Michael H. Flint

The Influence of Mechanical Forces on the Glycosaminoglycan Content of the Rabbit Flexor Digitorum Profundus Tendon

The physical forces acting on the flexor digitorum profundus tendon of the rabbit were altered by anterior translocation of the tendon. The glycosaminoglycan (GAG) content was determined in regions of the tendon previously under tension or previously subjected to pressure. There was an increase in the GAG content in the original tension transmitting region. Initially this was due to increased hyaluronic acid and chondroitin sulfate content during the early remodeling phase. Later when tension was restored to the translocated tendon, the content of these two GAG decreased to normal values while the high overall GAG concentration was maintained by increased amounts of dermatan sulfate. Finally the dermatan sulfate content and the total GAG content returned to normal values.The original pressure bearing region showed a rapid loss of total GAG. This was mainly due to a loss of chondroitin sulfate component, and eventually the region showed a GAG composition similar to that of normal tension transmitting tendo...

Collagen Fibril Diameters and Glycosaminoglycan Content of Skins— Indices of Tissue Maturity and Function

Samples of anatomically and functionally distinct regions of the skin of a variety of altricial and precocial animals were taken at various stages of development from birth to beyond maturity. The glycosaminoglycan (GAG) content and composition of the tissues were determined by chemical analysis and the collagen fibril diameters measured by transmission electron microscopy. The fibril diameters of the skins of two fish and a bird were also assessed for comparison. Analysis and comparison of the data collected show that there was a significant correlation between collagen fibril diameter distribution, GAG type and amount, and functional load-bearing of the various skins, and that the variations in the biochemical and physical composition of the tissues at different stages of development could be related to both their post-conceptual maturity and their prospective functional loading.

A role for glycosaminoglycans in the development of collagen fibrils

Extensive data on the glycosaminoglycan (GAG) composition and the collagen fibril diameter distribution have been collected for a diverse range of connective tissues. It is shown that tissues with the smallest diameter collagen fibrils (mass‐average diameter < 60 nm) have high concentrations of hyaluronic acid and that tissues with the largest diameter collagen fibrils (mass‐average diameter ∼200 nm) have high concentrations of dermatan sulphate. It is suggested that the lateral growth of fibrils beyond a diameter of about 60 nm is inhibited by the presence of an excess of hyaluronic acid but that this inhibitory effect may be removed by an increasing concentration of chondroitin sulphate and/or dermatan sulphate. It is also postulated that high concentrations of chondroitin sulphate will inhibit fibril growth beyond a mass‐average diameter of ∼150 nm. Such an inhibition may in turn be removed by an increasing concentration of dermatan sulphate such that it becomes the dominant GAG present in the tissue.

The Effect of Tensional Load on Isolated Embryonic Chick Tendons in Organ Culture

Digital flexor tendons isolated from 17-18 day embryonic chickens were cultured intact, either on steel mesh grids, or in an apparatus designed to apply a mechanical load to the tissue. Tendons cultured without an applied load continued to synthesize protein and glycosaminoglycans throughout a 7-day period, but DNA synthesis decreased during this time. Increases in both protein and DNA synthesis were observed in tendons experimentally loaded for 48-72 h. Glycosaminoglycan production by tendons isolated from 17-day embryos was also increased in loaded tendons, sulfated GAG being increased more than hyaluronic acid. The same loading regime applied to tendons from 18-day embryos produced a smaller, yet significant increase in sulfated glycosaminoglycans but hyaluronate production was reduced. These investigations demonstrate that embryonic chicken tendons can be maintained in a viable state in organ culture and may provide a useful model for studies of the effects of mechanical forces on the synthetic capability and structure of connective tissue cells.

The Masson staining of collagen — an explanation of an apparent paradox

SynopsisAlthough collagen of either tendon or dermis can be stained equally well with Ponceau 2R/Acid Fuchsin or Light Green SF if the dyes are used independently, tendon collagen retains the red dye mixture and dermal collagen the green counterstain when the dyes are used sequentially in the Masson trichrome procedure. The results of experiments designed to assess differences in the penetration, retention and displacement of these arylmethane dyes have demonstrated that they are retained more firmly by the tensioned collagen of tendon or stretched dermis, and are more easily displaced from the collagen of relaxed tendon or dermis.Experiments designed to test the basis of these differences in dye retention indicate that more positively-charged amino dye-binding sites are available in the tensioned collagen than in relaxed collagen, where they appear to be closely associated with adjacent carboxyl groups on the collagen fibres. The possibility that the carboxyl groups of associated acid mucopolysaccharides are implicated in the differences in staining propensity has been investigated and discounted. It is suggested that whereas the binding of arylmethane dyes to collagen under tension is through strong ionic linkages to amino groups, the binding of these and other dyes to relaxed collagen is through weaker hydrogen bonds. It is proposed that these differences in charge distribution on the collagen of the two situations is related to the previously described piezo-electric effect demonstrable on stretched collagen.

The effect of heating and denaturation on the staining of collagen by the Masson trichrome procedure

SynopsisAlthough normally, dermal collagen fails to retain the Ponceau 2R/Acid Fuchsin component of the Masson trichrome procedure and is stained green by the counterstain, the collagen of sections of human dermis which have been heated above its denaturation temperature (Ts) in moist conditions will retain the initial red stain. This difference in dye retention appears to be related to conformational changes of the collagen molecule which are associated with denaturation, for the change in staining is directly related to the shrinkage temperature, is reversed by rapid cooling after heating, is profoundly affected by differences in environmental conditions during heating which affect molecular rearrangement, and is age-dependent.Analogous changes in staining propensity have been observed in heated keratin and epidermis. However, whereas the retention of the initial red dye mixture is increased in denatured dermis, it is diminished in heated epidermis. It is suggested that the subtle dye substrate interaction of the Masson trichrome staining procedure can be utilized to demonstrate changes in molecular configuration which are associated with thermal, as well as other forms of denaturation of collagen and other proteins.

The glycosaminoglycans of Dupuytren's disease.

The total and individual glycosaminoglycan (GAG) content at various stages of the Dupuytren disease process and in samples of normal palmar connective tissue (palmar dermis, palmar fascia and digital flexor tendon) from the hands of uninvolved age-matched controls have been assayed and compared. Morphological comparisons between the different tissues were made by histological examination of sections stained to demonstrate collagen fiber patterns and glycosaminoglycan distribution. Significant differences in the type and amount of GAG were found between the various manifestations of the disease process, i.e., nodules, cellular and fibrous bands, and between these and the normal palmar connective tissues. In the most actively proliferating cellular regions chondroitin sulfate levels were 11 times greater than those of the normal palmar connective tissues, whereas dermatan sulfate tissue levels showed a fourfold increase. On the other hand, tissue concentrations of hyaluronate were similar to those of normal palmar connective tissue. The relationship of these differences in GAG levels to the development and maturation of the normal palmar connective tissues and the Dupuytrens process is discussed.

The adverse effects of HEPES, TES, and BES zwitterion buffers on the ultrastructure of cultured chick embryo epiphyseal chondrocytes

SummaryChick embryo epiphyseal chondrocytes cultured in media containing HEPES, TES, and BES zwitterion buffers, used in combination or independently, consistently developed cytoplasmic vacuoles. This cytoplasmic vacuolation was resolved when the zwitterion buffered media was replaced by media containing bicarbonate:CO2 enriched air buffer. Vacuoles were infrequent or absent in cultures grown in bicarbonate:CO2 enriched air. Chondrocytes with an established extracellular matrix showed less vacuolation than fibroblastlike and polygonal shaped cells that lacked such a matrix. The granular endoplasmic reticulum and Golgi dictyosomes of zwitterion buffered chondrocytes were distended and contained a flocculent amorphous material. Cytoplasmic vacuoles (0.5 to 3.0 μm diam) formed by the fusion and intracellular accumulation of Golgi vesicles and vacuoles also contained a flocculent material enhanced by ruthenium red. Membrane bound extracellular vacuoles containing ruthenium red stained proteoglycan aggregates were common in the extracellular matrix of zwitterion buffered cultures but were generally absent from bicarbonate treated cultures. Electron dense calcium deposits seemed much larger and more numerous in the presence of zwitterion buffers.It is suggested that HEPES, TES, and BES buffers, used alone or in combination, may adversely affect cell membrane systems, and thus the transport or secretory mechanisms operative in cultured chondrocytes, or both, resulting in vacuole formation and the intracellular accumulation of synthesized export material. Although the mechanism by which HEPES, TES, and BES induce these changes remains unclear, the use of zwitterion buffers in biological preparations should be treated with caution.

The Efficacy of Subcutaneous Goretex Implants in Monitoring Wound Healing Response in Experimental Protein Deficiency

This combined biochemical and histological study demonstrated that subcutaneously implanted Goretex tubing can be used to monitor and detect variations in wound healing potential in rats subjected to experimental hypoproteinaemic and normal refeeding conditions. Induced hypoproteinaemia was observed to be associated with a marked diminution in cellular infiltration, collagen synthesis and fibrous deposition within the implant. All these effects were completely reversed by subsequent refeeding of normal diet. Although regional variations in fibroblastic response attributable to biologic variability, were observed within individual control implants, or between paired controls, they were relatively minor as compared to the marked differences observed at the macroscopic, microscopic and biochemical level between implants removed from normally fed and protein deficient animals.

Relationship between the axial periodicity and staining of collagen by the Masson trichrome procedure

SynopsisAlthough wide variations in the axial periodicity of collagen fibrils in tissue sections have been reported previously, the comparative study presented in this paper of the axial periodicity and the Masson trichrome staining of collagen fibres from different tensional situations demonstrates that marked variations in collagen axial periodicity can be directly correlated with the tensional state of the tissues before fixation and with their Masson trichrome staining reaction.Collagen fibres from tensional situations consistently exhibit a longer periodicity and retain the Acid Fuchsin component of the Masson trichrome procedure, whilst collagen fibres from non-tensional situations have a shorter periodicity, fail to retain the Acid Fuchsin component, and are coloured by the Light Green counterstain.