- Email: [email protected]
Synergistic action of protease-modulating matrix and autologous growth factors in healing of diabetic foot ulcers. A prospective randomized trial
Journal of Diabetes and Its Complications 21 (2007) 387 – 391
Synergistic action of protease-modulating matrix and autologous growth factors in healing of diabetic foot ulcers. A prospective randomized trial Despoina D. Kakagiaa,4, Konstantinos J. Kazakosb, Konstantinos C. Xarchasb, Michael Karanikasa, George S. Georgiadisc, Gregory Tripsiannisd, Constantinos Manolasa a
1st Department of Surgery, Democritus University Hospital, Alexandroupolis, Greece Department of Orthopaedics, Democritus University Hospital, Alexandroupolis, Greece c Department of Vascular Surgery, Democritus University Hospital, Alexandroupolis, Greece d Department of Medical Statistics, Democritus University in Thrace, Alexandroupolis, Greece b
Received 25 April 2006; received in revised form 12 February 2007; accepted 26 March 2007
Abstract This study tests the hypothesis that addition of a protease-modulating matrix enhances the efficacy of autologous growth factors in diabetic ulcers. Fifty-one patients with chronic diabetic foot ulcers were managed as outpatients at the Democritus University Hospital of Alexandroupolis and followed up for 8 weeks. All target ulcers were z2.5 cm in any one dimension and had been previously treated only with moist gauze. Patients were randomly allocated in three groups of 17 patients each: Group A was treated only with the oxidized regenerated cellulose/collagen biomaterial (Promogran, Johnson & Johnson, New Brunswick, NJ), Group B was treated only with autologous growth factors delivered by Gravitational Platelet Separation System (GPSk, Biomet), and Group C was managed by a combination of both. All ulcers were digitally photographed at initiation of the study and then at change of dressings once weekly. Computerized planimetry (Texas Health Science Center ImageTool, Version 3.0) was used to assess ulcer dimensions that were analyzed for homogeneity and significance using the Statistical Package for Social Sciences, Version 13.0. Post hoc analysis revealed that there was significantly greater reduction of all three dimensions of the ulcers in Group C compared to Groups A and B (all Pb.001). Although reduction of ulcer dimensions was greater in Group A than in Group B, these differences did not reach statistical significance. It is concluded that protease-modulating dressings act synergistically with autologous growth factors and enhance their efficacy in diabetic foot ulcers. D 2007 Elsevier Inc. All rights reserved. Keywords: Autologous growth factors; Diabetic ulcers; Protease-modulating matrix
1. Introduction Diabetic ulcers represent a serious clinical problem with a significant medical and economic impact on health systems worldwide (Ramsey, Newton, & Blough, 1999; Ruckley, 1997). Otherwise productive patients often undergo amputation and may experience prolonged disability. It has been estimated that up to 80% of lower leg
4 Corresponding author. 1st Department of Surgery, Democritus University in Thrace Hospital, Dragana, Alexandroupolis, Greece. Tel.: +30 255 102 3334; fax: +30 255 102 3334. E-mail address: [email protected] (D.D. Kakagia). 1056-8727/07/$ – see front matter D 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.jdiacomp.2007.03.006
amputations in diabetic patients could have been prevented with better patient education on foot care and application of novel, more effective therapies (Larsson, Apelquist, Agardh, & Stenstrfm, 1995; Ramsey et al., 1999). Increasing knowledge on chronic wound pathophysiology and alterations in wound microenvironment is of great clinical value, as combination protocols including new emerging treatment modalities are used to combat the causes of slow healing (Nwomeh, Yager, & Cohen, 1998). Normal wound healing is the result of balanced tissue degradation and synthesis. Extracellular matrix degradation is one of the healing processes that, if left uncontrolled, may result in nonhealing or chronic wounds. It has been shown
388
D.D. Kakagia et al. / Journal of Diabetes and Its Complications 21 (2007) 387 – 391
that increased proteolytic activity due to increased protease release is significantly contributing to delayed healing (Rao et al., 1995; Wysocki, Staiano-Coico, & Grinnell, 1993). Autologous plasma concentrate is a popular therapy that has been used to actively stimulate repair of chronic, nonhealing wounds, including diabetic ulcers (Cenni et al., 2005; Crovetti et al., 2004; Henderson et al., 2003; Mazzucco et al., 2004; Saldalamacchia et al., 2004; Slater, Patava, Kingham, & Mason, 1995). There are several centrifugation systems that allow point-of-care preparation of a highly concentrated platelet formula, rich in growth factors that are reintroduced to the wound in order to accelerate healing (Kevy & Jacobson, 2004; O’Neill et al., 2001; Waters & Roberts, 2004). In chronic wounds, however, impaired healing is attributed not only to direct degradation of extracellular matrix components but also to degradation of significant healing regulators, such as peptide growth factors and endogenous protease inhibitors (Lauer et al., 2000; Mast & Schultz, 1996; Yager et al., 1997). This may explain the discouraging results often observed in the treatment of chronic wounds with growth factors (Brown, Kao, & Greenhalgh, 1997; Robson, 1997). Refinements in topical wound management aim to modifying the chronic wound microenvironment and restoring the balance between tissue synthesis and degradation. A new sterile biomaterial, consisting of 55% collagen and 45% oxidized regenerated cellulose (ORC), has been shown to be efficient in the management of diabetic ulcers. This dressing, which forms a gel on contact with wound exudate, was designed to modify the hostile chronic wound environment by binding and inactivating proteases, oxygen free radicals, and excess metal ions, while protecting growth factors from degradation by temporary binding and redelivering more than 70% of them back to the rebalanced wound bed (Cullen et al., 2002; Veves, Sheehan, & Pham, 2002). Theoretically, the use of this biomaterial in combination with autologous growth factors would optimize the wound bed microenvironment and would be more efficacious than each of these treatment modalities alone. The purpose of this prospective randomized study is to test this hypothesis
comparing the efficacy of combined protease-modulating matrix and autologous growth factors to the use of either method alone and determine whether this combination is of additional benefit in the treatment of diabetic foot ulcers. 2. Patients and methods Diabetic patients with significant soft tissue defects of the foot that had been present for at least 3 months were considered for enrollment in this study. All patients had undergone debridement of the ulcer followed by standard moist gauze treatment for at least 4 weeks, resulting in no more than 15% reduction in ulcer dimensions. All target ulcers had to be z2.5 cm in any one dimension after debridement. From December 2004 to December 2006, 72 patients were assessed for eligibility for this trial; however, 18 patients were excluded due to at least one of the following exclusion criteria: previous treatment with vacuum (n=3), hyperbaric oxygen (n=1), corticosteroid (n=2), immunosuppressive agents (n=1), radiation (n=1) and growth factors (n=3), anemia (n=3), presence of cellulitis (n=3), venous stasis (n=4), inadequate perfusion determined by toe pulses of b40 (n=5), osteomyelitis (n=5), malignancy in the wound (n=1), and patient’s inability to attend clinics for follow-up (n=2). All included patients received thorough explanation of the study and provided an informed consent. The study was conducted in accordance with the guidelines of Democritus University in Alexandroupolis for clinical trials and the Declaration of Helsinki. Fifty-four patients were initially enrolled in the trial; however, three of them did not succeed in completing the protocol: one due to incomplete data, one due to unforeseen inability to attend the clinic, and one due to death in a car accident. At enrollment, patients were randomly assigned by the use of a random number generator to receive treatment for 8 weeks: only with the ORC/collagen biomaterial (Promogran, Johnson & Johnson; Group A), only with autologous growth factors delivered by Gravitational Platelet Separation
Table 1 Patient’s data (values are expressed as meanFstandard deviation) Variable
Group A
Group B
Group C
P value
Age (years) Wound duration (weeks) Wound area (cm2) Leukocyte count (M/Al) Hemoglobin (g/dl) Platelet count (K/Al) Sodium (mmol/l) Potassium (mmol/l) Glucose (mg/dl) Creatinine (mg/dl) Albumin (g/dl) Glycosylated hemoglobin (g/dl)
58F10 17F11 25.8F15.2 7.7F1.9 13.4F1.9 289F63.5 140F1.6 4.4F0.4 129F69 1.6F0.9 3.7F0.7 8.9F3.1
57F12 20F6 28.4F13.6 7.9F1.7 13.9F1.2 270F101.0 140F1.7 4.3F0.6 140F67 1.3F0.7 3.6F0.9 8.1F2.8
61F9 19F8 27.6F17.5 8.1F1.3 14.2F1.5 268.9F96 139F2.2 4.6F0.3 134F72 2.0F1.1 3.7F0.6 8.5F4.0
.511 .587 .883 .781 .328 .759 .202 .153 .899 .092 .903 .784
D.D. Kakagia et al. / Journal of Diabetes and Its Complications 21 (2007) 387 – 391
System (GPS, Biomet; Group B), or a combination of both by means of covering the plasma-centrifuged concentrate that was produced by the GPS and applied at the ulcer bed with the ORC/collagen biomaterial (Group C). Each group included 17 patients. All ulcers were covered by vaporpermeable film (Tegaderm, 3M). Initial laboratory analysis consisted of leukocyte count, hemoglobin, platelet count, sodium, potassium, glucose, creatinine, albumin, and glycosylated hemoglobin. All wounds were sharply debrided prior to first application of dressings and were followed up by weekly assessments for 8 weeks. Ulcer culture swabs were obtained before debridement and at every weekly assessment to determine bacterial contamination and load. All wounds were photographed digitally at initial debridement and then once weekly with a reference marker of scale in three dimensions. Computerized planimetry was used (Texas Health Science Center at San Antonio ImageTool, Version 3.0, as downloaded at http://ddsdx.uthsca.edu/dig/ itdesc.html) to compare the progression of wound healing in the three groups. Wound dimensions were calculated in a blinded fashion and analyzed for homogeneity and significance using the Statistical Package for the Social Sciences (SPSS), Version 13.0 (SPSS, Inc., Chicago, IL, USA). All continuous variables were expressed as meanFstandard deviation. A Shapiro–Wilk test for normality was performed. One-way analysis of variance (ANOVA) was used to assess differences of a continuous variable between the three groups of patients. Post hoc analysis was performed using Tukey’s test. All tests were two tailed, and statistical significance was considered for P values b.05.
389
Fig. 2. Percentage of change in width of diabetic ulcers in the three groups.
Fifty-one patients, 29 female and 22 male, completed the 8-week study protocol. The final outcome criterion in this study was the change in ulcer dimensions within the 8-week follow-up.
There were no significant differences in demographic and clinical data between patients of the three groups (Table 1). One-way ANOVA showed a statistically significant difference among the methods used in this trial on the decrease of the length [ F(2, 48)=14.216, Pb.001; Fig. 1], width [ F(2, 48)=32.776, Pb.001; Fig. 2], and depth [ F(2, 48)=20.533, Pb.001; Fig. 3] of the diabetic ulcers. Post hoc analysis using Tukey’s test revealed that combined use of autologous plasma concentrate and the ORC/collagen biomaterial resulted in significantly greater reduction of all three dimensions of the wound compared to the use of either autologous growth factors or the biomaterial ORC alone (all Pb.001). Furthermore, although reduction of ulcer dimensions by the use of the ORC/collagen biomaterial alone was greater than in ulcers managed by autologous plasma concentrate alone, these differences did not reach statistical significance (length: P=.507; width: P=.194; depth: P=.979; Table 2). Complete healing occurred in two patients in each group at the end of the 8-week follow-up period. Ulcers that were not adequately healed were eventually managed surgically
Fig. 1. Percentage of change in length of diabetic ulcers in the three groups.
Fig. 3. Percentage of change in depth of diabetic ulcers in the three groups.
3. Results
390
D.D. Kakagia et al. / Journal of Diabetes and Its Complications 21 (2007) 387 – 391
shown that both collagen and ORC, the two components of the biomaterial used in this study, have the ability to physically bind proteases; however, maximal binding ability was observed with the combined material (Cullen et al., 2002). Furthermore, the biomaterial has the ability to bind and protect growth factors and deliver more than 70% of them back to the wound when protease activity has been adequately controlled (Cullen et al., 2002). In a preliminary study conducted on 40 patients with chronic venous ulcers, the wound score was improved by 4.92F1.68 by the use of the ORC/collagen matrix compared to 3.22F1.30 by standard good care in 2 weeks (Wollina et al., 2005). It has been suggested that the efficacy of the ORC/ collagen biomaterial is significantly higher in diabetic wounds of no more than 6 months in duration (Veves et al., 2002); nevertheless, in the present study, some ulcers in the three groups had already been present for a longer period at enrollment in the trial. In the present study, the most dramatic reduction was observed in wound depth in all three groups. The computerized planimetry image tool used in the study has been previously validated and is advantageous compared to other image tools as all wound dimensions and not only wound surface area can be determined (Langemo, Melland, & Olson, 2001). This study has certain limitations. The small number of ulcers studied is a potential source of bias, but the statistically significant differences in healing rates among the three groups as well as the careful design of the protocol may negate this comment. Furthermore, the design of the trial does not allow any suggestion of an optimal length of treatment for diabetic wounds, as all ulcers were followed up for 8 weeks only and not until full healing occurs. Also, the system used for centrifugation of the blood provides an eightfold plateletrich plasma concentrate. As it has been estimated that approximately 30% of the growth factors are bound by the biomaterial used in the study (Cullen et al., 2002), we may not assume that another platelet concentration system, achieving lower platelet concentration, would be as effective if combined with this biomaterial. Also, the fact that at least 55 ml of whole blood is needed for each application of the GPS may raise skepticism as to whether the method can be safely used if more frequent than weekly dressing changes are necessary. Pain and patient discomfort during the wound changes were not assessed, and cost analysis of the treatments employed was not performed. Further studies may assess the efficacy of this combined treatment in other types of chronic wounds as venous, pressure, or arterial ulcers.
by local flaps or autologous skin grafts. No complications or side effects were recorded in any patient during the followup period.
4. Discussion Results from this prospective randomized trial demonstrate that combined topical treatment with wound environment modulators, such as the ORC/collagen biomaterial, and autologous growth factors significantly accelerates healing of diabetic ulcers. The efficacy of this combination was found significantly superior to the treatment with either the ORC/collagen complex or autologous growth factors alone. In recent literature, both methods have been separately assessed for their efficacy in diabetic foot ulcers. Autologous plasma concentrate has been successfully used in difficult-to-heal ulcers (Crovetti et al., 2004; Mazzucco et al., 2004; Saldalamacchia et al., 2004). It is created by differential centrifugation of autologous whole blood. The GPS System used in this study provides plasma concentrate with up to eightfold the baseline platelet count (Kevy & Jacobson, 2004). Once activated in the presence of thrombin, platelets release growth factors that are proteinaceous molecules with a pivotal role in wound healing, acting as mitogens, chemoattractants, and stimulators of angiogenesis, fibroplasia, extracellular matrix synthesis, and epithelialization. Growth factors known to be expressed by platelet a granules are platelet-derived growth factor (PDGF-aa, PDGF-ab, and PDGF-bb), transforming growth factor (TGF-b1 and TGF-b2), vascular endothelial growth factor, epithelial growth factor, and insulin-like growth factor (Henderson et al., 2003; Mazzucco et al., 2004; Robson, 1997). However, it is known that prolonged inflammation in chronic wounds causes overproduction of proteases and free radicals, resulting in excess degradation of extracellular matrix, growth factors, and their surface receptors, thus generating a vicious cycle. In this hostile microenvironment, treatment with growth factors—exogenous or autologous— may be of limited success because they are susceptible to proteolytic degradation as are their receptors (Brown et al., 1997; Lauer et al., 2000; Mast & Schultz, 1996; Robson, 1997; Yager et al., 1997). This may explain the significantly higher healing rates observed in Group C of this study, where the ORC/ collagen biomaterial was used in combination with the autologous plasma concentrate, and therefore, a protective environment for growth factors was achieved. It has been Table 2 Percentages of change in wound dimensions Dimension Length Width Depth
Group A 18.59F10.36 23.94F10.75 35.59F10.64
Group B 14.29F7.13 17.41F8.04 34.88F9.85
Group C 33.76F14.74 46.06F13.06 55.12F10.83
P value b.001 b.001 b.001
D.D. Kakagia et al. / Journal of Diabetes and Its Complications 21 (2007) 387 – 391
Extensive debridement, adequate off-loading of the ulcerated foot, control and management of infection, and lower extremity revascularization, whenever required, are of vital significance in the diabetic foot treatment (Armstrong & Frykberg, 2003; Wu, Crews, & Armstrong, 2005). The treatments used in this study should be considered as valuable adjuncts to and not as substitutes for these principles. In summary, we have shown that dressing nonhealing diabetic foot ulcers with modulators of the wound environment in combination with the administration of autologous growth factors significantly accelerates the healing rate. It is suggested that rebalancing of the wound microenvironment by using dressings that inhibit proteases should initiate the repair process and increases the healing potential of autologous growth factors.
References Armstrong, D. G., & Frykberg, R. G. (2003). Classifying diabetic foot surgery: Toward a rational definition. Diabetic Medicine, 20, 329 – 331. Brown, D. L., Kao, D. D., & Greenhalgh, D. G. (1997). Apoptosis downregulates inflammation under the advancing epithelial wound edge: Delayed patterns in diabetes and improvement with topical growth factors. Surgery, 121, 372 – 380. Cenni, E., Ciapetti, G., Pagani, S., Perut, F., Giunti, A., & Baldini, N. (2005). Effects of activated platelet concentrates on human primary cultures of fibroblasts and osteoblasts. Journal of Periodontology, 76, 323 – 328. Crovetti, G., Martinelli, G., Issi, M., Barone, M., Guizzardi, M., Campanati, B., Moroni, M., & Carabelli, A. (2004). Platelet gel for healing cutaneous chronic wounds. Transfus Apheresis Sci, 30, 145 – 151. Cullen, B., Watt, P. W., Lundqvist, C., Silcock, D., Schmidt, R. J., Bogan, D., & Light, N. (2002). The role of oxidized regenerated cellulose/collagen in chronic wound repair and its potential mechanism of action. International Journal of Biochemistry & Cell Biology, 1307, 1 – 13. Henderson, J. L., Cupp, C. L., Ross, E. V., Shick, P. C., Keefe, M. A., Wester, D. C., Hannon, T., & McConnell, D. (2003). The effects of autologous platelet gel on wound healing. Ear, Nose, & Throat Journal, 82, 598 – 602. Kevy, S. V., & Jacobson, M. S. (2004). Comparison of methods for point of care preparation of autologous platelet gel. Journal of Extra-corporeal Technology, 36, 28 – 35. Langemo, D. K., Melland, H., & Olson, B. (2001). Comparison of 2 wound volume measurement methods. Advances in Skin & Wound Care, 14, 190 – 196. Larsson, J., Apelquist, J., Agardh, G. D., & Stenstrfm, A. (1995). Decreasing incidence of major amputations in diabetic patients: A consequence of a multi-disciplinary foot care team approach? Diabetic Medicine, 12, 770 – 776.
391
Lauer, G., Sollberg, S., Cole, M., Flamme, I., Sturzebecher, J., Mann, K., Krieg, T., & Eming, S. A. (2000). Expression and proteolysis of vascular endothelial growth factor is increased in chronic wounds. Journal of Investigative Dermatology, 115, 12 – 18. Mast, B. A., & Schultz, G. S. (1996). Interactions of cytokines, growth factors and proteases in acute and chronic wounds. Wound Repair and Regeneration, 4, 411 – 420. Mazzucco, L., Medici, D., Serra, M., Panizza, R., Rivara, G., Orecchia, S., Libener, R., Cattana, E., Levis, A., Betta, P. G., & Borzini, P. (2004). The use of autologous platelet gel to treat difficult-to-heal wounds: A pilot study. Transfusion, 44, 1013 – 1018. Nwomeh, B. C., Yager, D. R., & Cohen, I. K. (1998). Physiology of the chronic wound. Clinics in Plastic Surgery, 25, 341 – 356. O’Neill, E. M., Zalewski, W. M., Eaton, L. J., Popovsky, M. A., Pivacek, L. E., Ragno, G., & Valeri, C. R. (2001). Autologous platelet-rich plasma isolated using the Haemonetics Cell Saver 5 and Haemonetics MCS+ for the preparation of platelet gel. Vox Sanguinis, 81, 172 – 175. Ramsey, S. D., Newton, K., & Blough, D. (1999). Incidence, outcomes and cost of foot ulcers in patients with diabetes. Diabetes Care, 22, 382 – 387. Rao, C. N., Ladin, D. A., Liu, Y. Y., Chilukuri, K., Hou, Z. Z., & Woodley, D. (1995). a-1 antitrypsin is degraded and non-functional in chronic wounds but intact and functional in acute wounds: The inhibitor protects fibronectin from degradation by chronic wound fluid enzymes. Journal of Investigative Dermatology, 105, 572 – 578. Robson, M. C. (1997). The role of growth factors in the healing of chronic wounds. Wound Repair and Regeneration, 5, 12 – 17. Ruckley, C. V. (1997). Socioeconomic impact of chronic venous insufficiency and leg ulcers. Angiology, 48, 67 – 69. Saldalamacchia, G., Lapice, E., Cuomo, V., De Feo, E., D’Agostino, E., Rivellese, A. A., & Vaccaro, O. (2004). A controlled study of the use of autologous platelet gel for the treatment of diabetic foot ulcers. Nutrition, Metabolism, and Cardiovascular Diseases, 14, 395 – 396. Slater, M., Patava, J., Kingham, M., & Mason, R. S. (1995). Involvement of platelets in stimulating osteogenic activity. Journal of Orthopaedic Research, 13, 655 – 663. Veves, A., Sheehan, P., & Pham, H. T. (2002). A randomized, controlled trial of Promogran (a Collagen/Oxidized Regenerated Cellulose dressing) vs standard treatment in the management of diabetic foot ulcers. Archives of Surgery, 137, 822 – 827. Waters, J. H., & Roberts, K. C. (2004). Database review of possible factors influencing point-of-care platelet gel manufacture. Journal of Extracorporeal Technology, 36, 250 – 254. Wollina, U., Schmidt, W. D., Kronert, C., Nelskamp, C., Scheibe, A., & Fassler, D. (2005). Some effects of a topical collagen-based matrix on the microcirculation and wound healing in patients with chronic venous leg ulcers: Preliminary observations. International Journal of Lower Extremity Wounds, 4, 214 – 224. Wu, S. C., Crews, R. T., & Armstrong, D. G. (2005). The pivotal role of offloading in the management of neuropathic foot ulceration. Current Diabetes Reports, 5, 423 – 429. Wysocki, A. B., Staiano-Coico, L., & Grinnell, F. (1993). Wound fluid from chronic leg ulcers contains elevated levels of metalloproteinases MMP2 and MMP-9. Journal of Investigative Dermatology, 101, 64 – 68. Yager, D. R., Chen, S. M., Ward, S. I., Oluyinka, O., Olytoye, M. D., Diegelmann, R. F., & Cohen, I. K. (1997). Ability of chronic wound fluids to degrade peptide growth factors is associated with increased levels of elastase activity and diminished levels of proteinase inhibitors. Wound Repair and Regeneration, 5, 23 – 32.
Synergistic action of protease-modulating matrix and autologous growth factors in healing of diabetic foot ulcers. A prospective randomized trial Despoina D. Kakagiaa,4, Konstantinos J. Kazakosb, Konstantinos C. Xarchasb, Michael Karanikasa, George S. Georgiadisc, Gregory Tripsiannisd, Constantinos Manolasa a
1st Department of Surgery, Democritus University Hospital, Alexandroupolis, Greece Department of Orthopaedics, Democritus University Hospital, Alexandroupolis, Greece c Department of Vascular Surgery, Democritus University Hospital, Alexandroupolis, Greece d Department of Medical Statistics, Democritus University in Thrace, Alexandroupolis, Greece b
Received 25 April 2006; received in revised form 12 February 2007; accepted 26 March 2007
Abstract This study tests the hypothesis that addition of a protease-modulating matrix enhances the efficacy of autologous growth factors in diabetic ulcers. Fifty-one patients with chronic diabetic foot ulcers were managed as outpatients at the Democritus University Hospital of Alexandroupolis and followed up for 8 weeks. All target ulcers were z2.5 cm in any one dimension and had been previously treated only with moist gauze. Patients were randomly allocated in three groups of 17 patients each: Group A was treated only with the oxidized regenerated cellulose/collagen biomaterial (Promogran, Johnson & Johnson, New Brunswick, NJ), Group B was treated only with autologous growth factors delivered by Gravitational Platelet Separation System (GPSk, Biomet), and Group C was managed by a combination of both. All ulcers were digitally photographed at initiation of the study and then at change of dressings once weekly. Computerized planimetry (Texas Health Science Center ImageTool, Version 3.0) was used to assess ulcer dimensions that were analyzed for homogeneity and significance using the Statistical Package for Social Sciences, Version 13.0. Post hoc analysis revealed that there was significantly greater reduction of all three dimensions of the ulcers in Group C compared to Groups A and B (all Pb.001). Although reduction of ulcer dimensions was greater in Group A than in Group B, these differences did not reach statistical significance. It is concluded that protease-modulating dressings act synergistically with autologous growth factors and enhance their efficacy in diabetic foot ulcers. D 2007 Elsevier Inc. All rights reserved. Keywords: Autologous growth factors; Diabetic ulcers; Protease-modulating matrix
1. Introduction Diabetic ulcers represent a serious clinical problem with a significant medical and economic impact on health systems worldwide (Ramsey, Newton, & Blough, 1999; Ruckley, 1997). Otherwise productive patients often undergo amputation and may experience prolonged disability. It has been estimated that up to 80% of lower leg
4 Corresponding author. 1st Department of Surgery, Democritus University in Thrace Hospital, Dragana, Alexandroupolis, Greece. Tel.: +30 255 102 3334; fax: +30 255 102 3334. E-mail address: [email protected] (D.D. Kakagia). 1056-8727/07/$ – see front matter D 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.jdiacomp.2007.03.006
amputations in diabetic patients could have been prevented with better patient education on foot care and application of novel, more effective therapies (Larsson, Apelquist, Agardh, & Stenstrfm, 1995; Ramsey et al., 1999). Increasing knowledge on chronic wound pathophysiology and alterations in wound microenvironment is of great clinical value, as combination protocols including new emerging treatment modalities are used to combat the causes of slow healing (Nwomeh, Yager, & Cohen, 1998). Normal wound healing is the result of balanced tissue degradation and synthesis. Extracellular matrix degradation is one of the healing processes that, if left uncontrolled, may result in nonhealing or chronic wounds. It has been shown
388
D.D. Kakagia et al. / Journal of Diabetes and Its Complications 21 (2007) 387 – 391
that increased proteolytic activity due to increased protease release is significantly contributing to delayed healing (Rao et al., 1995; Wysocki, Staiano-Coico, & Grinnell, 1993). Autologous plasma concentrate is a popular therapy that has been used to actively stimulate repair of chronic, nonhealing wounds, including diabetic ulcers (Cenni et al., 2005; Crovetti et al., 2004; Henderson et al., 2003; Mazzucco et al., 2004; Saldalamacchia et al., 2004; Slater, Patava, Kingham, & Mason, 1995). There are several centrifugation systems that allow point-of-care preparation of a highly concentrated platelet formula, rich in growth factors that are reintroduced to the wound in order to accelerate healing (Kevy & Jacobson, 2004; O’Neill et al., 2001; Waters & Roberts, 2004). In chronic wounds, however, impaired healing is attributed not only to direct degradation of extracellular matrix components but also to degradation of significant healing regulators, such as peptide growth factors and endogenous protease inhibitors (Lauer et al., 2000; Mast & Schultz, 1996; Yager et al., 1997). This may explain the discouraging results often observed in the treatment of chronic wounds with growth factors (Brown, Kao, & Greenhalgh, 1997; Robson, 1997). Refinements in topical wound management aim to modifying the chronic wound microenvironment and restoring the balance between tissue synthesis and degradation. A new sterile biomaterial, consisting of 55% collagen and 45% oxidized regenerated cellulose (ORC), has been shown to be efficient in the management of diabetic ulcers. This dressing, which forms a gel on contact with wound exudate, was designed to modify the hostile chronic wound environment by binding and inactivating proteases, oxygen free radicals, and excess metal ions, while protecting growth factors from degradation by temporary binding and redelivering more than 70% of them back to the rebalanced wound bed (Cullen et al., 2002; Veves, Sheehan, & Pham, 2002). Theoretically, the use of this biomaterial in combination with autologous growth factors would optimize the wound bed microenvironment and would be more efficacious than each of these treatment modalities alone. The purpose of this prospective randomized study is to test this hypothesis
comparing the efficacy of combined protease-modulating matrix and autologous growth factors to the use of either method alone and determine whether this combination is of additional benefit in the treatment of diabetic foot ulcers. 2. Patients and methods Diabetic patients with significant soft tissue defects of the foot that had been present for at least 3 months were considered for enrollment in this study. All patients had undergone debridement of the ulcer followed by standard moist gauze treatment for at least 4 weeks, resulting in no more than 15% reduction in ulcer dimensions. All target ulcers had to be z2.5 cm in any one dimension after debridement. From December 2004 to December 2006, 72 patients were assessed for eligibility for this trial; however, 18 patients were excluded due to at least one of the following exclusion criteria: previous treatment with vacuum (n=3), hyperbaric oxygen (n=1), corticosteroid (n=2), immunosuppressive agents (n=1), radiation (n=1) and growth factors (n=3), anemia (n=3), presence of cellulitis (n=3), venous stasis (n=4), inadequate perfusion determined by toe pulses of b40 (n=5), osteomyelitis (n=5), malignancy in the wound (n=1), and patient’s inability to attend clinics for follow-up (n=2). All included patients received thorough explanation of the study and provided an informed consent. The study was conducted in accordance with the guidelines of Democritus University in Alexandroupolis for clinical trials and the Declaration of Helsinki. Fifty-four patients were initially enrolled in the trial; however, three of them did not succeed in completing the protocol: one due to incomplete data, one due to unforeseen inability to attend the clinic, and one due to death in a car accident. At enrollment, patients were randomly assigned by the use of a random number generator to receive treatment for 8 weeks: only with the ORC/collagen biomaterial (Promogran, Johnson & Johnson; Group A), only with autologous growth factors delivered by Gravitational Platelet Separation
Table 1 Patient’s data (values are expressed as meanFstandard deviation) Variable
Group A
Group B
Group C
P value
Age (years) Wound duration (weeks) Wound area (cm2) Leukocyte count (M/Al) Hemoglobin (g/dl) Platelet count (K/Al) Sodium (mmol/l) Potassium (mmol/l) Glucose (mg/dl) Creatinine (mg/dl) Albumin (g/dl) Glycosylated hemoglobin (g/dl)
58F10 17F11 25.8F15.2 7.7F1.9 13.4F1.9 289F63.5 140F1.6 4.4F0.4 129F69 1.6F0.9 3.7F0.7 8.9F3.1
57F12 20F6 28.4F13.6 7.9F1.7 13.9F1.2 270F101.0 140F1.7 4.3F0.6 140F67 1.3F0.7 3.6F0.9 8.1F2.8
61F9 19F8 27.6F17.5 8.1F1.3 14.2F1.5 268.9F96 139F2.2 4.6F0.3 134F72 2.0F1.1 3.7F0.6 8.5F4.0
.511 .587 .883 .781 .328 .759 .202 .153 .899 .092 .903 .784
D.D. Kakagia et al. / Journal of Diabetes and Its Complications 21 (2007) 387 – 391
System (GPS, Biomet; Group B), or a combination of both by means of covering the plasma-centrifuged concentrate that was produced by the GPS and applied at the ulcer bed with the ORC/collagen biomaterial (Group C). Each group included 17 patients. All ulcers were covered by vaporpermeable film (Tegaderm, 3M). Initial laboratory analysis consisted of leukocyte count, hemoglobin, platelet count, sodium, potassium, glucose, creatinine, albumin, and glycosylated hemoglobin. All wounds were sharply debrided prior to first application of dressings and were followed up by weekly assessments for 8 weeks. Ulcer culture swabs were obtained before debridement and at every weekly assessment to determine bacterial contamination and load. All wounds were photographed digitally at initial debridement and then once weekly with a reference marker of scale in three dimensions. Computerized planimetry was used (Texas Health Science Center at San Antonio ImageTool, Version 3.0, as downloaded at http://ddsdx.uthsca.edu/dig/ itdesc.html) to compare the progression of wound healing in the three groups. Wound dimensions were calculated in a blinded fashion and analyzed for homogeneity and significance using the Statistical Package for the Social Sciences (SPSS), Version 13.0 (SPSS, Inc., Chicago, IL, USA). All continuous variables were expressed as meanFstandard deviation. A Shapiro–Wilk test for normality was performed. One-way analysis of variance (ANOVA) was used to assess differences of a continuous variable between the three groups of patients. Post hoc analysis was performed using Tukey’s test. All tests were two tailed, and statistical significance was considered for P values b.05.
389
Fig. 2. Percentage of change in width of diabetic ulcers in the three groups.
Fifty-one patients, 29 female and 22 male, completed the 8-week study protocol. The final outcome criterion in this study was the change in ulcer dimensions within the 8-week follow-up.
There were no significant differences in demographic and clinical data between patients of the three groups (Table 1). One-way ANOVA showed a statistically significant difference among the methods used in this trial on the decrease of the length [ F(2, 48)=14.216, Pb.001; Fig. 1], width [ F(2, 48)=32.776, Pb.001; Fig. 2], and depth [ F(2, 48)=20.533, Pb.001; Fig. 3] of the diabetic ulcers. Post hoc analysis using Tukey’s test revealed that combined use of autologous plasma concentrate and the ORC/collagen biomaterial resulted in significantly greater reduction of all three dimensions of the wound compared to the use of either autologous growth factors or the biomaterial ORC alone (all Pb.001). Furthermore, although reduction of ulcer dimensions by the use of the ORC/collagen biomaterial alone was greater than in ulcers managed by autologous plasma concentrate alone, these differences did not reach statistical significance (length: P=.507; width: P=.194; depth: P=.979; Table 2). Complete healing occurred in two patients in each group at the end of the 8-week follow-up period. Ulcers that were not adequately healed were eventually managed surgically
Fig. 1. Percentage of change in length of diabetic ulcers in the three groups.
Fig. 3. Percentage of change in depth of diabetic ulcers in the three groups.
3. Results
390
D.D. Kakagia et al. / Journal of Diabetes and Its Complications 21 (2007) 387 – 391
shown that both collagen and ORC, the two components of the biomaterial used in this study, have the ability to physically bind proteases; however, maximal binding ability was observed with the combined material (Cullen et al., 2002). Furthermore, the biomaterial has the ability to bind and protect growth factors and deliver more than 70% of them back to the wound when protease activity has been adequately controlled (Cullen et al., 2002). In a preliminary study conducted on 40 patients with chronic venous ulcers, the wound score was improved by 4.92F1.68 by the use of the ORC/collagen matrix compared to 3.22F1.30 by standard good care in 2 weeks (Wollina et al., 2005). It has been suggested that the efficacy of the ORC/ collagen biomaterial is significantly higher in diabetic wounds of no more than 6 months in duration (Veves et al., 2002); nevertheless, in the present study, some ulcers in the three groups had already been present for a longer period at enrollment in the trial. In the present study, the most dramatic reduction was observed in wound depth in all three groups. The computerized planimetry image tool used in the study has been previously validated and is advantageous compared to other image tools as all wound dimensions and not only wound surface area can be determined (Langemo, Melland, & Olson, 2001). This study has certain limitations. The small number of ulcers studied is a potential source of bias, but the statistically significant differences in healing rates among the three groups as well as the careful design of the protocol may negate this comment. Furthermore, the design of the trial does not allow any suggestion of an optimal length of treatment for diabetic wounds, as all ulcers were followed up for 8 weeks only and not until full healing occurs. Also, the system used for centrifugation of the blood provides an eightfold plateletrich plasma concentrate. As it has been estimated that approximately 30% of the growth factors are bound by the biomaterial used in the study (Cullen et al., 2002), we may not assume that another platelet concentration system, achieving lower platelet concentration, would be as effective if combined with this biomaterial. Also, the fact that at least 55 ml of whole blood is needed for each application of the GPS may raise skepticism as to whether the method can be safely used if more frequent than weekly dressing changes are necessary. Pain and patient discomfort during the wound changes were not assessed, and cost analysis of the treatments employed was not performed. Further studies may assess the efficacy of this combined treatment in other types of chronic wounds as venous, pressure, or arterial ulcers.
by local flaps or autologous skin grafts. No complications or side effects were recorded in any patient during the followup period.
4. Discussion Results from this prospective randomized trial demonstrate that combined topical treatment with wound environment modulators, such as the ORC/collagen biomaterial, and autologous growth factors significantly accelerates healing of diabetic ulcers. The efficacy of this combination was found significantly superior to the treatment with either the ORC/collagen complex or autologous growth factors alone. In recent literature, both methods have been separately assessed for their efficacy in diabetic foot ulcers. Autologous plasma concentrate has been successfully used in difficult-to-heal ulcers (Crovetti et al., 2004; Mazzucco et al., 2004; Saldalamacchia et al., 2004). It is created by differential centrifugation of autologous whole blood. The GPS System used in this study provides plasma concentrate with up to eightfold the baseline platelet count (Kevy & Jacobson, 2004). Once activated in the presence of thrombin, platelets release growth factors that are proteinaceous molecules with a pivotal role in wound healing, acting as mitogens, chemoattractants, and stimulators of angiogenesis, fibroplasia, extracellular matrix synthesis, and epithelialization. Growth factors known to be expressed by platelet a granules are platelet-derived growth factor (PDGF-aa, PDGF-ab, and PDGF-bb), transforming growth factor (TGF-b1 and TGF-b2), vascular endothelial growth factor, epithelial growth factor, and insulin-like growth factor (Henderson et al., 2003; Mazzucco et al., 2004; Robson, 1997). However, it is known that prolonged inflammation in chronic wounds causes overproduction of proteases and free radicals, resulting in excess degradation of extracellular matrix, growth factors, and their surface receptors, thus generating a vicious cycle. In this hostile microenvironment, treatment with growth factors—exogenous or autologous— may be of limited success because they are susceptible to proteolytic degradation as are their receptors (Brown et al., 1997; Lauer et al., 2000; Mast & Schultz, 1996; Robson, 1997; Yager et al., 1997). This may explain the significantly higher healing rates observed in Group C of this study, where the ORC/ collagen biomaterial was used in combination with the autologous plasma concentrate, and therefore, a protective environment for growth factors was achieved. It has been Table 2 Percentages of change in wound dimensions Dimension Length Width Depth
Group A 18.59F10.36 23.94F10.75 35.59F10.64
Group B 14.29F7.13 17.41F8.04 34.88F9.85
Group C 33.76F14.74 46.06F13.06 55.12F10.83
P value b.001 b.001 b.001
D.D. Kakagia et al. / Journal of Diabetes and Its Complications 21 (2007) 387 – 391
Extensive debridement, adequate off-loading of the ulcerated foot, control and management of infection, and lower extremity revascularization, whenever required, are of vital significance in the diabetic foot treatment (Armstrong & Frykberg, 2003; Wu, Crews, & Armstrong, 2005). The treatments used in this study should be considered as valuable adjuncts to and not as substitutes for these principles. In summary, we have shown that dressing nonhealing diabetic foot ulcers with modulators of the wound environment in combination with the administration of autologous growth factors significantly accelerates the healing rate. It is suggested that rebalancing of the wound microenvironment by using dressings that inhibit proteases should initiate the repair process and increases the healing potential of autologous growth factors.
References Armstrong, D. G., & Frykberg, R. G. (2003). Classifying diabetic foot surgery: Toward a rational definition. Diabetic Medicine, 20, 329 – 331. Brown, D. L., Kao, D. D., & Greenhalgh, D. G. (1997). Apoptosis downregulates inflammation under the advancing epithelial wound edge: Delayed patterns in diabetes and improvement with topical growth factors. Surgery, 121, 372 – 380. Cenni, E., Ciapetti, G., Pagani, S., Perut, F., Giunti, A., & Baldini, N. (2005). Effects of activated platelet concentrates on human primary cultures of fibroblasts and osteoblasts. Journal of Periodontology, 76, 323 – 328. Crovetti, G., Martinelli, G., Issi, M., Barone, M., Guizzardi, M., Campanati, B., Moroni, M., & Carabelli, A. (2004). Platelet gel for healing cutaneous chronic wounds. Transfus Apheresis Sci, 30, 145 – 151. Cullen, B., Watt, P. W., Lundqvist, C., Silcock, D., Schmidt, R. J., Bogan, D., & Light, N. (2002). The role of oxidized regenerated cellulose/collagen in chronic wound repair and its potential mechanism of action. International Journal of Biochemistry & Cell Biology, 1307, 1 – 13. Henderson, J. L., Cupp, C. L., Ross, E. V., Shick, P. C., Keefe, M. A., Wester, D. C., Hannon, T., & McConnell, D. (2003). The effects of autologous platelet gel on wound healing. Ear, Nose, & Throat Journal, 82, 598 – 602. Kevy, S. V., & Jacobson, M. S. (2004). Comparison of methods for point of care preparation of autologous platelet gel. Journal of Extra-corporeal Technology, 36, 28 – 35. Langemo, D. K., Melland, H., & Olson, B. (2001). Comparison of 2 wound volume measurement methods. Advances in Skin & Wound Care, 14, 190 – 196. Larsson, J., Apelquist, J., Agardh, G. D., & Stenstrfm, A. (1995). Decreasing incidence of major amputations in diabetic patients: A consequence of a multi-disciplinary foot care team approach? Diabetic Medicine, 12, 770 – 776.
391
Lauer, G., Sollberg, S., Cole, M., Flamme, I., Sturzebecher, J., Mann, K., Krieg, T., & Eming, S. A. (2000). Expression and proteolysis of vascular endothelial growth factor is increased in chronic wounds. Journal of Investigative Dermatology, 115, 12 – 18. Mast, B. A., & Schultz, G. S. (1996). Interactions of cytokines, growth factors and proteases in acute and chronic wounds. Wound Repair and Regeneration, 4, 411 – 420. Mazzucco, L., Medici, D., Serra, M., Panizza, R., Rivara, G., Orecchia, S., Libener, R., Cattana, E., Levis, A., Betta, P. G., & Borzini, P. (2004). The use of autologous platelet gel to treat difficult-to-heal wounds: A pilot study. Transfusion, 44, 1013 – 1018. Nwomeh, B. C., Yager, D. R., & Cohen, I. K. (1998). Physiology of the chronic wound. Clinics in Plastic Surgery, 25, 341 – 356. O’Neill, E. M., Zalewski, W. M., Eaton, L. J., Popovsky, M. A., Pivacek, L. E., Ragno, G., & Valeri, C. R. (2001). Autologous platelet-rich plasma isolated using the Haemonetics Cell Saver 5 and Haemonetics MCS+ for the preparation of platelet gel. Vox Sanguinis, 81, 172 – 175. Ramsey, S. D., Newton, K., & Blough, D. (1999). Incidence, outcomes and cost of foot ulcers in patients with diabetes. Diabetes Care, 22, 382 – 387. Rao, C. N., Ladin, D. A., Liu, Y. Y., Chilukuri, K., Hou, Z. Z., & Woodley, D. (1995). a-1 antitrypsin is degraded and non-functional in chronic wounds but intact and functional in acute wounds: The inhibitor protects fibronectin from degradation by chronic wound fluid enzymes. Journal of Investigative Dermatology, 105, 572 – 578. Robson, M. C. (1997). The role of growth factors in the healing of chronic wounds. Wound Repair and Regeneration, 5, 12 – 17. Ruckley, C. V. (1997). Socioeconomic impact of chronic venous insufficiency and leg ulcers. Angiology, 48, 67 – 69. Saldalamacchia, G., Lapice, E., Cuomo, V., De Feo, E., D’Agostino, E., Rivellese, A. A., & Vaccaro, O. (2004). A controlled study of the use of autologous platelet gel for the treatment of diabetic foot ulcers. Nutrition, Metabolism, and Cardiovascular Diseases, 14, 395 – 396. Slater, M., Patava, J., Kingham, M., & Mason, R. S. (1995). Involvement of platelets in stimulating osteogenic activity. Journal of Orthopaedic Research, 13, 655 – 663. Veves, A., Sheehan, P., & Pham, H. T. (2002). A randomized, controlled trial of Promogran (a Collagen/Oxidized Regenerated Cellulose dressing) vs standard treatment in the management of diabetic foot ulcers. Archives of Surgery, 137, 822 – 827. Waters, J. H., & Roberts, K. C. (2004). Database review of possible factors influencing point-of-care platelet gel manufacture. Journal of Extracorporeal Technology, 36, 250 – 254. Wollina, U., Schmidt, W. D., Kronert, C., Nelskamp, C., Scheibe, A., & Fassler, D. (2005). Some effects of a topical collagen-based matrix on the microcirculation and wound healing in patients with chronic venous leg ulcers: Preliminary observations. International Journal of Lower Extremity Wounds, 4, 214 – 224. Wu, S. C., Crews, R. T., & Armstrong, D. G. (2005). The pivotal role of offloading in the management of neuropathic foot ulceration. Current Diabetes Reports, 5, 423 – 429. Wysocki, A. B., Staiano-Coico, L., & Grinnell, F. (1993). Wound fluid from chronic leg ulcers contains elevated levels of metalloproteinases MMP2 and MMP-9. Journal of Investigative Dermatology, 101, 64 – 68. Yager, D. R., Chen, S. M., Ward, S. I., Oluyinka, O., Olytoye, M. D., Diegelmann, R. F., & Cohen, I. K. (1997). Ability of chronic wound fluids to degrade peptide growth factors is associated with increased levels of elastase activity and diminished levels of proteinase inhibitors. Wound Repair and Regeneration, 5, 23 – 32.