Clinical, mechanical and histological evaluation of antifreeze peptide type 1M cryopreserved skin in tissue transplantation
Cryopreservation of tissues and cells has witnessed a magnificent attention in the scientific society since 1949, when Polge had discovered the cryoprotective properties of glycerol to preserve sperm. Later with the progress of using of autogenic and allogeneic tissues, the need for developing n...
Saved in:
| Main Author: | |
|---|---|
| Format: | Thesis |
| Language: | English |
| Published: |
2017
|
| Online Access: | http://psasir.upm.edu.my/id/eprint/68608/1/FPV%202018%207%20IR.pdf http://psasir.upm.edu.my/id/eprint/68608/ |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| Summary: | Cryopreservation of tissues and cells has witnessed a magnificent attention in
the scientific society since 1949, when Polge had discovered the
cryoprotective properties of glycerol to preserve sperm. Later with the
progress of using of autogenic and allogeneic tissues, the need for developing
new compounds and techniques for the cryopreservation of living tissues is
increasing but the freezing damage represent one of the biggest challenging
issues facing the maintenance of functional and structural integrity of living
tissues after cryopreservation. All approaches for cryopreservation aim to
overcome the biological, chemical, mechanical and thermal stresses of ice
crystal formation and re-crystallization associated with subzero
cryopreservation. The objectives of our study is to establish a cryopreservation
technique to preserve living tissues by using antifreeze peptide type 1m
(Afp1m) and to evaluate its ex vivo in vivo and in vitro effects on the
mechanical, clinical, and histological properties of skin grafts by using different
microscopic imaging techniques. This study was able to determine the effects
of freezing on skin samples, skin fibroblasts (M.dunni clone (1118C), and skin
grafts cryopreserved with Afp1m in different concentrations (3 and 5 mg/ml;
at -4 and/or -8 ᵒC for up to 72 hrs). Histological and immunohistological
examination of ex vivo skin samples showed that skin graft cryopreserved in
5mg/ml at 24, 48 and 72 hrs. have the best expression of TGF and VEGF and
also have the best preserved morphological appearance as compared to
3mg/ml and control (80% glycerolized tissue) at -4 and -8 ᵒC. Cell viability
studies showed that the viability of skin fibroblasts cryopreserved in 5mg/ml at
24, 48 and 72 hrs. was better as compared to 3mg and control at -4 and -8ᵒC.
Cell toxicity assessment indicates that Afp1m do not have toxic effects on skin
fibroblasts growth. While the level of DNA damage in skin fibroblast cryopreserved with AFP was higher at -8 ᵒC especially at lower concentration
as compared to -4 ᵒC .There was significant difference (P ˂0.05) between DNA
damage at two temperatures, the level of DNA damage in cell cryopreserved
with 3 mg/ml of AFP was higher than those cryopreserved with 5 mg/ml of AFP
at -8 ᵒC. The in vivo study was performed on 24 rats with 208 ± 31 g body
weight (mean ± SD), and age eight- weeks old. Rats (n=24) were divided into
four groups. One-two circular full-thickness 1.5-2.5 cm diameter wounds were
created on the backs of rats. Non-cryopreserved and cryopreserved auto skin
grafting were placed onto the wound area and stitched. Grafts were bandages.
Wounds were monitored macroscopically and evaluated clinically at days 5, 8,
11, 14, 17, 20 and 22 post-operation. All skin grafts were subjected to
histological and mechanical examinations at the end of experiment, while
blood samples were collected from all rats at week one and at end of
experiment for toxicity analysis. The clinical results showed a significant
difference (P ˂0.05) among 4 groups in pliability of skin grafts ,adherence to
the underling bed, and in the color of skin grafts (P <0.05), (P <0.05), and (P
<0.05) respectively. The results of mechanical properties showed no
significant difference (P ˃0.05) among the 4 experimental groups in maximum
load, tensile stress at maximum load, tensile strain at maximum load, and
elasticity (P >0.05), (P >0.05), (P >0.05), and (P >0.05) respectively .However
the results of non transplanted skin strips cryopreserved in glycerol, Afp1m
showed significant difference (P ˂0.05) in maximum load only. The histological
results showed a significant difference among 4 groups in epidermal integrity,
dermal-epidermal junction (P <0.05) and (P <0.05) respectively. The results of
liver enzymes ALT & AST and total bilirubin showed no significant difference
between weeks 1 and 3 (P > 0.05), (P >0.05), and (P >0.05) respectively and
among the 4 experimental groups as compared to normal values ( P >0.05),
(P >0.05), and (P >0.05) respectively. In conclusion, Afp1m was found
beneficial that could serve as an efficient agent for skin grafts
cryopreservation. |
|---|