Endogenous Thymosin β4 Expression in Sacrococcygeal Pilonidal Sinus Disease: A Retrospective, Immunohistochemical Analysis of Excisional Skin Biopsy Samples

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Ostomy Wound Management 2017;63(4):30–40
Sevinc Sahin, MD

Abstract

Thymosin beta 4 (Tβ4) is a peptide that has been shown in dermal, corneal, and cardiac preclinical injury models to potentially affect tissue protection, regeneration, and repair. Sacrococcygeal pilonidal sinus disease (SPSD) is a chronic inflammatory disorder associated with a high incidence of recurrence, chronic fistulation, and a challenging postoperative surgical wound healing process.

Retrospectively, an immunohistochemical analysis was conducted to evaluate endogenous Tβ4 expression in excisional skin biopsies from patients with SPSD. Patient demographics (age, gender) and surgical procedure data were obtained from their electronic medical records. Two (2) samples were cut from each specimen and prepared for histopathological assessment: 1 from the inflamed sinus tracts containing hair and granulation tissue (chronic wound group) and 1 from the normal tissue at least 1 cm away from the sinus tract (control group). Tβ4 expression was evaluated in the epidermal, dermal/subcutaneous collagen, and vascular structures of the samples from the sinus tract and healthy tissue. Inflamed sinus tract tissue and noninflamed normal tissue adjacent to the sinus tract were sampled from each specimen to confirm the diagnosis of SPSD and to determine distribution and intensity of Tβ4 expression. Presence of cytoplasmic staining for Tβ4 was considered in favor of positive Tβ4 expression; intensity of Tβ4 expression was scored as 0 = no staining, 1 = mild, 2 = moderate, and 3 = strong level of expression. A total of 31 excisional skin biopsy specimens were available from 31 patients with SPSD (mean age 26.0 ± 7.6 years, 25 [80.6%] men, 6 [19.4%] women) who underwent primary surgical closure. Demographic variables were analyzed using descriptive statistics. Data compliance with normal distribution was evaluated using the Kolmogorov-Smirnov and Shapiro-Wilk tests, and the Mann-Whitney U test was used for comparison of numerical data. P <.05 was considered statistically significant. Inflamed sinus tract tissue had significantly higher Tβ4 expression scores than noninflamed tissue samples in the epidermis (2.4 ± 0.8 [1.0–3.0] versus 0.8 ± 0.5 [0.0–2.0], P = .000), dermal/subcutaneous collagen (2.6 ± 0.5 [2.0–3.0] versus 1.6 ± 0.5 [1.0-2.0], P = .000), and vascular structure (2.6 ± 0.5 [2.0-3.0] versus 1.1 ± 0.3 [1.0-2.0], P = .000). Study findings indicate Tβ4 is endogenously expressed in normal skin tissue and is overexpressed in inflamed sinus tract tissue in patients with SPSD. Preclinical studies with a larger sample size are needed to enhance understanding of the potential role of Tβ4 in the inflammatory and tissue remodeling processes of SPSD by elucidating its mechanism of action at the molecular level, physiological role, and the therapeutic potential in dermal healing.

 

Thymosin beta 4 (Tβ4), a major actin monomer-binding peptide in mammalian cells, has diverse biological effects that include promotion of cell migration, proliferation, and survival; stem cell differentiation; and migration and modulation of cytokines, chemokines, and proteases, as shown in biochemical and molecular studies in humans and rodent models.1-7 Accordingly, several experimental and in vitro studies found Tβ4 was associated with anti-inflammatory properties and was shown to promote angiogenesis, hair growth, immunomodulation, and wound healing/repair processes.2-5,8-11 

Given its potential effect on tissue protection, regeneration, and repair, Tβ4 has been investigated in preclinical dermal, corneal, and cardiac injury models as well as in clinical trials for potential therapeutic applications in several diseases.1,12 Efficacy of chemically synthesized and recombinant Tβ4 on dermal healing was shown in preclinical models as well as in human patients.9,12-17 Thus, Tβ4 is considered to be an endogenous repair/regeneration factor naturally present in wounds and wound fluids, which may lead to faster healing and limited scar formation when added exogenously.12

Sacrococcygeal pilonidal sinus disease (SPSD) is a chronic inflammatory disorder of skin and subcutaneous tissue. As reported in clinical studies,18-22 SPSD primarily affects young men and is associated with a high incidence of recurrence and chronic fistulation. SPSD currently is considered to be an acquired disease caused by the penetration of loose hair follicles and cellular debris to the epithelium of the deep intergluteal sulcus and the generation of a subcutaneous cavitation in the presence of repeated microtrauma, rubbing, and/or crushing among predisposed patients.19-24 The subcutaneous cavitation may remain asymptomatic or lead to abscess formation and fistulas, with recurrences and multiple microabscesses that eventually extend deeper into the subcutaneous tissue.19,22,25,26

Complete surgical excision and drainage with healing by primary intention is the most direct treatment of SPSD in most cases, while wider excision with healing by secondary intention is required in complicated and recurrent cases.22,27-30 Thus, prolonged postoperative surgical wound healing is considered a primary challenge in patients with SPSD, particularly following wide excisions in complex cases.22,27,28

Accordingly, new therapeutic approaches to reduce the time to wound granulation healing and cutaneous regeneration in patients with SPSD have become a focus of scientific interest.22

To the author’s knowledge, no data are available in the literature concerning the distribution of endogenous Tβ4 in SPSD-related chronic wounds. Therefore, the present study was designed to evaluate endogenous Tβ4 expression in SPSD tissues based on retrospective immunohistochemical analysis of excisional skin biopsies obtained from operated SPSD patients. 

Methods

Study population. Excisional skin biopsy specimens obtained from patients with SPSD who consecutively underwent primary surgical closure at Bozok University Hospital, Yozgat, Turkey between November 2015 and April 2016 were included in this retrospective study. Inflamed sinus tract tissue and noninflamed normal tissue adjacent to the sinus tract were sampled from each specimen to confirm the diagnosis of SPSD and to determine distribution and intensity of Tβ4 expression. The study was conducted in full accordance with local Good Clinical Practice guidelines and current legislation with permission obtained from the author’s institutional ethics committee for the use of patient data for publication purposes. 

Assessments. Data on patient demographics (age, gender) and surgical procedure were obtained from the hospital’s electronic records. Following histopathological confirmation of the diagnosis of SPSD in excisional biopsy samples, distribution and intensity of Tβ4 expression were studied for both the wound and its adjacent tissue. 

Histopathological and immunohistochemical analysis. All specimens were transported to the pathology laboratory in 10% neutral formalin. Two (2) samples were cut from each specimen, 1 from the inflamed sinus tracts containing hair and granulation tissue (chronic wound group) and 1 from the normal tissue at least 1 cm away from the sinus tract (control group). All samples from the chronic wound and control groups were processed in an automated system (Excelsior ES; Thermo Scientific, Rockford, IL), and paraffin blocks were prepared using the HistoStar embedding station (Thermo Scientific). Two (2) tissue sections, each 4 µm thick, were obtained from each paraffin block using a microtome (Shandon-Finesse ME+; Thermo Scientific). One (1) was stained with hematoxylin and eosin (H&E) using an automated slide staining machine (Varistain Gemini; Thermo Scientific), and the other was immunostained for Tβ4 antibody (1:1000 dilution; anti Tβ4 antibody [ab14335], abcam, Cambridge, United Kingdom), using the labeled streptavidin-biotin complex system in a Leica-Bond Max autostainer (Leica biosystems, Wetzlar, Germany). Diaminobenzidine was used as chromogen. Fetal heart tissue, known for immunohistochemical positivity for anti-Tβ4 antibody as per product data sheet test results,31 was concomitantly studied with skin tissue samples to control and ensure the quality the immunohistochemical analysis.

The H&E-stained and Tβ4-stained slides were evaluated under a light microscope (BX53F, Olympus, Tokyo, Japan). Histopathological diagnosis of SPSD was made via visualization of the sinus tract, hair nidus, chronic granulation tissue, inflammation, and multinucleated giant cell reaction in H&E stained samples. Tβ4-stained slides were evaluated for Tβ4 expression in epidermis, dermal/subcutaneous collagen, and vascular structures of the samples from the chronic wound and control groups. Presence of cytoplasmic staining for Tβ4 was considered in favor of positive Tβ4 expression, and intensity of Tβ4 expression  was scored as follows: 0 = no staining, 1 = mild, 2 = moderate, and 3 = strong level of expression. A single referral pathologist blinded to the study groups reviewed the slides and performed scoring.

Statistical analysis. Data analysis was performed using IBM SPSS Statistics version 23 (IBM Corp, Chicago, IL). Descriptive statistics were used to analyze demographic variables. The compliance of data with normal distribution was evaluated with Kolmogorov-Smirnov and Shapiro-Wilk tests. The Mann-Whitney U test was used for comparison of numerical data. Data were expressed as mean ± standard deviation (SD), median (minimum-maximum), and percent (%) where appropriate. P <.05 was considered statistically significant.

Results

Thirty-one (31) specimens were obtained from 31 patients (mean age 26.0 ± 7.6 years, 25 [80.6%] men, 6 [19.4%] women). Epidermis, dermal/subcutaneous collagen, and vascular structures showed mild-to-moderate Tβ4 expression in the majority of cases in the adjacent, noninflamed tissue (96.8%, 100.0%, and 100.0%, respectively); whereas, in the sinus tract tissue strong Tβ4 positivity was noted in 58.1%, 61.3%, and 61.3% of samples from epidermal, dermal/subcutaneous collagen, and vascular structures, respectively (see Table 1). Photographs of the sinus tract and adjacent, noninflamed tissue specimens are shown in Figure 1 and Figure 2, respectively. 

owm_0417_sahin_table1owm_0417_sahin_figure1owm_0417_sahin_figure2

The sinus tract tissue had significantly higher mean ± SD (minimum-maximum) Tβ4 expression scores than the control group in the epidermis (2.4 ± 0.8 [1.0–3.0] versus 0.8 ± 0.5 [0.0–2.0], P = .000), dermal/subcutaneous collagen (2.6 ± 0.5 [2.0–3.0] versus 1.6 ± 0.5 [1.0–2.0], P = .000), and vascular structure (2.6 ± 0.5 [2.0–3.0] versus 1.1 ± 0.3 [1.0–2.0], P = .000) (see Table 2). owm_0417_sahin_table2

Discussion

This retrospective immunohistochemical analysis of excisional skin biopsies obtained from patients operated for SPSD revealed endogenous Tβ4 was expressed in both the chronic wound and adjacent, healthy tissue and overexpressed in the chronic wound tissue. These data provide the first evidence of the distribution of endogenous Tβ4 expression in SPSD, indicating overexpression of Tβ4 in epidermal, dermal/subcutaneous collagen, and vascular structures in excisional biopsies from inflamed sinus tracts.

Tβ4-mediated dermal healing has been reported in several animal cutaneous wound models,12 including rat skin flaps,32 mouse burn,14 and rat punch wound,17 while the trend of increased rate of dermal healing also was shown in phase 2 clinical trials in humans with use of topical Tβ4 in optimal doses of 0.02%–0.03% (w/w) in pressure ulcers and stasis ulcers.16

Increased angiogenesis, keratinocyte migration, collagen deposition, and wound contraction are considered to be among the mechanisms underlying wound healing and repair in Tβ4-treated dermal wounds per the full-thickness excisional wound model in rats and impaired-healing models in diabetic, steroid-immunosuppressed, and aged mice.1,6,9,12,33,34 Thus, Tβ4 is considered to be active in all 3 stages of the complex cascade of healing dermal wounds, including inflammation, proliferation, and remodeling, and contributes to dermal repair and regeneration.12  

In an analysis34 of the effect of Tβ4 on wound healing in a full-thickness cutaneous wound in a rat model, Tβ4-treated wounds were shown to heal faster than the control wounds. The authors suggested Tβ4 enhances wound healing by increasing neovascularization, while enhanced keratinocyte migration and increased deposition of well-organized collagen also were noted in Tβ4-treated wounds and associated with contraction and tensile strength of the wound. 

Similarly in the current study, along with higher amount of collagen around the sinus tracts (implying a chronic wound), higher expression of Tβ4 was noted in inflamed sinus tracts including epidermal keratinocytes, collagen, and vascular structures. Thus, overexpression of endogenous Tβ4 in epidermal, collagen, and vascular structures in inflamed sinus tract samples in the current study seem consistent with Tβ4-dependent mechanisms of dermal wound healing and repair shown in cutaneous wound models,6,12 while also emphasizing the potential beneficial effects of naturally elevated endogenous Tβ4 levels in wound and wound fluids.12

However, it should be noted that the current findings provide data on endogenous distribution of Tβ4 in SPSD rather than its wound healing properties. Thus, overexpression of endogenous Tβ4 in an inflamed sinus tract does not necessarily indicate its wound healing potential as an endogenous repair/regeneration factor nor does it confirm the likelihood of increased rate of healing if much higher Tβ4 levels are obtained in the wounded area. Histopathologically, chronic granulation tissue usually lines the sinus tract in SPSD, often infiltrated by neutrophils, lymphocytes, plasma cells, hemosiderin-laden macrophages, and multinucleated giant cells of foreign body type, usually a nest of hair.35-37 Spontaneous wound healing does not occur in SPSD, and secondary events such as reaction to a foreign body complicate the healing process. Therefore, treatment is dependent on complete surgical excision and drainage with healing by primary intention in most cases, and a wide surgical incision with healing by second intention following the laying open technique in complicated and recurrent cases.22,27-29 Thus, based on unique healing characteristics, it seems difficult to clearly assess the dermal wound healing function, if any, of endogenous Tβ4 overexpression in SPSD-related chronic wounds.

In addition to lack of clinical consensus on the optimal management of the disease and high recurrence rates,20-22,37,38 prolonged postoperative wound healing remains a major challenge in the management of SPSD, necessitating development of effective treatment modalities to avoid impaired healing and reduce the time required for both wound granulation healing and cutaneous regeneration.22,39-41 Based on the efficacy of Tβ4 on wound healing shown in preclinical studies and data from a phase-I clinical trial on favorable safety and tolerability in healthy volunteers (as well as several nonclinical toxicology and dermal-sensitization studies), clinical introduction of Tβ4 is considered likely in patients with chronic cutaneous wounds.6 Thus, Tβ4, formulated as topical dermal gel (RGN-137), has the potential to be studied in patients with chronic cutaneous wounds such as pressure ulcers, venous stasis ulcers, and diabetic lower-extremity ulcers, and other types of dermal wounds such as cases of epidermolysis bullosa.1,6,42

However, while Tβ4 efficacy and mechanisms of action are more clearly defined in cardiac injury models than in dermal healing, further preclinical studies are needed to determine the optimal dosing and treatment duration based on etiology, type, and severity of underlying injury as well as to identify the functional receptor for Tβ4-mediated activities in dermal repair.12

Limitations

Certain limitations to this study should be considered. First, due to the retrospective, single-center study design and small sample size, the ability to generalize the findings to the general SPSD population is limited. Nonetheless, power analysis revealed the power of the study to be 0.86 (α = 0.05, d = 0.8). Second, the study was not designed to examine cause and effect, nor can conclusion regarding potential wound healing properties of endogenous Tβ4 overexpression be made. Nevertheless, these findings provide the first evidence of endogenous Tβ4 expression in SPSD.

Conclusion

A retrospective immunohistochemical analysis of excisional skin biopsies obtained from operated SPSD patients revealed Tβ4 was uniformly distributed in epidermal, dermal/subcutaneous collagen, and vascular structures, was endogenously expressed in normal skin tissue, and was overexpressed in inflamed sinus tracts. Further preclinical studies in larger series are needed to elucidate the potential role of Tβ4 in the inflammatory and tissue remodeling processes of SPSD by addressing its mechanism of action at the molecular level, its physiological role, and its therapeutic potential in dermal healing. n

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Potential Conflicts of Interest: none disclosed

 

Dr. Sahin is an Assistant Professor, Department of Pathology, Bozok University School of Medicine, Yozgat, Turkey. Please address correspondence to: Sevinc Sahin, MD, Bozok Üniversitesi Araştırma ve Uygulama Hastanesi, Tıbbi Patoloji Anabilim Dalı, 66100, Yozgat, Turkey; email: sevcelik82@gmail.com.

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