牙骨质基质胍提取物促牙龈成纤维细胞、成骨细胞粘附研究

目的 :鉴定牙骨质基质胍提取物内牙骨质蛋白的生物活性。方法 :制取牙骨质基质胍提取物 ,检测人牙龈成纤维细胞、MC3T3 E1成骨细胞两种细胞在含牙骨质基质提取物浓度分别为 2 .5、5、10、2 0μg/ml的DMEM培养液中孵化 1h的贴壁率并以加牛血清蛋白 1mg/ml为空白对照组。检测两种细胞在含牙骨质基质提取物浓度 10 μg/ml的DMEM培养液中孵化 30、60、90、12 0min贴壁率。 结果 :不同浓度的牙骨质基质提取物均可促进牙龈成纤维细胞和成骨细胞的粘附 ,并呈浓度依赖性 ,尤以 10 μg/ml的牙骨质基质提取物的浓度为较佳浓度。细胞的贴壁率随牙骨质基质提取物作用时间的延长逐步升高 ,并以 90min为较理想的作用时间。结论 :人牙骨质基质胍提取物含有可促进牙龈成纤维细胞和成骨细胞粘附的牙骨质活性蛋白     

牙骨质基质提取物附着于牙根表面的实验研究

目的：观察牙骨质基质提取物是否能良好的附着在牙根表面上。方法：应用同位素标记原理用１３１Ｉ标记法标记牙骨质基质提取物，将牙片放置于含已标记的牙骨质基质提取物的培养液中培养。再将牙片取出，干燥后放置在Ｘ线感光胶片上，然后对感光照片作灰度值分析。结果：实验组的灰度值平均为１０８，而对照组为６４，实验组感光照片灰度值显著高于对照组。结论：牙骨基质提取物能较好的附着到牙根表面上，这就为牙骨质基质提取物作为牙根表面生物材料的更深入的研究奠定了一定的基础。     

牙骨质基质提取物促进牙周细胞在牙根表面附着的研究

目的 明确附着的牙根表面的牙骨质基质提取物具有促进牙周细胞附着的方法。方法 采集健康的牙龋组织和牙周膜,体外培养牙龈成纤维细胞和牙周膜成纤维细胞。从因正畸需拔除的健康牙体表面获得牙骨质基质提取物,分别观察不同作用时间,不同浓度的牙骨质基质提取物对牙龈成纤维细胞和牙周膜成纤维细胞在牙体表面附着的影响,结果 牙龈成纤维细胞,牙周膜成纤维细胞均对牙骨质基质提取物有浓度和时间依赖性,最佳作用时间为２小时,     

牙骨质基质提取物对两种细胞在牙根表面的细胞生物学研究

目的：观察牙骨质基质提取物能否促进牙周膜成纤维细胞、牙龈成纤维细胞向牙根表面移行、附着和趋向。方法：用细胞培养法和图像分析法分析细胞的附着和趋向。结果：发现牙骨质基质提取物能明显提高牙龈成纤维细胞和牙周膜成纤维细胞在牙根表面的附着，而且，随着培养时间的延长，牙骨质基质提取物促牙龈成纤维细胞和牙周膜成纤维细胞附着的功能更加显著。结论：牙骨质基质提取物可较好地促进牙龈成纤维细胞和牙周膜成纤维细胞在未脱矿的牙根表面上的附着、移行和趋向     

牙骨质基质提取物促进牙周结缔组织细胞在牙根表面上增殖的实验研究

目的：证实牙骨质基质提取物可促进牙周结缔组织细胞在牙根表面上增殖。方法：用细胞培养法和增殖细胞记数法。结果：加入牙骨质基质提取物的实验组无论是牙龈成纤维细胞还是牙周膜细胞的增殖能力均有显著的提高。结论：牙骨质基质提取物可促进牙龈成纤维细胞和牙周膜成纤维细胞在牙根表面上的增殖，牙骨质基质提取物对牙龈成纤维细胞的促有丝分裂作用强于对牙周膜成纤维细胞的作用     

牙骨质基质提取物促进牙周结缔组织细胞在牙根表面上增？…

目的：证实牙骨质基质提取物可是牙周结缔组织细胞在牙根表面上增殖。方法：用细胞培养法和增殖细胞记数法。结果：加入牙骨质基质提取物的实验组无认是牙龈成纤维细胞还是牙周膜细胞的增殖能力均有显著的提高。结论：牙骨质基质提取物可促进牙龈成纤维细胞和牙周膜成纤维细胞在牙根表面上的增殖，牙骨质基质提取物对牙龈成纤维细胞的促有丝分裂作用强于对牙周膜成纤维细胞的作用。     

牙骨质基质提取物吸附于光滑钛表面上的实验研究

目的:探讨牙骨质提取物能否良好地吸附在光滑钛表面上.方法:以含131I标记的牙骨质基质胍提取物的DMEM培养液为实验组,以131I处理的DMEM培养液为对照组,将金属钛片放置在以上两组溶液中孵化1h,钛片干燥后放置X线感光胶片上曝光约3h,分析感光照片的灰度值.结果:牙骨质基质提取物处理组的图象灰度面积和灰度值均显著高于仅用碘化物处理的空白对照组(P＜0.01).结论:牙骨质基质提取物能较好地吸附在光滑的钛表面上.     

牙骨质活性蛋白的组成,提取及其作用机理

牙骨质基质中有机成分主要由胶原和糖蛋白组成，其中糖蛋白对有质周围牙龈组织和牙周组织的形成及再生起着非常重要的作用。近年来，国外不少学者对牙骨质基质中活性蛋白的组成和提取以及牙骨质活性蛋白对成纤维细胞的生成、移行，粘附等作用进行了深入的研究，本文就目前牙骨质活性蛋白的组成，来源，提取，活性测定，作用和作用研究状况作一综述。     

牙骨质基质和骨基质对骨髓基质细胞体外钙化的影响

目的：研究骨髓基质细咆任牙骨质和骨片表面生长细胞表型发育的情况。方法：将骨髓基质细胞与牙骨质片和股骨片在体外共同培养，分别在第7d、第28d进行形态学观察，并用放射免疫的方法测定上清液中骨钙素的含量。结果：骨髓基质细胞在牙骨质片和骨片表面生长良好，但细胞形态不同，两组上清液中均有骨钙素的生成，骨片组较牙骨质片组的含量高，第28d骨钙素分泌量高于7d。结论：结果表明生长于牙骨质片和骨片表面的骨髓基质细胞在体外矿化液培养条件下可向成骨样细胞转化，但细胞表现型有所区别。     

牙骨质活性蛋白的组成、提取及其作用机理

牙骨质基质中有机成分主要由胶原和糖蛋白组成,其中糖蛋白对牙骨质周围牙龈组织和牙周组织的形成及再生起着非常重要的作用。近年来,国外不少学者对牙骨质基质中活性蛋白的组成和提取以及牙骨质活性蛋白对成纤维细胞的生成、移行、粘附等作用进行了深入的研究,本文就目前牙骨质活性蛋白的组成、来源、提取、活性测定、作用和作用机理研究状况作一综述。     

Cell attachment activity of cementum proteins and mechanism of endotoxin inhibition.

Cementum occupies a unique anatomical location where soft connective tissues of the periodontium are attached to root surfaces. Cell attachment properties of proteins present in cementum were studied. Human and bovine cementum were extracted with 0.5 mol/L CH3COOH followed by 4 mol/L guanidine, and proteins were separated by ion-exchange chromatography and SDS-polyacrylamide gel electrophoresis. Cells were labeled with radioactive amino acids and added to tissue-culture plastic plates incubated with cementum proteins, and attachment was measured. Results showed that cementum proteins promoted the attachment of smooth muscle cells, endothelial cells, and fibroblasts, but not epithelial cells. Fibroblasts attached more efficiently than other cell types, and they manifested spreading with re-organization of actin filaments. No attachment occurred to plates incubated with endotoxin from A. actinomycetemcomitans. Fewer fibroblasts attached to plates treated with cementum proteins in the presence of endotoxin, but cells pre-treated with endotoxin attached normally. Attachment was not inhibited when plates were incubated first with attachment proteins and then with endotoxin; however, it was decreased when endotoxin or bovine serum albumin preceded cementum proteins. Cementum proteins with Mr 68,000, 61,000, 55,000, and 36,000 (p68, p61, p55, and p36, respectively) manifested attachment activity, while protein(s) with Mr 23,000-24,000 did not. Western blots revealed that guanidine extracts contained three bands cross-reacting with anti-bovine sialoprotein-II antibody, but the p61, p55, and p36 were negative. We conclude that cementum contains bovine sialoprotein-II and at least four other fibroblast attachment proteins, and that they do not support epithelial cell attachment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of a fibroblast attachment protein from cementum

Cementum forms the interface through which soft connective tissue of the periodontium is attached to the root surface. The interactions between cementum and connective tissue are not completely understood and whether cementum influences periodontal connective tissue formation and regeneration is not clear. We have examined the effect of cementum components on the attachment of gingival fibroblasts. Cementum was harvested from healthy human and bovine teeth and extracted sequentially in 0.5 M CH3COOH, 4 M guanidine and bacterial collagenase. Fibroblast attachment was measured using 51Cr-labelled human gingival fibroblasts on tissue culture plates previously incubated with cementum components. Results showed that all three extracts mediated fibroblast attachment and attachment was dependent on concentration and incubation time. The attachment activity was not destroyed by digestion with bacterial collagenase or by antibodies to fibronectin and laminin. However, it was inhibited by a peptide containing the amino acid sequence RGD. By gel filtration or HPLC using a DEAE-cellulose column several proteins with attachment activity were fractionated. SDS-polyacrylamide gel electrophoresis revealed that HPLC fraction eluted by 0.2-0.3 M NaCl contained a protein with molecular weight 55 kDa as a major component. This protein was isolated and shown to promote fibroblast attachment, and optimal attachment occurred at a concentration of 2 micrograms/ml. We conclude that cementum contains substances capable of mediating fibroblast attachment and that these substances play an important role in periodontal connective tissue formation and regeneration by facilitating fibroblast attachment to root surfaces.     

Protein production by human gingival fibroblasts is enhanced by guanidine EDTA extracts of cementum

Nonconfluent cultures of human gingival fibroblasts were exposed to both guanidine and guanidine EDTA extracts of cementum for 48 hours. To compare the effects of cementum extracts on fibroblasts with other mineralized tissue extracts, cells were also exposed to guanidine and guanidine EDTA extracts of dentin and alveolar bone. The cells were radioactively labeled during the last 24 h. Total protein production was measured via the incorporation of radioactive proline. Collagen production was estimated by digestion of the radioactive protein mixture with bacterial collagenase. All guanidine EDTA extracts elicited statistically significant increases in total protein production compared to controls. At 50 μg/ml of extract, the increases in protein production were 340%, 143% and 338% for bone, cementum and dentin, respectively. Similar results were obtained for collagen production. In contrast, the guanidine extracts had no effect on either protein or collagen production by human gingival fibroblasts. These data indicate that the functions of gingival fibroblasts can be altered by proteins from associated mineralized tissues. Identifying such proteins and understanding their biological functions will enhance our knowledge of the mechanisms that regulate connective tissue regeneration.     

Cell attachment activity of cementum: bone sialoprotein _ identified in cementum

Considerable research effort has been directed at preparing root surfaces in a fashion that would promote cell attachment leading to periodontal regeneration; however, no methods have proven to be clinically predictable. Identification of attachment protein(s) associated with the root surface matrix of cementum may prove valuable for developing effective clinical treatments. In this study cementum proteins were extracted from bovine and human teeth by sequential chaotropic extraction using guanidine followed by guanidine/EDTA. The guanidine/EDTA extract, but not guanidine extract, was found to promote attachment of fibroblasts. This attachment activity was inhibitable with synthetic peptide containing the attachment sequence arginine-glycine-aspartic acid (RGD). Fractionation of the guanidine/EDTA extract revealed several fractions with attachment activity. Immunoblot analysis demonstrated that two of these fractions contain the bone-associated RGD containing attachment protein, bone sialoprotein-II (BSP-II). In addition, attachment activity was also noted in other fractions that could not be attributed to BSP-II or fibronectin. These studies indicate that a component of the attachment activity of cementum is likely to be due to BSP-II and that cementum contains additional, as yet undetermined, attachment proteins.     

Enhancement by extracts of mineralized tissues of protein production by human gingival fibroblasts in vitro

Non-confluent cell cultures were exposed to both guanidine and guanidine-EDTA extracts of cementum, dentine and alveolar bone, at concentrations from 2 to 50 μg/ml for 48 h. The cells were radioactively labelled during the last 24 h. Total protein production was measured via incorporation of radioactive proline; collagen production was estimated by digestion of the radioactive protein mixture with bacterial collagenase. All guanidine-EDTA extracts elicited statistically-significant increases in total protein production when compared to controls. At 50 μg/ml of extract, the increase in protein production was 340, 143 and 338 per cent for bone, cementum and dentine, respectively. Similar results were obtained for collagen production. Guanidine-EDTA extracts also stimulated an increase in the production of specific proteins, as ascertained by gel electrophoresis. In contrast, the guanidine extracts had no effect on either protein or collagen production. Thus the functions of gingival fibroblasts can be altered by proteins from associated mineralized tissues. Identification of such proteins and their biological functions would enhance knowledge of the mechanisms that regulate connective-tissue regeneration.     

Bacteroides gingivalis antigens and bone resorbing activity in root surface fractions of periodontally involved teeth

Bone resorbing activity and the presence of antigens of Bacteroides gingivalis were assessed in plaque, calculus, cementum, and dentin obtained from roots of teeth previously exposed to periodontitis. Each fraction was obtained by scaling the root surface. The fractions were extracted by stirring and sonication, and the soluble extracts centrifuged, sterilized, dialyzed, and adjusted to equivalent protein concentrations. Cementum and dentin extracts from impacted teeth were prepared similarly and served as controls. Stimulation of bone resorption by each extract was assessed in organ cultures of fetal rat bones by measurement of release of previously-incorporated ⁴⁵Ca from the bone into the medium. In some groups of teeth, calculus and cementum were treated with citric acid prior to scaling. Citric acid washes were recovered and dialyzed. An enzyme-linked immunosorbent assay (ELISA) was used to assess the extracts for the presence of antigens reactive with an anti-serum to B. gingivalis .
Significant stimulation of bone resorption was found in all calculus and periodontally-involved cementum preparations. ELISA showed significant levels of B, gingivalis antigens in plaque, calculus, and cementum of periodontally-involved teeth, but not in involved dentin nor in cementum or dentin of impacted teeth. Treatment with citric acid removed essentially all B. gingivalis antigens from cementum but not calculus.
The results suggest that substances which stimulate bone resorption and substances which react with B. gingivalis antiserum are present in surface plaque, calculus, and cementum of periodontally-involved teeth. These substances are not present in cementum and dentin of impacted teeth nor in dentin of periodontally-involved teeth. Treatment by both scaling and citric acid demineralization will remove most of these substances from cementum of teeth previously exposed to periodontitis.     

Inhibitory effect of periodontally diseased root extracts on the growth of human gingival fibroblasts

Cementum shavings obtained from periodontally diseased and nondiseased areas of 100 removed, single-rooted teeth were extracted with either pyrogen-free water (PFW) for 5 minutes, 1 M citric acid for 5 minutes or 45% phenol-PFW for 90 minutes at 65 degrees C. The extracts were membrane-filtered, dialyzed exhaustively versus PFW, lyophilized, weighed and then dissolved in complete growth medium. The phenol-water or citric acid extracts of cementum shavings from periodontally diseased roots were positive for endotoxin by the limulus lysate assay (LLA). Pyrogen-free water extracts of diseased or phenol-water extracts of nondiseased cementum shavings were negative, or only slightly positive, respectively, for endotoxin by LLA. Media containing the various extracts were added to logarithmically growing cultures of human gingival fibroblasts (HGF). Separate cultures of HGF were exposed to Escherichia coli endotoxin at concentrations of 50, 100, 250 and 500 micrograms/ml to determine the growth-inhibitory effects of a known endotoxin. Cell growth was analyzed by measuring the incorporation of tritiated thymidine into cells. Suppression of HGF growth from 30 to 49% by E. coli endotoxin was concentration-dependent and linear over the concentration range of endotoxin tested. Pyrogen-free water extracts of diseased (endotoxin negative) or phenol-water extracts of nondiseased cementum shavings (slightly endotoxin positive) did not effect HGF growth. However, citric acid or phenol-water extracts of diseased cementum shavings (highly endotoxin positive) significantly suppressed HGF growth 58% and 61%, respectively. These results indicate that citric acid is effective in removing cytotoxic substances, presumably endotoxin, from cementum shavings and suggest that citric acid treatment is effective clinically in detoxifying periodontally diseased root surfaces.     

Tetracycline conditioning augments the in vivo inflammatory response induced by cementum extracts

BACKGROUND: Studies have shown that extracts of cementum from periodontally involved teeth stimulated cytokine secretion from cultured human monocytes and that this stimulatory effect is inhibited by conditioning of the cementum with tetracycline. Using the subcutaneous chamber model in mice, the present study was designed to test the ability of cementum extracts from periodontally diseased teeth to induce an inflammatory response in vivo and to evaluate the effect of cementum conditioning with tetracycline. METHODS: Subcutaneous chambers were implanted in 24 mice. Two weeks later, the animals received intrachamber injection of one of the following: diseased-cementum extract, healthy-cementum extract, diseased-cementum extract preconditioned with tetracycline, or medium alone. Chamber exudates were harvested and analyzed for leukocyte levels, tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and interleukin-10 (IL-10). RESULTS: Injection of healthy- or diseased-cementum extracts increased the intrachamber levels of leukocytes. Extracts of diseased cementum were found to significantly increase the levels of TNF-alpha, IFN-gamma, and IL-10, compared with extracts of healthy cementum or media alone. Peak cytokine levels were observed 2 hours postinjection. Conditioning of diseased cementum with tetracycline before extraction resulted in augmented levels of TNF-alpha and IFN-gamma, and reduced levels of IL-10, compared with untreated diseased cementum. CONCLUSIONS: The present results demonstrate that conditioning of diseased cementum with tetracycline may induce an intense inflammatory response in a mouse model, and they suggest that local application of tetracycline for root conditioning should be carefully reinvestigated.     

Biological effects of cementum and bone extracts on human periodontal fibroblasts

BACKGROUND: Non-collagenous proteins of mineralized tissues play important roles in bone induction during mineralization and in regulating the activity of many types of mesenchymal cells. This study was conducted to determine the effects of acetic acid extracts of bone and cementum on alkaline phosphatase (ALPase) activity and in vitro mineralization of cultured human periodontal fibroblasts (hPF). METHODS: Alveolar bone and cementum obtained from clinically healthy subjects were extracted by a solution containing 0.5 M acetic acid and enzyme inhibitors. Osteoblastic phenotypes of hPF were assayed by ALPase activity, gene expression of bone marker proteins, and the ability to produce in vitro mineralization in culture media containing 50 microg/ml ascorbic acid, 10 mM sodium beta-glycerophosphate, and 10(-7) M dexamethasone. The effects of cementum and bone extracts on the expression of osteoblastic phenotypes in hPF were also determined. RESULTS: Many protein components, varying in molecular weight from 10 to 14 to 120 kDa, were detectable in 10% SDS-PAGE of both cementum and alveolar bone extracts. The hPF cells were found to exhibit a moderate ALPase activity when compared with rat osteosarcoma (ROS) 17/2.8 cells under the same experimental conditions. Gene expression for ALPase, osteocalcin bone sialoprotein, osteopontin, and BMP-7 at mRNA message was detected by RT-PCR in hPF and ROS 17/2.8 cells. The confluent hPF and ROS 17/2.8 cells showed evidence of calcium deposition in the extracellular milieu at 30 and 15 to 30 days' cultures, respectively, under a mineralization medium. The hPF appeared to form mineralized foci with morphological characteristics different from the mineralized nodules produced by ROS 17/2.8 cells. The addition of low concentrations (5 microg/ml) of either cementum or bone extract produced an increase in the size and number of mineralization spots, as well as greater ALPase activity in both hPF and ROS 17/2.8 cultures during the observation periods. CONCLUSIONS: These results suggest that hPF possess certain mineralizing phenotypes, and that acetic acid extracts of bone and cementum contain components capable of stimulating osteogenic differentiation of hPF.     

Glycosaminoglycans of human cementum

The glycosaminoglycans in human cementum have been studied. Following proteolytic digestion of guanidine/EDTA and collagenase extracts of cementum, glycosaminoglycans were isolated and then separated by cellulose acetate membrane electrophoresis. After specific elimination by enzymatic and chemical treatments the glycosaminoglycans were identified as hyaluronic acid, chondroitin sulfate and dermatan sulfate. Neither heparan sulfate nor keratan sulfate were observed. Quantitation of the glycosaminoglycans in both extracts revealed chondroitin sulfate to represent the major species present. Hyaluronic acid was observed predominantly in the guanidine/EDTA extract while dermatan sulfate was a quantitative minor component of both extracts.