Optimizing Non-Healing Venous Leg Ulcers and Diabetic Foot Ulcers: Standard of Care vs Amniotic Mem

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Optimizing Non-Healing Venous Leg Ulcers and Diabetic Foot Ulcers: Standard of Care vs Amniotic Membrane Editorial Summary This study is a retrospective review of comparative wound size changes when using standard of care (SOC) versus amniotic membrane (AM). The patient group had either non-healing venous leg ulcers (VLU) or diabetic foot ulcers (DFU). The inclusion criteria for this study was patients who had experienced minimal wound size change after 5-weeks of SOC, in effect considering amniotic membrane as an alternative treatment for a further 5-weeks of treatment. The wound sizes were measured at three points: 1) the initial treatment phase with SOC, 2) the end of SOC (after 5 weeks) or the beginning of AM treatment and 3) 5 weeks after the amniotic membrane treatment, as the comparative modality.

Introduction

S

tandard of Care (SOC) for non-healing wounds typically comprises of debridement of necrotic and infected tissue, establishing adequate circulation, maintaining a moist periwound environment, infection control, and offloading or compression dependent on the etiology of the wound itself.1 Standard of Care can vary due to clinician judgement and wound type. It is acknowledged as a time consuming and potentially less efficient method for treating chronic wounds which is why there has been a recent push for evidence based-innovation. One of which is Amniotic Membrane (AM) or amnion. AM has been evidenced in consideration of reconstruction due to the pluripotent properties of AM cells.2 AM has an avascular structure comprising of three layers containing collagen, extracellular matrix, and biologically active cells (mostly stem cells). Collagen is a naturally occurring matrix polymer and provides a structure to the amniotic membrane. Regulated by growth factors such as cytokines, chemokines, and other endogenous cells that are contained in the matrix of AM, this allows for epithelialization.

Dr Alton R. Johnson University of Michigan Ann Arbor MI, United States

The largest organ of the human body, skin is fundamentally the first line of defense, so when we consider the implication of chronic ulcers there is a severe infection risk with any breach of the epithelial surfaces leaving the patient vulnerable to cross contamination of bacteria and a possible site of sepsis. When this protective surface is compromised, it can lead to increased morbidity and mortality and increase the challenge of wound care.

Mr Shenlone Wu

Ms Briana Lay

University of Nevada, Las Vegas (UNLV)

University of California, Los Angeles (UCLA)

Las Vegas NV, United States

Los Angeles CA, United States

The classification of what constitutes a ‘chronic wound' is a wound persisting for more than 6-weeks, where no sign of healing has been highlighted.3 When a patient presents with a chronic wound, a clinician can identify this as they exhibit an stalled healing process that is different to an acute wound. This usually presents in the form of inflammation, wound infection, hypoxia, poor nutrition or possibly a biofilm element. Some of the factors that cause chronic wounds to persist include diabetes, weakened immune systems, and poor blood circulation.

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Optimizing Non-Healing Venous Leg Ulcers and Diabetic Foot Ulcers: Standard of Care vs Amniotic Membrane

“One study found that the combination method of SOC and AM resulted in an improved healing rate for patients with diabetic foot ulcers, in comparison to the use of SOC alone. However, not all chronic wounds result in a complete healing when using amniotic membrane as a method of treatment despite the advantages.”

When we consider non-healing chronic wounds, this is likely due to complications such as infections like cellulitis and/or osteomyelitis.4 In the context of current standard of care for chronic wounds this involves a number of steps, including; wound swabs, debridement of the wound to remove necrotic tissue, and dressing the wound and maintaining a moist environment to encourage healthy tissue re-epithelialization. There has also been advancements and developing technologies in wound care strategies that are now being implemented across the world. During the late 20th Century physicians began to experiment with AM as a form of wound treatment which provided various benefits like anti-inflammatory responses, bacteriostatic response (prevention of reproduction of microorganisms, not necessarily killing the bacteria), and scarprevention properties. These advantages of AM are generated from the cytokines that promote cell proliferation and differentiation.5 The Food and Drug Administration (FDA) has approved the use of AM for venous leg ulcers and diabetic foot ulcers. One main function of the AM is to provide tissue regeneration where there is a balance between the extracellular matrix (ECM), metabolically active cells and cellular signal mediators.6 The membrane itself is rich in collagen which is a protein responsible

for development of healthy joints and skin elasticity, and as we age our bodies produce less and find it equally difficult to continuously produce collagen. Therefore, with chronic wounds in older adults, AM offers a healthy and rich amount of collagen for skin regrowth and elasticity. This includes collagens I, III, IV, V, and VII. In addition, AM provides ECM, elastin, laminin, fibronectin, proteoglycans, and glycosaminoglycans as well as non-viable cells.7 As a matter of fact, AM provides over 200 natural bioactive proteins that are preserved within.8-10 AM itself is a thin and transparent lining of the chorionic layer of the placenta which comprises of two primary layers; an outer layer formed by the trophoblast and an inner layer that is formed by the somatic mesoderm. This allows AM to behave as a bioactive matrix that promotes fibroblasts and endothelial cell production. AM also promotes hematopoietic, mesenchymal and diabetic adipose stem cell migration.11 Combining all efforts, this results in cell proliferation, migration and biosynthesis.12-13 One study found that the combination method of SOC and AM resulted in an improved healing rate for patients with diabetic foot ulcers, in comparison to the use of SOC alone.14 However, not all chronic wounds result in a complete healing when using amniotic membrane as a method of treatment despite the advantages.

Methods Figure 1: 1a: Diabetic foot ulcer. 1b: Venous leg ulcer. 1a

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1b

In this retrospective study between 2018 2021, 23 patients treated by practitioners from Professional Wound Specialists were identified as meeting the inclusion criteria. The age range was from 33 to 89 years of age, 11 of the ulcers were proven to be diabetic foot ulcers, and a further twelve were identified as Venous Leg Ulcer (VLU) patients. The ratio of male to female patients was 18:5. All participants received 5-weeks of SOC followed by 5-weeks of AM applications. They were excluded if any of the following criteria were present:

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Optimizing Non-Healing Venous Leg Ulcers and Diabetic Foot Ulcers: Standard of Care vs Amniotic Membrane The administration of SOC treatment over the first 5-weeks is performed according to the guidelines suggested by the National Center for Biotechnology Information. The treatment options include silver gel, silver or calcium alginate, medical honey, collagenase, antibiotic ointment, negative pressure wound therapy (NPWT) amongst other treatment modalities. The choice of dressing is determined based on drainage percentage and whether the wound is emitting an odor. If the wound size change is insignificant after 5 weeks with SOC, AM is performed once weekly with no local or general anesthetic required for administration.

Exclusion Criteria Wound healing occurred within less than 5-weeks of AM applications Non-compliant patients resulting in an early termination of the study HbA1c of >10 in DFU patients Wounds presenting outwith the proliferation phase (i.e., infections) Disruption in the study such as acute changes to the participants medical status that resulted in a transfer of care

Table 4

Table 1 DFU Patients

Measurement 1

Measurement 2

Measurement 3

1

9 cm

8.7 cm

5.2 cm

2

2

2

15 cm

2

29.2 cm

27 cm2

3

12.5 cm2

14.3 cm2

5.1 cm2

4

16 cm2

16.8 cm2

14 cm2

5

37.8 cm2

39.1 cm2

15.6 cm2

6

12.3 cm2

12.3 cm2

7.8 cm2

7

36 cm2

38.2 cm2

31.5 cm2

8

29.9 cm2

34 cm2

4 cm2

9

19.5 cm2

18.6 cm2

9 cm2

10

12.3 cm2

16 cm2

3.6 cm2

11

9.9 cm2

12 cm2

8.4 cm2

Total Average

19.1 cm2

21.6 cm2

11.9 cm2

2

VLU Patients

% size change after SOC

% size change after AM treatment

1

-3%

-40%

2

195%

-7%

3

114%

-64%

4

105%

-17%

5

103%

-60%

6

100%

-37%

7

106%

-17%

8

114%

-88%

9

-5%

-52%

10

130%

-77%

11

121%

-30%

Total Average

113%

-45%

DFU Patients

% size change after SOC

% size change after AM treatment

1

100%

-60%

2

Table 2

Table 5

VLU Patients

Measurement 1

Measurement 2

Measurement 3

1

36 cm2

36 cm2

14.4 cm2

2

12.3 cm2

12 cm2

6 cm2

3

21 cm2

23.4 cm2

8.6 cm2

2

-2%

-50%

111%

-63%

4

33.1 cm2

31.3 cm2

4 cm2

3

5

38 cm2

28.5 cm2

15 cm2

4

-5%

-88%

6

28 cm2

15 cm2

8.4 cm2

5

-25%

-47%

7

28.6 cm2

27.6 cm2

16.8 cm2

6

-46%

-44%

8

28 cm2

18 cm2

7.6 cm2

7

-3%

-39%

9

26.9 cm2

30 cm2

18 cm2

8

-36%

-58%

10

14 cm2

22.1 cm2

10.5 cm2

9

112%

-40%

11

18 cm2

18 cm2

1.3 cm2

10

158%

-52%

12

7.3 cm2

8.4 cm2

1.8 cm2

11

100%

-93%

Total Average

19.1 cm2

21.7 cm2

9.4 cm2

12

115%

79%

Total Average

-11%

-57%

Table 3

10

DFU + VLU

Measurement 1

Measurement 2

Measurement 3

Total Average

21.8 cm

21.6 cm

10.6 cm2

2

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Optimizing Non-Healing Venous Leg Ulcers and Diabetic Foot Ulcers: Standard of Care vs Amniotic Membrane Figure 2: Amniotic product.

Table 6 DFU + VLU

% size change after SOC

% size change after AM treatment

Total Average

-1%

-51%

Table 7 Wound Type

N t score Two-tailed P-value

DFU

9 -3.63 0.0055

VLU

12 -4.66 0.0006

Total

21 -5.93 0.0001

Table 8 DFU Wound Area Measurements (cm2)

VLU Wound Area Measurements (cm2)

Patient

Initial size

After 5 weeks of SOC Treatment and Start of AM Treatment

5 weeks after AM & SOC Treatment

Patient

Prior to SOC Treatment

After 5 weeks of SOC Treatment and Start of AM Treatment

5 Weeks after AM & SOC Treatment

1

9

8.7

5.226

10

36

36

14.4

2

15

29.16

27

11

12.25

12

6

3

12.5

14.31

5.1

12

21

23.4

8.55

4

16

16.8

14.04

13

33.06

31.27

4

5

37.82

39.06

15.6

14

37.96

28.52

15

6

12.25

12.25

7.84

15

28

15

8.4

7

36

38.22

31.54

16

17.64

16.8

28.6

8

29.88

34

4

17

28

18

7.6

9

19.5

18.6

9

18

26.88

30

18

19

14

22.08

10.5

20

18

18

1.25

21

7.29

8.4

1.8

13.26

Mean

23.34

22.41

9.16

9.96

SD

9.81

8.43

Mean

20.88

23.46

SD

10.86

11.73

Total (N=23) Mean

22.29

The treatment protocol for AM application is to clean the wound bed and prepare the area for administration of the product. Debris was then removed, and curettage of the site was performed to ensure a clean and moist wound bed. AM was placed followed by a mesh and secured with steristrips. A secondary dressing was then applied to secure and maintain a balanced moisture environment for the wound bed. The procedure was repeated once weekly for the 5-week duration period. When collecting the data for this study, the initial measurements with SOC was recorded followed by 5 weeks of SOC treatment or the start of the AM treatment and then finally after 5 weeks of the subsequent AM treatment.

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22.41

5.44 10.92

Figure 3: Wound area measurement.

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Optimizing Non-Healing Venous Leg Ulcers and Diabetic Foot Ulcers: Standard of Care vs Amniotic Membrane

“When we look at the matrix of AM as a material with the epithelial and mesenchymal cells that possess characteristics of pluripotent stem cells (a capability to differentiate into all three germ layers), there is a distinctive indication for its usage.”

Data Analysis After the data was collected for the three measurements, the ulcer healing process was accessed using a percentage of healed wound area for the measurable outcome. The comparison was between the percentage of healing when SOC was administered, and the difference after AM application. When reviewing the patients with diabetic foot ulcers, the average initial size of wound was 19.1 cm2. After the initial treatment with SOC, the findings indicated an increase in measurement on average to around 21.6 cm2. However, after the administration of AM, there was a substantial decrease in the average to 9.4 cm2. decrease in the average to 9.4 cm2. This indicated an overall reduction percentage of -45%, thus proving the effectiveness of AM treatment for this type of chronic wound. In the VLU patients, the initial average size of the wound was around 24.3 cm2. There was then a slight decrease to 21.6 cm2 after the treatment with SOC. This indicated a reduction percentage of -11%. AM treatment was then administered, and a significant reduction was seen again as the wounds measured – on average – 9.4 cm2. Another substantial decrease in the wound size of around-57% after treatment course.

Discussion In this study, the experts intentionally chose challenging wounds to evaluate the efficacy of AM treatment, as demonstrated in the inclusion/ exclusion criteria for those with DFU and those with VLU. In both clinical wound types, the patients responded to the SOC treatment at a refractory rate that showed minimal change, and in some instances the wound responded by increasing in size. This is a clear indication for considering new modalities for treatment as the efficacy of SOC cannot be reproduced, or continued with in most cases as that will result in further wound deterioration and potential

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risk factors presenting like inflammation or proliferation of the wound itself. However, comparatively once the patients received the AM treatment in combination with SOC there was a significant improvement and overall greater response that led to wound healing and a decrease in the total surface area of the wound; seen in both DFU and VLU patients. This substantial difference therefore supports the efficacy of the treatment. Another discussion around the usage of AM treatment proposed that it can be used as a skin substitute and reconstructive option for treating deeper dermal wounds and full thickness wounds.15 In this study, the purpose of the AM is to scaffold and provide a cell source that has the same histological and physiological components as the skin. Therefore, when we look at the matrix of AM as a material with the epithelial and mesenchymal cells that possess characteristics of pluripotent stem cells (a capability to differentiate into all three germ layers), there is a distinctive indication for its usage. The study approved and acknowledged the properties of AM and its likeness to skin as an enriched cell that contains a scaffolding structure that could be translated into clinical management.

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Optimizing Non-Healing Venous Leg Ulcers and Diabetic Foot Ulcers: Standard of Care vs Amniotic Membrane

References 1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6798798/#:~:text=Current%20 standard%20 of %20care%20(SOC,depending%20on%20wound%20location%20and 2. https://pubmed.ncbi.nlm.nih.gov/22592624/ 3. Dreifke, M. B., Jayasuriya, A. A., & Jayasuriya, A. C. (2015). Current wound healing procedures and potential care. In Materials Science and Engineering C (Vol. 48) https:// doi.org/10.1016/j.msec.2014.12.068 4. Elheneidy, H., Omran, E., Halwagy, A., Al-Inany, H., Al-Ansary, M., & Gad, A. (2016). Amniotic membrane can be a valid source for wound healing. International Journal of Women’s Health, 8. https://doi.org/10.2147/IJWH.S96636 5. Kogan, S., Sood, A., & Granick, M. S. (2018). Amniotic Membrane Adjuncts and Clinical Applications in Wound Healing: A Review of the Literature. In Wounds : a compendium of clinical research and practice (Vol. 30, Issue 6) 6. Schultz GS, Davidson JM 7. Koob TJ, Lim JJ, Zabek N, Massee M. Cytokines in single layer amnion allografts compared to multilayer amnion/chorion allografts for wound healing. J Biomed Mater Res B Appl Biomater 2015 Jul;103(5):1133-40. 8. Koob TJ, Rennert R, Zabek N, Massee M, Lim JJ, Temenoff JS, Li WW, Gurtner G. Biological properties of dehydrated human amnion/chorion composite graft: implications for chronic wound healing. Int Wound J. 2013 Oct;10(5):493-500. 9. Koob TJ, Lim JJ, Massee M, Zabek N, Denoziere G. Properties of dehydrated human amnion/ chorion composite grafts: Implications for wound repair and soft tissue regeneration. J Biomed Mater Res B Appl Biomater. 2014 Aug;201(6):1353-62.

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10. Koob TJ, Lim JJ, Massee M Zabek N, Rennert R, Gurtner G, Li WW. Angiogenic properties of dehydrated human amnion/chorion allografts: therapeutic potential for soft tissue repair and regeneration. Vasc Cell. 2014 May 1;6:10. 11. Wu Qianqian et al. Comparison of the proliferation, migration and angiogenic properties of human amniotic epithelial and mesenchymal stem cells and their effects on endothelial cells. Int J Molecular Med Feb-2017 Vol 39 Issue 4 918-26. 12. Maan ZN, Rennert RC, Koob TJ, Januszyk M, Li WW, Gurtner GC, Cell recruitment by amnion chorion grafts promotes neovascularization. J Surg Res. 2015 Feb;193(2):953-62. 13. Massee M, Chinn K, Lim JJ, Godwin L, Young CS, Koob TJ. Type I and II Diabetic Adipose-Derived Stem Cells Respond In Vitro to Dehydrated Human Amnion/Chorion Membrane Allograft Treatment by Increasing Proliferation, Migration, and Altering Cytokine Secretion. Adv Wound Care (New Rochelle). 2016 Feb 1;5(2):43-54. 14. Irakoze L, et al. Efficacy and time sensitivity of amniotic membrane treatment in patients with diabetic foot ulcers: a systematic review and meta-analysis. Diabetes Therapy. 2017;8(5):967-79 15. Farhadihosseinabadi B, Farahani M, Tayebi T, Jafari A, Biniazan F, Modaresifar K, Moravvej H, Bahrami S, Redl H, Tayebi L, Niknejad H. Amniotic membrane and its epithelial and mesenchymal stem cells as an appropriate source for skin tissue engineering and regenerative medicine. Artif Cells Nanomed Biotechnol. 2018;46(sup2):431-440. doi: 10.1080/21691401.2018.1458730. Epub 2018 Apr 24. PMID: 29687742.

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