An Open Label Prospective Pilot Study to Evaluate the Efficacy of Cryopreserved Amniotic Tissue Grafts for Chronic Nonhealing Ulcers

Purpose. To evaluate the efficacy of cryopreserved amniotic tissue (Neox Cord 1K Wound Matrix, Amniox Medical, Inc, Atlanta, GA) in treating chronic nonhealing leg ulcers. Material and Methods. Ten patients with chronic leg ulcers, 7 with venous stasis, and 3 that were idiopathic, were consecutively enrolled after being unresponsive to conventional therapies for at least 6 months. The amniotic tissue was applied on days 0, 3, 4, and 5, and at each weekly visit until week 7 when wound closure was < 50%. When wound reduction of > 50% was obtained, the amniotic tissue application was discontinued. At day 28, patients demonstrating no wound area reduction were discontinued and considered failed cases, while those who showed improvement continued follow up until complete healing or end of the 6-month study period. Results. Four cases were considered successful, as they reached 50% wound closure at day 4 through day 35; of these 4 cases, 3 cases demonstrated complete wound healing on day 10, day 49, and day 84, respectively. Four patients showed no wound area reduction while 2 patients were excluded because of wound infections unrelated to the graft. Conclusion. This pilot study suggests that cryopreserved amniotic tissue is safe, and may be considered as an alternative graft for treating chronic nonhealing leg ulcers of > 6 month’s duration. Further investigation into the mechanism and potential application to diabetic foot ulcers is warranted.

  Loss of a skin barrier in nonhealing wounds can lead to pain; fluid, heat, and nutrient loss as a result of exposure; decreased mobility; and, most importantly, infection, which can then escalate morbidity for the patient. Hence, it is highly desirable to promote wound healing in the shortest time possible. Conventional wound treatment for chronic leg ulcers include debridement, synthetic dressings, compression bandaging, autologous/allogenic skin grafts, and tissue engineered skin substitutes.¹

  Leg ulcers of different origins that do not heal after 6 months of treatment are considered to have a poor healing outcome.^2,3 The majority (ie, 70%-90%) of lower extremity ulcers are associated with venous stasis,⁴ in which the likelihood of healing by compression bandage alone is > 93% if ulcers are < 6 months old and < 5 cm² in size, but drops to < 15% if ulcers are > 6 months old and > 5 cm² in size.^2,5 For venous stasis ulcers > 6 months duration, the median time to wound closure is 190 days when treated with compression bandages alone; this is reduced to 92 days when an allogeneic skin substitute is added.⁶ For venous stasis ulcers > 1 year duration, the wound healing rate is 19% when treated with compression bandages alone, but improves to 47% when an allogeneic skin substitute is added.⁶ However, 53% of the ulcers still remained unhealed at the end of the 6 month study period. Collectively, the previously mentioned studies indicate a great challenge to heal chronic leg ulcers that are nonhealing for > 6 months.

  One potential alternative treatment may be to apply cryopreserved amniotic tissue, which adheres well to the wound as an effective skin barrier, reduces pain facilitating early mobilization, has an antimicrobial effect, and does not exhibit any immunological reaction. As a result of these qualities, cryopreserved amniotic tissue has been used to treat wounds in different types of tissue.^6-8 The use of amniotic membrane (AM) to cover an open wound was first reported by Davis⁹ as early as 1910; its use on wounds later expanded to include many other etiologies such as venous stasis,^10-13 decubitus,^10,14-16 diabetes mellitus,¹⁶ trauma,^16,17,18 and burns.^19-25 These studies suggest that AM is effective in promoting granulation and epithelialization, suppressing excessive fibrosis, imparting an antimicrobial and analgesic effect, adhering well to wound surfaces, and remaining versatile in regard to handling.^{11,12,17,20,26-28} However, only 1 study has tested cryopreserved AM in 15 patients with chronic venous leg ulcers with no tendency for healing for at least 3 months duration.¹¹ In this study, 12 out of 15 (80%) patients achieved at least 50% reduction of the baseline ulcer surface area at 3 months follow up; 3 out of 15 (20%) patients had complete healing.¹¹ These reported clinical results encouraged the authors to further evaluate the efficacy of cryopreserved AM on chronic nonhealing leg ulcers of > 6 months duration.

  Different forms of AM, (ie, fresh,¹⁰ boiled,¹² frozen,¹⁴ maintained in tissue cultures,¹³ lyophilized,¹³ or cryopreserved¹¹) have been used in the treatment of skin wounds caused by venous stasis. Theoretically, fresh AM has the least manipulation but contains living cells²⁹ that may exert additional negative systemic metabolic effects. However, in order to meet US Food and Drug Administration (FDA) guidelines regarding Current Good Tissue Practice (cGTP) to manufacturers, it is impossible to use fresh AM because it is necessary to quarantine procured placental tissues for vigorous screening against possible transmissible diseases. Cryopreserved AM processed with the CryoTek Method (Bio-Tissue, Inc, Doral, FL) ensures cells are devitalized and prevents transmission of communicable diseases.³⁰ Amniotic membrane prepared in this way has been widely used for ocular surface reconstruction for ophthalmology in a variety of clinical situations.^8,31-34 Cryopreserved AM promotes ocular surface wound healing with minimal inflammation and scarring, resembling “scarless” fetal wound healing; whether it can also deliver similar efficacy on chronic nonhealing skin wounds deserves exploration. As a first step toward this goal, an open label prospective pilot study to evaluate its safety and efficacy in treating chronic leg ulcers of > 6 month’s duration was carried out.

  Materials. Neox Cord 1K Wound Matrix (Amniox Medical, Inc, Atlanta, GA) is a cryopreserved amniotic tissue product procured from the umbilical cord of eligible pregnant women following elective cesarean section delivery under full informed consent and processed by the CryoTek Method according to cGTP. This cryopreserved amniotic tissue is designated by the FDA as a tissue product under Section 361 Human Cells, Tissues and Cellular, and Tissue-Based Products (HCT/Ps) because it is minimally manipulated; does not include any other article except water, crystalloids, or a sterilizing, preserving, or storage agent; and does not exert systemic effect as its action is not dependent upon the metabolic activity of living cells for its primary function.

  Patient enrollment. Ten consecutive patients with chronic nonhealing ulcers of either venous stasis or other etiologies were prospectively recruited by physicians through the private practice of Reiss, Kang, Burks, Jayanetti, and Pereda, MD, PA (South Miami, FL) between May 2011 and November 2011 based on the inclusion and exclusion criteria listed in Table 1. The study was conducted following written informed consent and according to the Declaration of Helsinki.

  Wound management method. After removal of previously applied dressings, topical lidocaine ointment was applied and/or periwound cleaning with gauze saturated with normal saline when necessary. Sharp debridement with curette was performed on day 0; thereafter, debridement with curette or gauze was only performed to remove purulence and graft residue. The cryopreserved amniotic tissue was placed on the wound, covered by a contact layer (Drynet Wound Veil, Smith & Nephew, Inc, St. Petersburg, FL), and if needed, secured with adhesive strips (3M Steri-Strip Wound Closure System, 3M Health Care, St. Paul, Minnesota) (Figure 1). This was followed by nonadherent absorbent dressing (Exu-Dry Wound Dressing, Smith & Nephew, Inc, St. Peteresburg, FL) and a regular bandage. For venous stasis ulcers, compression wrap (Profore, Smith & Nephew, Inc, St. Petersburg, Florida) was applied in place of a regular bandage.

  Treatment Plan and Evaluation. On day 0 immediately following sharp debridement, the cryopreserved amniotic tissue was applied for the first time. The patient was seen again on days 3, 4, and 5 to ensure there was no adverse reaction. At each follow-up visit up, through to complete healing or the end of the 6 month study period, wound size was documented by photographs with a ruler placed at the same surface level as the wound. The surface area was measured by tracing the wound perimeter of the photograph using ImageJ software (National Institutes of Health, Bethesda, MD). In addition, changes in each patient’s pain level and in other wound parameters including exudates, graft outcome, and surrounding skin, were noted. During each weekly visit until week 7, ulcers that showed < 50% wound closure compared to day 0 received another application of the cryoperesevered amniotic tissue, while ulcers that showed > 50% wound closure did not receive another application. Treatment was considered successful if ulcers showed > 50% closure compared to day 0 by the end of the study period. Treatment was considered unsuccessful if ulcers showed no wound reduction compared to day 0 by day 28, or showed < 50% wound closure by the end of the study period. If patients developed a clinically apparent infection they were excluded from further study.

 The 10 patients in this study consisted of 4 males and 6 females with an average age of 61.8 ± 14.3 years (Table 2). Of the 10 ulcers, 7 were associated with venous stasis while the remaining 3 were idiopathic in nature. Despite previous treatment, these ulcers remained nonhealing for an average of 1.9 ± 0.9 years and presented with an average surface area of 7.8 ± 5.0 cm² (Table 2). Seven ulcers were located on the ankle while the rest were located either on the shin, calf, or dorsum of the foot. All ulcers were singular except for one case, which had satellite ulcers.

  During the study, no adverse effects, such as increase in pain or in wound area, were observed. The graft showed different degrees of disintergration 1 week after application (Figure 2), when complete or near complete graft disintegration was observed in 5 patients, partial graft disintegration was observed in 3 patients, and minimal graft disintegration was observed in 2 patients. For all ulcers with at least partial graft disintegration, the wound bed had minimal purulence.

  By the end of the study, cases 5, 6, 8, and 9 were considered successful, having reached 50% wound closure at day 4 (case 9), day 14 (case 8), day 28 (case 6), and day 35 (case 5). Cases 6, 8, and 9 subsequently achieved complete wound healing at day 84, day 49, and day 10, respectively. The pre- and poststudy treatment wound appearances of these successful cases are shown in Figure 3. Cases 1, 3, 7, and 10 were considered failures because 3 of them (cases 1, 7, and 10) showed no wound size reduction by day 28, and the remaining case showed no wound size reduction by day 98, despite an initial slight reduction in wound size. All 4 ulcers had not healed by the end of the 6 month study period. Their pre- and post- treatment wound appearances are shown in Figure 4. The remaining 2 cases (2 and 4) were excluded because the ulcers became infected at day 28 and day 21, respectively. Although the average baseline ulcer surface area was larger in the failure cases compared to that in the success cases (10.6 ± 3.3 cm² vs. 5.3 ± 3.3 cm²), the difference was not statistically significant (P = 0.08). The total number of grafts used in the successful cases was less than in the failed cases (4.5 ± 2.5 units vs. 6.3± 1.9 units) but the difference was not statistically significant (P = 0.31). Due to the small sample size, no correlation between the outcome and other clinical parameters such as demographic, wound etiology, nonhealing period, previous treatment, and the number of applications of cryopreserved amniotic tissue graft was observed.

  In this pilot study, the authors were assured from clinical results that the use of cryopreserved amniotic tissue processed by CryoTek Method on nonhealing wounds was safe as no adverse event was noted. In the cases excluded due to infection, the microbial agents were identified as Staphylococcus, Acetobacter, and Prevotella oris in case 2, and Enterococcus in case 4 by their respective wound culturing. These microbial agents were not introduced by the graft as verified by the negative cultures of the post-processing sampling. The overall safety of using the CryoTek Method has been reported when cGTP guidelines were followed.³⁵

  In this study, the authors chose to test the efficacy of treatment in ulcers of more than 6 months duration, which is a known risk factor for poor wound healing outcomes even if limb-compression bandaging alone,^2,5,6 with or without allogeneic skin substitutes,⁶ is instituted. In fact, the average nonhealing duration was 1.9 ± 0.9 years for the 10 patients despite having received previous treatments that included debridement, synthetic dressings, and compression bandaging for venous ulcers. Additionally, 5 (50%) of the patients had received at least 1 form of skin substitute.

  After excluding the 2 patients with confirmed microbial infections, complete healing was noted in 3 of the remaining 8 patients (37.5%). This outcome is comparable to previous reports, in which 2 different allogeneic skin substitutes achieved complete healing in 38% (36/95) and 47% (34/72) of venous leg ulcers that were > 6 weeks old³⁶ and > 1 year old,³⁷ respectively.

  Limitations. The small sample size of this pilot study precluded the authors from correlating the outcome with other clinical parameters such as underlying cause and severity, which might be reflected by the extent of previous treatments, including the use of allogeneic skin substitutes. The authors also do not know whether the treatment regimen of applying another graft when the wound closure is < 50%—a protocol used for other allogeneic skin substitutes³⁷—is appropriate for cryopreserved AM. The application regimen is particularly noteworthy since the majority of the tissue grafts disintegrated after their first application, in contrast to what was observed with the allogenic skin grafts, which achieved engraftment, or “graft take,” with revascularization in 42% of the patients.⁶ While the previously mentioned allogeneic skin substitute consists of a bilayered tissue engineered skin construct of human and bovine origin, the amniotic tissue used in the current study consists of cryopreserved human amniotic tissue that is minimally manipulated during processing and hence may employ a different healing mechanism that may be effective for wounds that had previously remained unresponsive to treatment, as observed in case 5. Future control investigation with a larger sample size is warranted to resolve the above issues and to confirm the encouraging results noted in this pilot study, especially in chronic leg ulcers that are hard to heal despite the use of other modalities. If proven, additional studies into the action mechanism are also needed to confirm that cryopreserved amniotic tissue represents a new class of allogeneic graft in which the clinical efficacy does not depend on engraftment.

  This pilot study suggests that cryopreserved amniotic tissue is safe and may be considered as an alternative graft for treating chronic nonhealing leg ulcers of greater than 6 month’s duration. Further investigation into the mechanism and potential application to diabetic foot ulcers is warranted.

Lorraine Siok May Chua, MS is from Tissue Tech, Inc, Miami, FL. Julie O’Connell, PhD is from Amniox Medical, Inc, Atlanta, GA. Steven Kang, MD; James Burks, MD; Chaminda Jayanetti, MD; Juan Carlos Pereda, MD; and Ian Reiss, MD are from Reiss, Kang, Burks, Jayanetti & Pereda, MD, PA, South Miami, FL.

Address correspondence to:
Lorraine Chua, MS
TissueTech, Inc.
7000 SW 97th Avenue, Suite 212
Miami, Florida 33173
lchua@tissuetechinc.com

Disclosure: The authors disclose Ms. Chua is an employee of Tissue Tech, Inc, Miami, FL, and Dr. O’Connell is a consultant for Amniox Medical, Inc, Atlanta, GA. This research was supported by a research grant from Amniox Medical, Inc, Atlanta, GA, which also provided Neox Cord1K Wound Matrix for the purposes of this study.