Efficacy of the Application of a Purified Native Collagen With Embedded Antimicrobial Barrier Followed by a Placental Allograft on a Diverse Group of Nonhealing Wounds of Various Etiologies

Acute wounds progress through stages of healing over a predictive time course and usually heal completely within 1 month. Occasionally, however, acute wounds may become chronic. Many factors can result in the stalling of the wound healing cycle in the inflammatory or proliferative stage.^1-3 These factors are divided into 2 broad categories—local (ie, oxygenation, infection, venous sufficiency, uncontrolled biofilm formation) and systemic (ie, age and sex, coexisting diseases, various immunocompromised states).¹ In particular, these factors can trigger the release of pro-inflammatory molecules, which, subsequently, can promote increased protease activity.^1,4 The upregulation of proteases (ie, collagenases, gelatinases A and B, serine proteases) in the wound bed can degrade the extracellular matrix (ECM) components, including fibronectin, as well as various pro-healing growth factors, all of which are essential in crosstalk between cells involved in ECM remodeling.^6,7 The final common denominators of all these factors are prolongation or full arrest of the wound at the inflammatory phase and chronicity.⁵ 

Biofilm is an example of a local factor that may stimulate an intense inflammatory response in a chronic wound.^1,8 Microbes, encapsulated by a defensive polysaccharide barrier, form biofilm communities that strongly attach to the wound bed.⁹ In response to the presence of persistent biofilm, host immune cells (eg, neutrophils) infiltrate the area of infection and release cytokines, oxygen species, and proteases in an attempt to eradicate biofilm.^2,8,10,11 At this stage, the release of pro-inflammatory molecules and the activation of proteases culminate in ECM degradation and a reduction in the pro-healing activities of keratinocytes and other cell types, thereby interrupting the normal course of wound healing.^8,10,12 Although debridement removes biofilm and necrotic tissue, debridement alone is not adequate, owing to rapid biofilm re-formation, usually within 24 hours, with full re-formation within 3 days.^13,14

Preclinical and clinical studies have demonstrated the effectiveness of the topical antimicrobial agent polyhexamethylene biguanide (PHMB).^15-17 As a cationic topical antimicrobial agent that interferes with catabolic functions in both gram-positive and gram-negative biofilm-forming bacteria, PHMB gains entry to bacterial cells by strongly binding to and disrupting the integrity of bacterial walls and membranes.^15,16,18,19 The broad-spectrum microbial activity of PHMB extends not only to both gram-positive and gram-negative biofilm-forming bacteria but also to methicillin-resistant Staphylococcus aureus and many fungal pathogens.^20,21 Polyhexamethylene biguanide has 2 extremely favorable clinical characteristics; it has no recorded resistance to any known bacterial species, and it does not exhibit significant local toxicity or systemic uptake.^16,22

Collagen matrices can effectively mitigate protease imbalances by serving as substrates for these enzymes.^23-25 Specifically, native type I collagen matrices can strongly bind proteolytic enzymes, such as matrix metalloproteinases and elastase, which are prevalent in nonhealing wounds, thereby effectively reducing both inflammation and destruction of native tissue.^24-26 

Purified native cross-linked ECM with PHMB is an antimicrobial barrier comprising native, bilayered, cross-linked type I collagen, which is saturated with topical antimicrobial PHMB (purified collagen matrix with embedded PHMB, further referenced as PCMP). It is believed that PCMP lessens pro-inflammatory protease activity and prevents biofilm recurrence through its native type I collagen and PHMB components.^19,27,28

One critical feature of a successful wound healing strategy is the quick transition to the proliferative and remodeling phases. Placental allografts, which have been used for more than a century for the treatment of various wound types and which have low immunogenicity, contain ECM proteins, regenerative growth factors, and cytokines essential for healing and promoting proliferation and remodeling.^29-34

Dehydrated amnion/chorion membrane (dACM) is a dehydrated placental allograft containing all native layers of placental membrane. In addition to regenerative growth factors and anti-inflammatory cytokines, dACM also includes the spongy layer, which has high concentrations of specific growth factors and provides proteoglycans, glycoproteins, and hyaluronic acid, as well as a rich collagen network.^31,33-35

Another type of placental allograft that has been applied as a wound covering for the treatment of both acute and chronic wounds is hypothermically stored amniotic membrane (HSAM). This placental allograft consists of a fresh amnion layer of the placenta, including the spongy layer, viable cell populations (ie, stem cells, epithelial cells, fibroblasts), and a vast array of angiogenic, regenerative, and anti-inflammatory factors and cytokines that are believed to assist in the wound healing process.^33,36,37 In addition, HSAM contains multiple ECM proteins important for scaffolding, including collagen types I, III, V, and VI, as well as proteoglycans.^36,37

In this case series, PCMP was used to treat 16 wounds of various etiologies prior to bridging to placental allografts. The approach was based on the treatment hypothesis that PCMP, in combination with aggressive debridements and optimal wound care practices, would reduce biofilm re-formation along with levels of local proteolytic enzymes, which would help transition the wound from the inflammatory phase, thus triggering the reparative process. This initial treatment with PCMP would be followed by applications of dACM and HSAM that would provide regenerative factors necessary for progression of the wound through the proliferative phase.

Sixteen wounds were treated with the aforementioned treatment algorithm, with most treatment provided by 1 vascular surgeon in a single wound care center. Treatment included PCMP (PuraPly AM; Organogenesis, Inc) followed by either dACM (NuShield; Organogenesis, Inc) or HSAM (Affinity; Organogenesis, Inc). In addition, each wound also was managed per the standard of care at the institution; at the discretion of the surgeon, adjunct tests were performed to detect and manage any arterial or venous insufficiency after initiation of wound care treatments.

Sixteen patients (7 males, 9 females) with a mean age (standard deviation [SD]) of 75.5 years (7.6), with several comorbidities and wounds that did not respond to prior traditional treatments, were retrospectively included in the case series. As depicted in Table 1, patients presented to the clinic with upper and lower extremity wounds of various etiologies (venous leg ulcer [VLU] [n = 2], trauma [n = 4], postoperative [n = 5], diabetic foot ulcer [DFU] [n = 2], pressure ulcer [n = 1], and complicated insect bite [n = 2]), with a baseline wound mean area (SD) of 20.16 cm² (21.84) and median area of 11.45 cm². The mean (SD) and median duration were 5.17 months (8.51) and 3 months, respectively (Table 1). After a detailed explanation of the treatment plan, informed consent was obtained. Institutional review board approval was not deemed necessary. Thus, this is a single center case series without controls, and with possible allergy or ineligibility to any of the products used as the only exclusion criteria. 

An entry arterial and venous examination was performed. Noninvasive arterial and venous insufficiency testing was completed for all patients with wounds and abnormal initial examinations. Any form of detected arterial insufficiency (as per the angiosome principle) thought to affect wound healing was corrected by either open or (more frequently) endovascular procedures. Similarly, any form of detected superficial venous insufficiency that was thought to result in ambulatory venous hypertension and that affected wound healing was addressed with endovenous or open techniques. These interventions were routinely performed in the first 2 to 3 weeks after initial patient evaluation. During these first 2 to 3 weeks, wounds were debrided weekly, followed by application of PCMP; after this initial period, weekly debridement was followed by application of placental allograft (HSAM or dACM) until healing was achieved. The change from using PCMP to using placental allografts was done if any of the following conditions was met: infection was controlled; drainage, periwound erythema, pain level, and inflammation were reduced; wound size was decreasing or improvement of wound healing ceased in the absence of infection; and granulation was present. 

For all wounds, debridement involved routine use of local anesthetics and consisted of 2 different phases, the first in which surgical curettes were used and the second involving aggressive brushing of the wound with a 3% chlorohexidine surgical brush, followed by normal saline rinsing. The PCMP was applied unmoistened on the wound after debridement and then, to avoid migration from the wound, the PCMP was stabilized with an adhesive perforated dressing (Versatel; Medline Industries, Inc), after which a foam product was placed for drainage control. The same products used for stabilization and drainage control with PCMP after debridement were used for application of the placental allograft (HSAM or dACM). Adjunctive treatments, such as medicated or nonmedicated compression to manage deep venous insufficiency and offloading or limb compression with a pneumatic garment to manage coexisting lymphatic dysfunction, were administered at the discretion of the clinician.

Patients received an average of 5.9 applications of PCMP, followed by an average of 3.2 applications of dACM or HSAM. Combined, an average of 9 applications of PCMP and dACM or HSAM placental allograft were done. Sixteen wounds were included in the study; however, 1 patient was excluded from the assessment of healing rates due to death at week 12 resulting from complications of cancer treatment. Of the remaining 15 wounds, 13 (86.67%) closed at or before week 12, with the remaining 2 wounds achieving complete wound closure (CWC) by week 17. For the patient who died, measurements taken at the patient’s last visit to the wound care center showed a 73% reduction from baseline during treatment. A summary of the clinical course for all patients is shown in Table 2. 

A subgroup analysis of the larger wounds (> 25 cm²) was conducted in addition to analysis of all wounds regardless of size. Of the 16 wounds, 6 (37.5%) were present for 8.5 weeks and ranged in size from 31 cm² to 78 cm². The mean baseline area (SD) and median baseline area of these larger wounds were 43.5 cm² (15.99) and 42 cm², respectively (Table 1). A large neck wound in a patient who died from complications related to cancer treatment at 12 weeks was included in these baseline area computations; however, this wound was excluded from calculating mean and median time to closure. Only the subset of large wounds were treated with an average of 7 PCMP applications followed by an average of 3.5 placental allograft applications. Using PCMP and placental allografts in combination resulted in an average of 10.5 total applications per patient. Following treatments (ie, first treatment with debridement followed by application of PCMP and placental allografts), the mean time (SD) and median time to CWC were 11.6 weeks (4.45) and 11.3 weeks, respectively. Of the 5 larger wounds, 3 (60%) closed before 12 weeks. 

Case 1: Skin squamous carcinoma excision 
An 80-year-old female with a history of hypertension, hyperlipidemia, and multiple skin squamous carcinoma excisions presented with a squamous carcinoma repeat excision wound of 1 month’s duration measuring 35 cm², with a cellulitic, infected, and necrotic dehisced flap at the excision site on the right calf. Wound biopsies were negative for malignancy. Arterial examination was normal, and duplex ultrasonography to assess for venous insufficiency showed no serious form of venous reflux. Serial wound debridements followed by once-weekly treatment with PCMP application were performed. This treatment continued for 6 weeks. When the wound bed demonstrated an evident granular base, no drainage, and reduced tenderness, PCMP application was stopped and dACM application was started. This new treatment was applied once-weekly for 5 weeks. Complete wound closure was achieved by week 11 (Figure 1). 

Case 2: Advanced squamous cell carcinoma
A 64-year-old female with a history of chronic smoking (25 years) presented with a progressively enlarging, foul-smelling, necrotic, and draining exophytic left neck mass of 2 years’ duration. She was immediately referred to an oncologist, who diagnosed the mass as a locally advanced squamous cell carcinoma with deep tissue penetration and invasion of the left carotid sheath. The patient refused radiation therapy but consented to and was started on a triple chemotherapy regimen. After initiation of chemotherapy, she was sent back to the wound care center for wound, odor, and drainage management. The wound was managed with active carbon dressings for odor control, highly absorbent foam for drainage control, and once-weekly serial debridement and PCMP applications for biofilm management and reduction of wound surface area. Because of the proximity of the wound bed to the lateral pharynx and esophagus as well as to the left common carotid artery, every effort was made to elevate the wound bed and heal the wound expeditiously, also taking into account the extensive cytotoxic effects of chemotherapy. Wound biopsies performed during chemotherapy were negative for malignancy. Upon completion of initial management of necrotic debris and odor, the baseline wound area measured 78 cm². The wound area decreased to 42 cm² after removal of necrotic tissue, at which time PCMP applications were started. The wound was managed with 7 applications of PCMP and 1 of dACM. The patient tolerated chemotherapy well; however, at week 11 she acquired pneumonia, and at week 12 she died. By the time of the patient’s death, the wound area had reduced by 73%, and the distance of the wound bed from the wall of the left common carotid artery had increased by almost 1 cm (Figure 2).

Case 3: Ischemic wound (medial mid-calf)
A 75-year-old female with a history of heavy smoking (28 years), coronary artery disease, previous non-ST-elevation myocardial infarction, aortic stenosis, congestive heart failure, poorly managed type 2 diabetes, hyperlipidemia, hypertension, rheumatic fever (in adolescence), previous aortobifemoral bypass graft for abdominal aortic occlusive disease, and short distance claudication in the right leg presented with fever, chills, and an ischemic, infected, draining, malodorous wound measuring 42 cm² at the right medial mid-calf. Antibiotic therapy was initiated. Arterial workup showed extensive arterial occlusive disease, with occlusion in the right common femoral artery and a 20-cm occlusion in the right superficial femoral artery in addition to severe infrapopliteal disease with occluded right peroneal and posterior tibial arteries. Venous mapping showed the saphenous veins to be inadequate for use as bypass conduits. Because of the severely infected, ischemic wound and also because of the occluded right common femoral artery and status post remote Dacron (INVISTA) aortobifemoral bypass, the patient was treated with right aortobifemoral–to-distal superficial femoral artery bypass with human cadaveric cryopreserved saphenous vein. Following these procedures and treatment, the patient underwent serial weekly wound debridement followed by 5 once-weekly applications of PCMP and then 3 once-weekly applications of HSAM. Complete wound healing was achieved by week 9 (Figure 3).

In this case series, acute wounds such as traumatic (n = 4), postoperative (n = 5), and insect bite (n = 2) exhibited chronicity, with wound duration ranging from 1.25 months to 9 months (Table 1, Table 2). As also depicted in Table 1 and Table 2, a duration of 3 months to 5 months was noted for chronic wounds such as VLUs (n = 2), DFUs (n = 2), and pressure ulcer (n = 1). The formation of biofilm contributes to wound chronicity by prolonging the inflammatory phase as a result of elevated pro-inflammatory cytokine and protease levels.^4,19 Regardless of the varied etiology of nonhealing wounds, experts currently believe that nearly all chronic wounds contain biofilm and that preventing its recurrence may be the common denominator in successfully managing these wound types.^19,38,39 A reduction in biofilm levels may help halt unmanageable inflammation and may prompt the wound regenerative phases of proliferation and remodeling, which is clinically manifested by reduced inflammation, reduced drainage, decreased pain, and the appearance of granulation tissue.

Wounds can progress to closure after the transition is made from the inflammatory to the proliferative phase. However, wounds in which the process stalls in the proliferative phase could potentially benefit from pro-healing cytokines and growth factors. The author’s clinical experience has shown that correcting for any coexisting arterial or/and venous insufficiency, reducing inflammation by managing biofilm, and then introducing a regenerative response in the wound bed are essential steps in accelerating wound healing. The approach and choice of product combination used in the algorithm reported here were selected based on the treatment hypothesis that after correcting for any coexisting arterial or/and venous insufficiency, PCMP in combination with aggressive debridements and optimal wound care practices, can potentially reduce biofilm re-formation and protease levels, which can in turn help transition the wound from an inflammatory phase to a reparative one. The addition of the application of placental allografts to the wounds would provide regenerative factors necessary for wound healing progression.

The results of this case series support this hypothesis. Most wounds (86.7%) achieved CWC by week 12, and the remaining 2 wounds (13.3%) closed by week 17. Complete wound closure, which in some cases was complicated by multiple comorbidities, was achieved in a varied group of wounds, such as VLUs, DFUs, pressure ulcers, postoperative wounds, and insect bites. The average size of the wounds was 20.16 cm²(Table 1). The mean time to complete closure (9.33 weeks) was less than half the mean wound duration (22.32 weeks [5.58 months]). A subgroup analysis of the larger wounds (ie, VLUs, DFUs, postoperative) showed favorable (considering wound size and complexity) mean time to CWC of 11.6 weeks. Additionally, 60% of the wounds closed before 12 weeks.

A few important points must be made concerning the treatment strategy presented herein. First, it is important to emphasize the prompt, early, and diligent approach in improving all amenable metrics concerning the arterial, venous, and lymphatic conditions of the wounds. Although improvement of these metrics alone will not result in wound healing, the author strongly believes that such improvement can maximize the wound healing potential of advanced wound care products, thereby making this treatment algorithm more effective.  

Second, all of the patients in this series underwent revascularization if occlusive disease was present in the artery responsible for supplying the wound or if the tissue pressure adjacent to an extremity wound was less than 60 mm Hg. Similarly, all patients with wounds associated with correctable forms of venous insufficiency that resulted in venous hypertension in the wound area (regardless of if the wound was a venous ulcer) were treated with open or endovenous intervention and/or microphlebectomy as well as sclerotherapy of the areas associated with the wounds. Coexisting lymphatic dysfunction was addressed with the use of inflatable extremity pumps.

From a clinical perspective, managing biofilm with proper debridements and prevention of biofilm re-formation by PCMP aids in the transition of a wound to the proliferative and remodeling stages of healing.¹⁹ Nevertheless, some wounds at this stage may require regenerative factors (ie, growth factor, angiogenic factor) that may enhance wound healing. After biofilm has been managed using an application of PCMP, the application of dACM and HSAM can provide regenerative factors and benefit the reparative process. 

Formulating a treatment strategy requires answers to pertinent questions specific to this strategy. At what point should the transition from the PCMP product to the amniotic products occur? What are the clinical indicators that signal depletion of the benefits PCMP to the wound? Should PCMP always be followed by a placental allograft or any other product to achieve wound closure? Can PCMP be used as a monotherapy for wound closure? 

In this case series, PCMP was used up to the point at which infection was controlled; drainage, periwound erythema, pain level, and inflammation were reduced; wound size was decreasing; and granulation was present. Moreover, if wound size ceased improving in the absence of infection, a transition to using amniotic products was made. For several patients (not included in this series), the wound continued to improve with application of PCMP and healed completely without the need to transition to an amniotic product. The author believes that the presence of systemic causes (eg, rheumatic diseases) of microvascular disease or microvascular disease resulting from diabetes probably increases the likelihood of placental allograft product utility to achieve complete wound healing.

From a safety perspective, multimodal applications of PCMP and either dACM or HSAM appear to be well tolerated. No systemic or local adverse events were attributed to these applications.  

A major limitation of this small case series is that statistical conclusions cannot be drawn as they would be in a randomized controlled trial or comparative study. Future studies are needed to further solidify outcomes of such treatment.

In this retrospective case series, the multimodal approach, consisting of initially improving all amendable metrics of arterial, venous, and lymphatic statuses of all wounds and then treating them with PCMP followed by dACM or HSAM, resulted in satisfactory wound closure rates in a diverse group of wounds in a patient group with multiple comorbidities. The author hypothesizes that PCMP, by serving as a sacrificial matrix for quenching proteases and by managing bioburden and preventing re-formation of biofilm, may aid in the progression of the wound from a stalled, pro-inflammatory state to one of wound healing. In addition, bridging to dACM or HSAM, either of which may serve as a source of regenerative cytokines and growth factors, may accelerate the proliferative and remodeling phases that may maximize the potential for wound healing.

Authors: George J. Koullias, MD, PhD

Affiliation: Division of Vascular and Endovascular Surgery, Stony Brook University Hospital and Stony Brook Southampton Hospital, Stony Brook School of Medicine, Stony Brook, NY 

Correspondence: George J. Koullias, MD, PhD, Division of Vascular and Endovascular Surgery, Stony Brook University Hospital and Stony Brook Southampton Hospital, Stony Brook School of Medicine, 101 Nicolls Road, Stony Brook, NY 11794-819; gkoullias@yahoo.com

Disclosure: The author serves on the Speaker’s Bureau and as a Consultant for Organogenesis, Inc.