Treatment of venous leg ulcers with cryopreserved cultured allogeneic keratinocytes: a prospective open controlled study

Twenty-seven patients with chronic venous leg ulcers were assigned to two treatment groups. Fifteen were treated with cryopreserved cultured allogeneic keratinocyte sheets and compression bandages and 12 with compression only. The observation time was 8 weeks and the keratinocyte grafting procedure was performed once weekly. The mean reduction of the initial wound area was 35% in the treatment group and 14% in the control group. This difference was not significant. The poor effect on wound healing given by the cryopreserved allogeneic keratinocyte sheets contrasts with our earlier experience using fresh keratinocyte sheets. We believe that this is due to a weakened condition of the cryostored cells, as we have shown a loss in protein synthesis capacity of at least 50%.     

Allogeneic cultured keratinocytes in the treatment of leg ulcers. A pilot study.

Forty-two patients (10 males and 32 females) with 52 chronic leg ulcers were treated with sheets of cultured allogeneic keratinocytes. Sixty-five % of the ulcers healed completely and the healing rate differed between various diagnostic groups. The best results were obtained in patients with venous ulcers and wounds with mixed etiology, whereas less improvement was observed with ischaemic ulcers. Rheumatic ulcers also responded well in combination with oral corticosteroids. The overall impression was that the grafting procedure markedly enhanced wound healing.     

Cultured allogeneic skin cells are effective in the treatment of chronic diabetic leg and foot ulcers.

Diabetic ulcers on the lower extremities present a difficult treatment problem, and some ulcers respond poorly to conventional topical and cast treatment. The purpose of this study was to assess the effect of cultured allogeneic keratinocyte epithelium and fibroblast-gelatin sponge on the healing of chronic, refractory diabetic leg and foot ulcers. Non-diabetic chronic leg ulcers were treated for comparison. This open study comprised 22 patients with type I or type II diabetes and 16 patients with leg or ankle ulcers of different aetiologies. A total of 26 diabetic and 25 non-diabetic ulcers were treated mainly with keratinocyte epithelium and/or fibroblast-gelatin sponge once weekly until complete healing or until no further healing could be observed despite several repeated treatments. The duration of diabetic ulcers was 10.3+/-15.8 (mean+/-SD) months and the size 3.1+/-6.6 cm2. The diabetic ulcers were located in the heel (7), toe (7), sole (5), leg (6) and Achilles (1). The mean duration of non-diabetic ulcers was 6.8+/-6.0 months and the size 10.5+/-11.8 cm2. A total of 12+/-11 skin cell transplantations were performed for the diabetic ulcers. All but 1 diabetic ulcer healed during the study. The time for 50% reduction in ulcer area was 32+/-32 days, but 99+/-110 days were needed for complete ulcer closure. The longer the ulcer had existed the longer was the healing time. Heel ulcers showed significantly slower healing response than leg, sole and toe ulcers. Preliminary results suggest that both keratinocytes and fibroblasts are equally effective in the healing process. The time required for healing of the diabetic ulcers did not differ markedly from that of the non-diabetic ulcers. The results suggest that cultured allogeneic skin cells used once weekly are effective in the treatment of recalcitrant diabetic ulcers.     

Allogeneic cultured epidermal grafts heal chronic ulcers although they do not remain as proved by DNA analysis

To investigate whether allogeneic cultured keratinocytes are rejected or not, and to find out how beneficial their effect on wound healing could be, patients with chronic ulcers were grafted with allogeneic cultured human keratinocytes. In order to examine the epidermal origin of the healed wound, DNA analysis was performed and compared to donor and recipient blood-cell DNA. Healing was observed in 84% of the grafted ulcers by granulation tissue stimulation and would edge effect. In little time 60% of the grafted chronic ulcers healed completely. Although no rejection was observed, DNA analysis revealed that the grafted allogeneic keratinocytes were finally replaced by the patient's own epidermis. This study confirmed that cultured allogeneic keratinocytes that have been grafted on ulcers, play an important role in the wound healing process.     

Clinical study with allogeneic cultured dermal substitutes for chronic leg ulcers

BACKGROUND: Clinical studies of the use of allogeneic cultured dermal substitutes (CDSs) have been conducted in 30 medical centers across Japan with the support of the Millennium Project of the Ministry of Health, Labor and Welfare. The CDS is prepared by plating cultured fibroblasts on a spongy matrix made from hyaluronic acid and atelo-collagen. The aim of the present clinical study was to evaluate an allogeneic CDS as cell therapy in which cytokines are released to promote wound healing. MATERIALS AND METHODS: The CDS was applied repeatedly at an interval of 5-7 days to 13 patients being treated with intractable chronic leg ulcers, using the CDS as a dermatological treatment. Intractable leg ulcers, whether venous or arterial, are difficult to heal because they occur as a secondary change of disease. RESULTS: None of these patients had responded to conventional therapies such as topical or surgical methods, but 77% of the patients had an efficacy score of > 80 points for wound healing with allogeneic CDS. A high safety level was obtained: A, 84.6%; B and C, 7.7%; D, 0%. In 92% of the patients, the final assessment of wound healing was good or very good. CONCLUSIONS: For promotion of wound healing, the present CDS is superior to existing topical agents and occlusive dressings.     

Autologous suction blister grafting for chronic leg ulcers

Background Non‐healing leg ulcers represent a treatment problem. Objective Investigate grafting of autologous suction blister roofs as treatment. Methods Twenty‐nine chronic, non‐healing leg ulcers of various aetiologies in 18 inpatients were treated by autologous epidermal grafting using the roofs of suction blisters. Results 55% of ulcers completely healed 2 to 6 weeks after grafting. A 50–90% reduction in size was documented in 34% and no change was observed in 11% of ulcers. Twelve weeks after grafting, 89% of ulcers were healed completely. In most ulcers, we observed a stimulation of reepithelialization from the wound edge (‘edge effect’) and an accelerated formation of healthy granulation tissue. During a follow‐up period of 12 months, 90% of the ulcers remained healed. Conclusion Grafting of autologous suction blister roofs is an effective treatment option for non‐healing leg ulcers. The advantages of the method are its lack of pain, low costs and immediate availability.     

Cryopreserved and lyophilized cultured epidermal allografts in the treatment of leg ulcers: a pilot study

BACKGROUND: In the conservative therapy of venous leg ulcers modern types of dressings are used most frequently. In the past 20 years 'active wound dressings' - cultured epidermal keratinocytes as autografts and allografts - were also introduced in the management of leg ulcers. METHODS: The aim of our study was to compare the effect of cryopreserved and lyophilized cultured epidermal allografts in the treatment of venous leg ulcers. Evaluation of the therapy was documented as photodocumentation, planimetry, healing time and evaluation of pain relief over a 3-month period after application. Fifty patients with venous leg ulcers were selected. Twenty-five patients (group I) were treated with cryopreserved keratinocytes and 25 (group II) with lyophilized keratinocytes. RESULTS: The final evaluation 3 months after the application of allografts showed 84% of healed ulcers in group I and 80% in group II. The number of healed ulcers and the healing rate both showed no statistically significant differences. The size of the ulcer was reduced by half during the first week in both groups. The size differences during the first week are statistically significant in both groups and they are comparable (P < 0.001). The intensity of the pain was statistically significantly reduced during the first week after application in both groups (P < 0.001). CONCLUSIONS: The cryopreserved and lyophilized cultured allografts are comparable in healing rate, course of healing and relief of pain, and also in planimetric changes during the healing of venous leg ulcers. Lyophilized allografts are more convenient for routine use than cryopreserved allografts as they can be stored at room temperature. These results could give rise to the more frequent use of lyophilized allografts in slow-healing venous leg ulcers.     

The longevity of a bilayered skin substitute after application to venous ulcers

BACKGROUND: A bilayered skin substitute composed of allogeneic keratinocytes and fibroblasts in a collagen gel has been approved by the US Food and Drug Administration for the treatment of venous and diabetic ulcers. Its mechanism of action has not been fully determined. OBJECTIVE: To determine the longevity of allogeneic fibroblasts and keratinocytes in a bilayered skin substitute in patients with venous leg ulcers. METHODS: Ten patients with venous leg ulcers were treated with a bilayered skin substitute on day 0, days 3 to 5, and weeks 1 through 3. Biopsy specimens of the grafted wound were taken. We used polymerase chain reaction analysis to determine whether allogeneic DNA was present in the biopsy specimens. RESULTS: We detected allogeneic DNA in 2 of 8 specimens at 1 month after initial grafting. Neither of the 2 patients showed persistence of allogeneic DNA at 2 months after initial grafting. CONCLUSIONS: Allogeneic cells from a bilayered skin substitute do not appear to survive permanently after grafting for treatment of venous leg ulcers. Other mechanisms of action might include cytokine release, structural support, or provision of a moist wound environment.     

Successful treatment of chronic venous leg ulcers with lyophilized cultured epidermal allografts

The case of a 67-year-old woman with chronic venous leg ulcers and severe gonarthrosis is described. In spite of intensive therapy, the leg ulcers had persisted for 4 years and made the intended orthopaedic operation of the right knee impossible. The patient was treated with lyophilized cultured epidermal allografts and her leg ulcers healed within 40 days. Lyophilized cultured epidermal allografts represent a modern type of active wound dressing that leads to rapid healing of chronic venous leg ulcers and enables patients to undergo surgical intervention.     

Healing effect of ketanserin on chronic leg ulcers in patients with diabetes

BACKGROUND: The treatment of chronic leg ulcers remains a stubborn problem in many patients. Topical 2% ketanserin ointment, a 5HT2-serotoninergic blocking agent, has been reported to improve healing of decubitus, venous, diabetic and ischaemic ulcers. METHOD: The present double-blind intra-individual comparative study was performed in 12 women with diabetes presenting with at least two similar leg ulcers. In each subject, the two lesions were randomly assigned to be treated for 8 weeks by 2% ketanserin ointment or its unmedicated vehicle. OBJECTIVE: assessments of the dynamics of wound healing were performed using computerized morphometry. Evaluations were performed at 2-week intervals for 8 weeks. RESULTS: A significant decrease in relative wound area was observed on the ketanserin-treated ulcers compared with the placebo group. CONCLUSION: Topical ketanserin is a valuable therapy for difficult-to-treat leg ulcers.     

Accelerated healing of pyoderma gangrenosum treated with bioengineered skin and concomitant immunosuppression

Pyoderma gangrenosum is a rare, destructive, neutrophilic dermatosis, the origin of which remains largely obscure. The ulcerative variant of this inflammatory disorder causes painful, necrotic, rapidly enlarging ulcers. Because of pathergy, many clinicians avoid managing these nonhealing ulcers with aggressive surgical debridement and autologous grafts. This article proposes that the application of an allogeneic cultured human skin equivalent (Graftskin) not only circumvents this problem, but also hastens re-epithelialization of the ulcer bed. An added benefit of the possible improvement of the cosmetic appearance of the final scar by preventing severe wound contracture is also postulated. We report a newly diagnosed case of ulcerative pyoderma gangrenosum; the use of bioengineered skin as an adjunct to concurrent immunosuppressive therapy with cyclosporine hastened the healing and diminished pain in a rapidly enlarging leg ulcer. Within 2 weeks, the ulcer was 30% to 40% healed, achieving 100% re-epithelialization within 6 weeks.     

A multicentre study of percentage change in venous leg ulcer area as a prognostic index of healing at 24 weeks

Venous leg ulcers, which may take months to heal, account for 40-70% of all lower extremity chronic wounds. New treatment options for venous leg ulcers have recently been proposed, and therefore deciding which patients are candidates for these novel-and often expensive-treatments is an important task. Moreover, researchers conducting clinical trials often wish to enroll patients who are unlikely to heal in order to minimize sample sizes needed and research costs. Our purpose was to assess the use of percentage change in venous leg ulcer area over the first few weeks of treatment as a prognostic indicator of healing or non-healing at 24 weeks. We conducted a cohort study based on an existing data set from a multicentre randomized clinical trial that enrolled 104 patients. Wounds were measured using digital planimetry for 4 consecutive weeks following the inception of good wound care. Utilizing the Wilcoxon rank sum (Mann-Whitney) test, we found that percentage change in area over time distinguished between those who healed and those who failed to heal after 24 weeks of good wound care (P < 0.05). The rate of healing, or area healed per week, did not differentiate between those who healed at 24 weeks and those who did not, as all patients had similar rates of healing over the first 4 weeks of treatment. Percentage change in area from baseline to week 4 provided the best combination of positive and negative predictive values (68.2%, 74.7%) and the largest area under the receiver operating characteristic curve (0.75). Thus, percentage change in area over the first 4 weeks of treatment represents a practical and predictive measure of complete wound healing by 24 weeks.     

The microbiology of infected and noninfected leg ulcers

BACKGROUND: A clinical study was undertaken to investigate and compare specifically the aerobic and anaerobic microbiology of infected and noninfected leg ulcers. METHODS: Leg ulcers, defined as being infected on the basis of clinical signs, were swab sampled and investigated for aerobic and anaerobic microorganisms using stringent isolation and identification techniques. RESULTS: Two hundred and twenty isolates were cultured from 44 infected leg ulcers, in comparison with 110 isolates from 30 noninfected leg ulcers. Statistical analysis indicated a significantly greater mean number of anaerobic bacteria per infected ulcer (particularly Peptostreptococcus spp. and Prevotella spp.) in comparison with the noninfected ulcer group (2.5 vs. 1.3, respectively) (P < 0.05). Also, anaerobes represented 49% of the total microbial composition in infected leg ulcers compared with 36% in noninfected leg ulcers. The mean numbers of aerobes per wound in the two ulcer groups were not statistically different (P > 0.05). The study failed to demonstrate a clear correlation between commonly implicated facultative pathogens and wound infection. The isolation rate of Pseudomonas aeruginosa was generally low and, although Staphylococcus aureus was a frequent isolate in both wound types, it was more prevalent in noninfected leg ulcers. CONCLUSIONS: This study has demonstrated the complex aerobic-anaerobic microflora which exists in leg ulcers, the prevalence of anaerobes in infected wounds, and a poor correlation between the presence of specific aerobic pathogens and wound infection. In view of these findings, the role of microbial synergistic interactions in the pathogenesis of chronic wound infection may be of greater clinical importance than the isolated involvement of any specific potential pathogen.     

Fresh versus cryopreserved cultured allografts for the treatment of chronic skin ulcers

Both fresh and cryopreserved cultured epithelial allografts were used to treat 20 patients with a total of 30 chronic ulcers. A profound stimulation of host epithelialization from the wound edges and epidermal appendages was observed. Our data suggest that allografts, prepared using a simple cryopreservation technique, retain their morphological and functional characteristics, and are as effective as fresh allografts in healing chronic ulceration.     

Repeated cultured epidermal allografts in the treatment of chronic leg ulcers of various origins.

Twelve patients with 16 leg ulcers, existing for at least 3 months and not responsive to conventional inpatient therapy of at least 3 weeks, were treated with repeated applications of cultured allogenic keratinocyte sheets. A marked decrease in size was seen in all ulcers but 2. Complete closure of the ulcer was seen in 62% of the ulcers within 8 weeks. Healing was due to enhanced granulation and increased epithelialization, starting from the periphery of the wound. This edge effect suggests that the epidermal allografts act by stimulation of migration and/or multiplication of the acceptor's keratinocytes, rather than by take of the allograft.     

Local treatment of chronic cutaneous leg ulcers with recombinant human granulocyte-macrophage colony-stimulating factor

AIM: To test the efficacy and safety of recombinant granulocyte-macrophage colony-stimulating factor (rHuGM-CSF) in the treatment of chronic cutaneous leg ulcers. METHODS: Five patients with chronic cutaneous leg ulcers were recruited for this 4-month study using only rHuGM-CSF to treat the ulcers. One patient had a neuropathic-diabetic ulcer, and four had long-standing vascular leg ulcers. RESULTS: The patient with the neuropathic diabetic ulcer showed complete healing after 1 month of treatment. The other four patients with vascular leg ulcers with a long history of ulceration had a poor prognosis for healing. The first, with three venous ulcerative lesions, presented complete resolution of one ulcer and stabilization of the other two; the second and third patients, with large vascular ulcers, improved with more then 50% reduction of the mean diameter of the ulcers; the fourth patient, with one large venous ulcer, did not show any improvement. CONCLUSIONS: Pathogenesis, size and duration of the ulcers seemed to be the most important parameters regarding wound repairing capability of rHuGM-CSF. None of the ulcers increased in size and none of the patients developed clinical side-effects or peripheral blood cell count abnormalities during the treatment. All the results described were stable after 6 months of follow up. The absence of peripheral leucocyte count variation and the size-dependent therapeutic effect indicate that the drug exercises local rather than systemic actions.     

Risk factors associated with the failure of a venous leg ulcer to heal.

BACKGROUND: Venous leg ulcers afflict a significant portion of the population. The most popular form of therapy for venous leg ulcers is a compression bandage (eg, Unna boot), a therapy that is frequently unsuccessful. OBJECTIVE: To describe risk factors associated with the failure of a wound to heal when treated with a limb-compression bandage for 24 weeks. DESIGN: A retrospective cohort study. SETTING: Single-center outpatient specialty clinic at an academic medical center. PARTICIPANTS: Two hundred sixty consecutive patients with chronic venous leg ulcers. MAIN OUTCOME MEASURE: The magnitude of the effect of a given risk factor on the probability that a wound will heal within 24 weeks of care. RESULTS: Based on an assessment of leg wounds during initial office visits, we observed that the failure of a wound to heal within 24 weeks was significantly associated with larger wound area, measured in square centimeters (odds ratio [OR], 1.19; 95% confidence interval [CI], 1.11-1.27), duration of the wound in months (OR, 1.09; 95% CI, 1.04-1.16), history of venous ligation or venous stripping (OR, 4.58; 95% CI, 1.84-11.36), history of hip or knee replacement surgery (OR, 3.52; 95% CI, 1.12-11.08), ankle brachial index of less than 0.80 (OR, 3.52; 95% CI, 1.12-11.08), and the presence of fibrin on more than 50% of the wound surface (OR, 3.42; 95% CI, 1.38-8.45). CONCLUSIONS: Several risk factors are associated with the failure of a patient's venous leg ulcer to heal while using limb-compression therapy. It is prudent to consider these factors when referring a patient to a wound care subspecialists or for alternative therapies.     

Wound teleconsultation in patients with chronic leg ulcers

BACKGROUND: The treatment of chronic leg ulcers requires frequent assessments of local wound status and adjustment of therapy. The availability of reasonably priced photographic equipment and quick electronic transfer of high-quality digital images should make it possible that the assessment of wound status can be made by remote experts. OBJECTIVE: This study examines the feasibility of using teledermatology for wound assessment and therapeutic suggestions for patients with chronic leg ulcers. METHODS: One hundred ten chronic leg ulcers of different origins were examined in face-to-face consultations. The examining doctor assessed the wound, made therapeutic recommendations and took 1-4 photographs of the wound using a digital camera. The digital images and relevant clinical information were then transmitted via a web application to an expert in wound care, who provided an independent teledermatological assessment of wound status and therapeutic recommendations. RESULTS: In our study, a high accordance between direct consultations and electronic consultations was found in the assessment of chronic leg ulcers, especially for important features like slough (concordance: 84.6%), necrosis (concordance: 98.2%) and granulation tissue formation (concordance: 76.4%). Furthermore, the teledermatologist generally felt confident in recommending further treatment strategies and in planning further wound assessments via the internet. CONCLUSIONS: Our results suggest that teledermatology offers great potential for the future in chronic wound care. By reducing the need to travel long distances to the hospital or to consult a physician with expertise in wound care, wound teleconsultation might lower health care costs and improve the quality of life for patients with chronic wounds, while still maintaining a high quality of wound care.     

An allogeneic cultured dermal substitute suitable for treating intractable skin ulcers and large skin defects prior to autologous skin grafting: three case reports

Intractable skin ulcers that arise as secondary lesions from disease and full-thickness skin defects that result from skin tumor excision often need autologous skin grafting to close the wound. We developed an allogeneic cultured dermal substitute (CDS) to shorten the time needed to prepare a wound bed suitable for autologous skin grafting. The CDS was prepared by plating normal human fibroblasts on a spongy matrix consisting of hyaluronic acid and atelo-collagen. The allogeneic CDS was then placed on the rinsed wound surface. This procedure was repeated twice a week for up to five weeks, until the wounds were closed by autologous skin grafting. In all three cases, after CDS treatment for two to five weeks, the wound conditions became suitable for skin grafting; these conditions had not been improved by conventional topical treatments, including topical basic fibroblast growth factor (bFGF). Healthy granulation tissue developed rapidly, concomitant with wound size reduction. The present results indicate that CDS is an excellent biological wound dressing for improving wound conditions so that they are suitable for subsequent autologous skin grafting as well as for shortening the treatment duration for skin ulcers and full-thickness skin defects.     

Contact sensitization to benzoyl peroxide following topical treatment of chronic leg ulcers

Sixteen patients with chronic leg ulcers were treated with 10% benzoyl peroxide gel for 6 weeks. At the end of the treatment, patch tests with 2 % benzoyl peroxide in petrolatum and 10 % in a gel showed that nine patients (56 %.| had become sensitized. This high sensitization rate is in good accord with the results obtained by experimental investigations using human maximization tests and repeated insult patch tests. A strong sensitizer such as benzoyl peroxide should not be used in the routine treatment of chronic leg ulcers, despite its good healing effect.