Oral mucosal carcinogenesis in hamsters: inhibition by vitamin E.

Eighty young adult male and female Syrian golden hamsters (Mesocricetus auratus) were divided into 4 equal experimental groups. In group I animals the left buccal pouch was painted three times weekly with a 0.5% solution of 7,12-dimethylbenz[a]anthracene (DMBA) in heavy mineral oil. In group 2 animals the left buccal pouch was similarly painted with DMBA, but the animals also received orally 10 mg vitamin E (dl-alpha-tocopherol) in peanut oil twice weekly on alternate days with DMBA painting. Group 1 animals received a similar amount of peanut oil vehicle, group 3 animals received only vitamin E (vitamin E controls) in peanut oil, and group 4 animals served as untreated controls receiving only peanut oil. Four animals in each group (2 males and 2 females) were killed at 8, 10, 12, 14, and 16 weeks. Buccal pouches were photographed and excised, and tumors were noted and measured in the left buccal pouches. In group 2 animals receiving vitamin E, tumor formation was significantly delayed, so that by 14-16 weeks there were fewer tumors and their average size was smaller than that of tumors in group 1 animals that were painted with DMBA but received no vitamin E supplement. In group 2 there was also less invasion of underlying tissues and less surface necrosis.

Prevention by vitamin E of experimental oral carcinogenesis.

In the standard model for hamster buccal pouch, using a 0.5% solution of 7,12-dimethylbenz[a]anthracene [(DMBA) CAS: 57-97-6], it was shown that vitamin E (alpha-tocopherol) inhibited carcinogenesis. With a less potent carcinogen (0.1% DMBA), vitamin E was shown to prevent tumor development. Eighty (total) male and female Syrian hamsters (Mesocricetus auratus) were divided into 4 equal groups. After 28 weeks, animals in group 2 that had left buccal pouches painted with 0.1% DMBA (in heavy mineral oil) three times/week and that had been given 10 mg DL-alpha-tocopherol on alternate days (i.e., two times/wk) showed no tumors there. However, the pouches of group 1 animals that had been similarly painted with DMBA but that had received no vitamin E demonstrated grossly and microscopically the presence of epidermoid carcinomas.

Regression by vitamin E of experimental oral cancer.

Vitamin E was shown to regress established epidermoid carcinomas of Syrian hamster buccal pouch in 20 experimental animals following tumor induction by applications three times a week of 0.5% 7,12-dimethylbenz[a]anthracene (CAS: 57-97-6) in mineral oil for 13 weeks. The vitamin E was injected into the tumor-bearing buccal pouch twice weekly for 4 weeks in a dose of 250 micrograms in minimum essential medium. Twenty animals were maintained as untreated controls, and another 20 animals were sham-inoculated vehicle controls. Microscopic examination of buccal pouches with regressed tumor showed small epidermoid carcinomas with degeneration of tumor cells and a dense infiltrate of leukocytes, lymphocytes, and histiocytes. Buccal pouches of control animals showed large well-differentiated or moderately differentiated epidermoid carcinomas. The hamster buccal pouch cancer model presents many similarities to human oral cancer, including expression of the same oncogene, and these results offer hope for the chemotherapy of human oral cancer with the use of a relatively nontoxic agent injected locally.

Retinoid inhibition of experimental lingual carcinogenesis: ultrastructural observations.

Sixty-four male and female Syrian golden hamsters (Mesocricetus auratus) were divided into 4 equal groups. Group 1 animals had the posterior lateral borders of their tongues painted three times weekly with a 0.5% solution of 7,12-dimethylbenz[a]anthracene (DMBA) in acetone. Group 2 animals were painted with DMBA and also received 10 mg of 13-cis-retinoic acid in peanut oil administered orally by pipette twice weekly. Group 3 animals received only retinoid, and group 4 animals were untreated controls. Four animals in each group were killed at 12, 14, 16, and 18 weeks. In the DMBA-painted animals receiving 13-cis-retinoic acid, the leukoplakia and epidermoid carcinomas developed more slowly and were better differentiated microscopically. The better degree of differentiation was demonstrated by ultrastructural studies. Less cellular separation and fewer microvilli-like structures were evident. No tubular mitochondria were observed. Less dispersion of heterochromatin occurred throughout nuclei, and the basal lamina tended to be regular and continuous.

An epidermoid carcinoma cell line derived from hamster 7,12-dimethylbenz[a]anthracene-induced buccal pouch tumors.

w cell line, HCPC I, was established from an epidermoid carcinoma of the Syrian golden hamster cheek pouch. The carcinoma was induced by applications three times a week of a 0.5% solution of 7,12-dimethylbenz[a]anthracene (DMBA) in heavy mineral oil. The HCPC I cell line has been maintained through 60 passages over 15 months. Electron microscopy of the cell line revealed tonofilaments and desmosomes, thereby confirming its epithelial nature. Presence of keratin in the HCPC I cells was demonstrated by both histochemical and indirect immunofluorescence studies. The population-doubling time of HCPC I, estimated from the exponential growth phase of the growth curve established for the cell line, was 12 hours. The plating efficiency of the cell line was estimated at 35%. When an inoculum size of HCPC I, numbering 2.4 X 10(7) cells, was transplanted to the cheek pouch of an immunosuppressed inbred hamster, a papillary tumor, measuring 5 X 1 mm and microscopically diagnosed as a well-differentiated epidermoid carcinoma, developed 1 week post transplantation. Further successful transplantation was achieved with 1.8 X 10(7) HCPC I cells inoculated into the cheek pouch of a nonimmunosuppressed inbred hamster. A raised plaquelike mass, microscopically diagnosed as invasive epidermoid carcinoma, developed in the hamster cheek pouch 5 weeks post transplantation. Furthermore, 5 nonimmunosuppressed hamsters that received 1 X 10(6) HCPC I cells on their cheek pouches developed tumors microscopically diagnosed as anaplastic carcinomas 14 weeks post transplantation. The largest of these tumors measured 2 cm in diameter.

Inhibition of oral carcinogenesis by a protease inhibitor.

The effect of the Bowman-Birk inhibitor (BBI) and soybean trypsin inhibitor (SBTI) on experimental 7,12-dimethylbenz[a]anthracene [(DMBA) CAS: 57-97-6]-induced oral carcinogenesis in Syrian male hamsters was examined. All treatments were applied topically on both cheek pouches for 20 weeks, and the animals were then sacrificed. Gross and microscopic evaluations revealed a statistically significant reduction in the number of invasive carcinomas, the total number of tumors, and the tumor mass for the DMBA + BBI treatment group compared to animals treated with DMBA alone, DMBA and autoclaved BBI (a preparation in which protease inhibitor activity is destroyed), or DMBA + SBTI. A protease activity (with the use of Boc-Val-Pro-Arg-MCA as substrate) was measured and found to be elevated about tenfold in tumorous and nontumorous tissue from DMBA-treated cheek pouches. This protease activity was found to be decreased in the DMBA and BBI treatment group but not in the DMBA + SBTI or DMBA and autoclaved BBI treatment groups, as compared to the protease activity in the DMBA treatment group. Partial characterization of the Boc-Val-Pro-Arg-MCA hydrolyzing activity with diisopropyl fluorophosphate suggests that the proteolytic activity is a serine protease. Iodoacetamide and diethyl pyrocarbonate also inhibit enzyme activity, suggesting that other residues may be necessary for catalysis, possibly including cysteine and histidine. Our results suggest that this protease activity may play a role in DMBA-induced cheek pouch carcinogenesis.

Rapid induction of gamma-glutamyl transpeptidase-rich intraepithelial clones in 7,12-dimethylbenz(a)anthracene-treated hamster buccal pouch.

Individual gamma-glutamyl transpeptidase (GGT)-stained cells and cell doublets were rapidly induced in the epithelium of hamster buccal pouch treated with biweekly topical applications of 0.5%, 7,12-dimethylbenz(a)anthracene (DMBA) in mineral oil. The cells were detected histochemically in whole mounts of pouch epithelium harvested as early as 3 days after the first application of this carcinogen. During 3 consecutive weeks of DMBA treatment, progressively larger GGT-stained epithelial cell populations (plaques) up to 0.5 mm in diameter were encountered. Similar GGT-stained lesions were not detected in whole mounts of untreated epithelium and were rarely seen in GGT-stained whole mounts prepared from mineral oil-treated pouch epithelium. In an experiment designed to assess the stability of the GGT-staining pattern, very few plaques could be detected 12 weeks after a 3-week regimen of six DMBA applications. However, data are presented suggesting that a brief series of three DMBA applications reinduced GGT histochemical activity in occult intraepithelial plaques, which had los enzyme activity but had persisted over an 11-week treatment-free interval. The clonal nature of the GGT-stained plaques for their apparent ability to persist in an occult form for several weeks or months lend further support to the hypothesis that these carcinogen-altered cell populations may be potential precursors for the development of squamous epithelial neoplasia.

Transforming growth factor alpha in chemically transformed hamster oral keratinocytes.

The cheek pouch of the Syrian hamster is an excellent tissue for the experimental induction of oral cancer by carcinogenic chemicals. Lysate prepared from a cell line (HCPC-1) derived from one of these hamster oral tumors greatly increased the growth of these oral tumor cells in vitro. We now show that the mitogenic substance, transforming growth factor alpha (TGF-alpha), is present in all of the chemically transformed hamster oral tumors examined (in vitro and in vivo). In no adult normal tissue of the Syrian hamster can we detect expression of TGF-alpha. TGF-alpha could be partly or wholly responsible for the mitogenic activity detected in the lysate of the chemically transformed hamster oral keratinocytes. Both normal and chemically transformed hamster oral keratinocytes express the receptor to epidermal growth factor. The consistent detection of TGF-alpha and epidermal growth factor receptor mRNAs in these hamster oral tumor cells suggests that an autocrine growth mechanism might be operative. This hamster cheek pouch oral cancer model can be used for the molecular analysis of how TGF-alpha and epidermal growth factor receptor might be involved in the malignant transformation of epithelial tissues.

Detection of Ki-ras messenger RNA in normal and chemically transformed hamster oral keratinocytes.

The cheek pouch of the Syrian hamster is an excellent model for the experimental study of oral carcinogenesis. The carcinogenic chemical 7,12-dimethylbenz[a]anthracene consistently produces epidermoid carcinomas in the cheek pouch of the Syrian hamster, giving rise to characteristic histopathological lesions in a time-dependent manner. We now present experimental evidence that c-Ki-ras mRNA can be detected in all 7,12-dimethylbenz[a]anthracene-induced tumors examined (in vivo and in vitro) in this experimental oral cancer model while no detectable c-Ki-ras mRNA can be found in the normal hamster cheek pouch epithelium. Cellular synchronization experiments using a cell line (hamster cheek pouch carcinoma cell line 1) derived from one of these 7,12-dimethylbenz[a]anthracene-induced hamster oral tumors revealed that the c-Ki-ras protooncogene is expressed during the G1 phase of the cell cycle (proliferation dependent). Serum starvation and RNA synthesis inhibition experiments using hamster cheek pouch carcinoma cell line 1 cells suggest that the c-Ki-ras protooncogene is indeed quiescent in the normal hamster cheek pouch epithelium and that failure to detect its mRNA is not related to the slower proliferation of the normal epithelial cells. These results suggest that the transcription of the c-Ki-ras protooncogene is associated with malignant transformation in the cheek pouch of the Syrian hamster.

A light and electron microscope study of aging parotid and submandibular salivary glands of Swiss-Webster mice.

Light and electron microscope comparisons were made of parotid and submandibular glands from male Swiss-Webster white mice 3, 13, and 18 months old. The glands from the 13- and 18-month-old mice were less organized and the parenchyma was not as dense. Fibrous connective tissue, intracellular lipofuscin granules, and residual body formation increased with age. In the cells of the parotid glands of 18-month-old mice, the nucleus-to-cytoplasm ratio was greater than in the specimens from the younger two ages. The granular convoluted tubules in submandibular glands of 18-month-old mice were the smallest of all age groups. The age changes appear comparable to those of rat and human salivary glands, yet this is an inexpensive animal model that achieves old age in less time than other animal models.

A common pathway for the destruction of cancer-cells - experimental-evidence and clinical implications.

Chemopreventatives derived from different classes of chemical inhibit the growth of various cancers. This review concentrates on the chemopreventative activities of carotenoids, retinoids, and tocopherols. In this review we outline a common pathway for the antitumor action of chemopreventative agents. To establish this pathway we reviewed previous in vivo and in vitro studies indicating that the underlying modes of actions for chemopreventative activities were oxidative-reduction changes that developed in the tumor cell. Some of these changes were selective and did not appear in normal cells. The oxidative redox characteristics of the agents interacted with the oxidative redox systems of the tumor cell. Many chemopreventative agents are highly hydrophobic and interact with thiol peptides, and/or metalloproteins, such as glutathione reductase, superoxide dismutases, or flavoproteins. These could then become linked to prenylated membrane associated protein complexes or oxidized proteins. Interactions of the chemopreventative agents with these proteins can result in changes in expression of transcription factors nuclear binding proteins, such as, heat shock proteins (hsps) and p53. Dramatic reductions in mutant p53 and increased expression of wild type (antioncogene) p53 protein product has been associated with some chemopreventative agent inhibition of oral carcinogenesis. Enhanced hsp 70 and 90 expression has also been observed. These changes in protein expression occurred early during the process of oral carcinogenesis. The tumor cells accumulated in GO/G1 of the cell cycle and the induction of some apoptotic like changes (programmed cell death) were noted. Cell to cell communication and differentiation has also been increased with carotenoid, retinoid, and tocopherol treatment of transformed cells. Enhanced cytotoxic responses by macrophages and T lymphocytes at developing tumor sites were found to produce the oxygen reactive cytokine, tumor necrosis factor. The experimental evidence and hypothesized common pathway of antitumor action for chemoprevention may point to possible biomarkers that could be utilized in clinical intervention trials.

Sequential mast cell infiltration and degranulation during experimental carcinogenesis.

Mast cell density, distribution, and ultrastructure were studied by light and electron microscopy in hamster buccal pouches undergoing chemically induced carcinogenesis. Epidermoid carcinomas in the pouches were induced by three topical applications per week of 0.5% 7,12-dimethylbenz[a]anthracene (DMBA) in oil using a brush. Four experimental, DMBA-treated and two normal, untreated hamsters were sacrificed after 8, 10, 12, 14, and 16 weeks. After 8 weeks of DMBA treatment, the epithelium showed the pathological signs of dysplasia and hyperkeratosis. In the dermis an increased number of mast cells were evident, some of which showed degranulation. A few mast cells had started to migrate upwards towards the dysplastic epithelium after 10 weeks of DMBA treatment. Rapid degranulation was also apparent in some mast cells. These processes of upward migration and degranulation continued progressively during the 12- and 14-week periods of DMBA application in correlation with the progression of the tumor. By 16 weeks of treatment with the carcinogen, more mast cells had migrated closer to the invasive carcinoma, and many had degranulated. In the connective tissue mast cells were fully packed with many granules, and some mast cells were in proximity to macrophages and eosinophils. Our observations demonstrate that there is a positive correlation between developing carcinomas and mast cell density. Mast cell migration towards the carcinoma and degranulation were also evident.

A histologic study of epithelial dysplasia in oral lichen planus.

One hundred cases of oral lichen planus were reviewed together with 100 nonspecific oral mucosal inflammatory lesions as a control group. The presence of dysplasia was noted, using well-established histologic criteria. Mild dysplasia was found in 57% of cases, moderate dysplasia in 9%, and severe dysplasia in 2% of cases. In the control group, mild dysplasia was observed in 32% of cases, moderate in 10%, and severe dysplasia was not present. It is suggested that, while mild or moderate dysplasia may not indicate precancerous potential, severe dysplasia in lichen planus may signify the development of a precancerous lesion.

Histopathology of oral lesions of discoid lupus erythematosus. A review of 25 cases.

In a study of 25 oral biopsy specimens of lesions of chronic discoid lupus erythematosus, a characteristic and diagnostic histopathological pattern was confirmed, which consisted of parakeratosis, hydropic degeneration of stratum germinativum, collagen degeneration and a lymphocytic infiltration in a perivascular orientation. It is suggested that more oral biopsies be used in the diagnosis of discoid lupus erythematosus because of the ease of oral mucosal biopsy technique and the absence of scarring and discomfort from oral biopsy lesions.

Alpha tocopherol alters the distribution of Langerhans cells in DMBA-treated hamster cheek pouch epithelium.

Thirty-seven adult male and female golden hamsters (Mesocricetus auratus) were divided into four experimental groups. In Group A, the animals served as untreated controls, having the left buccal pouches painted with mineral oil. In Group B, the animals received 10 mg vitamin E (alpha tocopherol) in peanut oil by the oral route, with a fine pipette, twice weekly. In Group C animals, the left buccal pouch was painted three times weekly with DMBA (0.5% solution of 7,12 dimethylbenz(a)anthracene in heavy mineral oil). Group D animals received both vitamin E and DMBA in the amounts indicated for Groups B and C, with the vitamin E being administered on days alternate to the DMBA painting, also in the manner described for the above groups. All animals were killed after eight weeks of treatment. Epithelial whole mounts were prepared from the left buccal pouches. These specimens were then stained for ATPase to demonstrate the presence of Langerhans cells (LCs). A notably decreased density of LCs was observed after treatment with DMBA. Vitamin E administration in addition to DMBA treatment resulted in a less dramatic decrease in LC density. Since vitamin E has been shown to retard experimental oral carcinogenesis, vitamin E may retard carcinogenesis by maintaining the number of Langerhans cells.

Mechanisms of cancer inhibition by anti-oxidant nutrients.

The cancer inhibitory properties of anti-oxidant micronutrients have been well established in experimental animal models and cell culture studies. Human studies have also tended to indicate an inhibition of various forms of cancer and the regression of some precancerous lesions. The biological mechanisms for cancer inhibition and regression are now gradually becoming understood, and the anti-oxidant nutrients appear to act through a number of pathways common to most of the agents studied. These various micronutrients appear to act through a complex group of "common pathways" of anticancer activity based upon three major mechanisms: (1) tumour inhibition by immune cytokines; (2) stimulation of cancer suppressor genes, such as "wild type" p53, and diminished expression or dysregulation of oncogenes such as mutant p53 and H-ras; (3) inhibition of tumour angiogenesis through the inhibition of angiogenesis-stimulating factors such as TGF alpha. Retinoid action differs, in some respects, from other micronutrient anticancer mechanisms and appears to relate to its stimulation of cellular differentiation and resultant apoptosis of neoplastic cells. Combinations of anti-oxidant nutrients have been shown to be synergistic in their anticancer activity, probably due to their optimal anticancer activity at different oxygen potentials. Selectivity in the action on cancer cells, as opposed to normal cells, is a major feature of the anti-oxidant micronutrients.

Verification in syngeneic hamsters of in vitro transformation of hamster oral mucosa by 7,12-dimethylbenz(a)anthracene.

The carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) has been used to induce oral carcinogenesis of the hamster buccal mucosa in an experimental model that exhibits many of the genetic, biochemical and pathological features of human oral squamous cell carcinoma. To complement this in vivo process we have established an in vitro transformation procedure that involved the treatment of normal hamster oral mucosal keratinocytes (NHKs) with DMBA. Uptake of DMBA in NHKs was verified by observing autofluorescence of DMBA in the oral mucosal cells. Treatment doses ranged from 5, 50 and 200 ng and the NHKs were generally treated with DMBA for 1-14 days. The 200 ng dose proved to be toxic to these cells. The 5 and 50 ng treatments were found to maintain the viability of the NHKs and demonstrate anchorage-independent agarose growth, producing 18 and 40 colonies, respectively, after 14 days of treatment. Characterisation assays included determinations for cellular growth through plating efficiency, counting of cell colony number, and 3H-thymidine incorporation. Differentiation was ascertained by counting cornified cells, specification of either high or low molecular weight keratins, the percentage of cells expressing gamma glutamyl-transpeptidase (GGT), the level of p53 expression, and a determination of cell cycle. After 24 h the plating efficiency of the NHKs was found to be slightly increased following treatment with a 5 or 50 ng dose of DMBA compared to the untreated NHKs. After 14 days of incubation these doses also enhanced the number of colonies formed by the NHKs (e.g. plating efficiency). In contrast, the number of cornified cells was reduced in these colonies, while immunohistochemistry disclosed an increase in the number of NHKs expressing low molecular weight keratins, significantly lower levels of high molecular weight keratins and high levels for GGT. Flow cytometric analysis verified an increase in p53 expression (e.g. p53 wild type, 19% and p53 mutant, 66%). Cell cycle analysis of NHKs treated with DMBA (5 ng) demonstrated a shift in the number of cells in S phase (17.2%) and G2 + mitosis (11.0%). Cells from this DMBA treatment group were injected into syngeneic hamster recipient buccal pouches (10 x 10(6) cells/0.25 ml). Squamous carcinomas grew in four of six hamster buccal pouches as determined by histopathological analysis. The in vitro assay system will enhance our ability to define the genetic and molecular changes related to chemical carcinogenesis and oral malignant transformation.

Experimental oral carcinoma of the tongue and buccal mucosa: possible biologic markers linked to cancers at two anatomic sites.

The application of the carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) can initiate and promote the development of oral squamous cell carcinoma of the tongue and buccal mucosa. In this study the level of expression of various markers related to the development of programmed cell death (PCD) in the respective oral carcinomas was analyzed. Sixteen male and female Syrian hamsters (Mesocrietus auratus) were treated with 0.05% DMBA for 16 weeks. Immunohistochemistry was used to observe the expression of p53, proliferating cell nuclear antigen (PCNA), Bcl-2, and nucleosome formation. Single-strand conformational polymorphism (SSCP) for exons 2-9 and sequence analysis of exon 9 of the p53 gene from normal buccal or tongue mucosa as well as the squamous cell carcinomas from the buccal mucosa or the tongue were determined. p53 (wild type) expression was significantly reduced in the tongue dysplastic mucosa or squamous cell carcinoma. The SSCP disclosed banding shifts or new bands in exons 2/3, 4, 8, and 9 for the tongue or buccal oral carcinomas (five of each). In exon 9 the mutation in codon 307 (ala)GCC-GTC(val) was present in the tongue but not in the buccal carcinoma. Other markers included the level of PCNA. PCNA was initially lower in the premalignant tongue lesions but increased in oral squamous cell carcinoma at both sites. In contrast, the amount of nucleosome formation in the tongue carcinomas was less than the level noted for buccal cancers but premalignant dysplasias in the tongue mucosa exhibited higher levels. The inhibitor of PCD, Bcl-2 was lower for dysplasias and carcinomas of the tongue compared to similar lesions of the buccal mucosa. These results indicate that oral carcinomas of different anatomical sites can exhibit differences in growth, oncogene mutation expression, and the development of PCD. The differences in Bcl-2 and nucleosome formation may signify their influence on oncogene expression and growth potential for developing transformed clones and established oral carcinomas.

The effect of vitamin E on the healing of gingival wounds in rats.

Forty male and female albino rats received a standardized gingival wound (gingivectomy) between the mandibular incisor teeth. One half of the animals received 60 I.U. of d-alpha-tocopheryl acetate daily, administered orally by pipette. An additional control group of 20 animals was not wounded and half of these animals received 60 I.U. of d-alpha-tocopheryl daily. Four animals in each of the two gingivectomy groups (Groups 1, 2) were sacrificed at periods of 1, 2, 4, 7 and 14 days following gingivectomy. Two animals in each of two control unwounded groups (Groups 3, 4) were sacrificed at similar times. Gingival healing was studied grossly and histologically. The animals receiving the vitamin E supplements healed more rapidly, with almost complete restoration of gingiva by 7 days. Complete healing was seen in both control and experimental groups by 14 days. Vitamin E was shown to accelerate gingival wound healing in experimental animals.

Healing of spontaneous periodontal defects in dogs treated with xenogeneic demineralized bone.

This study was undertaken to histologically, clinically and radiographically evaluate the sequence of healing following implantation of bovine demineralized bone powder (DBP) into severe, spontaneous periodontal defects in beagle dogs. Eight dogs with documented severe periodontitis were treated surgically following initial debridement. One quadrant in each arch was treated with conventional flap surgery and the others were treated with surgery followed by DBP implantation. Animals received postoperative debridement and clinical and radiographic evaluation. Two dogs were sacrificed at 1, 3, 6 or 12 months postoperatively, and the jaws were evaluated histologically. Clinically, DBP was well tolerated by recipients. No evidence of localized inflammatory response or delayed hypersensitivity reaction was noted. Significant reductions in gingival inflammation were noted in both experimental and control sites at 1 month postoperatively compared to preoperative scores. Equivalent periodontal pocket reduction was noted between test and experimental sites and remained significant at 12 months. Radiographically, no differences were noted in the rate of bone loss between control and test sites. Histologic evaluation demonstrated the presence of DBP at 1 month following implantation, but the material was replaced with new bone by the next sacrifice period. Periodontal ligament fibers of standard orientation were seen extending from DBP-induced bone to the root surface by 1 month after implantation. An intact epithelial attachment appeared to be present 1 month after the implantation of DBP. No differences in root surfaces were detected between test and control groups. Ankylosis was a rare finding, noted equally between test and control sites. DBP did not appear to predispose to external root resorption. In later stages, histologic evidence of advancing periodontitis was noted equally in both control and experimental groups. While DBP successfully induced new bone formation, the inability to adequately maintain the periodontal tissues due to bacterial accumulation in this model combined with recurrent pocket formation, precluded any conclusion regarding long-term advantage. Based on these findings, clinical trials of this or similar materials are recommended.