To compare the therapeutic efficacy of cryopreserved amniotic membrane (AM) grafts and standard of care (SOC) in treating nonhealing wounds (NHW) through a prospective multicenter clinical trial, 42 patients (76% polymorbid) with 54 nonhealing wounds of various etiologies (mainly venous) and an average baseline size of 20 cm2 were included. All patients were treated for at least 6 weeks in the center before they were involved in the study. In the SOC group, 29 patients (36 wounds) were treated. If the wound healed less than 20% of the baseline size after 6 weeks, the patient was transferred to the AM group (35 patients, 43 wounds). Weekly visits included an assessment of the patient's condition, photo documentation, wound debridement, and dressing. Quality of life and the pain degree were subjectively reported by patients. After SOC, 7 wounds were healed completely, 1 defect partially, and 28 defects remained unhealed. AM application led to the complete closure of 24 wounds, partial healing occurred in 10, and 9 remained unhealed. The degree of pain and the quality of life improved significantly in all patients after AM application. This study demonstrates the effectiveness of cryopreserved AM grafts in the healing of NHW of polymorbid patients and associated pain reduction.

2nd Department of Surgery Department of Cardiovascular Surgery 1st Faculty of Medicine Charles University and General University Hospital Prague 128 08 Prague Czech Republic

Clinic of Dermatovenerology General Teaching Hospital and 1st Faculty of Medicine Charles University 121 08 Prague Czech Republic

Department of Cardiovascular Surgery Motol University Hospital 150 06 Prague Czech Republic

Department of Statistical Modeling Institute of Computer Science The Czech Academy of Sciences 182 07 Prague Czech Republic

Department of Transplantation and Tissue Bank Motol University Hospital 150 06 Prague Czech Republic

Department of Vascular Surgery Na Homolce Hospital 150 30 Prague Czech Republic

Laboratory of the Biology and Pathology of the Eye Institute of Biology and Medical Genetics 1st Faculty of Medicine Charles University 128 00 Prague Czech Republic

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Jarbrink K., Ni G., Sonnergren H., Schmidtchen A., Pang C., Bajpai R., Car J. The humanistic and economic burden of chronic wounds: A protocol for a systematic review. Syst. Rev. 2017;6:15. doi: 10.1186/s13643-016-0400-8. PubMed DOI PMC

Maita K.C., Avila F.R., Torres-Guzman R.A., Garcia J.P., Eldaly A.S., Palmieri L., Emam O.S., Ho O., Forte A.J. Local anti-inflammatory effect and immunomodulatory activity of chitosan-based dressing in skin wound healing: A systematic review. J. Clin. Transl. Res. 2022;8:488–498. PubMed PMC

Posnett J., Gottrup F., Lundgren H., Saal G. The resource impact of wounds on health-care providers in Europe. J. Wound Care. 2009;18:154–161. doi: 10.12968/jowc.2009.18.4.41607. PubMed DOI

Gottrup F., Apelqvist J., Bjarnsholt T., Cooper R., Moore Z., Peters E.J., Probst S. EWMA document: Antimicrobials and non-healing wounds. Evidence, controversies and suggestions. J. Wound Care. 2013;22:S1–S89. doi: 10.12968/jowc.2013.22.Sup5.S1. PubMed DOI

Zelen C.M., Serena T.E., Denoziere G., Fetterolf D.E. A prospective randomised comparative parallel study of amniotic membrane wound graft in the management of diabetic foot ulcers. Int. Wound J. 2013;10:502–507. doi: 10.1111/iwj.12097. PubMed DOI PMC

Mekkes J.R., Loots M.A., Van Der Wal A.C., Bos J.D. Causes, investigation and treatment of leg ulceration. Br. J. Dermatol. 2003;148:388–401. doi: 10.1046/j.1365-2133.2003.05222.x. PubMed DOI

Mustoe T.A., O’Shaughnessy K., Kloeters O. Chronic wound pathogenesis and current treatment strategies: A unifying hypothesis. Plast. Reconstr. Surg. 2006;117:35S–41S. doi: 10.1097/01.prs.0000225431.63010.1b. PubMed DOI

Frykberg R.G., Banks J. Challenges in the Treatment of Chronic Wounds. Adv. Wound Care. 2015;4:560–582. doi: 10.1089/wound.2015.0635. PubMed DOI PMC

Price D.D., McGrath P.A., Rafii A., Buckingham B. The validation of visual analogue scales as ratio scale measures for chronic and experimental pain. Pain. 1983;17:45–56. doi: 10.1016/0304-3959(83)90126-4. PubMed DOI

Gomes F., Schuetz P., Bounoure L., Austin P., Ballesteros-Pomar M., Cederholm T., Fletcher J., Laviano A., Norman K., Poulia K.A., et al. ESPEN guidelines on nutritional support for polymorbid internal medicine patients. Clin. Nutr. 2018;37:336–353. doi: 10.1016/j.clnu.2017.06.025. PubMed DOI

Forbes J., Fetterolf D.E. Dehydrated amniotic membrane allografts for the treatment of chronic wounds: A case series. J. Wound Care. 2012;21:294–296. doi: 10.12968/jowc.2012.21.6.290. PubMed DOI

El Heneidy H., Omran E., Halwagy A., Al-Inany H., Al-Ansary M., Gad A. Amniotic membrane can be a valid source for wound healing. Int. J. Womens Health. 2016;8:225–231. doi: 10.2147/IJWH.S96636. PubMed DOI PMC

Sathyaraj W.V., Prabakaran L., Bhoopathy J., Dharmalingam S., Karthikeyan R., Atchudan R. Therapeutic Efficacy of Polymeric Biomaterials in Treating Diabetic Wounds-An Upcoming Wound Healing Technology. Polymers. 2023;15:1205. doi: 10.3390/polym15051205. PubMed DOI PMC

Pourmoussa A., Gardner D.J., Johnson M.B., Wong A.K. An update and review of cell-based wound dressings and their integration into clinical practice. Ann. Transl. Med. 2016;4:457. doi: 10.21037/atm.2016.12.44. PubMed DOI PMC

Huerta C.T., Voza F.A., Ortiz Y.Y., Liu Z.J., Velazquez O.C. Mesenchymal stem cell-based therapy for non-healing wounds due to chronic limb-threatening ischemia: A review of preclinical and clinical studies. Front. Cardiovasc. Med. 2023;10:1113982. doi: 10.3389/fcvm.2023.1113982. PubMed DOI PMC

Wei Q., Liu X., Su J.L., Wang Y.X., Chu Z.Q., Ma K., Huang Q.L., Li H.H., Fu X.B., Zhang C.P. Small extracellular vesicles from mesenchymal stem cells: A potential Weapon for chronic non-healing wound treatment. Front. Bioeng. Biotechnol. 2023;10:1083459. doi: 10.3389/fbioe.2022.1083459. PubMed DOI PMC

Shaabani E., Sharifiaghdam M., Faridi-Majidi R., De Smedt S.C., Braeckmans K., Fraire J.C. Gene therapy to enhance angiogenesis in chronic wounds. Mol. Ther. Nucleic Acids. 2022;29:871–899. doi: 10.1016/j.omtn.2022.08.020. PubMed DOI PMC

Dadkhah Tehrani F., Firouzeh A., Shabani I., Shabani A. A Review on Modifications of Amniotic Membrane for Biomedical Applications. Front. Bioeng. Biotechnol. 2020;8:606982. doi: 10.3389/fbioe.2020.606982. PubMed DOI PMC

Leal-Marin S., Thomas K., Nicola H., Olena P., Carsten F., Martin B., Constanca F., Birgit G., Oleksandr G. Human Amniotic Membrane: A review on tissue engineering, application, and storage. J. Biomed. Mater. Res. B Appl. Biomater. 2020;109:1198–1215. doi: 10.1002/jbm.b.34782. PubMed DOI

Koizumi N.J., Inatomi T.J., Sotozono C.J., Fullwood N.J., Quantock A.J., Kinoshita S. Growth factor mRNA and protein in preserved human amniotic membrane. Curr. Eye Res. 2000;20:173–177. doi: 10.1076/0271-3683(200003)2031-9FT173. PubMed DOI

Malhotra C., Jain A.K. Human amniotic membrane transplantation: Different modalities of its use in ophthalmology. World J. Transplant. 2014;4:111–121. doi: 10.5500/wjt.v4.i2.111. PubMed DOI PMC

Litwiniuk M., Krejner A., Speyrer M.S., Gauto A.R., Grzela T. Hyaluronic Acid in Inflammation and Tissue Regeneration. Wounds. 2016;28:78–88. PubMed

Litwiniuk M., Radowicka M., Krejner A., Grzela T. The influence of amniotic membrane extracts on cell growth depends on the part of membrane and childbirth mode selected: A proof-of-concept study. J. Wound Care. 2017;26:498–503. doi: 10.12968/jowc.2017.26.8.498. PubMed DOI

Nejad A.R., Hamidieh A.A., Amirkhani M.A., Sisakht M.M. Update review on five top clinical applications of human amniotic membrane in regenerative medicine. Placenta. 2021;103:104–119. doi: 10.1016/j.placenta.2020.10.026. PubMed DOI

Svobodova A., Vrkoslav V., Smeringaiova I., Jirsova K. Distribution of an analgesic palmitoylethanolamide and other N-acylethanolamines in human placental membranes. PLoS ONE. 2023;18:e0279863. doi: 10.1371/journal.pone.0279863. PubMed DOI PMC

Farivar B.S., Toursavadkohi S., Monahan T.S., Sharma J., Ucuzian A.A., Kundi R., Sarkar R., Lal B.K. Prospective study of cryopreserved placental tissue wound matrix in the management of chronic venous leg ulcers. J. Vasc. Surg. Venous Lymphat. Disord. 2019;7:228–233. doi: 10.1016/j.jvsv.2018.09.016. PubMed DOI

Serena T.E., Orgill D.P., Armstrong D.G., Galiano R.D., Glat P.M., Carter M.J., Kaufman J.P., Li W.W., Zelen C.M. A Multicenter, Randomized, Controlled, Clinical Trial Evaluating Dehydrated Human Amniotic Membrane in the Treatment of Venous Leg Ulcers. Plast. Reconstr. Surg. 2022;150:1128–1136. doi: 10.1097/PRS.0000000000009650. PubMed DOI PMC

Ananian C.E., Dhillon Y.S., Van Gils C.C., Lindsey D.C., Otto R.J., Dove C.R., Pierce J.T., Saunders M.C. A multicenter, randomized, single-blind trial comparing the efficacy of viable cryopreserved placental membrane to human fibroblast-derived dermal substitute for the treatment of chronic diabetic foot ulcers. Wound Repair. Regen. 2018;26:274–283. doi: 10.1111/wrr.12645. PubMed DOI

Lavery L., Fulmer J., Shebetka K.A., Regulski M., Vayser D., Fried D., Kashefsky H., Owings T.M., Nadarajah J., Hesp Z. Open-label Extension Phase of a Chronic Diabetic Foot Ulcer Multicenter, Controlled, Randomized Clinical Trial Using Cryopreserved Placental Membrane. Wounds. 2018;30:283–289. PubMed

Eskandarlou M., Azimi M., Rabiee S., Seif Rabiee M.A. The Healing Effect of Amniotic Membrane in Burn Patients. World J. Plast. Surg. 2016;5:39–44. PubMed PMC

Loeffelbein D.J., Rohleder N.H., Eddicks M., Baumann C.M., Stoeckelhuber M., Wolff K.D., Drecoll E., Steinstraesser L., Hennerbichler S., Kesting M.R. Evaluation of human amniotic membrane as a wound dressing for split-thickness skin-graft donor sites. Biomed. Res. Int. 2014;2014:572183. doi: 10.1155/2014/572183. PubMed DOI PMC

Tettelbach W., Cazzell S., Reyzelman A.M., Sigal F., Caporusso J.M., Agnew P.S. A confirmatory study on the efficacy of dehydrated human amnion/chorion membrane dHACM allograft in the management of diabetic foot ulcers: A prospective, multicentre, randomised, controlled study of 110 patients from 14 wound clinics. Int. Wound J. 2019;16:19–29. doi: 10.1111/iwj.12976. PubMed DOI PMC

Svobodova A., Horvath V., Smeringaiova I., Cabral J.V., Zemlickova M., Fiala R., Burkert J., Nemetova D., Stadler P., Lindner J., et al. The healing dynamics of non-healing wounds using cryo-preserved amniotic membrane. Int. Wound J. 2022;19:1243–1252. doi: 10.1111/iwj.13719. PubMed DOI PMC

Horvath V., Svobodova A., Cabral J.V., Fiala R., Burkert J., Stadler P., Lindner J., Bednar J., Zemlickova M., Jirsova K. Inter-placental variability is not a major factor affecting the healing efficiency of amniotic membrane when used for treating chronic non-healing wounds. Cell Tissue Bank. 2023 doi: 10.1007/s10561-023-10096-y. PubMed DOI PMC

Blome C., Baade K., Price P., Debus E.S., Augustin M. The ‘Wound-QoL’: A Short Questionnaire Measuring Quality of Life in Patients with Chronic Wounds. Wound Repair. Regen. 2014;22:504–514. doi: 10.1111/wrr.12193. PubMed DOI

Snyder D., Sullivan N., Margolis D., Schoelles K. Skin Substitutes for Treating Chronic Wounds. Agency for Healthcare Research and Quality; Rockville, MD, USA: 2020. Technology Assessment Program—Technical Brief; Project ID: WNDT0818. PubMed

Fitzmaurice G.M., Laird N.M., Ware J.H. Applied Longitudinal Analysis. 2nd ed. John Wiley & Sons; Hoboken, NJ, USA: 2004.

Laird N.M., Ware J.H. Random-effects models for longitudinal data. Biometrics. 1982;38:963–974. PubMed

Dmitrienko A., Tamhane A.C., Bretz F. Multiple Testing Problems in Pharmaceutical Statistics. 1st ed. CRC Press; New York, NY, USA: 2009.

Kaplan E.L., Meier P. Nonparametric Estimation from Incomplete Observations. J. Amer. Statist. Assoc. 1958;53:457–481. doi: 10.1080/01621459.1958.10501452. DOI

Aalen O.O., Borgan Ø., Gjessing H.K. Survival and Event History Analysis: A Process Point of View. Springer; Berlin/Heidelberg, Germany: 2008. pp. 90–104.

Wood S.N. Generalized Additive Models: An Introduction with R. 2nd ed. Chapman and Hall/CRC; Boca Raton, FL, USA: 2017.

Wood S.N., Pya N., Säfken B. Smoothing Parameter and Model Selection for General Smooth Models. J. Am. Stat. Assoc. 2017;111:1548–1563. doi: 10.1080/01621459.2016.1180986. DOI

R Core Team . R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing; Vienna, Austria: 2020. [(accessed on 15 January 2023)]. Available online: http://www.r-project.org/index.html.

Beyene R.T., Derryberry S.L., Jr., Barbul A. The Effect of Comorbidities on Wound Healing. Surg. Clin. N. Am. 2020;100:695–705. doi: 10.1016/j.suc.2020.05.002. PubMed DOI

Raspovic K.M., Wukich D.K., Naiman D.Q., Lavery L.A., Kirsner R.S., Kim P.J., Steinberg J.S., Attinger C.E., Danilkovitch A. Effectiveness of viable cryopreserved placental membranes for management of diabetic foot ulcers in a real world setting. Wound Repair. Regen. 2018;26:213–220. doi: 10.1111/wrr.12635. PubMed DOI

Ananian C.E., Davis R.D., Johnson E.L., Regulski M.J., Reyzelman A.M., Saunders M.C., Danilkovitch A. Wound Closure Outcomes Suggest Clinical Equivalency between Lyopreserved and Cryopreserved Placental Membranes Containing Viable Cells. Adv. Wound Care. 2019;8:546–554. doi: 10.1089/wound.2019.1028. PubMed DOI PMC

Lavery L.A., Fulmer J., Shebetka K.A., Regulski M., Vayser D., Fried D., Kashefsky H., Owings T.M., Nadarajah J., Grafix Diabetic Foot Ulcer Study G. The efficacy and safety of Grafix((R)) for the treatment of chronic diabetic foot ulcers: Results of a multi-centre, controlled, randomised, blinded, clinical trial. Int. Wound J. 2014;11:554–560. doi: 10.1111/iwj.12329. PubMed DOI PMC

Zelen C.M., Serena T.E., Gould L., Le L., Carter M.J., Keller J., Li W.W. Treatment of chronic diabetic lower extremity ulcers with advanced therapies: A prospective, randomised, controlled, multi-centre comparative study examining clinical efficacy and cost. Int. Wound J. 2015;13:272–282. doi: 10.1111/iwj.12566. PubMed DOI PMC

Valiente M.R., Nicolas F.J., Garcia-Hernandez A.M., Fuente Mora C., Blanquer M., Alcaraz P.J., Almansa S., Merino G.R., Lucas M.D.L., Alguero M.C., et al. Cryopreserved amniotic membrane in the treatment of diabetic foot ulcers: A case series. J. Wound Care. 2018;27:806–815. doi: 10.12968/jowc.2018.27.12.806. PubMed DOI

Zidan S.M., Eleowa S.A., Nasef M.A., Abd-Almoktader M.A., Elbatawy A.M., Borhamy A.G., Aboliela M.A., Ali A.M., Algamal M.R. Maximizing the safety of glycerol preserved human amniotic membrane as a biological dressing. Burns. 2015;41:1498–1503. doi: 10.1016/j.burns.2015.03.009. PubMed DOI

Kadkhoda Z., Tavakoli A., Chokami Rafiei S., Zolfaghari F., Akbari S. Effect of Amniotic Membrane Dressing on Pain and Healing of Palatal Donor Site: A Randomized Controlled Trial. Int. J. Organ. Transplant. Med. 2020;11:55–62. PubMed PMC

Vaheb M., Kohestani B.M., Karrabi M., Khosrojerdi M., Khajeh M., Shahrestanaki E., Sahebkar M. Evaluation of Dried Amniotic Membrane on Wound Healing at Split-Thickness Skin Graft Donor Sites: A Randomized, Placebo-Controlled, Double-blind Trial. Adv. Skin. Wound Care. 2020;33:636–641. doi: 10.1097/01.ASW.0000695752.52235.e3. PubMed DOI

Tseng S.C. HC-HA/PTX3 Purified from Amniotic Membrane as Novel Regenerative Matrix: Insight into Relationship between Inflammation and Regeneration. Investig. Ophthalmol. Vis. Sci. 2016;57:ORSFh1–ORSFh8. doi: 10.1167/iovs.15-17637. PubMed DOI PMC

Vrkoslav V., Smeringaiova I., Smorodinova N., Svobodova A., Strnad S., Jackson C.J., Burkert J., Jirsova K. Quantification of Analgesic and Anti-Inflammatory Lipid Mediators in Long-Term Cryopreserved and Freeze-Dried Preserved Human Amniotic Membrane. Bioengineering. 2023;10:740. doi: 10.3390/bioengineering10060740. PubMed DOI PMC

Cryopreserved amniotic membrane in chronic nonhealing wounds: a series of case reports