Injection of Cryopreserved Amniotic Membrane and Umbilical Cord Particulate for Pressure Injuries: A Retrospective Case Series

AMUC, amniotic membrane and umbilical cord; HIPAA, Health Insurance Portability and Accountability Act of 1996; IRB, institutional review board; NPIAP, National Pressure Injury Advisory Panel; NPWT, negative pressure wound therapy; PI, pressure injury; SIS, small intestine submucosa.

Affecting nearly 3 million people in the United States each year,PIs are a prevalent clinical problem.^1,2 Like other chronic wounds, PIs often do not progress through the orderly stages of the wound healing cascade and remain in a state of pathological inflammation.³ It is estimated that only 43% to 50% of PIs heal.^4,5 As a result, these wounds pose significant clinical and economic challenges to both the patient and the health care system. Padula and Delarmente reported that hospital-acquired PIs alone cost more than $26 billion annually in the United States and result in an increased hospital length of stay.⁶ Additionally, these wounds often affect patients’ physical, social, and psychological well-being.^7-10

The slow and often unattainable closure of PIs has prompted the development of innovative treatment modalities to improve wound healing. Cryopreserved AMUC is one potential modality that has been used for a variety of indications, including burn and complex wound dressings, owing to its anti-inflammatory, anti-scarring, and proregenerative properties.^11-13 Although AMUC grafts are more commonly used in the sheet configuration that is applied as a dressing over wounds,^11,12,14,15 AMUC also comes in morselized particulate powders that can be sprinkled or injected.¹³ Published data are limited, but a recent case series demonstrated complete healing in 5 chronic foot and ankle wounds treated with a single dose of AMUC particulate within 5 weeks in patients with medical conditions (smoking, arthritis, gout, and hypertension).¹³

Patients included in the current case series were treated for PIs in both the outpatient and long-term care settings. Many of these patients live in rural areas and have difficulty traveling, resulting in issues with irregular follow-up visits after graft placement and episodes of graft being discarded by outside providers. AMUC particulate offers the advantages of providing more surface area for biological interaction, the ability to penetrate deeper in the recipient site, and no potential for graft dislodgement. The particulate can also be stored in the clinic at room temperature. For these reasons, the authors’ institution started using AMUC injection in the clinic as a treatment for chronic PIs in March 2017. To evaluate the safety and effectiveness of these injections, a retrospective chart review of these cases was conducted.

A HIPAA-compliant, IRB-approved, single-center, retrospective chart review was conducted of patients with nonhealing PIs that were treated with AMUC particulate (NEOX FLO; Amniox Medical, Inc.) between March 2017 and November 2018. Patients were treated either in the clinic or in a long-term care setting by the authors’ team under the supervision of a single physician. Patients were eligible for inclusion if they were 18 years or older and had objective wound measurements at the time of injection and at follow-up visits. Patients were excluded if they were lost to follow-up before 12 weeks or did not adhere to physician orders. No patients with active osteomyelitis or who required surgical debridement were included.

AMUC particulate

The AMUC is derived from donated human placental tissue following healthy, live, full-term caesarean section births. AMUC is commercially attainable and is processed into a particulate powder. It is manufactured using a cryopreservation process at low temperatures that devitalizes the living cells but retains the biological characteristics relevant to the fresh AMUC tissue.¹⁶ The AM and UC tissue are morselized, lyophilized, and terminally sterilized. The AMUC particulate is packaged as a dry powder in a small vial and is stored at room temperature.

Treatment procedures

Although several clinicians performed these injections, all followed the same basic injection protocol. The index wound was cleaned with saline and, if necessary, debrided of slough or minimal necrotic tissue at bedside with sharp instruments (eg, scalpel or scissors) to achieve a healthy base. None of the patients underwent surgical debridement in the operating room. Following sharp debridement, wound measurements (length, width, and depth) were obtained with a ruler. PI length was defined as the longest edge-to-edge measurement of the PI from the 12-o’clock to the 6-o’clock position, width was measured perpendicular to the length at the widest point, and depth was the deepest vertical measurement perpendicular to the base obtained using a cotton tip swab. Undermining or tunneling was also measured, using the deepest point as the depth. AMUC particulate was reconstituted in normal saline at a concentration of 10 mg/mL to 15 mg/mL and then injected around the wound periphery and base. If there was a tunnel or tract, the tissue to each side of the tract and the tissue overlying the tract was injected if there was adequate thickness. A standard absorbent dressing was then applied, and wounds were offloaded as determined by the investigator. For 3 NPIAP stage 4 PIs larger than 100 cm² NPWT was applied after injection.

All patients were followed up until complete wound healing was achieved. During each follow-up visit, wounds were cleansed with a sterile saline solution, dressings were changed as needed, and measurements were obtained. When the wound was considered stalled based on appearance and clinical judgment, another AMUC injection was administered.

Outcome measures

Data collected from the medical charts included patient age, date of first visit, significant medical comorbidities, wound location, NPIAP stage, previous therapy, injection date and dosage, and wound dimensions at the time of injection and follow-up. The primary outcome measure of this study was the incidence of wound healing at 12 weeks. Incidence of wound healing was also measured at 24 weeks, 36 weeks, and 52 weeks. Time to wound healing was defined as the number of weeks until the wound volume was zero and complete reepithelialization was achieved. Average reduction in wound volume by 12 weeks was also calculated, and the proportion of patients with at least 90% reduction in wound volume was reported. Wound volume (length × width × depth) was used instead of area owing to the prevalence of undermining and tunneling. The tunnel volume was calculated and added to the calculated wound volume. Adverse events were also recorded and collected.

Statistical analysis

Study data were collected and entered in an Excel spreadsheet (Microsoft). Categorical variables were expressed as frequencies and percentages, whereas continuous variables were described as mean ± standard deviation or median (range). The Mann-Whitney U test was used to assess differences among continuous variables. The Fisher exact test was used to compare healing rates by patient characteristics. Two-sided statistical analyses were conducted with an α level of .05 to determine statistical significance using SPSS (version 20; IBM Corporation).

A total of 30 PIs in 24 patients were injected with AMUC particulate. Four PIs were excluded from analysis, either because the patients did not adhere to physician orders or because they transferred to other units and were lost to follow-up before 12 weeks. As a result, 26 wounds in 21 patients were included for retrospective analysis. Of these 26 PIs, 22 (85%) were classified as stage 4 PIs, and the remainder were classified as stage 2 (2 [8%]) or stage 3 (2 [8%]) PIs (Table 1). The majority of these wounds were located on the sacrum (35%), trochanter (23%), or buttocks (19%). In addition, prior treatment (NPWT, standard wound care, debridement, and/or surgical repair with skin flap) had been unsuccessful for all PIs (Table 1). During AMUC treatment, 3 stage 4 PIs larger than 100 cm³ also were managed using NPWT.

No complications or adverse reactions related to AMUC particulate injection occurred. Eleven patients (42%) received a second injection during the follow-up period, and 3 patients (11%) received a total of 3 or more injections. The median time between any 2 injections in a single patient was 8.1 weeks (range, 1–31 weeks).

Two patients who received 3 or more injections had wound volumes that were considerably larger than the average wound size of 47.1 cm³ ± 81.9. The third patient had a smaller wound, but that patient did not return for follow-up until 31 weeks after the first injection, by which time the wound volume had increased by 20%. A second injection resulted in reduced wound size to the baseline measurement at 6 weeks after that second injection, so a third injection was administered.

The average PI volume at initial injection was 47.1 cm³ ± 81.9 (range, 0.1–316 cm³). After AMUC treatment, complete wound healing was observed in 7 PIs (27%) at 12 weeks, 10 PIs (38%) at 24 weeks, 13 PIs (50%) at 36 weeks, and 14 PIs (54%) at 52 weeks. By 52 weeks, healing had occurred in 1 stage 2 PI (50%), 2 stage 3 PIs (100%), and 11 stage 4 PIs (50%). Wounds that achieved complete closure by 12 weeks were significantly smaller in volume at baseline compared with those that did not heal by 12 weeks (P =.008). Because of the large variation in initial wound size, the sample was separated into 2 groups based on the median initial wound size, whether less than or equal to 6.3 cm³ or greater than 6.3 cm³. Seven of 14 wounds (50%) with a baseline volume of less than or equal to 6.3 cm³ (ie, the median initial wound volume) healed by 12 weeks, compared with 0% of wounds (0 of 12) with a baseline volume greater than 6.3 cm³ (P =.004).

The median time to wound closure was 12.4 weeks (range, 5–52 weeks) (Figure; Table 2). Wounds with a volume greater than 6.3 cm³ required 29.6 weeks ± 14.3 to heal, compared with 9.0 weeks ± 3.8 for wounds with a volume less than or equal to 6.3 cm³ (P =.01). On average, wounds reduced in size by 88% ± 14 at 9.3 weeks ± 2.6; additionally, in 61% (14 of 23) of these wounds at least 90% reduction in wound volume was achieved. The mean number of office visits within 12 weeks of initial injection was 1.2 ± 0.8.

There was no statistical difference in healing based on the anatomic location of PI. Complete healing was achieved in 4 of 9 sacral wounds (44%), 5 of 6 trochanteric wounds (83%), 3 of 5 buttock wounds (60%), 1 of 4 ischial wounds (25%), and 1 of 2 other wounds (50%).

Three adverse events were observed in this retrospective analysis, although none of these complications is believed to be related to AMUC treatment. One patient required amputation owing to significant vascular issues despite 99% reduction in wound volume at 8 weeks post-injection. A second patient was admitted to the hospital with sepsis 3 months after the second injection and, as a result, at the fourth follow-up visit presented with an increase in PI volume. The third patient was transferred to a nursing home in another state and is now deceased.

This single-center, retrospective study evaluated the safety and effectiveness of AMUC particulate injection in promoting PI wound healing. No complications or adverse events directly attributable to treatment occurred. A total of 26 wounds (initial size, 47.1 cm³ ± 81.9), most of which were stage 4 PIs (85%), were treated with 1.8 injections ± 1.2 of AMUC. The injections resulted in complete wound healing in 7 (27%) wounds at 12 weeks, 10 wounds (38%) at 24 weeks, 13 wounds (50%) at 36 weeks, and 14 wounds (54%) at 52 weeks.

Comparatively, data from the US Wound Registry showed complete healing in 30% of PIs at 12 weeks and a 43% healing rate at a mean follow-up of 24.5 weeks ± 49.0 depending on the treatment used.⁴ A recent cohort analysis of wound outcomes obtained from a database of The Health Improvement Network reported similar results, with 50% of all PIs healed within 12 months from initial presentation.⁵ However, in that study only 21% of stage 4 PIs healed within 1 year, and complete wound closure was achieved at an average of 7.7 months. A study assessing the use of a radiant heat bandage for wound healing found that none of the stage 4 PIs in either the control group or the treatment group healed during the 12-week observation period.¹⁷ In this regard, the results of the present study are encouraging because 23% of stage 4 PIs (5 of 22) healed within 12 weeks and 50% (11 of 22) healed by 1 year, with an overall median time to wound healing of 3.8 months.

The AMUC membrane has been shown to be successful as an adjunct to healing in diabetic foot ulcers and venous leg ulcers,^11-15,18 and use of dehydrated amnion/chorion membrane on PI has been studied.¹⁹ To the knowledge of the authors of the present study, only 1 prior study has assessed the use of cryopreserved human AM membrane as a biologic dressing for PIs.²⁰ In that study, 75% of wounds in the AM group achieved wound closure (9 of 12) at 1-month follow-up, compared with 0% (0 of 12) in the control group. AM allografts were changed every 2 to 3 days, with an average use of 6.1 allografts ± 1.75 to achieve wound closure. Additionally, 88% of PIs were classified as stage 2 (21 of 24). In contrast, in the present study an average of 1.7 injections ± 1.6 of AMUC particulate were used for 85% of stage 4 PIs.

Other therapeutic modalities have also been evaluated for improving PI healing. The use of SIS wound matrix resulted in complete healing in 40% of PIs (27 of 67) at 12 weeks versus 29% (18 of 63) receiving standard of care alone.²¹ For stage 4 PIs, however, the healing rate dropped to 29% and 21% in the SIS group and the control group, respectively. A different study found that normothermic wound therapy achieved complete wound healing in 48% of PIs (10 of 21) at 12 weeks compared with 36% of control wounds (8 of 22), although the prevalence of stage 4 PIs in the study sample was not specified.²²

It is important to note that the aforementioned randomized clinical trials excluded patients with significant wound depth, undermining or tunneling, or comorbid diseases such as diabetes. These strict inclusion and exclusion criteria prompted the US Wound Registry to report a discrepancy between actual healing rates and those that are reported in randomized clinical trials.⁴ Although the healing rates were lower in the present real-world study (ie, 27% healed by 12 weeks), no patients were excluded from this study based on wound characteristics or comorbidities, and 10 PIs (42%) had extensive undermining/tunneling. In fact, injection of AMUC particulate led to a 90% reduction in PI volume in 61% of patients within 12 weeks. This is comparable to a 2019 study that reported 90% healing in 55% of patients treated with SIS wound matrix and in 38% of patients treated with the standard of care.²¹ This is important because moderate reductions in PI dimensions, even in the absence of complete healing, can decrease morbidity and substantially improve quality of life.²³ Additionally, 90% healing generally reflects wound contraction and granulation tissue formation and has been shown to significantly benefit patients.^21,24,25

The overall results of this retrospective study are promising considering that the majority of wounds were classified as stage 4 PIs, which have full-thickness skin and tissue loss with exposed bone, tendon, or muscle (as defined by the NPIAP classification²⁶),and nearly all PIs were located on the posterior (n = 19) or lateral (n = 6) pelvic region, presenting considerable challenges for pressure redistribution and offloading. Furthermore, these patients also had comorbidities, including severe malnutrition, pancreatitis, infection, osteomyelitis, and vascular disease.

Injection of AMUC particulate may help promote healing of recalcitrant PIs via several mechanisms. Stage 3 and 4 PIs have extensive inflammatory cell infiltration, which appears to be responsible for collagen matrix dissociation and wound chronicity.^27,28 Cryopreserved AMUC has been shown to reduce infiltration and induce apoptosis of proinflammatory neutrophils^29,30 and macrophages.^31-33 Additionally, PIs have elevated levels of inflammatory cytokines,²⁸ which have been shown to be downregulated or suppressed by AM membrane.^34-37 AMUC also promotes polarization of M2 macrophages,^38-40 which has been shown to promote wound healing and resolve inflammation.^41-43 In addition, AMUC exerts a direct anti-scarring effect by suppressing transforming growth factor β signaling at the transcriptional level.^35,44 Collectively, these actions may provide an optimal wound healing environment to promote regenerative healing.⁴⁴

Similar to other retrospective reviews, the current study is limited by the inability to control confounding variables and the lack of control group or randomization. Because most patients were outpatients, there was no control over nutrition, pressure redistribution, or repositioning strategies. Treatment and follow-up were not standardized, owing to absence of a prospective protocol; thus, frequency and dosage of AMUC particulate injections varied among patients, and wound measurements could not be consistently obtained on a weekly basis. The latter is especially important because weekly PI evaluations with thorough documentation have been recommended as critical to the successful treatment of PIs.^45,46 The average time between PI evaluations in the present study was 8.8 weeks. More frequent assessment might have allowed for more frequent injection and improved healing rates.

In the current retrospective review, injection of AMUC particulate was shown to promote healing of all stages of PI with efficacy similar to that of AMUC membranes. In this series, 54% of all PIs and 50% of stage 4 PIs healed by 1 year. This healing occurred even in the presence of comorbidities, and with limited follow-up or control of nutritional and pressure offloading. AMUC injection can be of benefit in managing PIs in outpatients, because there is no risk of inadvertent removal of the biologic graft by the patient or caregiver during dressing change. Future studies should consist of controlled, randomized, prospective evaluations and should enforce weekly follow-up visits with standard-of-care treatment.