Using Hyaluronic Acid Derivatives and Cultured Autologous Fibroblasts and Keratinocytes in a Lower Limb Wound in a Patient with

Luca Dalla Paola, MD; Alberto Cogo, PhD; Walter Deanesi, MD; Cristina Stocchiero, MD; and Valerie Carmela Colletta

C hronic lower extremity ulcers are a common and serious complication of diabetes mellitus. Moss1 estimates that patients with diabetes have a five to 15 times higher risk of requiring a lower extremity amputation than people who do not have diabetes. Foot ulcers are an important predictor of future lower extremity amputations in patients with diabetes and, despite efforts to prevent these ulcers from occurring, the incidence of lower extremity amputations continues to rise.2
Data are scant on the use of bioengineered tissue in the treatment of chronic wounds in patients with diabetes.3-5 This case study describes the use of an autologous tissue engineering approach to close a severe ulcer in the Achilles' tendon region of the lower limb of a patient with diabetes. The technique is based on the use of biodegradable scaffolds composed of an ester derivative of hyaluronic acid onto which autologous laboratory grown fibroblasts and keratinocytes (Hyalograft 3D® and Laserskin®, respectively, Fidia Advanced Biopolymers SrL, Abano Terme, Italy) are seeded.6 The ester derivatives of hyaluronic acid are used because they offer optimal biocompatibility and biodegradabilty in vitro and in vivo. Moreover, cultivated fibroblasts need a three-dimensional structure into which to grow; whereas, keratinocytes need a two-dimensional structure. The ester derivative has the necessary physical properties to be processed into different forms to obtain appropriate scaffolds for these different cell types. This two-stage approach has been used successfully to heal other chronic and acute wounds.7-8

Case History

The patient was a 65-year-old male, nonsmoker, with type 2 diabetes mellitus (treated with intensive insulin therapy), peripheral neuropathy, and infrapopliteal arteriopathy of the lower limbs. He also had a history of arterial hypertension and myocardial ischemic disease that was treated with warfarin, ACE-inhibitors, diltiazem, and nitrates. Diffuse infrapopliteal stenosis and the absence of a suitable vessel in the tibial and pedal region for distal arterial bypass run-off eliminated the possibility of revascularization. Due to the incorrigible critical ischemia and worsening of his infected lower leg wound, amputation was recommended.
The patient changed providers and visited the authors' clinic in October 1998. He presented with an extensive ulcer of the left lower limb (diagnosed 7 months earlier) caused by a minor trauma. The wound had remained unhealed despite ancillary treatments (see Figure 1). Up to this point, local treatment with saline solution-soaked gauze was performed daily. No surgical debridement had been performed. The patient received cyclic treatment with antibiotics. No orthesis or therapeutic shoes were used. Metabolic control measured at the first visit was discrete; his HbA1c was 6.9%.
The patient's ulcer measured 103.49 cm2 and was full thickness with exposure of a necrotic Achilles' tendon. Transcutaneous oxygen pressure (TcPO2) measured on the dorsal surface of the midfoot was 24 mm Hg and his ankle brachial index (ABI) was 0.3. Venous insufficiency was ruled out on the basis of the ultrasonography results. Distal neuropathy was also present.
The wound was clinically infected; Pseudomonas aeruginosa was isolated from microbiological specimens and intravenous antibiotic therapy (piperacillin plus tazobactam) was initiated. Treatment commenced on an outpatient basis. The wound was surgically debrided and the necrotic Achilles' tendon was removed. In this first phase, the lesion was treated with antiseptics (povidone-iodine and silver sulfadiazine) and subsequently with collagenase covered with a polyurethane foam dressing. During the week preceding the autologous graft, dressings with hyaluronic acid covered with a polyurethane foam were used.
A 2-cm x 1-cm full-thickness cutaneous biopsy was taken from the inner part of the upper arm of the patient and sent to the laboratory for the extraction and further separate cultivation of the fibroblasts and keratinocytes. The isolated autologous fibroblasts were grown on a three-dimensional nonwoven fleece of hyaluronic acid; whereas, the autologous keratinocytes were grown on a thin, laser-drilled microperforated transparent membrane of hyaluronic acid.9
After thorough cleansing of the ulcer, the tissue-engineered autologous dermal-like substitute was placed in direct contact with the wound bed (see Figure 2). After two applications (7 days apart), the tissue-engineered autologous epidermal-like substitute was placed on the wound (see Figure 3). A paraffin gauze and a gauze soaked with saline solution were used as secondary dressings.
During treatment, the patient was seen twice a week at the clinic to change the secondary dressings and assess the wound (see Figure 4). A transparent acetate grid was used to measure the dimensions of the wound. The depth of the wound also was measured.
A fiberglass splint was required to immobilize the ankle throughout the treatment period. In addition, the patient was asked to rest the day the grafts were applied and for 2 days after application.
On the tenth day following the last application of the autologous keratinocytes, the dressing regimen of hyaluronic acid covered with polyurethane foam was resumed. These dressings were changed twice a week.
Complete wound closure was achieved 60 days following the first fibroblast application. The application of fibroblasts appeared to allow the formation of well-vascularized granulation tissue in a wound bed that contained no granulation tissue when only advanced medications were being used. Rapid activation of re-epithelialization from the wound margins was observed after keratinocyte application.
The patient continued to come to the clinic for regular monthly check-ups. Rigid (rocker bottom) shoes and insoles made from plantar molds were provided. The treated lesion remained re-epithelialized. The newly formed epithelium integrated completely with the surrounding skin (see Figure 5). At this time, exact information about the cost of treatment with these autologous cells is not available but, for this specific case, the cost was estimated to be approximately $1,000 (US).


The technique described requires time to culture the cells. Furthermore, this case study represents only one successful approach to a clinically challenging situation. The results obtained may be due more to the effect of the hyaluronic acid or the cultured fibroblasts and keratinocytes; this has not been ascertained.


This experience suggests that the use of autologous fibroblasts and keratinocytes cultured on biodegradable hyaluronic acid-derived scaffolds could be a rational approach for the treatment of severe chronic wounds of the lower limbs in patients with diabetes. The use of this novel tissue-engineering technique is currently under investigation in randomized controlled clinical studies. - OWM


1. Moss SE, et al. The prevalence and incidence of lower extremity amputation in a diabetic population. Arch Intern Med. 1992;152:610-616
2. American Diabetes Association's Consensus Report. Special Report. Wounds. 1999;11(5):93-104.
3. Gentzkow GD, Iwasaki SD, Hershon KS, et al. Use of Dermagraft, a cultured human dermis, to treat diabetic foot ulcers. Diabetes Care. 1996;19:350-354.
4. Pollak R, Edington H, Jensen J, Kroeker R, Gentzkow G. Dermagraft Diabetic Ulcer Study Group. A human dermal replacement for the treatment of diabetic foot ulcers. Wounds. 1997;9(1):175-183.
5. Pham HT, Rosenblum BI, Lyons TE, et al. Evaluation of Graftskin (Apligraft), a human skin equivalent, for the treatment of diabetic foot ulcers. Wounds. 1999;11(4):79-86.
6. Campoccia D, Doherty P, Radice M, Brun P, Abatangelo G, Williams DF. Semisynthetic resorbable materials from hyaluronan esterification. Biomaterials. 1998;19:2101-2127.
7. Brun P, Grosso F, Galassi G, et al. Use of dermal-like tissue in the management of chronic and acute full-thickness cutaneous wounds. Wounds. 2000;46(9):44-48.
8. Faglia E, Mantero M, Gino M, Quarantiello A, Signorini M. A combined conservative approach in the treatment of a severe Achilles' tendon region ulcer in a patient with diabetes: a case report. Wounds. 1999;11(5):105-109.
9. Zacchi V, Soranzo C, Cortivo R, Radice M, Brun P, Abatangelo G. In vitro engineering of human skin-like tissue. J Biomed Mater Res. 1988;40(2):187-194.


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