Morphological Changes in Diseased Cementum Layers: A Scanning Electron Microscopy Study

The aim of this study was to compare the morphological changes that occurred in root cementum layers due to periodontal disease by using scanning electron microscopy (SEM). Ninety-two periodontally hopeless teeth extracted from 29 patients were studied. Measurements of probing depth (PD) and clinical attachment loss (CAL) were taken prior to extractions. After the longitudinal fracturing process of root specimens, healthy and diseased cementum layers of roots were evaluated by SEM for the thickness of the cementum and the morphological changes in collagen fibers. The result of SEM evaluation revealed a significant (P < 0.001) decrease in the thickness of cementum layer on the diseased root surfaces compared to the healthy surfaces. There were denser and conspicuous collagen fibers with their interfibrillar matrix in cementum layers on the healthy root surfaces compared to the diseased surfaces. Within the limits of this study, the thickness of cementum layers in diseased areas was found to be significantly less than that in the healthy areas of root surfaces. However, there exist variations in the density and visibility of cemental fibers between individuals and within the individual.     

A preliminary characterization of human cementum collagen

Summary Human teeth were used to obtain cementum. Collagen could not be significantly solubilized from the cementum by salt and acetic acid extraction or by pepsin digestion. CNBr digestion (86%) of cementum and subsequent carboxymethyl cellulose chromatography suggests that human cementum consists of type I collagen only as identified by amino acid and hexose analyses.     

Resorption of the mouse incisor after the application of cold to the periodontal attachment apparatus

Summary In order to study in detail the processes leading to the resorption and ankylosis of teeth after trauma, the effects of cold application on the periodontal tissues were studied in the mouse. Liquid nitrogen was applied locally to the outer surface of the lower jaw which resulted in a freezing of the incisor and its surrounding tissues. The healing processes in the damaged periodontal ligament and the accompanying phenomena of ankylosis and dental root resorption were investigated histologically at both the light and electron microscopic levels. As a result of cold application, the cells in the periodontal ligament were killed. After a few days, the ligament started to be repopulated with cells like fibroblasts and macrophages. From 3 days on, mineral crystallites were deposited along the cementum covering the lingual, mesial, and lateral surfaces of the incisor, finally resulting in a 4–6 μm thick layer. During the period of 7–12 days following cold application, this layer of mineralized material started to be phagocytosed and degraded, presumably by mononuclear cells. Finally, extensive root resorption and some ankylosis between the tooth and the laveolar bone were observed. In the resorbed areas, cells were seen which could not be distinguished from osteoclasts. In some instances, their ruffled border was in close apposition with each of the three mineralized tissues—dentin, cementum, and alveolar bone. It is hypothesized that the deposition and subsequent phagocytosis of mineralized material along the root surface may be an important factor in the initiation of dental root resorption.     

A sulphated glycopeptide in human supragingival calculus extracts

Summary A method is described for the isolation and purification of a sulphated glycopeptide from human supragingival calculus. The compound was isolated after using EDTA treatment, 2 M CaCl₂ extraction, proteolytic digestion, ethanol precipitation, and finally purified by DEAE cellulose chromatography. It migrated as a single component on cellulose acetate electrophoresis, and chemical and infrared spectral analysis showed the presence of covalently attached sulphate groups. The sulphated glycopeptide was distinguished from being a sulphated glycosaminoglycan.     

薄基底膜肾病基因点突变后蛋白空间二级结构的改变及与表型的关系

目的 分析薄基底膜肾病（TBMN）患者COL4A4基因点突变后导致编码蛋白中甘氨酸被其它类型氨基酸替代后的结构;探讨基因突变对编码蛋白二级结构的影响及与表型的关系。 方法 以临床确诊的1例常染色体显性连锁遗传型TBMN并发局灶节段性肾小球硬化症（FSGS）患者为研究对象,该患者症状较重,临床表现为血尿、大量蛋白尿,基因检测确定为COL4A4中的g. 1214G>A导致p．G405E。对照选取1例健康人和1例文献报道的单纯TBMN患者（基因检测确定为COL4A4中的g. 1550G>A导致p．G448S）。应用E. coli分别表达患者α4（Ⅳ）链的含有突变位点的结构域及对照α4（Ⅳ）链的相同结构域,圆二色谱检测并比较它们二级结构的差异。 结果 TBMN并发FSGS患者重组蛋白的圆二色谱最低峰所在的波长由正常对照的208 nm变为约220 nm处,而且峰度降低。单纯TBMN对照重组蛋白的检测结果与健康对照相比改变较轻微,最低峰所在的波长不变,峰度仅轻度降低。二级结构分析显示,来自健康对照的重组蛋白中α螺旋、β折叠、转角和无规卷曲均存在,其中前二者各占约1/4。与健康对照蛋白相比,来自TBMN并发FSGS患者的重组蛋白中α螺旋结构增多,约占1/3,无β折叠结构;单纯TBMN对照的重组蛋白与健康对照相似,α螺旋的比例下降而β折叠增多。 结论 位于α4(Ⅳ)链相邻结构域的两个不同位置的甘氨酸被不同的氨基酸替代,它们的临床表型不同,α4(Ⅳ)链的二级结构也存在显著差异。而且,二级结构的改变程度与临床表型的严重性一致。     

Isolation and properties of fluorescent components associated with calcified tissue collagen

Fluorescent components in bone and dentine were separated from alkaline hydrolysates of their matrices on Sephadex C25 CM cationic exchange columns. The fluorescence levels, and the excitation ( max 330 nm) and emission ( max 395 nm) spectra, were the same as those observed in the intact and gelatinised matrices. The fluorescence parameters were unaltered by the hydrolysis procedure. Gel filtration on Sephadex G. 10 columns further resolved the isolated material into two components with the same fluorescence and UV absorption properties. The fluorescence was independent of pH over the range 3.5–9.5. Dialysis and gel filtration studies on the gelatinised matrices indicated a firmly-bonded association of the fluorescent material with the collagen polypeptide chains.

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Zusammenfassung Fluorescierende Bestandteile aus Knochen und Dentin wurden in Sephadex C25 CM Kationen-Austauschersäulen von alkalischen Hydrolysaten ihrer Matrices getrennt. Die Fluorescenzintensitäten sowie die Erregungs- ( max 330 nm) und Emissions- ( max 395 nm) Spektren waren dieselben wie bei intakten und gelatinisierten Matrices. Die Fluorescenzparameter wurden durch die Hydrolyse nicht verändert. Eine Gelfiltration über Sephadex-G10-Säulen trennte das isolierte Material in 2 Komponenten auf, welche gleiche Fluorescenz- und UV-Absorptionseigenschaften zeigten. Im pH-Bereich zwischen 3,5 und 9,5 war die Fluorescenz unabhängig vom pH. Dialysierversuche sowie Gelfiltrationsexperimente mitden gelatinisierten Matrices zeigten eine starkgefügte Bindung des fluorescierenden Materials mit den Polypeptidketten des Kollagens.

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Résumé Des composants fluorescents de l'os et la dentine sont séparés des hydrolysats alcalins de leur marice sur des colonnes Sephadex C25 CM d'échange cationique. Les concentrations en fluorescence et le spectre d'excitation ( max 330 nm) et d'émission ( max 395 nm) sont les mêmes que ceux observés au niveau des matrices intactes et gélatinisées. Les paramètres de fluorescence ne sont pas altérés par hydrolyse. La filtration sur gel à l'aide de colonnes Sephadex G 10 perment de différencier le matériel isolé en deux composants, ayant la même fluorescence et la même absorption UV. La fluorescence est indépendante de pH de 3.5–9.5. Des études de dialyse et de filtration sur gel de matrices gélatinisées indiquent une association étroite du matériel fluorescent avec les chaines polypeptidiques de collagène.

     

Electrophoretic and sephadex gel filtration studies of bovine foetal enamel matrix at acid pH

Demineralised bovine foetal enamel matrix was subjected to polyacrylamide gel disc electrophoresis at acid pH. This technique revealed a protein composition more complex than hitherto described with a minimum of six principal and twelve minor components. Sephadex G. 50 gel filtration chromatography at pH 3.0 and low ionic strength produced a partial separation of these components in accord with a fractionation based on molecular weight differences. Polyacrylamide electrophoresis of these column fractions both in the presence and absence of 5 M urea, showed that at acid pH the components seen in the acrylamide gels were not present as labile aggregates. These results are discussed in relation to current concepts of the matrix composition and its possible role in the calcification of enamel.

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Zusammenfassung Die fetale Zahnschmelzmatrix von Rindern wurde demineralisiert und einer Polyacrylamid-Gel Dise-Elektrophorese bei saurem pH unterworfen. Diese Technik zeigte eine Proteinzusammensetzung, die komplexer ist als bisher beschrieben wurde, nämlich mit mindestens 6 Haupt- und 12 Nebenkomponenten. Sephadex G 50 Gel-Filtrations-Chromatographie bei einem pH von 3.0 und schwacher Ionenstärke ergab eine teilweise Trennung dieser Komponenten, in Übereinstimmung mit einer Fraktionierung, welche sich auf Unterschiede im Molekulargewicht stützt. Eine Polyacrylamid- Elektrophorese dieser Säulenfraktionen sowohl mit als auch ohne 5 M Harnstoff zeigte, daß bei saurem pH die Acrylamid-Gel sichtbaren Komponenten nicht als labile Aggregate vorhanden sind. Diese Resultate werden im Zusammenhang mit den aktuellen Begriffen über die Zusammensetzung der Matrix und ihrer möglichen Rolle bei der Verkalkung des Zahnschmelzes besprochen.

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Résumé De la matrice d'émail de buf déminéralisé est soumise à l'électrophorèse à gel de polyacrylamide, à pH acide. Une composition protéique beaucoup plus complexe que celle décrite jusqu'à présent est mise en évidence. Elle est constituée par 6 composés principaux et 12 composés mineurs. La chromatographie par filtration sur gel Sephadex G. 50, à pH 3.0 et sous force ionique faible, donne une séparation partielle de ces composants, qui concorde avec le fractionnement obtenu par différences de poids moléculaire. L'électrophorèse à polyacrylamide de ces fractions, avec et sans 5 M d'urée, montre qu'à pH acide, les composés isolés dans les gels d'acrylamide ne constituent pas des substances labiles. Ces résultats sont envisagés en fonction des théories actuelles concernant la composition matricielle et son rôle possible au cours de la calcification de l'émail.

     

Oxytalan fibers of the periodontium

Oxytalan fibers have been described for the first time in 1958 by FULLMER and LILLIE; they seem to be different from collagen fibers and elastic ones (the last being scare in the alveolar ligament). Either their biochemical structure or their specific functions remains to be known. This first work tries to describe the maturation and the organisation of the oxytalanic system. Molars of rats are observed and use is made of both photonic and electronic transmission microscopies.     

双向凝胶电泳和质谱技术在睾丸蛋白表达研究中的应用

目的 :探讨双向凝胶电泳和质谱技术在睾丸蛋白表达研究中的应用。　方法 :用双向凝胶电泳分离成年雄性ICR小鼠睾丸总蛋白 ;从胶上切下 2个蛋白点 ,经胶内原位酶解后 ,用质谱仪测得其肽质量指纹谱 ;再通过数据库检索鉴定蛋白质。　结果 :从胶上切下的 2个蛋白点数据库检索结果是小鼠血清白蛋白和蛋白质二硫化物异构酶。　结论 :用双向凝胶电泳分离睾丸蛋白样品取得了很高的分辨率 ,质谱技术鉴定蛋白快速方便 ,可用于从蛋白质组水平上研究睾丸表达的蛋白     

Rabbit cranial sutures in vitro: A new experimental model for studying the response of fibrous joints to mechanical stress

Summary An organ culture system has been developed whereby mechanical stress can be applied to cranial sutures under controlled experimental conditions. The application of a continuous tensile mechanical stress (30 g) to cranial sutures from newborn rabbits (1–2 days) was accompanied by a significant increase in the incorporation of³H-leucine and³H-proline into suture protein. The specific activities of³H-hydroxyproline indicated that mechanical stress produced a two-fold increase in the incorporation of³H-proline into collagen. However, the proportion of the total radioactivity recoverable in collagen (45.63±2.33% for nonstressed; 40.58±2.17% for stressed sutures) was not significantly different. These data suggest that the increase in collagen synthesis occurs as part of a general stimulation of protein synthesis, and do not support the view that mechanical stress is the principal mechanism regulating the turnover of collagen in fibrous joints. These initial studies demonstrate that an in vitro experimental model has considerable potential for investigating the morphological and metabolic response of fibrous joints to mechanical deformation.     

Proteins Adsorbed on Hemodialysis Membranes Modulate Neutrophil Activation

The rapid adsorption of plasma proteins is one of the initial events that occur when blood enters into contact with an artificial surface. This study investigates adsorption of plasma proteins in vitro by different types of dialysis membranes and how it influences neutrophil oxygen radical production. The recovery of proteins varied between the membranes and was by far the largest on polysulfone. Electrophoresis of the proteins removed indicated that albumin was present on all of the membranes. A specific band at 45 kD was observed on polysulfone, whereas a band at 12-14 kD was seen on polysulfone and polyacrilonitrile. The adsorbed proteins enhanced or reduced the ability of the membranes to stimulate neutrophil superoxide production, as measured by cytochrome-C reduction. The complement system was involved in this stimulation only on some membranes. Therefore, protein adsorption and neutrophil activation appear to take part in the membrane bioincompatibility process.     

Dental pulp stem cell-derived chondrogenic cells demonstrate differential cell motility in type I and type II collagen hydrogels

Background Context

Advances in the development of biomaterials and stem cell therapy provide a promising approach to regenerating degenerated discs. The normal nucleus pulposus (NP) cells exhibit similar phenotype to chondrocytes. Because dental pulp stem cells (DPSCs) can be differentiated into chondrogenic cells, the DPSCs and DPSCs-derived chondrogenic cells encapsulated in type I and type II collagen hydrogels can potentially be transplanted into degenerated NP to repair damaged tissue. The motility of transplanted cells is critical because the cells need to migrate away from the hydrogels containing the cells of high density and disperse through the NP tissue after implantation.

胶原基纳米骨修复拔牙创及颌骨囊肿术后骨腔的疗效观察

目的 研究胶原基纳米骨(nHAC)修复智齿拔牙创及颌骨囊肿术后骨腔的治疗效果。方法 66例患者按照自愿原则分为3组：nHAC组30例，阻生齿21例，根端囊肿9例，骨创植入nHAC；多孔矿化骨(Bio-Oss)组6例，阻生齿2例，根端囊肿3例，含牙囊肿1例，骨创植入Bio-Oss；对照组30例，阻生齿27例，根端囊肿3例，骨创不植入材料。结果 nHAC与Bio-Oss组，12周时X线显示两者骨密度基本一致。nHAC与空白对照组临床疗效差异有显著性。结论 nHAC生物相容性好，是一种较为理想的骨替代材料，可以在临床推广应用。     

慢性肾衰竭患者血液透析前后氨基酸谱和蛋白质水平的变化

目的：观察慢性肾衰竭（CRF）患者血液透析（HD）前后血清蛋白质和氨基酸（AA）谱变化。方法：对20例CRF患者HD前后蛋白质和氨基酸浓度进行比较。结果：透析后血清视黄醇结合蛋白（RBP）、白蛋白（Alb)和前白蛋白（PA）较透析前有所下降，而转铁蛋白有所增高，但均无统计学意义。血清丝氨酸（Ser)、苏氨酸（Thr)、谷氨酸（Glu)、脯氨酸（Pro)、甘氨酸（Gly)、丙氨酸（Ala)、半胱氨酸（Cys)、赖氨酸（Lys)浓度，透析后下降显著（P＜0．01），余无显著性改变。结论：HD影响机体蛋白质、氨基酸代谢，可使部分血清氨基酸蛋白质丢失，加重机体营养不良。     

Differences in the Peritoneal Transport of Water, Solutes and Proteins Between Dialysis with Two- and with Three-Litre Exchanges

In eight. CAPD patients who either had insufficient resultsof dialysis treatment (six) or loss of ultrafiltration (two)on a normal scheme (4x2–1;). the effects of a 3-1 dialysateexchange on the in situ intraperitoneal volume, solute masstransfer, and mass transfer area coefficients were comparedwith a 2-1 exchange. The solutes investigated were urea, lactate,creatinine. glucose. kanamycin, inulin, ß₂-microglobulinalbumin and IgG. The time course of the intraperitoneal volumeshowed marked interindividual variations. However, mean volumewas lower with the 3-1 exchanges than with 2-1. This was theresult of an increased water reabsorption rate during 3-1 (2.18ml/min vs 0.94 ml/min. P<0.01), probably caused by increasedlymphatic absorption. Peritoneal mass transfer of low-molecular-weightsolutes (up to 500) was increased with the 3-1 exchange, butthere was no evidence of any alteration in effective peritonealsurface area or peritoneal permeability. By comparing the masstransfer area coefficients of the various solutes to their freediffusion coefficients, it appeared likely that the diffusionof the low-molecular-weight solutes was unrestricted by membranepermeability and only hampered by the effective peritoneal surfacearea. For the proteins a size-selective restricted diffusionwas found.     

The wound healing potential of collagen peptides derived from the jellyfish Rhopilema esculentum

Purpose: Wound represents a major health challenge as they consume a large amount of healthcare resources to improve patient''s quality of life. Many scientific studies have been conducted in search of ideal biomaterials with wound-healing activity for clinical use and collagen has been proven to be a suitable candidate biomaterial. This study intended to investigate the wound healing activity of collagen peptides derived from jellyfish following oral administration. Methods: In this study, collagen was extracted from the jellyfish–Rhopilema esculentum using 1% pepsin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fourier transform infrared (FTIR) were used to identify and determine the molecular weight of the jellyfish collagen. Collagenase II, papain and alkaline proteinase were used to breakdown jellyfish collagen into collagen peptides. Wound scratch assay (in vitro) was done to determine migration potential of human umbilical vein endothelial cells (HUVEC) covering the artificial wound created on the cell monolayer following treatment with collagen peptides. In vivo studies were conducted to determine the effects of collagen peptides on wound healing by examining wound contraction, re-epithelialization, tissue regeneration and collagen deposition on the wounded skin of mice. Confidence level (p < 0.05) was considered significant using GraphPad Prism software. Results: The yield of collagen was 4.31%. The SDS-PAGE and FTIR showed that extracted collagen from jellyfish was type I. Enzymatic hydrolysis of this collagen using collagenase II produced collagen peptides (CP1) and hydrolysis with alkaline proteinase/papain resulted into collagen peptides (CP2). Tricine SDSPAGE revealed that collagen peptides consisted of protein fragments with molecular weight <25 kDa. Wound scratch assay showed that there were significant effects on the scratch closure on cells treated with collagen peptides at a concentration of 6.25 mg/mL for 48 h as compared to the vehicle treated cells. Overall treatment with collagen peptide on mice with full thickness excised wounds had a positive result in wound contraction as compared with the control. Histological assessment of peptides treated mice models showed remarkable sign of re-epithelialization, tissue regeneration and increased collagen deposition. Immunohistochemistry of the skin sections showed a significant increase in b-fibroblast growth factor (b-FGF) and the transforming growth factor-b1 (TGF-b1) expression on collagen peptides treated group. Conclusion: Collagen peptides derived from the jellyfisheRhopilema esculentum can accelerate the wound healing process thus could be a therapeutic potential product that may be beneficial in wound clinics in the future.     

The Essential Role of Fetuin in the Serum-Induced Calcification of Collagen

The mineral in bone is located primarily within the collagen fibril, and during mineralization the fibril is formed first and then water within the fibril is replaced with mineral. Our goal is to understand the mechanism of fibril mineralization, and as a first step we recently determined the size exclusion characteristics of the fibril. This study indicates that apatite crystals up to 12 unit cells in size can access the water within the fibril while molecules larger than a 40-kDa protein are excluded. We proposed a novel mechanism for fibril mineralization based on these observations, one that relies exclusively on agents excluded from the fibril. One agent generates crystals outside the fibril, some of which diffuse into the fibril and grow, and the other selectively inhibits crystal growth outside of the fibril. We have tested this mechanism by examining the impact of removing the major serum inhibitor of apatite growth, fetuin, on the serum-induced calcification of collagen. The results of this test show that fetuin determines the location of serum-driven mineralization: in fetuin’s presence, mineral forms only within collagen fibrils; in fetuin’s absence, mineral forms only in solution outside the fibrils. The X-ray diffraction spectrum of serum-induced mineral is comparable to the spectrum of bone crystals. These observations show that serum calcification activity consists of an as yet unidentified agent that generates crystal nuclei, some of which diffuse into the fibril, and fetuin, which favors fibril mineralization by selectively inhibiting the growth of crystals outside the fibril.     

Enhanced prolylhydroxylase activity in the posterior annulus fibrosus of canine intervertebral discs following long-term running exercise

Summary The effect of long-term exercise on the intervertebral disc collagen concentration (hydroxyproline), collagen-synthesizing enzymes (prolyl-4-hydroxylase, PH, and galactosyl-hydroxylysyl glucosyltransferase, GGT) and hydroxypyridinium crosslinks was studied in ten female beagle dogs. The dogs were run on a treadmill for 1 year starting at the age of 15 weeks. The daily running distance was gradually increased to 40 km, which distance the dogs ran for the final 15 weeks. Ten untrained dogs from the same breeding colony served as controls. The nucleus pulposus and anterior and posterior halves of the annulus fibrosus of C2–3, T10–12, L4–5 disc segments were analysed. Crosslinks were measured from the anterior annulus fibrosus of the T10–11 disc. Hydroxyproline and hydroxypyridinium concentrations remained similar in both groups. PH and GGT were significantly elevated by running in the posterior annulus fibrosus of the thoracic and lumbar discs and in the lumbar nucleus pulposus. In the thoracic nucleus pulposus GGT was reduced significantly. The results suggest activated collagen metabolism in the posterior annulus fibrosus of the thoracic and lumbar discs as a result of locally increased strains on the spine.The research was carried out at the Department of Anatomy, University of Kuopio     

The history of injectable biomaterials and the biology of collagen

The authors discuss the history and use of injections of paraffin, silicone, and collagen for soft-tissue contouring. The structure and uses of collagen are described with particular reference to Zyderm^® Collagen Implant, a highly purified bovine collagen.