The role of root surface demineralization in periodontal wound healing

Introduction

Periodontitis causes substantial alterations of the periodontium including loss of supporting alveolar bone, loss of connective tissue attachment to the tooth and apical migration of the junctional epithelium along the root surface. Therapy of periodontitis involves ridding the subgingival area of pathological microorganisms since the primary etiology for the disease is bacterial plaque. This will leave soft tissue and bony defects in the peridontium which may compromise function and prognosis. Furthermore, substantial pathological alterations of the root surface also occur with periodontitis which can detrimentally affect periodontal wound healing.

Surgical management of the anatomical defects produced by periodontitis can be resective, reparative or regenerative in nature. Regeneration of a new periodontium has been an elusive goal and the management of the periodontitis-affected root surface seems to be important to this regenerative process.

The periodontitis-affected root surface

The pathologically exposed root surface is not an appropriate substrate for cell attachment, cementum formation and fiber development, all of which are necessary to achieve periodontal regeneration (^{Polson and Caton, 1982} ; ^{Polson and Proye, 1983} ; ^{Polson et al., 1984} ; ^{Proye and Polson, 1982a} ; ^{Proye and Polson, 1982b}). Pathologic root surface alterations include loss of inserting connective tissue fibers (Sharpey's fibers) (^{Selvig, 1969}), contamination by bacteria and bacterial products (endotoxin) (^{Adriaens et al., 1988}) and changes in normal mineral density and composition (^{Selvig, 1969} ; ^{Selvig and Zander, 1962} ; ^{Selvig and Hals, 1977}).

The normal root is rich in collagen, with extrinsic and intrinsic fibers forming a renewable connection to the adjacent alveolar bone. Plaque induced inflammation destroys these Sharpey's fibers, allowing downgrowth of junctional and pocket epithelium. The root surface is thus exposed to the periodontal pocket and the oral environment. With loss of collagen, the root surface becomes hypermineralized. Bacterial plaque and calculus penetrate into the cementum and dentin of the root. Decalcification and caries can also occur. The root surface thus becomes toxic and unsuitable to support the formation of a new connective tissue attachment necessary for periodontal regeneration.

A critical step in periodontal regenerative therapy is to alter the periodontitis-affected root surface to make it a hospitable substrate to support and encourage migration, attachment, proliferation and proper phenotypic expression of periodontal connective tissue progenitor cells. It has been demonstrated that mechanical instrumentation (scaling and root planning) of the diseased root surface can effectively remove bacteria, calculus and endotoxin contaminated cementum and dentin (^{Garrett et al., 1978} ; ^{Jones and O'Leary, 1978}). However, scaling and root planning leave a smear layer on the root surface which does not favor fibroblast (^{Boyko et al., 1980} ; ^{Boyko et al., 1981}) or connective tissue attachment (^{Proye and Polson, 1982a} ; ^{Proye and Polson, 1982b}). When the smear layer is removed by decalcification the root surface favors both the attachment of fibroblasts (^{Boyko et al., 1981} ; ^{Nalbandian and Cote, 1982}) and new connective tissue attachment (^{Abair et al., 1982} ; ^{Proye and Polson, 1982b}).

Historical perspectives

Acid demineralization in periodontal therapy was first introduced in the 1800's as a substitute for scaling and calculus removal and several prominent dentists advocated its use for this purpose and for removal of hypermineralized cementum (^{Register, 1973} ; ^{Stewart, 1895}). It was also used to aid in pocket eradication (^{Box, 1952} ; ^{Marshall, 1883}). An early histological study by Box indicated that treatment of the root with a citric acid-antiformin combination resulted in new attachment to the root surface by facilitating epithelium exclusion (^{Box, 1952}).

The resurgence of interest in root surface demineralization to promote periodontal wound healing was due to studies by Urist and coworkers (^{Urist, 1965a} ; ^{Urist, 1965b}). These studies indicated that acid demineralized dentin possessed inductive properties. ^{Urist et al. (1967} ; ¹⁹⁷⁰ ; ¹⁹⁷³ ; ¹⁹⁷⁹) demonstrated in a series of experiments that allogeneic dentin matrix, following partial or total demineralization with 0,6 N HCl and transplanted in vivo in various animal models, possessed the ability to induce differentiation of cells. The inductive property was only available after acid demineralization, suggesting that the inorganic component of dentin may obscure potential inductive proteins associated with the organic component.

The results of the studies by Urist encouraged further research to investigate the potential of acid demineralization of root surfaces for periodontal regeneration in in vivo model systems (^{Register, 1973}). Register investigated whether new attachment, cementogenesis and osteogenesis could be induced adjacent to tooth roots demineralized in vivo. On the roots of 21 teeth in various animals, the root dentin was surgically exposed. Using a split-mouth design, the dentin on one-half of the teeth was treated with HCl for 15 minutes and the teeth on the contralateral side were treated with saline and covered with a split thickness flap. Histological analysis demonstrated that the teeth treated with acid healed by connective tissue reattachment, with evidence of accelerated cementogenesis and osteogenesis. By comparison, non-acid-treated teeth demonstrated artifactual separation of the flap from the root surface, incomplete cementogenesis and little or no new osteogenesis. ^{Register and Burdick (1975)}, in a later study, evaluated various acids by their potential to promote new connective tissue attachment occurred when roots were demineralized with citric acid pH 1,0 for 2-3 minutes. These findings have provided the basis for later studies using root surface demineralization in periodontal regeneration attempts in both in vitro and in vivo model systems.

It has been shown that acid demineralization of the instrumented root surface with citric acid (^{Polson et al., 1984}) and phosphoric acid effectively removed the smear layer resulting from instrumentation of the root surface, exposed and enlarged the openings of dentinal tubules (^{Garberoglio and Brannström, 1976} ; ^{Garrett et al., 1978} ; ^{Polson et al., 1984}) and demineralized the intertubular dentin surface (^{Garrett et al., 1978}). The resultant dentin surface exhibited a fibrillar surface texture (^{Polson et al., 1984}), with exposure of the collagen matrix to a depth of 3-10 µm (^{Garrett et al., 1978}). The exposed acid-treated dentin surface was verified by using transmission electron microscopy to be collagenous in nature (^{Garrett et al., 1978}). Citric acid demineralization has also been shown to remove limuluslysate positive endotoxin from the root surface (^{Fine et al., 1980}) and to act as a potent anti-bacterial agent (^{Daly, 1982}).

The acid-exposed collagen matrix of dentin consists primarily of type I collagen (^{Osborn and Ten Cate, 1983}), and along with its degeneration products, type I collagen is chemotactic for polymorphonuclear neutrophilis, macrophages, and fibroblasts (^{Postlethwaite et al., 1978}), provides a substrate for fibrin linkage (^{Lopez, 1984} ; ^{Stahl, 1977}) and can support the attachment and migration of fibroblasts (^{Fardal and Lowenberg, 1990} ; ^{Gauss-Muller et al., 180}). In a wound-healing environment, the presence of a collagen substrate subsequent to acid demineralization of the dentin surface could influence the wound-healing response and serve to enhance chemotaxis, migration and attachment of the cells necessary for connective tissue regeneration.

Studies in animal models

The studies by ^{Register (1973)} and ^{Register and Burdick (1975} ; ¹⁹⁷⁶) were done in surgically created periodontal defects and stimulated other investigators to perform studies to determine if demineralized periodontitis-affected root surfaces behaved similary. ^{Crigger et al. (1978)} and ^{Nilveús et al. (1980)} did flap debridement with and without decalcification on furcation defects in dogs. They reported significant periodontal regeneration within the furcations and complete connective tissue attachment in 55 % and 71 % of sites, respectively. In a similar model, ^{Bogle et al. (1981)} was unable to duplicate these results and reported a frequent occurrence of ankylosis and resorption. The inconsistent findings in this study were attributed by these authors to inadequate flap adaptation and coverage and the chronicity of the lesions.

^{Selvig et al. (1981)} examined the root surface soft tissue interface at various postoperative timepoints following flap surgery with root surface decalcification. By two weeks, the attachment consisted of an interdigitation of exposed fibrils on the demineralized dentin surface with the fibers of the gingival connective tissue. New cementum was formed on the demineralized surface by 42 days.

^{Gottlow et al. (1984)} implanted decalcified periodontitis-affected roots into osseous grooves in edentuous areas of the jaws of dogs. Histological analysis revealed no new connective tissue attachment to either demineralized or non-demineralized root surfaces, but extensive root resorption was a consistent finding. The authors proposed that the lack of new connective tissue attachment was due to the absence of periodontal progenitor cells (^{Melcher, 1976} ; ^{Melcher et al., 1987}). Root resorption was attributed to the repopulation of these roots by cells of gingival and bone origin.

Another study in dogs (^{Pettersson and Aukhil, 1986}) evaluated the effects of citric acid demineralization of the roots in guided tissue regeneration procedures, where cells from the adjacent periodontal ligament were allowed to populate the root surface. After 3 months of healing, the citric acid treated group had more resorption and ankylosis and less cementum formation than the control group. The authors suggested that the application of the citric acid may have damaged the adjacent periodontal ligament cells.

A series of studies by ^{Polson and Proye (1982} ; ¹⁹⁸³⁾ and ^{Proye and Polson (1982a} ; ^1982b) examined the effects of root surface decalcification on the wound healing response. An extraction-reimplanatation model was used in the squirrel monkey. When extracted teeth were root planed and reimplanted, epithelium migrated into the wound from the gingival margin and resulted in the formation of a long junctional epithelium. However, when the planed roots were decalcified with citric acid for 3 minutes prior to reimplantation, a new connective tissue attachment was formed. These authors also described the phenomenon of fibrin linkage which attached the blood clot to the exposed collagen of the dentin, thereby preventing apical migration of the marginal epithelium and allowing connective tissue cells to repopulate the root surface. These results were supported by a series of other studies (^{Hanes et al., 1985} ; ^{Hanes et al., 1987} ; ^{Polson et al., 1986} ; ^{Polson and Hanes, 1987} ; ^{Frantz and Polson, 1988} ; ^{Lowenguth et al., 1993}) in which demineralized and nondemineralized root fragments were inserted into incisional wounds in the backs of rats. A connective tissue attachment formed on the demineralized fragment while nondemineralized fragments were surrounded by epithelium.

Agents other than citric acid have been evaluated for root surface demineralization and have been shown to be safe and efficacious. These include tetracycline HCl (^{Baker et al., 1983} ; ^{Bjorvatn and Olsen, 1982} ; ^{Wikesjö et al., 1986} ; ^{Labahn et al., 1992} ; ^{Claffey et al., 1987} ; ^{Wikesjö et al., 1988}) and EDTA (^{Blomlöf et al., 1995} ; ^{Blomlöf et al., 1995} ; ^{Blomlöf et al., 1996}).

Human studies

Positive results with root demineralization in in vitro and in vivo animal studies stimulated number of human clinical investigations. Most of the studies used a control of root planning alone and showed a wide variation in acid treated sites ranging form marked regeneration to no regeneration. Much of this variation has been attributed to study design, flap management, early wound healing disruption and interpretation of clinical attachment level measurements (^{Gantes et al., 1988} ; ^{Martin et al., 1988} ; ^{Garrett et al., 1990} ; ^{Wikesjö et al., 1992}).

^{Stahl and Froum (1977)} utilized topical application of a citric acid solution on the teeth of two subjects with advanced chronic periodontitis. The surgical areas were managed with a mucoperiosteal flap, scaling and root planing, citric acid demineralization of the roots, and flap replacement. After four months of healing, tissue blocks were taken and evaluated histologically. There was no evidence of new connective tissue attachment or cementogenesis and healing occurred by means of a long junctional epithelium. These disappointing results were attributed to incomplete removal of cementum, since another report (^{Garrett et al., 1978}) suggested periodontitis-affected root cementum may inhibit the effectiveness of acid treatment.

^{Cole et al. (1980)} examined 10 sites in 6 patients with pocket depths greater than 6 mm and subgingival calculus. A notch was placed in the root surface at the apical extent of calculus in order to demonstrate the pathologically exposed root surface. After flap elevation, the roots were thoroughly scaled and treated with pH 1,0 citric acid for 5 minutes.

Histological analysis showed inhibition of epithelial migration along the root surface with new connective tissue attachment and cementum deposition coronal to the notch in all sites. In the majority of sites, new alveolar bone was observed at a level coronal to the notch. In a separate study from the same lab, ^{Steiner et al. (1981)} duplicated the ^{Cole et al. (1980)} protocol with the exception that the roots were not demineralized and thus, provided the control group. Histological results revealed that epithelium migrated to the level of or apical to the notch, with no evidence of new connective tissue attachment or new cementum formation. Together, these two studies form the most compelling evidence for utilizing root surface demineralization was provided by ^{Frank et al. (1983)} and ^{Albair et al. (1982)}, using ultrastructural techniques. Histological studies from another lab (^{Stahl et al., 1983} ; ^{Froum et al., 1983}) did not support these findings, and a human clinical trial (^{Marks and Mehta, 1986}) found no significant differences in probing measurements when acid treated roots were compared to those treated with root planing alone.

Conclusion

Current information indicated a role for root surface demineralization to enhance regeneration of periodontal supporting tissues. The periodontitis-affected root surface does not favor regeneration of the periodontium due to its substrate characteristics.

Demineralization has been shown to alter the diseased root surface creating a more acceptable surface for fibrin (clot) and cell attachment, cell attraction and fiber generation.

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