A Prospective, Single-center, Open-label Case Series Evaluating the Clinical Outcomes of Lyopreserved Placental Membrane Containing Viable Cells in the Treatment of Chronic Wounds

Introduction. Cryopreserved placental membrane containing viable cells (vCPM) in conjunction with standard of care (SOC) has shown clinical effectiveness in several studies for the management of acute and chronic wounds. Recently, a new lyophilization technique has been developed that allows viable tissues to be stored at room temperature as a structural and functional equivalent to vCPM. Objective. This case series evaluates the clinical outcomes of a lyopreserved placental membrane containing viable cells (vLPM) for the management of chronic wounds of various etiologies. Materials and Methods. Eleven patients (11 wounds: 5 diabetic foot ulcers, 5 venous leg ulcers, and 1 pressure ulcer) received weekly vLPM applications adjunct to SOC. Results. By week 12 of treatment, 63.6% (7/11) of patients achieved complete wound closure, with a mean time to closure of 47.1 days and a mean of 6 vLPM applications. Further, there were no adverse events attributed to vLPM. Conclusions. This is the first case series reporting the clinical outcomes of vLPM for the management of chronic wounds. Results of this study demonstrate similar closure rates to those previously reported for vCPM. These results suggest potential clinical equivalence between the 2 formulations, with vLPM providing the added convenience of long-term room-temperature storage (current shelf life of 12 months). 

About 2% of the US population suffers from a chronic wound, defined as a wound with the inability to proceed through the normal phases of wound healing in a timely manner.^1,2 These wounds prove challenging to treat, as they are often linked to advanced patient age and underlying comorbidities such as diabetes, venous insufficiency, and peripheral arterial disease (PAD).³ Generally, the standard of care (SOC) for chronic wounds includes cleaning and debridement, maintenance of a moist wound environment, nutritional support, establishment of adequate blood circulation, infection management, and compression or offloading based on wound etiology.⁴ Chronic wounds often do not respond to this first-line SOC treatment and require advanced adjunctive therapies.³ 

Skin substitutes have become a widespread option in the treatment of wounds that are unresponsive to SOC. As a broad category, skin substitutes include acellular, devitalized, and viable products.^5,6 Examples of products containing viable cells are a cryopreserved human fibroblast-derived dermal substitute (hFDS), a bilayered skin substitute, and a cryopreserved placental membrane containing viable cells (vCPM).^7-9 

The beneficial properties of placental membranes in wound care have been reported for more than 100 years.¹⁰ A vCPM graft retains all of the components of fresh placental tissue, such as the 3-dimensional collagen-rich matrix, endogenous growth factors, and viable cells.⁹ A commercially available product, vCPM has shown positive outcomes in clinical trials for various types of wounds such as diabetic foot ulcers (DFUs), venous leg ulcers (VLUs), and pressure ulcers.^11-14 

For many years, cryopreservation was the only method for long-term storage of living cells and tissues. While effective, cryopreservation requires ultra-low temperature freezers and dry ice or liquid nitrogen for storage and shipment, limiting the widespread use of vCPM. Recently, a novel lyophilization technique has been developed that allows living tissue to be stored at room temperature. Similar to vCPM, lyopreserved placental membrane containing viable cells (vLPM) is a placental tissue allograft that retains the components of fresh placental membrane.¹⁵ The vLPM graft is categorized as a tissue allograft skin substitute and is intended for the management of acute and chronic wounds without restriction by wound etiology or location. Both vLPM and vCPM are 2 formulations of the same product with identical composition, structure, and properties.¹⁵ The only difference between vLPM and vCPM is the required storage and shipment temperature. 

The current study evaluated clinical outcomes of the vLPM for the treatment of chronic wounds. Here, the authors present the results of the first case series utilizing vLPM, a commercially available lyopreserved tissue allograft that retains the extracellular matrix, growth factors, and endogenous viable cells of fresh tissue. 

This was a single-center, prospective case series evaluating 11 patients receiving vLPM (GrafixPL PRIME; Osiris Therapeutics, Inc, Columbia, MD) for the treatment of chronic wounds. The vLPM graft is aseptically processed from donated human placental tissue following rigorous quality assurance standards and is stored and distributed for use in accordance with the regulations outlined in 21 Code of Federal Regulations 1271 and the standard of the American Association of Tissue Banks (AATB). All donors have been extensively screened, and all tissues have been recovered, processed, stored, tested, and distributed in accordance with current US Federal Regulations, current AATB standards, and state/local regulations as required. Every lot is tested per United States Pharmacopeia (USP) <71> sterility tests, residual moisture content analysis per USP <921> water determination, and custom in vitro assays to determine the presence of epidermal growth factor and the presence of viable cells across ≥ 70% of the tissue tested.¹⁶ Currently, vLPM has a 12-month shelf life.

Institutional Review Board approval was not required per the Center for Clinical Research (San Francisco, CA). Written informed consent was obtained from each patient before receiving the vLPM, and all patient data were de-identified. 

In order to be eligible for vLPM application, patients were required to have a chronic wound of at least 4-weeks’ duration, be at least 18 years of age or older, and not be a participant in any other skin substitute trials during the course of this study. All patients had to have previously failed SOC treatment, at which point the investigator elected to use vLPM. 

Prior to product application, SOC consisted of cleaning and debriding the wound bed as needed. A vLPM graft then was applied and secured per the manufacturer’s specifications.¹⁶ Patients could receive up to 12 weekly applications of vLPM. The treatment regimen frequency could be changed or discontinued per investigator discretion. Following vLPM application, a nonadherent dressing was applied on top of the graft and secondary dressings were applied as needed. All DFUs were offloaded with a fixed ankle walker, and VLUs received 3M Coban 2 Two-Layer Compression System (3M, St Paul, MN).
 

Analysis of clinical outcomes
The primary outcome of the study was the proportion of patients who achieved complete wound closure by the end of treatment. Complete wound closure was defined as 100% reepithelialization, as determined by the investigator. Additional outcomes included mean time to closure, mean number of vLPM applications, the proportion of patients who achieved ≥ 50% wound area reduction by day 28, and number of product-related adverse events.

Patient demographics and wound characteristics are outlined in the Table. Of the 11 patients selected to receive vLPM, 8 were men and 3 were women with an average age of 59.9 years (range, 38-74 years). There were 5 patients with DFUs, 5 with VLUs, and 1 with a pressure ulcer. Six patients had a history of hypertension, 2 had a history of deep vein thrombosis (DVT), and 7 patients had diabetes. Other collective medical history included hyperlipidemia, hepatitis C, anemia, gastroesophageal reflux disease, stage 3 chronic kidney disease, end-stage renal disease (ESRD), and rectal cancer. The average wound size was 6.2 cm² with an average wound duration of about 17.8 months.

Seven of the 11 patients (63.6%; 3 DFUs, 3 VLUs, 1 pressure ulcer) who received vLPM achieved complete wound closure with a mean time to closure of 47.1 days (range, 15-85 days) and a mean of 6 vLPM applications (range, 2-12 applications). The proportion of patients who achieved ≥ 50% wound area reduction by day 28 was also 63.6%. Of these 7 patients, 6 achieved complete wound closure. The 4 patients who did not achieve complete closure were considered treatment failures, as the wounds were not progressing towards closure in a timely manner. These patients were removed from the study early to pursue other treatment options. There were no adverse events attributed to vLPM application. Clinical outcomes for each patient are included in the Table. Detailed summaries for 3 representative cases are presented below. 

Case 1
A 54-year-old man with a history of hypertension, hyperlipidemia, type 2 diabetes mellitus, a right transmetatarsal amputation, and a left fifth toe amputation presented with a chronic right dorsal DFU of more than 2-years’ duration. The ulcer had previously been treated with Enluxtra(hydrogel fiber dressing [HFD]; OSNovative Systems, Inc, Santa Clara, CA) alone. 

The patient was seen weekly for treatment. Prior to application, the wound was cleaned and debrided. After vLPM application, the wound was dressed with HFD, Kling conforming bandage (Johnson & Johnson, Skillman, NJ), and paper tape and then offloaded. At the initial treatment application, the wound measured 4.75 cm². At day 28, the wound measured 2.0 cm², resulting in a percent area reduction of 57.9%. The ulcer achieved complete closure at day 85 after receiving 10 applications of vLPM (Figure 1).

Case 2
A 62-year-old man with a history of venous insufficiency, DVT, human immunodeficiency virus, and anticoagulant therapy presented with a VLU of 1-month duration on the right leg. The ulcer had previously been treated with collagen powder and HFD.

The patient was seen for weekly treatment. Prior to application, the ulcer was cleaned and debrided. After application, the ulcer received HFD, Kerlix(gauze dressing; Covidien, Mansfield, MA) and 2-layer compression wrap. At the initial treatment application, the ulcer measured 3.9 cm². The ulcer achieved complete closure at day 30 after receiving only 3 applications of vLPM (Figure 2). 

Case 3
A 59-year-old man with a history of type 2 diabetes mellitus, ESRD, hyperlipidemia, DVT, diabetic peripheral neuropathy, and obesity presented with a chronic pressure ulcer of 12-months’ duration on the left posterior medial heel. The ulcer had previously been treated with HFD, SANTYL (Smith & Nephew, Inc, Fort Worth, TX), and Apligraf (Organogenesis, Canton, MA).

The patient was seen for weekly treatment. Prior to vLPM application, the ulcer was cleaned and debrided. After application, the ulcer received HFDand gauzedressing. At the initial treatment application, the ulcer measured 1.0 cm². The ulcer achieved complete closure at day 35 after receiving 3 applications of vLPM (Figure 3).

As a lyopreserved amniotic membrane, vLPM retains the extracellular matrix, growth factors, and endogenous neonatal mesenchymal stem cells, fibroblasts, and epithelial cells of the native tissue.¹⁵ In this prospective, single-center case series, the authors evaluated clinical outcomes of the vLPM product for the treatment of chronic wounds. Five DFUs, 5 VLUs, and 1 pressure ulcer were treated with weekly applications of vLPM, demonstrating an overall closure rate of 63.6%. 

Previously, positive clinical outcomes had been demonstrated for the cryopreserved formulation of placental membrane. In a multicenter, pivotal, randomized controlled trial,¹¹ vCPM was used as an adjunct to SOC in the treatment of chronic DFUs. Of the 97 enrolled patients, 50 received vCPM plus SOC and 47 received SOC treatment alone. The proportion of patients who achieved complete wound closure was significantly higher in the vCPM arm compared with the SOC arm (62% vs. 21%).¹¹ 

Similar closure rates have been seen in various other clinical studies using cryopreserved placental membrane. Such studies include a multicenter, prospective, open-label study¹³ in which complex DFUs were treated with vCPM. The results demonstrated a closure rate of 59.3% at 16 weeks.¹³ Additionally, in a retrospective, single-center comparative-effectiveness study,¹⁴ treatment of various types of wounds with vCPM resulted in a 63% closure rate.Regulski et al¹² demonstrated in a retrospective, single-center, open-label study that treatment with vCPM resulted in a 76% closure rate for multiple wound types. Also, in a retrospective analysis evaluating the effectiveness of vCPM, Raspovic et al¹⁷ found 59.4% of 350 chronic DFUs achieved complete closure at the end of treatment. Finally, Farivar et al¹⁸ conducted a single-center, prospective study evaluating clinical outcomes of vCPM in the treatment of chronic VLUs. Thirty VLUs were evaluated, and 53% achieving complete closure.¹⁸ Figure 4 is a graphic representation of wound closure rates across multiple studies utilizing vCPM.^11-14,17-19 These rates are in line with the 63.6% closure rate achieved with the lyopreserved formulation of the product in the current study. The benefits of vLPM adjunct to SOC for the management of chronic wounds reported in this study are also in line with a recently published clinical case¹⁹ reporting successful wound closure of a chronic radiation wound.

Until recently, cryopreservation was the only method that allowed preservation of living tissues and cells for a prolonged period of time. While cryopreserved products containing viable cells, such as vCPM, hFDS, and a cryopreserved split-thickness allograft, have demonstrated clinical effectiveness, widespread usage of such products has been limited by the need to have ultra-low temperature freezers and/or access to dry ice for storage. The vLPM graft was processed using a novel tissue lyophilization technology that allows for the storage of viable tissues at room temperature. 

A recent publication showed that vLPM is equivalent to vCPM with the convenience of room-temperature storage.¹⁵ In addition, the preparation and application process for the lyopreserved product is faster than the cryopreserved formulation, as the graft does not require thawing or reconstitution prior to application. Because vLPM is applied directly onto the wound, vLPM can be rehydrated with wound fluid or with sterile saline in the case of dry wounds. The current results also demonstrate that the wound closure rate using vLPM for wound management as an adjunct to SOC is not different from closure rates reported previously^11-14,17-19 for the cryopreserved formulation of the product. 

The main limitation of the present case series is the small sample size as well as the limited eligibility criteria. Future prospective, randomized clinical trials with a more standardized protocol will be needed to further evaluate clinical effectiveness. 

In vitro and preclinical studies have shown that vLPM and vCPM are 2 formulations of the same product. The vLPM formulation provides room-temperature shipping and storage advantages as well as ease of preparation and application with no pre-application thawing or reconstitution required. This is the first case series in which clinical performance of vLPM in chronic wound management has been evaluated. Notably, clinical outcomes were similar to those reported in clinical studies with vCPM.

Note: The authors would like to thank the Osiris Therapeutics Clinical and Medical Affairs team for their assistance with preparation and review of the manuscript. 

Authors: Alexander M. Reyzelman, DPM¹; Mher Vartivarian, DPM¹; Alla Danilkovitch, PhD²; and Molly C. Saunders²

Affiliations: ¹Center for Clinical Research, San Francisco, CA; and ²Osiris Therapeutics, Inc, Columbia, MD

Correspondence: Molly Saunders, Osiris Therapeutics, Inc, 7015 Albert Einstein Drive, Columbia, MD 21046; MSaunders@Osiris.com

Disclosures: This case series was funded by Osiris Therapeutics, Inc (Columbia, MD). Dr. Reyzelman received research funding from Osiris Therapeutics for his participation in the study; Dr. Reyzelman has previously received honoraria for speaking on Osiris Therapeutics' behalf. Ms. Saunders and Dr. Danilkovitch are employees of Osiris Therapeutics.