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A Comparative Study of Cell Therapy and Fibrin Glue Applied to Chronic Venous Ulcers
Available online at www.sciencedirect.com
Procedia Engineering 59 (2013) 85 – 91
3rd International Conference on Tissue Engineering, ICTE2013
A comparative study of cell therapy and fibrin glue applied to chronic venous ulcers Jussara Rehder1, Carla Aparecida Faccio Bosnardo1,3, Maria Beatriz de Paula Leite Kraft1, Marco Andrey Cipriano Frade4,5, Ana Terezinha Guillaumon3, Fabiana Regina Xavier Batista6, Maria Beatriz Puzzi1,2* 1
Pediatrics’ Investigagion Center (CIPED), Faculty of Medical Sciences, University of Campinas, Tessália Vieira de Camargo street, Campinas CEP 13083-887, Brazil. 2 Clinical Medicine department’s discipline of Dermatology, Facuty of medical Sciences, University of Campinas, Tessália Vieira de Camargo street, Campinas CEP 13083-887, Brazil. 3 Surgery Department’s discipline of Vascular Surgery, Faculty of Medical Sciences, University of Campina , Tessália Vieira de Camargo street, Campinas CEP 13083-887, Brazil. 4 Division of Dermatology of Internal Medicine Department, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Bandeirantes Avenue, Ribeirão Preto, Brazil. 5 Neurovascular Ulcers Outpatient Clinic of Clinical Hospital of Ribeirão Preto,University of São Paulo, Bandeirantes Avenue, Ribeirão Preto, Brazil. 6 School of Chemical Engineering, Federal University of Uberlândia, João Naves de Ávila Avenue, Uberlândia, Brazil.
Abstract The hypothesis is that cell therapy with autologous keratinocytes promotes healing of chronic venous ulcers that failed to respond to other forms of treatment. This is a randomized study in which 31 patients with chronic venous ulcers participated, totaling 38 venous ulcers. Thirty one ulcers were included in Group 1 (G1) treated with autologous keratinocytes applied at the ulcers’ base with autologous fibrin scaffold. Group 2 (G2) included 7 ulcers treated with autologous fibrin glue only. The ulcers were evaluated with digital photography and computer image analysis [area and wound healing rate (WHR)] over 90 days. The duration of ulcers from the G1 was 118 months on average and in G2 the average was 114 months without any difference. In relation to the initial size of ulcers, both groups were similar considering the mean, SD and median respectively: G1 (18; 27; 5.6 cm2) and G2 (19.5; 0.11; 0.29 cm2). The medians of WHR in G1 and G2 have got significant differences between groups. Considering only the wounds that reached WHR larger than 0.4 (40% rate of healing), in the G1, 29% of ulcers reached it at
* Corresponding author. Tel.: +55-19-9285-9798; fax: +55-19-3521-8964. E-mail address: [email protected]
1877-7058 © 2013 The Authors. Published by Elsevier Ltd. Selection and peer-review under responsibility of the Centre for Rapid and Sustainable Product Development, Polytechnic Institute of Leiria, Centro Empresarial da Marinha Grande doi:10.1016/j.proeng.2013.05.097
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the 30th day, 58% at the 45th, 77% at the 60th, 84% at the 75th and 90% at the 90th day while in the G2 only 14% (one ulcer) reached it at the 30th day remaining steady until the 90th day when only one more ulcer reached this rate (28.6%). The median of procedures was 2 for G1 and 3 for G2. These findings showed the relevant efficacy of cell therapy with autologous keratinocytes applied with autologous fibrin glue scaffold achieving high rates of healing from the thirtieth day of follow-up and becoming larger by the ninetieth day. In contrast, the autologous fibrin glue applied lonely in G2 did not improve the wound healing significantly. The cell therapy with autologous keratinocytes applied with autologous fibrin scaffold promotes greater healing of chronic venous ulcers than autologous fibrin glue only, particularly large recalcitrant ulcers that do not respond to conventional treatment. © 2013 The Authors. Published by Elsevier Ltd. Selection and peer-review under responsibility of the Centre for Rapid and Sustainable Product Development, Polytechnic Institute of Leiria, Centro Empresarial da Marinha Grande. Keywords: Fibrin scaffold; Fibrin Glue; Keratinocytes; Cell therapy.
1. Introduction Leg ulcers are characterized by circumscribed or irregular loss of the epidermis of the lower limbs and possibly the dermis and subcutaneous tissue. It has many different pathogenic mechanisms and is frequently caused by diabetes mellitus, cardiac insufficiency and chronic venous hypertension complications. They can also be caused by autoimmune diseases and local infections. A total of 75% of lower limb ulcers are venous ulcers, which means that they are caused by a chronic venous insufficiency due to superficial and deep veins abnormal function [1]. According to Van Bemmelen et al. the presence of multiple refluxing foot vein segments is often associated with forefoot and toe ulceration [2]. Lower limb ulcers, secondary to chronic venous disease in European and Western populations, are estimated to be from 0,5 to 1% prevalent. In the United Kingdom, 1,3% of the health budget is spent on venous ulcers. Tassiopoulos et al. analysed thirteen studies that included 1249 ulcerated limb. Of all these ulcerated limbs, reflux was seen in 1153 limbs (92%) [3]. Frade et al. evaluated 124 patients with leg ulcers in Juiz de Fora (Minas Gerais – Brazil), analysing their clinical and epidemiological profile. The average age was 62,6 years old and 65,3% were female. The venous insufficiency, Systemic arterial hypertension and diabetes mellitus were associated with the leg ulcers in 74,2%, 51,6% and 20,2% of the cases, respectively. Therefore, 67,8% of the wounds were classified as venous ulcers. They were located mainly on the distal third segment of legs, 40% of them were considered as medium size (25cm) and 35,6% as large size (ulcers larger than 5cm) [4]. In this matter, venous ulcers are an important public health issue for many developed and developeding countries when taking in consideration the increase in life expectancy of the world’s population. With its increase, there will also be an increase of diseases associated with ulcers, such as chronic venous insufficiency, cardiac insufficiency and diabetes mellitus. Therefore, in the coming years, a higher incidence of venous ulcers is expected. This study tested the hypothesis that cell therapy with autologous keratinocytes applied with autologous fibrin glue promotes faster healing of chronic venous ulcers that failed to respond to other forms of treatment. 2. Materials and Methods This is a case-controlled randomized study. 2.1. Patients and ulcers A total of 31 patients with chronic venous ulcers were selected randomly from the Vascular Surgery Outpatient Clinic of the University of Campinas’ Clinical Hospital (Campinas, Brazil) and from the Ribeirão Preto’s Clinical Hospital’s Neurovascular Ulcers Outpatient Clinic (Ribeirão Preto, Brazil). These patients were selected considering the clinical aspects of venous damage diagnosed by surgeons and dermatologists. The treatments
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started after the patients signed the informed consent term, approved by the Ethical Committee of the University of Campinas’ Clinical Hospital (Protocol number 0076/2001). 2.2. Groups Thirty eight ulcers were selected for treatment and follow up. Thirty one of these ulcers were included in group one (G1) and were treated with autologous keratinocytes applied with autologous fibrin glue. The other seven ulcers were included in group 2 (G2) and were treated with autologous fibrin glue only. 2.3. Surgical procedures After local anesthesia, 1cm2 samples of skin from the inguinal region of the patients were taken in order to form keratinocytes culture. 2.4. Cell culture According to Rehder et al., the skin fragments were placed on Petri dishes with trypsin 2,5% and Ethylenediamine tetraacetic acid (EDTA) 0,1% (Trypsin EDTA – GIBCO cat. 25200-056) and then were kept in incubator at 37oC with 5% of CO2 tension for 3 hours, followed by dermal-epidermal separation. Trypsin was then neutralized with serum collected from the patient and the mixture was filtered. The filtrate contained the skin cells and was centrifuged at 1.200 rotations per minute (rpm) for 10 minutes. After cell count, the cells were distributed in culture bottles (1x105 cells/cm2) with keratinocyte culture medium supplemented with 10% of serum collected from the patient. The culture bottles were then incubated at 37oC with 5% CO2 tension. The culture medium was changed three times a week. When the cells reached confluence, cell passage occurred. After 21 days there was a quantity of 10x106 keratinocytes [5]. 2.5. Fibrin Glue The autologous fibrin glue was prepared according to the technique described by Moraes et al.; venous blood was collected from each patient and placed in individual tubes containing 3,8% of sodium citrate (9:1). The plasma was obtained after centrifugation for 10 minutes at 3.000 rpm. Plasma fibrinogen was purified through the glycine precipitation technique. Autologous fibrin, calcium chloride (40mM) and autologous thrombin (300 IU) were applied concomitantly with cell cultures to the lesions [6]. 2.6. Patient treatment The G1 ulcers were covered with 2 mL of the cultivated keratinocyte cells applied with autologous fibrin glue (10x106 cells/mL) regardless of the wounded area. The G2 ulcers were treated only with 2 mL of autologous fibrin glue. Both groups received a framework of hardened plastic and nylon cover to avoid contact between the wound bed and the outside environment. 2.7. Clinical follow-up by image The wounds were photographed every 15 days and analyzed by Image J software to calculate the wound area and the wound healing rate (WHR) over 90 days. WHR = (Ai-Af)/Ai Ai: Initial area; Af: Final area.
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3. Results The patients were 61,5 years old on average (Standard Deviation=14.1; Median=63 years old). 24 of all 31 patients were female and 7 were male, totaling 38 venous ulcers clinically characterized by specialists in vascular surgery and dermatology. The ulcers initial size was similar in both groups (p>0.05) and the average, the standard deviation (SD) and the median were 18; 27 and 5.6 cm2 respectively for G1. For G2, the average, the SD and the median were 19.5; 0.11 and 0.29 cm2, respectively (figure 1A) The duration of the ulcers in the G1 was 118 months on average (SD=144 months; Median= 72 months). The duration of ulcers in the G2 was 114 months on average (SD=85 months; Median=96 months). There was practically no difference (p>0.05) as shown in figure 1B.
Fig. 1. (a) Comparison between ulcers initial size in G1 and G2 before treatment; (b) Comparison between the duration of the ulcers in months in G1 and G2.
During the follow up the ulcers treated with autologous keratinocytes and fibrin glue (Group 1) quickly formed a thick crust, which covered the wound bed. The crust formation was followed by wound reepithelization and therefore the ulcer size was reduced. The ulcers in G2 also formed a thick crust, but no reepithelization was detected (figure 2).
Fig. 2. (a), (b), (c) and (d) G1’s patient follow up after treatment; (e), (f), (g) and (h) G2’s patient follow up after treatment.
The medians of WHR for G1 were 0.28; 0.43; 0.60; 0.76 and 0.87; while for G2 they were 0.05; 0.14; 0.18; 0.08 and 0.14 at the 30th, 45th, 60th, 75th and 90th follow up day, respectively with significant differences between groups in all days with p=0.01; p=0.003; p=0.0004; p=0.0004; p=0.0001, respectively (figure 3).
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Fig.3. G1 and G2’s WHR.
Considering the effective treatment when the wounds reach WHR larger than 0.4 (40% healing rate) in group one 29% of the ulcers reached it at the 30th day, 58% at the 45th , 77% at the 60th, 84% at the 75th and 90% at the 90th day. In group 2 only 14% (one ulcer) reached it at the 30th day maintaining this WHR until the 75th day when only one more ulcer reached this rate. At the 90th day 14% had 40% of healing rate. The median of procedures was 2 for G1 and 3 for G2 (figure 4).
Fig. 4. Percentage of ulcers that reached at least 40% of wound healing rate.
4. Discussion Nowadays, the cell therapy has shown an attractive alternative to treat skin wounds such as chronic venous ulcers. This form of therapy has been used to create skin substitutes [5, 7], aid wound healing, treat burns, among other purposes [8]. This technique could be used with autologous cells, which are cultured and expanded in vitro outside the body, and reused into the damaged place. The use of autologous cells may provide the minimization of risks from systemic immunological reactions, as well as disease transmission associated with grafts or cells that are not cultivated from the individual. To ensure the complete cell adhesion, autologous fibrin glue is commonly used. The development of tissue adhesives and sealants is a rapidly expanding area of clinical use. On the whole, the fibrin was recognized as the scaffold for tissue regeneration and the use of fibrin tampons was proposed to accelerate local hemostasis. As discussed by Valbonesi, tissue injury can cause the disruption of blood vessels that is responsible for extravasation of blood constituents and the first step of the process in the platelet activation after exposure of collagen. Platelets initiate clotting through the coagulation system. When thrombin is formed, fibrinogen is transformed into fibrin. This can be characterized as the real first step of wound healing. In addition, the fibrin glue reproduces this process and has been widely used in surgery to obtain hemostasis and expedite the process of wound healing [9].
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Our findings showed that the combination of the autologous keratinocytes with the autologous fibrin glue resulted in faster wound healing. This might have happened because of the increase of growth factors delivered by the implanted keratinocytes and its subsequent interactions with other cells, such as the dermal fibroblasts. It is known that keratinocytes produce Interleukin 1 (IL-1), Vascular Endothelial growth factors (VEGF), plateletderived growth factor (PDGF) and beta transforming growth factor (TGF- ), among others [10]. The upper levels of IL-1, combined with the environment’s tumor necrosis factor-alpha (TNF- ) are biomarkers of the inflammatory phase of the wound healing. They stimulate even more the local endothelial cells to increase the expression of cell adhesion molecules (CAMs). In this way, several blood cells migrate through the endothelium and reach the wound area. These cells include neutrophils and monocytes. The monocytes differentiate onto macrophages, which release other growth factors that help to induce the beginning of the proliferative phase. Besides that, it is known that IL-1 induces fibroblasts to produce and secrete growth factors such as keratinocyte growth factor (KGF), fibroblast growth factor 7 (FGF7) and interleukin 6 (IL-6) [10, 11, 12]. In this regard, the application of the autologous keratinocytes with autologous fibrin glue might have accelerated the healing process. The other growth factors secreted by the keratinocytes could also be involved in the better wound healing showed in this study. The VEGF induces angiogenesis, which is responsible for the formation of the granulation tissue, enhancing the new entered proliferative phase [10, 11]. The application of autologous keratinocytes might have increased the levels of VEGF, which could have altered positively the number of neo-synthetized vasculature vases. This could also be involved in the greater healing of the chronic venous ulcers in G1. In the proliferative phase, the PDGF also plays an important role. Right after the tissue injury the levels of PDGF increase because of the platelets degranulation. Later it is locally synthetized, and keratinocytes may be involved in this production, once these cells do produce PDGF and once it can be detected in the epithelium. According to Eming et al, the overexpression of PDGF-A promotes a faster fibro-vascular ingrowth and also cause deposition of mouse collagen type I. This probably happened because the collagen type I is the most abundant collagen fiber of the connective tissue [12, 13]. Another important growth factor secreted by keratinocytes is the TGF- . It is related to the cellular proliferation and migration, especially in wound healing. Studies show that in the first moment of the healing process the keratinocytes of the wound limits migrate towards it, reaching its center, while the fibroblasts do not proliferate nor migrate at this period. The migration of keratinocytes happens within hours, while the migration of fibroblasts do not happen until days after the injury has happened. Among other factors, this delay is also due to the different actions of the TGF- in these cells: It is known that TGF- inhibits proliferation and migration of fibroblast and the proliferation keratinocytes only. Co-cultured keratinocytes and fibroblasts show an overexpression of TGF- . The wounds in G1 could have had their TGF- levels higher than the wounds in G2, which is very likely to have enhanced its healing process [12, 14, 15]. Lis et al (2012) studied the use of cultured keratinocytes suspension in fibrin glue on the healing of oral mucosa wounds surgically produced using a rabbit model. Those authors concluded that the suspension of cultured autologous oral keratinocytes in fibrin glue significantly accelerates oral wound healing in the rabbit model and could be beneficial in the treatment of oral wounds in patients. According to these researchers, this data showed the direct effect of cells and their growth factors spread on the ulcer bottoms activating the indolent wounds and stimulating their healing [16]. Myers remarked that the keratinocyte-derived factors are essential to various stages of wound healing, such as inflammation, cell migration, matrix deposition, and maturation. This action can be explained taking into consideration the cytokines produced by these cells. In addition, considering the high morbidity of chronic venous ulcers in both groups, the results showed the relevant efficacy of cell therapy with autologous keratinocytes applied with fibrin glue scaffold achieving high rates of healing since the thirtieth day of follow-up becoming higher until the ninetieth day. In contrast, the fibrin glue alone applied in G2 did not improve the wound healing significantly, even receiving the greatest number of procedures [17].
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5. Conclusion Cell therapy is efficient in the treatment of chronic venous ulcers that do not respond to usual treatments. Autologous keratinocytes applied with autologous fibrin glue promotes greater healing of chronic venous ulcers than fibrin glue only, especially in ulcers that do not respond to conventional treatment. References [1] Augustin, M. Vanscheidt, W. 2012. Chronic venous leg ulcers: the future of cell-based therapies. Lancet. 2012 Sep 15;380(9846):953-5. Doi: 10.1016/S0140-6736(12)61255-0. Epub 2012 Aug 3. [2] Van Bemmele, PS. Spivack, D. Kelly, P. 2012. Reflux in foot veins is associated with venous toe and forefoot ulcerations. J Vasc Surg. 2011 Feb;53(2):394-8. Doi 10.1016/j.jvs.2010.08.079. Epub 2010 Nov 3. [3] Tassiopoulos, AK. Golts, E. Oh, DS. Labropoulos, N. 2000. Current concepts in chronic venous ulceration. Eur J Endovasc Surf. 2000 Sep;20(3):227-32. [4] Frade, MAC. et al. 2005. Úlcera de perna: um estudo de casos de Juiz de For a – MG (Brasil) e região. Anais Brasileiros de Dermatologia, V.80, N.1, Pág. 41-46. 2005. [5] Rehder, J. Souto, LR. Issa, CMBM. Puzzi, MB. 2004. Modelo of human epidermis reconstructed in vitro with keratinocytes and melanocytes on dead de-epidermized human dermir. São Paulo Med J. 122: 22-25. 2004. [6] Moraes, AM. Annichino-bizzacchi, JM. Rossi, AB. 1998. Use of autologous fibrin glue in dermatologic surgery: application of skin graft and second intention healing. São Paulo Med J. 1998 Jul-Aug;116(4):1747-52. [7] Souto, LR. Vassallo, J. Rehder, J. Pinto, GA. Puzzi, MB. 2009. Immunoarchitectural characterization of a human skin model reconstructed in vitro. Sao Paulo Med J 127: 28-33. [8] Dinato, M. Puzzi, MB. Rehder, J. Batista, FRX. 2012. Tissue therapy with autologous dermal and epidermal culture cells for diabetic foot ulcers. Cell Tissue Bank (2012) 13:241–249. [9] Valbonesi, M. 2006. Fibrin glues of human origin. Best Practice & Research Clinical Haematology, Vol. 19, No. 1, pp. 191–203, 2006. [10] Isaac, C. Ladeira, PRS. Rêgo, FMP. Aldunate, JCB. Ferreira, MC. 2010. Processo de cura das feridas: cicatrização fisiológica. Rev Med (São Paulo). 2010 jul-dez.; 89(3/4): 125-31. [11] Shah, JMY. Omar, E. Pai, DR. Sood, S. 2012. Cellular events and biomarkers of wound healing. Indian J Plast Surg. 2012 May-Aug; 45(2): 220-228. [12] Werner, S. Krieg, T. Smola, H. 2007. Keratinocyte-Fibroblast Interactions in Wound Healing. Journal of Investigative Dermatology (2007) 127, 998-1008. [13] Eming, SA. Medalie, DA. 1998. Genetically modified human keratinocytes overexpressimg PDGF-A enhance the performance of a composite skin graft. Hum Gene Ther; 9(4):529-39,1998. [14] Han, A. Bandyopadhyay, B. Jayaprakash, P. Lua, I. Sahu, D. Chen, M. Woodley, D. Li, W. 2012. The anti-mobility signaling mechanisms of TGF- 3 that controls cell traffic during skin wound healing. Biol Open, 2012, December 15; 1(12): 1169-1177. [15] Singer, AJ. Clark, RAF. 1999. Cutaneous wound healing. New Engl J. Med. 341, 738-746 [16] Lis, GJ. Zarzecka, J. Litwin, JA. Jasek, E. Cichocki, T. Zapa a, J. 2012. Effect of cultured autologous oral keratinocyte suspension in fibrin glue on oral wound healing in rabbits. Int. J. Oral Maxillofac. Surg. 2012; 41: 1146–1152 [17] Myers, S. Navsaria, H. Sanders, R. Green, C. Leigh, I. 1995. Transplantation of keratinocytes in the treatment of wounds. The American Journal of Surgery, Volume 170, Issue 1, July 1995, Pages 75-83.
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3rd International Conference on Tissue Engineering, ICTE2013
A comparative study of cell therapy and fibrin glue applied to chronic venous ulcers Jussara Rehder1, Carla Aparecida Faccio Bosnardo1,3, Maria Beatriz de Paula Leite Kraft1, Marco Andrey Cipriano Frade4,5, Ana Terezinha Guillaumon3, Fabiana Regina Xavier Batista6, Maria Beatriz Puzzi1,2* 1
Pediatrics’ Investigagion Center (CIPED), Faculty of Medical Sciences, University of Campinas, Tessália Vieira de Camargo street, Campinas CEP 13083-887, Brazil. 2 Clinical Medicine department’s discipline of Dermatology, Facuty of medical Sciences, University of Campinas, Tessália Vieira de Camargo street, Campinas CEP 13083-887, Brazil. 3 Surgery Department’s discipline of Vascular Surgery, Faculty of Medical Sciences, University of Campina , Tessália Vieira de Camargo street, Campinas CEP 13083-887, Brazil. 4 Division of Dermatology of Internal Medicine Department, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Bandeirantes Avenue, Ribeirão Preto, Brazil. 5 Neurovascular Ulcers Outpatient Clinic of Clinical Hospital of Ribeirão Preto,University of São Paulo, Bandeirantes Avenue, Ribeirão Preto, Brazil. 6 School of Chemical Engineering, Federal University of Uberlândia, João Naves de Ávila Avenue, Uberlândia, Brazil.
Abstract The hypothesis is that cell therapy with autologous keratinocytes promotes healing of chronic venous ulcers that failed to respond to other forms of treatment. This is a randomized study in which 31 patients with chronic venous ulcers participated, totaling 38 venous ulcers. Thirty one ulcers were included in Group 1 (G1) treated with autologous keratinocytes applied at the ulcers’ base with autologous fibrin scaffold. Group 2 (G2) included 7 ulcers treated with autologous fibrin glue only. The ulcers were evaluated with digital photography and computer image analysis [area and wound healing rate (WHR)] over 90 days. The duration of ulcers from the G1 was 118 months on average and in G2 the average was 114 months without any difference. In relation to the initial size of ulcers, both groups were similar considering the mean, SD and median respectively: G1 (18; 27; 5.6 cm2) and G2 (19.5; 0.11; 0.29 cm2). The medians of WHR in G1 and G2 have got significant differences between groups. Considering only the wounds that reached WHR larger than 0.4 (40% rate of healing), in the G1, 29% of ulcers reached it at
* Corresponding author. Tel.: +55-19-9285-9798; fax: +55-19-3521-8964. E-mail address: [email protected]
1877-7058 © 2013 The Authors. Published by Elsevier Ltd. Selection and peer-review under responsibility of the Centre for Rapid and Sustainable Product Development, Polytechnic Institute of Leiria, Centro Empresarial da Marinha Grande doi:10.1016/j.proeng.2013.05.097
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the 30th day, 58% at the 45th, 77% at the 60th, 84% at the 75th and 90% at the 90th day while in the G2 only 14% (one ulcer) reached it at the 30th day remaining steady until the 90th day when only one more ulcer reached this rate (28.6%). The median of procedures was 2 for G1 and 3 for G2. These findings showed the relevant efficacy of cell therapy with autologous keratinocytes applied with autologous fibrin glue scaffold achieving high rates of healing from the thirtieth day of follow-up and becoming larger by the ninetieth day. In contrast, the autologous fibrin glue applied lonely in G2 did not improve the wound healing significantly. The cell therapy with autologous keratinocytes applied with autologous fibrin scaffold promotes greater healing of chronic venous ulcers than autologous fibrin glue only, particularly large recalcitrant ulcers that do not respond to conventional treatment. © 2013 The Authors. Published by Elsevier Ltd. Selection and peer-review under responsibility of the Centre for Rapid and Sustainable Product Development, Polytechnic Institute of Leiria, Centro Empresarial da Marinha Grande. Keywords: Fibrin scaffold; Fibrin Glue; Keratinocytes; Cell therapy.
1. Introduction Leg ulcers are characterized by circumscribed or irregular loss of the epidermis of the lower limbs and possibly the dermis and subcutaneous tissue. It has many different pathogenic mechanisms and is frequently caused by diabetes mellitus, cardiac insufficiency and chronic venous hypertension complications. They can also be caused by autoimmune diseases and local infections. A total of 75% of lower limb ulcers are venous ulcers, which means that they are caused by a chronic venous insufficiency due to superficial and deep veins abnormal function [1]. According to Van Bemmelen et al. the presence of multiple refluxing foot vein segments is often associated with forefoot and toe ulceration [2]. Lower limb ulcers, secondary to chronic venous disease in European and Western populations, are estimated to be from 0,5 to 1% prevalent. In the United Kingdom, 1,3% of the health budget is spent on venous ulcers. Tassiopoulos et al. analysed thirteen studies that included 1249 ulcerated limb. Of all these ulcerated limbs, reflux was seen in 1153 limbs (92%) [3]. Frade et al. evaluated 124 patients with leg ulcers in Juiz de Fora (Minas Gerais – Brazil), analysing their clinical and epidemiological profile. The average age was 62,6 years old and 65,3% were female. The venous insufficiency, Systemic arterial hypertension and diabetes mellitus were associated with the leg ulcers in 74,2%, 51,6% and 20,2% of the cases, respectively. Therefore, 67,8% of the wounds were classified as venous ulcers. They were located mainly on the distal third segment of legs, 40% of them were considered as medium size (25cm) and 35,6% as large size (ulcers larger than 5cm) [4]. In this matter, venous ulcers are an important public health issue for many developed and developeding countries when taking in consideration the increase in life expectancy of the world’s population. With its increase, there will also be an increase of diseases associated with ulcers, such as chronic venous insufficiency, cardiac insufficiency and diabetes mellitus. Therefore, in the coming years, a higher incidence of venous ulcers is expected. This study tested the hypothesis that cell therapy with autologous keratinocytes applied with autologous fibrin glue promotes faster healing of chronic venous ulcers that failed to respond to other forms of treatment. 2. Materials and Methods This is a case-controlled randomized study. 2.1. Patients and ulcers A total of 31 patients with chronic venous ulcers were selected randomly from the Vascular Surgery Outpatient Clinic of the University of Campinas’ Clinical Hospital (Campinas, Brazil) and from the Ribeirão Preto’s Clinical Hospital’s Neurovascular Ulcers Outpatient Clinic (Ribeirão Preto, Brazil). These patients were selected considering the clinical aspects of venous damage diagnosed by surgeons and dermatologists. The treatments
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started after the patients signed the informed consent term, approved by the Ethical Committee of the University of Campinas’ Clinical Hospital (Protocol number 0076/2001). 2.2. Groups Thirty eight ulcers were selected for treatment and follow up. Thirty one of these ulcers were included in group one (G1) and were treated with autologous keratinocytes applied with autologous fibrin glue. The other seven ulcers were included in group 2 (G2) and were treated with autologous fibrin glue only. 2.3. Surgical procedures After local anesthesia, 1cm2 samples of skin from the inguinal region of the patients were taken in order to form keratinocytes culture. 2.4. Cell culture According to Rehder et al., the skin fragments were placed on Petri dishes with trypsin 2,5% and Ethylenediamine tetraacetic acid (EDTA) 0,1% (Trypsin EDTA – GIBCO cat. 25200-056) and then were kept in incubator at 37oC with 5% of CO2 tension for 3 hours, followed by dermal-epidermal separation. Trypsin was then neutralized with serum collected from the patient and the mixture was filtered. The filtrate contained the skin cells and was centrifuged at 1.200 rotations per minute (rpm) for 10 minutes. After cell count, the cells were distributed in culture bottles (1x105 cells/cm2) with keratinocyte culture medium supplemented with 10% of serum collected from the patient. The culture bottles were then incubated at 37oC with 5% CO2 tension. The culture medium was changed three times a week. When the cells reached confluence, cell passage occurred. After 21 days there was a quantity of 10x106 keratinocytes [5]. 2.5. Fibrin Glue The autologous fibrin glue was prepared according to the technique described by Moraes et al.; venous blood was collected from each patient and placed in individual tubes containing 3,8% of sodium citrate (9:1). The plasma was obtained after centrifugation for 10 minutes at 3.000 rpm. Plasma fibrinogen was purified through the glycine precipitation technique. Autologous fibrin, calcium chloride (40mM) and autologous thrombin (300 IU) were applied concomitantly with cell cultures to the lesions [6]. 2.6. Patient treatment The G1 ulcers were covered with 2 mL of the cultivated keratinocyte cells applied with autologous fibrin glue (10x106 cells/mL) regardless of the wounded area. The G2 ulcers were treated only with 2 mL of autologous fibrin glue. Both groups received a framework of hardened plastic and nylon cover to avoid contact between the wound bed and the outside environment. 2.7. Clinical follow-up by image The wounds were photographed every 15 days and analyzed by Image J software to calculate the wound area and the wound healing rate (WHR) over 90 days. WHR = (Ai-Af)/Ai Ai: Initial area; Af: Final area.
(1)
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3. Results The patients were 61,5 years old on average (Standard Deviation=14.1; Median=63 years old). 24 of all 31 patients were female and 7 were male, totaling 38 venous ulcers clinically characterized by specialists in vascular surgery and dermatology. The ulcers initial size was similar in both groups (p>0.05) and the average, the standard deviation (SD) and the median were 18; 27 and 5.6 cm2 respectively for G1. For G2, the average, the SD and the median were 19.5; 0.11 and 0.29 cm2, respectively (figure 1A) The duration of the ulcers in the G1 was 118 months on average (SD=144 months; Median= 72 months). The duration of ulcers in the G2 was 114 months on average (SD=85 months; Median=96 months). There was practically no difference (p>0.05) as shown in figure 1B.
Fig. 1. (a) Comparison between ulcers initial size in G1 and G2 before treatment; (b) Comparison between the duration of the ulcers in months in G1 and G2.
During the follow up the ulcers treated with autologous keratinocytes and fibrin glue (Group 1) quickly formed a thick crust, which covered the wound bed. The crust formation was followed by wound reepithelization and therefore the ulcer size was reduced. The ulcers in G2 also formed a thick crust, but no reepithelization was detected (figure 2).
Fig. 2. (a), (b), (c) and (d) G1’s patient follow up after treatment; (e), (f), (g) and (h) G2’s patient follow up after treatment.
The medians of WHR for G1 were 0.28; 0.43; 0.60; 0.76 and 0.87; while for G2 they were 0.05; 0.14; 0.18; 0.08 and 0.14 at the 30th, 45th, 60th, 75th and 90th follow up day, respectively with significant differences between groups in all days with p=0.01; p=0.003; p=0.0004; p=0.0004; p=0.0001, respectively (figure 3).
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Fig.3. G1 and G2’s WHR.
Considering the effective treatment when the wounds reach WHR larger than 0.4 (40% healing rate) in group one 29% of the ulcers reached it at the 30th day, 58% at the 45th , 77% at the 60th, 84% at the 75th and 90% at the 90th day. In group 2 only 14% (one ulcer) reached it at the 30th day maintaining this WHR until the 75th day when only one more ulcer reached this rate. At the 90th day 14% had 40% of healing rate. The median of procedures was 2 for G1 and 3 for G2 (figure 4).
Fig. 4. Percentage of ulcers that reached at least 40% of wound healing rate.
4. Discussion Nowadays, the cell therapy has shown an attractive alternative to treat skin wounds such as chronic venous ulcers. This form of therapy has been used to create skin substitutes [5, 7], aid wound healing, treat burns, among other purposes [8]. This technique could be used with autologous cells, which are cultured and expanded in vitro outside the body, and reused into the damaged place. The use of autologous cells may provide the minimization of risks from systemic immunological reactions, as well as disease transmission associated with grafts or cells that are not cultivated from the individual. To ensure the complete cell adhesion, autologous fibrin glue is commonly used. The development of tissue adhesives and sealants is a rapidly expanding area of clinical use. On the whole, the fibrin was recognized as the scaffold for tissue regeneration and the use of fibrin tampons was proposed to accelerate local hemostasis. As discussed by Valbonesi, tissue injury can cause the disruption of blood vessels that is responsible for extravasation of blood constituents and the first step of the process in the platelet activation after exposure of collagen. Platelets initiate clotting through the coagulation system. When thrombin is formed, fibrinogen is transformed into fibrin. This can be characterized as the real first step of wound healing. In addition, the fibrin glue reproduces this process and has been widely used in surgery to obtain hemostasis and expedite the process of wound healing [9].
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Our findings showed that the combination of the autologous keratinocytes with the autologous fibrin glue resulted in faster wound healing. This might have happened because of the increase of growth factors delivered by the implanted keratinocytes and its subsequent interactions with other cells, such as the dermal fibroblasts. It is known that keratinocytes produce Interleukin 1 (IL-1), Vascular Endothelial growth factors (VEGF), plateletderived growth factor (PDGF) and beta transforming growth factor (TGF- ), among others [10]. The upper levels of IL-1, combined with the environment’s tumor necrosis factor-alpha (TNF- ) are biomarkers of the inflammatory phase of the wound healing. They stimulate even more the local endothelial cells to increase the expression of cell adhesion molecules (CAMs). In this way, several blood cells migrate through the endothelium and reach the wound area. These cells include neutrophils and monocytes. The monocytes differentiate onto macrophages, which release other growth factors that help to induce the beginning of the proliferative phase. Besides that, it is known that IL-1 induces fibroblasts to produce and secrete growth factors such as keratinocyte growth factor (KGF), fibroblast growth factor 7 (FGF7) and interleukin 6 (IL-6) [10, 11, 12]. In this regard, the application of the autologous keratinocytes with autologous fibrin glue might have accelerated the healing process. The other growth factors secreted by the keratinocytes could also be involved in the better wound healing showed in this study. The VEGF induces angiogenesis, which is responsible for the formation of the granulation tissue, enhancing the new entered proliferative phase [10, 11]. The application of autologous keratinocytes might have increased the levels of VEGF, which could have altered positively the number of neo-synthetized vasculature vases. This could also be involved in the greater healing of the chronic venous ulcers in G1. In the proliferative phase, the PDGF also plays an important role. Right after the tissue injury the levels of PDGF increase because of the platelets degranulation. Later it is locally synthetized, and keratinocytes may be involved in this production, once these cells do produce PDGF and once it can be detected in the epithelium. According to Eming et al, the overexpression of PDGF-A promotes a faster fibro-vascular ingrowth and also cause deposition of mouse collagen type I. This probably happened because the collagen type I is the most abundant collagen fiber of the connective tissue [12, 13]. Another important growth factor secreted by keratinocytes is the TGF- . It is related to the cellular proliferation and migration, especially in wound healing. Studies show that in the first moment of the healing process the keratinocytes of the wound limits migrate towards it, reaching its center, while the fibroblasts do not proliferate nor migrate at this period. The migration of keratinocytes happens within hours, while the migration of fibroblasts do not happen until days after the injury has happened. Among other factors, this delay is also due to the different actions of the TGF- in these cells: It is known that TGF- inhibits proliferation and migration of fibroblast and the proliferation keratinocytes only. Co-cultured keratinocytes and fibroblasts show an overexpression of TGF- . The wounds in G1 could have had their TGF- levels higher than the wounds in G2, which is very likely to have enhanced its healing process [12, 14, 15]. Lis et al (2012) studied the use of cultured keratinocytes suspension in fibrin glue on the healing of oral mucosa wounds surgically produced using a rabbit model. Those authors concluded that the suspension of cultured autologous oral keratinocytes in fibrin glue significantly accelerates oral wound healing in the rabbit model and could be beneficial in the treatment of oral wounds in patients. According to these researchers, this data showed the direct effect of cells and their growth factors spread on the ulcer bottoms activating the indolent wounds and stimulating their healing [16]. Myers remarked that the keratinocyte-derived factors are essential to various stages of wound healing, such as inflammation, cell migration, matrix deposition, and maturation. This action can be explained taking into consideration the cytokines produced by these cells. In addition, considering the high morbidity of chronic venous ulcers in both groups, the results showed the relevant efficacy of cell therapy with autologous keratinocytes applied with fibrin glue scaffold achieving high rates of healing since the thirtieth day of follow-up becoming higher until the ninetieth day. In contrast, the fibrin glue alone applied in G2 did not improve the wound healing significantly, even receiving the greatest number of procedures [17].
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5. Conclusion Cell therapy is efficient in the treatment of chronic venous ulcers that do not respond to usual treatments. Autologous keratinocytes applied with autologous fibrin glue promotes greater healing of chronic venous ulcers than fibrin glue only, especially in ulcers that do not respond to conventional treatment. References [1] Augustin, M. Vanscheidt, W. 2012. Chronic venous leg ulcers: the future of cell-based therapies. Lancet. 2012 Sep 15;380(9846):953-5. Doi: 10.1016/S0140-6736(12)61255-0. Epub 2012 Aug 3. [2] Van Bemmele, PS. Spivack, D. Kelly, P. 2012. Reflux in foot veins is associated with venous toe and forefoot ulcerations. J Vasc Surg. 2011 Feb;53(2):394-8. Doi 10.1016/j.jvs.2010.08.079. Epub 2010 Nov 3. [3] Tassiopoulos, AK. Golts, E. Oh, DS. Labropoulos, N. 2000. Current concepts in chronic venous ulceration. Eur J Endovasc Surf. 2000 Sep;20(3):227-32. [4] Frade, MAC. et al. 2005. Úlcera de perna: um estudo de casos de Juiz de For a – MG (Brasil) e região. Anais Brasileiros de Dermatologia, V.80, N.1, Pág. 41-46. 2005. [5] Rehder, J. Souto, LR. Issa, CMBM. Puzzi, MB. 2004. Modelo of human epidermis reconstructed in vitro with keratinocytes and melanocytes on dead de-epidermized human dermir. São Paulo Med J. 122: 22-25. 2004. [6] Moraes, AM. Annichino-bizzacchi, JM. Rossi, AB. 1998. Use of autologous fibrin glue in dermatologic surgery: application of skin graft and second intention healing. São Paulo Med J. 1998 Jul-Aug;116(4):1747-52. [7] Souto, LR. Vassallo, J. Rehder, J. Pinto, GA. Puzzi, MB. 2009. Immunoarchitectural characterization of a human skin model reconstructed in vitro. Sao Paulo Med J 127: 28-33. [8] Dinato, M. Puzzi, MB. Rehder, J. Batista, FRX. 2012. Tissue therapy with autologous dermal and epidermal culture cells for diabetic foot ulcers. Cell Tissue Bank (2012) 13:241–249. [9] Valbonesi, M. 2006. Fibrin glues of human origin. Best Practice & Research Clinical Haematology, Vol. 19, No. 1, pp. 191–203, 2006. [10] Isaac, C. Ladeira, PRS. Rêgo, FMP. Aldunate, JCB. Ferreira, MC. 2010. Processo de cura das feridas: cicatrização fisiológica. Rev Med (São Paulo). 2010 jul-dez.; 89(3/4): 125-31. [11] Shah, JMY. Omar, E. Pai, DR. Sood, S. 2012. Cellular events and biomarkers of wound healing. Indian J Plast Surg. 2012 May-Aug; 45(2): 220-228. [12] Werner, S. Krieg, T. Smola, H. 2007. Keratinocyte-Fibroblast Interactions in Wound Healing. Journal of Investigative Dermatology (2007) 127, 998-1008. [13] Eming, SA. Medalie, DA. 1998. Genetically modified human keratinocytes overexpressimg PDGF-A enhance the performance of a composite skin graft. Hum Gene Ther; 9(4):529-39,1998. [14] Han, A. Bandyopadhyay, B. Jayaprakash, P. Lua, I. Sahu, D. Chen, M. Woodley, D. Li, W. 2012. The anti-mobility signaling mechanisms of TGF- 3 that controls cell traffic during skin wound healing. Biol Open, 2012, December 15; 1(12): 1169-1177. [15] Singer, AJ. Clark, RAF. 1999. Cutaneous wound healing. New Engl J. Med. 341, 738-746 [16] Lis, GJ. Zarzecka, J. Litwin, JA. Jasek, E. Cichocki, T. Zapa a, J. 2012. Effect of cultured autologous oral keratinocyte suspension in fibrin glue on oral wound healing in rabbits. Int. J. Oral Maxillofac. Surg. 2012; 41: 1146–1152 [17] Myers, S. Navsaria, H. Sanders, R. Green, C. Leigh, I. 1995. Transplantation of keratinocytes in the treatment of wounds. The American Journal of Surgery, Volume 170, Issue 1, July 1995, Pages 75-83.
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