Clinical Results Related to the Use of the TissueTech Autograft System in the Treatment of Diabetic Foot Ulceration

Introduction Chronic wounds, which can include leg ulcers, pressure ulcers, and diabetic foot ulcers, are a major health burden and represent an expensive drain on healthcare resources.[1] Diabetic foot ulcers in particular represent a recurrent and expensive problem in terms of morbidity, mortality, and healthcare costs.[2,3] In recent years, standards of care have been accepted for diabetic foot ulcer treatment,[4,5] which include restoration of adequate vascular supply, treatment of infection, and offloading.[6] However, despite an optimum standard of care, many factors can affect wound healing and time of healing in the diabetic foot, such as patient’s age, gender, type and duration of diabetes, and ulcer size and duration.[7–9] In the last few years, several new therapies have been developed in wound healing that take advantage of the advancements made in the understanding of wound physiology and the healing process.[10,11] Unique among these are treatments based on living skin equivalents.[12–27] These bioengineered tissues for replacement may be dermal, dermal and epidermal combinations, allograft, or autograft. It is thought that they exert their effects by providing an immediate skin coverage of the wound and by influencing the profile of growth factors and cytokines within the wound.[20] Autologous tissue replacements have the distinct advantage of immunological compatibility. Furthermore, in some cases the scaffold on which the cells are grown may exert a positive effect on wound healing, as seen with the use hyaluronan-based scaffolds.[21,24,25] The TissueTech® Autograft System (TTAS) (FIDIA Advanced Biopolymers, Abano Terme, Italy) comprises a autologous dermal substitute (ADS) (Hyalograft® 3D, FIDIA Advanced Biopolymers) made of a resorbable three-dimensional matrix derived from hyaluronic acid (MHA) (HYAFF®, FIDIA Advanced Polymers)[25,28] onto which autologous fibroblasts (laboratory grown) are seeded and expanded, and autologous epidermal replacement (AER) (Laserskin®, FIDIA Advanced Polymers), a transparent biodegradable microperforated membrane of the three-dimensional matrix containing preconfluent autologous keratinocytes, applied seven days after the application of fibroblasts.[21,22,25,29] The use of TTAS has shown positive results in the treatment of diabetic foot ulcers. An observational study on 60 patients with diabetic foot ulcers treated with ADS and AER showed a healing rate of 91.3 percent in a mean healing time of 72.7 ± 48.2 days without the occurrence of treatment-related side effects.[30] These findings have been recently confirmed by a randomized, controlled clinical trial where a 65-percent healing rate has been observed.[24] Other clinical experiences have shown the usefulness of the TTAS for the treatment of particularly difficult-to-heal diabetic ulcers.[22,31,32] The clinical experience gathered on the use of TTAS led to the development of a protocol of use of this system on the treatment of diabetic foot ulcers,[33] which defines the indications, timing of application, and overall ulcer management, both at a local and at a general level. Based on this experience, a large retrospective, observational investigation has been recently undertaken in order to gain extensive data on the characteristics and outcomes of the chronic ulcers treated with the TTAS in everyday clinical practice from January 1997 to December 2000.[34] In the present study, the authors present in detail data on the diabetic foot ulcers, comprising 401 wounds in 346 patients. Materials and Methods This large retrospective, observational survey included all chronic ulcers of the foot affecting diabetic patients treated with the TTAS in the participating clinics between January 1, 1997, and December 31, 2000. All physicians who treated patients with TTAS during this time frame were contacted and asked to voluntarily fill in a short case report form for collection of data of each foot ulcer treated based on information recorded in clinical records. Therefore, no preselection or screening process was used, and all treated patients identified with essential information available were entered into the database for final analysis. The following data were recorded: 1) patient demographics, including relevant pathology and concomitant treatments known to negatively affect the wound healing process (e.g., use of steroids or radiotherapy); 2) wound type defined, according to physician’s classification, as neuropathic, ischemic, neuroischemic, or post-surgical (i.e., wounds that required a more aggressive surgical debridement, including extensive removal of necrotic tissue or minor amputations of the foot); 3) wound location, area, depth, and duration at the time of the first TTAS application; 4) details of ADS and AER treatments (i.e., number of applications of each graft); 5) outcome of treatment in terms of healing (complete closure of the ulcer), time to healing, and recurrence; and 6) adverse events judged to be related to treatment. A clinical monitor who performed the data cleaning process reviewed the returned questionnaires and checked for and amended any missing or incongruent data as is standard procedure for prospective clinical trials. The data was entered in a database (CLINICS) resident on Digital UNIX V4.0F. This comprehensive data set was then subjected to statistical analysis as detailed next. Tissue-Tech Autograft System. Preparation of autologous dermal substitute (ADS) and autologous epidermal replacement (AER). Skin biopsies obtained from each patient were sent to the TTAS laboratories for autologous fibroblast and keratinocyte cell culturing according to standardized procedures. Briefly, skin specimens are enzymatically digested to separate epidermis from dermis. The fibroblasts and keratinocytes obtained are propagated for subsequent passaging after which the cells are seeded on the ADS (a MHA three-dimensional nonwoven scaffold) and AER (a MHA membrane with laser-drilled microperforations), respectively.[37] Once seeded, autologous fibroblasts adhere to the MHA fibers, proliferate, and begin the production of extracellular matrix components (Figure 1A). The presence of microperforations on the AER surface facilitates colonization of the membrane by keratinocytes and the subsequent cell proliferation and migration once grafted in the lesion area (Figure 1B). Seven days after seeding, the dermal grafts or the epidermal sheets are ready for transplantation (Figure 2A and 2B, respectively). Clinical application. A standard treatment is recommended for the application of the TTAS components to the ulcer bed. This includes an appropriate wound bed preparation with debridement of the necrotic tissue, hemostasis, and treatment of infection, if needed. ADS is applied directly on the clean, noninfected lesion to stimulate granulation tissue formation. Should a second graft be required, the wound is cleansed with saline solution and a second graft applied. AER is applied 7 to 10 days after ADS grafting following washing with saline solution. A second autologous keratinocyte graft can be performed if required. The application of both engineered tissues has to be performed with sterile forceps. A non-adherent dressing is subsequently applied in contact with the graft followed by application of a secondary dressing (sterile cotton gauze or semicompressive elastic bandage). Grafts and the nonadherent dressing must be left undisturbed for at least the first week after grafting. The secondary dressing may be changed after five days from graft application or earlier if the wound is heavily exuding. Correct patient management is necessary for TTAS, which includes offloading the ulcer in the case of neuropathic foot ulcers or treatment of peripheral vascular disease by revascularization procedures when arterial circulation is compromised. Statistical analysis. The data collected on special forms were entered into a database at the company on Digital UNIX V4.0F. Statistical analysis was performed with SAS statistical package (SAS Institute, Cary, NC, USA). The outcome of treatment was assessed by means of the following three key parameters: 1) number of healed cases (complete closure of the ulcer); 2) healing time; and 3) number of recurrences, both in the overall population and according to the type of ulcer. The parameters were described by frequency distribution. Comparisons among groups of ulcers with different diagnoses were performed by the chi-square test. For explanatory purposes, the predictability of some ulcer-related variables that might have affected the outcome of treatment was verified by logistic regression analysis. The three above outcome parameters were therefore considered in relation to the size, depth, and duration of the ulcer, considering the whole ulcer population and the sub-groups of ulcers with different diagnoses. Results Physicians from 60 different centers contributed to the project, describing 401 diabetic foot ulcers affecting 346 patients. The patients had a mean age of 64.4±12 years; the majority (81.9%) had type 2 diabetes. The age and gender distribution of these patients (Table 1) in the different ulcer sub-groups was found to correspond well with those of patients affected by diabetic foot ulcers as reported in literature,[7,17] and therefore, this treated patient population may be regarded as “typical” or representative for this kind of pathology. Most of the patients were older than 60 years. Nine patients were receiving concomitant therapy likely to adversely affect healing, particularly treatment with steroids. The baseline characteristics of the 401 ulcers treated and of all the ulcers in each category of diagnosis are summarized in Table 2. The ulcers were neuropathic, neuroischemic, and ischemic in a fairly equal proportion. Additionally, 58 ulcers were classified as post-surgical, as described in the Methods section. A large proportion (mean 44.7%) was greater than 15cm2. A sub-analysis of the data revealed that of the 179 total ulcers greater than 15cm2, the majority were greater than 30cm2 (65.4%). The groups of neuroischemic and ischemic ulcers included the largest proportion of lesions higher than 30cm2 (76.5 and 67.3%, respectively). It is of interest to note that a large proportion of the ulcers (85%) were full thickness or deep, and only a minority were superficial ulcers. The percentage of deep ulcers was particularly high in the neuroischemic and ischemic ulcer groups (more than 48%). This data, combined with that of ulcer area, accounts for the high severity of the ulcers belonging to these two groups, particularly the neuroischemic. All the 401 ulcers treated received at least one applications of autologous fibroblasts on ADS (mean 1.8 applications) and at least one application of AER (mean 1.3 applications). Table 3 shows the outcome of treatment of the ulcers stratified on the basis of their diagnosis. The 70.3 percent of the total ulcer population reached complete closure at a mean observation time of 330 days, with 63 percent of these healed within four months. There was an equal proportion of ulcers healed among the different groups, although the post-surgical ulcers healed in a slightly higher proportion. Moreover, the neuropathic ulcer group showed the highest percentage of ulcers healed in less than four months (76.1%). The recurrence rate observed was low (mean 8.2%). This value was 11.3 percent in the group of 62 healed ulcers followed up for over one year. No statistically significant differences emerged in the subgroup analysis among the ulcers with different diagnoses with regard to the three outcome parameters considered. The multivariate logistic regression analysis revealed that ulcer dimension greater than 5cm2 was the only factor negatively related to healing (Table 4). The same variable was found to be predictive of healing in more than four months (Table 5). Any of the ulcer characteristics considered was found to significantly affect the recurrence rate (Table 6). Interestingly, ulcer depth and duration were not negatively associated with any of the three outcomes of treatment. Analysis by subgroups confirmed the results obtained by the logistic regression analysis when considering the whole ulcer population. No adverse events possibly related to treatment were recorded by the physicians in any of the treated diabetic foot ulcers over the follow up. Discussion In this large retrospective study, the authors reported the characteristics and outcomes of diabetic foot ulcers treated with TTAS, as it is currently utilized in routine clinical practice. In the authors’ ulcer population, ulcers of different etiologies (neuropathic, neuroischemic, and ischemic) were equally distributed, while the post-surgical ones were fairly less. Despite the different etiologies, no difference in the healing rate, healing time, and recurrence rate was observed among the various ulcer sub-groups. Moreover, ulcer depth and duration did not affect the healing rate or the time of healing in the total population and in the different sub-groups. In the last few years, the use of modern dressing technology has paved the way to a more physiological approach to the repair process. In particular, allogeneic skin substitutes have been recently made available by tissue engineering techniques and shown to significantly reduce healing times in neuropathic diabetic foot ulcers. A dermal replacement (Dermagraft®, Smith & Nephew, Largo, Florida) has been studied in 281 patients with diabetic foot ulcers by Pollak, et al.,[13] in a randomized, controlled, clinical trial. A total of 50.8 percent of the dermal replacement-treated group exhibited complete wound healing at 12 weeks compared to 31.7 percent in the control group. Similarly, a living skin equivalent (Apligraf®, now distributed by PDI, Inc., Upper Saddle River, New Jersey) has been evaluated in 208 diabetic patients by Veves, et al.,[18] showing a healing rate of 56 percent at 12 weeks compared to 38 percent of the control patients. Previous studies with the TTAS on diabetic foot ulcers have shown healing rates comparable to the previously described tissue-engineered treatments. In a multicenter, randomized, controlled clinical trial, Caravaggi, et al.,[24] found a 65 percent healing rate at 11 weeks on a population of 79 ulcers. The clinical outcomes obtained in the present retrospective investigation, in terms of healing rates, are substantially higher than those achieved in “standard of care” studies11 and are comparable with those observed in the previous clinical trials with living skin equivalents.[13,18,24] However, this ulcer population is not the typical one enrolled in the published clinical trials on wound healing, since even neuroischemic and ischemic ulcers, i.e., ulcers without an adequate vascular circulation, were included and showed healing rate, healing time, and recurrence rate comparable to the nonischemic ulcers. This makes the results of this study even more promising. In this study, a low recurrence rate was observed compared to those reported in studies conducted with traditional treatments.[35,36] This finding suggests that the newly formed cutaneous tissue obtained following TTAS treatment is functionally valid. The authors believe that this may be related to the contribution of hyaluronic acid, a molecule known for its essential role in the processes of wound healing, which is released from the MHA.[29] This is further supported by the observation that the recurrence rate was not affected by baseline ulcer size and depth, which represent the amount of tissue replaced. Wound characteristics, in particular ulcer duration, have been proven to be prognostic for healing outcomes, as reported in previous studies.[7,11] In this study, an ulcer area above 5cm2 was the only predictive factor of faulty healing and of healing in more than four months. Interestingly, ulcer depth and duration did not affect either healing rate or time in the total ulcer population and in the different ulcer sub-groups. Therefore, it is possible to speculate that the use of the TTAS might have abolished the negative impact of ulcer depth and duration on the healing process. Further study will be necessary to confirm these findings. This retrospective study was intended to represent actual outcomes, as might be anticipated from routine clinical practice. The population of ulcers, as presented in the demographic baseline data, is a typical clinical cross-section, being distributed over a wide range of areas, depths, durations, and etiologies. Therefore, despite the limitation of not being randomized and controlled, this study has the advantage of considering foot ulcers with a wide spectrum of clinical characteristics without being restricted by more rigorous inclusion and exclusion criteria. The authors admit that many factors involved in this study might have influenced the results, not least the freedom to enroll or not. In this retrospective study, the authors believe that this was not the case, since the authors asked the physicians to enroll all diabetic foot ulcers that had been treated with the TTAS. Moreover, data came from 60 different centers, allowing the probability of a selection bias to be diluted. In this retrospective study on diabetic patients with chronic foot ulcers of different etiologies, the TTAS proved to be an effective and safe treatment and may be used as an integral part of a standard-of-care approach,[34] particularly for severe ulcers. *Study Contributors Expert panel. E. Faglia,(1) A. Bertani,(2) C. Caravaggi,(3) G. Clerici,(4) L. Dalla Paola,(5) D. Dioguardi,(6) M. Gargiulo,(7) G. Ghirlanda,(8) G. Maggio,(6) A. Motolese,(9) L. Pedrini,(10) A. Piaggesi,(11) E. Pisano,(10) L. Ricci,(12) F. Romagnoli,(13) A. Scalise,(2) A. Stella(7) 1)Unità Operativa di Medicina Interna, Policlinico Multimedia, Sesto S. Giovanni (MI) 2)Clinica di Chirurgia Plastica e Ricostruttiva, Università degli Studi, Ancona 3)Centro per lo Studio e la Cura del Piede Diabetico, Presidio Ospedaliero "C. Cantù", Abbiategrasso (MI) 4)Centro per la Prevenzione e la Cura del Piede Diabetico, ICRSS Fondazione "S. Maugeri", Pavia 5)Unità di Diabetologia, Casa di Cura, Abano Terme (PD) 6) Clinica di Chirurgia Plastica e Ricostruttiva, Università degli Studi, Bari 7)Cattedra di Chirurgia Vascolare, Università degli Studi di Modena e Reggio Emilia, Policlinico di Modena, Modena 8) Servizio di Diabetologia, Università Cattolica del Sacro Cuore, Policlinico "A. Gemelli", Roma 9) Unità Operativa di Dermatologia, Ospedale "A. Manzoni", Lecco 10) Unità Operativa di Chirurgia Vascolare, Ospedale Maggiore, Bologna 11) Unità Operativa di Malattie Metaboliche e Diabetologia, Azienda Ospedaliera Pisana, Pisa 12) Sezione di Diabetologia, Ospedale San Donato, Arezzo 13) Unità Operativa di Malattie Metaboliche e Diabetologia, INRCA, Ancona Other contributors. Addeo, MD (Bra, CN); Allochis, MD (Novara); Anichini, MD (Pistoia); Arzini, MD (Garbagnate, MI); Astolfi, Mrs (Torino); Barbieri, MD (Pavia); Benvegnù, BSc (Abano Terme, PD); Bellan, MD (Torino); Bertone, MD (Bergamo); Bidoli, MD (Treviso); Borrione, BSc (Abano Terme, PD); Bortolotti, MD (Lucca); Brauzzi, MD (Grosseto); Brunetti, MD (Perugia); Brustia, MD (Biella); Buniato, MD (Garbagnate, MI); Calandra, MD (Ravenna); Caminiti, MD (Sesto S.Giovanni, MI); Caselli, MD (Roma); Cianci, MD (Foggia); Colombo, MD (Garbagnate, MI); Costelli, MD (Parma); Cusaro, MD (Novara); D’ Agostino, MD (Milano); D’ Angelo, MD (Garbagnate, MI); Damiano, MD (Massacra, TA); De Angeli, MD (Biella); De Giglio, MD (Abbiategrasso, MI); De Luca, BSc, (Abano Terme, PD); Donelli, MD (Piacenza); Fanello, MD (Pavia); Fanti, MD (Bologna); Felici, MD (Scafati, SA); Floris, MD (Cagliari); Galassi, MD (Ferrara); Gibellini, MD (Pavia); Giurato, MD (Roma); Grispigni, MD (Milano); Grosso, MD (Rovigo); Iurlaro, MD (Francavilla, BR); Lonardi, MD (Modena); Magagnali, MD (Bologna); Malvicini, MD (Alessandria); Manara, MD (Cremona); Mantovani, MD (Grosseto); Manzoni, MD (Modena); Mariani, MD (Milano); Mascitelli, MD (Lucca); Mazzoleni, MD (Padova); Mingardi, MD (Vicenza); Miselli, MD (Scandiano, RE); Morisi, MD (Ravenna); Moroni, MD (Busto Arsizio, VA); Murgolo, MD (Cerignola, FG); Muscogiuri, MD (Francavilla, BR) Nasole, MD (Casalecchio di Reno, BO); Nigri, MD (Cerignola, FG); Palasciano, MD (Siena); Pavesio, BSc (Abano Terme, PD); Petrolati, MD (Legnano, MI); Pierangeli, MD (Ancona); Pigini, MD (Bologna); Piva, StSc (Abano Terme, PD); Pizzola, , MD (Fidenza PR); Pritelli, MD (Abbiategrasso, MI); Rheo, MD (Legnano, MI); Ricci, MD (Torino); Sciarrone, MD (Massacra, TA); Somaglino, MD (Alessandria); Soro, MD (Sassari); Spina, MD (Pelago, FI); Stocchiero, MD (Vicenza); Strazzabosco, MD (Vicenza); Tarantini, MD (Rimini); Targher, MD (Negrar, VR); Telleschi, MD (Lucca); Tiengo, MD (Padova); Torasso, BSc (Abano Terme, PD); Uccheddu, MD (Cagliari); Vermigli, MD (Perugia); Vezzani, MD (Fidenza, PR); Zappavigna, MD (Scandiano, RE); Zeccara, MD (Vigevano, PV); Zenari, MD (Negrar, VR); Zurrino, MD (Pavia)