Rat Periodontal Tissues Research Articles

A single application of hydrogen sulphide induces a transient osteoclast differentiation with RANKL expression in the rat model

Objective Oral malodor is mainly attributed to volatile sulphur compounds (VSCs) such as hydrogen sulphide (H 2S), methyl mercaptan and dimethyl sulphide. VSC accelerate periodontal soft tissue destruction. However, there is little information about the potential role of H 2S in alveolar bone loss. The purpose of this animal study was to examine the effects of sodium hydrogen sulphide (NaHS), H 2S donor drug, on osteoclast differentiation in rat periodontal tissue. Design Twenty-four male Wistar rats (8 weeks old) were divided into four groups: a control group and three experimental groups, which were examined at 3 h, 1 day, and 3 days after topical application of 3 μl NaHS (l M in physiological saline) into the gingival sulcus of rat first molar. Expression of tumour necrosis factor (TNF)-α, RANKL, NF-κB and tartrate-resistant acid phosphatase (TRAP) was evaluated in the periodontal tissue. Results Three hours after NaHS application, TNF-α expression increased in the periodontal ligament. The numbers of RANKL-positive osteoblasts and TRAP-positive osteoclasts significantly increased progressively with time and reached a maximum level after 1 day. Significant up-regulation of RANKL and NF-κB mRNA was observed at 3 h after NaHS application. Conclusions H 2S application caused a transient increase of osteoclast differentiation with up-regulation of RANKL expression in osteoblasts. H 2S, which is primarily responsible for halitosis, may also contribute to alveolar bone resorption through RANKL expression.

Central nitric oxide participates in the lipopolysaccharide‐induced tolerance in periodontal tissues of rats

Endotoxin tolerance is defined as an attenuation of the febrile response to repeated administrations of LPS. In this study, we tested the hypothesis that LPS tolerance occurs after repeated local injection of LPS into the periodontium of rats and that nitric oxide plays a role in this tolerance. Rats were given a gingival injection of saline or LPS (100 μg/kg) on 3 days. Body temperature was measured with a datalogger. Another group was treated with L‐NMMA, 500μg/rat i.c.v. 30 min before LPS in the 3rd day. Fos immunohistochemistry was performed 3 h after the injections in non‐tolerant rats (day 1) and in tolerant rats (day 3), after i.c.v. saline or L‐NMMA. On day 1 we observed a polyphasic febrile response after LPS injection, on day 2 this response was sensitized and on day 3 it was completely abolished, characterizing the development of LPS tolerance. This tolerance could be prevented with the pretreatment with L‐NMMA (p<0.05). Additionally, immunohistochemistry detected a reduction in LPS‐induced Fos‐like expression in the caudal subnuclei of the spinal trigeminal nucleus and in the preoptic area of hypothalamus in tolerant rats compared with non‐tolerant rats, and an enhanced of Fos positive cells in both areas after the reversion of tolerance with L‐NMMA (p<0.05). These results indicate that the LPS tolerance may occur locally in the oral cavity and central NO plays a role in this mechanism. Finantial support: FAPESP.

Alloxan-Induced Diabetes Triggers the Development of Periodontal Disease in Rats

BackgroundPeriodontal disease in diabetic patients presents higher severity and prevalence; and increased severity of ligature-induced periodontal disease has been verified in diabetic rats. However, in absence of aggressive stimuli such as ligatures, the influence of diabetes on rat periodontal tissues is incompletely explored. The aim of this study was to evaluate the establishment and progression of periodontal diseases in rats only with diabetes induction.Methodology/Principal FindingsDiabetes was induced in Wistar rats (n = 25) by intravenous administration of alloxan (42 mg/kg) and were analyzed at 1, 3, 6, 9 and 12 months after diabetes induction. The hemimandibles were removed and submitted to radiographical and histopathological procedures. A significant reduction was observed in height of bone crest in diabetic animals at 3, 6, 9 and 12 months, which was associated with increased numbers of osteoclasts and inflammatory cells. The histopathological analyses of diabetic rats also showed a reduction in density of collagen fibers, fibroblasts and blood vessels. Severe caries were also detected in the diabetic group.Conclusions/SignificanceThe results demonstrate that diabetes induction triggers, or even co-induces the onset of alterations which are typical of periodontal diseases even in the absence of aggressive factors such as ligatures. Therefore, diabetes induction renders a previously resistant host into a susceptible phenotype, and hence diabetes can be considered a very important risk factor to the development of periodontal disease.

Reduced central c-fos expression and febrile response to repeated LPS injection into periodontal tissue of rats

Systemic injection of repeated doses of Escherichia coli lipopolysaccharide (LPS) results in attenuation of the febrile response, i.e. endotoxin tolerance, which has been fairly well characterized in rats. In the present study, we tested the hypothesis that endotoxin tolerance also occurs after repeated local injection of LPS into periodontal protection tissue. Male Wistar rats were given a gingival intra-pouch injection of sterile saline or LPS at dose of 100 μg/kg on three consecutive days. Body core temperature (Tb) was measured with a miniature datalogger. Another group of animals were used for Fos immunohistochemistry 3 h after the injections in both non-tolerant and tolerant animals. On day one we observed a polyphasic febrile response after LPS injection. The increase in body temperature started about 2 h after LPS administration and lasted 5 h. On day two this response was sensitized and on day three the febrile response was completely abolished. These data suggest that rats develop endotoxin tolerance after repeated LPS administrations into tissues within the oral cavity. Moreover, immunohistochemistry detected a reduction in LPS-induced Fos-like immunoreactivity expression in the subnucleus caudalis of spinal trigeminal nucleus and in the preoptic area of the hypothalamus (POA) in tolerant rats compared with non-tolerant animals, indicating that the endotoxin tolerance may be locally mediated in the periodontal protection tissues of rats.

Appearance of tartrate-resistant acid phosphatase-positive cells exposed to enamel matrix derivative during healing of rat periodontal tissue defects

Appearance of tartrate-resistant acid phosphatase-positive cells exposed to enamel matrix derivative during healing of rat periodontal tissue defects

Immunohistochemical localization and expression of fibromodulin in adult rat periodontium and inflamed human gingiva.

The aim of this study was to determine the distribution and expression of fibromodulin in adult rat periodontal tissues and inflamed human gingiva. The distribution of fibromodulin in rat molar periodontium and human gingival tissue was studied by immunohistochemistry. The expression of fibromodulin mRNA from human gingival fibroblasts, periodontal ligament fibroblasts and osteoblasts was studied by reverse transcription-polymerase chain reaction (RT-PCR). For comparative purposes, the distribution and mRNA expression of collagen types I and III, as well as the two small leucine-rich proteoglycans decorin and biglycan were also studied. In the adult rat periodontium, fibromodulin was distributed in the suprabasal gingival epithelium, gingival and periodontal fibroblasts as well as their surrounding extracellular matrices. Strong expression was noted in the palatal gingival tissues and the interfaces of the periodontal ligament with alveolar bone and cementum. In human gingival tissues, staining of fibromodulin was detected in the connective tissue of inflamed gingiva associated with both gingivitis and periodontitis; whereas, weak staining for this molecule was noted in healthy gingival tissues. The expression of mRNA for fibromodulin was strongest in the cultured osteoblasts. Periodontal ligament fibroblasts showed only a weak level of expression for fibromodulin mRNA. Fibromodulin is differentially expressed throughout the periodontium being primarily associated with collagen type I in non-mineralized sites. In addition fibromodulin showed an upregulation in inflamed gingival tissue.

Immuno-localization of COX-1 and COX-2 in the rat molar periodontal tissue after topical application of lipopolysaccharide

Up-regulation of prostaglandin E 2 (PGE 2) production in the periodontal tissue is considered to be important for periodontal tissue destruction. The purpose of the study was to demonstrate the dynamic changes of immuno-localization of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) in rat periodontal tissue after topical application of lipopolysaccharide (LPS: 5 mg/ml in physiological saline) from Escherichia coli into the rat molar gingival sulcus. In the normal periodontal tissue, small numbers of junctional epithelium (JE) cells and numerous osteocytes embedded in alveolar bone constitutively expressed COX-1. The COX-1 expression was not effected by LPS application. JE cells, especially in the coronal portion of JE also expressed COX-2. LPS application induced the JE cells with consequent transient expression of COX-2 with a peak at day 1. These findings suggest that JE cells may play a critical role in first defense line against LPS challenge and PGE 2 from JE cells may be responsible for the initiation of periodontal inflammation. In the deep periodontal tissue, cementoblasts and osteoblasts showed constitutive expression of COX-2, which may be induced by continuous cyclic tension force due to occlusal pressure. LPS application caused a transient up-regulation of COX-2 expression in periodontal ligament fibroblasts, cementoblasts and osteoblasts. It is suggested that the inducible production of PGE 2 via COX-2 by these cells may be associated with connective tissue destruction and alveolar bone resorption.

Immunolocalization of CXC chemokine and recruitment of polymorphonuclear leukocytes in the rat molar periodontal tissue after topical application of lipopolysaccharide.

This study investigated the recruitment of polymorphonuclear leukocytes (PMNs) and the immunolocalization of CXC chemokines, including macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant-2 (CINC-2) in rat periodontal tissue after topical application of lipopolysaccharide (LPS; 5 mg/ml) from Escherichia coli into the rat molar gingival sulcus. In normal periodontal tissues, a small number of MIP-2- and CINC-2-positive cells were seen in junctional epithelium (JE), especially in its coronal half. After topical application of LPS, a prominent increase of MIP-2- and CINC-2-positive JE cells was observed. Almost all JE cells strongly expressed them at day 1 and day 2, and then the number of chemokine-positive cells returned to normal at day 7. Corresponding to these chemokine expressions, LPS application induced a significant increase in the number of PMNs in the sub-JE area from 1 h to 2 days and a significant increase in JE area from 3 h to 5 days, indicating a dynamic flow of PMNs from the sub-JE area into JE. These findings indicated that JE cells produced MIP-2 and CINC-2 in response to LPS stimulation and suggested that MIP-2 and CINC-2 may be responsible for PMN migration toward the periodontal pathogen and may play an important role in the initiation of inflammation and subsequent periodontal tissue destruction.

Localization of Cathepsin B and L in Rat Periodontal Tissues During Experimental Tooth Movement

Cathepsins are common and well-characterized lysosomal cysteine proteinases involved in tissue destruction under pathological conditions. A recent immunocytochemical study reported that cathepsins B (CAB) and L (CAL) were localized in the periodontal ligament (PDL) of the rat molar. Further, our laboratory demonstrated that the levels of CAB and CAL in gingival crevicular fluid increased during orthodontic tooth movement. The present study focused on the localization of CAB and CAL during experimental tooth movement as well as participation by these cathepsins in the alteration of the periodontal tissues. Tooth movement was performed with the Waldo method, and the time points used for elastic band extraction were 1, 3, and 7 days after tooth movement. To determine localization of CAB and CAL using immunohistochemical analysis was used. Immunoreactivity for CAB and CAL was detected in osteoclasts and PDL fibroblasts. One day after tooth movement, the PDL fiber arrangement became coarse on both sides, and regressive lesions formed while the degree of staining for CAB and CAL increased in those cells as a whole. However, 3 days after tooth movement, many osteoclasts appeared in the compression and tension sides, and the immunoreactivity of CAB and CAL was increased in osteoclasts and fibroblasts. The present study demonstrated that CAB and CAL could be detected in PDL fibroblasts and osteoclasts and were increased in those cells following experimental tooth movement. Therefore, mechanical stress may be involved in extracellular matrix degradation and bone resorption through the stimulation of cathepsins, which is one of the important factors in the periodontal tissues.

In situ expression of RANKL, RANK, osteoprotegerin and cytokines in osteoclasts of rat periodontal tissue.

This study examined the in situ expression of receptor activator of nuclear factor-kappaB ligand (RANKL), receptor activator of nuclear factor-kappaB (RANK), osteoprotegerin, interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha) in the osteoclasts of rat periodontal tissue. In periodontal disease, osteoclasts cause resorption of the alveolar bone. The function of osteoclasts is regulated by interaction with periodontal ligament cells (PDLs). Furthermore, various kinds of molecules such as RANKL, RANK, osteoprotegerin, IL-1beta and TNFalpha are known to be related to the osteoclasts differentiation and function. It is therefore important to observe the expression of RANKL, RANK, osteoprotegerin and cytokines in osteoclasts and PDLs. Four-week-old Wistar rats were used. Tooth movement was performed by the Waldo method, and the pathological bone resorption was induced. The demineralized maxillae and mandiblae were embedded with paraffin. In situ hybridization was performed to detect RANKL, RANK, osteoprotegerin, IL-1beta, and TNFalpha mRNAs in osteoclasts and other cells using the specific RNA probes, respectively. Both RANKL and RANK were concomitantly expressed in some osteoclasts. RANKL was also positive in osteoblasts and PDLs. No IL-1beta- and TNFalpha-positive osteoclast was noted. The positive signals of osteoprotegerin were detected in almost all osteoblasts, PDLs and odontoblasts. No osteoprotegerin-positive osteoclasts were observed. The number and the distribution pattern of RANKL- and RANK-expressing osteoclasts changed when orthodontic excessive force was applied to periodontal tissue. In addition, IL-1beta and TNFalpha were shown to be expressed in osteoclasts under pathological status. These findings suggest that an autocrine mechanism of RANKL-RANK exists in osteoclast, which is heightened in the pathological conditions. Furthermore, the autocrine mechanism of IL-1beta and TNFalpha is also provided in osteoclast under pathological condition. These autocrine mechanisms therefore seem to regulate the osteoclast function in both physiological and pathological conditions.

Histopathological Study of the Effect of Periodontopathic Bacterial Lipopolysaccharide on Changes in Rat Periodontal Tissue after Penetration through the Gingival Sulcus.

This study was performed to clarify the in vivo damage to periodontal tissue induced by lipopolysaccharide (LPS) from Actinobacillus actinomycetemcomitans Y 4 and Porphyromonas gingivalis 381, which penetrate the junctional epithelium. Each LPS solution (5 mg/ml) was continuously dropped into the gingival sulcus of the lower first molar of a Lewis rat for 30min every 24h. The animals were sacrificed 30min after the 1st, 3 rd, 7th, 10th and 15th administration and their mandibles were examined histopathologically and histomorphometrically (n=25 in each group). A gradual increase in inflammatory findings was observed in both LPS groups. However, A. actinomycetemcomitans LPS induced more marked inflammation and degeneration of periodontal tissue. Loss of connective tissue attachment and rete peg elongation were significantly greater in rats administered with A. actinomycetemcomitans LPS compared to those administered with P. gingivalis LPS. Degeneration of junctional epithelium and subepithelial connective tissue was found at the 1st and 3rd administrations. At the 7th and 10th administrations, epithelial cells and collagen fibers were detached from root surfaces. Rete pegs showed deep invasion toward alveolar bone and epithelial detachment downward migration. At the 15 th administration, osteoclastic bone resorption was in some cases observed on the alveolar bone crest in the A. actinomycetemcomitans LPS group. These findings suggest that A. actinomycetemcomitans LPS had a greater effect on periodontal tissue destruction than P. gingivalis LPS.

ラット歯周組織の神経線維の分布，動態に関する免疫組織化学的研究 外科用メス及び電気メスによる歯肉切除後の神経線維の再生

歯周外科処置後の神経線維の再生を検索する目的で, 以下の実験を行った。ラット口蓋歯肉に, 外科用メス及び電気メスによる歯肉切除術を施し, 1日, 3日, 7日, 14日後の歯周組織および同組織内の神経線維の再生について, 病理組織学的観察及びNeurofilament Protein (NFP), Calcitonin Gene-Related Peptide CGRP), 各種抗体を使用した免疫組織学的観察を行った。その結果, 外科用メス使用群では術後3日か (ら神経線維の新生が観察され始め, 7日ではほぼ対照群と同様な所見を呈した。一方電気メス使用群では7日で神経線維の新生が観察されたが, 外科用メス使用群と比べ, 周囲組織の再生と同様に神経線維の再生は遅延していた。以上の結果から, 神経線維の再生は侵襲の種類, あるいは周囲の組織の再生状態に影響されることが示唆された。

Inhibitory effect of aspirin on root resorption induced by mechanical injury of the soft periodontal tissues in rats

The purpose of the present study was to study histologically and histometrically the inhibitory effect of aspirin on root resorption induced by mechanical injury of the periodontal soft tissues in rats. Resorption lacuna in the root surface of the molar in animals given both mechanical injury and aspirin administration contained fewer odontoclasts and was smaller in length and area than that in animals given only mechanical injury. The result of the present investigation indicated that the administration of aspirin might suppress root resorption induced by mechanical injury of the periodontal soft tissues.

Immunohistochemical detection of prostaglandin I 2 synthase in various calcified tissue-forming cells in rat

Localization of prostaglandin (PG) I 2 synthase immunoreactivity was examined in demineralized sections of rat pulpal, periodontal and skeletal tissues using isn-1, a monoclonal antibody raised against the enzyme. Various calcified tissue-forming cells, i.e. odontoblasts, osteoblasts, osteocytes, cementoblasts, cementocytes and chondrocytes, were similarly immunoreactive for PGI 2 synthase, suggesting that they are capable of producing PGI 2. In odontoblasts and chondrocytes, the reactivity increased gradually with maturation. Weak immunoreactivity was also observed in endothelial cells and fibroblast-like cells in pulpal and periodontal tissues. However, no reactivity was seen in ameloblasts. These results suggest the possible involvement of PGI 2 in the regulation of the metabolism of various calcified tissues. Monoclonal antibodies such as isn-1 may become useful markers of the maturation of calcified tissue-forming cells of mesenchymal origin.

Ultrastructural and ultracytochemical characteristics of multinucleated cells after hydroxyapatite implantation into rat periodontal tissue.

Multinucleated cells (MNCs) that appeared after hydroxyapatite (HAP) implantation into experimentally-produced bone defects in rat periodontal tissues were investigated both ultrastructurally and ultracytochemically. At day 5 after implantation, MNCs first appeared along the HAP surface. They had no features of typical osteoclasts such as ruffled border and clear zone. By d 14, these cells acquired features similar to osteoclasts, including ruffled border and clear zone. With the appearance of ruffled borders in MNCs, new bone deposited around the implanted HAP. MNCs appeared to excavate both newly-formed bone and implanted HAP simultaneously. Ingested HAP particles were observed not only in MNCs but also in macrophages. MNCs contained both tartrate-resistant acid phosphatase (ACPase) and carbonic anhydrase (CAase). ACPase activity was detected along all the biosynthesizing pathways in MNCs. Extracellular ACPase activity around the ruffled border region was also demonstrable. CAase activity could be detected only in the cytosol, vesicles and mitochondrial cristae of the MNCs. These cytochemical characteristics were almost the same regardless of the time elapsed after implantation.

Segregation of NGF receptor in sensory receptors, nerves and local cells of teeth and periodontium demonstrated by EM immunocytochemistry

Nerve growth factor receptor immunoreactivity (NGFR-IR) in sensory nerves and somatosensory receptors of adult rat dental and periodontal tissue was analysed using a monoclonal antibody (192-IgG) and electron microscopy. In dental and periodontal nerves, the unmyelinated axons and their Schwann cells had occasional labelling of their cell membranes, and myelinated axons had none. Dental free nerve endings in predentin had varied NGFR-IR: 15% were unlabelled, 25% had some axonal membrane NGFR-IR, and 60% had intense membrane label and cytoplasmic staining. In periodontal ligament there were two types of NGFR-IR somatosensory receptors: Ruffini mechanoreceptors had extensive NGFR-IR on apposed membranes of the terminal Schwann cell and nerve endings, but no labelling of the neural fingers which extended out into the ligament tissue; and thin fibres had intense membrane NGFR-IR and cytoplasmic stain. Non-neuronal NGFR-IR had cell specific patterns: perineurial and endoneurial cells and Ruffini terminal Schwann cells had NGFR-IR on cell membranes and inside numerous pinocytotic vesicles; Schwann cells along unmyelinated axons had NGFR-IR cell membrane intensities which varied depending on the NGFR-IR intensity of the enclosed axons; odontoblasts were unlabelled except at sites of contact with the NGFR-IR pulpal or neural cells; pulp fibroblasts in the subodontoblast zone had intense NGFR-IR all along their cell membrane; and ligament fibroblasts were unlabelled. The diverse NGFR-IR patterns described here suggest that there are specific categories of cellular expression and localization which correlate with somatosensory receptor type, and that specific patterns also characterize various non-neuronal cells in dental and periodontal tissue. Only the endoneurial cells, perineurial cells, and Ruffini terminal Schwann cells had NGFR-IR endocytotic vesicles, suggesting NGF internalization by high-affinity receptors.

ラット脱灰歯周組織におけるコンドロイチン硫酸プロテオグリカンの免疫組織化学的検討

We investigated the immunohistochemical localization of the chondroitin sulfates (chondroitin-4 sulfate, -6 sulfate and dermatan sulfate) in demineralized rat periodontal tissue using monoclonal antibodies (2-B-6, 3-B-3). Also, fixative and demineralized methods were established using these monoclonal antibodies. The result showed that the most effective combination of fixative and demineralized methods was 2% glutaraldehyde 1% para-formaldehyde and 5% EDTA. Chondroitin-4 sulfate and dermatan sulfate were widespread in gingival connective tissue and periodontal membrane, with an especially strong response of dermatan sulfate shown along collagen fiber bundles. Chondroitin-6 sulfate was located in peripheral blood vessels. In alveolar bone, chondroitin-4 sulfate and dermatan sulfate were found inside Hareversion canals, Volkman's canals and lacunae. Chondroitin-6 sulfate was localized at peripheral blood in alveolar bone. In cementum, chondroitin-4 sulfate and dermatan sulfate were found at lacunae of cellular cementum and a part of embedding Sharpey's fiber.

Appearance of osteoclasts by injections of lipopolysaccharides in rat periodontal tissue

The effect of lipopolysaccharides (LPS) on periodontal tissue was studied in 11-week-old Wistar rats. Four injections of 500 micrograms LPS induced marked osteoclastic alveolar bone resorption, whereas no alveolar bone resorption occurred after four injections of physiological saline. The osteoclast count increased progressively during the four injections of 500 micrograms LPS. After eight injections, however, the osteoclast count fell from the maximum level after four injections, although osteoclasts continued to increase in size and demonstrate an increased number of nuclei. Comparison of the osteoclast number between a series of four injections of 5, 50 or 500 micrograms LPS each revealed a dose-dependent increase. This strongly suggests LPS induction of osteoclastic bone resorption in vivo. Combined use of indomethacin, a prostaglandin synthesis inhibitor, with LPS injections inhibited both the LPS-induced alveolar bone resorption and osteoclast increase, suggesting a possible participation of prostaglandins in osteoclast-mediated bone resorption.

Long junctional epithelium produced by application of bacterial protease in rats

In the present study, the effect of bacterial protease on the periodontal tissues in rats was investigated histologically and histometrically. In the control group, 0.03 ml distilled water was applied to the lingual marginal gingiva of the mandibular incisor while, in the experimental group, 0.03 ml bacterial protease (distilled water with 300 units, Sigma type XIV) was applied to the same area. Both groups were treated once a day for 1, 3, 6, 9, 15 and 21 d. Histologically, the junctional epithelium in the experimental group showed a marked apical proliferation along the tooth surface, and the gingival connective tissue displayed also a slight inflammatory cell infiltration subepithelially. The remaining periodontal tissues were almost similar to those of the control group. Histometrically, the length of the junctional epithelium between the base of the gingival crevice and the most apical portion of the junctional epithelium in the experimental group at 9, 15 and 21 d were significantly greater (p less than 0.01) than those of the control group, respectively.

Immunohistological study of wound healing in periodontal tissue of rats. Distribution of fibronectin and laminin after flap operation

This study describes the distribution of fibronectin and laminin in periodontal tissue of rats after a flap operation. After full thickness flaps were raised, the roots were surgically exposed and planed. Animals were sacrificed at 12 hours, and 1, 3, 5, 7, 14, 28 and 56 days after the wounding. The block specimens were fixed in formalin, decalcified with EDTA, made into serial paraffin sections, and examined after hematoxylin and eosin staining, after Masson-trichrome staining and by indirect immunofluorescence for the presence of fibronectin and laminin. After the wounding, fibronectin was detected in the fibrin clot, and the migratory epithelial cells crossed over this fibrin clot (12 hours-5 days). Fibronectin was deposited heavily in the granulation tissue. When the gingival connective tissue had matured, fibronectin diminished (5-14 days). On the root surface, a layer of fibronectin was present in the region where connective tissue fibers were oriented parallel to the root surface, while no fibronectin was seen at the site of reattachment of the regenerated collagen bundle (14-56 days). Laminin was present in the basement membrane of normal epithelium and blood vessels, but was absent from the internal basal lamina. After the wounding, laminin was absent from the basement membrane zone of the distal site of the migrating epithelium (1-3 days). Upon completion of wound reepithelialization at 5-7 days after wounding, laminin reappeared throughout the basement membrane except the internal basal lamina. These results suggest that fibronectin may be important in the regeneration of epithelium, connective tissue and connective tissue attachment during repair by functioning as an extracellular provisional matrix for migrating cells. On the other hand laminin may be important in maintaining the normal epithelium.