From appraising the literature apparent gaps were identified in the broad overview of keloid aetiopathology; hypotheses were then formulated within these gaps around pertinent related aspects and focussed into different studies for investigation. The logical approach to ultrastructural morphological investigations of keloid formation was to comprehensively interpret the light microscopical features and complement this with prospective immunocytochemical and electronmicroscopical studies. It is imperative that all recognisable features are deciphered and explained before postulating the pathological processes involved.
Piecing together the various parts of the study should eventually result in valuable information on the pathogenesis of keloid formation
1.23.1 Paper 1: Keloids show regional distribution of proliferative and degenerative connective tissue elements
In reviewing the literature on keloid morphology, there are many contradictory reports. These contradictions range from keloids exhibiting a hyperproliferation of dermal fibroblasts (87) to
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few fibroblasts (169); from the presence of a higher number of blood vessels in keloids when compared with normal skin (170), to the presence of a reduced vascular component (171). Other conflicting reports on keloid morphology were about the patency of vessels: when compared to normal skin and normal scars, it was reported that in keloids, the blood vessels were dilated and longer (170), while other reports showed an increase in the number of occluded microvessels (172). The absence of distinct nodules in keloids and their conspicuous presence in hypertrophic scars was a major histological difference between the two types of excessive scar tissue formation reported by Ehrlich et al (123) and this was contradicted by Luo et al (63) who reported that a hallmark feature of keloids was the presence of “collagen nodules”. Another contradictory report on collagen arrangement informed that “discrete bundle formation was virtually absent” in keloids. Also, there are conflicting reports on the collagen content of keloids:
Clore et al (173) report the quantity of types I and III collagen in keloids to be the same as in normal dermis, whereas other researchers showed elevated levels to be present in keloids (174).
The variable expression of α-SMA expression reported in keloid scars suggested that this feature could not reliably help in distinguishing the two types of scars and implies that the pathogenesis of keloid scars is multifactorial, repudiating the clonal theory of origin (57).
Hypothesis: Thorough in depth ultrastructural examination of the entire keloid lesion and subsequent establishment of baseline morphological characteristics of keloids should explain many of the past and present contradictions, regarding morphological features. Also such a study was essential for future ultrastructural research investigating the pathogenesis of keloid formation.
This resulted in the publication of the following original article:
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1.23.2 Paper 2: Does the paucity of elastic fibres contribute to the process of keloidogenesis?
During excessive wound healing, as in keloids, in addition to the constant production and progressive accumulation of scar tissue, traction force is continuously generated by migrating fibroblasts. Excessive, prolonged traction forces deform the extracellular matrix up to their elastic limit; beyond this limit the friction force that the tissue can withstand is exceeded resulting in movement of the matrix (175). Stress applied beyond the elastic limit, results in plastic changes that occur at a rate greater than the tissue can tolerate causing injury and permanent deformation; this is called plasticity stress (175). Beyond the plasticity level, tissue cannot tolerate more stress and it ruptures (175). Could the disfiguring dermal deformations in keloids be a manifestation of gradually accumulated effects of stress from many lesser loads exerted by continuous traction force generated by wound healing fibroblastic cells, especially during the granulation, angiogenic and tissue remodeling stages? Supportive evidence for this includes: the formation of keloids months to years post injury, after apparent successful healing and the use of pressure therapy in the effective treatment of keloids (176). Pressure therapy compensates for the elastic insufficiency of the affected dermal tissue and reduces scar tissue formation by inducing localised hypoxia and subsequent fibroblastic degeneration (176).
However, preexisting hypoxia was confirmed in keloids where the accumulation of hypoxia- inducible factor-1 protein was found (177). The effectiveness of pressure therapy treatment of keloids may, therefore, be by exacerbation of the existing hypoxic state. This corroborates an association between stresses (force applied), strain (deformation when stress is applied), elastic insufficiency, hypoxia and keloid formation; thus reinforcing the necessity to investigate the role of traction forces in the pathogenesis of keloids.
Hypothesis: Keloids are growths formed by excessive wound healing where traction force generated by migrating fibroblasts deforms the extracellular matrix up to their elastic limit.
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Beyond this plastic changes occur and cause permanent deformation. As permanent deformation of tissue occurs in keloids and as elastic recoil is the principal function responsible for recovery of tissue deformed by traction forces, we hypothesise that, in keloids, connective tissue elasticity is decreased, reducing the elastic limit and resulting in augmented tissue deformation which progresses to keloid formation.
Morphological assessment of the elastic fiber content of keloids resulted in the publication of the following original article:
• Does the paucity of elastic fibres contribute to the process of keloidogenesis?
1.23.3 Paper 3:
There is a definite tendency for keloids to form in the upper torso of the body, the main areas being the head (earlobes and mandibular ridge), neck, back, presternal and deltoid areas (178).
The reasons for the propensity of skin in these regions to form keloids may be related to the inherent characteristics of skin and the effects of intrinsic and extrinsic factors on it.
Hypotheses: The predilection for keloids to form in skin covering the upper torso of the body is related to elevated dermal stress caused by:
1. stress-amplification at the soft and hard supportive connective tissue interface
2. inherent skin characteristics such as skin thickness, the bundled arrangement of collagen in the reticular dermis, fat content, the existent high tension, the low elastic modulus and low stretch ability
3. contractile forces generated by wound healing fibroblastic cells 4. action of external forces
Augmented internal stress causes dermal distortion and compression resulting in increased loading on cells; this triggers anabolic effects, with amplification of gene expression, collagen
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synthesis and mitosis. Microvessels that become compressed and occluded cause ischemia and reperfusion injury; reperfusing blood is rich in growth factors which stimulate growth when released into the extracellular matrix of the healing tissue. Brainstorming and substantiating the possible reasons that support these hypotheses resulted in the publication of the following original article:
1.23.4 Paper 4: The significance of hyalinized tissue in the pathogenesis of keloids Hyalinised collagen bundles (HCB) are pathognomonic of keloids where they are a characteristic and diagnostic feature (179).
Hypothesis: The hyalinization process is crucial to the understanding of the pathogenesis of keloids and possible reasons for the occurrence are hypothesised below:
• To provide a protective role by sequestrating cytokines and proteases produced by mast cells
• To prevent autoimmune attack by providing a safely isolated region where injured cells, that might not have gained prior tolerance, can demise without the danger of exposure to immune reactivity. These cells include degenerative and necrotic myofibroblastic cells and other fibroblastic phenotypes.
Investigations into these hypotheses resulted in the preparation and submission of the following original article:
• The significance of hyalinized tissue in the pathogenesis of keloids
43 1.24 Expected outcome
In this study it is envisaged that verification or elucidation of the above hypotheses, using a holistic approach where all areas of the keloid are examined in routine wax sections stained with haematoxylin and eosin and various differential and immunocytochemical stains. Light
microscopy is the gold standard for the examination of pathological tissue and the “maintenance and reinforcement of good quality microscopy remains a priority” not only at the health center level, but also at the research level (180). Light microscopy should therefore, be the main tool of examination of specimens and this should be complemented by electron microscopy. Integration of critically, comprehensively and accurately interpreted results, with pertinent information from previous investigations, should permit the formulation of novel concepts that would contribute to a better understanding of the pathology and aetiopathogenesis of keloid formation. It is anticipated that prospective microscopical investigations arising from the morphological work covered thus far, will direct biochemists and molecular biologists ultimately, to the invention of efficient, unfailing treatment.
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PUBLICATIONS AND SUBMITTED PAPER
2. 2 Does the paucity of elastic fibres contribute to the process of keloidogenesis?