Effect of low intensity photobiomodulation associated with norbixin-based poly (hydroxybutyrate) membrane on post-tenotomy tendon repair. In vivo study.

PURPOSE: To evaluate the in vivo response of photobiomodulation therapy associated with norbixin-based poly(hydroxybutyrate) membrane (PHB) in tenotomized calcaneal tendon. METHODS: Thirty rats were randomly allocated to six groups (n=5 each): LED groups (L1, L2 and L3) and membrane + LED groups (ML1, ML2 and ML3). The right calcaneal tendons of all animals were sectioned transversely and were irradiated with LED daily, one hour after surgery every 24 hours, until the day of euthanasia. At the end of the experiments the tendons were removed for histological analysis. RESULTS: The histological analysis showed a significant reduction in inflammatory cells in the ML1, ML2 and ML3 groups (p=0.0056, p=0.0018 and p<0.0001, respectively) compared to those in the LED group. There was greater proliferation of fibroblasts in the ML1 (p<0.0001) and L3 (p<0.0001) groups. A higher concentration of type I collagen was also observed in the ML1 group (p=0.0043) replacing type III collagen. CONCLUSION: Photobiomodulation in association with norbixin-based PHB membrane led to control of the inflammatory process. However, it did not favor fibroblast proliferation and did not optimize type I collagen formation in the expected stage of the repair process.

Effect of low intensity photobiomodulation associated with norbixin-based poly (hydroxybutyrate) membrane on post-tenotomy tendon repair. In vivo study

Abstract Purpose: To evaluate the in vivo response of photobiomodulation therapy associated with norbixin-based poly(hydroxybutyrate) membrane (PHB) in tenotomized calcaneal tendon. Methods: Thirty rats were randomly allocated to six groups (n=5 each): LED groups (L1, L2 and L3) and membrane + LED groups (ML1, ML2 and ML3). The right calcaneal tendons of all animals were sectioned transversely and were irradiated with LED daily, one hour after surgery every 24 hours, until the day of euthanasia. At the end of the experiments the tendons were removed for histological analysis. Results: The histological analysis showed a significant reduction in inflammatory cells in the ML1, ML2 and ML3 groups (p=0.0056, p=0.0018 and p<0.0001, respectively) compared to those in the LED group. There was greater proliferation of fibroblasts in the ML1 (p<0.0001) and L3 (p<0.0001) groups. A higher concentration of type I collagen was also observed in the ML1 group (p=0.0043) replacing type III collagen. Conclusion: Photobiomodulation in association with norbixin-based PHB membrane led to control of the inflammatory process. However, it did not favor fibroblast proliferation and did not optimize type I collagen formation in the expected stage of the repair process.

Healing effect of andiroba-based emulsion in cutaneous wound healing via modulation of inflammation and transforming growth factor beta 31.

PURPOSE: To evaluate the effects and mechanisms of andiroba-based emulsion (ABE) topical treatment on full-thickness cutaneous wounds in rats. METHODS: The wounds were harvested on days 3, 7, 15, and 20 post-surgery. Wound contraction rate, quantitative immunohistochemistry [macrophages, myofibroblasts, capillaries, collagens (col) I and III, transforming growth factor ß3ß (TGFß3)], and tensile strength were assessed. RESULTS: Treated wounds were smaller, contracted earlier and had increased angiogenesis, fewer CD68+ and M2 macrophages on days 7 and 15, but higher on day 20. Myofibroblasts appeared on days 3 to 7 in untreated wounds and on days 7 to 15 in treated wounds. TGFß3 levels were higher in the treated wounds, less dense collagen fibers, lower col I/III ratios and a higher tensile strength. CONCLUSION: These results demonstrate the important anti-inflammatory role of treatment and the associated modulation of macrophages, myofibroblasts, and TGFß3 levels. Collagen fibers in the treated wounds were more organized and less dense, similar to unwounded skin, which likely contributed to the higher tensile strength.

Healing effect of andiroba-based emulsion in cutaneous wound healing via modulation of inflammation and transforming growth factor beta 3

Abstract Purpose: To evaluate the effects and mechanisms of andiroba-based emulsion (ABE) topical treatment on full-thickness cutaneous wounds in rats. Methods: The wounds were harvested on days 3, 7, 15, and 20 post-surgery. Wound contraction rate, quantitative immunohistochemistry [macrophages, myofibroblasts, capillaries, collagens (col) I and III, transforming growth factor β3β (TGFβ3)], and tensile strength were assessed. Results: Treated wounds were smaller, contracted earlier and had increased angiogenesis, fewer CD68+ and M2 macrophages on days 7 and 15, but higher on day 20. Myofibroblasts appeared on days 3 to 7 in untreated wounds and on days 7 to 15 in treated wounds. TGFβ3 levels were higher in the treated wounds, less dense collagen fibers, lower col I/III ratios and a higher tensile strength. Conclusion: These results demonstrate the important anti-inflammatory role of treatment and the associated modulation of macrophages, myofibroblasts, and TGFβ3 levels. Collagen fibers in the treated wounds were more organized and less dense, similar to unwounded skin, which likely contributed to the higher tensile strength.

Sorafenib and praziquantel synergistically attenuate Schistosoma japonicum-induced liver fibrosis in mice.

Liver fibrosis is an important process that occurs in most types of chronic liver diseases and often results in the end stage of liver diseases, such as cirrhosis, portal hypertension, and hepatocellular carcinoma. Sorafenib, a multiple tyrosine kinase inhibitor, has been shown to inhibit liver fibrosis in multiple experimental fibrosis mouse and rat models. The aim of this study was to test the therapeutic effect of sorafenib on liver fibrosis induced by infection with a parasite, Schistosoma japonicum, in mice. Mice were percutaneously infected through the abdomen with Schistosoma cercariae to develop a schistosomula liver fibrosis model. Eight weeks after infection, infected mice were treated with the anti-parasitic agent praziquantel for 2 days and sorafenib for 2 weeks. Hepatic histopathological changes were assessed using hematoxylin and eosin (HE) and Masson's trichome staining. The hepatic expression levels of collagen I, collagen III, alpha-smooth muscle actin (α-SMA), platelet-derived growth factor (PDGF), and PDGF receptor-beta (PDGFR-ß) were analyzed by immunohistochemistry and western blot. Praziquantel administration alone but not sorafenib reduced liver fibrosis, and the combination of praziquantel and sorafenib significantly attenuated liver fibrosis in S. japonicum-infected mice. Moreover, sorafenib plus praziquantel markedly decreased the hepatic deposition of collagen and expression of fibrogenic genes in these mice. In conclusion, the use of sorafenib following praziquantel treatment may represent a potential therapeutic strategy for liver fibrosis induced by S. japonicum in patients.

Investigation of the biomechanical and histopathological effects of autologous conditioned serum on healing of Achilles tendon.

OBJECTIVE: The aim of this study to evaluate the effects of autologous conditioned serum (ACS) on the healing of transected rat Achilles tendons via the assessment of biomechanical and histological parameters. METHODS: The study was conducted on 45 male Sprague-Dawley rats. Five rats were used as donors for ACS preparation. Animals were randomly assigned to the experimental or control group. In both groups, the Achilles tendon was cut transversally and then sutured. In the placebo control and ACS-treated groups, saline or ACS, respectively, was injected into the repair zone three times after surgery. Ten rats from each group (ACS group, n = 20; control group, n = 20) were euthanized at days 15 and 30 after surgery for histopathological (n = 5) and biomechanical (n = 5) testing. The histopathological findings were interpreted using the Bonar and Movin scales. Tendon remodelling was evaluated via the immunohistochemical staining of collagen type 3. Biomechanical effects were assessed by tensile testing. RESULTS: The Bonar and Movin scale scores were significantly better in the ACS-treated group on both day 15 (p = 0.003 and p = 0.003, respectively) and day 30 (p = 0.005 and p = 0.004, respectively). The immunohistochemical density of collagen type 3 was significantly lower in the ACS-treated group on day 30 (p = 0.018). The type 1/3 collagen ratios of the groups were similar on days 15 and 30, as determined by Sirius Red staining (p = 0.910 and p = 0.133, respectively). In the biomechanical assessment results, the ACS-treated group's maximum load to failure values were significantly higher on day 15 (p = 0.049). CONCLUSION: Injection of ACS had a positive effect on the histopathological healing of rat Achilles tendons on days 15 and 30 and on biomechanical healing on day 15. ACS treatment contributed to lowering the collagen type 3 density by day 30. According to our study, ACS may be favourable for the treatment of human Achilles tendon injuries and tendinopathies.

Effect of laser phototherapy (λ660 nm) on type I and III collagen expression during wound healing in hypothyroid rats: an immunohistochemical study in a rodent model.

OBJECTIVE: The aim of this study was to assess, immunohistochemically, the impact of hypothyroidism and the effect of laser phototherapy on the expression of type I and III collagen during wound healing. BACKGROUND DATA: Hypothyroidism has been associated with the disruption of the body's metabolism, including the healing process. Laser phototherapy has been shown to be effective in improving wound healing, but its usefulness on enhancing wound healing under hypothyroid condition remains unknown. MATERIALS AND METHODS: Using general anesthesia, a standard surgical wound (1 cm(2)) was created on the dorsa of 48 Wistar rats divided into four groups of 12 animals each: control euthyroid (EC), euthyroid plus laser (EL), control hypothyroid (HC), and hypothyroid plus laser (HL). The irradiation with laser GaAlAs [λ660 nm, 40 mW, 1 W/cm(2), continuous wave (CW), ø=0.04 cm(2)] started immediately after surgery and was repeated every other day until end-point of study was reached, and animals were euthanized (i.e., 7 and 14 days). Laser light was applied on four different points (6 J, 150 sec and 150 J/cm(2) per point). Hypothyroidism was induced in rats with propylthiouracil (0.05 g/100 mL) administered orally for 4 weeks and maintained until the end of the experiment. Immunohistochemistry for collagen I and III was performed with EnVision(™) in the specimens removed. RESULTS: Seven days after the surgery EC, EL, and HL groups showed higher immunoexpression of collagen I and lower immunoexpression of collagen III in the newly formed tissue. There was increased immunoexpression of collagen I in EC when compared with HC (p=0.019). The immunoexpression of collagen III was significantly lower in EL than in EC (p=0.047) and HL (p=0.019). No significant difference was found in the experimental period of 14 days among the groups. CONCLUSIONS: Laser light therapy performed with the parameters of this investigation increased immunoexpression of collagen type I during tissue repair, and improved the quality of newly formed tissue in the presence of hypothyroidism.

Increased collagen III in culled chicken meat after feeding dietary wood charcoal and vinegar contributes to palatability and tenderness.

We comprehensively evaluated meat quality in chickens fed a diet consisting of wood charcoal and vinegar (WCV) using food scientific and histological approaches. In culled hens, lipid and fatty acid in Musculus semimembranosus, cooking loss and sensory tests of whole thigh meat, and meat texture of breast meat were observed. In male broilers, cross section of M. semimembranosus was used for observations on muscle area, perimysium, non-collagen total protein and total collagen content, and anti-collagen I and III reactions. In frozen male broilers, conventional morphology of M. semimembranosus as well as chicken anti-collagen III reaction to selected muscles of thigh meat and breast meat were compared between the control and WCV-fed birds. Increased lipid and fatty acids, decreased cooking loss, high score in total evaluation for sensory test of thigh meat, and decreased meat texture values were observed for culled hens fed WCV. The higher values of muscle area, total collagen and collagen III were observed for broilers fed WCV. No perimysium collapse for M. semitendinosus or increased collagen III reactions of M. tensor fasciae latae, the flexor muscle group and M. pectoralis superficialis were observed for frozen muscles in the WCV group. These total results suggest that WCV produces palatable and tender meat by increasing collagen III.

The effects of conjugated estrogen, raloxifene and soy extract on collagen in rat bones.

OBJECTIVE: To evaluate the action of conjugated equine estrogen, raloxifene and isolated or combined genistein-rich soy extracts on collagen fibers in the bones of oophorectomized rats. MATERIALS AND METHODS: Seventy female rats received testosterone propionate (0.1 µg/g) on the 9th day after birth. At 6 months of age, the rats were administered the vehicle (propylene glycol, 0.5 ml/day), and ten of the rats were randomly chosen to comprise the non-oophorectomized control group (GI). The other 60 rats were ovariectomized and randomized into six groups of ten as follows: GII, vehicle; GIII, conjugated equine estrogen (CEE), 50 µg/kg/day; GIV, raloxifene (RAL), 0.75 mg/kg/day; GV, genistein-rich soy extract (GSE), 300 mg/kg/day; GVI, CEE + GSE, 50 µg/kg/day + 300 mg/kg/day; and GVII, CEE + RAL, 50 µg/kg/day + 0.75 mg/kg/day. Three months after surgery, the drugs were administered for 60 consecutive days. All rats were euthanized, and their left tibiae were removed for histological routine. The histological sections were stained with hematoxylin-eosin, and picrosirius for evaluating bone microarchitecture. Types I and II collagen fibers were analyzed by immunofluorescence. Data analysis was carried out with ANOVA and Tukey's test. RESULTS: Collagen reduction was significant in the GIII animals when compared to the other groups (p < 0.05). There was no significant difference in the thickness of collagen fibers among the groups. There was a greater quantity of type III collagen in GVI than in the other groups. CONCLUSION: Our data indicate that conjugated equine estrogen improves bone quality because it increases the quantity of type I collagen while reducing the quantity of thin collagen fibers. In addition, the combination of CEE and raloxifene or genistein-rich soy extract is not as efficient as CEE itself to improve bone quality.

Electrical stimulation inhibits neointimal hyperplasia after abdominal aorta balloon injury through the PTEN/p27Kip1 pathway.

Electric fields (EFs) exert biological effects on promoting wound healing by facilitating cell division, cell proliferation, and cell directional migration toward the wound. In this study, we examined the inhibitory effect of direct-current (DC) EFs on the formation of neointimal hyperplasia and the possible mechanism in an abdominal aorta balloon injury rabbit model. Sixty rabbits were divided into normal, control, and experimental groups. After establishment of the abdominal aorta balloon injury model, electrodes were implanted into the bilateral psoas major muscle in control and experimental groups. Only the experimental group received electric stimulation (EFs applied at 3 or 4 V/cm for 30 min/day) for 1, 2, and 4 weeks, respectively. Neointimal hyperplasia of the abdominal aorta and proliferation of vascular smooth muscle cells (VSMCs) were measured. Expressions of collagen, p27(Kip1), and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) were detected. Results showed that the ratio of the tunica intima area to the tunica media area, the expression of type-I collagen in the neointimal, and the proliferating cell nuclear antigen index in experimental groups were significantly less than those in control groups 2 weeks post-operation (P< 0.01). Expressions of p27(Kip1) and PTEN were increased in experimental groups compared with control groups (P< 0.01). In conclusion, our results suggested that the application of DC EFs could inhibit neointimal hyperplasia and reduce collagen expression after abdominal aorta balloon injury. This was probably induced by upregulation of PTEN/p27(Kip1) expression, thereby inhibiting VSMC proliferation.

Paeoniflorin: a monomer from traditional Chinese medical herb ameliorates Schistosoma japonicum egg-induced hepatic fibrosis in mice.

Treatment of liver fibrosis associated with Schistosoma japonicum ova-induced granulomas remains a challenging proposition. There is a close relationship between high levels of interleukin-13 (IL-13) and the development of severe schistosome fibrosis. In contrast, IL-13 receptor (R) α2 has an effective role in attenuation of profibrosis. Several Chinese herbs have significant beneficial effects in liver disease. Accordingly, the purpose of the present study was to investigate the therapeutic effect of Paeoniflorin (PAE) on liver fibrosis. A mouse model for liver fibrosis was established, using infection with S. japonicum cercariae via the skin. Liver tissue was used to examine the effect of PAE on hydroxyproline, collagen I and III, and IL-13 and IL-13Rα2. The results showed that PAE has significant suppressive effect on the increase of both hepatic hydroxyproline and collagen I and III, which are the main components of extracellular matrix (ECM). Meanwhile, PAE not only inhibits IL-13 production, it also elevates IL-13Rα2 in PAE-pretreated groups compared with controls. These results suggested that PAE can improve liver fibrosis due to S. japonicum infection. The effect of PAE appears to depend on a decrease of IL-13 and an increase of IL-13Rα2.

Prevention of colonic fibrosis by Boswellia and Scutellaria extracts in rats with colitis induced by 2,4,5-trinitrobenzene sulphonic acid.

BACKGROUND: Currently, no effective preventive measures or medical therapies are available for intestinal fibrosis and, thus, surgery remains the only available strategy in the management of fibrostenotic enteropathies, especially Crohn's disease. The aim of this study was to evaluate the efficacy of a combined therapy of anti-inflammatory Boswellia and antifibrotic Scutellaria extracts on the development of colonic fibrosis in rats. MATERIALS AND METHODS: Chronic colonic inflammation-associated fibrosis was induced in rats by intracolonic administration of 2,4,5-trinitrobenzene sulphonic acid (TNBS). Sixty-four healthy male Sprague-Dawley rats were assigned to five groups: 8 controls, 14 TNBS, 14 TNBS orally treated with Boswellia extracts (50 mg kg(-1) day(-1)), 14 TNBS orally treated with Scutellaria extracts (150 mg kg(-1) day(-1)), and 14 TNBS orally treated with both Boswellia (50 mg kg(-1) day(-1)) and Scutellaria extracts (150 mg kg(-1) day(-1)). The colon was removed after 21 days of treatment and assessed by macroscopic, histological, morphometric and immunohistochemical analyses. For immunohistochemical analysis, alpha-smooth muscle actin (alpha-SMA), collagen types I-III, connective tissue growth factor (CTGF), transforming growth factor-beta1 (TGF-beta1), Smad3, Smad7 and CD3 antibodies were used. RESULTS: Combined oral administration of Boswellia and Scutellaria significantly improved the course and macroscopic findings of TNBS-induced chronic colitis assessed by disease activity index, colon weight, length, adhesions, strictures, dilatation, thickness, oedema, ulcerations and extension of damage. The histological severity of the colonic fibrosis was also notably improved by the treatment and associated with a significant reduction in the colonic expression of alpha-SMA, collagen I-III, CTGF, TGF-beta1, Smad3, and Smad7. CONCLUSIONS: These data demonstrate that the prophylactic administration of anti-inflammatory Boswellia and antifibrotic Scutellaria extracts is effective in preventing colonic fibrosis in TNBS-induced colitis. Their antifibrotic mechanism of action seems to be mediated by the inhibition of TGF-beta1/Smad3 pathway.

[Effects of rejuvenation by intense pulsed light and basic mechanism thereof].

OBJECTIVE: To evaluate the effects of rejuvenation by intense pulsed light (IPL) and the mechanism thereof. METHODS: Fifty-eight patients with photo aging were treated with IPL of single, double, or triple pulse pattern for 3 - 5 times with the intervals of 3 - 4 weeks. Three weeks after the last treatment, photography was conducted and the pictures underwent grading by the physicians and patients according to blind method. Skin specimens of the posteroinferior ear lobe or the nape were obtained from 4 patients to undergo HE staining, Uana orcein staining of elastin, immunohistochemical staining for collagenous fibers of types I and III, and transmission electron microscopy was conducted in 2 of the 4 patients. Skin digitalized image analysis was conducted on 34 female patients to measure and analyze the depth and width of dermatographs, roughness of skin. RESULTS: After the third treatment, the wrinkles and skin texture of 62.1% of the patients showed improvement, and 84.60% of the pigmented lesions and 81.25% of the vascular lesions showed improvement. Pathology showed that type I and type III collagen increased while elastin decreased, and the fibers were orderly re-arranged. Transmission electron microscopy showed that after treatment the fibroblasts increased in number and became more active in secretion and there were more collagen fibers orderly re-arranged in the stroma. Digitalized image analysis showed significant improvement in skin smoothness, depth, arithmetic average roughness and average roughness of skin texture (all P < 0.01). CONCLUSION: IPL is effective to improve the skin texture. The mechanism may be the increasing of the activity of the fibroblasts, and hyperplasia and re-arrangement of collagen and elastin.

[Effects of Feixianping on collagen type I and III in bleomycin induced pulmonary fibrosis rats].

OBJECTIVE: To study the effect of Feixianping (FXP) on collagen type I and II in rats with pulmonary fibrosis (PF). METHODS: Sixty healthy male SD rats were randomly divided into 5 groups, the normal group (A), the model group (B), the positive control group (C) and the two FXP groups (D and E) treated respectively with high and low dose of FXP. Except those in Group A (they were not modeled and administered with normal saline), all rats were established into PF model by intra-tracheal instillation of bleomycin and administered with respective medicines starting from the 1st day after modeling. Rats were sacrificed in batches at 3 time points, the 7th, 14th, and 28th day for observing the pathological changes of lung under light microscope with HE staining and to identify collagen type I and III in lung tissue by immunohistochemical stain and image quantitative analysis. RESULTS: Light-dyeing proliferative collagen fiber was presented in the slightly thickened alveolar wall in lung of modeled rats from the 14th day on, and the pathological changes became more distinct on the 28th day. The highest amount of collagen appeared in the group B, correspondingly, that in all the other groups was much lower (P < 0.05). Reduction of collagen type I and III revealed in both FXP treated groups, but better effect was shown in the high dose FXP group. The effect of FXP was superior to that of positive control on the 14 th day (P <0.05). CONCLUSION: FXP can effectively reduce the abnormal proliferation of collagen in experimental rats with PF.

[Function of MMP/TIMP on airway remodeling of bronchial asthma and treating effects of Zhichuan capsule].

OBJECTIVE: To explore the mechanism of the bronchial asthma and to study the treating effects of Zhichuan Capsule on the airway remodeling of asthmatic model rats. METHODS: The rat model was established by being sensitized and activated with different density of ovalbumin through prolonged and repeated exposure for 8 weeks. The rats were randomly divided into model group, Zhichuan Capsule treated group, dexameson treated group, and Zhichuan Capsule and dexameson treated group. Another group of normal rats were taken as control. General histological changes were observed by hematoxylin and eosin stained sections. Being standardized by internal perimeter (Pi), the wall thickness (d), internal area (Ai), outer area (Ao) and wall area (WA) of the airway were quantified by computer-assisted image analysis system. The express of MMP-9, TIMP-1, Col I, Col III and ColV in the airway were examined by immunocytochemical methods. During the course of airway remodeling, the dynamic changes of model rats were observed at different time points (2, 4, 6 and 8 weeks after the activating). Statistical comparison was performed by ANOVA followed by Fisher LSD test. RESULTS: (1) Histologic examination showed eosinophil infiltration within the airway walls, epithelial damage, excessive mucus in the lumen and edema in the submucosa of the airways in model rats, and that the collagen deposition increased accompanied by increasing of TIMP-1. In the model rats, MMP-9 increased at the time point of 2 weeks, but it decreased in the late stage (8 weeks after activating) of airway remodeling. And the level of TIMP-1 was far higher than MMP-9 at the time point of 8 weeks. (2) Zhichuan Capsule could down-regulate the level of TIMP-1 in the airway wall, as well as the thickness of airway wall and the collagen deposition. And there were progressing effects when it was used together with dexameson. CONCLUSION: (1) The early increase of MMP-9 is a key point to start remodeling; and the increase of TIMP-1 in the late stage, which inhibits collagenase activity, may play an important role in developing airway fibrosis. Imbalance between MMP-9 and TIMP-1 is a marker of airway remodeling. (2) Zhichuan Capsule can decrease the deposition of collagen and suppress the airway remodeling by inhibiting the TIMP-1 expression.

Immunohistochemical evaluation of matrix molecules associated with wound healing following treatment with an enamel matrix protein derivative in humans.

Application of enamel matrix protein derivative (EMD) onto a debrided and conditioned root surface has been shown to promote periodontal regeneration in animals and humans. However, until now there is virtually no information from humans describing the expression of different matrix molecules in the newly formed periodontal tissues following treatment with EMD. This study investigated immunohistochemically in humans the expression of matrix molecules associated with periodontal tissues reformed after treatment with EMD. Eight patients with intrabony defects were treated with EMD. Six months after surgery teeth together with some of their surrounding soft and hard tissues were removed, fixed in buffered formalin, decalcified in EDTA, and embedded in paraffin. Serial sections of 6 micro m were cut in mesiodistal direction. Sections were evaluated immunohistochemically by means of polyclonal antibodies against osteopontin, collagen I and collagen III. The original (non-treated) parts of the periodontium served as controls. In all specimens the healing resulted to a varying extent in formation of cementum, periodontal ligament and alveolar bone. In all specimens the expression of the investigated matrix molecules was stronger at the reformed than at the original sites. Osteopontin expression was most intense at the border near the newly formed cementum and bone. In the regenerated periodontal ligament, collagen I and III were localized throughout the entire periodontal ligament connective tissue. Within the newly formed PDL connective tissue the immunohistochemical staining appeared stronger for collagen III than for collagen I. The present findings suggest that (a) treatment of human intrabony defects with EMD creates an environment favourable for periodontal regeneration and, (b) in humans the healing and/or remodelling process of the reformed tissues may be followed immunohistochemically for a period of 6 months.