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 Table of Contents  
MUMBAI NABICON RESEARCH AWARD 2013
Year : 2014  |  Volume : 22  |  Issue : 1  |  Page : 3-9

Scar biology: Fibroblast the key modulator


1 Department of Burns and Plastic Surgery, CHILDS Trust Medical Research Foundation, Kanchi Kamakoti CHILDS Trust Hospital, Chennai, India
2 Research Office, CHILDS Trust Medical Research Foundation, Kanchi Kamakoti CHILDS Trust Hospital, Chennai, India
3 Consultant Plastic Surgeon, Kanchi Kamakoti CHILDS Trust Hospital, Nungambakkam, Chennai, India
4 Department of Burns and Plastic Surgery, Chennai Medical College and Research Institute, Thiruchirapalli, Tamilnadu, India

Date of Web Publication15-Dec-2014

Correspondence Address:
Mathangi K Ramakrishnan
H.O.D, Department of Burns and Plastic Surgery, CHILDS Trust Medical Research Foundation, Kanchi Kamakoti CHILDS Trust Hospital, Chennai
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0971-653X.146990

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  Abstract 

Introduction: Normal wound repair of human skin involves several well-orchestrated phases, which are overlapping, yet distinct in their own way. This presentation aims to define, identify, and classify scars, irrespective of the etiology, describe the methods available to differentiate the various types of scar, and describe the current management strategies available to prevent scar formation. From our studies we conclude that for a good scar outcome early intervention during the evolution of wound healing is required. Meticulous surgery is also essential. Various interventions could be with drugs, chemicals, growth factors, or ionizing radiation. Future holds for continued research in cellular signals.

Keywords: Fibroblasts, hypertrophic scars, keloids


How to cite this article:
Ramakrishnan MK, Babu M, Mathivanan T, Jayaraman V. Scar biology: Fibroblast the key modulator. Indian J Burns 2014;22:3-9

How to cite this URL:
Ramakrishnan MK, Babu M, Mathivanan T, Jayaraman V. Scar biology: Fibroblast the key modulator. Indian J Burns [serial online] 2014 [cited 2019 Dec 8];22:3-9. Available from: http://www.ijburns.com/text.asp?2014/22/1/3/146990


  Introduction Top


Normal wound repair of human skin involves several well-orchestrated phases, which are overlapping, yet distinct in their own way. The initial phases immediately after wounding are coagulation and platelet degranulation. This is responsible for the release of important cytokines and growth factors which function as chemotactic agents. This leads to the recruitment of neutrophils, macrophages, endothelial cells, and fibroblasts to the wound region. This is the inflammatory phase, which is followed by the proliferation phase. This phase involves the proliferation and differentiation of the cells. [1],[2]

Wound healing



Aims and objectives of the study

  • To define, identify, and classify scars, irrespective of the etiology.
  • To describe the methods available to differentiate the various types of scar.
  • To describe the current management strategies available to prevent scar formation.


Definition of scar

Scar is an abnormal area of collagenization after wound repair as compared with the surrounding tissue. An ideal scar has good texture with normal pigmentation neither depressed nor exuberant without contracture. The healed area should look like the normal surrounding tissue [Figure 1].
Figure 1: Deep-partial thickness burns with healed wound

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Abnormal scars

Hypertrophic scars: [Figure 2]
Figure 2: Post burn hypertrophic scar

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  • Thick fibrous structure.
  • Hypercellular matrix.
  • Increased collagen expression with myofibroblasts.
{Figure 2}

These scars occurred due to lack of skin resurfacing and second intention wound healing resulting in Hypertrophic scars.

Keloid: [Figure 3]
Figure 3: Post burn keloidal scar

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  • Florid growth of fibroblasts.
  • Excessive collagen in the dermal matrix.
  • Lesion spreads beyond the margins of the original wound.
{Figure 3}

Components of scar



Over activity of growth factors may be responsible for modulating the components of the skin to form abnormal scarring. [3]

Differentiation of scars

Scars can be differentiated by water content, histochemical analysis, biochemical studies in cultured fibroblasts, ultrastructural examination, and ultrasound studies.

Water content

Biochemical studies show that there was increased water content in the vertices of scar tissue. As we cut these scars, tissue fluids ooze out, particularly in biopsy-performed specimen. This fact was validated by estimating T1 relaxation studies with Magnetic Resonance Imaging between normal skin and scar tissue.



Water content is high clinically and in MRI studies. MRI detects the movement of floating water particles reflected and expressed as relaxation time T1. When there is more water, the T1 (relaxation time) will be longer as seen in the chart.

Histochemistry

By using Vangeison's stain, which is specific for collagen, tissues can be stained. Massive collagen deposition is seen in scars due to the fact that more number of fibroblasts that are present in the matrix secretes more collagen [Figure 4].
Figure 4: Histochemistry of scars

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Cytokine estimations

  • PMN leucocytes secrete the proinflammatory IL6 and ILβ1 cytokines at the wound site. IL6 and IL β1 are increased in the stage of inflammation. Matrix synthesis, degradation, cell proliferation, and cell recruitment at the wound site are influenced by these cytokines. This increase affect the scar outcome




Biochemical studies on cultured fibroblasts

Fibroblasts from normal skin hypertrophic scar and keloid were cultured in DMEM with 10% fetal calf serum. [Figure 5] The cultured fibroblasts were visualized for any morphological changes. The rate of proliferation of fibroblasts was estimated by MTT assay.
Figure 5: Biochemical studies on fibroblasts

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Quantitative estimation of fibroblast proliferation



Morphology of fibroblasts remains the same. Excessive proliferation rate is seen in keloids and hypertrophic scars.

Type I collagen biosynthesis was studied in normal and keloid fibroblast by immunoblotting with antibody to Type I collagen [Figure 6].
Figure 6: Type I collagen expression is 3-4 fold higher in keloids

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Fibronectin, which is the second important matrix protein expression, was studied by immunofluorescence in normal and keloid fibroblasts with antibody to fibronectin. Fibronectin expression is increased in Keloid fibroblasts compared to normal skin fibroblasts [Figure 7].
Figure 7: Phase contrast fibronectin immunoflorescence

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Apart from changes in dermal matrix proteins, some changes were observed in the epidermis of keloids compared to normal skin. Keratins were isolated from normal skin hypertrophic scar and keloids, and SDS Page Electrophoresis was carried out, and using marker proteins the molecular weight of Keratins were analyzed [Figure 8].
Figure 8: Analysis of molecular weight of keratins

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The immunoblot of keratins [Figure 9] was carried out with isolated keratins from normal skin, hypertrophic scar and keloids with antibody to proliferating type of keratins. From both of the above Figures [Figure 8] and [Figure 9] the increased quantities of proliferating type of keratin of molecular weight 50 KD is confirmed.
Figure 9: Immunoblot of keratins

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[Figure 10] shows the Ultra structural studies of fibroblast by Electron microscopy. The increased endoplasmic reticulum in keloid fibroblasts signifies increased protein synthesis.
Figure 10: Ultra structural studies of fibroblast by electron microscopy

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Differentiation of scars by ultrasound studies

High resolution ultrasound scanning [Figure 11] can differentiate scars. The third dimension of spread into the depth of the dermis by collagen fibers can be seen. Keloidal scars have maximum invasion of the depth by the collagen bundles.
Figure 11: Ultrasound of scars

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Management strategies

  • Prevention.
  • Pressure garments.
  • Surgical excisions and grafting or flap surgery.
  • Drug modulation with a low molecular weight heparin and corticosteroids.
  • Antihistamine.
  • Ionizing radiation.
  • Therapy with decorin.


Closure of the wound at the earliest, will help in preventing scar formation. [4] Meticulous care of the wound with appropriate dressing will not form a bad scar. Avoidance of infection will help early wound closure. Anti-inflammatory agents like low molecular weight heparin will enable the wound to heal faster by reducing chronic wound healing phase. Management strategies are directed toward the phases of wound healing, which in turn predicts the scar outcome.

Prevention

Meticulous wound care, excision of devitalized tissue, and the use of proper suture materials gives a good scar. In the case of skin grafting, proper massage with moisturizing creams and Jobst pressure garments to give pressure.

Pressure garments

To reduce the interstitial edema occurring during post-burn period treatment can be done by pressure dressings, which can be custom-made after surgery. In the field of plastic and reconstructive surgery, every step, planning, execution, postoperative care, and managing of the scars are very important. [5],[6],[7] Reduction in scar hypertrophy can be achieved by pressure garments, hydrogel therapy, and silicone incorporated pressure dressings. This reduces interstitial edema and induces apoptosis.

Surgical treatment

[Figure 12] shows how treatment is achieved by excision of scar and realignment, skin grafting, and scar therapy.
Figure 12: Surgical management of scars

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Inhibition of scar growth with anti-inflammatory drugs, namely low molecular weight heparin and corticosteroids

Inhibition of scar growth by low molecular weight heparin is through modulation of pro-inflammatory cytokines - IL6.

Heparin protocol

Low molecular weight heparin fragmin is usually used.

Adult

Loading dose: 2500-4000 I.U (as per cardiac protocol) or 400 I.U/Kg/15 %/TBSA 12th hourly for 7 days.

Pediatric

Loading dose: 400-1000 I.U intravenouslyThen 40 units/kg /15% TSBA 12 th hrly for 7 days.

Topical spray

400 I.U. or 40mg/Kg/15% TSBA, Sprayed 12th hourly for 7 days.

Biological membrane

Harvested in 2500 I.U. in 500 ml of normal saline with Gentamycin in the regular refrigerator. Used within 12 hours.

Monitoring

Estimation of platelet count should not fall below 50,000.Nitric oxide synthase is increased in acute burns. This is responsible for prolonged inflammatory response and ultimately results in deranged wound healing and scar production. Low molecular heparin effectively reduces the NOS by its anti-NOS activity. This is shown in the two immunohistochemistry pictures where the NOS expression was increased in control and NOS levels decreased in treated cases. Monoclonal antibody for NOS was used for this work [Figure 13].
Figure 13: (a and b) Immunohistochemistry showing levels of NOS in control and treated cases with Heparin protocol

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Treatment with corticosteroids

Long acting synthetic glucocorticoids such as triamcinolone are most commonly used for treatment of hypertrophic and Keloid scars. Solution of triamcinolone is injected in 10-40 mg/ml concentrations into the scar. [8] However, the morbidity of repeated injection on a monthly basis, fat atrophy and the depigmentation of skin are major drawbacks to these treatments. Steroids are often useful for the treatment of older scars that are less metabolically active and also are not routinely used as a scar preventive measure after wound closure.

Effect of antihistamine Avil on cultured fibroblasts [Figure 14]

We investigated the effect of avil (pheniramine maleate) on fibroblasts cultured from keloids and hypertrophic scars. We observed a decrease in the rate of proliferation and total DNA synthesis by keloid fibroblasts in comparison to normal skin fibroblasts. There was also corresponding decrease in the rate of collagen synthesis observed in scar fibroblasts. Hence avil may prove useful in the treatment of keloids and hypertrophic scars. [9]
Figure 14: Effect of antihistamine avil on cultured fibroblasts

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Radiotherapy

Radiotherapy after surgical excision of keloids [Figure 15] had shown signs of promise by earlier workers. [10]
Figure 15: Effect of radiation on cultured cells. Both keloid and normal skin fibroblasts show arrest of growth but not death after radiation

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Low-dose ionizing radiation given within 24 hours following excision of small area of isolated keloid in the earlobe gives 80% success in preventing the recurrence.

Under general anesthesia, keloid was carefully excised and within 24 hrs the patient was sent for radiation. Fifteen Gray electron beam radiation of 1600 RADS were given in 3 divided doses of 5 Gray each (every other day). The long-term results have been very good as we have followed for a period of 1-7 years and found that no case in the group recurred.

To establish the scientific basis for the postoperative radiotherapy, the fibroblasts from excised keloid and normal skin biopsies were isolated and cultured in vitro. Confluent cultures were subjected to 5 Gray gamma radiations for 5 minutes. The proliferations of the irradiated cells were assessed for a period of 5 days. Interestingly the cell proliferation got arrested, but no actual cell death occurred. This clearly shows that irradiation inhibits the growth of dermal fibroblasts but does not kill them leading to prevention of excess matrix production thereby arrest keloid growth. [11]

Role of decorin [Figure 16]
Figure 16: Role of decorin

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Non-collagenous matrix proteins, such as proteoglycans are associated with the surface of the collagen fibrils and bring about their proper assembly. Such proteoglycans may sometimes substitute for collagen species at the fibrillar surface or perform auxiliary functions. [12],[13] Some of these contain leucine-rich repeats and have been shown to affect collagen fibrillogenesis and spacing between the mature fibrils. In remodeling phase of wound healing, decorin is an important member of small leucine-rich proteoglycan family. Decorin is indispensable modulator of collagen alignment in wound healing. We have observed that scar fibroblasts have less decorin to normal skin fibroblast. Molecular RNA studies were done in both abnormal scar tissue and cultured fibroblasts. Densitometry was used as a mode of measurement to visualize the intensity of the bands. [GAPDH] is an enzyme that is constant in normal skin and scars. As its intensity does not change, this was used as a marker.{Figure 16}

Immunofluorescence

Immunofluorescence in fibroblasts was visualized by a fluorescent microscope after instilling the immunofluorescent antibodies to normal skin and keloid fibroblasts. [Figure 17] As the scar fibroblasts have less decorin, the fluorescence is less in keloid tissue when compared to normal skin. [14]
Figure 17: Immunofluorescence in fibroblasts

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Decorin binds with TGFb in the cultured fibroblast and reduces cell proliferation and synthesis of collagen in wound healing. Our study indicates that decorin that appears to be a natural antidote for fibrotic disorders can be exploited for therapeutic intervention. Decorin may be incorporated in reconstituted collagen dressing, but cannot be given to the patient either orally or even directly applied on the burn.{Figure 17}


  Conclusion Top


For a good scar outcome, early intervention during the evolution of wound healing is required. Meticulous surgery is also essential. Various interventions could be with drugs, chemicals, growth factors, or ionizing radiation. Future holds for continued research in cellular signals.


  Acknowledgement Top


This work was sponsored by the "CHILDS Trust Medical Research Foundation and Kanchi Kamakoti CHILDS Trust Hospital Chennai".

 
  References Top

1.
Meenakshi J, Jayaraman V, Ramakrishnan KM, Babu M. Keloids and hypertrophic scars: A review. Indian J Plast Surg 2005;38:175-9.  Back to cited text no. 1
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2.
Ehrlich HP, Desmoulier A, Diegelmann RF, Cohen IK, Compton CC, Garner WL, et al. Morphological and immunochemical differences between keloid and hypertrophic scar. Am J Pathol 1994;145:105-13.  Back to cited text no. 2
    
3.
Raghow R. The role of extracellular matrix in postinflammatory wound healing and fibrosis. FASEB J 1994;8:823-13.  Back to cited text no. 3
    
4.
Roseborough IE, Grevious MA, Lee RC. Prevention and treatment of excessive dermal Scarring. J Natl Med Assoc 2004;96:108-16.  Back to cited text no. 4
    
5.
Kischer CW, Shetler MR, Shetlar CL. Alteration of hypertrophic scars induced by mechanical pressure. Arch Dermatol 1975;111:60-4.  Back to cited text no. 5
    
6.
Ricketts CH, Martin L, Faria DT, Saed GM, Fivenson DP. Cytokine mRNA changes during the treatment of hypertrophic scars with silicone and nonsilicone gel dressings. Dermatol Surg 1996;22:955-9.  Back to cited text no. 6
    
7.
Phillips TJ, Gerstein AD, Lordon V. A randomized controlled trial of hydrocolloid dressing in the treatment of hypertrophic scars and keloids. Dermatol Surg 1996;22:775-8.  Back to cited text no. 7
    
8.
Sclafani AP, Gordon L, Chadha M, Roma T 3 rd . Prevention of earlobe keloid recurrence with postoperative corticosteroid injection versus radiation therapy: A randomized, prospective study and review of the literature. Dermatol Surg 1996;22:569-74.  Back to cited text no. 8
    
9.
Venugopal J, Ramakrishnan M. Habibullah CM, Babu M. The effect of the anti-allergic agent avil on abnormal scar fibroblasts. Burns 1999;25:223-8.  Back to cited text no. 9
    
10.
Ragoowansi R, Cornes PG, Moss AL, Glees JP. Treatment of keloids by surgical excision and immediate postoperative single-fraction radiotherapy. Plast Reconstr Surg 2003;111:1853-9.  Back to cited text no. 10
    
11.
Janakiraman M, Ramakrishnan KM, Jayaraman V, Chandrashekar S, Babu M. Etiology and management of ear lobule keloid in south India. Plast Reconst Sur 2007;119:495-7.   Back to cited text no. 11
    
12.
Roughly PJ. Articular cartilage and changes in arthritis: Noncollagenous proteins and protoglycans in the extracelular matrix of cartilage. Arthritis Res 2001;3:342-7.  Back to cited text no. 12
    
13.
Vogel KG, Paulson M, Heingard D. Specific inhibition of type I and type II collagen fibrillogenesis by the small proteoglycan of tendon. Biochem J 1984;223:587-97.  Back to cited text no. 13
    
14.
Meenakshi J, Vidyameenakshi S, Ananthram D, Ramakrishnan KM, Jayaraman V, Babu M. Low Decorin expression along with inherent activation of ERK1,2 in ear lobe keloids. Burns 2009;35:519-26.  Back to cited text no. 14
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12], [Figure 13], [Figure 14], [Figure 15], [Figure 16], [Figure 17]



 

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