| Platelets(PLTs) have been shown to actively promote and participate in angiogenesis and blood vessel maturation and accelerate wound healing. PLTs also play acritical role in wound healing and matrix remodeling, tissue regeneration, and recruitment of marrow progenitor cells to the site of injury.Inclusion of platelets, especially various growth factors, play important roles in the course of wound healing. As a kind of biological active substance, growth factors derivative from platelets can enhance a lot of wound-repair mechanisms such as chemotaxis, proliferation, vascular generation and platelet rich plasma (PRP) is the normally using form of platelets, which will release abundant growth-factors like PDGF, TGF, VEGF, IGF and EGF after being activated. They'll impact cellular differentiation and ability of karyokinesis greatly during the whole wound-repair period. Hurt cicatrization and tissue repair are biological process with regulation and cooperation of multifarious factors. So compared with single gene-recombined growth-factor, the ratio of growth-factors in platelets is closer to what in vivo circumstance needs. They have better effects of repairing and no toxicity. However, it's very easy to be contaminated for PRP stored under condition of shaking at 22士2℃. Difficulties in preservation and transportation make it unavailable to use platelets in repairing. To the opposition, freezing-dried platelets can be stored for long term and easy to be transported due to their stable biological activities, smaller volume and lighter weight. In the study, small molecule sugar was used to lyophilize platelets as a stabilizing agent. Morpha and function of the freeze-dried platelets were evaluated. Species and contents of growth-factor in the platelets were analyzed. Human fibroblast cells were cultured with the lyophilized platelets to observe whether the proliferation activity was enhanced. Furthermore, rat model with excision of the whole layer skin was applied to assess the wound-repair effect of the fresh platelets and the lyophilized platelets. Such experiments will be helpful to develop novel type of wound-repair agents.To obtain the lyophilized platelets preserved well in morpha and function, sorts of pre-freezing protect agents for lyophilizing platelets were screened. The result shows that pre-treated platelets in group 3 were much better than in group1, group2, with recoveries of 57.2%±8.1%,63.6%±11.7% and 80.6%±13.9% respectively (P<0.05). Platelets which are Lyophilized in group 3 were stored for 0d, 30d, and 90d at -20℃, also stored at room temperature for 20d. After rehydration, the recoveries of platelets have no significant difference among the groups. Observed under electronic microscope, the lyophilized platelets have normal morpha and intact membrane.α- granule, compact granule and open canalicular system ( OCS) existed stably, which are involved in release of growth-factors. The result of in vitro aggregation suggests that lyophilized platelets preserved at different temperature and stored for different period shows no significant difference with the fresh platelets. Contents of the total proteins from lyophilized platelets stored at -20℃for 0d, 30d, 90d and stored at room temperature for 20d are 64.2±9.7,65.4±6.6,63.5±6.7,61.7±10.1 separately, which show no significant difference with contents of the fresh platelets'total proteins. ELISA tests demonstrate that concentration of rhPDGF-BB(pg/ml)are 497.74±98.54,517.26±95.45,534.17±102.38,501.97±98.37,concentration of TGF-β1 are 5.74±1.27,5.57±0.96,6.04±1.38,5.87±1.21 from lyophilized platelets stored at -20℃for 0d,30d,90d and stored at room temperature have no significant difference with those from fresh platelets(503.81±101.13pg/ml and 5.67±1.28 ng/ml). According to the above results, we conclude that the contents of growth-factors are stable when the lyophilized platelets are preserved at -20℃for 0d,30d and 90d or preserved at room temperature for 20d.On the basis of the study about lyophilizing platelets and detection of the growth-factors, we assessed the wound-repair effect when lyophilized platelets were used in vitro and in vivo. The experimental groups were divided into lyophilized platelets group, fresh platelets group and rhPDGF group. It was found that when the concentration of platelets are increased, proliferations of the human skin fibroblast cells are enhanced and achieve to the top value when the concentration,The top values are 168.4%±4.9% and 167.6%±6.9%,separately in fresh platelets group and lyophilized platelets group, with no significant difference. In the rhPDGF group, we also found that proliferation value of the cells increases with the higher concentration of rhPDGF. the value is 135.4%±5.1% at the same concentration. For in vivo experiment, the whole skin lost model of rat is established. Then the wound-repair effects of lyophilized platelets, fresh platelets and rhPDGF were tested. Healing time are(17.76±1.61)d,(17.34±1.89)d, and(20.88±1.34)d when fresh platelets, lyophilized platelets and nothing used on the wounds. In the two groups of treatment, healing time has no significant difference. But in the group with no treatment, wounds need more time to heal. Immunity histology analysis demonstrates that in the 9th day after hurt, quantities of the proliferated cells all achieve the top value in the different groups. PCNA positive cells in the lyophilized platelets group, fresh platelets group, and no treatment group are 167.22±7.25,173.45±4.16 and 123.6±5.84 respectively. The values of treatment groups are higher than the no treatment group with significant difference, which suggests that lyophilized platelets can promote the wound-repair of the skin.To conclude, in this study, we screened the available pre-treatment protect agent for lyophilizing platelets and freeze dry platelets successfully. After rehydration, the morpha, function and the growth factors of lyophilized platelets are all maintained in a good situation,similar to fresh platelets. Lyophilized platelets can promote wound-repair of skin observably either in vivo and in vitro, having no significant difference with fresh platelets and better effect than single growth-factor rhPDGF-BB...
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