All PhD Theses

A.M.H. Cornelissen

Effects of interferons on palatal wound healing.

11-02-2000

A scientific essay in Medical Sciences

DOCTORAL THESIS defended in public on 11th of February 2000

SUMMARY

The objective of this study was to analyse the changes in tissue architecture and matrix composition during healing of palatal wounds of immature rats, and to compare this with rats of the same age that did not receive mucoperiosteal wounds. Wounds were made in the mucoperiosteum of the palate of 35-days-old rats. Samples were evaluated histologically at numerous points in time after wounding. The DNA, hydroxyproline, and sulphated-glycosaminoglycancontents were determined at 8 , 15 , 30 , and 60 days post-wounding. Eight-days-old granulation tissue contained 43% less hydroxyproline , and 100% more glycosaminoglycans and cells than unwounded palatal tissue of 43-day-old rats. Sixty-day-old wounds contained 100% more DNA and 39% more hydroxyproline than unwounded tissue of 95-day-old rats. At the same time , densely packed and transversely aligned collagen fibres were present. It is concluded that palatalmucoperiosteal wounds made in 35-day-old rats heal with distinct scar tissue formation. The scar contains more collagen than non-wounded palatal tissue of rats of the same age. Therefore, this model may be of use for the development of therapies aiming to reduce palatal scarrinng.

In order to identify wound contraction and scar formation during palatal mucoperiosteal wound healing in growing rats, the temporal and spatial distribution of myofibroblasts and matrix components were determined immunohistochemically. Myofibroblasts were found in the mucosal part of the palatal wound tissue between 4 and 22 days , with the highest density at 8 days post-wounding. The number of collagen type I and III fibres gradually increased until about 8 days post-wounding and thereafter the staining intensity of collagen type III decreased. At 60 days post-wounding, there were more transversally orientated collagen type I fibres, and less type III and lastin present in the submucosa than in normal tissue. The results suggest that in this model contraction mainly takes place in the mucosa between 4 and 22 days post-wounding. Furthermore , palatal wounds made in young rats heal with distinct scar tissue formation. Therefore this model is useful to test the effects of therapies aiming to reduce wound contraction and scarring after cleft palate surgery.

The purpose of this study was to select drugs that specifically reduce the collagen synthesis of palatal granulation fibroblasts without affecting their proliferation. Granulation tissue fibroblasts were obtained from 8-day-old palatal mucoperiosteal wounds and normal fibroblasts from palatal tissue of unwounded rats. Cultured cells were treated with interferon-a2b , interferon-b, interferon-g (0, 100 , 1000 and 10000 U/ml). Cell proliferation was measured by ³H-thymidine incorporation. Collagen synthesis and non-collagenous protein synthesis were determined from the incorporation of ³H-proline. None of the interferons significantly inhibited the proliferation of either type of fibroblasts. Interferon-a2b had no effect on the studied parameters at te dosages used. Interferon-b reduced collagen synthesis of granulation fibroblasts without affecting non-collagenous protein synthesis or the protein synthesis of normal fibroblasts. Interferon-g reduced collagen synthesis of granulation and normal fibroblasts and non-collagenous protein synthesis of granulation fibroblasts.These data show that interferon-b specifically reduces the collagen synthesis of oral granulation fibroblasts without affecting normal palatal fibroblasts.

Wound contraction and scar formation in the palatal mucoperiosteum after cleft palate surgery impair maxillary growth and dento-alveolar development. The aim of this study was to determine the effects of IFN-b on palatal mucoperiosteal wound healing in rats. Standardised wounds were made in the palatal mucoperiosteum of young rats. Either IFN-b or vehicle were injected at the wound site between 4 and 29 days after wounding. The results were compared with unmanipulated control wounds. Samples of wound tissue were collected at 8 , 15 , 30, and 60 days after wounding, and were prepared for biochemical and microscopical analysis. The hydroxyproline, sulphated-glycosaminoglycan, and DNA contents of the wound tissue were analysed biochemically. The degree of re-epithelialisation, the number of myofibroblasts, the tissue thickness, elastin, and collagen type I and type III fibres were evaluated microscopically. Injection of vehicle alone affected almost all healing parameters compared with the controls and delayed the healing process. IFN-b simulated re-epithelialisation when compared with the vehicle treated wounds but had no significant effect on the other parameters evaluated. It is concluded that treatment with IFN-b does not reduce the number of myofibroblasts and scar tissue formation during palatal wound healing. Other techniques need to be developed to locally deliver pharmacological agents to palatal wounds.

Chapter 6 [contents] Standardised wounds were made in the palatal mucoperiosteum of young rats. Either IFN-g or vehicle was injected locally to the wound site between 4 and 29 days post-wounding. The results were compared wih unmanipulated control wounds. Samples of wound tissue were collected at 8,15,30, and 60 days post-wounding, and were prepared for biochemical and microscopical analysis. The hydroxyproline , sulphated-glycosaminoglycan, and DNA contents of the wound tissues were analysed biochemically. The degree of re-epithelialisation, tissue thickness, the number of myofibroblasts, elastin, and collagen type I and III were evaluated in histological sections. Injection of vehicle alone affected almost all healing parameters compared with the controls, and delayed the wound healing process. IFN-g stimulated re-epithelialisation and decreased 1the number of myofibroblasts when compared with vehicle-treated wounds. It also decreased the hydroxyproline and glycosaminoglycan content of 60-day-old wound tissue but the histological characteristics of scar tissue persisted. It is concluded that IFN-g reduces the number of myofibroblasts and the collagen content of scar tissue after palatal wound healing. Therefore, it may be a promising pharmaceutical agent to reduce wound contraction and scarring after cleft palate surgery. However, other techniques need to be developed to locally deliver pharmaceutical agents to palatal wounds.

Chapter 7 [contents] The results of the previous chapters are discussed as a whole, and suggestions for future research are made. Combining the results of chapter 2 and 3 a diagram illustrating the subsequent phases of palatal wound healing in young rats is given. It is concluded that the animal model described in this thesis can be of use to test the effects of pharmaceutical agents on palatal wound healing. It has become apparent that IFN-beta and IFN-gamma reduce the collagen synthesis of palatal wound fibroblasts in vitro.