Experimental Induction of "Functionally" Oriented

The

Fibers Attached to Cementum*

cavity or filling barely piercing the dentinocemental junction. 2 They were cut and nicked as previously described1, and preserved at —5° Celsius. Autogenous bone was obtained by amputating the femur of the host rat. After scraping off all soft tissue, it was broken into small pieces about 5 to 6 mm long and

decalcified in 5% EDTA at 3.3° Celsius until it was radiographically invisible and soft to the insertion of pins. The bone was preserved at —5° Celsius until the time of implantation. The root and bone were then inserted together into the abdominal subcutaneous tissues of the host 200 gm white female Sherman strain rat. Twelve animals, with two sets of implants apiece, were sacrificed with chloroform after 1, 2, 3, 4, and 6 weeks and 2,3,4,5,6,7 and 8 months. Eight controls containing two implants each of bone without root were sacrificed at 1,2, 3 and 4 weeks and 2, 4, 6 and 8 months. Specimens were fixed in 10% formalin solution, decalcified in a mixture of EDTA, potassium sodium tartrate and hydrochloric acid, embedded in paraffin, and stained with haemotoxylin and eosin. Groups of serial sections 8 µ thick were examined at intervals of 20 slices.

by Melvin L. Morris, b.s., m.a.,

d.d.s.f

This paper is part of a series in which the physiologic interactions of dentin and cementum with connective tissues have been studied by implanting small pieces of human tooth root into the subcutaneous tissues of the rat. To

provide variety in the roots, they have been obtained both from healthy and periodontally diseased teeth and have been given selective physical and chem-

ical treatments. The connective tissue environment has been changed by using bone-forming combinations of bone and/or marrow. The bone also has been modified by physical and chemical treatments. One of the purposes has been to find a combination that might induce regeneration of the periodontium. Marrow alone and marrow with bone both produced profuse quantities of new bone which ankylosed to the root at about 6 months and then proceeded to resorb and replace it with bone.1'2 Simply growing bone adjacent to a root did not necessarily produce a periodontal attachment. It was apparent that one must grow all the com-

ponents of a periodontium:

cementum and

Results Bone

(Decalcified Autogenous Bone Without Root). specimen showed recalcification of the inner borders of cancellous spaces (Fig. 1). At the second and third weeks (Fig. 2) recalcification appeared as individControls

A 1-week

periodontal

fibers as well as bone. It was reasoned that perhaps boneformers of a lower order might produce these elements without invasion of the root. Indeed, autogenous bone without marrow produced a cellular cementum-like substance on the root surface that was separated from the bone by a "periodontal" space containing fibers parallel to bone and root. This lasted for 2 months when ankylosis again occurred.3 Decalcified allogeneic bone produced new bone to a much lesser degree.4 All bone-forming activity stopped by 6 weeks and there never was any effect on the root. The present experiment utilized decalcified autogenous bone. Materials and Methods Roots were selected from teeth that had little or no bone loss and were either caries-free or had a one-surface * Presented at the American Academy of Periodontology meeting in Phoenix, Arizona on September 29, 1978. These investigations were supported, in part, by grants from the following foundations: Sergei Zlinkoff, Ralph and Frances DeJur, Max and Eva Deitmer, Goldman Sachs, and Lewittes. f Clinical Professor of Dentistry, Director, Laboratory for Periodontal Research, Columbia University School of Dental and Oral Surgery, 630 West 168th Street, New York, NY 10032.

Figure 1. One week, control. Decalcified bone (DB) showing recalcification (REC) of inner border of cancellous space.

467

468

Morris

J. Periodonlol. 1979

September.

Figure 3A. Four weeks, control. Recalcification as foci (F) and inner borders of canaliculi (REC). Cartilage cells (CAR). B. Four months, control. Diffuse recalcification (REC). New bone (NB) growing on side of implant facing adjacent implant surface. C. Four months, control. Ossicles (OS) formed by new bone (NB) lining cancellous spaces. Reticular network (RET) in ossicle cavity. D. Six months, control. Part of implant recalcified (REC), the remainder is unchanged (DB).

Volume 50 Number 9

ual foci, as well as the lining of inner borders of canaliculi. Cartilage cells were present within the canaliculi. Recalcification continued during the 4th and 8th weeks. Cartilage cells were seen but to a much lesser

degree (Fig. 3A).

At 4 months individual foci

were

present but many of

them had apparently merged into larger solidly recalcified areas. New bone was seen lining several cancellous spaces forming ossicles (Fig. 3B). Occasional sites of new bone were on the sides of bony spicules facing adjacent similar implants (Fig. 3C). At the 6th and 8th months continuing recalcification

"Functionally" Oriented Fibers 469 was observed, although many individual implanted pieces of bone were unchanged. No new bone or cartilage cells were seen (Fig. 3D). Experimental (DecalcifiedAutogenous Bone with Root). The first 2 weeks showed no changes in implanted bone. At the third week foci of recalcification

were seen

in the of

pattern described above, as well as several small new bone formation and cartilage cells (Figs. 4A, 4B). areas

The 4th and 6th weeks showed little or no change except for foci of recalcification. At 2 months the recalcification continued. New bone lined small cancellous spaces forming ossicles. The ossicle spaces were filled

Figure 4A. Three weeks, experimental. Decalcified bone (DB) recalicifed as foci (F) and (REC) as lining of canaliculi (CL). B. Three weeks, experimental. New bone (NB) growing on side of implant near recalcified canalicular border (REC) enclosing cartilage cells

(CAR).

Figure 5A. Two months, experimental. Formation of new bone (NB) on lumen of cancellous space in decalcified bone (DB), forming an ossicle (OS). A reticular network (RET) is present. B. Three months, experimental. Recalcification (REC) of inner border of cancellous space in decalcified bone (DB). A reticular network is present (RET).

470

with a reticular network (Fig. 5A). Similar bony changes were noted at the 3rd, 4th and 5th months (Figs. 5B, 6,

7A).

The 6th and 7th month specimens showed progressive recalcification. Bone formation seemed to have ceased,

Figure 6. Three months, experimental. Foci of recalcification and growth of new bone (NB) on adjacent surfaces of

(F)

j. Periodontol.

Morris

implants.

September,

1979

neither osteocytes nor osteoblasts being present (Figs. 7B, 8). The 8th month trabeculae showed no changes at all. Root The roots were surrounded by a connective tissue capsule which usually appeared to be composed of inner and outer layers. Fibers from the inner capsule were attached to adjacent cementum entering the cementai surface at angles varying from 45 to 90°. This was a consistent finding for the entire postoperative period. Representative sections are described below. At 1 week the double capsule could be seen (Fig. 9A). The inner one (IC) was closely attached to cementum (CE) above the dentinal nick (N). A higher power of the area (Fig. 9B) showed a loose cellular connective tissue with fiber bundles (CTF) attached to cementum (CE) on both comparatively smooth surfaces and in résorption bays (RB). The depth of the bays appeared amorphous, pink in the H & E sections. A 2-week specimen (Fig. 10A) also showed a loose cellular connective tissue (CT) with fine reticulin-like fibers (RF) radiating out from cementum (CE). The cementai surface was irregular with a thin amorphous border appearing in many spots. At the third week (Fig. 10B) connective tissue fibers (CTF) were now thicker and attached at right angles to cementum (CE). Connective tissue cells were prominent. At 4 weeks (Fig. 11) connective tissue fibers (CTF) appeared as thicker bundles inserting well into cementum (CE). The connective tissue (CT) seemed a bit less cellular. A section at 2 months (Fig. 12A) showed decalcified bone (DB) adjacent to cementum (CE). Near the top of the bone there were several areas of right angled connective tissue fibers (CTF) attached to the cementum

Figure 7A. Five months, experimental. Ossicle formed by new bone (NB) within the implant to form ossicles (OS). A reticular network (RET) is present with nonhemopoietic connective tissue cells (CTC). B. Six months, experimental. Diffuse and focal areas of recalcification (REC) of decalcified bone (DB). Cellular lacunae are empty except for a few remaining pyknotic nuclei (N).

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'Functionally" Oriented Fibers

(CE). A higher power view (Fig. 12B) showed the fibers, (CTF) attached to a rough cementai surface (CE). At 5 months (Fig. 13A) a longer area of obliquely attached fibers (CTF) was seen in relation to cementum

471

(CE). The cellular content of the connective tissue fiber (CTF) was further diminished. A higher power section

(Fig. 13B) showed the oblique fibers attached to an irregular cementai surface (CE). Only a few connective

tissue cells (CTC) were apparent. It was characteristic throughout this series to see fiber connections with the root in separate small areas. Serial sections showed that these connections disappeared while others then appeared in different planes. The threedimensional concept suggested is that of stalks of connective tissue, each stalk composed of collagenous fiber bundles. Small patches of attachment were seen at the 6th month (Fig. 14A). Here thick fiber bundles (CTF) were attached to the cementum (CE). At 7 months new bone (NB) has grown toward and fused with cementum (CE) to effect an ankylosis (Fig. 14B). This was the only section exhibiting this phenomenon.

Discussion

Figure 8. Seven months, experimental. Wide No vital cells are evident.

fication (REC).

areas

of recalci-

Past studies of autogenous bone implants showed marked bone formation at 1 week and hemopoietic ossicles by 4 weeks.3 By the 2nd week, decalcified allogeneic bone had induced a new bone growth to form ossicles which contained a reticular network with an occasional normoblast.4 In both instances the ossicles were formed by trabecular outgrowths from the implants. By contrast, controls in the present study showed sparse growth on outer surfaces and inner borders of cancellous spaces. Thus, the ossicles in this instance were formed within the cancellous spaces of the implants. The ossicle spaces contained a reticular network with no sign of hemopoietic cells. A major activity was a recalcifica-

Figure 9A. One week. A double capsule may be seen, the outer (OC) and the inner one (IC), which is attached to cementum (CE). D is dentin. B. One week. A higher power view showing dentin (D), cementum (CE) and connective tissue fibers (CTF). The fibers are attached to a surface that is smooth in some parts and roughened in others. Some fiber bundles are thick and some are rather fine. The connective tissue (CT) is loose and cellular.

472

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1979

Figure 10A. Two weeks. A loose cellular connective tissue (CT) shows thin reticulin-like fibers (RF) attached to an irregular cementai . Three weeks. A high power of a cellular connective tissue (CT) with fiber bundles attached to cementum (CE).

surface (CE).

shown that the maximal amount of bone formation produced by marrow-free fresh allografts occurs when they are implanted in daylight, when host immunocompetence is at its circadian peak. They suggested that the accompanying increase in inflammatory response would bring with it more cells with osteogenic potential. The complete lack of immune response in the present experiment may have played a role in the sparse production of new bone. It should be noted, however, that a possibly salutary effect of the immune response operates over a limited range. The response to xenogeneic grafts destroys

Osteogenesis.

The bone of the experimental group (with roots) changes similar to those of the controls, with perhaps a bit less cartilage and a bit more ossicle formation. The differences were not great enough to make any quantitative distinctions. However, the most exciting finding is the presence of connective tissue fibers attached to and exiting from cementum at angles ranging from 45 to 90°. This is the first time that this has been reported, and it has never been clearly demonstrated in reattachment, bone graft or synthetic root implant experiments. These fibers certainly resemble the fiber system of a periodontium. It seems reasonable to hypothesize that in the clinical situation such cementai periodontal fibers would unite with fibers growing from adjacent bone to then constitute

showed

Figure 11. Four weeks. Thick connective tissue fiber bundles attached to cementum (CE) at right angles and inserted into the cementai substance. Cells are present but to a lesser

(CTF)

degree.

periodontal organ. possibility exists that these fibers may be artefacts or they may have been left as a result of incomplete root planing before implantation. However, the roots were treated in exactly the same manner and by the same laboratory assistants as for all previous experiments in this series. Other evidence against the possibility of artefact includes the consistency of the findings throughout a new

tion process starting with many tiny foci clustered about the canaliculi in which cartilage cells were seen. Decalcified autogenous bone produced the least amount of new bone in this entire series of bone implants. It is of particular interest that decalcified autogenous bone induces far less new bone than does decalcified allogeneic bone.4 Simmons, Sherman and Lesker5 have

The

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"Functionally" Oriented Fibers

473

Figure 12A. Two months. Decalcified bone (DB) separatedfrom cementum (CE) by a connective tissue fiber attachment (CTF) near the "crest" of the bone. B. A higher power of the right angled connective tissue attachment (CTF) to an irregular surface of cementum (CE) near the "crest" of decalcified bone (DB). The area of attachment is acellular although the more distant connective tissue (CT) is cellular.

Figure 13 A. Five months. Oblique fiber bundles (CTF) are attached to cementum (CE). Dentin is D. B. A higher power section shows the oblique fiber bundles (CTF) in an almost acellular connective tissue inserting into an irregular border of cementum (CE). D is dentin.

the 8-month period and the maturing of the fibers from the slender reticulin type in the early specimens to thicker less cellular collagenous bundles later on. Two questions present themselves. Why did the fibers form in a functional arrangement? What is the manner of attachment? The functional arrangement has been noted, in the

absence of function, in many tooth germ implantation and developmental studies.6"10 Thus, the fibroblasts are either genetically programmed or induced by adjacent structures. In the present situation the autogenous bone matrix may contain a self-specific inducer which would not operate in an allogeneic situation or it may have a metabolic level slightly higher than cementum. This

J. Periodonlol. 1979

474 Morris

September.

Figure 14A. Six months. A small stalk of connective tissue fibers (CTF) attached and inserted into cementum New bone (NB) has grown towards and fused with cementum (CE) to effect an ankylosis.

latter alternative would mimic the natural situation.11"16 Perhaps this biological gradient would deliver the proper message to the adjacent fibroblasts. The manner of attachment to cementum is still obscure. Very thin pink amorphous borders were seen but no discrete cementai layers could be identified. Presumably such a layer would form eventually. Reattachment has been shown in electromicroscopic studies to be effected by the release of surface apatite crystals followed by the insertion of collagen fibers into the resultant

ultramicroscopic recesses.17, 18 The fibers were attached only

to

cementum, not to

dentin, and not even in the nicks which have been shown to be the most favorable sites for both bone and cementum formation.19 This indicates that the clinician should in order to preserve cemenavoid excessive root

contamination

by

or

. Seven months.

possible epithelial

interference, and it occurred on a xenogeneic, periodon-

tally healthy root surface. Thus one cannot extrapolate directly from this to the clinical situation. Yet it did occur under these experimental conditions and under no other previous ones. It certainly warrants the clinical trials which

are now

in progress.

Summary Decalcified autogenous bone induced the least amount of new bone yet observed. It also apparently helped to induce the formation of functionally oriented connective tissue fibers attached to cementum. Clincal trials are indicated.

planing

Endotoxins found in the cementum of periodontal pockets 20-23 are another facet of this problem. It would also seem logical to eliminate the endotoxins to make the root surface favorable for reattachment. This could be done by chemical extraction or by removal of cementum. The latter procedure had been recommended for diverse reasons in the past by this author24 and by Riffle.25 In the light of the present information it would now seem more reasonable to curette lightly to preserve the cementum and then to chemically extract the endotoxins. Another interesting finding is the ankylosis in the 7month slide. Melcher26 has shown that the periodontal membrane acts to prevent the adjacent bone from crossing the space to fuse with the root. It is possible that such fibers did not form in the particular area where ankylosis occurred. In the clinical situation, adjacent periodontal ligament cells from below the base of the pocket might help inhibit the invasion of the low-grade bone former. This fiber growth occurred in a closed system without tum.

oral bacteria

(CE).

Acknowledgments

the following people whose this study possible: Drs. Irwin Mandel, Louis Mandel, George Minervint, Abbe Selman, George Armstrong, Paul Schneider, Indulal Nagrecha, and Imha Park, and Mrs. Rose Tarantino and Anne Himmelstein.

Appreciation expressed cooperation helped to make is

to

References

Morris, M. L.: The implantation of human dentin and cementum with autogenous red marrow into the subcutaneous tissues of the rat. J Periodontol 40: 571, 1969. 2. Morris, M. L.: The implantation of human dentin and cementum with autogenous bone and red marrow into the subcutaneous tissues of the rat. J Periodontol 40: 259, 1969. 3. Morris, M. L.: The implantation of human dentin and 1.

bone into the subcutaneous tissues 286, 1971. 4. Morris, M. L.: The effects of homologous bone and matrix, with and without marrow, on implanted dentin and cementum. J Periodontol 44: 667, 1973. 5. Simmons, D. J., Sherman, N. E., and Lesker, P. .: Allograft induced osteo induction in rats—a circadian rhythm. Clin Ortho 103: 252, 1974. cementum with autogenous of the rat. J Periodontol 42:

Volume 50 Number 9

"Functionally" Oriented Fibers

6. Barton, J. M., and Keenan, R. M.: The formation of fibers in the hamster under non-functional conditions. Arch Oral Biol 12: 1331, 1967. 7. Hoffman, R. L.: Tissue alterations in intramuscularly transplanted developing molars. Arch Oral Biol 12: 713, 1967. 8. Ten Cate, A. R., Mills, C, and Solomon, G.: The development of the periodontium: a transplantation and autoradiographic study. Anat Res 170: 365, 1971. 9. Atkinson, M. E.: The development of the mouse molar periodontium. J Periodont Res 7: 255, 1972. 10. Ten Cate, A. R., and Mills, C: The development of the periodontium: the origin of alveolar bone. Anat Res 173: 69, 1972. 11. Muhlemann, H. R., Zander, . ., and Halberg, F.: Mitotic activity in the periodontal tissues of the rat molar. J Dent Res 33: 459, 1954. 12. Stallard, R. E.: The utilization of H'-proline by the connective tissue elements of the periodontium. Periodontics 1: 185, 1963. 13. Waerhaug, J.: The effect of C-avitaminosis in the supporting structures of the teeth. J Periodontol 29: 87, 1958. 14. Fava-de-Moraes, F., and Villa, N.: The effects of inanition on the periodontal tissues of the rat. J Periodont Res 3: 223, 1969. 15. Melcher, A. H., and Correia, . .: Remodeling of periodontal ligament in erupting molars of mature rats. J Periodont Res 6: 118, 1971. 16. Kameyama, Y.: An autoradiographic investigation of the developing rat periodontal membrane. Arch Oral Biol 18:

Sharpey's

473, 1973. 17. Frank, R., Fiore-Donne, G., Cimasoni, G., and Ogilvie, .: Gingival reattachment after surgery microscopic study. J Periodontol 43: 597,

in man: 1972.

Jeffrey A.

Severson 1946-1978

A. Severson, an active member in the Academy, has been presumed dead his disappearance in a light plane. Dr. and Mrs. Severson departed Homer, November 19, 1978. When they failed to arrive in Anchorage, an intensive on Alaska, search was conducted but no trace of the plane or passengers was ever found. eight-day The court ruling was a presumption of death. Dr. Severson completed his graduate training at the University of Washington in 1975. His practice was limited to periodontics in Anchorage and he was a founding member of the Alaskan Society of Periodontology. Although he had finished his training only three years ago, he had contributed to the literature, including an article in the Journal of

Periodontology.

an

electron

18. Frank, R., Fiore-Donne, G., Cimasoni, G., and Matter, J.: Ultrastructural study of epithelial and connective gingival reattachment in man. J Periodontol 45: 626, 1974. 19. Morris, M. L.: The effects of root shape and biology on bone growth. J Periodontol 49: 33, 1978. 20. Aleo, J. J., DeRenzis, F. ., Farber, P. ., and Varboncoeur, A. P.: The presence and biologic activity of cementumbound endotoxin. J Periodontol 45: 672, 1974. 21. Aleo, J. J., DeRenzis, F. ., and Färber, P. .: In vitro attachment of hyman gingival fibroblasts to root surfaces. J Periodontol 46: 639, 1975. 22. Jones, W. ., and O'Leary, T. J.: The effectiveness of in vivo root planing in removing bacterial endotoxin from the roots of periodontally involved teeth. J Periodontol 49: 337, 1978. 23. Fine, D. H., Morris, M. L., and Tabak, L.: The characterization and location of endotoxins in periodontally diseased roots (Submitted for Publication) J Periodont Res 1978. 24. Morris, M. L.: Reattachment of periodontal tissue, a critical study. Oral Surg 2: 1194, 1949. 25. Riffle, A. B.: The cementum during curettage. J Periodontol 23: 170, 1952. 26. Melcher, A. H.: Repair of wounds in the periodontium of the rat. Influence of periodontal ligament of Osteogenesis. Arch Oral Biol 15: 1183, 1970.

In Memóriám

Jeffrey following

475

The experimental induction of "functionally" oriented fibers attached to cementum.

Experimental Induction of "Functionally" Oriented The Fibers Attached to Cementum* cavity or filling barely piercing the dentinocemental junction...
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