Biochemical studies of the material of intercellular spaces

The thesis is based on some physical investigations of the properties and organization of some components of intercellular space. The work was divided into two parts: (l) an investigation of the contribution of hyaluronic acid and collagen to the structure of Wharton's jelly found in human umbilical cord, and (2) an investigation of neutral salt soluble collagen.
(1) Investigation of the contribution of hyaluronic acid and collagen to the structure of Wharton's jelly found in human umbilical cord.
After a study of the swelling of the interstitial tissue of the cord in various media, and the effect of swelling on the extractability of hyaluronic acid, a method was adopted for extracting the material. Care was taken to obtain fresh cords and to avoid degradation during extraction. The Wharton's jelly was dissected out, removing blood vessels and as far as possible the fibrous tissue associated with them. The Wharton's jelly was finely minced and the mince was treated with small volumes of solvent (water and buffer solutions) and, after being left to stand, was cleared by centrifugation. The conditions of extraction were studied and the stability of the extract on storage. A number of successive extractions were required to remove the bull of the polysaccharide, and more severe treatment could yield still further extracts.
Sedimentation and viscosity studies on polysaccharide extracted by mild methods showed that its physicochemical properties resemble those of the hyaluronic acid of the synovial fluid of human knew joints fairly closely. An investigation of these synovial fluids was made in collaboration with Drs. J. E. Stanier and A. G. Ogston.
The structure of Wharton's jelly was investigated by visible and electron microscopy. This suggested that there is a network of unbranched collagen fibres, many of them very fine (down to 100A diameter), which lie freely in the jelly. Similar fibres were found in cords which had not reached full term. Material was examined after digestion with hyaluronidase and with collagenase. Electron micrographs of hyaluronic acid suggested the possibility of a fibrous structure; but doubts were cast upon this interpretation by studies of the appearance of "pearl-strings" in random distributions of small glass spheres.
These findings led to a hypothesis, of the structure of intercellular spaces, and specifically of the parts played by water, hyaluronic acid, and collagen fibres. Theoretical considerations show that a combination of a fibrous network and an incompressible fluid can provide an important contribution to the mechanical strength of the tissue, provided that the incompressible fluid, water, is not permitted to move freely through the network. Experimentally it was shown that hyaluronic acids a considerable resistance to water moving through them, and calculation showed that a collagen network could in turn offer sufficient resistance to a solution of hyaluronic acid, to hinder the movement of water. Sedimentation and viscosity studies on native and degraded hyaluronic acids showed the importance of a high degree of polymerisation in producing this effect, and suggested a mode of action of hyaluronidase as it is employed in clinical practice.
Further investigations, by means of model systems, were obstructed both by the lack of a suitable fibre-forming system and by the difficulty in determining the proportions of collagen and hyaluronic acid in tissues.
(2) Investigation of neutral salt soluble collagen.
This was first undertaken as a simple investigation of the physico-cheimical properties of neutral salt soluble collagen in solution. The study was hampered by the ease with which solutions forced precipitates under the conditions of investigation. An investigation of this precipitate formation was therefore undertaken, which showed that the precipitates are fibrous and have the typical cross striations of collagen as examined in the electron microscope. Above a certain temperature, determined by the other conditions such pH, fibres were rapidly precipitated from the solution, and with care the process could be partly reversed by cooling.
By these means it was shown that the original material consists of three components: A, which reversibly precipitates as fibres on warming and re-dissolves on cooling; B, which does not precipitate; and C which precipitates but does not readily re-dissolve on cooling. Polarimetric studies showed a method of distinguishing between native material and gelatin formed from it. A method was devised for separating the three components of neutral salt soluble collagen.
Sedimentation, viscosity, flow birefringence and ultraviolet absorbtion studies were made on solutions of the unfractionated material, of the separate fractions, and of a control solution which had undergone the fractionation procedure but whose fractions had not been separated. These studies indicated that all the materials are similar and consist of elongated particles having a "molecular weight" of 5 × 105. All the fractions except A were found to be heterogeneous. Electron microscope studies were also made on these solutions, and these suggested that fraction C consists of only partly disintegrated fibres.