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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 6  |  Issue : 2  |  Page : 127-132

A study to compare efficacy of non cultured autologus melanocyte transfer versus punch grafting technique for stable vitiligo patients


1 Department of Dermatology, Mahadevappa Rampure Medical College, Kalaburagi, Karnataka, India
2 Department of Ophthalmology, GIMS, Kalaburagi, Karnataka, India
3 Department of Dermatology, Command Hospital Air Force Bangalore, Bengaluru, Karnataka, India

Date of Submission01-Sep-2021
Date of Decision10-Feb-2022
Date of Acceptance14-May-2022
Date of Web Publication26-Aug-2022

Correspondence Address:
Shruti Ambresh Badad
Department of Ophthalmology, GIMS, Dr Badad's Skin, Eye and Laser Care Centre, Sharan Nagar, Kalaburagi - 585 103, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/cdr.cdr_66_21

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  Abstract 


Background: Vitiligo is a common acquired idiopathic hypomelanosis, which is characterized by loss of melanocytes in the epidermis associated with autoimmune and hereditary causes. The various surgical modalities available are tissue grafting techniques such as punch grafts, suction blister grafts, and split-thickness grafts as well as cellular grafts such as autologous noncultured epidermal cell suspensions and cultured melanocyte suspensions. Objective: We aimed to study and compare the efficacy of noncultured melanocyte transfer technique and punch grafting technique in the management of stable vitiligo. Materials and Methods: Fifty patients of both sexes between the ages of 15 and 50 years having stable vitiligo for minimum 1 year were included in the study and divided in two groups. Group 1 was treated with autologous noncultured melanocyte-rich cell suspension and Group 2 was treated by punch grafting technique. Response to treatment was evaluated on the basis of degree of repigmentation (both subjective and objective assessment), which was carried out at monthly intervals for a period of 6 months along with clinical photographs. Results: The results were statistically analyzed using “Chi-square test” and “statistical significance” (p-value) methods. An excellent (>75% repigmentation) result was obtained in 77.27% of patients by NCMT (noncultured melanocyte transfer) technique whereas good (60%–75% repigmentation) result was obtained in 78.57% of patients by punch grafting technique after 6 months. Conclusion: Noncultured melanocyte transfer is a simple and effective technique to produce uniform pigmentation in short duration and is suitable to cover large body surface areas with a cosmetically better outcome and is better than punch grafting.

Keywords: Noncultured melanocyte transfer technique, punch grafting technique, vitiligo


How to cite this article:
Badad AS, Bhatia AS, Hogade A, Badad SA, Mitra D. A study to compare efficacy of non cultured autologus melanocyte transfer versus punch grafting technique for stable vitiligo patients. Clin Dermatol Rev 2022;6:127-32

How to cite this URL:
Badad AS, Bhatia AS, Hogade A, Badad SA, Mitra D. A study to compare efficacy of non cultured autologus melanocyte transfer versus punch grafting technique for stable vitiligo patients. Clin Dermatol Rev [serial online] 2022 [cited 2022 Sep 26];6:127-32. Available from: https://www.cdriadvlkn.org/text.asp?2022/6/2/127/354759




  Introduction Top


Vitiligo is a common acquired idiopathic and often familial hypomelanosis, which is characterized by pale white macules that enlarges centrifugally over time.[1] Vitiligo affects 1% of the world's population and significantly impacts the quality of life of patients. It causes cosmetic problems as well as sociopsychological concerns in the patients and their relatives.[2]

Out of various methods of treatment of vitiligo, there are many cases of vitiligo who either fail to respond or only partially respond to medical line of treatment, indicating that melanocyte reservoir is no more available for repigmentation in these areas.[3] The various medical therapeutic modalities used for treatment of vitiligo have certain limitations. These are the cases which can be taken up for surgery. However, stability in vitiligo is a key factor in deciding for surgical intervention in vitiligo. “Stable vitiligo” is defined as disease inactivity and no new patch development in the past 1 year.[4] Dermatosurgical modalities are thereby advised in stable or nonprogressive type of vitiligo, refractory to medical therapies. Various surgical methods used to treat stable vitiligo are mini punch grafting,[5] suction blister epidermal grafting,[6] thin split-thickness grafting,[7] transplantation of basal cell layer-enriched suspension (noncultured melanocyte transfer),[8] cultured epithelial/melanocyte transplantation,[9],[10] single-hair follicle transplant, and recently developed “modified dermabrasion” which utilizes dermabraded epidermal material (melanocytes and keratinocytes) entangled in an antibiotic paste.

There are many dermatosurgical modalities for treating stable vitiligo. A comparative evaluation of noncultured melanocyte transfer technique and punch grafting technique was done in this study.

Non culture melanocyte transfer technique is a procedure in which donor skin is harvested and melanocyte basal cell suspension prepared.The melanocyte suspension is then applied over the dermabraded recipient area followed by psoralene ultraviolet light A obtained by sunlight (PUVASOL) therapy.

Punch grafting is a technique in which miniature thin split-thickness, 1–3 mm in size punch grafts are taken from donor area and are then transplanted to the recipient vitiliginous area in which chambers are made by punches followed by PUVA-SOL therapy for further enhancing repigmentation.

We aimed to compare the efficacy of noncultured melanocyte transfer technique and punch grafting technique in the management of stable vitiligo. The objective was to assess the efficacy of both the treatment modalities in repigmentation of the vitiligo lesions and their safety profile.


  Materials and Methods Top


A comparative study was done in which 50 stable patients (22 of NCMT and 28 of punch grafting) were operated over a period of 1 year after obtaining Institutional Ethics Committee clearance.

It was a prospective interventional comparative study wherein fifty stable vitiligo patients were treated by two surgical modalities: punch grafting and nonculture melanocyte transplant over a period of 1 year. Inferential statistics such as Chi-square test/Fisher's exact test were used. P < 0.05 was considered to be statistically significant.

Inclusion criteria

  1. Patients willing for treatment
  2. Patients not responding adequately to medical treatment
  3. Patients with stable vitiligo for 12 months (no new lesions and no expansion of old ones)
  4. Patients aged 15–50 years.


Exclusion criteria

  1. Patients receiving any concomitant medical treatment for vitiligo
  2. Patients who are positive to infectious disease HIV, hepatitis C virus, and hepatitis B virus
  3. Patients who have a history of koebnerization, keloid tendency, and coagulative disorders.


Procedure/methods

Two techniques were employed: the autologous melanocyte-rich cell suspension (noncultured) technique and the punch grafting technique. Both these techniques share a common principle of selective replenishment of melanocytes at the recipient stable vitiligo macules.

Noncultured melanocyte transfer technique

Non culture melanocyte transfer technique The autologus melanocyte basal cell suspension harvested from the donor split skin graft is transplanted to dermabraded reciepient area.

Donor site

About one-tenth the size of the recipient area was selected as the donor site, usually on noncosmetically important sites such as the thighs, buttocks, or waist. It was cleaned with povidone-iodine and 70% ethanol and draped. The site was anesthetized with 2% lidocaine infiltrated in the subcutis. The skin was stretched and a very superficial sample was obtained with Silver's skin grafting knife or a sterile razor blade on a straight hemostat forceps. The superficial wound was then dressed with paraffin gauze dressing 10 cm × 10 cm.

Laboratory procedure for cell separation

The skin graft was immediately transferred to 6 ml of 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) solution in a Petri dish. This mixture of skin sample with trypsin-EDTA solution was incubated at 37°C for 50 min. The grafts were then transferred into a Petri dish containing 8 ml of melanocyte nourishment medium, i.e., Dulbecco's modified Eagle's medium (DMEM). This media also acted as a diluting agent to wean off the trypsin action. The entire subsequent step was performed in a laminar air flow bench under strict aseptic conditions. The epidermis was teased gently and separated from the dermis with the help of curved forceps. The dermal pieces were discarded and epidermal pieces were retained. The epidermal pieces were then scrapped, so that there was no pigment left on their surface. The contents of Petri dish was then transferred into a centrifuge tube and centrifuged for 6 min at 3000 rpm. The cell pellet settled down at the bottom. The supernatant was discarded and pellet containing cells from the stratum basale and lower half of the stratum spinosum that was rich in melanocytes was taken. The pellet was then resuspended in a total volume of 0.8 ml of DMEM medium and transferred gently in steps to a pipette.

Recipient site

It was cleaned, painted, and draped with Betadine O, spirit, and Savlon and washed thoroughly with normal saline. It was anesthetized with the use of 2% lignocaine infiltration.

Transplantation

The recipient area was abraded with a dermabrader until tiny pinpoint bleeding spots were seen, which implied that the dermoepidermal junction had been reached. The denuded area was covered with saline-moistened gauze pieces. The suspension was poured evenly from the pipette to the denuded surface, which was then covered with a collagen dressing. This was then covered by a small gauze piece dipped in trypsin medium and held in place by Tegaderm transparent dressing. The patient was allowed to be ambulant 30 min later with the instructions to avoid vigorous activities and to carry out only restricted movements for the next 7 days. The dressing was removed at their first follow-up visit after a week.

Postoperative care

Oral antibiotics and analgesics were prescribed for 5 days. Dressing of the donor area was changed on alternate days, and for the recipient areas, it was removed after 7 days. PUVA-SOL (1:10 solution of TRIOXSALENE) sun exposure for 10 min during morning hours was added for accelerating the repigmentation and was started 2 weeks after erythema subsided. The patients were followed up at the 1st, 3rd, and 6th months after procedure for assessing repigmentation in 3 visits.

Punch grafting

Punch grafting was carried out in 28 cases of stable vitiligo. The donor site chosen in all patients was lateral side of thigh. The donor area was shaved properly and part prepared with strict aseptic precautions. Inj 2% xylocaine with adrenaline was infiltrated after a test dose. A wheal was raised for l cm × 1 cm size to take approximately 8–12 grafts. Commonly used size of skin punch at donor area was 2.5 mm.

Donor site

  1. Punch (2.5 mm) was rotated till the cutting edge descended up to the depth of upper dermis
  2. Edge was lifted up with the help of nontraumatizing forceps and was cut close to the epidermis with sharp scissor to free the graft from the base
  3. The graft was transferred to a sterile bowl containing normal saline
  4. Hemostasis by pressure was achieved and dressing was done with double layer of framycetin tulle and bandage.


Recipient site

  1. Part preparation was done and local anesthesia (1% Xylocaine) was intradermally injected
  2. Punch used was 0.5 mm smaller than donor size graft.


  • Similarly punches were rotated till it reached mid-dermis and edges were lifted and cut
  • After achieving hemostasis, the grafts taken from donor area are placed in these newly prepared chambers
  • Care was taken that the dermal end of donor area's graft was in direct contact with the recipient dermis
  • Firm pressure with moist gauze to achieve hemostasis was applied. Dressing was done with double-layered framycetin tulle.


Follow-up dressing was done in 24 h. If any grafts had shifted, it was replaced by fresh stored graft and a second dressing was done after 8–10 days. Donor site dressing was removed after 8–10 days. All the patients were covered with a broad-spectrum antibiotic orally for a week.

All patients of punch graft were put on postoperatively PUVA-SOL therapy (1:10 topical Trioxsalene) followed by sun exposure for 10 min during morning hours. Patients were observed during the postoperative recovery period for any complication like superficial scarring at donor area, cobblestoning, sinking pits, blotchy skin appearance and contact dermatitis due to framycetin tulle, rejection of graft due to movements, and secondary infection. Pre- and postprocedure photographs and follow-up photographs were taken. Data were collected and analyzed by appropriate statistical method.


  Results Top


The present study was conducted on 50 patients of stable vitiligo. Out of 50 patients, females were 32 (64%) and males were 18 (36%). The maximum number of patients was in the age group of 21–30 years, while the minimum number of patients was seen in the age group of 41–50 years. Out of the 50 stable patients selected for the study, 2 (4%) had diabetes and 1 (2%) had hypothyroidism. Maximum number 31 (62%) of patients had vitiligo of 6–10 years duration, and minimum number 6 (12%) of patients had vitiligo of more than 11-year duration.

Vitiligo vulgaris was seen as the most common condition (28 [56%]) among all the vitiligo patients. Most of the patients 19 (38%) had lesions on the lower extremities followed by trunk 12 (24%), upper extremities 12 (24%), and face and neck 7 (14%). Surgical procedure was used in various patients in this study; 22 (44%) were treated by NCMT (noncultured melanocyte transfer) and 28 (56%) by punch grafting. The procedure and results of NCMT are shown in [Figure 1], [Figure 2], [Figure 3]. Punch grafting technique and result are shown in [Figure 4].
Figure 1: Steps of noncultured melanocyte transfer: (a) Graft harvesting with silver knife, (b) Graft separated, (c) Graft in DMEM medium in Petri dish, (d) Incubation for 55 min. DMEM: Dulbecco's modified Eagle's medium

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Figure 2: Steps of noncultured melanocyte transfer

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Figure 3: Dressing of recipient area and results of noncultured melanocyte transfer

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Figure 4: Results and procedure of punch grafting technique

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Postoperative evaluation

Inference

A statistically significant difference was observed at 1 month, 3 months, and 6 months. [Table 1] shows the comparison between NCMT (noncultured melanocyte transfer) technique and punch grafting technique based on subjective observations. It shows that excellent result was obtained in 17 (77.27%) patients by NCMT (noncultured melanocyte transfer) technique and poor response was seen in 2 (9.09%) patients after 6 months of treatment whereas good result was obtained in 22 (78.57%) patients by punch grafting technique and poor response was seen in 2 (7.15%) patients after 6 months of treatment.
Table 1: Comparison based on subjective observations between two groups at different time intervals

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The findings are tabulated in [Table 1] and represented graphically in [Figure 5].
Figure 5: Comparison based on subjective observations between two groups at different time intervals

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Complications (donor site)

Superficial scarring at the donor site was seen in 21 (75%) cases of punch grafting technique and 2 (9.09%) cases of NCMT technique. Depigmentation was seen in 1 (4.55%) case and keloid formation was seen in 1 (4.55%) case of NCMT at the donor site.

Complications (recipient site)

In punch grafting technique, complications at the recipient site were seen which included cobblestoning in 6 (21.42%) cases, infection in 3 (10.71%) cases, sinking pits in 1 (3.57%) case, and variegated pigmentation in 2 (7.15%) cases. In NCMT technique, complications at the recipient site were seen which include erythema in 3 (13.64%) cases and infection in 2 (9.09%) cases. Hence, overall NCMT technique had lesser complications as compared to punch grafting technique.

Hence, among the two methods, NCMT shows good results and better repigmentation as compared to punch grafting.


  Discussion Top


Vitiligo is an autoimmune disorder of depigmentation known for centuries. PUVA therapy has been widely accepted as it is effective in majority of vitiligo patients, however prolonged course and partial response are disadvantages. These vitiligo lesions are known to be relatively refractory and require shifting to other modes of medical or surgical therapies such as skin grafting or grafting of in vitro cultured and noncultured melanocytes.

Various surgical methods were used for treatment of stable vitiligo: suction blister grafting, split-thickness skin grafting, punch grafting, tattooing, and dermabrasion. These procedures have various shortcomings in the form of pigment mismatch, cobblestoning, and stuck-on appearance. Thus, there is an increasing demand toward the use of advanced technology in the treatment of vitiligo. Replenishing melanocytes selectively in depigmented macules by autologous melanocytes transfer is one such promising treatment. Both the NCMT and punch grafting technique are based on the principle of seeding of melanocytes, i.e., introduction of melanocytes from normal skin into a region of depigmented skin. The advantage of NCMT technique is that a wide recipient area can be covered by a small donor area, with no cobblestoning, and shows good color match and good efficacy.

In the present study, the patients were divided in two broad groups; the first group constituting 28 patients (56%) underwent punch grafting, and the second group constituting 22 patients (44%) underwent noncultured melanocyte transfer technique.

Out of 22 patients who underwent noncultured melanocyte transfer technique, 17 patients (77.27%) showed excellent results (>75% improvement) while 3 (13.64%) showed good results ans two patients (9.09%) showed poor results due to secondary infection and immobilization.

Verma et al.[11] reported that a total of 25 patients were enrolled in their study, which included 50 sites of depigmentation for autologous nonculture melanocyte keratinocyte transfer (NCMT) technique and 50 sites for autologous cultured melanocyte transfer (CMT). In the NCMT method, 31 (62%) patches showed good repigmentation, i.e., >70% repigmentation; 10 (20%) patches showed fair repigmentation, i.e., 30%–69% repigmentation; and 9 (18%) patches showed poor repigmentation, i.e., <30% repigmentation. While, in the CMT method, 26 (52%) patches showed good repigmentation, i.e., >70% repigmentation; 5 (10%) patches showed fair repigmentation, i.e., 30%–69% repigmentation; and 19 (38%) patches showed poor repigmentation, i.e., <30% repigmentation. The method is time-consuming, but the results achieved are good.

Paul[12] did autologous noncultured basal cell-enriched epidermal cell suspension in 58 patients and reported that out of 49 patients who came for follow-up, thirty-two (65%) had excellent (>90%) repigmentation, 9 (18%) had good (70%–89%), 4 (8%) had fair (30%–69%), and 4 (8%) patients had poor (<30%) repigmentation. During the follow-up, eight patients (16%) showed relapse of the disease.

Out of 28 patients who underwent punch grafting, 22 patients could reach the result of 78.57% re-pigmentation (good result) by the end of 6 months, only 4 patients (14.28%) out of 28 could achieve fair result, and 2 patients (7.15%) showed poor result by the end of 6 months.

Lahiri and Sengupta[13] reported that out of 60 patients who underwent punch grafting, only 31 of them achieve 70%–100% repigmentation. Five patients could repigment by 30%–50% and three patients showed 0%–30% improvement. Cobblestoning was noted in 28 patients.

Singh,[14] did punch grafting in 55 cases, out of which 36 showed complete response, 8 patients showed good response, while 11 patients showed poor results. Cobblestoning was noticed in one-third of cases.

Savant[15] reported after doing punch grafting in 62 cases, the complications seen were graft rejection due to improper immobilization in 6 cases, graft rejection due to secondary infection in 1, contact allergic dermatitis of framycetin in 3, and reactivation of vitiligo in 2, side effects seen were cobblestoning in 32, sinking pits in 12, variegated appearance in 4, superficial scaring at donor sites in all 62 cases.

Superficial scarring at donor site was seen in 21 (75%) cases of punch grafting, while in NCMT, 2 (9.09%) cases with hyperpigmentation and superficial scarring were found. Keloid/hypertrophic scar was seen in 1 case of NCMT at the donor site. Depigmentation was seen in 1 case of NCMT at donor site.

Cobblestoning and sinking pits were associated with punch grafting but were not associated with NCMT at the recipient site. Infection was seen in 3 cases of punch grafting and 2 cases of NCMT. Erythema was seen in 3 cases of NCMT at the recipient site. Loss of pigment and variegated hyperpigmentation were associated with punch grafting, while no case was found with NCMT. Hence, on the basis of adverse effects and compared with efficacy of punch grafting, NCMT is better than punch grafting.

In a similar study done by Kumar et al.,[16] it was found that out of 125 patches operated upon, good results were seen in 91.20% of patches by using NCMT method and 82.40% by using punch grafting technique. Similarly fair results were seen in 1.6% of patches by NCMT method and 4.8% of patches by punch grafting technique. Poor results were seen in 7.2% of patches by NCMT method and 12.80% of patches by punch grafting technique.


  Conclusion Top


Autologous nonculture melanocyte transfer is a simple and effective technique to produce homogeneous pigmentation quickly in stable vitiligo patients. It has an advantage over punch grafting as it requires very little donor skin. Although Punch grafting is simpler, less time consuming and requires less technical expertise but has disadvantages like large donor surface and cobblestoning. Patients were satisfied by the results to both the methods, however the quality and homogenous repigmentation was by far superior with NCMT than punch grafting technique. Hence, NCMT should be the first choice technique for stable vitiligo patients.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Mosher DB, OrtonneJP, Fitzpatrick TB. Disorder of pigmentation. In: Fitzpatrick's TB, Eisen AZ, Wolff K, et al editors. Dermatology in General medicine. 3rd edition. New York: Mc Graw Hill; 1987: 794-876.  Back to cited text no. 1
    
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Lerner AB, Nordlund JJ. Vitiligo. What is it? Is it important? JAMA 1978;239:1183-7.  Back to cited text no. 2
    
3.
Suvanprakorn P, Dee-Ananlap S, Pongsomboon C, Klaus SN. Melanocyte autologous grafting for treatment of leukoderma. J Am Acad Dermatol 1985;13:968-74.  Back to cited text no. 3
    
4.
Lahiri K. Evolution and evaluation of autologous mini punch grafting in vitiligo. Indian J Dermatol 2009;54:159-67.  Back to cited text no. 4
[PUBMED]  [Full text]  
5.
Falabella R. Repigmentation of stable leukoderma by autologous minigrafting. J Dermatol Surg Oncol 1986;12:172-9.  Back to cited text no. 5
    
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Mutalik S. Transplantation of melanocytes by epidermal grafting. An Indian experience. J Dermatol Surg Oncol 1993;19:231-4.  Back to cited text no. 6
    
7.
Kahn AM, Cohen MJ. Repigmentation in vitiligo patients. Melanocyte transfer via ultra-thin grafts. Dermatol Surg 1998;24:365-7.  Back to cited text no. 7
    
8.
Olsson MJ, Juhlin L. Leucoderma treated by transplantation of a basal cell layer enriched suspension. Br J Dermatol 1998;138:644-8.  Back to cited text no. 8
    
9.
Lerner AB, Halaban R, Klaus SN, Moellmann GE. Transplantation of human melanocytes. J Invest Dermatol 1987;89:219-24.  Back to cited text no. 9
    
10.
Plott RT, Brysk MM, Newton RC, Raimer SS, Rajaraman S. A surgical treatment for vitiligo: Autologous cultured-epithelial grafts. J Dermatol Surg Oncol 1989;15:1161-6.  Back to cited text no. 10
    
11.
Verma R, Grewal RS, Chatterjee M, Pragasam V, Vasudevan B, Mitra D. A comparative study of efficacy of cultured versus non cultured melanocyte transfer in the management of stable vitiligo. Med J Armed Forces India 2014;70:26-31.  Back to cited text no. 11
    
12.
Paul M. Autologous non-cultured basal cell-enriched epidermal cell suspension transplantation in vitiligo: Indian experience. J Cutan Aesthet Surg 2011;4:23-8.  Back to cited text no. 12
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13.
Lahiri K, Sengupta SR. Treatment of stable and recalcitrant depigmented skin conditions by autologous punch grafting. Indian J Dermatol Venereol Leprol 1997;63:11-4.  Back to cited text no. 13
[PUBMED]  [Full text]  
14.
Singh SK. Punch grafting in vitiligo: Refinements and case selection. Indian J Dermatol Venereol Leprol 1997;63:296-300.  Back to cited text no. 14
    
15.
Savant SS. Autologous miniature punch skin grafting in stable vitiligo. Indian J Dermatol Venereol Leprol 1992;58:310-4.  Back to cited text no. 15
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16.
Kumar R, Singh SK, Gupta AK, Patel PK, Chhachhi H, Patel RK. “Comparative study of efficacy of non cultured melanocyte transfer technique and punch grafting technique in the management of stable vitiligo.” J Evol Med Dent Sci 2015;4:16754-8.  Back to cited text no. 16
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
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