Comparison of the impact of preservation methods on amniotic membrane properties for tissue engineering applications

Mater Sci Eng C Mater Biol Appl. 2019 Nov:104:109903. doi: 10.1016/j.msec.2019.109903. Epub 2019 Jun 24.


Human amniotic membrane (hAM) is considered as an attractive biological scaffold for tissue engineering. For this application, hAM has been mainly processed using cryopreservation, lyophilization and/or decellularization. However, no study has formally compared the influence of these treatments on hAM properties. The aim of this study was to develop a new decellularization-preservation process of hAM, and to compare it with other conventional treatments (fresh, cryopreserved and lyophilized). The hAM was decellularized (D-hAM) using an enzymatic method followed by a detergent decellularization method, and was then lyophilized and gamma-sterilized. Decellularization was assessed using DNA staining and quantification. D-hAM was compared to fresh (F-hAM), cryopreserved (C-hAM) and lyophilized/gamma-sterilized (L-hAM) hAM. Their cytotoxicity on human bone marrow mesenchymal stem cells (hBMSCs) and their biocompatibility in a rat subcutaneous model were also evaluated. The protocol was effective as judged by the absence of nuclei staining and the residual DNA lower than 50 ng/mg. Histological staining showed a disruption of the D-hAM architecture, and its thickness was 84% lower than fresh hAM (p < 0.001). Despite this, the labeling of type IV and type V collagen, elastin and laminin were preserved on D-hAM. Maximal force before rupture of D-hAM was 92% higher than C-hAM and L-hAM (p < 0.01), and D-hAM was 37% more stretchable than F-hAM (p < 0.05). None of the four hAM were cytotoxic, and D-hAM was the most suitable scaffold for hBMSCs proliferation. Finally, D-hAM was well integrated in vivo. In conclusion, this new hAM decellularization process appears promising for tissue engineering applications.

Keywords: Acellular scaffold; Amniotic membrane; Cryopreservation; Freeze-drying; Processed amnion; Rat; in vivo biocompatibility.

Publication types

  • Comparative Study

MeSH terms

  • Amnion / drug effects
  • Amnion / physiology*
  • Animals
  • Biocompatible Materials / pharmacology
  • Cell Death / drug effects
  • Cryopreservation*
  • DNA / metabolism
  • Extracellular Matrix / drug effects
  • Extracellular Matrix / metabolism
  • Female
  • Humans
  • Implants, Experimental
  • Inflammation / pathology
  • Rats, Wistar
  • Subcutaneous Tissue / drug effects
  • Tissue Engineering / methods*


  • Biocompatible Materials
  • DNA