Investigation On The Promotive Effects Of Drug-loaded Three-dimensional Fibrous Skin Tissue Engineering Scaffolds On Capillary Restoration And Wound Healing In Diabetic Rats

Objective: Three-dimensional fibrous skin tissue engineering scaffolds(TES) with Astragalus Polysaccharide(APS) or Lovastatin(Lov) loading were prepared by electrospinning. The morphology, drug loading, encapsulation efficiency and in vitro release behavior of drug- loaded scaffolds were investigated. The promotive effects of the drug- loaded skin tissue scaffolds on skin capillary restoration and skin wound healing were investigated on the skin defect model of diabetic rats.Methods:(1) A series of APS- loaded TES, with low(APSL-F), middle(APSM-F), and high(APSH-F) APS loading amount respectively, were prepared by technique of electrospinning. The TES morphology was investigated by scanning electron microscope(SEM). The drug loading and encapsulation efficiency were detected by phenol-sulfuric acid method. The skin wound model was established by full thickness excision in streptozocin(STZ) induced diabete rats. TES of APSL-F, APSM-F, APSH-F and blank fibers(Blank-F) were covered on the skin wound, with natural healing wound(NH group) as control. Immunohistochemistry(IHC) staining for CD105 and e NOS expression in regenerated skin tissue and Doppler blood flowmeter for skin blook flow around wound were used to evaluate the skin capillary restoration. Processes of skin regeneration, collagen secetion, and collagen type were investigated by hematoxylin-eosin(HE) and sirius red(SR) staining. The healing rates were assessed by calculating the unhealing area.(2) A series of Lov-loaded TES, with low(LovL-F), middle(LovM-F), and high(LovH-F) Lov loading amount respectively, were prepared by technique of electrospinning. The TES morphology was investigated by SEM. The drug loading, encapsulation efficiency and in vitro release behavior were detected by high performance liquid chromatography(HPLC) method. The skin wound model was established by full thickness excision in STZ induced diabete rats. TES of LovL-F, LovM-F, LovH-F and Blank-F were covered on the skin wound, with NH groupas control. IHC staining for CD105 expression in regenerated skin tissue and Doppler blood flowmeter for skin blook flow around wound were used to evaluate the skin capillary restoration. Processes of skin regeneration, collagen secetion, and collagen type were investigated by HE and SR staining. The healing rates were assessed by calculating the unhealing area. Results:(1) The scaffolds of APSL-F, APSM-F, APSH-F and Blank-F were all formed by randomly arrayed and bifurcation- free polymeric fibers, with diameters of(720±110),(570±120),(650±180), and(660±130) nm, respectivey. The fiber d iameters exhibited no significant difference between each scaffold(P>0.05). Drug loading of APSH-F, APSM-F, APSL-F was(5.22±0.08)%,(1.29±0.07)%, and(0.127±0.006)%, respectively. The encapsulation efficiencies of all the samples were over 80%. No obvious difference was shown between Blank-F group and NH group in wound healing rates(P>0.05). APS-loaded scaffolds could significantly increase the CD105 and e NOS expression in regenerated skin tissues, enhance the angiogenesis and skin blood flow value, promote the fibroblasts proliferation and migration, the collagen secretion, the epidermis coverage and differentiation, and wound closure(P<0.05). Healing time of APSL-F, APSM-F, APSH-F, Blank-F, and NH group were(23.1±4.7),(21.2±4.0),(17.7±2.6),(27.3±5.8),(27.2±5.0) days. The promotive effects of APS-loaded scaffolds on microvessel restoration and wound healing in diabetic rats were positive correlated with the APS loading amount.(2) The scaffolds of LovL-F, LovM-F, LovH-F and Blank-F were all formed by randomly arrayed, uniform thickness, and bifurcation- free polymeric fibers, with diameters of(500±80) nm,(460±70) nm,(470±100) nm,(470±50) nm, respectivey. The fiber diameters exhibited no significant difference between each scaffold(P >0.05). Drug loading of LovH-F, LovM-F, LovL-F was(7.39±0.40)%,(4.13±0.10)%,(0.898±0.002)%, respectively. The encapsulation efficiencies of all the samples were over 80%; In vitro release behaviors showed the sudden release amounts of LovH-F, LovM-F, LovL-F at the first 10 h was(41.99±2.05)%,(41.99±2.05)%,(37.56±1.70)%, respectively, and the accumulated drug releaseamounts were more than 80% in 30 days. No obvious difference was shown between Blank-F group and NH group in wound healing rates(P>0.05). Lov- loaded scaffolds could significantly increase the C D105 expression in regenerated skin tissues, promote the angiogenesis, enhance the skin blood flow value, promote the collagen secretion, the epidermis coverage and differentiation, and wound closure(P<0.05). Healing time of LovL-F, LovM-F, LovH-F, Blank-F, and N H group were(24.1±4.7),(22.4±5.1),(19.0±2.2),(27.5±4.8),(28.1±5.2) days. The promotive effects of Lov-loaded scaffolds on microvessel restoration and wound healing in diabetic rats were pos itive correlated with the Lov loading amount.Conclusion: The APS-loaded and Lov- loaded skin TES could promote the skin capillary restoration and wound healing in diabetic rats by not only simulating the three dimensional structure of extracellular matrix but also releasing the angiogenic substances. The present study could provide new ideas and experimental basis for the diabetic skin ulcer treatment.