Cryopreserved Placental Membranes Containing Viable Cells Result in High Closure Rate of Nonhealing Upper and Lower Extremity Wounds of Non-Diabetic and Non-Venous Pathophysiology

Nonhealing wounds affect approximately 2% of the US population, with the majority of patients affected being at least 65 years of age.¹ In an analysis of a 2014 Medicare database, wound prevalence among beneficiaries was determined to be nearly 15%. Of these, the most prevalent types of wounds were surgical and traumatic. However, the majority of published clinical studies have evaluated wound closure outcomes for diabetic foot ulcers (DFUs) and venous leg ulcers (VLUs), and there is a lack of data for the more prevalent types of wounds (ie, surgical and traumatic).²

Nonhealing wounds continue to present a therapeutic challenge to physicians. Typical standard of care (SOC) for such wounds includes thorough evaluation of systemic issues that may affect wound healing, cleansing and debridement, maintenance of a moist wound environment, infection and biofilm management, nutritional support, and offloading and/or compression dependent on wound location. For burns specifically, hemodynamic resuscitation, pain control, prevention of scarring, and rehabilitation are also part of the SOC regimen.³

For difficult-to-heal wounds, typically in patients with significant comorbidities, SOC alone is not enough to achieve closure.⁴ Subsequently, advanced therapies such as skin substitutes are often recommended for use adjunct to SOC.^5,6 One such skin substitute is cryopreserved placental membrane containing viable cells (vCPM). A vCPM allograft is a cryopreserved placental membrane, derived from amnion or chorion, that retains the extracellular matrix, growth factors, and endogenous neonatal cells, including mesenchymal stem cells of the native tissue.^7,8 The main difference between the 2 products is that vCPM-amnion contains an epithelial layer, whereas the vCPM-chorion does not.

Previously, positive clinical outcomes with the use of vCPM have been demonstrated in various DFU and VLU studies. In 2013, a retrospective single-center study showed an 85.2% closure rate for 27 chronic DFUs and a 67.6% closure rate for 34 chronic VLUs when vCPM-amnion was used adjunct to SOC.⁹ In a multicenter, prospective, randomized controlled trial of 97 total patients, Lavery et al¹⁰ demonstrated a 62% closure rate in the treatment of chronic DFUs using vCPM-amnion adjunct to SOC with a faster time to closure compared to SOC alone. In a retrospective analysis of 350 chronic DFUs in a real-world setting, Raspovic et al¹¹ showed a 59.4% closure rate by the end of treatment using vCPM-amnion and vCPM-chorion. Lastly, Farivar et al¹² showed a 53% closure rate in 30 chronic VLUs with vCPM-amnion treatment after failing 12 weeks of standard therapy.

With published vCPM data for DFU and VLU studies demonstrating safety and efficacy, vCPM coverage for these types of wounds among different payers is fairly widespread. The use of vCPM in the treatment of other wound types is considered investigational and is not considered medically appropriate due to lack of clinical evidence. However, vCPM has been used in the treatment of other types of wounds. Based on previously published data, the authors hypothesized that positive clinical outcomes would be seen in the treatment of nonhealing upper-extremity and lower-extremity wounds of nondiabetic and nonvenous pathophysiology with vCPM adjunct to SOC.

Study design
This was a single-center retrospective analysis evaluating the clinical outcomes of vCPM plus SOC in the treatment of upper-extremity and lower-extremity wounds of nondiabetic and nonvenous pathophysiology. To be included in the study, patients were required to have a nonhealing wound that was neither a DFU nor a VLU and had to be receiving vCPM as part of their treatment regimen. Nonhealing wounds were defined as either present for at least 4 weeks with no progression toward closure, or those in patients with significant comorbidities that put them at a high risk of nonclosure. Patients could not be receiving any other skin substitutes during the course of vCPM treatment. Data were deidentified and retrospectively collected through chart review. This study was conducted in compliance with the principles outlined in the Declaration of Helsinki. Due to the retrospective nature of this study, Institutional Review Board approval was waived.

Graft description and treatment regimen
Both vCPM-amnion (Grafix PRIME; Osiris Therapeutics, Inc.) and vCPM-chorion (Grafix CORE; Osiris Therapeutics, Inc.) are aseptically processed from donated human placental tissue following rigorous quality-assurance standards. The vCPM product is a minimally manipulated tissue allograft indicated for use as a cover for acute and chronic wounds without restriction to etiology or location. It is supplied in sheet form and packaged within a cryobag contained within a heat-sealed pouch. To thaw, the cryobag is removed from the pouch and placed into room temperature sterile saline or water. Once thawed, the graft is removed from the pouch and placed into a second sterile basin with saline to rinse. After rinsing, the graft is removed from the plastic backing and placed onto the wound. It is supplied in several different sizes (3 cm x 4 cm, 2 cm x 3 cm, 1.5 cm x 2 cm, 16 mm disc) and can be stored between -75ºC and -85ºC.^7,8

Wounds were treated and dressed according to the Bozeman Health Wound and Hyperbaric Center SOC which is in accordance with nationally accepted guidelines. Such guidelines state that SOC consists of evaluating wounds for any systemic issues that would impair closure, cleansing and debridement, maintenance of a moist wound environment, infection and biofilm management, nutritional support, management for edema and exudate, and offloading and/or compression dependent on wound location.³ At the Bozeman Health Wound and Hyperbaric Center, it is routine to use DNA sequencing to identify biofilm and address the findings prior to using any advanced skin substitutes. Based on physician interpretation of the molecular findings, topical antibiofilm agents were utilized before any vCPM allograft was placed. Prior to application of vCPM, wounds were cleaned and debrided with a curette to remove dead and necrotic tissue. Cleansing depended on provider preference, but usual practice involved a hypochlorous acid/sodium chloride cleanser. Patients received vCPM-amnion, vCPM-chorion, or a combination of both. The lack of the epithelial layer makes chorion-derived vCPM “jelly-like,” which makes it better suited for deep or tunneling wounds. On the other hand, vCPM-amnion is recommended for more superficial wounds. For patients who received both types, these grafts were not applied simultaneously; rather, the graft type was chosen on a week-to-week basis. The graft was applied weekly as recommended for most cases; however, in some cases, the frequency of applications deviated at the investigator’s discretion. The number of grafts used per patient varied based on the size of the wound. Graft was applied to cover the entire wound. Following vCPM application, wounds were typically dressed with either Mepitel(MölnlyckeHealth Care) or ADAPTIC TOUCH (KCI, now part of 3M Company) in accordance with manufacturer’s instructions. Patients typically returned to the clinic on a weekly or biweekly basis for ulcer assessments, which included ruler measurements and photography of the wound, as well as adverse event (AE) assessments. Patients were followed for clinical and safety outcomes until complete wound closure, or until the investigator terminated vCPM application.

Clinical outcomes and statistical analyses
The primary endpoint was complete wound closure, defined as 100% re-epithelialization, as determined by the investigator. Additional endpoints included time to closure, number of applications, percent area reduction (PAR) for nonclosed wounds, and number of vCPM-related AEs.

Separate analyses were conducted of vCPM-chorion and vCPM-amnion treatment groups as well as of upper-extremity and lower-extremity wounds. Amnion and chorion have been shown to have similar structural and functional properties.¹³ Due to this fact, the authors hypothesized that results would suggest no difference in closure outcomes between the 2 comparator groups.

Descriptive statistics for categorical variables are presented as frequencies and percentages, and as means, standard deviations, medians, and ranges for continuous variables. P values were determined using a X² test and Kruskal-Wallis test for nonparametric samples for categorical and continuous variables, respectively. A P value of less than or equal to .05 was considered significant.

Patient demographics and wound characteristics
Ninety-two patients with 104 wounds treated between January 2016 and May 2019 with vCPM plus SOC satisfied the eligibility criteria. Patient demographics and wound characteristics are presented in Table 1 and Table 2. Of the 92 patients treated with vCPM, 42 were male and 50 were female, and the average age was 60.1 years. Significant comorbidities included hypertension (32.6%), diabetes mellitus (18.5%), hyperlipidemia (16.3%), coronary artery disease (10.9%), venous insufficiency (6.5%), congestive heart failure (5.4%), and chronic obstructive pulmonary disease (3.3%). Sixty-seven of the 104 wounds (64.4%) were surgical, 17 were traumatic (16.3%), 10 were burns (9.6%), and 10 were considered other wounds, which consisted of chronic ulcers, pressure ulcers, necrotizing fasciitis, and a pilonidal cyst (9.6%). Sixty-four wounds were upper-extremity wounds (61.5%) and 40 were lower-extremity wounds (38.5%). The median wound size was 3.15 cm² (range, 0.02 cm²–210 cm²) with a median duration of 1.50 months (range, 0.03–180 months). Of the 104 wounds analyzed, 16 wounds received only vCPM-amnion applications, 78 received vCPM-chorion applications, and 10 received a combination of both.

Clinical outcomes
Eighty-seven of the 104 wounds (83.7%) achieved complete wound closure with vCPM application in a median time of 41 days and with 3 applications (Figure 1). Eighty-five percent of surgical wounds, 82% of traumatic wounds, 90% of burns, and 60% of other wounds achieved complete closure. Fifty-three of the 64 (82.8%) upper-extremity wounds and 34 of the 40 (85.0%) lower-extremity wounds achieved complete closure (Figure 2). Further, 15 of the 16 wounds (93.3%) that received vCPM-amnion, 64 of the 78 wounds (82.1%) that received vCPM-chorion, and 8 of the 10 wounds (80.0%) that received both products achieved complete wound closure (Figure 3). There were no statistically significant differences in closure rates between the upper and lower extremities, nor between graft types. Further, there were no AEs related to vCPM application.

Forty-five of 94 wounds (48%; duration missing for 10 wounds) were treated within 1 month of onset. Of these, 40 wounds (89%) achieved complete closure in a median time of 43.5 days (range, 8.0–177 days). Forty-nine wounds had greater than 1-month duration, and of these, 38 achieved closure in an average of 36 days (range, 6.0–409 days). These results were not statistically significant (P = .77).

Seventeen of 104 wounds (16%) did not achieve complete wound closure. These wounds had a median baseline wound size of 5.85 cm² (range, 0.2–210 cm²) and a median duration of 2 months (range, 0.35–180 months). With vCPM application, nonclosed wounds achieved a median PAR of 54.2, and 4 wounds increased in size.

The main objective of this retrospective, single-center study was to evaluate clinical outcomes of vCPM, a cryopreserved placental allograft, in the treatment of upper-extremity and lower-extremity wounds of nondiabetic and nonvenous pathophysiology. Currently, there are several advanced options often used in the treatment of nonhealing wounds. One such treatment is negative pressure wound therapy (NPWT), which has been shown to promote granulation tissue and tissue perfusion, as well as to decrease edema, bacterial colonization, and wound drainage.¹⁴ Hyperbaric oxygen (HBO) therapy has also been used in the treatment of nonhealing wounds, often in conjunction with a flap or graft. It is hypothesized that HBO therapy increases oxygen supply to the wound area, which is essential for wound healing.¹⁵ Additionally, split-thickness skin grafts (STSG) can provide permanent coverage for larger surface areas and can be meshed with variable expansion ratios. However, the success of STSGs relies on a well-vascularized wound bed with healthy granulation tissue.¹⁶ Moreover, NPWT, HBO, and STSG treatments are typically reserved for larger and more complex wounds. More recently, skin substitutes have been used in the treatment of non-DFU and non-VLU wounds. Such products include a bilayered dermal regenerative therapy, fetal bovine dermis, and porcine intestinal submucosa.^17-19 Although the literature supports the use of these treatment modalities, overall, data from larger, well-designed randomized, controlled trials are limited. Further, most institutions do not have an established protocol for non-DFUs and non-VLUs, and physicians are choosing treatment based on personal preference.

Many patients with non-DFU and non-VLU wounds also have significant comorbidities that negatively affect wound healing. In the present study, 58 of 92 patients (63%) had a significant comorbidity and/or were a current smoker. Previously, vCPM has prospectively shown clinical benefits in the treatment of nonhealing wounds in patients with multiple comorbidities.^10,20-22 Because the mechanisms of wound healing and factors/comorbidities precluding wounds from closure are universal for wounds of various etiologies and locations, it was logical to select vCPM to be evaluated and analyzed in the treatment of non-DFU and non-VLU wounds. Here, 83.7% of wounds achieved complete closure in a median time of 41 days (mean, 76 days). These results are similar to those seen in smaller studies using vCPM in the treatment of non-DFU and non-VLU wounds.

In 2016, Johnson et al²³ reported outcomes of amnion and chorion vCPM applications in the treatment of various types of wounds. They showed a closure rate of 81.8% (9 of 11) for surgical wounds and 83.3% (10 of 12) for other types of wounds, which consisted of traumatic wounds, burns, pyoderma gangrenosum, and others.²³ In 2 case reports describing the management of full-thickness thermal burns, patients received weekly debridement and vCPM-amnion application along with occupational therapy. The first patient sustained a 55.4 cm² thermal burn with exposed bone and tendon and the second patient sustained a 4.7 cm² crush burn. Both patients achieved complete wound closure in an average of 63.5 days and 7.5 applications, and they regained full range of motion in the affected limb and the hand digit.²⁴ Typically, these patients would receive an STSG in the operating room. In these cases, the patients were able to avoid the OR and receive serial applications of vCPM in the outpatient setting. Anselmo et al²⁵ also evaluated weekly application of vCPM-amnion in the treatment of an arterial ulcer, a pressure ulcer, and a reoccurring pyoderma gangrenosum ulcer. The pressure and arterial ulcers achieved closure at 4 and 5 weeks, respectively. The pyoderma gangrenosum ulcer achieved a 64% reduction in size after 9 applications.²⁵ In a case report describing the use of vCPM-chorion in the treatment of a large necrotic nasal tip wound, the patient achieved aesthetic wound closure in 21 days with 2 vCPM applications.²⁶ Lastly, Golla et al²⁷ evaluated outcomes of vCPM-chorion for augmentation with surgical flap closure in 4 nonhealing pressure ulcers. All 4 patients achieved complete closure in an average of 7 weeks without recurrence at an average 12-month follow-up.²⁷ The above-referenced studies all demonstrate clinical benefits of vCPM application in the treatment of wounds of different etiologies and locations. These studies also suggest no differences in clinical outcomes between vCPM-amnion and vCPM-chorion.

Overall, there is a lack of sufficient clinical evidence for skin substitutes in the treatment of some of the most prevalent types of wounds. The present study suggests that vCPM can result in positive clinical outcomes for wounds of any location. Fifty-three of the 64 (82.8%) upper-extremity wounds and 34 of the 40 (85.0%) lower-extremity wounds achieved complete closure. This study also showed similar closure outcomes in patients treated with vCPM-amnion, vCPM-chorion, and their combination. Closure rates for wounds managed with vCPM-amnion, vCPM-chorion, or their combination were 93.3%, 82.1%, and 80.0%, respectively, suggesting clinical equivalence between amnion and chorion vCPMs.

The high closure rate reported in this study could potentially be explained by the fact that nearly half of the wounds received vCPM applications earlier in treatment. For DFUs and VLUs, advanced therapies such as vCPM are not indicated for use until the wound has failed at least 4 weeks of SOC. Forty-five of 94 wounds in the current study (48%) were treated within a month of onset. Of these, 40 wounds (88.9%) achieved complete closure by the end of the study in a median time of 43.5 days. Of wounds with greater than 1-month duration, 38 of 49 (77.5%) achieved complete closure in a median time of 36 days. The results here suggest that there could be clinical benefit to using vCPM early in patient care. This is the same conclusion drawn from an expert wound-care panel, which suggested that early intervention with vCPM could contribute to efficient wound closure, especially in patients who are high-risk.²⁸

This study reports outcomes for upper-extremity and lower-extremity wounds of nondiabetic and nonvenous pathophysiology. Results of this study demonstrate benefits of utilizing vCPM plus SOC for difficult-to-treat wounds. These data provide valuable insight for providers, hospitals, and payers as it relates to medically appropriate and necessary patient care for patients with nonhealing wounds.

The main limitations of this study include the retrospective nature of the study and the lack of a control group. This study was not powered to show statistical clinical significance. Additional limitations include lack of long-term outcome evaluation, such as durability and scarring, and lack of information regarding degrees for burn wounds in the study.

Many wound-care studies evaluate outcomes of skin substitutes in the management of chronic DFUs and VLUs. Unfortunately, there are limited published data regarding closure outcomes for wounds of other etiologies, such as surgical and traumatic, which tend to be more prevalent. This is the first large retrospective analysis of clinical outcomes utilizing vCPM, a placental tissue allograft type of skin substitute, for non-DFU and non-VLU wounds. This initial retrospective study provides evidence that suggests the use of vCPM may be beneficial in the treatment of nonhealing upper-extremity and lower-extremity wounds of nondiabetic and nonvenous pathophysiology, although larger controlled trials are needed to confirm these preliminary findings.

Authors: Eric L. Johnson, MD¹; Molly Saunders, BS²; Tanushree Thote, PhD²; and Alla Danilkovitch, PhD²

Affiliations: ¹Bozeman Health Deaconess Hospital, Wound and Hyperbaric Center, Bozeman, MT; and ²Osiris Therapeutics, Columbia, MD (Osiris Therapeutics is now operating as a subsidiary of Smith+Nephew, Inc.)

Correspondence: Eric Johnson, MD, Bozeman Health Deaconess Hospital, Wound and Hyperbaric Center, 905 Highland Boulevard, Bozeman, MT 59715; ejohnson@bozemanhealth.org

Disclosure: Dr Johnson previously received honoraria as a speaker for Osiris Therapeutics, Inc./Smith+Nephew. Ms. Saunders is a full-time employee of Osiris Therapeutics, Inc./Smith+Nephew. Drs Thote and Danilkovitch were previous full-time employees of Osiris Therapeutics, Inc./Smith+Nephew.