Swift Downregulation of Gelatinases (MMP-2, MMP-9) in Neuropathic Diabetic Foot Ulcers Treated With Total Contact Cast

Objective. The aim of this study is to evaluate how treatment with total contact cast (TCC) affects the balance of proteases in diabetic foot ulcers (DFUs) as they heal. Materials and Methods. This was a prospective observational study of 22 eligible patients with neuropathic plantar DFUs in a hospital-based wound care center. All patients treated with TCC had adequate arterial circulation (ankle-brachial index > 0.75), no sign of infection, and all DFUs were grade 1A according to the University of Texas Diabetic Wound Classification System. Patients had weekly follow-up visits for wound evaluation and reapplication of the TCC. Wound tissues were obtained at baseline (week 0 prior to initial treatment), week 3, week 6, and week 12. Tissue homogenates were analyzed for matrix metalloprotease (MMP) 2, MMP-9, tissue inhibitor matrix metalloproteinase (TIMP) 1, and TIMP-2. Wound measurements were obtained at weekly follow-up visits, and healing rates were calculated by photodigital planimetry. Results. Treatment with TCC for 3 weeks resulted in a 20% decrease in MMP-2 (P = .031) and 44% decrease in MMP-9 (P = .018). By week 6, MMP-2 and MMP-9 levels were reduced by 37% and 55%, respectively. Tissue inhibitor matrix metalloproteinase 1 increased by 42% (P = .033) and TIMP-2 by 44% (P = .04) after 6 weeks of therapy with TCC. Conclusions. This significant and rapid drop of both MMP-2 and MMP-9 strongly suggests a decline of the inflammatory phase and initiation of the proliferation phase. 

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How To: Application of a Total Contact Cast

A common unifying hypothesis of chronic wounds is that they have a prolonged (exaggerated) inflammatory phase. In the uninfected wound, this is likely due to an imbalance of matrix metalloproteases (MMPs) and their specific inhibitors (tissue inhibitor matrix metalloproteinase [TIMP]) together with inflammatory cytokines that break down components of the provisional matrix.¹ Levels of MMP-2 and MMP-9 are significantly higher in diabetic foot ulcers (DFUs), while TIMP-2 is lower in DFUs than in acute wounds in patients without diabetes.² In addition, an excess of MMP-9 has been shown to be deleterious to DFU healing and high levels are considered a risk factor to nonhealing.³ It is well established that the gold standard for the treatment of uninfected DFUs is total contact casting (TCC), as it is recognized to clinically decrease inflammation by providing effective offloading.⁴ However, there is little or no evidence about the role of TCC in the regulation of MMPs in wounded tissue. In light of this, the authors sought to evaluate how treatment with TCC affects the balance of proteases in DFUs as they heal.

Study design
All patients (N = 22) were treated with TCC. Follow-up visits were performed weekly for wound evaluation and reapplication of the TCC. Wound tissues were obtained at baseline (week 0 prior to initial treatment), week 3, week 6, and week 12. Tissue homogenates were analyzed using a custom multiplex kit (Bio-Rad; Hercules CA) for MMP-2, MMP-9, TIMP-1, and TIMP-2.⁵ Wound measurements were obtained at weekly follow-up visits, and healing rates were calculated using a digital planimetry software (PicZar Digital Planimetry Software; Biovisual Technologies LLC, Elmwood Park NJ).⁶ 

Inclusion criteria
Either male or female patients aged between 18 and 85 years with a diagnosis of diabetes mellitus (type 1 or 2) were eligible for enrollment. The DFU had to be on the plantar surface of the foot with a grade 1A score according to the University of Texas Diabetic Wound Classification System.⁷ Study patients had to have adequate arterial circulation to the foot as determined by an ankle-brachial index (ABI) > 0.75, toe-brachial index (TBI) > 0.65, or toe systolic pressure > 50 mm Hg. 

Exclusion criteria
Subjects were excluded if the DFU was not a grade 1A, there were any clinical signs of infection, evidence of osteomyelitis, or if the ulcer was due to a nondiabetic etiology. Patients also were excluded if they had or previously had cancer (other than cutaneous epithelioma) not in remission, were receiving oral or parenteral corticosteroids, had other advanced wound therapies (eg, autologous platelet-rich plasma gel, becaplermin, bilayered cell therapy, dermal substitute, extracellular matrix) or received topical collagenase, or currently enrolled or participated in another device, drug, or biological trial within 30 days prior to enrollment. 

Ethics
The governing institutional review board (IRB) (Calvary Hospital IRB, Bronx, NY) ruled this evaluation of an existing patient was exempt from IRB review in accordance with 45CFR46.101. Data collected do not include identifiers or link to identifiers. In addition, the records review was conducted by investigators who normally have access to the records as part of the patient’s routine clinical care. 

Statistical analysis
Multiple regression analyses and Spearman’s Rank correlation coefficient tests were performed to detect correlations between the percentage decrease in wound area and changes in the levels of MMPs or TIMPs. An alpha of 0.05 was used as the cutoff for significance. SPSS for Windows Version 11.5 (SPSS Inc, Chicago, IL) was used for all statistical calculations.

Levels of MMP-2 and MMP-9 in DFU tissues at baseline, week 3, week 6, and week 12 are shown in Figure 1. Treatment with TCC for 3 weeks resulted in a 20% decrease in MMP-2 (P = .031) and 44% decrease in MMP-9 (P = .018). By week 6, MMP-2 and MMP-9 levels were reduced by 37% and 55%, respectively. Levels of TIMP-1 and TIMP-2 in DFU tissues at baseline, week 3, week 6, and week 12 are shown in Figure 2. Tissue inhibitor matrix metalloproteinase 1 increased by 42% (P = .033) and TIMP-2 by 44% (P = .04) after 6 weeks of treatment with TCC. Representative photographs of a DFU with a 10-year history of nonhealing after treatment with TCC at week 3, week 6, and week-12 are provided in Figure 3. 

A limitation of this study is that the authors did not measure inflammatory cytokines, which together with MMPs also play a role in the destruction of the provisional matrix. 

This significant and rapid drop of both MMP-2 (made constitutively) and MMP-9 (produced by inflammatory cells) strongly suggests a decline of the inflammatory phase and initiation of the proliferation phase. The high levels of MMP-9 observed prior to TCC treatment supports the idea that an excess of this gelatinase is harmful to DFU healing.

Authors: Oscar M. Alvarez, PhD^1,2; Lee Markowitz, DPM¹; Neh Onumah, MD¹; and Martin Wendelken, RN, DPM¹

Affiliations: ¹Center for Curative and Palliative Wound Care, Calvary Hospital, Bronx NY; and ²Vascular and Wound Care Center, University Hospital, Newark, NJ 

Correspondence: Oscar M. Alvarez, PhD, Vascular and Wound Care Center, University Hospital, 150 Bergen Street (Room D-106), PO Box 27050, Newark, NJ 07101-6750; alvareom@uhnj.org 

Disclosure: This study was funded in part by the New York State Department of Health and the RTS Family Foundation. A poster abstract of this work was presented at the 2017 Symposium on Advanced Wound Care Spring in San Diego, CA.