Abstract
Purpose: :
To date, there has been no comprehensive comparative study of histological, biochemical, and functional differences among cryopreserved, glutaradehyde-treated, and dried amniotic membrane (AM).
Methods: :
Six AMs, (1) fresh AM (F-AM), (2) cryopreserved AM (C-AM), (3) cryopreserved & thick AM (CT-AM), (4) dried & epithelially intact AM (DI-AM), (5) dried & epithelially denuded AM (DD-AM), and (6) glutaldehyde-fixed AM (G-AM) were prepared for histological sections and stained with Hematoxylin & Eosin. Additional samples were homogenized, extracted with 4M Guanidine Hydrochloride, dialyzed against PBS, and analyzed for total protein and hyaluronic acid (HA) content. Sample extracts, with or without prior NaOH treatment, were examined by western blot using an antibody against inter-alpha-trypsin inhibitor (IαI).
Results: :
Histologically, amniotic epithelia cells and the basement membrane were present in all samples except DD-AM. Only the stroma of F-AM and C-AM retained a spongy layer, while DI-AM contained chorion. There was no extractable protein or HA in G-AM presumably due to protein crosslinking. The western blot showed that the HC•HA complex, an critical active component recently identified in AM, (He et al., 2008) was only present in F-AM, C-AM, and CT-AM .
Conclusions: :
Dramatic differences in histology, biochemical compositions, and functional properties were noted in different methods of preparing AM. Cryopreservation clearly preserves the stromal spongy layer and the active component of HC•HA. We expect these differences to be reflected by functional assays measuring macrophage and osteoclast suppression, as well as cell adhesion studies that are currently under investigation in our laboratory.
Keywords: cornea: clinical science • growth factors/growth factor receptors • conjunctiva