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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 28  |  Issue : 1  |  Page : 69-73

Use of epidermal cell suspension in burns wound management: A pilot study


1 Plastic Surgeon, Department of Plastic Surgery, Army Hospital Research and Referral, New Delhi, India
2 Classified Specialist (Community Medicine), Directorate General of Medical Services (Army), New Delhi, India

Date of Submission29-Jan-2020
Date of Acceptance08-Aug-2020
Date of Web Publication21-May-2021

Correspondence Address:
Dr. Vikas Singh
Army Hospital Research and Referral, New Delhi
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijb.ijb_4_20

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  Abstract 


Introduction: It is a standard practice to cover the burn wounds with skin grafts at the earliest. Deficiency of donor skin seriously limits timely cover of extensive burns. Studies have shown that these autologous cells enhance the wound-healing process by reducing the time needed for the host cells to invade the wound tissue and by early synthesis of new skin this technique of enzymatically digesting small skin grafts, centrifuging the suspended cells and spraying the epidermal cells over burn raw areas may allow early healing.
Aims and Objective: The aim of this study is to assess feasibility and role of epidermal cell suspension as wound cover in burns.
Materials and Methods: This study was conducted at the burn center of a tertiary Hospital in India over a period of 1 year. Patients with postburn raw areas were included. Similar size two sites were identified in the patients. On one site cell suspension was applied, while other areas were managed with conventional dressings. Small piece of split-skin graft (SSG) was used to make cell suspension. Under anesthesia, required size of SSG was harvested from healthy donor site. Trypsinization of graft was done; epidermis was separated from the dermis. The epidermis was cut into tiny pieces, and patient's serum was added and centrifuged. Epidermal cell suspension was made, and viability of cells is confirmed using an inverted microscope. Meanwhile, the wound is thoroughly cleaned by soda bicarbonate solution. The freshly prepared keratinocytes suspension was applied on day 1, 3, and 5. Similar standard dressing protocol was followed on the control side. Wound assessment was done based on the percentage of reepithelization on the 7th and 14th day.
Results: Twenty-eight patients (18 males and 10 females with a mean age of 34 years) were included the study. Cell suspension was sprayed on 4735 cm2 area. Wound size reduced to 2247 cm2 and 923 cm2 on the 10th and 14th day, respectively. While, at control site wound reduced to 3882 cm2 and 2134 cm2 on the 7th and 14th day, respectively. The difference in cell suspension and control treated area was found to be significant.
Conclusion: Keratinocytes cell suspension is feasible in burns and improves the healing of wounds. However, larger studies are required to standardize and validate its use in major burns.

Keywords: Burns wound management, epidermal cell suspension, pilot study


How to cite this article:
Mishra B, Singh V, Arora C. Use of epidermal cell suspension in burns wound management: A pilot study. Indian J Burns 2020;28:69-73

How to cite this URL:
Mishra B, Singh V, Arora C. Use of epidermal cell suspension in burns wound management: A pilot study. Indian J Burns [serial online] 2020 [cited 2021 Jun 18];28:69-73. Available from: https://www.ijburns.com/text.asp?2020/28/1/69/316579




  Introduction Top


Wound healing is always of paramount importance for survival and decreasing morbidity in burns patients. Covering wounds with split-skin grafts (SSGs) after early tangential excision is the standard management in burns. In extensive injury, there are large raw areas and limited donor available site for skin grafting. Large raw areas are directly related to wound-related complications, septicemia, prolonged hospitalization, and death. Limited harvested grafts can be practically expanded to 1:4 but are usually insufficient to provide cover. Meek micro grafting though useful is also not readily available. Micro grafting, while resulting in an effective skin expansion ratio approaching 1:18, has been shown to be associated with severe wound contraction that is often worse than that noted following the application of meshed skin autografts.[1] Newer advanced dressing material and skin substitutes provide only temporary cover. Facilities of skin banks and allograft are having limited reach and virtually not existing in most of the places in our country. Hence, very few options are available to cover the large raw areas. An unexplored option of permanent cover is the use of patients own epidermal cells in suspension rather than graft sheet. These can be harvested from small skin graft taken under local anesthesia following simple laboratory processing and can be made available for wound cover. This keratinocytes suspension also contains various salutary and growth factors which promote wound healing and wound contraction. Keratinocytes mainly release growth factors (transforming growth factor α [TGF-α], platelet-derived growth factor [PDGF], basic fibroblast growth factor [bFGF], vascular endothelial growth factor [VEGF], and TGF-β) and cytokines interleukin (IL-1,-6,-8, and-10), but lack the ability to secrete extracellular matrices.[2],[3],[4] With this premise, a preliminary study was conducted to assess the feasibility and efficacy of cell suspension in the Indian setup.


  Materials and Methods Top


This study was conducted at the burn center of tertiary care teaching hospital of New Delhi from August 1, 2017, to July 31, 2018. After taking well informed written consent, patients admitted during this period irrespective of burn area were included in the study. However, patients with frank sepsis, multiple organ dysfunction syndrome, and wounds with the sign of local invasive infection were excluded from the study. Patients with autoimmune diseases or taking chemotherapy or immunosuppressants were also excluded.

Procedure of making cell suspension

Under anesthesia required size of SSG was harvested from healthy donor site. A parameter of 1 cm2 of SSG for every 80 cm2 raw area was used. The skin graft was immediately transferred to the laboratory. Trypsinization of graft was done in 0.25% trypsin −0.05% ethylenediaminetetraacetic acid solution. It was kept in a 90-mm petridish in the incubator for 45 min at 37°C. After this, the epidermis was separated from the dermis. The epidermis was cut into tiny pieces, and patient's serum was added to neutralize the action of trypsin. Lysed epidermis was transferred to sterile 15 ml conical tubes for centrifugation for 10 min at 2000. The supernatant was discarded, and this step was repeated two times for proper washing of the cells. Viability of cells is confirmed using an inverted microscope. Meanwhile, the wound is thoroughly cleaned by soda bicarbonate solution. The freshly prepared keratinocytes suspension was applied using Tisseel Fibrin Sealant spray machine (Baxter AG, Vienna, Austria) or by spraying with syringe in case of small areas.

Two similar size areas were identified, and the cell suspension was applied on healthy post burn raw area on day 1, 3, and 5. No local antibiotic was applied, and it was covered by paraffin gauze, and secondary dressings were applied. Standard dressing protocol was followed on the control side. Wound assessment was done by an independent observer based on the percentage of re-epithelization on the 7th and 14th day.

Patient's perception of treatment (pain and discomfort) on the application of cell suspension and adverse effects were also noted.


  Results Top


This study was conducted over a period of 18 months. Seventy-seven patients were admitted in the burn center during this period. Twenty- eight patients were included in the study. There were 18 males and 10 females with a mean age of 34.3 years.

Cell suspension was sprayed on 4735 cm2 area. Wound size reduced to 2247 cm2 and 923 cm2 on the 7th and 14th day, respectively. While, at control site surface area was 4670 and wound reduced to 3882 cm2 and 2134 cm2 on the 7th and 14th day, respectively [Table 1]. The surface area (mean), in each group, before treatment, was comparable and was shown to be nonsignificant with a P = 0.90. Posttreatment, the difference in cell suspension and control treated area was found to be significant.
Table 1: Comparison of outcome

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The application of spray was painless, and no adverse reaction or side effects were noticed. Photos of representative cases are shown in illustration 1–5. [Figure 1], [Figure 2], [Figure 3] are showing application of epithelial cell suspension on day 1 and assessment on day 7 and complete healing on day 14. [Figure 4] showed a child with residual post burn raw areas following skin grafting. Epithelial cell suspension was applied and good response was noted on day 14 [Figure 5].
Figure 1: Case 1, 14th day of cell suspension application

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Figure 2: Case 1, 7th day of cell suspension application

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Figure 3: Case 1, 7th day of cell suspension application

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Figure 4: Case 2, 14th day of cell suspension application showing healed areas on which cell suspension was

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Figure 5: Case 2, I sitting of cell suspension application

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  Discussion Top


The estimated annual burn incidence in India is approximately 6–7 million per year. Nearly 1–1.5 lac people get crippled and require multiple surgeries and prolonged rehabilitation. Seventy percent of the burn victims are in the most productive age group of 15–40 years, and most of the patients belong to poor socioeconomic strata.[5] Economic uplift and shift from kerosene to safer LPG stoves have brought down annual burn admission by 43% in a major burn unit of Delhi.[6] Timely coverage of the burns wound is most critical for the survival, lesser morbidity, and post burn sequelae. Autologous skin graft has been recognized as the best definitive burn wound coverage, but it is constrained by the limited available sources, especially in major burns. It is also associated with additional donor site morbidities in terms of additional wounds and scarring.[7] Bates-Jensen wound Assessment tool is used for assessment of chronic wounds, but no such tool is used for acute burn wounds.[8],[9]Limited harvested grafts can practically be expanded to 1:4 but are usually insufficient to provide adequate cover. Meek micrografting provides more expansion, but it is associated with severe wound contraction that is often worse than that noted following the application of meshed skin autograft. Skin substitutes, though are an attractive alternative, but their availability, long-term utility (mostly provide temporary cover), and cost make it a less preferred option. At present, there is no ideal substitute in the market that provides an effective and scar-free wound healing.[7] Allografts have also been used to provide temporary cover, but this facility has a very limited reach, especially in vast country like India. At present, India has <10 fully functional skin banks, and those are mostly located in metro cities. Hence, all these alternatives, though look promising but have inherent problems to provide a viable permanent solution to hasten wound healing and provide wound cover. An underutilized option of permanent cover is the use of autologous epidermal cells in suspension rather than graft sheet. Cell therapy has been used for the management of skin loss caused by the variety of etiology, including burn wounds, congenital melanocytic nevus and donor sites of split-thickness skin.[10],[11] In contrary to cell culture, keratinocytes cell suspension is easier to prepare and use. It can be prepared in an hour, and there is no requirement for storage also. Freshly prepared keratinocytes suspension is not only a source of viable cells but also various salutary and growth factors. Keratinocytes mainly release growth factors (TGF-α, PDGF, bFGF, VEGF, and TGF-β) and cytokines (IL-1,-6,-8, and-100).[2],[3],[4] Many studies have shown that these autologous cells enhance the wound healing process by reducing the time needed for the host cells to invade the wound tissue and by early synthesis of new skin.[12],[13],[14] However, no study is available from India, thus it was a feasibility and preliminary study to assess the role of keratinocytes suspension in major burns. In this study, spraying of keratinocytes suspension was found to have beneficial in the healing of wounds. It was found that better reepithelization was noticed in the raw areas treated with the freshly prepared cell suspension. Cell suspension was sprayed on 4735 cm2 area. Wound size reduced to 2247 cm2 and 923 cm2 on the 7th and 14th day, respectively. While, at control site, surface area was 4670 and wound reduced to 3882 cm2 and 2134 cm2 on the 7th and 14th day, respectively. Re-epithelization was noted in approximately 80.5% of treated area with cell suspension on the 14th day, while control area showed reepithelization in only 54.3% of treated areas with standard dressings. Allogenic cell suspension has also been used and found to be useful in in wound healing.[15],[16] Allogeneic cells promote migration and proliferation of host cells from the wound beds and edges because and release growth factors.[4] Kirsner et al. in a randomized trial showed that venous ulcers can be healed with a spray formulation of allogeneic neonatal keratinocytes and fibroblasts without the need for tissue engineering.[17],[18] The use of single cell suspension by employing epidermal cells for skin cover in extensive burns is a biotechnological advance and will contribute to better healing and improved survival.


  Conclusion Top


To conclude, this study proves that the use of keratinocytes cell suspension is feasible in normal set up of burn center. Keratinocytes cell suspension is useful in burns and improves healing of wounds. However, larger studies are required to standardize and validate its use in major burns.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Kagan RJ, Robb EC, Plessinger RT. The Skin bank, Total Burn Care IIIe. In: Herndon DN, editor. Ch. 15. Philadelphia: Saunders Elsevier; 2007. p. 232.  Back to cited text no. 1
    
2.
Yates CC, Whaley D, Wells A. Transplanted fibroblasts prevents dysfunctional repair in a murine CXCR3-deficient scarring model. Cell Transplant 2012;21:919-31.  Back to cited text no. 2
    
3.
El-Ghalbzouri A, Gibbs S, Lamme E, Van Blitterswijk CA, Ponec M. Effect of fibroblasts on epidermal regeneration. Br J Dermatol 2002;147:230-43.  Back to cited text no. 3
    
4.
You HJ, Han SK. Cell therapy for wound healing. J Korean Med Sci 2014;29:311-9.  Back to cited text no. 4
    
5.
Gupta JL, Makhija LK, Bajaj SP. National programme for prevention of burn injuries. Indian J Plast Surg 2010;43:S6-S10.  Back to cited text no. 5
[PUBMED]  [Full text]  
6.
Ahuja RB, Bhattacharya S, Rai A. Changing trends of an endemic trauma. Burns 2009;35:650-6.  Back to cited text no. 6
    
7.
Halim AS, Khoo TL, Mohd Yussof SJ. Biologic and synthetic skin substitutes: An overview. Indian J Plast Surg 2010;43:S23-8.  Back to cited text no. 7
    
8.
Anna and harry borun center for gerontological research. The bates-jensen wound assessment tool page. Available from: borun.medsch.ucla.edu/modules/Pressure_ulcer_prevention/puBWAT.pdf. [Last accessed on 2017 Jul 07].  Back to cited text no. 8
    
9.
Harris C, Bates-Jensen B, Parslow N, Raizman R, Singh M, Ketchen R. Bates-Jensen wound assessment tool: Pictorial guide validation project. J Wound Ostomy Continence Nurs 2010;37:253-9.  Back to cited text no. 9
    
10.
Sood R, Roggy D, Zieger M, Balledux J, Chaudhari S, Koumanis DJ, et al. Cultured epithelial autografts for coverage of large burn wounds in eighty-eight patients: The Indiana University experience. J Burn Care Res 2010;31:559-68.  Back to cited text no. 10
    
11.
Whang KK, Kim MJ, Song WK, Cho S. Comparative treatment of giant congenital melanocytic nevi with curettage or Er: YAG laser ablation alone versus with cultured epithelial autografts. Dermatol Surg 2005;31:1660-7.  Back to cited text no. 11
    
12.
Morimoto N, Saso Y, Tomihata K, Taira T, Takahashi Y, Ohta M, et al. Viability and function of autologous and allogeneic fibroblasts seeded in dermal substitutes after implantation. J Surg Res 2005;125:56-67.  Back to cited text no. 12
    
13.
Seo YK, Song KY, Kim YJ, Park JK. Wound healing effect of acellular artificial dermis containing extracellular matrix secreted by human skin fibroblasts. Artif Organs 2007;31:509-20.  Back to cited text no. 13
    
14.
Erdag G, Sheridan RL. Fibroblasts improve performance of cultured composite skin substitutes on athymic mice. Burns 2004;30:322-8.  Back to cited text no. 14
    
15.
Yanaga H, Udoh Y, Yamauchi T, Yamamoto M, Kiyokawa K, Inoue Y, et al. Cryopreserved cultured epidermal allografts achieved early closure of wounds and reduced scar formation in deep partial-thickness burn wounds (DDB) and split-thickness skin donor sites of pediatric patients. Burns 2001;27:689-98.  Back to cited text no. 15
    
16.
Gallego L, Junquera L, Villarreal P, Peña I, Meana A. Use of cultured human epithelium for coverage: A defect of radial forearm free flap donor site. Med Oral Patol Oral Cir Bucal 2010;15:e58-60.  Back to cited text no. 16
    
17.
Kirsner RS, Marston WA, Snyder RJ, Lee TD, Cargill DI, Slade HB. Spray-applied cell therapy with human allogeneic fibroblasts and keratinocytes for the treatment of chronic venous leg ulcers: A phase 2, multicentre, double-blind, randomised, placebo-controlled trial. Lancet 2012;380:977-85.  Back to cited text no. 17
    
18.
Lindgren C, Toftgard M. Treatment of venous ulcer with cryopreserved cultured allogenic keratinocytes. Br J Dermatol 1998;139(2):271-5.  Back to cited text no. 18
    


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