The Distribution of the Protein-Polysaccharide Complex in the Nucleus Pulposus Matrix in Young Rabbits

1968 ◽  
Vol 3 (1) ◽  
pp. 33-40
Author(s):  
J. W. SMITH ◽  
A. SERAFINI-FRACASSINI
Keyword(s):  
Nucleus Pulposus ◽  
Osmium Tetroxide ◽  
Lead Nitrate ◽  
Vitreous Body ◽  
Collagen Fibrils ◽  
Bismuth Nitrate ◽  
Young Rabbit ◽  
Native Collagen ◽  
The Matrix ◽  
Concentrated Solution

The distribution and relationship of the collagen and the proteinpolysaccharide complex in the extracellular matrix of the nucleus pulposus of the young rabbit were studied by electron microscopy. The polysaccharide was demonstrated by treatment of the tissue with either 0.5% bismuth nitrate at pH 2 or a mixture of equal volumes of 1% lead nitrate and 2% osmium tetroxide at pH 5. The protein part of the complex was observed in tissue fixed in osmium tetroxide at pH 7.2 and stained with 1% phosphotungstic acid. The collagen of the tissue was apparent in all preparations. The water-extracted proteinpolysaccharide was studied after coagulation of a concentrated solution in 0.5% bismuth nitrate, whereas the isolated fibrous fraction was examined by negative staining of sprayed material. Collagen is present in the intact tissues as native collagen fibrils of about 300 Å diameter, and as sheets, 4OO-Å thick, of parallel, separate, filaments of 50-60 Å diameter. Usually several such sheets lie close, and approximately parallel, to one another. After extraction of the proteinpolysaccharide from the tissue with water, all the collagenous components tend to aggregate into large native collagen fibrils. Some of the proteinpolysaccharide complex of the intact tissue is free in the matrix. The polysaccharide moiety is visualized as 270 Å particles, serially attached to a protein core in the form of beaded filaments about 40Å in diameter. The rest of the complex is attached to the sheets of collagen filaments in regular periodic zones which are 270 Å wide and centred 670 Å apart. None of the complex appears to be associated with the larger collagen fibrils. The staining reactions of the polysaccharides in several connective tissues are discussed and the relationships of the proteinpolysaccharide complexes to collagen in cartilage, the vitreous body and the nucleus pulposus are compared.

Banded Structures in the Connective Tissue of Meningioma

1976 ◽  
Vol 34 ◽  
pp. 234-235
Author(s):  
C. N. Sun ◽  
H. J. White
Keyword(s):  
Connective Tissue ◽  
Osmium Tetroxide ◽  
Uranyl Acetate ◽  
Collagen Fibrils ◽  
Lead Citrate ◽  
Connective Tissues ◽  
Native Collagen ◽  
Routine Procedures

Previously, we have reported on extracellular cross-striated banded structures in human connective tissues of a variety of organs (1). Since then, more material has been examined and other techniques applied. Recently, we studied a fibrocytic meningioma of the falx. After the specimen was fixed in 4% buffered glutaraldehyde and post-fixed in 1% buffered osmium tetroxide, other routine procedures were followed for embedding in Epon 812. Sections were stained with uranyl acetate and lead citrate. There were numerous cross striated banded structures in aggregated bundle forms found in the connecfive tissue of the tumor. The banded material has a periodicity of about 450 Å and where it assumes a filamentous arrangement, appears to be about 800 Å in diameter. In comparison with the vicinal native collagen fibrils, the banded material Is sometimes about twice the diameter of native collagen.

Electron Microscopy of Wound Healing in Patients with Hernia

1975 ◽  
Vol 33 ◽  
pp. 352-353
Author(s):  
C.N. Sun ◽  
H.J. White ◽  
R.C. Read
Keyword(s):  
Electron Microscopy ◽  
Wound Healing ◽  
Phosphate Buffer ◽  
Osmium Tetroxide ◽  
Phosphotungstic Acid ◽  
Rectus Sheath ◽  
Uranyl Acetate ◽  
Collagen Fibrils ◽  
Lead Citrate ◽  
Native Collagen

Previously we have reported the defect of collagen fibrils from herniated rectus sheath. This presentation includes additional sections from postsurgical incisions (10 days) from both control and hernia patients. Small pieces of rectus sheath were fixed in 3% glutaraldehyde in phosphate buffer (pH 7.2) and post fixed with buffered 2% osmium tetroxide. The tissues were then dehydrated in serially increasing concentrations of alcohol and embedded in Epon 812. Sections were stained with 2.5% phosphotungstic acid or uranyl acetate and lead citrate.Previously we found that collagen fibrils from "non-herniated" rectus sheath have uniform diameters and 640 Å periodicity with seven or more intraperiodic bands resembling typical native collagen fibrils, while the fibrils from fascia obtained from patients with direct herniation show considerable variation in diameter. These variations are often found in the same individual fibers with a range from 300 Å to 3000 Å.

The Disposition of Proteinpoly-Saccharide in the Epiphysial Plate Cartilage of the Young Rabbit

1970 ◽  
Vol 6 (3) ◽  
pp. 843-864
Author(s):  
J. W. SMITH
Keyword(s):  
Uranyl Acetate ◽  
Bismuth Nitrate ◽  
Young Rabbit ◽  
Proliferative Zone ◽  
Attachment Sites ◽  
Central Regions ◽  
The Matrix ◽  
And Function ◽  
Initial Mineral ◽  
Positively Charged

Although acidic bismuth nitrate stains the polysaccharide in the unfixed bovine epiphysial plate, it fails to do so in that of the rabbit. After fixation in glutaraldehyde, however, Araldite sections stained with bismuth nitrate exhibit 3-nm spots which are interpreted as polysaccharide chains in coiled conformation. In the matrix of the proliferative zone, and in the large Golgi vesicles of the associated chondrocytes, the polysaccharide spots are arranged in closely packed groups. Lead citrate and uranyl acetate staining shows the non-fibrillar material in these situations as a continuous network of particles. The centre of each particle is translucent, and double staining with phosphotungstic acid and bismuth indicates that these central regions contain the groups of polysaccharide spots. It is suggested that in the unfixed tissue bismuth binding is prevented by screening of the polysaccharide sulphates by positively charged non-collagenous protein, and that fixation, by precipitating this protein as a network around groups of proteinpolysaccharide molecules, frees the sulphate groups to react with bismuth. Proteinpolysaccharide molecules appear to be tangentially attached to each period of the small collagen fibrils of the matrix. Fixation precipitation of the associated protein tends to aggregate these molecules into groups which are often somewhat eccentric to their original attachment sites, and consequently the fibrillar attachment of proteinpolysaccharide usually appears to be rather irregular. In the zone of early calcification single mineral crystals are associated with dense bodies of unknown nature and function. Many are also associated with short linear rows of polysaccharide spots. This and the similarity in the lengths of the two structures suggest that initial mineral crystals may grow along a proteinpolysaccharide molecule from one end to the other. Absence of polysaccharide spots in relation to crystal clusters suggest that these clusters are formed as a result of the seeding effect of the initial single crystals.

scholarly journals LOCALIZATION OF ADENOSINE TRIPHOSPHATASE ACTIVITY IN THE RAT SPERM TAIL AS REVEALED BY ELECTRON MICROSCOPY

1965 ◽  
Vol 25 (2) ◽  
pp. 101-112 ◽  
Author(s):  
Toshio Nagano
Keyword(s):  
Osmium Tetroxide ◽  
Surface Membrane ◽  
Nitrate Solution ◽  
Lead Nitrate ◽  
Reaction Products ◽  
Lead Phosphate ◽  
Sperm Tail ◽  
Middle Piece ◽  
The Matrix ◽  
Coarse Fibers

The epididymides of rat testis were fixed in glutaraldehyde and cut as frozen sections. The sections were incubated in lead nitrate solution containing as a substrate either ATP, AMP, creatinine phosphate, beta glycerophosphate, or phenyl phosphate. Then they were postfixed in osmium tetroxide, embedded, sectioned, and examined with the electron microscope. In the sperm tail, when ATP is used as a substrate the reaction product (lead phosphate) is observed both in the tail filament complex and on the surface membrane of the mitochondrial helix of the middle piece. In the tail filament complex, this product is seen near the nine paired peripheral and two central filaments, and in the matrix between the outer coarse fibers. But the product is not observed within these filaments and fibers. In longitudinal sections, no periodicity of the deposits in the complex is observed. When the other phosphate compounds are used as substrates the reaction products appear on the surface membrane of the mitochondrial helix, and are not found in the tail filament complex. No distinctly different localization of the reaction products is observed when substrates other than ATP are used. Possible relationships between the structure and the function of the sperm tail are discussed in the light of these findings.

Effects of EDTA, Hyaluronidase and Collagenase on Epiphyseal Cartilage Matrix

1968 ◽  
Vol 26 ◽  
pp. 56-57
Author(s):  
H. Clarke Anderson ◽  
Priscilla R. Coulter
Keyword(s):  
Light And Electron Microscopy ◽  
Matrix Vesicles ◽  
Cartilage Matrix ◽  
Collagen Fibrils ◽  
Epiphyseal Cartilage ◽  
Dense Matrix ◽  
Type Iv ◽  
Bovine Testicular Hyaluronidase ◽  
The Matrix ◽  
Testicular Hyaluronidase

Epiphyseal cartilage matrix contains fibrils and particles of at least 5 different types: 1. Banded collagen fibrils, present throughout the matrix, but not seen in the lacunae. 2. Non-periodic fine fibrils <100Å in diameter (Fig. 1), which are most notable in the lacunae, and may represent immature collagen. 3. Electron dense matrix granules (Fig. 1) which are often attached to fine fibrils and collagen fibrils, and probably contain protein-polysaccharide although the possibility of a mineral content has not been excluded. 4. Matrix vesicles (Fig. 2) which show a selective distribution throughout the epiphysis, and may play a role in calcification. 5. Needle-like apatite crystals (Fig. 2).Blocks of formalin-fixed epiphysis from weanling mice were digested with the following agents in 0.1M phosphate buffer: a) 5% ethylenediaminetetraacetate (EDTA) at pH 8.3, b) 0.015% bovine testicular hyaluronidase (Sigma, type IV, 750 units/mg) at pH 5.5, and c) 0.1% collagenase (Worthington, chromatograhically pure, 200 units/mg) at pH 7.4. All digestions were carried out at 37°C overnight. Following digestion tissues were examined by light and electron microscopy to determine changes in the various fibrils and particles of the matrix.

scholarly journals Ultrastructural localization of the C-propeptide released from type II procollagen in fetal bovine growth plate cartilage.

1996 ◽  
Vol 44 (5) ◽  
pp. 433-443 ◽  
Author(s):  
E R Lee ◽  
C E Smith ◽  
R Poole
Keyword(s):  
Growth Plate ◽  
Ultrastructural Localization ◽  
Collagen Fibrils ◽  
Type Ii ◽  
Guanidinium Hydrochloride ◽  
High Affinity ◽  
Sds Page ◽  
The Matrix ◽  
Fetal Bovine ◽  
Mineralized Matrix

We used immunochemical and immunoelectron gold techniques to determine whether the C-propeptide previously identified in the matrix of endochondral cartilage (CPII) was still a part of the Type 11 procollagen molecule or had been released from it. Guanidinium hydrochloride extraction, followed by SDS-PAGE and Western blotting techniques and immunoelectron localization, revealed that predominantly only the released form (hereafter referred to as released CPII) was detected. The ultrastructural distribution of this CPII was examined with affinity-purified antibodies and with immunogold or immunoperoxidase localization techniques in the presence or absence of embedding resins. These methods yielded similar results. Although no significant amount of this CPII was retained in the matrix after guanidinium hydrochloride extraction, it was present in two recognizable sites under normal conditions, i.e., locally concentrated in a random association with collagen fibrils in the nonmineralized matrix and mainly concentrated in interfibrillar mineralizing sites in the mineralized matrix. These results suggest that the C-propeptide that has been released from Type II procollagen associates with collagen fibrils and then preferentially associates with mineralizing sites when these form in the endochondral cartilage. The significance of this preference for mineral is not known but may have something to do with its high affinity for hydroxyapatite.

Compartmentalization of the matrix formed by nucleus pulposus and annulus fibrosus cells in alginate gel

2002 ◽  
Vol 30 (6) ◽  
pp. 874-878 ◽  
Author(s):  
E. Thonar ◽  
H. An ◽  
K. Masuda
Keyword(s):  
Nucleus Pulposus ◽  
Annulus Fibrosus ◽  
Articular Chondrocyte ◽  
Alginate Gel ◽  
Age Related ◽  
The Matrix ◽  
Disc Cells ◽  
A Cell ◽  
Associated Matrix

Intervertebral disc cells cultured in alginate gel are capable of reforming in alginate, a matrix that consists of two compartments: a rim of metabolically active cell-associated matrix and a more abundant, but metabolically less active, further removed matrix. At any one age and in most species, the cell-associated matrix formed by a nucleus pulposus or annulus fibrosus cell cultured in this way is less abundant than that formed by an articular chondrocyte. In both the cell-associated matrix and further removed matrix, the ratio of aggrecan to collagen is significantly higher in the case of nucleus pulposus than of annulus fibrosus, a feature that also distinguishes the matrices of the nucleus pulposus and annulus fibrosus in vivo. Nucleus pulposus and annulus fibrosus cells from older donors show a decreased ability to reform a cell-associated matrix rich in aggrecan. There is, however, some evidence that gene therapy and/or exposure of the cells to defined stimulatory factors can help overcome some of these age-related limitations. This contention is supported by recent evidence that nucleus pulposus and annulus fibrosus cells from adult donors can be manipulated to form, using the recently developed alginate-recovered chondrocyte system, a resilient tissue that bears many of the characteristics of the tissue in which these cells reside in vivo.

scholarly journals Dynamic Compression Promotes the Matrix Synthesis of Nucleus Pulposus Cells Through Up-Regulating N-CDH Expression in a Perfusion Bioreactor Culture

2018 ◽  
Vol 46 (2) ◽  
pp. 482-491 ◽  
Author(s):  
Yichun Xu ◽  
Hui Yao ◽  
Pei Li ◽  
Wenbin Xu ◽  
Junbin Zhang ◽  
...  
Keyword(s):  
Nucleus Pulposus ◽  
Dynamic Compression ◽  
Akt Pathway ◽  
Bioreactor Culture ◽  
Nucleus Pulposus Cells ◽  
Matrix Synthesis ◽  
Static Compression ◽  
Matrix Deposition ◽  
The Matrix

Background/Aims: An adequate matrix production of nucleus pulposus (NP) cells is an important tissue engineering-based strategy to regenerate degenerative discs. Here, we mainly aimed to investigate the effects and mechanism of mechanical compression (i.e., static compression vs. dynamic compression) on the matrix synthesis of three-dimensional (3D) cultured NP cells in vitro. Methods: Rat NP cells seeded on small intestinal submucosa (SIS) cryogel scaffolds were cultured in the chambers of a self-developed, mechanically active bioreactor for 10 days. Meanwhile, the NP cells were subjected to compression (static compression or dynamic compression at a 10% scaffold deformation) for 6 hours once per day. Unloaded NP cells were used as controls. The cellular phenotype and matrix biosynthesis of NP cells were investigated by real-time PCR and Western blotting assays. Lentivirus-mediated N-cadherin (N-CDH) knockdown and an inhibitor, LY294002, were used to further investigate the role of N-CDH and the PI3K/Akt pathway in this process. Results: Dynamic compression better maintained the expression of cell-specific markers (keratin-19, FOXF1 and PAX1) and matrix macromolecules (aggrecan and collagen II), as well as N-CDH expression and the activity of the PI3K/Akt pathway, in the 3D-cultured NP cells compared with those expression levels and activity in the cells grown under static compression. Further analysis showed that the N-CDH knockdown significantly down-regulated the expression of NP cell-specific markers and matrix macromolecules and inhibited the activation of the PI3K/Akt pathway under dynamic compression. However, inhibition of the PI3K/Akt pathway had no effects on N-CDH expression but down-regulated the expression of NP cell-specific markers and matrix macromolecules under dynamic compression. Conclusion: Dynamic compression increases the matrix synthesis of 3D-cultured NP cells compared with that of the cells under static compression, and the N-CDH-PI3K/Akt pathway is involved in this regulatory process. This study provides a promising strategy to promote the matrix deposition of tissue-engineered NP tissue in vitro prior to clinical transplantation.

scholarly journals Comparison of the Structural Characteristics of Native Collagen Fibrils Derived from Bovine Tendons Using Two Different Methods: Modified Acid-Solubilized and Pepsin-Aided Extraction

Materials ◽  
2020 ◽  
Vol 13 (2) ◽  
pp. 358 ◽  
Author(s):  
Haiyan Ju ◽  
Xiuying Liu ◽  
Gang Zhang ◽  
Dezheng Liu ◽  
Yongsheng Yang
Keyword(s):  
Type I Collagen ◽  
Structural Characteristics ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Dry Weight ◽  
Collagen Fibrils ◽  
Type I ◽  
X Ray Diffraction ◽  
Aggregation Structure ◽  
Native Collagen

Native collagen fibrils (CF) were successfully extracted from bovine tendons using two different methods: modified acid-solubilized extraction for A-CF and pepsin-aided method for P-CF. The yields of A-CF and P-CF were up to 64.91% (±1.07% SD) and 56.78% (±1.22% SD) (dry weight basis), respectively. The analyses of both amino acid composition and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed that A-CF and P-CF were type I collagen fibrils. Both A-CF and P-CF retained the intact crystallinity and integrity of type I collagen’s natural structure by FTIR spectra, circular dichroism spectroscopy (CD) and X-ray diffraction detection. The aggregation structures of A-CF and P-CF were displayed by UV–Vis. However, A-CF showed more intact aggregation structure than P-CF. Microstructure and D-periodicities of A-CF and P-CF were observed (SEM and TEM). The diameters of A-CF and P-CF are about 386 and 282 nm, respectively. Although both A-CF and P-CF were theoretically concordant with the Schmitt hypothesis, A-CF was of evener thickness and higher integrity in terms of aggregation structure than P-CF. Modified acid-solubilized method provides a potential non-enzyme alternative to extract native collagen fibrils with uniform thickness and integral aggregation structure.

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