Collagen scaffolds implanted in the palatal mucosa

Scar formation after repair of the cleft palate leads to growth impairment of the upper jaw and midface. The implantation of a suitable scaffold during surgery may reduce this adverse effect. However, little is known about tissue reactions to scaffolds implanted in the oral cavity. Our goal was to analyze the tissue reactions to cross-linked type I collagen scaffolds after submucoperiosteal implantation in the palate of rats. Collagen type I scaffolds were implanted in the palate of 25 male Wistar rats. Groups of 5 rats were killed consecutively after 1, 2, 4, 8, and 16 weeks and were processed for histologic and immunohistochemical analyses. After 1 and 2 weeks, 3 rats from the sham group were also killed. On hematoxylin and eosin-stained sections, the cell density and the number of giant cells were determined. Blood vessels, inflammation, and the presence of myofibroblasts were detected by immunohistochemistry. An influx of inflammatory cells started after 1 week but had completely subsided after 8 weeks. Myofibroblasts were observed within the scaffolds only in the first 2 weeks. Angiogenesis already started after 1 week and showed a peak after 4 weeks, slowly declining afterward. The scaffolds were gradually integrated within the host tissue and only elicited a mild and transient inflammatory response. The scaffolds were biocompatible and seemed to be promising for future applications in cleft palate surgery.     

Electron-immunocytochemistry of laminin and type-IV collagen in the junctional epithelium of rat molar gingiva

The present study was carried out to examine the ultrastructural localization of laminin and type-IV collagen in the junctional epithelium of rat molar gingiva by means of the indirect immunoperoxidase method. Intense laminin reaction occurred in both the internal and the external basal laminae. Laminin reaction products were observed within the cisternae of the rough endoplasmic reticulum of junctional epithelium cells and in concavities formed at the distal plasma membranes adjacent to the basal lamina. Small spherical bodies occurring in the concavities also reacted positively to laminin. Type-IV collagen reaction was intense in the external basal lamina. The internal basal lamina, however, showed no reaction for type-IV collagen. These results indicate that the internal basal lamina contains laminin but no type-IV collagen and that junctional epithelium cells seem very likely to be involved in the production of laminin.     

Biodegradation of differently cross-linked collagen membranes: an experimental study in the rat

The aim of the present study was to compare the biodegradation of differently cross-linked collagen membranes in rats. Five commercially available and three experimental membranes (VN) were included: (1) BioGide (BG) (non-cross-linked porcine type I and III collagens), (2) BioMend (BM), (3) BioMendExtend (BME) (glutaraldehyde cross-linked bovine type I collagen), (4) Ossix (OS) (enzymatic-cross-linked bovine type I collagen), (5) TutoDent (TD) (non-cross-linked bovine type I collagen, and (6-8) VN(1-3) (chemical cross-linked porcine type I and III collagens). Specimens were randomly allocated in unconnected subcutaneous pouches separated surgically on the back of 40 wistar rats, which were divided into five groups (2, 4, 8, 16, and 24 weeks), including eight animals each. After 2, 4, 8, 16, and 24 weeks of healing, the rats were sacrificed and explanted specimens were prepared for histologic and histometric analysis. The following parameters were evaluated: biodegradation over time, vascularization, tissue integration, and foreign body reaction. Highest vascularization and tissue integration was noted for BG followed by BM, BME, and VN(1); TD, VN(2), and VN(3) showed prolongated, while OS exhibited no vascularization. Subsequently, biodegradation of BG, BM, BME and VN(1) was faster than TD, VN(2), and VN(3). OS showed only a minute amount of superficial biodegradation 24 weeks following implantation. Biodegradation of TD, BM, BME, VN(2), and VN(3) was associated with the presence of inflammatory cells. Within the limits of the present study, it was concluded that cross-linking of bovine and porcine-derived collagen types I and III was associated with (i) prolonged biodegradation, (ii) decreased tissue integration and vascularization, and (iii) in case of TD, BM, BME, VN(2), and VN(3) foreign body reactions.     

Biochemical analysis of total collagen content and collagen types I,III, IV,V and VI in gingiva of various periodontitis categories

Collagen is one of the chief components of the extracellular matrix of gingival connective tissue, where five different types have been identified to date. The molecular mechanism of collagen loss in periodontitis still needs to be explored. In the present study total collagen content was investigated in gingival connective tissue of adult periodontitis (AP) as well as early onset periodontitis patients (EOP) and clinically healthy subjects. Furthermore, collagen type I, III, IV, V andVI content was evaluated in gingival biopsies obtained from periodontitis patients. There was a statistically significant difference between AP (25.1 +/- 8.1 microg/mg) and EOP (15.6 +/- 4.0microg/mg) groups with regard to the total collagen content (P < 0.05). In the clinically healthy control group the total collagen content was 20.7 +/- 4.6microg/mg. Moreover, the distribution of collagen types exhibited variations in pooled homogenates of each periodontitis group. The total collagen loss seemed to be greater in the EOP patients than in the AP patients. When the ratio of fibril forming collagens to nonfibrillar collagens was evaluated, it seems to be decreased in AP patients in comparison to EOP patients. The findings of the present study suggest that different collagen types present in various periodontitis categories may be related with diverse pathogenic mechanisms acting in these diseases.     

Effects of interferons on proliferation and collagen synthesis of rat palatal wound fibroblasts.

The purpose was to select drugs that specifically reduce collagen synthesis by palatal granulation fibroblasts without affecting their proliferation. Granulation fibroblasts were obtained from 8-day-old palatal mucoperiosteal wounds and normal fibroblasts from palatal tissue of unwounded rats. Cultured cells were treated with interferon-alpha2b, interferon-beta and interferon-gamma (0, 100, 1000, and 10000 U/ml). Cell proliferation was measured by [3H]thymidine incorporation. Collagen synthesis and non-collagenous protein synthesis were determined from the incorporation of [3H]proline. None of the interferons significantly inhibited the proliferation of either type of fibroblasts. Interferon-alpha2b had no effect on the variables studied at the dosages used. Interferon-beta reduced collagen synthesis of granulation fibroblasts without affecting their non-collagenous protein synthesis or protein synthesis by normal fibroblasts. Interferon-gamma reduced collagen synthesis of both types of fibroblast and the non-collagenous protein synthesis of granulation fibroblasts. These data show that interferon-beta specifically reduces collagen synthesis by oral granulation fibroblasts without affecting normal palatal fibroblasts.     

Angiogenesis pattern of native and cross-linked collagen membranes: an immunohistochemical study in the rat

The aim of the present study was to immunohistochemically evaluate angiogenesis pattern of native and cross-linked collagen membranes after subcutaneous implantation in rats. Five commercially available and three experimental membranes (VN) were included: (1) BioGide (BG), (2) BioMend (BM), (3) BioMend Extend (BME), (4) Ossix (OS), (5) TutoDent (TD), and (6-8) VN(1-3). Specimens were randomly allocated in unconnected subcutaneous pouches (n=4) separated surgically on the back of 40 wistar rats, which were divided into five groups (2, 4, 8, 16, and 24 weeks), including eight animals each. Pattern of angiogenesis was labelled using primary mouse monoclonal antibody to transglutaminase II. For each membrane, the period of time, needed for a complete and homogeneous transmembraneous vascularization, was assessed immunohistomorhometrically. Differences between the membranes were found regarding the initial pattern of transmembraneous angiogenesis, as evaluated 2 weeks following implantation. Mean cross- and longitudinal-sectional area of blood vessels (%) was highest for VN(3) (5.27+/-2.73), followed by BG (2.45+/-0.88), VN(1) (2.07+/-0.29), VN(2) (1.91+/-0.55), TD (1.44+/-0.53), BME (0.35+/-0.29) and BM (0.25+/-0.4). In contrast to BG and VN(1-3), BM, BME and TD exhibited a homogeneous transmembraneous formation of blood vessels merely 4-8 weeks following implantation. OS, however, exhibited no signs of angiogenesis throughout the whole study period. Within the limits of the present study, it may be concluded that pattern of transmembraneous angiogenesis markedly differs among the membranes investigated.     

Edema-preventing mechanisms in rat gingiva

The long-term effect of increased local venous pressure (Pv) on interstitial fluid pressure (Pi), colloid osmotic pressure (COPi), and fractional removal rate of 125I-labeled human serum albumin (kAlb) was studied in rat gingiva. Measurements were performed on experimental animals and sham-operated controls up to 4 days after ligation of jugular veins. On the day of ligation Pv in the facial veins rose from 2.5 +/- 0.3 (SD) to 15.8 +/- 2.8 mm Hg and stayed at about this level for 2 days before a decrease to 7.4 +/- 0.9 mm Hg on day 4. In free gingiva Pi rose from an average of 3.5 +/- 0.4 to a maximum of 6.3 +/- 0.7 mm Hg, whereas in attached gingiva the corresponding increase in Pi was from 6.0 +/- 0.7 to 11.1 +/- 2.1 mm Hg. One day after the ligation COPi in wick fluid from gingiva was reduced from the control level of 10.6 +/- 1.4 to 4.5 +/- 0.9 mm Hg. COP in plasma and COPi in subcutaneous tissue on the back were unaffected. The removal rate of 125I-labeled albumin (kAlb) from the gingiva showed a nearly threefold increase after venous ligation, from 0.073 +/- 0.01 to 0.211 +/- 0.06 h-1. It is concluded that in free and attached gingiva, both a rise in Pi and a decrease in COPi will counteract the increased filtration pressure and thus prevent edema formation during venous stasis. The fall in COPi is most likely due to increased lymph flow and not dilution, as venous stasis significantly increased kAlb without any visible increase in gingival volume.     

Use of palatal mucosa and lyophilized dura mater to create attached gingiva

Gingival extension procedures using free palatal mucosal autografts or homologous lyophilized dura were compared clinically. Palatal mucosa was transplanted in 68 cases and lyophilized dura in 16 cases. The width of the zone of attached gingiva was measured before surgery and at regular intervals up to 2 years postoperatively. The width of the attached gingiva remained relatively constant in the mucosal transplant group from 1 month postoperatively, with an average relapse of about 20%. In the dura group the width of attached gingiva showed a marked decrease until 3 months after surgery with an average relapse of approximately 63%. If a preset minimal width of the attached gingiva is considered to be the criterion of success, the superiority of the palatal mucosa grafting procedure is demonstrated.     

不对称牵引对成年SD大鼠髁突软骨中胶原类型影响的研究

目的 观察不对称牵引作用下,成年SD大鼠髁突软骨中胶原类型的变化。方法 选用10周龄雄性SD大鼠220只(随机分为轻力组104只、重力组104只、对照组12只),实验组动物使用关节外固定加力装置,分别施加0.39N和1.18N(轻力组和重力组),加力第28天拆除加力装置。在加力后3、7、14、28d以及停止牵引后3、7、14、28d分别取得标本,进行苦味素-天狼星红染色,用偏振光显微镜进行观察。结果 不同类型胶原的主要分布位置和排列形态不同,Ⅰ、Ⅱ、Ⅲ型胶原出现的部位和数量随着加力的大小和不同的实验阶段出现变化。Ⅲ型胶原较多出现在受力较大、负荷出现时间较长的部位。结论 成年个体的髁突软骨在外力刺激下,仍然出现胶原合成和类型的变化,重力组的胶原类型更替在经历了较长的时间后仍然持续进行。?     

Laminin and collagen IV distribution and ultrastructure of the basement membrane of the gingiva of the rat incisor

The continuous growth of the rat incisor is associated with renovation of the junctional epithelium and resorption of the periodontal ligament. The circumdental papilla separates the connective tissue suffering resorption from the rest of the gingiva. Laminin and collagen IV were detected by the immunoperoxidase technique on the basement membrane of all regions of the gingival epithelium of the rat incisor, except the internal basal lamina and the internal surface of the circumdental papilla. The internal basal lamina is formed by a granular electron-dense material, without the organization of a typical basal lamina. Areas of the internal surface of the circumdental papilla, negative for laminin and collagen IV, lack the basal lamina. These data suggest that these molecules are not components of the dento-epithelial junction of the distal surface of the rat incisor. In addition, the basal lamina is absent or fragmented on the internal surface of the circumdental papilla, adjacent to the areas of the connective tissue undergoing resorption.     

自体游离硬腭黏膜移植在牙种植体周附着龈增量中的应用评价

目的：应用自体游离硬腭黏膜移植,进行种植体周附着龈增量,评价其临床效果。方法：选择21例种植术后种植体周附着龈严重不足患者．在种植术后1个月行自体游离硬腭黏膜移植,术后6个月测量附着龈宽度增加量。采用SPSS11．5软件包中的配对t检验分析手术前、后的附着龈宽度值变化。结果：种植体周附着龈宽度术后6个月平均增加量为（3．77±0．43）mm,手术前、后比较,差异显著（P〈0．05）。结论：自体游离硬腭黏膜移植是种植体周附着龈增量的有效方法。     

Immune responses to implanted human collagen graft in rats

Immunity to collagen implants may be mediated by cellular and humoral immune responses. To examine the possibility of such immunological reactivity and crossreactivity to collagen, 39 Sprague-Dawley rats (female, 10 weeks old, approximately 250 g wt) were implanted subcutaneously at thigh sites with crosslinked, freeze-dried human placental type I collagen grafts (4 x 4 x 2 mm) which had been irradiated (520 Gray) or left untreated. Blood was obtained by intracardiac sampling prior to implantation or from normal rats, and at various times afterwards when the animals were sacrificed. The sera from these animals were examined for circulating antibodies to human, bovine and rat tail (type I) collagens by enzyme-linked immunosorbent assay (ELISA). Also, the lymphoblastogenic responses of spleen lymphocytes from the irradiated collagen-implanted animals were assessed in culture by measuring thymidine uptake with autologous and normal rat sera in the presence of human and bovine type I collagens. Implantation of the irradiated and non-irradiated collagen grafts in rats led to a significant increase in the level of circulating antibodies to human collagen. Also antibody to bovine and rat tail collagens was detectable in the animals implanted with irradiated collagen grafts but at a lower level than the human collagen. There was a raised lymphoblastogenic response to both human and bovine collagens. The antibody level and lymphoblastogenesis to the tested collagens gradually decreased towards the end of the post-implantation period.     

Response of rat and guinea-pig subcutaneous tissue to implanted human roots containing collagen/hydroxyapatite preparations

Summary. The root canals of extracted human roots were cleaned and enlarged. The roots were sterilized before insertion of test materials of collagen, hydroxyapatite or a mixture; saline was used as a control. The roots were implanted subcutaneously in rats or guinea-pigs. After 30 and 180 days, the embedded roots were prepared for histological examination. In the rat, the tissue reaction was similar whether the roots were filled with collagen, collagen/hydroxyapatite or saline; there was ingrowth of granulation tissue. The canals of teeth filled with hydroxyapadte showed mature fibrous tissue around islands of the material. The response in guinea-pigs was similar, except that the roots stimulated some bone formation.     

Induced palatal candidosis in the Wistar rat

Palatal candidosis was induced by inoculating Candida albicans 3091 (serotype A) under an acrylic appliance. After 1 week, the palatal epithelium showed acute inflammatory cell infiltration and surface colonization by blastospores. Two weeks after inoculation, the inflammation had increased, micro-abscesses were observed and fungal hyphae penetrated the keratin layer. After 4 weeks, these changes were further intensified and the epithelium was markedly hyperplastic. Six weeks after inoculation, some areas of the epithelium were atrophic and others markedly hyperplastic, with fungal hyphal penetration. The histological changes in the epithelium were dependent on the presence of the palatal acrylic plate. When the appliance was removed 4 weeks after inoculation, the epithelial changes resolved within a further 2 weeks. Microbiological sampling of the mucosal surface revealed, however, that the organisms persisted.