Proteomic analysis of protein components in periodontal ligament fibroblasts.

BACKGROUND: Characterization of periodontal ligament (PDL) fibroblast proteome is an important tool for understanding PDL physiology and regulation and for identifying disease-related protein markers. PDL fibroblast protein expression has been studied using immunological methods, although limited to previously identified proteins for which specific antibodies are available. METHODS: We applied proteomic analysis coupled with mass spectrometry and database knowledge to human PDL fibroblasts. RESULTS: We detected 900 spots and identified 117 protein spots originating in 74 different genes. In addition to scaffold cytoskeletal proteins, e.g., actin, tubulin, and vimentin, we identified proteins implicated with cellular motility and membrane trafficking, chaparonine, stress and folding proteins, metabolic enzymes, proteins associated with detoxification and membrane activity, biodegradative metabolism, translation and transduction, extracellular proteins, and cell cycle regulation proteins. CONCLUSIONS: Most of these identified proteins are closely related to the extensive PDL fibroblasts' functions and homeostasis. Our PDL fibroblast proteome map can serve as a reference map for future clinical studies as well as basic research.

The effect of centrifugal force on mRNA levels of collagenase, collagen type-I, tissue inhibitors of metalloproteinases and beta-actin in cultured human periodontal ligament fibroblasts.

BACKGROUND: The aim of orthodontic treatment is to relocate teeth abnormally positioned in the jaws. This is achieved by application of continuous force on the tooth, which is immediately being sensed by the periodontal ligament (PDL), bone and the gingiva. Since the bony response is mediated by the PDL, tooth movement is primarily a PDL phenomenon. OBJECTIVES: Thus, the purpose of the present study was to evaluate the direct effect of force (excluding the in vivo tissue response) on the molecular level of matrix metalloproteinase-1 (MMP-1) and collagen type-I (Col-I) in human PDL fibroblasts. METHODS: PDL cell culture flasks were centrifuged for 10, 20, 30, 60, 90 and 120 min by horizontal microplate rotor. The effect of force on mRNA levels of beta-actin, MMP-1, Col-I, tissue inhibitors-1 and -2 (TIMPs) genes was analyzed by RT-PCR. RESULTS: The results showed that force had no effect on the mRNA levels of beta-actin during the first 90 min of application of force, indicating for the first time the use of beta-actin gene as an internal invariant control. It increased the mRNA levels of MMP-1 while almost no effect on Col-I and TIMPs was observed. CONCLUSIONS: The results indicate that PDL remodeling following application of orthodontic force could be partly attributed to the direct effect of the force on MMP-1 gene expression in fibroblasts.

Bone morphogenetic protein 2 induces the expression of cementum attachment protein in human periodontal ligament clones.

Cementum is continuously formed during the lifetime of a tooth. The paravascular zones in the adult periodontal ligament (PL) comprise the progenitors for the fibroblastic (Fb) lineage and mineralized tissue-forming (MTF) cell lineages--the osteoblastic (Ob) and cementoblastic (Cb) lineages. Recent studies indicate that cementum attachment protein (CAP) is related to the differentiation of the Cb lineage and is instrumental in differentiating between the three periodontal cell lineages. The purpose of this study was to assess the effect of bone morphogenetic protein 2 (BMP2) on the expression of cementum attachment protein (CAP) and on the differentiation of cloned PL progenitors. The effect of BMP2 on CAP expression and on the differentiation of cloned Fb and MTF progenitors was tested by assessing the expression of alkaline phosphatase (ALP), CAP, and bone sialoprotein (BSP) by immunochemistry and by determining the CAP-binding capacity of these clones. Untreated Fb clones were negative for all tested markers and had low CAP-binding capacity. Untreated MTF clones had a high CAP-binding capacity and were positive for the three markers. BMP2 enhanced the CAP-binding potential of both Fb and MTF clones. BMP2 induced the expression of CAP, ALP, and BSP in the Fb clones and enhanced the expression of CAP and BSP in the MTF clones. These results indicate for the first time that BMP2 can recruit progenitors to the Cb lineage and regulate the differentiation of the Cb lineage by inducing and enhancing the expression of CAP, a cell lineage-specific regulator. Furthermore, the results suggest that the MTF and Fb lineages may originate from a common early progenitor cell.

Immunolocalization of a human cementoblastoma-conditioned medium-derived protein.

Little is known about the molecular mechanisms that regulate the cementogenesis process, because specific cementum markers are not yet available. To investigate whether a cementoblastoma-conditioned medium-derived protein (CP) could be useful as a cementum biological marker, we studied its expression and distribution in human periodontal tissues, human periodontal ligament, alveolar bone, and cementoblastoma-derived cells. In human periodontal tissues, immunoreactivity to anti-CP was observed throughout the cementoid phase of acellular and cellular cementum, cementoblasts, cementocytes, cells located in the endosteal spaces of human alveolar bone, and in cells in the periodontal ligament located near the blood vessels. Immunopurified CP promoted cell attachment on human periodontal ligament, alveolar bone-derived cells, and gingival fibroblasts. A monoclonal antibody against bovine cementum attachment protein (CAP) cross-reacted with CP. These findings indicate that CP identifies potential cementoblast progenitor cells, is immunologically related to CAP species, and serves as a biological marker for cementum.

The inhibitory effects of colchicine on cell proliferation and mineralisation in culture.

Colchicine is often used in the treatment of diseases such as familial Mediterranean fever (FMF) and gout. We have previously reported that patients with FMF who had colchicine on a daily basis and who had a total hip arthroplasty showed no heterotopic ossification after surgery. The mechanism by which colchicine causes this clinical phenomenon has never been elucidated. We therefore evaluated the effect of various concentrations of colchicine on cell proliferation and mineralisation in tissue culture, using rat and human cells with and without osteogenic potential. Cell proliferation was assessed by direct cell counts and uptake of (3H)thymidine, and mineralisation by measuring the amount of staining by Alizarin Red. Our findings indicate that concentrations of colchicine of up to 3 ng/ml did not affect cell proliferation but inhibition was observed at 10 to 30 ng/ml. Mineralisation decreased to almost 50%, which was the maximum inhibition observed, at concentrations of colchicine of 2.5 ng/ml. These results indicate that colchicine at low concentrations, of up to 3 ng/ml, has the capacity to inhibit selectively bone-like cell mineralisation in culture, without affecting cell proliferation. Further clinical and laboratory studies are necessary to evaluate the effects of colchicine on biological processes involving the proliferation of osteoblasts and tissue mineralisation in vivo, such as the healing of fractures, the formation of heterotopic bone and neoplastic bone growth.

Basic fibroblast growth factor suppresses tropoelastin gene expression in cultured human periodontal fibroblasts.

Growth factors are known to play a major role in the regeneration of the periodontium. Basic fibroblast growth factor (bFGF) is a polypeptide growth factor considered to have a role in chemotaxis and mitogenesis of periodontal ligament (PDL) cells. The aim of this study was to assess the effect of bFGF on the transcription level of tropoelastin. As known controls, we assessed the transcription levels of collagen type I, collagen type II and the housekeeping gene, actin. Initially, PDL cells were cultured without bFGF for 3, 7 and 14 days. At each time point. total RNA was extracted and the levels of transcription were assessed by semiquantitative reverse transcription polymerase chain reaction (RT-PCR) assay. The results showed that tropoelastin mRNA is transcribed in PDL cells and its levels increased from minimal amounts by day 3 to maximal amounts by day 14 of culture. We further examined the effect of the addition of 10 ng/ml bFGF to the culture media by day 14. The results showed that the addition of bFGF suppressed the transcription level of tropoelastin. At that time, as expected, a decrease in collagen type I transcription level was shown, while the transcription level of collagen type III was not affected. The findings that elastin is transcribed in vitro by PDL cells, but only negligibly in vivo, imply mechanisms that downregulate or even shut down the expression of the elastin gene in the functioning PDL. Basic FGF might be one of the cytokines involved in control of elastin expression in vivo.

Observations on a new collagen barrier membrane in 16 consecutively treated patients. Clinical and histological findings.

BACKGROUND: Space-maintaining capacity, cell disclusive potential, and stability over time are crucial factors to achieving sufficient bone augmentation with membrane barriers. The case series presented here assessed a new collagen barrier used in bone augmentation. Clinically, the healing pattern, especially in cases of secondary healing, was studied. METHODS: Soft tissue healing was documented by photographs, and the size of the dehiscences calculated by image analysis. The measurements were performed on digitized photographs. During reentry, barrier remnants were dissected and histologically evaluated. RESULTS: The mean value for dehiscences was 35.5 mm2; all dehiscences healed within 4 weeks after the exposure became evident. The difference was statistically significant between the week 2 and week 6 visits (P = 0.008) for each previously exposed site. The histologic observation of barrier remnants revealed direct apposition of fibrous and bone tissues on the membrane surface. CONCLUSION: In cases of membrane exposure, gingival dehiscences always disappeared in the following weeks without affecting the healing process. Histologic results showed barrier stability over a 6-month period, promoting bone regeneration.

Effect of maturation on the osteogenic response of cultured stromal bone marrow cells to basic fibroblast growth factor.

Formation of bone-like tissue in culture by stromal bone marrow cells (SBMC) derived from young growing rats is dependent on dexamethasone (Dex) (Cell Tissue Res 254:317; 1988) and is significantly enhanced by basic fibroblast growth factor (bFGF) (J Bone Miner Res 8:919; 1993). The aim of this study was to examine the effect of maturation on the osteogenic potential and the response to Dex and bFGF of SBMC by using cultures derived from young growing (6 weeks old) and adult (9 months old) rats. SBMC cultures were grown in the presence of Dex (10(-8) or 10(-7) mol/L) at both P(0) and P(1) and either in the presence or absence of bFGF. The effect of Dex and bFGF on mineralized bone-like tissue (MBT) formation was assessed at P(1). The highest levels of mineralized tissue formation in P(1) subcultures in the absence of bFGF were obtained when cultures derived from young rats (6 weeks old) were treated with Dex 10(-7) and 10(-8) mol/L at P(0) and P(1), respectively, and when cultures derived from adult rats were exposed to Dex 10(-8) mol/L both at P(0) and P(1). Under these optimal Dex concentrations, the amount of MBT formed by adult rat-derived cultures was 15-fold lower than that of young rat-derived ones. The addition of bFGF to P(0) cultures or to P(1) cultures grown under optimal Dex conditions enhanced MBT formation in P(1) cultures derived from both young and adult rats, but this effect was considerably more pronounced in the adult rat-derived cultures. The maximal levels of MBT formation were produced by cultures derived from adult rats treated with bFGF at both P(0) and P(1), whereas in cultures derived from young rats, the addition of bFGF at P(0) was not necessary for maximal MBT production. This stimulating effect of bFGF on MBT formation by adult rat-derived cultures was accompanied by a 2.2-, 1.8-, and 4.3-fold increase in proliferation, alkaline phosphatase activity, and Ca(2+) deposition rate, respectively. bFGF increased the level of glucocorticoid receptor by approximately 2. 3-fold in Dex-treated cultures derived from young animals. These results indicate that maturation is associated with a decrease in the proportion of osteoprogenitor cells in the stromal bone marrow and in their capacity to express the osteogenic phenotype. They further point to the significant role of bFGF in stimulating proliferation and osteogenic expression of stromal bone marrow osteoprogenitors derived from adult rats.

Cementum attachment protein enriches putative cementoblastic populations on root surfaces in vitro.

We tested the capacity of cementum attachment protein (CAP) to recruit putative cementoblastic populations to root surfaces in vitro by determining the phenotypic expression of periodontal ligament cloned cell populations. The clones were derived from cells that attached to either CAP-coated (experimental) or uncoated (control) root slices. Root slices were co-cultured with primary human periodontal ligament cells. Cloned and parent populations were analyzed for their capacity to express alkaline phosphatase (AP), osteopontin, bone sialoprotein (BSP), and CAP and to form mineralized tissue in vitro. The percentage of CAP- and BSP-positive clones was significantly higher in the experimental clones than in the controls. The percentage of cells positive for AP, BSP, and CAP was higher in the experimental clones than in their control counterparts. Mineralized tissue formation was observed only in the cell populations derived from the CAP-coated root slices. These results indicate that CAP is capable of recruiting putative cementoblastic populations on root slices in vitro and therefore might play an important role in cementogenesis during periodontal homeostasis and wound healing.

Inverse dose- and time-dependent effect of basic fibroblast growth factor on the gene expression of collagen type I and matrix metalloproteinase-1 by periodontal ligament cells in culture.

BACKGROUND: Growth factors are known to play a major role in the regeneration of the periodontium. Basic fibroblast growth factor (bFGF) is a polypeptide growth factor considered to have a role in chemotaxis and mitogenesis of periodontal ligament cells (PLC). The aim of this study was to assess the dose-dependent effect of bFGF administration on the levels of gene expression of collagen type I (a1) (col I), collagen type III (col III), and collagenase-1 (MMP-1) in PLC. METHODS: PLC were cultured in different concentrations of bFGF (0.1 to 10 ng of bFGF) for 14 and 21 days. At each time point, the gene expression of the examined molecules was assessed semi-quantitatively by reverse transcription-polymerase chain reaction (RT-PCR) assay. RESULTS: The results indicated that bFGF exhibits an inverse time- and dose-dependent effect on the gene expression of col I and MMP-1: it simultaneously downregulates the gene expression of col I and upregulates the gene expression of MMP-1. On the other hand, bFGF had no dose-dependent effect on col III gene expression. The effect of bFGF on the expression of the three genes was modulated by the time of incubation with bFGF. CONCLUSIONS: These results suggest that bFGF is one of the important regulators involved in the active remodeling of col I in the periodontal ligament and possibly in other connective tissues.

Basic fibroblast growth factor enhances the growth and expression of the osteogenic phenotype of dexamethasone-treated human bone marrow-derived bone-like cells in culture.

Basic fibroblast growth factor (bFGF) was shown to enhance rat stromal bone marrow cells in culture to produce mineralized bone-like tissue in response to dexamethasone (Dex) treatment (Pitaru et al., J Bone Miner Res 8:919; 1993). The purpose of this study was to explore the effect of bFGF on Dex-treated human stromal bone marrow cells (hSBMC) in culture. Human SBMC from 6 patients were cultured for 14 days (P0) and then subcultured and grown for 28 days in the presence of Dex (10(-8) mol/L). The effect of bFGF on cell proliferation at P0 and protein content, DNA content, alkaline phosphatase activity (ALP), osteocalcin secretion, and formation of mineralized bone-like tissue (MBT) at P1 was analyzed. bFGF treatment resulted in a 2.4-fold increase in cell number at P0 and a concentration-dependent increase in [3H]-thymidine incorporation at P1, reaching a maximum increase of 3.7-fold at a concentration of 0.3 ng/mL. Furthermore, bFGF significantly increased both DNA content (two- to threefold), protein content (five- to sixfold), and the amount of MBT (up to 20-fold) at P1 cultures. Morphological evaluation of the MBT at the electron microscope level revealed a mineralization process along collagen fibrils similar to the natural process. The osteogenic nature of the bFGF-treated cultures was further shown by their ALP activity, as well as osteocalcin secretion in response to 1,25-dihydroxyvitamin D3. In conclusion, bFGF demonstrated a stimulatory effect on the proliferation of Dex-treated hSBMC-derived osteoprogenitors while maintaining their capacity to fully differentiate and form bone-like tissue in culture.

Rapid palatal expansion. Part 2: Dentoskeletal changes in cats with patent versus synostosed midpalatal suture.

Intercanine expansion (C-C) following rapid palatal expansion is made up of sutural displacement (Sd-Sd), tooth tip (Tt-Tt), tooth displacement (Td-Td), and alveolar process tipping and bending (At+b-At+b). The involvement of these four components was studied on 10 rapid palatal expansion treated and two control cats during an active phase (25 days), a retention phase (60 days), and a relapse phase (60 days). The midpalatal suture was analyzed for linear measurements, radiopaque versus radiolucent zones and optical density from occlusal radiographs. Nine treated cats exhibited sutural split and one treated cat showed no split as a result of synostosis of the suture. The contribution of the four constituents [(Sd-Sd):(Tt-Tt):(Td-Td):(At+b-At+b)] to the C-C expansion changed from active to relapse phase from [45%:15%:25%:15%] to [50%:25%:25%:0%] in the animals with sutural split and from [0%:40%:60%:0%] to [0%:0%:100%:0%] in the cat without sutural split, implying the major role of sutural displacement in patent suture, and tooth displacement in synostosed suture. The latter indicates the potential buccal corticalis fenestration, dehiscence or perforation in synostosed suture undergoing RPE. In patent suture (animals with sutural split), optical density increased during rapid palatal expansion (soft tissue build-up) and decreased during retention (remineralization) and relapse phases (medial convergence of the palatal processes). In the animal without sutural split, a continuous decrease in the optical density (predetermined ossification) was found. The progressive six-fold surge in coefficient of variation of C-C expansion during the relapse phase indicates limitation in predicting rapid palatal expansion stability. Clinically, the use of serial occlusal radiographs during rapid palatal expansion is recommended to evaluate patency and extent of retention period.

Rapid palatal expansion: Part 1. Mineralization pattern of the midpalatal suture in cats.

The mineralization pattern of the midpalatal suture after rapid palatal expansion was investigated in 10 treated and 2 control cats, in light of the tendency of RPE to relapse. The rapid palatal expansion treatment consisted of active (25 days), retention (60 days), and relapse (60 days) phases. Standardized occlusal radiographs were taken periodically and analyzed for suture width, suture optical density in anterior vs. posterior regions, and suture area measurements of radiopaque vs. radiolucent zones. Nine cats exhibited suture splitting. During the active phase, the radiolucent zone (nonmineralized tissue) increased 12-fold and the increase in optical density was 50% greater in the anterior over the posterior suture region, demonstrating increased formation of loose connective tissue at the anterior region. During the retention period, the suture's radiopaque zone (mineralized tissue) increased by 62%, the radiolucent zone declined (64%) and the suture width decreased (65%) indicating reorganization of mineralized tissue. The decrease in optical density (increased mineralization) was 2.5 times greater in the posterior over the anterior suture region, indicating that the remineralization (closure) pattern of the expanded suture is analogous to a zipper closed in a posteroanterior direction. During the relapse phase, the reduction in total suture area (41%) and in the radiopaque zone (32%) indicates medial convergence of the maxillary horizontal processes. From our findings we extrapolated that the retention of the suture anterior region should be longer than the posterior region to catch up the lag in rebuilding and maturation of the newly deposited hard tissue.

Differential chemotactic effect of cementum attachment protein on periodontal cells.

Selective re-population of the root surface by periodontal ligament cells is considered a key factor in periodontal regeneration. A recently isolated cementum attachment protein (CAP) has been shown to enhance fibroblast attachment. In the present study the potential of CAP to selectively attract periodontal ligament cells (PLC) was studied in vitro in a micro-chemotaxis system. Human periodontal ligament cells and gingival fibroblasts (GF) were compared for their chemotactic response to either cementum attachment protein or to fibronectin. Murine dermal fibroblasts (MDF) served as control, irrelevant to the periodontium. The chemotactic response of PLC to fibronectin at 10(-8) M was of a similar magnitude as that of GF (16 +/- 5 and 11 +/- 3 cells/field, respectively), but both were significantly lower than the response of MDF (28 +/- 3 cells/field). The chemotactic response of periodontal ligament cells to the cementum attachment protein at 10(-7) M was higher (36 +/- 5 cells/field) than that of gingival fibroblasts or murine dermal fibroblasts (14 +/- 2 and 16 +/- 2 cells/field, respectively). These results suggest that cementum attachment protein can influence the selective re-population of root surfaces by periodontal ligament cells.

Cementum attachment protein manifestation is restricted to the mineralized tissue forming cells of the periodontium.

The mechanisms that regulate cementogenesis are mainly unknown. A specific cementum attachment protein (CAP) has been recently partially characterized and found to be more efficient in supporting the attachment of alveolar bone cells (ABC) and periodontal ligament cells (PLC) than that of gingival fibroblasts (GF). The purpose of this study was to determine the capacity of human periodontal-derived cells to bind and express CAP and to relate these properties to their capacity to express alkaline phosphatase (AlP) and form mineralized tissue (MTF). ABC, PLC and GF were tested. Human stromal bone marrow cells (SBMC) and a cementoma-derived cell line (CC) served as controls. CAP binding was determined using 125I-CAP. The amount of MTF was assessed by alizarin red staining and image analysis determination of the amount of red-stained material. AlP and CAP expression were examined by histochemistry and immunochemistry, respectively. The highest expression of CAP was observed in CC, followed by PLC and ABC in decreasing order, whereas SBMC and GF did not express CAP. SBMC manifested the highest CAP binding capacity followed by CC, ABC, PLC and GF. MTF and AlP manifestation were greatest in SBMC, followed by ABC, PLC and CC. Collectively the results indicate that CAP binding and secretion are not linked and that CAP manifestation is restricted to periodontal derived cell lineages with the potential of forming mineralized tissues.

Changes in the shape and orientation of periodontal ligament fibroblasts in the continuously erupting rat incisor following removal of the occlusal load.

One of the main theories which attempts to explain the phenomenon of tooth eruption suggests that periodontal ligament (PDL) fibroblasts move actively and pull the tooth with them out of its socket. To find further support for this theory, we determined the changes in the shape and orientation of PDL fibroblasts induced by a transition from impeded to unimpeded eruption. We measured nuclear area, elongation (length-to-width ratio), and orientation (angulation in relation to the eruption axis) of PDL fibroblasts in impeded (functionally loaded) and unimpeded (hypoloaded) rat incisors. The mean cross-sectional nuclear area did not differ between fibroblasts in the two groups. In contrast, unimpeded eruption resulted in a marked increase in the mean nuclear elongation (from about 2 to 2.56) and a significant increase in the mean nuclear orientation (from 25.6 to 14.0 degrees). Bivariate analysis suggested that these changes occurred in the same cells. Analysis of nuclear elongation and orientation at various distances from the cementum toward the alveolar bone revealed a profile of both parameters, such that cells located 20 to 80 microns away from the cemental surface were more elongated and more frequently oriented toward the eruption axis, while cells at 0 to 20 and 80 to 100 microns were more round/oval and had a greater angulation with the eruption axis. These findings, together with other observations of changes in cell number, number of microtubules, and migration velocity which occur on the shift to unimpeded eruption, support the theory of active movement of PDL fibroblasts as an important component of tooth eruption.

A collagenous cementum-derived attachment protein is a marker for progenitors of the mineralized tissue-forming cell lineage of the periodontal ligament.

The periodontal ligament (PDL) is a fibrous and cellular connective tissue that mediates tooth attachment to bone, and it comprises fibroblastic and mineralized tissue-forming (MTF) progenitors. The MTF progenitors are believed to give rise to the cementoblastic and osteoblastic lineages. Cementum attachment protein (CAP) is a collagenous cementum-derived protein which binds strongly to osteoblasts, moderately to PDL cells, and weakly to gingival fibroblasts. The aim of the present study was to determine the relationship between the capacity of PDL progenitors to bind CAP and their potential to express alkaline phosphatase (ALP) and form mineralized-like tissue in culture. Cloned human PDL progenitor populations obtained from nine human donors were assayed for their constitutive capacity to bind CAP and express ALP, and for the dexamethasone-induced potential to form mineralized-like tissue in culture in the presence of ascorbic acid and beta-glycerophosphate. Forty percent of the progenitor clones produced mineralized-like tissue. Two patterns of mineralization were observed: a spread and flat pattern similar to that produced by human bone cells in culture and a nodular ridge-like type resembling that formed by human cementoma-derived cells. A direct correlation was found between the percentage of ALP positive cells in each progenitor clone and the amount of mineralized-like tissue formed (r = 0.565). Similar correlations were found between the number of ALP positive cells and the binding capacity of each clone (r = 0.392) and between the CAP binding capacity and mineralized-like tissue formation (r = 0.584). Multiple regression analysis indicated that the constitutive capacity of a clone to bind CAP and express ALP is directly correlated to its dexamethasone-induced potential to form mineralized tissue (r = 0.675). These results indicate that CAP binding and ALP expression can serve as markers for the identification of MTF progenitors in the heterogeneous cultured population of the human periodontal ligament. These data show for the first time that binding capacity to extracellular components of mineralized tissues can be a marker for mineralized tissue-forming progenitors.

Effect of non-erupted third molars on roots of approximal teeth. A radiographic, clinical and histologic study.

This study was undertaken to evaluate clinically and histologically root resorption in extracted human second molars in close proximity to non-erupted third molars. The control group consisted of extracted second molars that were proximal to fully erupted third molars. Eight out of the 11 teeth in the study group presented different degrees of radiographic root resorption, nine presented clinical resorption, and all 11 had histologic evidence of root resorption. In the control group, no signs of root resorption were seen radiographically or clinically. Histologically, limited sites of resorption were identified in all teeth, which were partially repaired by cellular cementum. Histologic observation of study specimens revealed root surface resorption in 10 out of the 11 teeth, one showing replacement resorption as well. Inflammatory resorption was observed in the three most advanced cases in the study group. Reparative cementum partially lining resorbed areas was evident in all teeth with surface resorption. Within the limits of this study, radiographic identification of distal root resorption of second molars in close proximity to non-erupted third molars appears reliable. The findings may support the hypothesis that the presence of a non-erupted third molar in close proximity to the distal root of the second results in root resorption.