696 WOUND HEALING 8 



Bensley in 1934 first noted that the ground substance of tissues stains meta- 

 chromatically. She also observed an increased metachromasia during fibrosis of 

 the pancreas following ligation of its duct. Sylven in 1941 found that increased 

 extracellular metachromasia also occurs in the granulation tissue of healing 

 wounds. This has been verified many times; the reaction is most intense at rela- 

 tively early stages of wound healing, with a decrease between the third and tenth 

 day, depending upon the type of wound produced (Penny and Balfour, 1949; 

 Bunting and White, 1950; Campani and Reggianini, 1950; Persson, 1953; Dunphy 

 and Udupa, 1955). This has suggested that the acid polysaccharide content of 

 newlv formed granulation tissue is elevated. In normal skin, metachromasia is 

 limited to the papillary layer of the dermis, the stroma about hair follicles, and 

 mast cells. Hydrolysis by various hyaluronidases has shed some light upon the 

 tvpe of polysaccharide involved. In vitro experiments show that streptococcal 

 hvaliironidase affects only hyaluronic acid (Meyer et al., 1938) while the testicular 

 enzyme breaks down both chondroitin sulfate and hyaluronic acid in vitro (Meyer. 

 1947). Campani and Reggianini (1950) noted that the metachromasia of wounds 

 varies in its response to hydrolysis by hyaluronidase. The metachromasia of 

 the first four days (unlike the later period) disappears following enzyme treat- 

 ment; this suggests that the initial reaction is due to hyaluronic acid which 

 probably becomes more polymerized and hence resistant to hydrolysis at a 

 later time. 



The scorbutic state characterized by failure in formation of extracellular material, 

 affords an interesting way to evaluate polysaccharides during wound healing (Hojer, 

 1924; Wolbach and Howe, 1926; Wolbach, 1933). In animals with scurvy the pre- 

 formed collagen content remains undisturbed (Elster, 1950) and fine collagen strands 

 are formed; however, the amount of reticulin fails to diminish and collagen strands 

 remain slender. Fibroblasts do not assume a mature morphology, and capillary 

 proliferation is limited with many endothelial columns failing to form a lumen. This 

 is associated with an almost complete absence of metachromasia (Penny and 

 Balfour, 1949). After injection of Vitamin C, metachromasia appears within 

 6-1 2h. and the wound soon reverts to a normal appearance. In a subscorbutic 

 state, Bunting and White (1950) observed increased metachromasia which was 

 readily removed by testicular but not streptococcal hyaluronidase, indicating 

 a svilfated mucopolysaccharide. Persson's studies (1953) support the view that 

 scurvy is characterized by an overproduction of incompletely polymerized ground 

 substance, which stains readily by the P.A.S. reaction but is not highly meta- 

 chromatic (Bradfield and Kodicek, 1951). Whether the lack of metachromasia 

 is due to a depolymerized state, the absence of sulfate groups, or to an abnormal 

 mucopolysaccharide is not known. 



Persson (1953) believes that only highly polymerized hyaluronic acid produces 

 significant metachromasia; in the depolymerized state it stains by the P.A.S. 

 method. A reciprocal relationship seems to exist between these two staining 

 reactions. Metachromatic substances are usually P.A.S. negative (Pearse, 1953; 



^ An excellent critical review of these techniques has been pubUshed recently by McMannus 

 (1957)- 



