Journal of Cutaneous and Aesthetic Surgery
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INNOVATION  
Year : 2020  |  Volume : 13  |  Issue : 1  |  Page : 38-42
Autologous smashed follicular dermal graft with epidermal cell suspension in chronic nonhealing leg ulcers


Department of Dermatology, Venereology and Leprology, RajaRajeswari Medical College and Hospital, Bengaluru, Karnataka, India

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Date of Web Publication17-Mar-2020
 

   Abstract 

Wound healing is a multifaceted process. Restoration of the epithelium may occur either by migration of activated epithelial keratinocytes from the adjoining epithelium or by centrifugal migration of putative stem cells found in the bulge region of the hair follicle. Dermal collagen and glycosaminoglycans also play a key role in laying down the scaffold for vascular in-growth and population of the matrix with host fibroblasts. Despite the availability of a wide range of treatment modalities, chronic nonhealing leg ulcers remain a therapeutic challenge and cause significant patient morbidity. Hence, there is a further need to develop novel techniques which would be effective, safe, easily available, and affordable for the patient.

Keywords: Chronic leg ulcer, epidermal cell suspension, smashed follicular dermal graft, wound healing

How to cite this article:
Nagaraju U, Kashyap P, Raveendra L. Autologous smashed follicular dermal graft with epidermal cell suspension in chronic nonhealing leg ulcers. J Cutan Aesthet Surg 2020;13:38-42

How to cite this URL:
Nagaraju U, Kashyap P, Raveendra L. Autologous smashed follicular dermal graft with epidermal cell suspension in chronic nonhealing leg ulcers. J Cutan Aesthet Surg [serial online] 2020 [cited 2020 Apr 1];13:38-42. Available from: http://www.jcasonline.com/text.asp?2020/13/1/38/280787





   Introduction Top


Chronic leg ulcers are chronic wounds of the leg that show no tendency to heal after 6 weeks despite appropriate treatment, thereby causing significant patient morbidity.[1] Wound healing is a multifaceted process involving clot formation, cell migration and proliferation, extra cellular matrix synthesis and deposition, contraction, and finally dermal and epidermal reconstitution.[2]

Hence, we took up 12 cases of chronic nonhealing leg ulcers for a novel procedure combining smashed follicular dermal grafts with autologous epidermal cell suspension, which contributes to wound healing by being a source of follicular stem cells, epidermal keratinocytes, and dermal collagen.


   Technique Top


After acquiring approval from the Institutional Ethics Committee and informed written consent from each subject, complete evaluation with relevant basic investigations were carried out to find out the primary cause of ulcer. The procedure was performed on only healthy leg ulcers under local anesthesia in an outpatient setting.

Equipments needed

The following are the list of the equipments used in the study: graft-harvesting dermatome, angular graft holding forceps, biopsy punches (3.5 mm), incubator, centrifuge, centrifuge tubes, prilocaine cream, compression bandage, paraffin gauze,  Petri dish More Detailses, 2-mL disposable syringe and sterile glass side, Reagents-Trypsin EDTA, and Dulbecco’s modified Eagle’s medium.

Steps of the procedure

Step 1: Preparation of epidermal cell suspension

The anterolateral aspect of thigh was taken as the donor site and anaesthetized using topical 2% lidocaine and prilocaine cream. An ultra-split thickness epidermal graft was harvested from the donor site with a ratio of donor-to-recipient area of 1:4, using graft-harvesting dermatome [Figure 1]. The graft was then trypsinized with the epidermis facing upward in a petri dish [Figure 2] and incubated at 37°C for 50min, following which it was recovered and transferred to Dulbecco’s Modified Eagle’s medium in another petri dish. Using graft-holding forceps, the graft was gently agitated to separate the epidermal components from the dermis [Figure 3]. Then the cell suspension was centrifuged at 3000 rpm for 10min. The cellular sediment obtained [Figure 4] was aspirated into a 2-mL syringe for application.
Figure 1: Ultra-split thickness epidermal graft harvested from donor area using graft-harvesting dermatome

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Figure 2: Graft trypsinized in a petridish

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Figure 3: Separation of epidermal component from dermis by gentle agitation in DMEM

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Figure 4: Cellular sediment after centrifugation at 3000 rpm for 10 min

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Step2: Preparation of smashed follicular dermal grafts

Dermal grafts were harvested using a 3.5-mm punch, spaced at least 5 mm from each other, making sure each graft consisted of a hair follicle [Figure 5]. Two grafts per cm2 of ulcer area were taken. The grafts were placed on a glass slide [Figure 6] and minced with a pair of scissors into a semisolid paste, and aspirated into the same syringe containing ECS. The donor area was closed with paraffin gauze dressing, which was opened after 7 days.
Figure 5: Follicular dermal grafts harvested using a 3.5-mm punch from the donor area

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Figure 6: Follicular dermal grafts placed on a glass slide and minced into a semisolid paste

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Step3: Application of dermal graft and ECS to wound bed

The final suspension consisting of smashed follicular dermal graft with ECS was applied evenly over the ulcer bed [Figure 7]. The ulcer was covered with paraffin gauze and elastic bandage.
Figure 7: Application of the suspension evenly over the ulcer bed

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The subject was put on antibiotics for 5 days. The dressing was removed on day 7 post-procedure.

Follow-up

The subjects were subsequently followed up with regular paraffin gauze dressing up to 8 weeks or until the ulcer healed, whichever was earlier. Ulcer area, volume, and photographic records with a high-resolution 20-megapixel digital camera were recorded weekly [Figure 8]A–C and [Figure 9]A–C.
Figure 8: A healing traumatic ulcer: (A) at baseline, (B) on day 7 post-procedure, and (C) at week 4 post-procedure

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Figure 9: Another healing ulcer: (A) at baseline, (B) on day 7 post-procedure, and (C) at week 4 post-procedure

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In our experience of this technique, 41.6% of the ulcers healed by 4 weeks post-procedure, and 100% ulcers had healed completely by the 8th week. At the end of 4 weeks, mean percentage of improvement in ulcer area was 98.33% and that in ulcer volume was 99.26%. Ulcers healed both from the periphery as well as in a centrifugal manner from a few cream-colored dome-shaped islands of keratinocytes measuring approximately 1–2 mm in size, which gradually merged to cover the ulcer bed completely [Figure 10]. Hypertrophic granulation tissue was seen in two of the ulcers, which subsided spontaneously within a week with regular normal saline dressing. The donor site in all cases showed complete healing by 7 days post-procedure with mild pigmentation. No other adverse effect because of the procedure was noted in any case.
Figure 10: A diabetic ulcer healing with few yellowish pin-head-sized dome-shaped colonies of cells

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   Discussion Top


Various studies using pure epidermal sheet grafting, cultured keratinocyte grafting, and engineered skin products have emphasized the pivotal role of keratinocytes in wound healing.[3] Our study was based on the scientific fact that if used in ulcers, ECS may promote migration or multiplication of acceptor keratinocytes and formation of extracellular matrix proteins. A previous study assessing the effectiveness of autologous non-cultured epidermal cell suspension in 15 cases of nonhealing ulcers found a success rate of 60% by 20 weeks post-procedure with 1 case of failure of therapy.[4]

Collagen and hair follicular stem cells found in dermal follicular grafts have been shown to initiate fibroblast formation and endothelial migration, as well as capillary sprouting, respectively, when implanted into ulcer tissue.[5],[6] Budamakuntla et al.[7] evaluated the use of autologous scalp hair follicular unit grafts in chronic ulcers and found a mean percentage improvement in ulcer area and volume of 48.8% and 71.98%, respectively, at 18 weeks post-procedure.

In a prior study, Nagaraju et al.[8] used autologous smashed dermal graft in acne scars and reported favorable results with neocollagenesis. The modification to use autologous follicular dermal grafts in our study was based on the fact that it would also make the follicular stem cells available for faster wound healing. However, this is the first study to our knowledge using autologous smashed follicular dermal graft with ECS in nonhealing ulcer.

There are many beneficial effects of combining autologous ECS and smashed dermal follicular grafts. The ratio of donor-to-recipient skin area required is significantly reduced compared to conventional skin grafts. There is no requirement of absolute immobilization for the patient. The smashed dermal graft is also soft and easily moldable, and thus it can be tailored to cover any ulcer irrespective of its shape and size. Finally, this method is cost-effective and safe with only minimal scarring of donor area resulting in good cosmetic appearance.


   Conclusion Top


This modified technique of combining autologous smashed follicular dermal graft with epidermal cell suspension can be an effective, simple, time-saving, and affordable modality of treatment for chronic nonhealing leg ulcers.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Reichenberg J, Davis M. Venous ulcers. Semin Cutan Med Surg 2005;24:216-26.  Back to cited text no. 1
    
2.
Brown JB, McDowell F. Epithelial healing and the transplantation of skin. Ann Surg 1942;115:1166-81.  Back to cited text no. 2
    
3.
Yamaguchi Y, Kubo T, Tarutani M, Sano S, Asada H, Kakibuchi M, et al. Epithelial-mesenchymal interactions in wounds: treatment of palmoplantar wounds by nonpalmoplantar pure epidermal sheet grafts. Arch Dermatol 2001;137:621-8.  Back to cited text no. 3
    
4.
Shukla VK, Tiwary SK, Barnwal S, Gulati AK, Pandey SS. Effect of autologous epidermal cell suspension transplantation in chronic nonhealing wounds: a pilot study. Can J Surg 2010;53:6-10.  Back to cited text no. 4
    
5.
Lough DM, Yang M, Blum A, Reichensperger JD, Cosenza NM, Wetter N, et al. Transplantation of the LGR6+ epithelial stem cell into full-thickness cutaneous wounds results in enhanced healing, nascent hair follicle development, and augmentation of angiogenic analytes. Plast Reconstr Surg 2014;133: 579-90.  Back to cited text no. 5
    
6.
Mian M, Beghè F, Mian E. Collagen as a pharmacological approach in wound healing. Int J Tissue React 1992;14:1-9.  Back to cited text no. 6
    
7.
Budamakuntla L, Loganathan E, Sarvajnamurthy SA, Nataraj HV. Follicular unit grafting in chronic nonhealing leg ulcers: a clinical study. J Cutan Aesthet Surg 2017;10:200-6.  Back to cited text no. 7
[PUBMED]  [Full text]  
8.
Nagaraju U, Chikkaiah MK, Raju BP, Agarwal P. Autologous smashed dermal graft with epidermal re-closure: modified technique for acne scars. J Cutan Aesthet Surg 2016;9: 258-62.  Back to cited text no. 8
[PUBMED]  [Full text]  

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Correspondence Address:
Pranami Kashyap
Department of Dermatology, Venereology and Leprology, RajaRajeswari Medical College and Hospital, Mysore Road, Bengaluru 560074, Karnataka.
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JCAS.JCAS_105_19

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10]



 

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