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.

Bone marrow from mechanically unloaded rat bones expresses reduced osteogenic capacity in vitro.

Bone formation during mechanical unloading is reduced, mainly as a result of osteoblastic hypofunction. At the same time, the total number of osteoblasts per long bone is also markedly reduced. We tested the hypothesis that the number of osteogenic precursors present in the bone marrow stroma was concomitantly diminished by using an in vitro cell culture system in which femoral adherent bone marrow cells differentiate into active osteoblasts and produce bone-like nodules. Hindlimbs of 32-day-old male rats were either immobilized (unloaded) by sciatic neurectomy (immo) or sham operated (sham) and animals were killed after 11 days. Femora were either ashed to determine bone mass or used to generate bone marrow cultures. Adherent marrow cells were cultured in the presence of ascorbic acid, beta-glycerophosphate, and dexamethasone. Bone mass was significantly reduced in unloaded femora (by 16%) and tibiae (by 18%). The number of adherent cells (determined on day 6) was reduced by 50% in the immo group. Reduced cell number did not result from slower proliferation in culture since [3H]thymidine incorporation on days 4 and 6 was similar in the two groups. The osteogenic potential in vitro of marrow from unloaded bones was diminished compared with that from loaded ones as evidenced by (1) lower alkaline phosphatase (ALP) activity per mg protein (by 25-40%, examined on days 6 and 12), and (2) reduced nodule formation (by 70%, expressed as percentage of the dish area stained with Alizarin Red S on day 21). None of these changes occurred in the contralateral limb of operated (immobilized) animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of basic fibroblast growth factor on the growth and differentiation of adult stromal bone marrow cells: enhanced development of mineralized bone-like tissue in culture.

Rat stromal bone marrow cells (SBMC) were shown to produce mineralized bone-like tissue in culture in the presence of dexamethasone, ascorbic acid, and beta-glycerophosphate. The addition of 3 ng/ml of basic fibroblast growth factor (bFGF) resulted in a significant increase in formation of mineralized tissue. The present study was aimed at assessing the effect of bFGF on the proliferation and differentiation of SBMC and on the sequential development of mineralized bone-like tissue in culture. Transmission electron microscopy of bFGF-treated cultures demonstrated the development of a multilayered structure resembling mineralized bone tissue consisting of cell layers embedded within a heavy extracellular matrix. The matrix was rich in bundles of collagen fibers associated with extensive mineral deposits consisting of hydroxyapatite as determined by infrared spectrophotometry. The addition of 3 ng/ml of bFGF resulted in significant enhancement of [3H]thymidine and [3H]proline incorporation and protein accumulation by 12-, 2.5-, and 2.5-fold, respectively. bFGF treatment increased cAMP responsiveness, alkaline phosphatase activity, osteocalcin level, 45Ca2+ deposition, and mineralized-like tissue formation and induced the earlier expression of these markers in the treated culture. A biphasic sequence of events was observed during the development of mineralized bone-like tissue in bFGF-treated and control cultures. The first phase is characterized by cell proliferation and matrix accumulation and is reflected by a progressive increase in [3H]thymidine and [3H]proline incorporation until day 11. The second phase, which follows, is characterized by a sharp decline in cell proliferation and matrix accumulation and a concomitant expression of osteoblast differentiation as reflected by the progressive increase in alkaline phosphatase activity, mineral deposition, and osteocalcin expression. Treatment of cultures with bFGF accentuated this biphasic sequence of events. These results indicate that bFGF has the capacity to stimulate both the growth and the biochemical functions of SBMC obtained from a young adult animal.

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.

Basic fibroblast growth factor enhances the capacity of bone marrow cells to form bone-like nodules in vitro.

The role of basic fibroblast growth factor (bFGF) in the proliferation and differentiation of rat bone marrow cells in culture was studied. bFGF stimulated [3H]thymidine incorporation into these cells by 4-fold at a concentration of 0.3 ng/ml and half-maximal effect was observed at a concentration of 15 pg/ml. In addition to its mitogenic effect, bFGF stimulated alkaline phosphatase activity by 3.6-fold. Continuous treatment with bFGF (for 21 days) resulted in a 6.3-fold increase in the culture dish surface area covered by bone-like mineralized tissue. Maximal bone-like tissue formation was observed in the presence of 3 ng/ml bFGF with half-maximal effect at a concentration of 0.3 ng/ml. These results indicate the possible role of bFGF in the proliferation of osteogenic rat bone marrow cells and their differentiation into cells of osteoblast-like phenotype.

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.

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.

The odontoblast process extends to the dentinoenamel junction: an immunocytochemical study of rat dentine.

The length and extent of the odontoblast cell process in dentine has been the subject of controversy for many years. Here an immunofluorescence technique has been applied at the light microscope level to rat coronal dentine to localize the intracellular components actin and tubulin. Adult rats were perfused with periodate-lysine-paraformaldehyde fixative, teeth were extracted, the molar crowns were demineralized, dehydrated, wax embedded, and 6 micron sections were prepared. The sections were postfixed in -20 degrees C acetone and then incubated with affinity-purified rabbit anti-actin or anti-tubulin antibodies, followed by fluorescein-conjugated goat anti-rabbit immunoglobulin. Intratubular immunofluorescence labeling for tubulin extended to the dentinoenamel junction, whereas labeling for actin, although extending to the dentinoenamel junction, was more prominent in the pulpal third of the rat dentine. Areas in which odontoblast processes are known not to occur, i.e., the atubular dentine, were not labeled by either antibody. The presence of actin- and tubulin-containing structures extending to the dentinoenamel junction is consistent with the hypothesis that the odontoblast process traverses the dentine for up to 3-4 mm, all the way to the dentinoenamel junction. Furthermore, the different staining patterns for actin-containing microfilaments as compared to tubulin-containing microtubules suggest that these two filamentous systems may have different roles in the function of the odontoblast process.

The effects of partial demineralization and fibronectin on migration and growth of gingival epithelial cells on cementum in vitro.

The capacity of mineralized cementum to support epithelial cell migration and growth and the effect that fibronectin and partial demineralization of cementum have on these processes were assessed in vitro. Dog gingival explants, 1 X 2 mm, were cultured on the cementum surfaces of pig root pieces in a defined medium consisting of DMEM and F12 (1V/1V), transferrin, insulin, epidermal growth factor, cortisone, selenium, and high-density lipoprotein. Sixty root pieces were divided into four equal groups according to the treatment: (1) untreated mineralized cementum; (2) treated with 5 micrograms of fibronectin; (3) partially demineralized in 18% EDTA for 30 min; and (4) both partially demineralized and fibronectin-treated as above. Epithelial cell migration and growth on each of the four differently treated cementum surfaces were assessed histomorphometrically by means of scanning electron microscopy. The defined culture medium supported the selective migration and growth of epithelial cells from the gingival explants onto the mineralized cementum. This was confirmed by the positive immunostaining of these cells with antikeratin antibodies. Partial demineralization of cementum inhibited epithelial migration and growth by 83% and 91%, respectively. Fibronectin treatment did not affect epithelial cell migration and growth on mineralized cementum, but it decreased the degree of epithelial cell migration and growth inhibition on partially demineralized cementum to 57% and 43%, respectively. The results indicate that: (i) mineralized cementum may consist of components that are recognized by gingival epithelial cells and support their migration and growth in vitro; (ii) these components can be removed by demineralization; and (iii) fibronectin partially restores epithelial cell migration and growth on partial demineralized cementum in vitro.

Bacterial endotoxin inhibits migration, attachment, and orientation of human gingival fibroblasts in vitro and delays collagen gel contraction.

The purpose of this study was to assess the effect of endotoxin adsorbed to dental surfaces and to collagen type I on the migration, attachment, and orientation of human gingival fibroblasts (HGF). Transversely cut porcine tooth root slices (RS), 200 micron thick, were prepared. Half of the RS obtained were partially demineralized in EDTA. Half of the demineralized and non-demineralized RS were incubated with 400 micrograms/mL of endotoxin for 24 hr, whereas the other half were maintained in PBS and served as controls. Experimental and control RS were placed on confluent layers of HFG and cultured for six days. Cell migration toward and cell attachment to the periphery of the RS and the formation of oriented cell sheets were assessed by means of photographic techniques. Additionally, six-day-old cultures were fixed and processed for SEM observation. In separate experiments, the effect of endotoxin on cell attachment to collagen type I and on contraction of three-dimensional collagen gels was assessed. It was found that: (i) bacterial endotoxin inhibited migration and attachment of HGF to both demineralized and non-demineralized cementum and interfered with the development of oriented cellular structure: (ii) the inhibitory effect was significantly more pronounced for non-demineralized than for demineralized cementum: (iii) the morphology of HGF attached to endotoxin-treated dental surfaces was altered compared with that of their controls: and (iv) bacterial endotoxin inhibited cell attachment to collagen type I and delayed the contraction of collagen gel.

Mechanism of endotoxin inhibition of human gingival fibroblast attachment to type I collagen.

Bacterial endotoxin inhibits the attachment of human gingival fibroblasts to collagen. The present study attempted to elucidate the possible mechanism of this inhibition. Two mechanisms were considered: direct toxicity to the cells and steric interference. Collagen substrates were prepared by rat type I collagen being air-dried in the wells of 24 multi-well plates. Experimental collagen substrates were treated with 50 micrograms of endotoxin/well, while untreated collagen substrates served as controls. Two mL of cell suspension (10(4) cells/mL) was added to each well, and these were incubated at 37 degrees C for two h. The average cell number/mm2 attached to experimental and control substrates was determined. Cell attachment to endotoxin-treated collagen was inhibited by 78%, compared with that to untreated collagen. The washing of the endotoxin-treated collagen for two h did not affect the inhibition of cell attachment, whereas after 24 h of washing, cell attachment was inhibited by 54%, compared with that to untreated collagen. Pre-incubation of the cells in endotoxin for two h did not affect their attachment to collagen. The addition of fetal calf serum (15%) to the experimental system completely reversed the inhibition of fibroblast attachment to endotoxin-treated collagen. These findings suggest that endotoxin interferes with fibroblast attachment to collagen through a steric phenomenon, possibly by blocking the binding sites on the collagen molecule recognized by the membrane receptor for collagen.

The effects of titanium and hydroxyapatite on osteoblastic expression and proliferation in rat parietal bone cultures.

Titanium and hydroxyapatite are used for the fabrication of dental and orthopedic implants. The longevity of these implants depends on the amount and rate of bone formation that occurs around their surfaces. In the present study, the effects of titanium, hydroxyapatite, and polystyrene (control) on the proliferation of rat calvarial cells, and on their capacity to express alkaline phosphatase and respond to parathyroid hormone (PTH) stimulation, were studied. The nature of the substrate did not affect the DNA and protein contents of experimental and control cultures throughout the experimental period. Alkaline phosphatase expression and PTH response, as assessed by DNA synthesis and adenylate cyclase activity, were higher in cultures grown on hydroxyapatite and polystyrene than in those grown on titanium. These results indicate that hydroxyapatite was a more favorable substrate than titanium for the growth and differentiation of osteoblast-like cells in vitro.

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.

Role of attrition and occlusal contact in the physiology of the rat incisor: IX. Impeded and unimpeded eruption in lathyritic rats.

In the rat, the administration of a lathyrogenic agent reduced both impeded and unimpeded eruption rates of incisors. Unimpeded eruption rates were greater than impeded eruption rates. The general eruption pattern in the experimental rats was, however, similar to that in the control rats. Thus, eruption was possible even in rats with a lathyrogenically impaired periodontal ligament.

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.

Role of attrition and occlusal contact in the physiology of the rat incisor: X. The part played by the periodontal ligament in the eruptive process.

Removal of the proliferating base of the rat incisor did not influence the rate of eruption which responded to changes in impediment to eruption in a fashion similar to that for control teeth. It is the peridontal ligament rather than the proliferating cells that is responsible for tooth eruption. The elements of the peridontal ligament apparently responsible for tooth movement are the mature fibroblasts.

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.

Growth and migration of gingival epithelial cells on mineralized and partially demineralized root surfaces in an in vitro system.

The capacity of epithelial cells to migrate and grow from gingival explants cultured on mineralized and partially demineralized root surfaces in an in vitro system was assessed. Explants of attached gingiva were obtained from mongrel dogs, cut into rectangular pieces (1 x 2 mm) and cultured either on mineralized or partially demineralized cementum in a defined culture medium containing transferrin, insulin, epidermal growth factor, cortisone, high density lipoprotein and selenium. After seven days of culture, the specimens were prepared for scanning electron microscopic examination. The amount of epithelial outgrowth from each explant was assessed by measuring the distances between the four aspects of the rectangular explant and the furthest epithelial cell located opposite to each of these aspects. The mean value obtained for epithelial outgrowth of explants grown on mineralized cementum was six times higher than that for explants cultured on partially demineralized cementum. These results indicate that partially demineralized cementum does not support epithelial growth and migration in vitro.

Partial regeneration of periodontal tissues using collagen barriers. Initial observations in the canine.

The capacity of collagen membranes to support guided regeneration of periodontal tissues in the dog was assessed. The mesiolabial, labial and distolabial aspects of the mesial root of the second and third mandibular premolar were surgically exposed in three beagle dogs. Collagen membranes, 0.5 to 0.7 mm thick, prepared from a purified solution of rat-Type I collagen were interposed between the gingival flap and the exposed root surfaces of the right premolars. The left premolars were sham-operated without the use of collagen membranes. Animals were killed one month after surgery. Tissue blocks, including the surgical sites, were removed and prepared for histological and histometric examination. Long epithelial attachment was the modality of healing in the control sites. The apical level of the junctional epithelium was located either at, or close to, the apical level of the defect. The experimental sites exhibited a combination of three healing modalities: (1) partial regeneration of periodontal tissues (new bone, periodontal ligament and cementum) occurred in the apical half of the defect, (2) long epithelial attachment developed in the coronal quarter of the defect and (3) connective tissue adhesion developed between the two. Pocket depth was similar in both the control and experimental sites. Collagen membranes could not be identified at the time of examination. The results indicate that: (1) collagen membranes have the capacity to support regeneration of periodontal tissues and (2) collagen membranes are either incorporated within the healing tissues or degraded by these during the healing process. These findings suggest that collagen membranes may be of value in reconstructive periodontal therapy.

Heparan sulfate and fibronectin improve the capacity of collagen barriers to prevent apical migration of the junctional epithelium.

The objective of the present study was to assess the effect of bilayered/collagen barriers enriched with fibronectin and heparan sulfate on the prevention of apical migration of the epithelium during the initial stage of periodontal wound healing. Experimental osseous defects were produced on the labial aspect of maxillary canines in dogs. Experimental sites were treated with either bilayered enriched collagen barriers or with non-enriched bilayered collagen barriers, using the guided tissue regeneration technique. Control sites were treated with monolayered collagen barriers that were not enriched with fibronectin and heparan sulfate. Histologic and histomorphometric examinations performed on specimens obtained 20 days post-operative indicate the formation of a short junctional epithelium in the experimental sites treated with enriched collagen barriers. In this group, 95% of the occlusal-apical length of the defects was repopulated by connective tissue cells. In the other 2 groups, a long junctional epithelium developed with only 65% of the occlusal-apical length of the defects being repopulated by connective tissue cells. These findings suggest that the enrichment of collagen barriers with fibronectin and heparan sulfate may be important to enhance the repopulation of exposed root surfaces by connective tissue cells and prevent the apical migration of the epithelium during the initial stages of periodontal wound healing.