A Single-center, Retrospective Study of Cryopreserved Umbilical Cord for Wound Healing in Patients Suffering From Chronic Wounds of the Foot and Ankle

Objective. Chronic wounds are a significant issue not only in wound care facilities, but also in daily practice for general practitioners and specialists across a wide variety of disciplines. These wounds are primarily foot or lower extremity ulcers and can result from a combination of factors including neuropathy, vascular insufficiency, and impaired wound healing. In addition to a significant health care cost, ulcerations have a devastating impact on virtually every aspect of the affected patient’s daily life such as extensive pain, sleep impairment, restricted mobility, and work capacity.The objective of this single-center, retrospective study was to evaluate the clinical effectiveness of a human cryopreserved umbilical cord (cUC) allograft as an advanced therapeutic treatment modality for chronic, nonhealing lower extremity wounds. Materials and Methods. Following Institutional Review Board approval, data from all qualifying patients who had received cUC tissue treatment during a period of 16 months was collected retrospectively. A total of 57 patients presenting with 64 chronic wounds who received treatment with cUC and were treated by the same surgeon at a single wound care center were analyzed. Results. The average initial wound area was 6.85 cm² ± 16.29 cm². Overall, 51 of 64 wounds achieved complete healing, resulting in an overall wound-healing rate of 79.7%. For wounds that healed, the average wound-healing time was 5.53 ± 3.93 weeks, and an average of 3.43 ± 2.42 applications of cUC were used to achieve healing. Conclusion. Overall, these results demonstrate cUC may be effective in promoting the healing of chronic, lower extremity ulcers. In addition, this study suggests cUC may be a useful advanced tissue treatment modality with the potential not only to improve patient quality of life, but also positively impact rising health care costs associated with long-term treatment of such ulcers. Further exploration, including prospective, randomized controlled trials, is warranted to better understand the effectiveness of cUC.

Wound healing consists of a complex series of processes at the molecular, cellular, and tissue levels that act to repair damaged tissue and restore function.¹ Any disruption of the wound-healing cascade may slow the course of healing, resulting in chronic, nonhealing wounds. In particular, it is thought that a pathologically extended inflammatory phase causes a delay in the formation of mature granulation tissue and a parallel reduction in wound tensile strength in the healing process due to decreased synthesis of collagen, abnormal synthesis of extracellular matrix proteins, and decreased fibroblast proliferation,² resulting in the development of chronic, nonhealing wounds.

Chronic lower extremity wounds or ulcers including venous leg ulcers (VLUs), diabetic foot ulcers (DFUs), and pressure ulcers (PUs) are a significant burden on the health care system. The incidence of lower extremity ulceration is rising as a result of an aging population and increased risk factors such as smoking and obesity. The prevalence of such wounds is approximately 0.18% to 2% in the general population, and up to 5% in patients over 65 years of age.³ In the United States, it is conservatively estimated that the total cost of chronic wound treatment exceeds $50 billion annually.⁴

Chronic venous insufficiency is the primary cause of 60% to 80% of chronic leg ulcers.⁵ In ulcerations of the foot and ankle however, the majority of ulcers are a result of complications related to diabetes. The cost of treating diabetes and its complications is approximately $116 billion in the United States each year, with up to 33% directly linked to the treatment of foot ulcers.⁶ Diabetic foot ulcers are responsible for approximately 80% of all diabetes-related lower leg amputations, with the risk of re-amputation being extremely high in this disease state. In a longitudinal study, Izumi et al⁷ found more than 50% of amputees with diabetes are at a cumulative risk of requiring re-amputation within 5 years of the initial surgery. With the 5-year survival rate following an amputation between 50% to 74%,⁸ research shows foot and ankle ulceration is a significant contributor to high morbidity and mortality.

Treatment options for chronic wounds of various etiologies include standard compression dressings, advanced moist wound therapy, and topical ointments, as well as more advanced treatment modalities such as bioengineered tissues or skin substitutes and growth factors. These treatments have been used with varying degrees of success depending on the wound environment. More recently, some success has been achieved using newer technologies that include negative pressure therapies, hyperbaric oxygen treatment, and sound wave technology.^9-11

Amniotic membrane (AM) extends from the fetal membrane to the placental proper and is the outermost lining of the umbilical cord (UC). In addition to the amniotic epithelium, the UC is comprised of a glycosaminoglycan-rich substance called Wharton’s jelly. Amniotic membrane was first used in surgical applications in 1910 during skin transplantation procedures.¹² However it wasn’t until later in the 20th century that clinical use of AM tissue began to be more widely used in ophthalmic indications and subsequently gained popularity for use in open wounds,^13,14 burns,¹⁵ and leg ulcers.^16,17 Nonresolving inflammation is a hallmark of nonhealing wounds, including chronic ulcers of multiple etiologies^18,19 and a common threat of many diverse acute and chronic diseases. Studies have shown direct AM and UC transplantation into the wound bed^20-23 modulates local inflammation by delivering anti-inflammatory and antiscarring properties to promote wound healing (reviewed in Tseng et al,²² Dua et al,²⁴ and Meller et al²⁵), and as a result, AM/UC transplantation has become the standard of care for numerous ocular reconstructive procedures.

The actions of AM/UC tissues have been found to be modulated through a unique glycoprotein complex called the HC-HA/PTX3 complex.^26,27 In particular, this complex inhibits cell adhesion and proliferation and induces apoptosis of pro-inflammatory cells, as well as modulates cytokine signaling by upregulating anti-inflammatory cytokines and downregulating pro-inflammatory cytokines.²⁸

Years of wound healing experience in ophthalmology²³ has led to clear opportunities for this science to come forward as a new advanced tissue treatment modality to address the pathological inflammation associated with chronic, nonhealing lower extremity ulcers of multiple etiologies. The objective of this retrospective study was to determine the clinical effectiveness of a cryopreserved umbilical cord (cUC) allograft in promoting wound healing in patients suffering from one or more chronic, nonhealing lower extremity ulcers of multiple etiologies (DFUs, VLUs, PUs, and wounds from postsurgical dehiscence) that demonstrated similar complexity to Wagner Grades 1-2 ulcers. The hypothesis was that treatment of lower extremity ulcers with cUC as an advanced wound tissue therapy would demonstrate enhanced wound-healing rates compared to historically published standard-of-care controls.

Study population. After approval by the Institutional Review Board of the Veterans Administration Hospital System, a retrospective chart review was performed for 77 patients presenting with chronic lower extremity ulcers treated at the Central Texas Veterans Health Care System - Olin E. Teague Veterans’ Center, Temple, TX.  All patients in the review underwent wound debridement and application of cUC allograft (cUC; NEOX CORD 1K, Amniox Medical, Inc, Atlanta, GA) between November 1, 2013 and March 1, 2015 to determine the safety and effectiveness of cUC in healing lower extremity ulcers of varying etiologies specifically in this challenging patient population. The primary exclusion criteria for the study was significant noncompliance with instructions from the principal investigator (PI), patients who developed an infection resulting in amputation, or patients lost to follow-up after application of cUC. After clinical chart review, 23 patients were excluded from the study. Seven patients were lost to follow-up, 9 underwent amputation as a result of an infection unrelated to the study product, and 7 were noncompliant, resulting in a total of 57 patients eligible for inclusion in the study population. Collected clinical data included demographic information, as well as past and current medical history. In addition, data were also retrieved regarding assessment of wound measurement to document wound healing changes in all ulcers during the entire follow-up period; any concomitant therapy data were collected.

Clinical management. All patients were treated by the same surgeon, the Principal Investigator (PI), and underwent the same procedure. Briefly, for all patients at the time of initial application of cUC, the wound was debrided in either the clinic or the operating room and cUC was placed directly over the debrided wound bed to completely cover the index wound. All wounds were then secured with a nonadherent layer (Mepitel One, Mölnlycke Health Care, Norcross, GA), followed by saline moist gauze or a sterile dry dressing for larger wounds, and finally with a standard dressing (Kerlix Bandage Roll, Medtronic Minimally Invasive Therapies, Minneapolis, MN; or Kling Conforming Sterile Bandage Dressing, DeRoyal Industries, Inc, Powell, TN). Every patient was given standard offloading instructions including the use of a total contact cast, Cam boot, surgical shoe, crutches, or knee scooter if necessary. At weekly follow-up visits, if the cUC was no longer present in the wound bed and the wound had not fully healed, adequate debridement of necrotic and nonviable tissue was performed and another piece of cUC was applied as described above. If cUC was still present in the wound bed, the exposed wound bed was debrided as deemed medically necessary by the PI, taking care to leave any cUC still in the wound bed in place, and the wound was redressed as described above. 

Outcome measures. At initial application of cUC and at all follow-up visits, wound length and width was measured using a ruler to determine wound area progression over time. Complete wound healing was defined as 100% reepithelialization as determined by the PI. For those wounds achieving complete healing, the total time needed to achieve initial wound closure was assessed and plotted using Microsoft Excel 2011. In addition to wound closure, the relationship between the initial wound area and the time needed to achieve closure was assessed by subdividing the initial wound area into quartiles similar to the method described by Regulski et al.²⁹ The mean wound area, time to achieve wound closure, and average number of applications of cUC were compared between quartiles using an unpaired t-test. P < 0.05 between groups were considered to be statistically significant. 

Clinical features. A total of 57 patients presenting with 64 wounds were identified for inclusion into the study population. A summary of patient demographics is provided in Table 1. Briefly, the average patient age was 66.2 ± 12.1 years, and the entire patient population was male. Overall, the patient population presented with multiple comorbidities. Among the most significant comorbidities were diabetes (49/57), hypertension (48/57), peripheral neuropathy (43/57), coronary artery disease (29/57), hyperlipidemia (29/57), congestive heart failure (14/57), peripheral vascular disease (11/57), and chronic kidney disease (10/57). At initial presentation, 21 ulcers were in the rearfoot, 34 in the forefoot, 8 in the midfoot, and 1 in the right leg. During the course of treatment with cUC, some patients received concomitant therapies for treatment of their index ulcers (Table 1). Concomitant therapies used were transcutaneous oxygen therapy (8/64), collagenase ointment treatment (3/64), and silver dressing treatment (8/64). 

Wound healing following cUC application. A summary of the wound characteristics is provided in Table 1. The average wound area for all wounds at the time of application of cUC was 6.85 cm² ± 16.29 cm². All wounds underwent debridement of the wound bed to achieve a healthy, bleeding base followed by application of cUC to completely cover the wound bed. Of the 64 wounds treated with at least 1 application of cUC, 51 achieved complete healing as determined by the PI for an overall wound healing rate of 79.7%. For wounds that achieved complete epithelialization, the average time for healing was 5.53 ± 3.93 weeks, and the average number of applications of cUC was 3.43 ± 2.42. An assessment of wound healing progression over time revealed 11.8% (6/51) of wounds healed within 1 week of cUC application, 51% (26/51) healed by week four, 80.4% (41/51) healed by week eight, 90.2% (46/51) of wounds achieved complete closure within 12 weeks of care, and all wounds completely healed by 16 weeks following the initial application of cUC (Figure 1). Of the 51 healed wounds, 12 received additional treatments in conjunction with cUC (23.5%). Due to the small number of patients analyzed in this study, no statistical analysis could be performed on data stratified by wound type or location; however, an overall summary of wound healing based on these parameters is outlined in Tables 2 and 3, respectively. 

To further examine the ability of cUC to promote healing of chronic, nonhealing ulcers, wounds were separated into quartiles based on initial wound area (Table 4); and the total number of healed wounds and the average rate of healing was determined. Fourteen of 16 wounds (87.5%) achieved complete healing in quartile 1; 16 of 16 wounds (100%) healed in quartile 2; 13 of 16 wounds (81.3%) healed in quartile 3; and 8 of 16 (50%) wounds completely healed in quartile 4. For completely healed wounds for each quartile, the average time to healing was determined (Figure 2A). In the smallest wounds (quartiles 1 and 2), the average time to reach closure was 4 ± 2.58 weeks and 3.23 ± 2.49 weeks, respectively. In larger wounds, the time to achieve wound healing increased, averaging 6.54 ± 3.62 weeks in quartile 3, and 8.58 ± 4.64 weeks in quartile 4. Finally, the average number of applications of cUC needed to achieve wound healing in each quartile was determined (Figure 2B). Wounds in quartiles 1 and 2 required significantly fewer applications of cUC (2.46 ± 1.27 and 2.23 ± 1.36, respectively) compared to wounds in quartiles 3 and 4 (3.85 ± 2.15 and 5.33 ± 3.28, respectively). 

This retrospective study evaluated the clinical effectiveness of a cUC allograft as an advanced tissue therapeutic modality to treat chronic, lower extremity ulcers of various etiologies (VLUs, DFUs, PUs, and postoperative wound dehiscence). Overall, 79.7% of wounds healed with an average of 3.4 cUC allograft applications and an average time to healing of 5.4 weeks. The majority of the patients had significant comorbidities and had failed prior wound treatment modalities including collagenase ointment, silver dressing, negative pressure wound therapy, as well as other advanced skin subsitutes (EpiFix, MiMedx, Marietta, GA; Dermagraft and Apligraf, Organogenesis Inc, Canton, MA) before going on to healing with the use of a cUC allograft.

These results are encouraging as chronic ulcer healing is a significant issue for the medical profession today. Chronic wounds represent a significant health care burden around the world. In the United States alone, chronic wounds affect around 6.5 million individuals,³⁰ with a conservative cost estimate of $50 billion annually.⁴ In addition to the cost associated with the treatment of chronic ulcers, lower extremity wounds and ulcers significantly impact a patient’s life. A failure to heal affects a patient’s general health and quality of life negatively and the patient’s subsequent ability to work, ultimately resulting in increasing health care and socioeconomic costs. 

Published data on the healing rates for such wounds employing traditional standard of care for wound care therapy is low, with only 24% of ulcers achieving complete healing within 12 weeks.³¹ Several prospective and retrospective studies^32-36 have been performed examining the ability of advanced tissue therapy modalities to heal chronic wounds. Studies examining the clinical efficacy of a bioengineered human fibroblast-derived dermal substitute and a bilayered cell-based product reported 30% and 56% healing of DFUs at 12 weeks, respectively.^32,33 A large, retrospective analysis of a cryopreserved human skin allograft reported an overall healing rate of approximately 60% after 12 weeks of care.³⁴ More recently, studies examining the efficacy of amniotic membrane products in promoting wound healing have been conducted. A comparative analysis of a bioengineered living cellular construct versus a dehydrated human amniotic membrane allograft for the treatment of DFUs found overall healing rates at weeks 12 and 24 were 48% and 72% for the bioengineered living cellular construct versus 28% and 47% for dehydrated human amniotic membrane allograft, respectively.³⁵ In a prospective, controlled trial examining the efficacy of a cryopreserved amniotic membrane product for the healing of DFUs, an overall healing rate of 62% was found at 12 weeks; however, the average number of applications required to achieve wound closure was 6, compared to 3.4 in this study.³⁶ A fewer number of applications to complete wound healing may translate directly to a decrease in the overall treatment cost per patient. The current study lends further supporting evidence for the use of amnion tissues for chronic wound therapy and extends the above-referenced study findings to include cUC (amniotic membrane + umbilical cord) in addition to amniotic membrane tissue alone. 

Further, the authors found wound size at the time of presentation impacted both the total time to healing as well as the number of applications of cUC to achieve closure. In this study, the smallest 50% of wounds had an average time of 3.6 weeks with an average of 2.3 applications of cUC for complete epithelialization of the index wound. In larger wounds, the mean time needed to heal was approximately 7.6 weeks with an average of 4.6 applications of cUC. Similar to the results found in this study, a direct relationship between initial wound size and the time to complete healing has been reported using both an acellular dermal matrix and a cryopreserved human skin allograft.^34,37 While larger wounds received more applications of cUC, this study was not designed to determine the minimum number of cUC allograft applications to achieve healing. The application frequency of cUC was based on the standard treatment regimen of the PI, where cUC was continually applied as it degraded in the wound bed.

The use of human AM or UC tissue to modulate wound healing in different parts of the body has been increasing over the past several years.²³ The use of fetal tissues such as AM and UC was first reported in 1910 during skin transplantation procedures.¹² However, the use of these tissues was not very widespread until later in the 20th century when the clinical use of AM/UC tissues in a variety of ophthalmic indications began to increase.²³ Since then, their use in other indications, including the treatment of chronic wounds of various etiologies, has expanded with impressive clinical success.^38-40 Within the extracellular matrix of AM and UC tissues, a unique glycoprotein complex (HC-HA/PTX3) has been identified. The HC-HA/PTX3 complex has been demonstrated to impart many of the anti-inflammatory and antiscarring actions observed clinically in these tissues. This complex is formed by a covalent linkage between hyaluronan (HA) and heavy chain 1 (HC1) of inter-α- trypsin inhibitor (IαI).^27,41 This covalent HC-HA complex then may be tightly associated with pentraxin 3 (PTX3) to form the HC-HA/PTX3 complex.²⁶ Previous studies demonstrated the HC-HA/PTX3 complex present in cryopreserved AM and UC tissues acts by inhibiting pro-inflammatory cell adhesion, spreading, and proliferation, as well as by inducing apoptosis of pro-inflammatory cells.²⁸ The HC-HA/PTX3 complex has also been demonstrated to modify the local inflammatory cytokine milieu by increasing the expression of anti-inflammatory cytokines such as IL-10, while decreasing the expression of pro-inflammatory cytokines including TNF-α and IL-12. Finally, the HC-HA/PTX3 complex has been found to polarize macrophages towards a more reparative M2 phenotype.^26-28 By modulating the local inflammatory environment, a cUC allograft may resolve pathological inflammation present in chronic wounds, thus allowing such wounds to continue through the wound healing cascade and achieve closure.

The overall wound-healing rate of 79.7% found in this study is higher than historically published standard-of-care wound healing rates (approximately 24%), confirming the hypothesis that application of a cUC allograft can improve the healing of chronic wounds. While the results of this study are promising, there are some limitations including the retrospective nature of the study, small sample size, and lack of a control group. In addition, due to the retrospective study design, a small number (23%) of patients received concomitant treatments for their wounds while receiving cUC allograft. A more strictly controlled prospective randomized trial could be helpful to specifically isolate the direct clinical effect of cUC in treating chronic ulcers in the absence of concomitant therapies. However, the small percentage of patients who healed with concomitant therapy in addition to cUC in this study suggests the impact of these additional treatments were minor components of the overall 79% healing rate. While prospective randomized controlled trials are beneficial to demonstrate product efficacy in a strictly defined patient population and setting of care, products may perform differently in general clinical practice. The results of this study indicate cUC may be effective in healing chronic ulcers of various etiologies in a broad population of patients in a real world clinical environment. The results of this retrospective analysis are encouraging and suggest further research, including performing controlled and comparative studies, which evaluate the efficacy of cUC in promoting the healing of historically difficult-to-heal chronic wounds.

Affiliation: Olin E. Teague Veterans’ Medical Center, Central Texas Veterans Health System, Temple, TX

Correspondence:
Mark Couture, DPM
Central Texas Veterans Health Care System
Olin E. Teague Veterans’ Medical Center
Temple, TX 76504
drmark100@gmail.com 

Disclosure: The author is a consultant for Amniox Medical, Inc.