|Year : 2014 | Volume
| Issue : 2 | Page : 68-74
Picrosirius red staining assessment of collagen after dermal roller application: A minimally invasive percutaneous collagen induction therapy
Fatma El-Zahraa Salah El-Deen Yassin1, Reham Ezz El-Dawela2, Mohammad Abel Kerim2
1 Department of Pathology, Venereology and Andrology, Sohag University, Sohag, Egypt
2 Department of Dermatology, Venereology and Andrology, Sohag University, Sohag, Egypt
|Date of Web Publication||18-Dec-2014|
Fatma El-Zahraa Salah El-Deen Yassin
Department of Pathology, Faculty of Medicine, Sohag University, Sohag
Source of Support: None, Conflict of Interest: None
Background: Percutaneous collagen induction (PCI) through dermal roller breaks old collagen strands, promotes removal of damaged collagen and induces more collagen formation. Collagen fibers can be assessed by traditional stains or by polarized light assessment of Picrosirius red stain. Objective: The purpose of the current study is the clinical and histopathological evaluation of percutaneous collagen formation in atrophic acne scars after dermal roller application. Patients and Methods: Total study duration was 26 weeks in which 12 patients received seven sessions of PCI at 3-weeks interval, 3 mm punch biopsy specimens of scars were obtained before and after treatment (at 18 and 26 weeks). Microscopic examination of pre and post operative biopsies were done, using routine stains and Picrosirius red stain. Results: PCI induced notable improvement in the appearance of acne scars with significant reduction in the score from 123.3 ± 24.5 to 74.16 ± 16.49 (P = 0.00) after 26 weeks. Polarized light assessment of Picrosirius red stain clarified the gradual replacement of old thick orange-red birefringent collagen fibers by newly synthesized thin green-yellow birefringent ones postoperatively. Conclusion: Skin needling is a simple and minimally invasive procedure. The polarized light assessment of Picrosirius red stain clarified the change of the optical properties of collagen fibers according to the maturation process.
Keywords: Collagen, dermal roller, Picrosirius red stain
|How to cite this article:|
Yassin FZS, El-Dawela RE, Kerim MA. Picrosirius red staining assessment of collagen after dermal roller application: A minimally invasive percutaneous collagen induction therapy. Indian J Dermatopathol Diagn Dermatol 2014;1:68-74
|How to cite this URL:|
Yassin FZS, El-Dawela RE, Kerim MA. Picrosirius red staining assessment of collagen after dermal roller application: A minimally invasive percutaneous collagen induction therapy. Indian J Dermatopathol Diagn Dermatol [serial online] 2014 [cited 2019 Jun 19];1:68-74. Available from: http://www.ijdpdd.com/text.asp?2014/1/2/68/147289
| Introduction|| |
Despite many advances in the treatment of post acne scarring, it still does not have a fully satisfactory treatment.  Percutaneous collagen induction (PCI) with dermal roller (a needling tool) has been described as an alternative maneuver for management of postacne scars; it protects the epidermis and stimulates natural collagen synthesis. ,
PCI with dermal roller produces thousands to tens of thousands of fine pricks which are close to each other, promoting the normal post-traumatic release of growth factors and infiltration of fibroblasts which are "instructed" to produce more collagen. 
Falabela and Falanga considered that skin needling- induced normal wound healing occurred in three phases: Inflammation phase, tissue formation and fibroblast proliferation phase and tissue remodeling phase, where collagen type III is laid down in the upper dermis and is gradually replaced by collagen type I over a period of a year or longer.  Another hypothesis proposed by Liebl, (2009), is the bioelectricity theory which triggers a cascade of growth factors leading to new collagen deposition in the upper dermis. 
The purpose of this study is the clinical and histopathological evaluation of percutaneous collagen formation in atrophic acne scars after dermal roller application.
| Patients and Methods|| |
This prospective study was conducted from January to October 2012, it was approved by the Ethics Committee and written informed consent was obtained from all participants before enrollment. Overall, 14 patients (6 men and 8 women) with different types of atrophic acne scars were enrolled in this study of which 12 completed the study (five men and seven women). Inclusion criteria were age ≥ 18 years and any type of facial atrophic scars (icepick, boxcar and rolling). Exclusion criteria were systemic retinoid or immunosuppressive drug intake during the previous 6 months, coagulation defects or hematological diseases, evidence or history of keloid scar formation and history of facial laser treatment, surgical procedure or dermabrasion 6 months before study enrollment, presence of skin cancer, warts, solar keratoses or any skin infection, patients with a medical condition that might have influenced the wound healing process, pregnancy and unrealistic expectations. Patients were allowed to continue previous acne medications during the study period, except isotretinoin. PCI treatment was performed for all patients. Each patient received seven sessions of treatment at 3-week interval. Patients were instructed to avoid medications such as aspirin and nonsteroidal anti-inflammatory drugs (NSAIDs) for at least 1 week before the session and to start using topical retinoid 2 weeks before each session, stopping 2 days before the session to avoid irritation to the skin.
Clinical outcome assessment
Patient follow-up was scheduled at 3 weeks interval during the 18-week treatment period and at 4 weeks interval for 8 weeks after the final session (total study duration, 26 weeks from treatment commencement). Acne scar improvements were quantified by assessing the degree of improvement according to scar types, and the echelle d'evaluation clinique des cicatrices d'acne [clinical evaluation scale for acne scarring] (ECCA) scores by two dermatologists. ECCA grading scales are based on semiquantitative, weighted assessments of six types of acne scars, namely V-shaped atrophic scars (icepick), U-shaped atrophic scars (boxcar), M-shaped atrophic scars (rolling), hypertrophic inflammatory scars, keloid scars, and superficial elastolysis, the degrees of improvement were evaluated as percentage improvement (0-100%). ,
Three millimeter punch biopsy specimens of untreated scars were obtained from the 12 patients before treatment commencement, and after the seventh session (at 18 weeks). A third biopsy was taken from two volunteer patients at 8 weeks after final treatment (after 26 weeks of study). Punch biopsy specimens were fixed in 10% neutral buffered formalin, embedded in paraffin and sectioned at a thickness of 5 μm; three stains were used: Standard Hematoxylin and Eosin (H and E), Masson's trichrome stain (Biotech, code KT034) and Picrosirius red (Via Marche, 19, DDK, Italia S.R.l).
Picrosirius red (PR) stain was applied as follows: The deparaffinized sections were hydrated, stained with Weigert`s hematoxylin (8 minutes for nuclei), and stained with PR (1 hour). Then all sections were dehydrated, cleared in xylene and mounted with DPX.
The PR-stained sections were analyzed using an Olympus microscope (U- MDOB3, Japan) equipped with Simple Polarizing Attachment (BX-POL, Olympus, Japan); it consists of two filters to provide circularly polarized illumination; A Polarizer (U-POT) (circular polarizer) and an Analyzer (U-ANT). These two filters were aligned to obtain a dark field; tissue images were obtained with different objective lenses and recorded on a digital Camera (E330-DC 7.4V, Olympus, Japan).
Statistical analysis was performed with Wilcoxon signed rank test (for comparison of before and after dermal roller treatment) and Student T test using SPSS software (version 16, SPSS Inc., Chicago, IL). Data were expressed as mean and standard deviation.
| Results|| |
Twelve patients (five men and seven women) were enrolled in this study. They completed the 26-week study without any significant adverse effects. Age of patients ranged from 20 to 31 years mean ± SD (25.9 ± 4.3 years). The mean duration of acne scars was 4.3 years (range 2-8 years). Detailed ECCA scores of all patients are listed in [Table 1].
|Table 1: Types of postacne atrophic scars and grading of score severity (echelle d'evaluation clinique des cicatrices d'acne; ECCA) before and after PCI |
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Dermal roller treatment induced a statistically significant improvement in acne scars. Consistently, ECCA scores were significantly reduced following PCI after 26 weeks (123.3 ± 24.5 → 74.16 ± 16.49) (P = 0.0.00). There was a highly significant decrease in the mean score of all types of atrophic acne scars after PCI treatment [Table 2]. The Wilcoxon signed rank test showed highly significant decline in icepick, boxcar and rolling scars, total ECCA grading score at 26 weeks treatment (P = 0.001, 0.005, 0.005, 0.002, respectively) with variable degree of improvement but not statistically significant difference in mean percentage of improvement between different types of atrophic scars (icepick, boxcar or rolling) (P = 0.16), [Table 2], [Figure 1] and [Figure 2]. No significant difference between male and female cases in their severity score (ECCA) both before and after treatment was observed (P = 0.65 and 0.45 respectively). No significant correlation was seen between the degree of improvement and scar duration.
|Figure 1: Degree of improvement in different types of atrophic acne scars and total ECCA grading scores after PCI|
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|Figure 2: Atrophic acne scars of representative patients showed notable improvement in all types. (a and d) before treatment (baseline), (b and e) after 18 weeks of PCI treatment, (c and f) after 26 weeks (at the end of study)|
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|Table 2: Mean ECCA grading scores and percentage improvement in atrophic acne scars before and after PCI |
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Examination of the pre-operative punch biopsies (old atrophic scars) of 12 patients revealed the following: Flat dermo-epidermal junction with unremarkable rete ridges and compact, poorly defined eosinophilic dermal collagen bundles [Figure 3]a. Masson's trichrome-stained sections showed the dermal collagen bundles in the form of dense blue thick fibers arranged in parallel scar pattern [Figure 3]b. Polarizing light assessment of PR stain identified the old thick collagen fibers of atrophic scars as orange to red birefringence [Figure 3]c
|Figure 3: (a and b) Thin epidermis with flat rete ridges and dense collagen bundles; (c) orange-red birefringence of old collagen fibers (original: ×400, ×200, ×100); (d) pin hole cutting the epidermis (arrow) with dermal infiltrate. (e) The recent collagen bundles laid down in lattice pattern. (f) A green birefringence fibers of recent fibers (arrow) dissecting the old red-orange ones (original: ×100, ×100, ×200). (g, h and i) Different collagen deposition with more mature yellow ones (original: ×100, ×100, ×100)|
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The post operative punch biopsies (at 18 and 26 weeks) recorded the following histopathological changes.
The epidermis, particularly the stratum corneum, remained intact except for minute holes, which were about 2-4 cells in diameter [Figure 3]d with more developed rete ridges and unremarkable changes in melanocytes. Masson's trichrome-stained sections revealed more collagen deposition in the dermis in the form of blue fibers arranged in a lattice pattern [Figure 3]e. A background of neovascularization, proliferating fibroblasts and scarce inflammatory infiltrate was noted in both previous sections. Discrimination between newly synthesized collagen fibers and old ones could be difficult to assess at both levels.
Polarizing light assessment of PR stain confirmed all previous findings with more detection and qualifications of dermal collagen content. Superficial dermis showed newly formed thin collagen bundles as green-colored birefringence, dissecting the old thick collagen bundles which appeared orange-red colored birefringent [Figure 3]f.
An interesting finding was noticed in post procedure punch biopsies done at 26 weeks; we could assess gradual replacement of thin green birefringent collagen fibers by more mature yellow birefringent ones. After PCI as collagen matured; the optical properties of the fibers showed signs of increase in birefringence from green to yellow [Figure 3]i and [Figure 4].
|Figure 4: Polarized light assessment of Picrosirius red stain revealing the maturation process of collagen fibers: (a) Preoperative biopsy of old acne scar showing orange-red birefringence collagen fibers; (b) postoperative 18 weeks biopsy showing very recently formed green birefringence collagen fibers; (c) postoperative 26 weeks biopsy revealing more mature yellow birefringence collagen fibers|
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| Discussion|| |
PCI therapy stimulates the natural posttraumatic inflammatory cascade by rolling the dermal roller over skin area to create thousands of closely neighboring microwounds in the dermis that results in a confluent zone of very superficial inflammation, triggering the release of growth factors that ultimately results in increasing the patient's own normal woven collagen. 
The present study revealed a statistically significant overall improvement of atrophic acne scars in all patients as the mean ECCA score was significantly reduced following PCI after 26 weeks (123.3 ± 24.5 → 74.16 ± 16.49) (P = 0.00) with 39.8% percentage improvement. This was in agreement with Majid (2009) who reported an excellent response in 72.2% of patients after four sessions of PCI. 
The present study showed highly significant decline in icepick, boxcar, rolling scars and total ECCA scores at 26 weeks of treatment. Rolling scars showed 58% improvement after PCI, followed by 33.5% in Boxcar and 31.4% in icepick scars. In comparison to these results, Majid (2009) reported an excellent response in rolling and boxcar scars, while moderate response in icepick scars. 
For collagen assessment, histopathological examination of pre- and post procedure scars was done, using three different stains (H and E, Masson's trichrome and PR stain).
Although previous studies , promoted the traditional stains such as Van Gieson and Masson's trichrome for the detection of collagen fibers in tissue section, we suggest that traditional stains may not be ideal for collagen detection because their mechanism depends mainly on differential binding of the combined anionic dyes to tissue components which lack precise selectivity. Moreover, the traditional stains would fail to reveal very thin collagen fibers, which may lead to underestimation of collagen content; this finding was demonstrated by Kiernan, 2002.  An additional disadvantage, may be the tendency of stain to fade as shown by Sweat and colleagues. 
To overcome all previous disadvantages, we used PR stain in addition to traditional ones to assess collagen fibers; PR could stain the thin collagen fibers, did not fade and was suitable for use by polarized light microscope as described by Rich and Whittaker, 2005.  Thus, the combined use of PR stain and simple polarized light enabled us to detect, analyze and qualify collagen content.
Although many studies ,, have employed bright field illumination to detect collagen content, we preferred to use polarized light system (dark field) rather than bright field illumination for PR-stained sections as collagen fiber color in bright field illumination appears deep red for thick fibers and bright pink for thin fibers, the difference being highly subjective and difficult to assess. Rich and Whittaker's results corroborated with ours, where they reported that the potential use of bright field illumination approach underestimated the collagen content, especially in tissues containing large amount of thin fibers. 
The use of circularly polarized light allows visualization of every portion of collagen fiber in contrast to the use of linearly polarized light as reported by Rich and Whittaker, therefore we used the first technique as it is easy, readily available and relatively inexpensive. ,,
In the present study, histopathological examination of the post-procedure punch biopsies of 12 patients (H and E stained sections) showed intact epidermis with well developed rete ridges. This is in agreement with Fernandes and Signorini, 2008 and Aust et al., 2008; they showed that PCI left the epidermis intact without any damage to stratum corneum or any other layers of the epidermis or the basal membrane. ,
The current study compared collagen deposition in acne scars pre- and post-operatively at the end of treatment (18 and 26 weeks); pre-operative collagen fibers were arranged in parallel pattern with lack of newly formed thin capillaries and fibroblasts while post-operative biopsies showed increased collagen deposition which was laid down in normal lattice pattern along with newly formed thin capillaries, proliferating fibroblasts and scarce inflammatory cells (H and E and Masson's trichrome stained sections). This was in agreement with several studies; ,, Fernandes and Signorini, 2008 reported considerable greater collagen deposition 4-6 months postoperatively.
We identified the recent thin collagen fibers at the end of treatment (18 weeks) as green to yellow birefringence in comparison to the old thick fibers of pre-operative acne scars that appeared orange-red birefringent as visualized by polarized light of PR-stained sections. The results were in accordance with Rich and Whittaker, 2005,  who found a time associated shift from green to orange in PR-stained fibers within myocardial scars.
El-Domyati et al. explained in more detail their and our results as follows; as collagen matures, the optical properties of fibers shows signs of increase in birefringence (the ability to change color under polarized light) with a consequent decrease in light penetration. They evaluated the non-ablative radiofrequency device in photoaging with PR stain, which showed a significant increase in newly synthesized collagen, as reflected by the presence of yellow birefringence at the end of treatment, compared with baseline. 
Fernandes, 2005,  reported that collagen type III is dominant in the early wound healing phase, becomes maximal 5-7 days after injury and gets laid in the upper dermis just below the basal layer of epidermis, collagen type III is gradually replaced by collagen type I over a period of one year or more. Junqueira et al. studied spectrum of mammalian tissues stained with sirus red stain and examined under polarized light; they described collagen type I having yellow, orange or red color while collagen type III appeared green. 
Hence we used PR stain for qualification of types III and I collagen fibers. Whereas type III recent collagen fibers appeared thin green to yellow birefringent in the superficial dermis, the thick old orange to red birefringent collagen fibers (in atrophic acne scars) represented type I collagen fibers, PR stain is therefore a good qualitative method for further classification of collagen fiber types (types III and I).
| Conclusions|| |
Skin needling is a simple and minimally invasive procedure with rapid healing. The combined use of PR stain and simple circular polarizing system could assess change in the optical properties of collagen fibers from green to yellow to orange to red birefringence according to their maturation process and fiber' thickness.
| Acknowledgement|| |
We thank all the patients who participated in this study.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]