The study was aimed to investigate the optimum conditions of freeze drying preservation of amniotic membrane (AM). The AM from the health puerperal woman was preserved by freeze drying at optimized way. The key factors of freeze drying process, including abstersion aqua, conservation liquor, the curve of freezing temperature, and the ingredient of protective agent, were optimized. All their morphologic structure was observed by light microscope and scanning electron microscope. The degradation rates by collagenase IV and the characterization of biomechanics were analyzed. The radio-immunologic method was used to investigate the cytokines quantity. All properties of freeze dried AM were compared with those of fresh AM. Light micrographs showed that the five structure-layers exist both in the fresh AM and in those preserved by freeze drying, while the fibro-material was tight-structured in the fresh AM, but loose slightly; the thickness of fibro-material was larger slightly in freeze dried AM. Scanning electron micrographs show that the micro-hairs of epithelial cells in fresh AM were decreased slightly in optimized drying AM, the collagen fibre of fresh AM and of optimized drying AM were well in morphological structures and arranged tightly. The degradation rate by collagenase IV was faster in optimized drying AM,compared with that of the fresh AM. There were insignificant diversity in biomechanical characters (tensile strength, elongation at break and elastic modulus) of the optimized drying AM compared with fresh AM. The cytokines quantity in optimized drying AM decreased significantly compared with fresh AM. The improved freeze drying process has better advantage in keeping the morphological structure, preferable biomechanics and biological vitality of AM, compared with the early research.
Amnion ; metabolism ; ultrastructure ; Biomechanical Phenomena ; Collagenases ; metabolism ; Cytokines ; metabolism ; Female ; Freeze Drying ; methods ; Humans

OBJECTIVETo repair rabbit tendon defects with tissue engineering method.

METHODSThe third passage of fetal skin fibroblast cells was labeled with 5-bromo-2' deoxyuridine (Brdu) and then seeded on human amnion extracellular matrix (HA-ECM). Using 1 cm-long-Achilles tendon defects as repairing models in the experimental group, tendon defects were core bridged with polydioxanone (PDS) and then capsulated with the complex of fibroblasts-HA-ECM. In the control group I, defective tendons were sutured with PDS following the former procedure and capsulated with HA-ECM (without fibroblasts). In the control group II, only PDS was applied to connect the defective tendons. Gross examination, light microscopy, scanning electronmicroscopy and biomechanical measurement of the repaired tendons were respectively performed at postoperative 1, 2, 3 month as well as immunohistochemical examination.

RESULTSThe optimal cell concentration for seeding fibroblasts was 3.5 x 10(6) cells/ml. Cells grew well and radiated or paralleled on HA-ECM. Immunohistochemistry showed that the labeled seed fibroblasts played an important role in tendonization. The results of light microscopy, electron microscopy, and biomechanical assessment suggested that the rate and quality of tendonization in the experimental group was superior to those of the control group I and II. The tensile strength in the experimental group was the greatest, the next was in the control group I, and the worst in the control group II (P<0.05).

CONCLUSIONSHA-ECM is the excellent carrier for fibroblasts. Fibroblasts-HA-ECM complex has the capability to repair tendon defect and to tendonize with rapid rate and good performance three months after operation. Its tensile strength is 81.8% of that of normal tendon.

Amnion ; transplantation ; Animals ; Cells, Cultured ; Extracellular Matrix ; Fibroblasts ; cytology ; Immunohistochemistry ; Microscopy, Electron, Scanning ; Polydioxanone ; Rabbits ; Suture Techniques ; Tendons ; surgery ; ultrastructure ; Tensile Strength ; Wound Healing ; physiology

The purpose of this study is to characterize and compare the ultrastructural changes occurring during the in vivo cultivation of corneal epithelium on amniotic membrane (AM) at several different time points. Corneal burn patients (n=7) with a corneal epithelial defect and severe limbal damage were selected. Initially, AM transplantation with limbal autograft was performed at the acute stage of corneal burn to reconstruct the damaged ocular surface. One to six (mean interval; 3.3+/-1.2) months later, the central part of AM containing an in vivo expanded corneal epithelium was excised and retransplanted in adjacent lesions. The excised epithelium with AM was examined by electron microscopy and immunohistochemical study. By electron microscopy, one and two months after expansion, cultivated epithelium on AM showed an undifferentiated epithelium and an incomplete basement membrane (BM). But, after three months, the cultivated epithelium began to differentiate into a multilayered epithelium with a continuous BM with increased hemidesmosomes. These findings were further confirmed by immunohistochemical study, that cytokeratin K3 was expressed in the cultivated corneal epithelium and newly formed BM was partially positive of collagen IV at three months. At least 3 months may be needed for the proliferation and differentiation of in vivo cultivated corneal epithelium on AM.
Stem Cells/cytology ; Stem Cell Transplantation/*methods ; Middle Aged ; Microscopy, Electron ; Male ; Keratin-3/biosynthesis ; Immunohistochemistry ; Humans ; Epithelium, Corneal/cytology/*metabolism/*pathology/*transplantation ; Corneal Diseases/*therapy ; Burns/*surgery/therapy ; Biological Dressings ; Amnion/*ultrastructure ; Adult

OBJECTIVETo observe the effect of intra-amniotic administration of pulmonary surfactant (PS) on the lung ultrastructure and expression of surfactant protein A (SP-A) in fetal rabbit with intrauterine infection.

METHODSIntra-amniotic PS injection was administered in a rabbit model of premature rupture of membrane and intrauterine infection induced by intrauterine colibacillus injection on the gestational days 24 and 26 days. The lung ultrastructural changes in the fetal rabbits were observed using electron microscope, and the expression of SP-A was measured with immunohistochemical staining and Western blotting 19.5 h after the PS administration.

RESULTSalveolar type I cell (AT I), alveolar type II cell (AT II). In fetal rabbits with intrauterine colibacillus injection, inflammatory cell infiltration was observed in the pulmonary alveolus, bronchus lumens and intracytoplasm irrespective of PS administration. Compared with those in normal fetal rabbits, the number of alveolar type II cells (AT II) in the lung tissue decreased in fetal rabbits with intrauterine infection, and vacuolization of the lamellar bodies occurred with evidence of cell apoptosis; PS administration resulted in increased number of the AT II cells and lamellar bodies and reduced the cell apoptosis. The expression of SP-A was significantly lower in the infection group than in normal control group (P<0.05), but comparable between the PS group and the control group (P>0.05).

CONCLUSIONChanges in pulmonary alveolar ultrastructure and decreased expression of SP-A occur in fetal rabbits after intrauterine infection, and intra-amniotic administration of PS can alleviate these changes to promote lung maturation.

Amnion ; Animals ; Female ; Fetal Membranes, Premature Rupture ; drug therapy ; Injections ; Lung ; embryology ; metabolism ; ultrastructure ; Pregnancy ; Pregnancy Complications, Infectious ; metabolism ; Pulmonary Surfactant-Associated Protein A ; metabolism ; Pulmonary Surfactants ; administration & dosage ; Rabbits