Indian Journal of Plastic Surgery Medknow Publications on behalf of Indian Journal of Plastic Surgery ISSN: 0970-0358 EISSN: 1998-376x Vol. 40, Num. 1, 2007, pp. 104-107

Indian Journal of Plastic Surgery, Vol. 40, No. 1, January-June, 2007, pp. 104-107

Letter To Editor

Early experience with autologous skin culture for wound therapy

Rajiv Agarwal, A.K. Singh, T.N. Dhole

Postgraduate Department of Plastic and Reconstructive Surgery, K.G. Medical University, Lucknow - 226 003
Correspondence Address:A-15 Nirala Nagar, Lucknow - 226 003 drrajivagarwal@rediffmail.com

Code Number: pl07020

Dear Sir,

Open wounds present unique clinical problems for the reconstructive surgeon. Skin wounds contract by stretching the surrounding skin to close the defect and not by the production of new skin. In areas subjected to significant skin loss exceeding the available supply, especially in burns, wound coverage becomes a challenge. In such situations many options for coverage including dressing materials ranging from nonbiological to biological products exist, ^[1],[2] but the current gold standard remains coverage by patient′s own skin.

Over the years, coverage after excision by cultured epidermal autografts (CEA) has become an established and standardized method practiced in several centers worldwide. ^[3],[4] Today, from a single biopsy measuring 2x3 cm, enough keratinocytes can be cultivated to cover the entire body of an adult in three to four weeks. Currently, use of skin culture and substitutes, though quite prevalent around the world has surprisingly not been developed enough and practiced in Indian plastic surgery and burn units. At this time to the best of our knowledge, it is limited to a handful of select centers. We would like to share our early experience with epidermal culture of skin in close collaboration with a state-of-the-art tissue culture laboratory with an aim to benefit our colleagues and their patients with difficult wounds.

The clinical work of harvesting skin specimens from patients and also later engraftment of cultured keratinocytes was performed at the Postgraduate Department of Plastic and Reconstructive Surgery, K.G. Medical University, Lucknow. The laboratory work of culturing keratinocytes was carried out in the facility at the Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow. The following groups of patients were included in the study- Patients with less than 40% total body surface area full thickness burns, post burn contractures, non healing diabetic wounds, pressure ulcers and unstable scars. All patients presenting with frank, purulent wounds were excluded from the study. The parameters and variables recorded for the individual cases included identification data of patient, raw area data including size, age, depth and cause of wound. Skin culture harvest data included size of skin harvested, duration of culture and total area of cultured skin engrafted. The quality of healing was observed by assessing the scar following cultured skin application. Photographic documentation was done at regular intervals at different stages of this procedure. Ethical clearance and a written informed consent were obtained from all patients undergoing this procedure. On admission of patient, within 48h, thin partial thickness biopsy specimens of skin were harvested from an unburned area or thigh under local anesthesia at the bedside using Humby′s knife and a rectangular segment of skin measuring 40 mm x 10 mm was harvested under aseptic conditions. The biopsy skin piece was divided into three or four aliquots and then transported immediately to tissue culture laboratory in a strictly sterile environment in special flasks containing transport media in ambient temperature-controlled containers. In the laboratory, the skin sample was minced and trypsinized to produce a single cell suspension. Aliquots of 2 x 10 ⁶ cells were stored in flasks with a surface area of 75 cm ² using modification of the fibroblast feeder layer method of Rheinwald and Green. ^[5] Cultured cell colonies as early as five days old in suspension were used for engraftment onto the patients. Prior to engraftment, all wounds were dressed using conventional methods dictated by wound nature, discharge and infection status. At the time of engraftment, patients were returned to the operating room, the designated wound bed was inspected and re-excised to muscle fascia. Acute excisional wounds were generated by layered excision of full thickness wounds to the level of muscle fascia so as to ensure that any epithelial coverage has come from the cultured grafts only and not from adjacent tissue. Reconstructive wounds were generated by incisional release. Haemostasis was ensured prior to the application of skin culture suspension. The suspension was painted onto the wound using regular brush, sterilized with flash autoclave. Following application of cultured suspension, the wound was allowed to dry so that the cultured cells settle on the wound surface. A protective no-touch dressing was used to achieve this objective. After two hours of suspension application, the dried up wound surface was covered by tulle dressings and then was inspected every six hours thereafter for 48h post-engraftment, supplemented by appropriate splintage of the part. Thereafter the wound was inspected every 12h and any collection or discharge was cleared. The patients were given appropriate antibiotic cover. Active range of motion exercises and ambulation was delayed for several days because of concerns about fragility of cultured epidermis suspension. Application of compression garments was also delayed by four to five weeks in the cultured epidermis recipients.

The initial skin specimens from the first eight patients met with failure evidenced by no culture in the laboratory. In our experience the first two samples were thick containing a lot of dermis preventing adequate separation of epidermis and dermis. The next two samples were harvested taking care to include less of dermis but in the laboratory culture was unsuccessful due to growth of bacteria. Strict aseptic conditions were subsequently ensured for skin specimen harvesting and adequate specimens were transported to the laboratory. This time the culture progressed for 48h but the epidermal cells failed to survive beyond this time period. A review of the laboratory media and various growth additives was performed and subsequently the medium was modified by addition of growth factors and change of pH. The last two samples were finally harvested from patients taken with adequate aseptic precautions and were processed in the modified medium. Successful epidermal cell culture was observed with confluent colonies of cells in the culture medium [Figure - 1]. These cells were isolated in the form of suspension and then prepared for engraftment onto the patients according to the technique detailed earlier. On wound inspection following engraftment, on Day 2 the wound did not look raw and a scab formed over the entire wound. The scab remained till day 7 and till that time no accurate wound evaluation was possible. At day 10 the scab over the entire wound separated leaving behind a thin layer of epithelium of yellowish hue. The epithelial hue started changing to pink at Day 15 and by Day 20 the entire wound was covered by this epithelium. At four weeks, the pink hue turned into light brown and the epithelium further looked to be stable. There was complete coverage of the wound by the cultured epithelium. At five weeks, the patient was advised gentle graft massage and emollients. At six weeks pressure garment and silicon sheeting therapy was instituted to prevent scar hypertrophy and un aesthetic sequelae. Successful engraftment was obtained at eight-month follow-up wound inspection [Figure - 2a], [Figure - 2b], [Figure - 2c], [Figure - 2d], [Figure - 2e], [Figure - 2f].

This is our first experience with indigenous cell culture and though the patient demonstrated may not be the ideal one for cell culture our initial experience has paved the way for further work and research in the technique of skin culture so that consistent results are obtained at cost-effective prices for most centers of plastic surgery requiring skin coverage for difficult wounds. We have been able to culture skin for the first time in the department with laboratory assistance and cooperation from an extra-institutional nearby tissue culture facility. We plan to further develop this technique on these lines and conduct larger series of cases with cultured epithelium ′take′ results matched with simultaneous controls of split thickness autografts. The engrafted cultured skin patches would also be subjected to detailed histological studies to determine their cell architecture and composition. The data so obtained would be analyzed and compared statistically for significance.

Cell culture currently is not cost-effective in the Indian scenario as the setting up of a laboratory and subsequent running entails huge costs. We need to develop indigenous cost-effective and economical technology for cell culture so that it is available to a greater percentage of patients. However, there is a need at this time to develop parallel tissue culture laboratories working in tandem with plastic surgery units.

Acknowledgement

References

Copyright 2007 - Indian Journal of Plastic Surgery

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