| BackgroundSkin is the largest organ in the human body and it is important for human life. Skin functions as a barrier which protects human body from mechanical damage, chemical damage and bacterial invasion. Meanwhile it maintains body fluid and electrolytes. For patients suffering from burn and trauma, the skin barrier is destroyed and the loss of body fluid, electrolytes and nutrients will eventually result in the death of human.Silicone rubber is a polymer material which posses many excellent physical and chemical properties such as good mechanical properties, chemical stability and gas permeability. Importantly, it posses good biocompatibility. Thus silicone rubber is an ideal material for wound dressing.Although silicone rubber is an ideal material for wound dressing, it is mainly used for plastic surgery in the current studies. In some other studies, silicone rubber membrane is just used as a cover sheet to protect the artificial skin/scaffold.In this study, we prepared silicone rubber membranes with different pore structures. As pore structure is critical important for cell proliferation and tissue regeneration, and silicone rubber has good mechanical property and biocompatibility, thus the prepared silicone rubber membrane may be used as a wound dressing.Although the prepared porous silicone rubber membrane may be used as a wound dressing, studies should be carried out to investigate the effects of porous silicone rubber membrane on the wound healing. And based on these studies, we can finally pre pare a novel bilayer wound dressing which mimics the "epidermis-dermis" structure of human skin.ObjectiveIn this study, silicone rubber membranes with different pore structures were prepared. The effects of porous silicone rubber membrane on the wound healing and the underlying mechanism were studied. Based on the data, we prepared a novel bilayer wound dressing which could potentially be used for skin wound protection in the future.Methods1. Preparation of silicone rubber membranes with different pore structures and observation of the pore structure.The porous silicone rubber membrane was prepared via the method of solvent evaporation-induced phase separation. And the pore structure was observed under a scanning electron microscope(SEM).2. Effects of the pore structure on the bacterial permeation.The bacterial suspension was instilled on the membrane. And the number of bacteria which permeated the membrane was determined by routine colony-forming units(CFU) analysis on an agar dish.3. Effects of the pore structure on the cell adhesion and proliferation.3.1 Green fluorescent protein(GFP) transgenic fibroblasts were isolated from GFP transgenic neonatal mice. The cells were seeded on the silicone membrane and the fibroblasts seeded on the membrane were observed under a fluorescent microscope and a SEM.3.2 Cell numbers on the membranes were counted with Cell Counting Kit-8(CCK8); cell cycle was examined via the method of propidium iodide(PI) staining; the m RNA level of wnt3 a, β-catenin and proliferating cell nuclear antigen(PCNA) were determined by the Real-time PCR test; wnt/β-catenin signaling pathway was further examined in our study; XAV939, an inhibitor of Wnt/β-catenin signaling was used to confirm the involvement of Wnt3a/β-catenin signaling pathway in the pore structure-dependent cell proliferation.4. The effects of silicone rubber membranes with different pore structures on the wound healing.A mouse full thickness skin defect wound model was used to study the effects of silicone rubber membrane on the wound healing and wound contraction; the length of the newly formed epithelium was determined based on the hematoxylin and eosin(HE) section using the Image Pro Plus 6.0(IPP6.0) software; the expression of wnt3 a, β-catenin and PCNA were detected by immunohistochemistry and western blot(WB).5. Preparation of bilayer wound dressing composed of silicone rubber.5.1 A bilayer wound dressing was prepared to mimic the epidermis-dermis structure of human skin. Briefly, a solution of liquid silicone rubber(LSR) precursor/liquid paraffin(LP)/hexane(25/20/55 w/w/w) was used to prepare the lower layer of wound dressing and the casting thickness was 1.5 mm, then a solution of LSR precursor/LP/hexane/Span-80(25/5/68/2 w/w/w/w) was used to prepare the upper layer of wound dressing and the casting thickness was 0.5 mm. The two-layer structure of the dressing was observed using a super depth of field microscope.5.2 Measurement of the mechanical properties and water vapor transmission rates(WVTR) of the membranes.5.3 Examination of the biocompatibility of the bilayer membrane in vivo.5.4 The influence of the bilayer membrane on the wound healing process was studied using a mouse full thickness skin defect wound model.5.5 The length of the newly formed epithelium and the granulation thickness were determined.5.6 The expression of PCNA and cluster of differentiation 31(CD 31) were detected by immunohistochemistry to investigate keratinocyte proliferation and wound angiogenesis.5.7 To investigate keratinocyte migration at the wound edge, E-cadherin was detected by immunofluorescence.Results1. Silicone rubber membrane with different pore structure was prepared. The pore size of large pore membrane(LPM), medium pore membrane(MPM) and small pore membrane(SPM) were 218.03 ± 17.95 μm, 110.47 ± 14.03 μm and 5.27 ± 1.44 μm, respectively. No pore formed on the nonporous membrane(NPM).2. The membrane with a small pore size could effectively protect the wound from bacterial invasion compared with vaseline gauze, LPM and MPM(**p < 0.01,n=3).3. Effects of the pore structure on the cell adhesion and proliferation.3.1 The GFP transgenic fibroblasts were isolated from GFP transgenic neonatal mouse and seeded on the silicone rubber membrane. It was found that more cells were adhered on the MPM. Meanwhile under the SEM, the morphology of the cell on the MPM was typical and a spindle like morphology was exhibited.3.2 The cell numbers on the membrane were counted with CCK8. It was found tha t the number of cells in MPM group was larger than that in the LPM, SPM and NPM at day 3 and day 6(**p < 0.01, n=3).3.3 The m RNA level of PCNA was highest in the MPM group, followed by the LPM and the SPM group, and the m RNA level of PCNA was lowest in the NPM group; The expression of Wnt3 a m RNA was significantly increased in the cells seeded on the MPM; And it was found that the m RNA level of β-catenin was highest in the MPM group, followed by the SPM group, and the m RNA level of β-catenin was lowest in the LPM and NPM group.3.4 The protein level of Wnt3 a and β-catenin in the MPM group was higher than that in the LPM, SPM and NPM. Meanwhile the expression of phospho-glycogen synthase kinase-3β(p-GSK-3β) and PCNA was increased in the cells seeded on the MPM, and the expression of glycogen synthase kinase-3β(GSK-3β) in the four groups was the similar without any significant difference. On the contrary, the expression of Axin2 was downregulated in the cells seeded on the MPM.3.5 Cell proliferation was suppressed in the MPM group after the treatment with XAV939(an inhibitor of wnt/β-catenin signailing). Meanwhile the WB results showed that the expression of wnt3 a,β-catenin and PCNA was downregulated. On the contrary, the protein level of Axin2 was increased in the MPM group after treatment with the inhibitor.3.6 It was found that the proportion of cells in S phase in the LPM and the MPM group were larger than that in the SPM and the NPM group at day 1 post-seeding. At day 3, the proportion of cells in S phase in the LPM group were larger than that in the NPM group.4. The effects of porous membranes on the wound healing.4.1 At day 3 post-wounding, the rates of wound healing in LPM, MPM, SPM and NPM group were 40.28%, 63.87%, 56.85% and 28.24%, respectively. Meanwhile the rates of wound healing at day 7 in the four groups were 63.42%, 89.25%, 68.59% and 67.28%. The data demonstrated that wound healing was accelerated in the MPM group.4.2 At day 3, the rates of wound contraction in LPM, MPM, SPM and NPM grou p were 36.30%, 60.09%, 51.78% and 24.67%, respectively. Meanwhile the rates of wound contraction at day 7 in the four groups were 52.35%, 56.58%, 55.23% and 57.93%. The data demonstrated that wound contraction was enhanced in the MPM group at day 3(*p < 0.05,n=5).4.3 It was found that 7 days post-wounding, the length of newly formed epithelium in MPM group was 1173.78 μm, which was significanty longer than that in the LPM, SPM and NPM group(761.9 μm, 825.13μm and 656.61μm, respectively; **p < 0.01,n=5).4.4 Immunohistochemistry and WB results revealed that the expression of Wnt3 a and PCNA was upregulated in the MPM group compared with that in the LPM, SPM and NPM group(*p < 0.05,n=3). Meanwhile β-catenin protein level in the MPM group was higher than that in the SPM and NPM group(*p < 0.05,n=3).5. Preparation of bilayer silicone rubber wound dressing.5.1 The two-layer structure could be identified under the super depth of field microscope. The pore sizes of the upper layer and lower layer were 1.12 ± 0.25 μm and 135.91 ± 30.75μm, respectively. And the thickness of the upper layer and lower layer were 0.5 ± 0.15 mm and 1.2 ± 0.5 mm, respectively.5.2 The average Young’s modulus, the elongations at break and the WVTR of the bilayer wound dressing were 0.16 Mpa, 2427.64% and 229.10g/m2/day.5.3 Favorable tissue compatibility was demonstrated for the bilayer wound dressing in a rat subcutaneous embedding model.5.4 The wound healing experiment results demonstrated that the wound healing was accelerated and the wound closure time was shortened when the bilayer wound dressing was applied on the wound(**p < 0.01,n=5).5.5 The wound reepithelialization was also demonstrated enhanced when the bilayer wound dressing was applied on the wound.5.6 Immunohistochemical staining of PCNA and CD31 demonstrated that cell proliferation and wound angiogenesis were enhanced when the wound was covered with the bilayer wound dressing.5.7 The expression of E-cadherin at the wound edge was down regulated with the coverage of bilayer wound dressing, which indicated that cell migration was enhanced when the bilayer wound dressing was applied on the wound.Conclusion1. The porous silicone rubber membranes with different pore structures were prepared in our study via the method of solvent evaporation-induced phase separation.2. The small pore membrane could protect the wound from bacterial invasion effectively.3. The membrane with a large pore size had a promoting effect on cell adhesion and proliferation. Wnt/β-catenin signaling pathway was involved in the pore size-dependent cell proliferation.4. The membrane with a large pore size had a promoting effect on wound healing.5. Based on the results, a bilayer porous silicone rubber wound dressing was prepared. The upper layer was thin and dense which inhibited bacterial entry effectively. The lower layer with a large pore size had a promoting effect on cell proliferation. And the entire membrane posses good mechanical property and biocompatibility, which could protect the wound effectively and eventually promoted wound regeneration.The study of the effect of the porous silicone membrane on the wound healing may be useful for wound dressing design in the future. And the bilayer porous silicone rubber membrane may serve as a kind of promising wound dressing. |
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