Transformation of Collagen Fibrils into "Elastin" 



231 



incubated in phosphate buffer (pH 7.4) containing 0.2 mg 

 elastase for 6 hours at 37 C. 



0.05 ml. of a penicillin and streptomycin mixture was 

 added to each test tube in the above experiments, and 

 this successfully prevented bacterial contamination. 



CI. histolyticiini collagenase was kindly supplied b\ 

 Dr. J. D. MacLennan; the elastase was prepared by Hall 

 and Gardiner (2). 



Samples of three of the substrates (aged 2, 9 and 78 

 years) were heated in sterile distilled water to 55 C 

 for 1 hour, to 75 C for a further hour, and finally to 

 100 C for an hour. Prior treatment with either collagenase 

 or alkaline buffer was of course omitted. 



Samples for electron microscope examination were 

 taken at each stage in the experiments, and ground gentl> 

 in a glass tissue-grinder until the material appeared 

 milky; drop preparations were made which were shad- 

 owed with chromium and examined in a Siemens electron 

 microscope, type UM 60 C. 



Counts of the different kinds of elastic structures were 

 made by carefully scanning two grids from each sample. 

 The grids contained 16 squares, and each part of each 

 square was examined in sequence. As about 20 fields 

 covered a square, a total of 640 fields was scrutinised 

 from each specimen. 



The wide spectrum of morphological change pro- 

 duced exhibited marked age-differences. 



1. Heat controls. Heat alone on the untreated 

 2-, 9- and 78-year-old prepared collagen samples 

 simply produced progressive gelatinisation as the 

 temperature increased, which was complete after 

 boiling for one hour. There was no age-difference, 

 no increase in elastic structures and none of the 

 new structures described below. The fully-formed 

 elastin present in the starting material was some- 

 what reduced in quantity, whilst the component 

 elastin filaments became particularly well defined. 



Fully-formed elastin. This is a normal component 

 of human fresh whole dermis and prepared dermal 

 collagen from all age groups. Natural elastin forms 

 about 1-5 "o of the histologically stained dermis, but 

 the amount normally present increases with age. 

 The following variants can be distinguished and 

 counted under the electron microscope: 



(a) Skin-type elastin (which forms about 95 % of 

 the elastin) denotes dense, irregular, structures 

 without any regular features (fig. 1). 



(h) Filamenting elastin (which constitutes about 

 5 "o of the elastin) is usually found in long, twisting, 

 ribbons. 



(c) Large natural networks (which account for 

 1 "^o of the elastin) differ from the manufactured 

 networks (MN) described below in being completely 

 free of amorphous material or dense bits and seem 

 to be a network variety of skin-type elastin. They 

 occurred sporadically in all preparations examined. 



This fully-formed elastin was unaffected by heat, 

 although in the 56- and 78-year-old preparations 

 there was a suggestion of concentration (i.e. higher 

 counts) as the collagen gelatinised. 



2. Effect of heat on collagenase-treated prepara- 

 tions. Over the age of 50 years collagenase only 

 produced slight macroscopic digestion of the col- 

 lagen samples without the production of true MEF. 



Fig. 1. Typical example of skin-lype elastin from llic fresh 

 dermis of an intlividiial aged 66 years. This variant forms 

 ')5"„ of nauiral (fully-formed) elastin and contains a fair 

 amount of dense amorphous material (elastomucin). The 

 other variants (filamenting and large networks) have less 

 elastomucin so their component filaments are better visua- 

 lised. Magnification 12.000. 



Fig. 2. Typical "moth-eaten" fibre (MEF) from the prepared 

 dermal collagen of a 2-ycar-old child after incubation for 

 24 hours at 37 C with collagenase (CI. histolyticiini) in phos- 

 phate bufter pH 7.4. Note the beaded fibrils and numerous 

 "beads" in the background. Magnification 10,000. 



Thus the starting material consisted simply of stri- 

 ated collagen showing slight "standard"" collagenase 

 change (7) and the usual ciuantity of fully-formed 

 elastin. These components responded to heat in 

 identical fashion to the heat controls described 

 above. However, samples from the child and young 

 adult substrates gave a very different picture. 

 85-90 "o of the collagen was digested, and the deposit 

 consisted mainly of typical MEF (fig. 2). Gentle 

 grinding fragmented these thick, dense fibres into 

 portions of different lengths accompanied by a 

 large number of angular, dense bits. These MEF 

 proved relatively heat-resistant, a considerable degree 

 of breakdown occurring only after boiling. In addi- 

 tion many portions of MEF were found to terminate 

 as "elastic networks" (MEFC) and a number of 

 isolated networks (MN) were also seen. 



MEF conversions (MEFC) (figs. 3 and 4) denote 

 moth-eaten fibres transforming into "elastin net- 

 works", identical with the manufactured networks 

 (MN) described below. In the two child substrates 

 some of the MEF appeared to be transforming into 

 sheet-like elastin in addition to the usual network 

 type and heat markedly increased the numbers so 

 transformed. The substrate from the 27-year-old 

 was outstanding in consisting almost entirely of 

 large MEF conversions throughout, in fact the 

 starting material resembled the heat-treated prepa- 

 rations from the 2- and 9-year-old substrates. The 

 28- and 43-year-old starting material showed no 

 definite MEFC or MN but both these structures 

 became definite and easily identifiable after heating. 

 No conversions were seen in material over the age 

 of 50. 



