Formation of Dental Hard Tissues and Periodontal Ligament Around

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Formation of Dental Hard Tissues and Periodontal Ligament Around Titanium Implants After Tooth-Bud Injury: A Pilot Study Adriano Piattelli, MD/Gian Piero Cordioli, MD/Piero Passi, MD/Paolo Trisi, DDS, PhD

This pilot study was done to evaluate the potential of the different cells of the forming tooth bud to induce formation of dental hard tissues and periodontal ligament around titanium implants. Deciduous premolar teeth were extracted in a 3⁄-month-old Landrace pig 1 month before implant placement. Four implants were placed in a pig's mandible near the tooth buds. A wide artificial bone defect was created around one of the implants; all implants were covered with Gore-Tex membranes. After 3 months, the animal was sacrificed, and the implants were retrieved and studied with the cutting-grinding system. Histologic analysis showed that all implants were covered by a mineralized tissue with a histologic appearance similar to cementum and separated from the bone by a periodontal-like space filled by collagen fibers. (INT J ORAL M AXILLOFAC IMPLANTS 1994;9:417-421) Key words: cementum, periodontal ligament, titanium implants

The role of cementum is very important in the physiology of healthy periodontium, since it functions as an anchoring substance for the tooth to the alveolar bone.1 Precursor cells for cementum or alveolar bone seem to originate from periodontal ligament cells1,2 that derive from mesenchymal cells in the dental sac.2 Follicle cells are able to produce alveolar bone, periodontal ligament, and cementum when transplanted in an extraoral site.3,4 McCullough and Melcher5 observed that wounding could raise the percentage of periodontal ligament cells up to 60%. Numerous authors now agree that regeneration of cementum and fiber attachment may originate from periodontal ligament cells.6,7 Melcher7 suggested that in the healing phase the root surface could be colonized by cells arising from different sources (epithelium, gingival connective tissue, periodontal ligament, and bone), and that the cells of the periodontal ligament were implicated in the integrity of fibers, bone, and cementum. Melcher7 also postulated that during healing, the cells present in the wound were extremely important in determining the nature of attachment. The same author also wrote that "if surgical procedures could be designed such that both periodontal ligament and bone would be allowed to migrate coronally, then the cells with the capacity to regenerate and maintain the periodontium would colonize the wound." Repopulation of the root surface by cells different from those arising from JOMI on CD-ROM, 1994 Apr (417-421 ): Formation of Dental Hard Tissues and Periodo… Copyrights © 1997 Quinte… the periodontal ligament would prevent the formation of new attachment; clearly, this should be avoided if surgical procedures aimed at the regeneration of new attachment are carried out.7 The absence of cementum progenitor cells from the sites prepared for implants can explain the lack of cementum on endosseous implants.2,8 Absence of a periodontal ligament around implants produces difficulties in connecting implants and natural teeth for prosthetic restorations because of their different mobilities. Even though it is possible to connect implant and teeth without any special attention, the bony ankylosis of implants does not give the patient contact and load sensations provided by the periodontal ligament, because of the lack of proprioceptors found in the periodontium. Previous studies demonstrated the formation of a periodontal ligament around different types of titanium implants.8-10 In these studies, periodontal ligament cells derived from the root tip of teeth in direct contact with the implant were able to produce cementum and collagen fibers on titanium. The aim of our study was to evaluate the potential of the different cells of the forming tooth bud to induce formation of dental hard tissues and periodontal ligament around titanium implants. Materials and Methods One 3⁄-month-old Landrace pig weighing approximately 40 kg was used in this pilot study. The deciduous premolar teeth were extracted one month before the implant placement. Under general anesthesia with Ketalar (Parke-Davis, Morris Plains, NJ), a mucoperiosteal flap was elevated in the middle of the alveolar crest and the implant sites were prepared using profuse saline irrigation. The underlying tooth buds were disrupted using a low-speed bur. Two titanium plasma-sprayed implants (10 mm, Biomax, Vicenza, Italy) were placed on each side of the mandible (Fig 1). One of the four sites, on the right side, was prepared with a wide artificial bone defect, but the implant did demonstrate primary stability. Expanded polytetrafluoroethylene (ePTFE) membranes (Gore-Tex, W.L. Gore) were placed over the crestal bone on both sides of the mandible (Fig 2) and the mucosa was sutured with Gore-Tex sutures (W.L. Gore). The animal was allowed to heal with a normal diet, and no antibiotics were provided. After 3 months, the animal was sacrificed with an overdose of pentothalsodium and block sections of the mandible were obtained to be processed for histology. The specimens were dehydrated in an ascending series of alcohols and embedded in Technovit 7200 methacrylate resin (Kulzer, Wehrheim, Germany). After polymerization, blocks were sectioned with a diamond saw at 200 µm and ground down to from 30 to 40 µm, according to the technique proposed by Donath and Breuner.11 The slides were then stained with basic fuchsin-methylene blue and von Kossa-basic fuchsin, the latter to evaluate mineralized tissues. Slides were then observed in a Laborlux S microscope (Leitz, Wetzlar, Germany) in normal JOMI on CD-ROM, 1994 Apr (417-421 ): Formation of Dental Hard Tissues and Periodo… Copyrights © 1997 Quinte… transmitted and polarized light. Results Clinically, all four implants healed without signs of inflammation, but the membranes were exposed in the oral cavity 1 week after placement. The implant with the bone defect was exposed in the oral cavity and was colonized by plaque; it showed a grade 2 mobility and a pocket depth of 4 mm. The other three implants were not exposed in the oral cavity. Histologic sections showed that none of the implants presented the features of osseointegration. The follicles of the permanent teeth present were almost completely disrupted and islands of dental tissues were randomly distributed in the jawbone. The titanium implants were surrounded by a mineralized tissue that attached directly to the metal surface, but was separated from the surrounding bone by a layer of connective tissue. In some areas, this mineralized tissue showed a structure similar to dentin, while in other areas, this tissue had the features of root cementum with a thickness varying from 50 to 60 µm to 1 mm (Fig 3 ). In other zones, this mineralized tissue contained small marrow spaces similar to bone marrow spaces. Cellular lacunae were always present. In the connective tissue surrounding the implant, oriented collagen fibers connected the mineralized tissue on the implant to the jawbone. In some areas, a periodontal ligament-like space was present, while in other areas, wider spaces were interposed between bone and the new cementum-like tissue formed on the implant surface (Fig 4). The implant with the large bone defect was open in the oral cavity and a long junctional epithelium lined the more coronal part of it. The deeper part of this implant showed the formation of a very thin layer of mineralized tissue attached to the implant surface with collagen fibers inserting perpendicularly on it. Trabecular bone was not in contact or at a distance from the implant and collagen fibers were directed towards the epithelium or were dispersed in the soft tissue. A striking finding was the observation of a periodontal-like connective attachment on the implant, at the bottom of the epithelial pocket. In fact, on one side, the epithelium penetrated deeply around the implant, while on the opposite side, the presence of a small layer of a mineralized tissue on the titanium surface, into which collagen fibers were inserted, blocked the apical migration of the epithelium (Fig 5). As not all of the titanium surface was surrounded by mineralized tissue, collagen fibers also seemed to insert perpendicularly on the bone free metal surface (Figs 4 to 6). Enamel was never seen attached directly on the titanium surface, but in one case ameloblasts and enamel coming from a tooth bud were separated from the metal by a small gap (Fig 7). Transverse sections of one of the four implants showed that half of it was in contact with a tooth bud. In this area, collagen fibers surrounded the titanium and inserted perpendicularly on the cementum-like tissue adherent to the metal. On the other half of the implant, bone trabeculae were in contact with the metal. No signs of resorption were seen in the mineralized tissues. Signs of inflammation, with the presence of polymorphonuclear leukocytes and lymphocytes, JOMI on CD-ROM, 1994 Apr (417-421 ): Formation of Dental Hard Tissues and Periodo… Copyrights © 1997 Quinte… were seen in the soft tissues underneath alveolar mucosa. Discussion The pig was used in this investigation because of its low cost and because the animal has a series of dental elements almost completely similar to humans. Our study was done to evaluate the potential role of tooth-forming cells in the healing around dental implants. Implants were placed in the bone near tooth buds disrupted during implant placement. The rationale for our experiment was based on the fact that, theoretically, all cells involved in tooth development and the cells derived from the bone were potentially able to participate in the healing process around implants. Gore-Tex membranes were placed to exclude epithelial and lamina propria cells from the healing process. Although trauma resulting from mastication produced membrane exposure, a dehiscence of one of the implants, and a slight inflammatory reaction in the soft tissues, the implants healed without clinical signs of infection. The histologic analysis showed that mineralized tissue covered an unpredictable area of the implant surface. The tissue was not alveolar bone, because bone was separated from the implant by an inconstant periodontal-like space filled by collagen fibers. Because of the variable thickness of this mineralized tissue, it could not be stated that a layer of cementum formed on the implant surface. Nevertheless, the histologic features of the tissue were very similar in many areas to root cementum, with no osteonic lamellar arrangement even if in some areas it was possible to observe small medullary cavity spaces characteristic of alveolar bone. Possible terms to describe this tissue formed on the implant surface could be "osteocementum" or "dystrophic cementum," similar to the tissue found in the cementum-producing lesions of the jawbones. Another interesting observation was the absence of bone ankylosis, probably because of production of a tissue inhibiting bone growth by cementoblasts. Since the implants had been placed with good primary stability, an initial direct bone contact should have been achieved. This bone could have been resorbed, as no direct bone contact was visible at the interface with the implants. An alternative explanation could be that the portions of the implants covered by bone were not in the slides examined. Moreover, it was possible to observe the presence of cementum and collagen fibers on the orally exposed loaded implant. This implant was probably mobilized as a result of mastication forces, but areas of mineralized tissue with oriented collagen fibers were able to maintain the implant in situ and inhibited epithelial downgrowth. Other studies have demonstrated the formation of periodontal tissue around dental implants. Buser et al8 were able to induce cementum migration on titanium adjacent to tooth roots. Knofler et al12 demonstrated the same in rabbits and Kirsch13 in humans. From the aforementioned studies, it seems that direct contact between an artificial implant and periodontal ligament cells should be reached to allow JOMI on CD-ROM, 1994 Apr (417-421 ): Formation of Dental Hard Tissues and Periodo… Copyrights © 1997 Quinte… cementum growth. From the results of our study, it would appear that periodontal ligament cells have the most rapid and active potential to repair the traumatic injury between the tooth-forming cells. These cells seem to inhibit direct bone contact on the implant, thereby allowing the formation of periodontal fibers. Once the structures were formed, they could withstand occlusal load and movements inhibiting epithelial downgrowth, as was observed in the implant exposed to the oral cavity. More studies are certainly needed to confirm these observations and to try to obtain a functional periodontal ligament around dental implants. Acknowledgments This work was partially supported by the Italian Ministry of University, Research, Science and Technology (MURST 60%), and the National Research Council. JOMI on CD-ROM, 1994 Apr (417-421 ): Formation of Dental Hard Tissues and Periodo… Copyrights © 1997 Quinte…

1. Engel LD, Page RC. The normal and diseased periodontium and periodontal disease activity. Curr Opin Dent 1991;1:4-11. 2. Listgarten MA, Lang NP, Schroeder HE, Schroeder A. Periodontal tissues and their counterparts around endosseous implants. Clin Oral Impl Res 1991;2:1-19. 3. Ten Cate AR, Mills C. The development of the periodontium: the origin of alveolar bone. Anat Rec 1971;173:69-77. 4. Ten Cate AR, Mills C, Salomon G. The development of the periodontium: a transplantation and autoradiographic study. Anat Rec 1971;170:365-379. 5. McCullough CA, Melcher AH. Continuous labelling of the periodontal ligament of mice. J Periodont Res 1983;18:231-244. 6. Melcher AH. On the repair potential of periodontal tissues. J Periodontol 1976;47:256-260. 7. Melcher AH. Repair of wounds in the periodontium of the rat. Influence of periodontal ligament on osteogenesis. Arch Oral Biol 1970;15:1183-1204. 8. Buser D, Warrer K, Karring T. Formation of a periodontal ligament around titanium implants. J Periodontol 1990;61:597-601. 9. Warrer K, Karring T, Gotfredsen K. Periodontal ligament formation around different types of dental titanium implants. I. The self-tapping screw type implant system. J Periodontol 1993;64:29-34. 10. Buser JD, Warrer K, Karring T, Stich H. Titanium implants with a true periodontal ligament: An alternative to osseointegrated implants? Int J Oral Maxillofac Implants 1990;5:113-116. 11. Donath K, Breuner G. A method for the study of undecalcified bones and teeth with attached soft tissues. J Oral Pathol 1982;11:318-326. 12. Knofler W, Knofler G, Hampel H, Bethmann W. Über die Bildung einer Zementschicht und eines regelrechten Periodontiums um eines enossales Implantat im Kaninchenkiefer. Zahn Mund Kieferheilkd 1983;71:349-357. 13. Kirsch A. Titan-spritzbeschichtetes Zahnwurzelimplantat unter physiologischer Belastung beim Menschen. Dtsch Zahnarztl Z 1980;35:112-114. JOMI on CD-ROM, 1994 Apr (417-421 ): Formation of Dental Hard Tissues and Periodo… Copyrights © 1997 Quinte…

Fig. 1 Titanium implants placed in pig mandible. The surgical defect created around one implant is visible.

Fig. 3 Polarized light micrograph of tissues surrounding the implant. On one side, the tissue has features similar to dentin, while on the other side, it resembles cementum. Bar = 250 µm.

Fig. 4 In the most apical region, oriented collagen fibers, originating from the alveolar bone, seem to penetrate perpendicularly the titanium plasma-sprayed surface. Bar = 250 µm. JOMI on CD-ROM, 1994 Apr (417-421 ): Formation of Dental Hard Tissues and Periodo… Copyrights © 1997 Quinte…

Fig. 5 High-power polarized micrograph of mineralized tissue, demonstrating oriented collagen fibers attached to titanium and to the newly formed tissue. Bar = 50 µm.

Fig. 6 High-power microscopic view of the mineralized tissue. Oriented Sharpey's collagen fibers penetrate perpendicularly in this mineralized tissue, enabling a functional orientation. Note that some fiber bundles come in direct contact with the titanium surface. Bar = 100 µm. JOMI on CD-ROM, 1994 Apr (417-421 ): Formation of Dental Hard Tissues and Periodo… Copyrights © 1997 Quinte…

Fig. 7 A transverse section demonstrates that, in some areas, the implant came in contact with the epithelial component of a tooth bud. Bar = 250 µm.