hydroxyproline and this step involves enzyme activity dependent upon ascorbic acid. Thus, in the absence of the acid, enzyme activity ceases and hydroxylation of the prolinc cannot occur. As hydroxyproline is essential for the stability of the classical triple helical structure of collagen, collagen does not form and instead precursor material accumulates within the cisternae of the rough endoplasmic ret,iculum. This is seen at the fine structural level as a dilation and rounding of the profiles of rough endoplasmic reticulum as collagen precursors accumulate within them. Protein deficiency halts collagen synthesis” by withholding the basic building materials for collagen from the fibroblast. Thus, if periodontal ligament remodeling involves phagocptosis of collagen, a loss of extracellular collagen should he expect.cd in both scorbutic and protein deficient animals. Fig. 1 illustrates a scorbutic fibroblast and its cytoplasm is characterized by the classic dilated cistcrnae of rough endoplasmic reticulum. It can be inferred, therefore, that collagen synthesis by this fibroblast has ceased. Another feature of this cell is the presence of many intracellular collagen profiles, which indicates a continuance of collagen degradation, and this is confirmed by the lesser amounts of extracellular collagen. An almost similar picture is seen in the periodontal ligament of the protein-deficient animal. There is again a marked loss of extracellular collagen (Fig. 5) and an accumulation of banded collagen within the fibroblasts. The only difference is that in protein-deficient fibroblast,s no dilation of the cisternae of the rough rndoplasmic reticulum occurs as collagen synthesis has never begun.
Fig. rough Fig.
4. A scorbutic
5. A fibroblast
protein-deficient of
fibroblast
(guinea
reticulum
ted.
endoplasmic
intracellular
endoplasmic
from diet. fibers.
reticulum
the
There
pig].
Note
periodontal is a loss
This
also
cell
the
ligament
is characterized
of a mouse
of extracellular
As there
is no accumulation
cisternae
are
not dilated
collagen of
in this
dilated
maintained fibrils
intermediary cell.
by
of collagen
accumulation
and collagen
profiles
within
for 4 days an
of
this cell. on a
accumulation products,
the
The other may we have attempted to dcmonst rate the unequivocal occ~~rrer~cc of collagen phagocptosis by the fibroblast is with the use of elccAtron-dense markers such as thorium dioxide. The rationale is that if fibroblasts active]) ingest extracellular collagen they map well also incorporate cxtrancous material lying in the extracellular compartment. Fig. 6 illustrates a coincidental uptake of marker and collagen by the fibroblast and this further substantiates our claim that this cell can ingest its own product, collagen. Evidence
indicating
that
the fibroblast
synthesizes
and
degrades
collagen
simultaneously
The findings we have presented so far may be interpreted in different ways. It is possible that, in the population of fibroblasts which constitute the periodontal ligament, some are involved in collagen synthesis while others are involved in collagen degradation. lt could also be that, our findings indicate a modulation of the fibroblast to a fibroclast. Finally, it could bc that t,he cells of the ligament are capable of synthesizing and degrading collagen simultaneously. The evidence indicates that the last possibility is the correct one. Not only do the protein and ascorbic acid deficiency experiments indicate that both processes are occurring simultaneously (for example, the dilation of the cisternac of the rough endoplasmic reticulum in the scorbutie fihroblast indicates its synthetic activity) but there is also strong supportive cvidencc~ from autoradiographp studies at the fine structural level. Weinstock (personal communication) has been able to show that collagen-containing vesicles in ligament fihrohlasts are not labeled with “II-prolinc 30 minutes after injection of the isotope but that the cell organelles associated sequentially with the pathway of collagen synthesis are. Not only does this finding indicate that the collagen-containing vesicles are not associated with collagen synthesis but also that both processes (synthesis and degradation) are occurring at the same time. Evidence
indicating
the widespread
occurrence
of fibroblastic
activity
It is worth noting that although our work began using the periodontal ligament as a model system, it has now been extended by ourselves8 and others12-14 to show that the ability of the fibroblast to degrade collagen is a widespread occurrence and is not confined to the cells of the ligament. Thus fibroblastic activity has now been described in man (periodontal ligament), monkey (gingiva), chick (embryonic skin), mouse (ligament and skin), rat (ligament and skin), guinea pig (ligament and skin), and rabbit (ligament and skin), and in such varying locations as in dermal wound repair,8 bone lesions,‘” and fracture repair, I5 tendon development,12 experimental arthritic some pathologic 1esions.l’ Also we have just begun to examine remodeling ill cranial sutures and here also collagen phagocytosis by fibroblasts has been identified (Fig. 7). Our experiments on repair of skin wound9 indicates that fibroblasts not normally degrading collagen map he “switched on” to assume this function bp some change in the local environment. This may he significant as it suggests the possibility of control.
Volume Number
69 2
Fig.
Electron
micrograph
injected
intravenously
6.
dioxide are
localized
Fig. readily
7.
Role
of
in a phagosome
Fibroblasts demonstrated
in
the
part 8 hours
of
(arrows)
of
within
fibroblasts
in
which
has
a fibroblast previously.
coincident suture
of
with the cells.
The
a collagen
3.day-old
thorium
PDL
been dioxide
renzodelkg
exposed particles
to
163
thorium (arrowed)
fibril.
mouse.
Intracellular
collagen
can
be
164
Ten Cnte, Deporter,
Quantitation
and
distribution
and Freemnv within
the
Ant. J. Orthod. Pebrzcary 197F
ligament
Before discussing the application of our findings to therapeutic tooth movement two further points need to be introduced : (1) the quantitation of ligament fibroblasts degrading collagen and (2) their distribution. Quantitation is cxtremely difficult at the electron-microscopic level. It must be realized that in sections prepared for electron microscopy only an extremely small field may be examined-indeed, only an extremely small part of any one cell can be examined-at any one time. At present we can only suggest that many cells in the periodontal ligament of the mouse first molar are involved in collagen remodeling. This suggestion is based on the fart that, in the thousands of sections that have been examined, it is always easy to demonst,ratc intracellular collagen within the fibroblast. If collagen phagocytosis were an unusual phenomenon, the sampling restrictions imposed by the techniques of electronmicroscopy would restrict its ready demonstration. Even so, the fact remains that, at the present time, WC have no good quantitative data concerning the occurrence of collagen phagocytosis in the periodontal ligament or, for that matter, in any other connective tissue. There is only slightly better information concerning the distribution of collagen phagocytosis within the ligament Again, after the examination of many sections, it can be stated that collagen phagocytosis is not confined to an intermediate zone or plexus in the periodontal ligament of the mouse first molar. As far as can be judged from nonserial sections, there seems to be a fairly random distribution of fibroblasts engaged in collagen degradation across the width of the periodontal ligament. A point worth mentioning here is that we have so far never observed collagencontaining profiles in bone cells lining the socket wall or in cementoblasts. We have, however, observed processes of fibroblast extensions interposed between cementoblasts which contain collagen profiles and this suggests that the collagen fiber bundles may be remodeled right up to their insertion into the cement (Fig. 8). Thus there is no evidence to suggest the existence of an intermediate plexus and, indeed, if the fibroblast’s role in collagen remodeling is accepted. there is no need to require such a plexus in the ligament during tooth movement. Another area of the periodontium where we have noted a significant number of fibroblasts synthesizing and degrading collagen is within the transseptal ligament (Figs. 9 and 10). This activity seemed to be confined to the cells in the lower part of the ligament. The significance
of remodeling
related
to therapeutic
tooth
movement
It must be emphasized that, so far, WC have been describing the activity of the fibroblast in the ligaments of teeth undergoing physiologic t,ooth movement. Given that the fibroblast has a ke,v role in the remodeling of the periodontal ligament during such movements, it should bc possible to utilize this function to bring about orderly remodeling of the pcriodontium during therapeutic tooth movement. Unfortunately, all the available evidence indicates that during tooth movement, even with light forces (5 g in the rat, 70 g in man) ,I*-“’ damage occurs to the periodontal ligament (hyalinization) which is followed b>
Role of fibroblnsts
ill
PDL
remodeling
165
Fig. 8. Electron micrograph of the periodontal ligament of the first molar of the mouse. This photograph illustrates the insertion of ligament fiber bundles into the cement (C). A process of the fibroblast interposed between two fiber bundles includes a collagen-containing phagosome.
repair so that the often-quoted statement that orthodontic tooth movement is “a pathologic process from which the tissue recovers” is most likely an apt description, From the work of Ryghzl it seems that most, if not all, the damaged material in an area of hyalinization is removed before repair can be initiated. Hyalinization is not the most desirable sequel to the application of force to the tooth as it has been shown, not suprisingly, that these zones delay tooth movement.22 The fact is that a cellular mechanism exists within the periodontal ligament to permit an orderly remodeling during physiologic tooth movement which suggests that this mechanism should be utilized to achieve therapeutic tooth movement. What is required is information concerning the factors which control the ligament fibroblast in its synthetic and degradative function. Finally, some of the problems of tooth rotation are worth considering. There is a large and somewhat confusing literature concerning retention after tooth rotation and this has been well reviewed in three fairly recent articles by Edwards,” Boese,23 and Parker.?’ Edwards” stated that, “Throughout the literature pertaining to the collagenous bundles of the periodontium, one observation is consistent-the supracrestal connective tissue fibers, whose length and direction are not altered by an ever-changing osseous attachment, are extremel: slow to adjust to orthodontic movements of the teeth.”
Fig.
9.
Electron
mandibular
micrograph
molars
of
the
of
the
mouse.
transseptal
Notice
the
ligament
between
uctive
fibroblasts
in the
transseptal
the
between
first the
and
second
collagen
fiber
bundles.
Fig. 10. A residual ing
Higher
in this
magnification
body (arrowed) region.
of a fibroblast containing
banded
collagen
indicates
ligament
shown
the occurrence
in
Fig.
of remodel-
9.
Role of fibroldasts
ha PDL
remodeling
167
Erikson, Kaplan, and AisenbergZ5 were satisfied that there is no physiologic process which shortens or removes the excess of supracrestal collagen fibers accumulated between two teeth which have been orthodontically approximated. In view of our findings we cannot agree with this statement. Our observations on the transseptal ligament clearly indicate the physiologic process which can achieve remodeling in this region-namely, fibroblastic activity. Our finding that only the deeper fibroblasts of this ligament are remodeling collagen supports the findings of Edwards3 who was able to show that, after 5 months of retention, the fibrous bundles of the periodontal ligament, as well as the transseptal fibers closest to the crest of the alveolar septum, appeared completely adapted to the new rotational position of the tooth. In other words, Edwards’3 results concur with our limited findings on the spatial distribution of fibroblasts engaged in the synthesis and degradation of collagen. Conclusion
Our findings indicate a cellular basis for the connective tissue remodeling which takes place during physiologic tooth movement. This cell is the fibroblast which is capable of synthesizing and degrading collagen simultaneously and, utilizing this ability, the orderly control of collagen remodeling within the periodontal ligament is possible. It is suggested that this cellular basis of connective remodeling will have a direct significance for orthodontic tooth movement once control mechanisms have been established. REFERENCES
1. Storey, E. : The nature of tooth movement, AM. J. ORTHOD. 63: 292314, 1973. The axial movement of continuously growing teeth, J. Dent. 2. Sicher, H.: Tooth eruption: Res. 21: 201-210, 1942. 3. Edwards, J. G.: A study of the periodontium during orthodontic rotation of teeth, AM. J. ORTHOD. 54: 441-461, 1968. 4. Carneiro, J., and Fava de Moraes, F.: Radioautographic visualization of collagen metabolism in the periodontal tissue of the mouse, Arch. Oral Biol. 10: 833-845, 1965. 5. Ten Cate, A. R.: Morphological studies of fibrocytes in connective tissue undergoing rapid remodelling, J. Anat. (Lond.) 112: 401-414, 1972. 6. Deporter, D. A., and Ten Cate, A. R.: Fine structural localization of acid and alkaline phosphatase in collagen-containing vesicles of fibroblasts, J. Anat. (Lond.) 114: 457-461, 1973. 7. Ten Cate, A. R., and Deporter, D. A.: The role of the fibroblast in collagen turnover in the functioning periodontal ligament of the mouse, Arch. Oral Biol. 19: 339-340, 1974. 8. Ten Cate, A. R., and Freeman, E.: Collagen remodelling by fibroblasts in wound repair. Preliminary observations, Anat. Rec. 179: 543-546, 1974. 9. Ten Cate, A. R., and Syrbu, S.: A relationship between alkaline phosphatase activity and the phagocytosis and degradation of collagen by the fibroblast, J. Anat. (Lond.) 117: 351-359, 1974. 10. Ten Cate, A. R., and Deporter, D. A.: The degradative role of the fibroblast in the remodelling and turnover of collagen in soft connective tissue, Anat. Rec. 182: l-14, 1975. 11. Bhuyan, V. N., Deo, M. G., Ramalingaswami, V., and Nayak, N. C.: Fibroplasia in experimental protein deficiency: A study of fibroblastic growth and of collagen formation and resorption in the rat, J. Pathol. 108: 191-197, 1972. 12. Oaks, B. W. : Intracellular collagen-phagocytosis or intracellular aggregation? VIII Int. Cong. Electron Microscopy, II: 368-369, 1974. 13. Listgarten, M. A.: Intracellular collagen fibrils in the periodontal ligament of the mouse, rat, hamster, guinea pig and rabbit, J. Periodont. Res. 8: 335-342, 1973.
14. Beersten, W., Everts, V., and van den Hooff, A.: Fine structure of fibroblnsts in the periodontal ligament of the rat incisor and their possible role in tooth eruption, Arch. Oral. Biol. 19: 1087-1098, 1974. 15. Gothlin, G., and Ericsson, J. L. E.: Electron microscopic studies of cytoplasmic filaments and fibers in different cell types of fracture callus, Acta Pathol. Microbial. &and., Se&ion A, Pathol. 81: 523-542, 1970. 16. Cullen, J. C.: Intracellular collagen in experimental arthritis in rats, J. Bone Joint Surg. 54B: 351-359, 1972. Intracytoplasmic collageno17. Allegra, S. R.., and Broderick, P. A.: Desmoid fibroblastoma. synthesis in a. peculiar fibroblastic tumour: Light and intrastructural study of a case, Hum. Pathol. 4: 419-429, 1973. 18. Rygh, P.: Ultrastructural cellular reactions in pressure zones of rat molar periodontium incident to orthodontic tooth movement, Acta Odont. Stand. 50: 575-593, 1972. 19. Rygh, P.: Ultrastructural changes in pressure zones of human periodontium incident to orthodontic tooth movement, Acta Odontol. Stand. 31: 109-122, 1973. 20. Buck, D. L., and Church, D. H.: A histological study of human tooth movement, AM. J.
ORTHOD.62: 507-516,1972. 21. Rygh, P.: Elimination of hyalinized periodontal tissues associated with orthodontic tooth movement, Stand. J. Dent. Res. 82: 57-73, 1974. rotation of teeth in the dog, 22. Skillen, W. G., and Rcitan, K.: Tissue changes following Angle Orthod. 10: 140-147, 1940. rotated teeth following gingivectomy 23. Boese, L. R.: Increased stability of orthodontically in Macaca nemestrina, AM. J. ORTHOD.56: 273.290, 1969. 24. Parker, G. R.: Transseptal fibers and relapse following bodily retraction of teeth: A histological study, AM. J. ORTHOD.61: 331-344, 1972. 25. Erikson, R. E., Kaplan, Il., and Aisenberg, M. : Orthodontics and trausseptal fitwrs, AM.
J. ORTHOD.31: l-26, 1945. 164 Edward
St. (M5GlG6)
Toronto,
Ontario,
D. A. Deporter,
D.D.S.,
Ph.D.,
Cnnndn
1t is probably
fair comment to state that, to most orthodontists, tooth movement is usually considered as its translocation through bone and it is on this biologic basis that, until recently, most orthodontic therapy has been c0nceived.l It is easy to understand why this should be so. The resorption and deposition of bone-in other words, bony remodeling-can be readily demonstrated in tissue sections by conventional histologic methods and with the aid of various bone-seeking labels such as the tetracyclines. Thus, the responses of bone during tooth movement have been fairly easy to analyze. On the other hand, the remodeling of the soft connective tissue intervening between bone and tooth cannot be demonstrated by conventional histology and this phenomenon is, therefore, not properly understood. To explain how the periodontal ligament is remodeled during physiologic tooth movement, especially during tooth eruption, Siche? introduced the concept of an intermediate plexus within the ligament. In this plexus the dissolution and reformation of collagen fibers supposedly occurred, thereby permitting tooth movement. Although the concept of an intermediate plexus is attractive to explain movements such as tooth eruption and tooth rotation, it is not adequate to explain how the ligament remodels during movement of the tooth in, for example, a mesial or distal direction. Furthermore, there is considerable debate as to whether the intermediate plexus exists at all. Edwards3 has recently reviewed the literature on the intermediate plexus and concluded that there is no such structure. Instead, he offered From the Toronto.
Division
This study was
of
Biological
Sciences,
Faculty
of
Dentistry,
University
of
supported by the Medical Research Council of Canada.
*1975 Milo Hellman
Research Award
of the American
Association
of Orthodontists.
155
the following alternative mechanism to explain how remodeling of the pcriotion tal ligament might occur in response to tooth movement. He proposed ( 1) that progressive osteogenic activity (and cementogenic act,irity to a far lesser degree 1 played an active role in the shortening of extended fibers and in the rcattadment of new fibers developed during tooth movement ; (2) that the stretching of the wavy collagen fibers and reorientation of their directional morphology coultl permit a certain amount of tooth movement; and (3) that the existence of a type of intermediate plexus might allow an elongation of fiber bundles by “slippage” of the fibers over one anot,hcr and subsequent reorientation of the fibers in a new position. Edwards also made the point that the final return of the slozo-nzetnbol~zi?zn connective tissue fibers to their original and stable relationship to the tooth and each other depended on the remodeling of osseous tissue. Unfortunately, these alternate suggestions explaining how the ligament remodels are also largely speculative. A real problem is that collagen is generally considered to be a very stable protein and any explanation of ligament remodeling has to accommodate this supposed fact; hence such terms as “slippage” and “stretching.” But is collagen a stable protein? Certainly some collagen in the body, for example in tendon, is relatively inert. On the other hand, experiments using radioactive proline indicate that there is a high rate of collagen turnover in the periodontal ligament, probably higher than anywhere else in the body.’ Given the possibility that thcrc is a rapid rate of’ collagen remodeling in the periodontal ligament, a further problem is to explain how the tlissolution and synthesis of collagen takes p1ac.e in orderly and cont,rolled fashion during physiologic tooth movement. With bone there is no problem in explaining how it is remodeled. The occurrence of two cell types, one producing bone (the osteoblast) and the other destroying bone (the osteoclast), and acting in concert, is well documented and understood. There would also be no problem in explaining how connective tissue remodeling occurs if ttio cell types, one synthesizing collagen and the other degrading it, were recognized. The work coming from this laborator> +I” has shown that there is indeed a cellular mechanism involved in collagen remodeling and that, unlike the bone analogy where two distinct cell types are involved, this is achieved by a single cell, the fibroblast-in other words, that the fibroblast is capable of both synthesizing and degrading collagen at any one time. The purpose of this article is to review some of the work from this laboratory which has led to the concept of the fibroblast as a cell capable of synthesizing and tlegrading collagen, to introduce some new information in support of this concept, and to discuss tooth movement, be it physiologic or therapeutic, in the light of this concept. The
macrophage
As there is a parallel between macrophage function and fibroblast function, it is convenient to begin by giving a description of the events that occur when a macrophage ingests foreign material because this is a well-understood (and therefore undisputed) physiologic phenomenon. The sequence of events
Volunle 69 Number 2
Role of fibroblasts
Fig. 1. Diagram grades In B the of
foreign
lysosomes
foreign
illustrating
a foreign
body
body with takes
the
sequence
of
In A pseudopodia
body.
is now
the
intracellular
phagosome
to
events
of the and form
as
PDL
the
macrophage
macrophage
surround
is located a
in
within
phagolysosome.
remodeli?tg
ingests the
foreign
a phagosome
after
In C degradation
157
and
debody.
fusion of
the
place.
which occur during and after phagocytosis is illustrated in diagrammatic form in Fig. 1. The material to be ingested is engulfed by pseudopodal processes of the cell so that it eventually comes to lie within it in a vesicle lined by cell membrane. This vesicular structure is termed the phagosome. The Golgi apparatus of the macrophage then synthesizes lysosomes, vesicles which contain a whole battery of catabolic enzymes. The lpsosomes fuse with the phagosome, exposing its ingested material to the enzymes, and it is in this structure, now termed the phagolysosome, that the final degradation of the ingested material occurs. The phagolysomes then disappear or persist as electron-dense residual bodies. Furthermore, it has been clearly demonstrated that the macrophage can, on occasions, ingest and degrade collagen. The classical situation where this occurs is in uterine involution after pregnancy. Here macrophages achieve the rapid removal of collagen from the uterine wall by ingesting collagen and degrading it intracellularly. The question is, can macrophages act in a similar manner to bring about remodeling of the periodontal ligament? Unfortunately, when the periodontal ligament is examined with the electron microscope, no macrophages can be demonstrated within its cell population. The bulk of the cells in the normal functioning periodontal ligament are fibroblasts and this, therefore, prompts the question whether or not the fibroblasts may have a phagocytic function similar to that of the macrophage.
Fig. 2. Electron
micrograph
dioxide
intravenously
dense some
injected particules)
has
been
of
part
of
8 hours taken
up
a fibroblast previously. !by the
which The
fibroblast
has
thorium and
been dioxide
sequestered
exposed
to
(seen in
as a
thorium electron-
phagolyso-
(arrowed).
Evidence indicating
that the fibroblast
may act as a macrophage
The standard way of demonstrating the occurrence of phagocytosis at the fine structural level is to inject into the vascular system of an experimental animal an electron-dense material, such as thorium dioxide. This marker rapidly leaves the vascular system and enters the extracellular compartment, where it is usually picked up by scavenger cells in the immediate vicinity. Because thorium dioxide is electron dense, its location can be readily discerned with the electron microscope. Fig. 2 illustrates a fibroblast from such an experimental animal and shows the accumulation of thorium dioxide within a phagolysosome: clear evidence that phagocytosis has occurred. Thus there can be no doubt that the fibroblast is capable of ingesting foreign material. The key question, however, is whether or not the fibroblast can ingest its own product, collagen. Evidence indicating
that the fibroblast
can ingest and degrade
collagen
When the fine structure of the periodontal ligament is examined it is a fairly easy matter to assemble a series. of pictures which appear to indicate the phagocytosis of collagen by ligament fibroblasts (Fig. 3), an assumption strengthened by the demonstration of acid phosphatase activity within the collagen-containing vesicles or phagolysosomes. Convincing as this pictorial evidence may seem, however, unequivocal proof of phagocptosis is still required,
Fig.
3. A series
demonstrates illustrates the
collagen
development
which is also
of the
banded present
pictures actual within
indicating
the
phagocytosis
of
a phugosome
of a phagolysosome collagen fibrils in this cell.
are
phagocytosis collagen and
this
(C in Fig. still
discernible.
ot collagen and
corresponds l),
and A
by
corresponds
the to
A
to B in Fig.
3, d illustrates phagolysosome
fibroblast. in
Fig.
3, a 1. 3,
b
1. 3, c illustrates residual
containing
bodies collagen
in
for it is just possible that the morphologic appearance of collagen partly within and partly without the fibroblast, as seen in Fig. 3, n, could bc caused b) plane of section. Also Fig. 3, b and c, could be interpreted as indicating an overproduction of collagen and its subsequent degradation without the collagen ever leaving the fibroblast. We have attempted to provide unequivocal evidence two ways and our results are reported hcrr in detail for the first time. ElsewherelO we have suggested the following equation : synthesis amino acids , ’ collagen degradation so that if the rates of synthesis and degradation are equal the amount of collagen should remain steady. Now, if this equation holds good for the rapidly remodeling collagen in the periodontal ligament, it should be possible to predict an over-all loss of extracellular collagen if its synthesis is arrested without interfering with the fibroblasts’ phagocytic function. There are two ways whereby the synthesis of collagen by the fibroblast can be halted: by depriving the cell either of vitamin C or of the basic amino acids required to make collagen. Vitamin C (ascorbic acid) has a specific action on a particular sequence in the several steps of collagen synthesis. In the assembly of the collagen molecule the amino acid prolinc is first incorporated, with other amino acids, at a ribosomal site. The collagen precursor molecule formed here is then transferred from the ribosomes to the cisternae of the rough endoplasmic reticulum. On the unit membrane of the rough endoplasmic reticulum the proline is hydroxylated to hydroxyproline and this step involves enzyme activity dependent upon ascorbic acid. Thus, in the absence of the acid, enzyme activity ceases and hydroxylation of the prolinc cannot occur. As hydroxyproline is essential for the stability of the classical triple helical structure of collagen, collagen does not form and instead precursor material accumulates within the cisternae of the rough endoplasmic ret,iculum. This is seen at the fine structural level as a dilation and rounding of the profiles of rough endoplasmic reticulum as collagen precursors accumulate within them. Protein deficiency halts collagen synthesis” by withholding the basic building materials for collagen from the fibroblast. Thus, if periodontal ligament remodeling involves phagocptosis of collagen, a loss of extracellular collagen should he expect.cd in both scorbutic and protein deficient animals. Fig. 1 illustrates a scorbutic fibroblast and its cytoplasm is characterized by the classic dilated cistcrnae of rough endoplasmic reticulum. It can be inferred, therefore, that collagen synthesis by this fibroblast has ceased. Another feature of this cell is the presence of many intracellular collagen profiles, which indicates a continuance of collagen degradation, and this is confirmed by the lesser amounts of extracellular collagen. An almost similar picture is seen in the periodontal ligament of the protein-deficient animal. There is again a marked loss of extracellular collagen (Fig. 5) and an accumulation of banded collagen within the fibroblasts. The only difference is that in protein-deficient fibroblast,s no dilation of the cisternae of the rough rndoplasmic reticulum occurs as collagen synthesis has never begun.
Fig. rough Fig.
4. A scorbutic
5. A fibroblast
protein-deficient of
fibroblast
(guinea
reticulum
ted.
endoplasmic
intracellular
endoplasmic
from diet. fibers.
reticulum
the
There
pig].
Note
periodontal is a loss
This
also
cell
the
ligament
is characterized
of a mouse
of extracellular
As there
is no accumulation
cisternae
are
not dilated
collagen of
in this
dilated
maintained fibrils
intermediary cell.
by
of collagen
accumulation
and collagen
profiles
within
for 4 days an
of
this cell. on a
accumulation products,
the
The other may we have attempted to dcmonst rate the unequivocal occ~~rrer~cc of collagen phagocptosis by the fibroblast is with the use of elccAtron-dense markers such as thorium dioxide. The rationale is that if fibroblasts active]) ingest extracellular collagen they map well also incorporate cxtrancous material lying in the extracellular compartment. Fig. 6 illustrates a coincidental uptake of marker and collagen by the fibroblast and this further substantiates our claim that this cell can ingest its own product, collagen. Evidence
indicating
that
the fibroblast
synthesizes
and
degrades
collagen
simultaneously
The findings we have presented so far may be interpreted in different ways. It is possible that, in the population of fibroblasts which constitute the periodontal ligament, some are involved in collagen synthesis while others are involved in collagen degradation. lt could also be that, our findings indicate a modulation of the fibroblast to a fibroclast. Finally, it could bc that t,he cells of the ligament are capable of synthesizing and degrading collagen simultaneously. The evidence indicates that the last possibility is the correct one. Not only do the protein and ascorbic acid deficiency experiments indicate that both processes are occurring simultaneously (for example, the dilation of the cisternac of the rough endoplasmic reticulum in the scorbutie fihroblast indicates its synthetic activity) but there is also strong supportive cvidencc~ from autoradiographp studies at the fine structural level. Weinstock (personal communication) has been able to show that collagen-containing vesicles in ligament fihrohlasts are not labeled with “II-prolinc 30 minutes after injection of the isotope but that the cell organelles associated sequentially with the pathway of collagen synthesis are. Not only does this finding indicate that the collagen-containing vesicles are not associated with collagen synthesis but also that both processes (synthesis and degradation) are occurring at the same time. Evidence
indicating
the widespread
occurrence
of fibroblastic
activity
It is worth noting that although our work began using the periodontal ligament as a model system, it has now been extended by ourselves8 and others12-14 to show that the ability of the fibroblast to degrade collagen is a widespread occurrence and is not confined to the cells of the ligament. Thus fibroblastic activity has now been described in man (periodontal ligament), monkey (gingiva), chick (embryonic skin), mouse (ligament and skin), rat (ligament and skin), guinea pig (ligament and skin), and rabbit (ligament and skin), and in such varying locations as in dermal wound repair,8 bone lesions,‘” and fracture repair, I5 tendon development,12 experimental arthritic some pathologic 1esions.l’ Also we have just begun to examine remodeling ill cranial sutures and here also collagen phagocytosis by fibroblasts has been identified (Fig. 7). Our experiments on repair of skin wound9 indicates that fibroblasts not normally degrading collagen map he “switched on” to assume this function bp some change in the local environment. This may he significant as it suggests the possibility of control.
Volume Number
69 2
Fig.
Electron
micrograph
injected
intravenously
6.
dioxide are
localized
Fig. readily
7.
Role
of
in a phagosome
Fibroblasts demonstrated
in
the
part 8 hours
of
(arrows)
of
within
fibroblasts
in
which
has
a fibroblast previously.
coincident suture
of
with the cells.
The
a collagen
3.day-old
thorium
PDL
been dioxide
renzodelkg
exposed particles
to
163
thorium (arrowed)
fibril.
mouse.
Intracellular
collagen
can
be
164
Ten Cnte, Deporter,
Quantitation
and
distribution
and Freemnv within
the
Ant. J. Orthod. Pebrzcary 197F
ligament
Before discussing the application of our findings to therapeutic tooth movement two further points need to be introduced : (1) the quantitation of ligament fibroblasts degrading collagen and (2) their distribution. Quantitation is cxtremely difficult at the electron-microscopic level. It must be realized that in sections prepared for electron microscopy only an extremely small field may be examined-indeed, only an extremely small part of any one cell can be examined-at any one time. At present we can only suggest that many cells in the periodontal ligament of the mouse first molar are involved in collagen remodeling. This suggestion is based on the fart that, in the thousands of sections that have been examined, it is always easy to demonst,ratc intracellular collagen within the fibroblast. If collagen phagocytosis were an unusual phenomenon, the sampling restrictions imposed by the techniques of electronmicroscopy would restrict its ready demonstration. Even so, the fact remains that, at the present time, WC have no good quantitative data concerning the occurrence of collagen phagocytosis in the periodontal ligament or, for that matter, in any other connective tissue. There is only slightly better information concerning the distribution of collagen phagocytosis within the ligament Again, after the examination of many sections, it can be stated that collagen phagocytosis is not confined to an intermediate zone or plexus in the periodontal ligament of the mouse first molar. As far as can be judged from nonserial sections, there seems to be a fairly random distribution of fibroblasts engaged in collagen degradation across the width of the periodontal ligament. A point worth mentioning here is that we have so far never observed collagencontaining profiles in bone cells lining the socket wall or in cementoblasts. We have, however, observed processes of fibroblast extensions interposed between cementoblasts which contain collagen profiles and this suggests that the collagen fiber bundles may be remodeled right up to their insertion into the cement (Fig. 8). Thus there is no evidence to suggest the existence of an intermediate plexus and, indeed, if the fibroblast’s role in collagen remodeling is accepted. there is no need to require such a plexus in the ligament during tooth movement. Another area of the periodontium where we have noted a significant number of fibroblasts synthesizing and degrading collagen is within the transseptal ligament (Figs. 9 and 10). This activity seemed to be confined to the cells in the lower part of the ligament. The significance
of remodeling
related
to therapeutic
tooth
movement
It must be emphasized that, so far, WC have been describing the activity of the fibroblast in the ligaments of teeth undergoing physiologic t,ooth movement. Given that the fibroblast has a ke,v role in the remodeling of the periodontal ligament during such movements, it should bc possible to utilize this function to bring about orderly remodeling of the pcriodontium during therapeutic tooth movement. Unfortunately, all the available evidence indicates that during tooth movement, even with light forces (5 g in the rat, 70 g in man) ,I*-“’ damage occurs to the periodontal ligament (hyalinization) which is followed b>
Role of fibroblnsts
ill
PDL
remodeling
165
Fig. 8. Electron micrograph of the periodontal ligament of the first molar of the mouse. This photograph illustrates the insertion of ligament fiber bundles into the cement (C). A process of the fibroblast interposed between two fiber bundles includes a collagen-containing phagosome.
repair so that the often-quoted statement that orthodontic tooth movement is “a pathologic process from which the tissue recovers” is most likely an apt description, From the work of Ryghzl it seems that most, if not all, the damaged material in an area of hyalinization is removed before repair can be initiated. Hyalinization is not the most desirable sequel to the application of force to the tooth as it has been shown, not suprisingly, that these zones delay tooth movement.22 The fact is that a cellular mechanism exists within the periodontal ligament to permit an orderly remodeling during physiologic tooth movement which suggests that this mechanism should be utilized to achieve therapeutic tooth movement. What is required is information concerning the factors which control the ligament fibroblast in its synthetic and degradative function. Finally, some of the problems of tooth rotation are worth considering. There is a large and somewhat confusing literature concerning retention after tooth rotation and this has been well reviewed in three fairly recent articles by Edwards,” Boese,23 and Parker.?’ Edwards” stated that, “Throughout the literature pertaining to the collagenous bundles of the periodontium, one observation is consistent-the supracrestal connective tissue fibers, whose length and direction are not altered by an ever-changing osseous attachment, are extremel: slow to adjust to orthodontic movements of the teeth.”
Fig.
9.
Electron
mandibular
micrograph
molars
of
the
of
the
mouse.
transseptal
Notice
the
ligament
between
uctive
fibroblasts
in the
transseptal
the
between
first the
and
second
collagen
fiber
bundles.
Fig. 10. A residual ing
Higher
in this
magnification
body (arrowed) region.
of a fibroblast containing
banded
collagen
indicates
ligament
shown
the occurrence
in
Fig.
of remodel-
9.
Role of fibroldasts
ha PDL
remodeling
167
Erikson, Kaplan, and AisenbergZ5 were satisfied that there is no physiologic process which shortens or removes the excess of supracrestal collagen fibers accumulated between two teeth which have been orthodontically approximated. In view of our findings we cannot agree with this statement. Our observations on the transseptal ligament clearly indicate the physiologic process which can achieve remodeling in this region-namely, fibroblastic activity. Our finding that only the deeper fibroblasts of this ligament are remodeling collagen supports the findings of Edwards3 who was able to show that, after 5 months of retention, the fibrous bundles of the periodontal ligament, as well as the transseptal fibers closest to the crest of the alveolar septum, appeared completely adapted to the new rotational position of the tooth. In other words, Edwards’3 results concur with our limited findings on the spatial distribution of fibroblasts engaged in the synthesis and degradation of collagen. Conclusion
Our findings indicate a cellular basis for the connective tissue remodeling which takes place during physiologic tooth movement. This cell is the fibroblast which is capable of synthesizing and degrading collagen simultaneously and, utilizing this ability, the orderly control of collagen remodeling within the periodontal ligament is possible. It is suggested that this cellular basis of connective remodeling will have a direct significance for orthodontic tooth movement once control mechanisms have been established. REFERENCES
1. Storey, E. : The nature of tooth movement, AM. J. ORTHOD. 63: 292314, 1973. The axial movement of continuously growing teeth, J. Dent. 2. Sicher, H.: Tooth eruption: Res. 21: 201-210, 1942. 3. Edwards, J. G.: A study of the periodontium during orthodontic rotation of teeth, AM. J. ORTHOD. 54: 441-461, 1968. 4. Carneiro, J., and Fava de Moraes, F.: Radioautographic visualization of collagen metabolism in the periodontal tissue of the mouse, Arch. Oral Biol. 10: 833-845, 1965. 5. Ten Cate, A. R.: Morphological studies of fibrocytes in connective tissue undergoing rapid remodelling, J. Anat. (Lond.) 112: 401-414, 1972. 6. Deporter, D. A., and Ten Cate, A. R.: Fine structural localization of acid and alkaline phosphatase in collagen-containing vesicles of fibroblasts, J. Anat. (Lond.) 114: 457-461, 1973. 7. Ten Cate, A. R., and Deporter, D. A.: The role of the fibroblast in collagen turnover in the functioning periodontal ligament of the mouse, Arch. Oral Biol. 19: 339-340, 1974. 8. Ten Cate, A. R., and Freeman, E.: Collagen remodelling by fibroblasts in wound repair. Preliminary observations, Anat. Rec. 179: 543-546, 1974. 9. Ten Cate, A. R., and Syrbu, S.: A relationship between alkaline phosphatase activity and the phagocytosis and degradation of collagen by the fibroblast, J. Anat. (Lond.) 117: 351-359, 1974. 10. Ten Cate, A. R., and Deporter, D. A.: The degradative role of the fibroblast in the remodelling and turnover of collagen in soft connective tissue, Anat. Rec. 182: l-14, 1975. 11. Bhuyan, V. N., Deo, M. G., Ramalingaswami, V., and Nayak, N. C.: Fibroplasia in experimental protein deficiency: A study of fibroblastic growth and of collagen formation and resorption in the rat, J. Pathol. 108: 191-197, 1972. 12. Oaks, B. W. : Intracellular collagen-phagocytosis or intracellular aggregation? VIII Int. Cong. Electron Microscopy, II: 368-369, 1974. 13. Listgarten, M. A.: Intracellular collagen fibrils in the periodontal ligament of the mouse, rat, hamster, guinea pig and rabbit, J. Periodont. Res. 8: 335-342, 1973.
14. Beersten, W., Everts, V., and van den Hooff, A.: Fine structure of fibroblnsts in the periodontal ligament of the rat incisor and their possible role in tooth eruption, Arch. Oral. Biol. 19: 1087-1098, 1974. 15. Gothlin, G., and Ericsson, J. L. E.: Electron microscopic studies of cytoplasmic filaments and fibers in different cell types of fracture callus, Acta Pathol. Microbial. &and., Se&ion A, Pathol. 81: 523-542, 1970. 16. Cullen, J. C.: Intracellular collagen in experimental arthritis in rats, J. Bone Joint Surg. 54B: 351-359, 1972. Intracytoplasmic collageno17. Allegra, S. R.., and Broderick, P. A.: Desmoid fibroblastoma. synthesis in a. peculiar fibroblastic tumour: Light and intrastructural study of a case, Hum. Pathol. 4: 419-429, 1973. 18. Rygh, P.: Ultrastructural cellular reactions in pressure zones of rat molar periodontium incident to orthodontic tooth movement, Acta Odont. Stand. 50: 575-593, 1972. 19. Rygh, P.: Ultrastructural changes in pressure zones of human periodontium incident to orthodontic tooth movement, Acta Odontol. Stand. 31: 109-122, 1973. 20. Buck, D. L., and Church, D. H.: A histological study of human tooth movement, AM. J.
ORTHOD.62: 507-516,1972. 21. Rygh, P.: Elimination of hyalinized periodontal tissues associated with orthodontic tooth movement, Stand. J. Dent. Res. 82: 57-73, 1974. rotation of teeth in the dog, 22. Skillen, W. G., and Rcitan, K.: Tissue changes following Angle Orthod. 10: 140-147, 1940. rotated teeth following gingivectomy 23. Boese, L. R.: Increased stability of orthodontically in Macaca nemestrina, AM. J. ORTHOD.56: 273.290, 1969. 24. Parker, G. R.: Transseptal fibers and relapse following bodily retraction of teeth: A histological study, AM. J. ORTHOD.61: 331-344, 1972. 25. Erikson, R. E., Kaplan, Il., and Aisenberg, M. : Orthodontics and trausseptal fitwrs, AM.
J. ORTHOD.31: l-26, 1945. 164 Edward
St. (M5GlG6)