C.A. Shuttleworth

The presence of type III collagen in the developing tooth.

Type I and type III collagens have been isolated from dental papilla and dental pulp of bovine tissues by enzymic digestion with pepsin and differential salt precipitation. Type III collagen was further purified by molecular sieve and ion-exchange chromatography. The relative proportions of type III and type I collagens were estimated by two different techniques, gel electrophoresis in the presence of mercaptoethanol, and direct estimation of the alpha1(III)CB3 peptide. Both techniques gave similar values. Type III collagen constituted 24% of papilla and 28% of pulp collagen by the first procedure, and 20% and 31% by the second. 18% of dental follicle collagen was type III.

Collagen synthesis in rabbit dental pulp fibroblast cultures

Enzyme dissociation of rabbit dental pulp enabled isolation of fibroblast-like cells. Confluent rabbit dental pulp fibroblasts maintained in Dulbeccos modification of Eagles medium incorporate L-[3H]proline in a linear manner with time into non-diffusable macro-molecules for up to 24 h. Assays of 4-hydroxyl[3H]proline indicate that collagen synthesis was also linear over this period. The nature of the secreted collagen molecules was investigated by molecular-sieve chromatography and sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The proportion of type-III collagen produced by the cells in culture was estimated from the pepsin-digest of the proteins in the medium before and after reduction and found to be 16 per cent by gel filtration and 15 per cent by SDS/gel electrophoresis.

The collagen composition of the mandibular joint of the foetal calf

Abstract The distribution of genetically distinct collagen molecules in the mandibular joint was investigated. Type II collagen was found only in the articular cartilage. Both the fibrous layer covering the articular cartilage and the articular disc were composed of type I collagen and no type II collagen was detected. The histological appearance of these tissues was consistent with the biochemical findings. Small amounts of type III collagen were associated with the fibrous layer covering the articular cartilage; its function is unknown.

Microfibrillar assemblies of foetal bovine skin. Developmental expression and relative abundance of type VI collagen and fibrillin.

Intact type VI collagen microfibrils and fibrillin-containing microfibrils were isolated from foetal bovine skin and investigated immunochemically and ultrastructurally. Substantial variations were detected in the abundance and macromolecular assembly of these structures at progressive stages of gestation. Microfibrils of collagen VI were increasingly abundant in skin through foetal development from late first trimester to term. The pattern of expression of fibrillin-containing microfibrils in foetal skin differed from that of collagen VI. Fibrillin-containing microfibrils were particularly sparse in first trimester skin, and present only as short assemblies. However, by early second trimester there had been a sharp increase in the abundance and length of these fibrillin-containing microfibrils. These observations are consistent with early second trimester being a key phase of fibrillin assembly. In the third trimester, fibrillin-containing microfibrils were frequently isolated in association with amorphous material. This information on the pattern of expression and assembly of collagen VI microfibrils and fibrillin-containing microfibrils in foetal skin implies temporally and functionally distinct contributions of these two components to the establishment of the fibrous dermal matrix.

Microfibrillar components in dental pulp: Presence of both type VI collagen- and fibrillin-containing microfibrils

Microfibrillar elements were isolated from developing and formed bovine dental pulp by a procedure involving bacterial collagenase tissue digestion and chromatography on Sepharose CL-2B. Two microfibrillar assemblies could be demonstrated. Type VI collagen microfibrils with a characteristic periodicity of about 100 nm appeared as long, thin, flexible filaments. In a number of cases these structures aggregated by lateral association. Microfibrils of 10-14 nm dia were identified as containing fibrillin on the basis of their distinctive, periodic, beaded morphology. In addition to long, single strands there were instances of chains coalescing to give amorphous aggregates. No differences in the type of microfibrillar assemblies were evident between developing and formed pulp, although fibrillin-containing microfibrils were more abundant in formed pulp.

Collagenolytic activity of the extracellular vesicles of Bacteroides gingivalis W50 and an avirulent variant W50/BE1.

The activities of the extracellular vesicle fractions of these two organisms were compared. Lytic activity against a native type I placental collagen substrate at 30 degrees C was assessed following sodium dodecyl sulphate-polyacrylamide gel electrophoresis and densitometry. A rapid rate of collagen depolymerization was achieved by the extracellular vesicle fraction of W50, yielding approx. 90% substrate degradation compared to 5% for W50/BE1 extracellular vesicles over 6 h incubation. The polypeptide digestion patterns produced by incubation with extracellular vesicle fractions of both organisms were identical, and similar to those yielded by incubation of substrate with whole W50 cells.

Biosynthesis of glycoproteins by rabbit dental pulp fibroblasts in culture.

Confluent dental pulp fibroblast cultures incorporated L-[3H]-fucose in a linear manner with time into non-diffusible macromolecules over 24 h. Ascorbate supplementation did not appear to alter the amount or type of macromolecules. Equilibrium CsCl-density-gradient centrifugation established that the [3H]-fucose-labelled macromolecules released into the medium were predominantly glycoproteins. Sodium dodecyl sulphate-polyacrylamide-gel electrophoresis showed that the major fucosylated glycoprotein had an apparent molecular weight of 230,000, but several other species were also seen. The major fucosylated glycoprotein was fibronectin by its affinity for gelatin and its immunoprecipitation with a specific anti-(cold-insoluble globulin).

Synthesis of sulphated glycosaminoglycans by rabbit dental pulp fibroblasts in culture

Abstract The synthesis of sulphated glycosaminoglycans was studied in dental pulp fibroblast cultures and in organ culture. The uptake of radioactive sulphate in both systems increased with time. Papain digestion and DEAE-cellulose chromatography showed that the major sulphated glycosaminoglycan synthesized by either cell or organ culture was chondroitin sulphate. This was confirmed by electrophoresis on cellulose-acetate strips, its susceptibility to chondroitinase AC and testicular hyaluronidase. Small amounts of heparan sulphate were synthesized in both cell and organ culture and identified by its electrophoretic mobility and by its susceptibility to nitrous acid degradation. The presence of dermatan sulphate was indicated by the resistance to chondroitinase AC and testicular hyaluronidase and the degradation by chondroitinase ABC. A fraction with an electrophoretic mobility identical to authentic dermatan sulphate could be isolated by ethanol precipitation in the presence of calcium acetate.

Extraction of collagen fractions from bovine and rabbit dental follicle, papilla and pulp

Abstract Age-related changes in collagen solubility were found in bovine dental papilla and dental pulp. Increased amounts of insoluble collagen in mature dental pulp were associated with completion of root formation. In the pulp of the continually erupting rabbit incisor tooth, increase in the amount of newly synthesized collagen led to an increase in soluble collagen.

In-vivo incorporation of [3H]-proline into rabbit dental pulp collagen.

Abstract Collagen metabolism of rabbit dental pulp was studied by following the incorporation of [3H]-proline into pulp collagen. The specific activity of hydroxyproline was studied in various collagen fractions in rabbit incisor and molar teeth of different ages. The specific activity of the collagen fractions was consistently higher in incisor than in molar pulps at all ages, except for the pepsin-resistant fraction. In the molar at most ages, the pepsin-resistant fraction had a specific activity equal to, or greater than, that in the fractions solubilized by either acid or pepsin. The results demonstrate the high metabolic activity of dental pulp, and rapid incorporation of new collagen into the insoluble fibre network.