皮膚修復之奈米複合生醫材料

曾向榮

doi:10.6845/NCHU.2011.00456

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皮膚修復之奈米複合生醫材料

Study of Nanobiomaterials for Skin Repair

曾向榮 , Ph.D　　Advisor：戴憲弘　　Co-advisor ：徐善慧

英文 DOI： 10.6845/NCHU.2011.00456

抑菌；生物相容性；奈米複合材料；銀奈米粒子；聚胺酯(PU) ；蒙脫土；矽片；幾丁聚醣；明膠；支架；真皮層；生物膠；人工皮膚. ； bacteriostasis ； biocompatibility ； nanocomposites ； silver nanoparticles (nano-Ag) ； polyurethane (PU) ； montmorillonite ； silicate platelet ； zeta potentials ； chitosan ； gelatin ； scaffold ； dermis ； bioglue ； artificial skin

Abstract │ Reference (169) │ Altmetrics

Abstract 〈TOP〉

皮膚受傷可造成身體功能失去平衡，臟器嚴重失調，甚至因而死亡，所以大面積的創傷就顯露出人工皮膚的需求。一般使用於燒傷或外傷之創傷敷料與人工皮膚表面需具有一層薄膜作為阻絕層保護薄膜底下之人工皮膚及組織，避免外部細菌侵入及感染，而人工皮膚本身須具備吸收體內滲出液、可供細胞進入及繁殖的特點。本研究中，水性聚胺酯(PU)與奈米尺寸材料(例奈米銀或奈米矽片)混摻提供暫時性的阻絕層，生物可降解支架則開發在阻絕層下。在論文第一部份，奈米複合材料顯示有良好的奈米粒子分佈，直到30 ppm的奈米銀導入，並由TEM確認。PU含有奈米銀粒子時氧化降解會減少，特別是在30 ppm時(PU-Ag 30 ppm)。PU-Ag 30 ppm相對於PU及其他濃度的奈米銀複合材料增加了纖維母細胞的貼附及內皮細胞的反應，減少單核球及血小板活化。大鼠的皮下植入印證了PU-Ag 30 ppm有較好的生物相容性。枯草桿菌、大腸桿菌及抗銀離子大腸桿菌黏著在所有濃度的PU奈米銀複合材料上都是較低的。所有的成果與奈米銀的高度分散有關。另一方面，由聚醚型水性PU混合了0.1 %矽片材料組成了奈米複合材料，這矽片材料有天然黏土，脫層黏土[奈米矽片(NSP)]及十八個碳鏈脂肪胺修飾的NSP (NSP-S)。奈米複合材料含NSP (PU-NSP)時有較佳的內皮細胞貼附及基因表現。PU-NSP和PU-NSP-S有較好的生物相容性是由大鼠皮下植入試驗證實，因其包覆的外來物膠囊厚度較薄。PU-NSP和PU-NSP-S有較強的抑菌率顯露出脫層黏土在高分子載體中可能與微生物會有交互影響。在第二部分中，幾丁聚醣溶液和明膠溶液混合並經過冷凍乾燥後，可製備多孔的皮膚組織工程支架。不同的交聯劑包括戊二醛、1-(3-二甲基胺丙基)-3-乙基碳二亞胺(EDC)、梔子素使用在幾丁聚醣和明膠的支架上，可使其生物穩定性增加。分析人類纖維母細胞在支架中生長，發現以EDC交聯的支架在第四天有最大量的細胞。EDC交聯的支架與市售膠原蛋白敷料產品在乾狀態有相似抗張模數、在濕狀態有相似壓縮模數。此支架同時顯示了適合的孔洞尺寸、高水吸收率及細胞培養期間具有優良尺寸安定性。一個以明膠為基底的特別生物膠適用於幾丁聚醣和明膠支架的上層，同時角質細胞植覆在這上面來模擬表皮。14天後生物膠降解，同時角質細胞在支架上面成長形成一個單層的構造。本研究證實了在EDC交聯的幾丁聚醣明膠支架上植覆人類纖維母細胞可以提供真皮結構，同時塗佈生物膠植覆角質細胞可以提供表皮結構。這種結合體結合了真皮層及表皮層可以幫助皮膚再生，也許有潛力可以使用在組織工程皮膚。

Parallel Abstract 〈TOP〉

Skin injury could make the body to lose equilibrium, to make the organ disorder seriously, or even to dead.  Therefore, large area trauma presented the demand of artificial skin.  Generally, the artificial skin or the trauma dressing used in the treatment of burn or trauma need cover a film as the barrier layer to protect the artificial skin or tissue.  This treatment could also prevent the bacteria invasion or infection.  Meanwhile, the artificial skin must have some characteristics such as the adsorption of body fluid penetration, the cell entering into skin or proliferation.  In this study, waterborne polyurethane (PU) was blended with nano-scale materials (e.g. nano silver or nano silicate platelets) to serve as the tentative barrier layer, the biodegradable scaffold under the barrier layer was developed.  In the first part, the PU-Ag nanocomposites exhibited good nanoparticle dispersion up to 30 ppm of nano Ag, confirmed by the transmission electron microscopy.  The oxidative degradation of PU-Ag was inhibited in all concentrations of nano Ag tested, especially at 30 ppm (”PU-Ag 30 ppm”).  PU-Ag 30 ppm showed enhanced fibroblast attachment and endothelial cell response, as well as reduced monocyte and platelet activation, relative to PU alone or nanocomposites at the other silver contents.  The rat subcutaneous implantation confirmed the better biocompatibility of the nanocomposites.  The adhesion of Bacillus subtilis, Escherichia (E.) coli or Ag+-resistant E. coli on PU-Ag nanocomposites was significantly lower at all concentrations of nano Ag tested.  The dispersion of nano Ag was highly associated with the overall performance.  On the other hand, nanocomposites from a polyether-type waterborne PU and 0.1 wt% of silicate materials were prepared.  The individual silicate materials were natural clays, their exfoliated clays [nano silicate platelets (NSP)], and NSP modified with C18 fatty amine (NSP-S).  The nanocomposite containing NSP (PU-NSP) showed better endothelial cell attachment and gene expression.  The better biocompatibility of PU-NSP and PU-NSP-S was evidenced by the lower thickness of foreign body capsules in rat subcutaneous implantation.  PU-NSP and PU-NSP-S showed strong bacteriostatic effects, which suggested that the nano clay in the polymer matrix may still interact with the microbes.  In the second part, porous scaffolds for dermal tissue engineering were fabricated by freeze-drying the mixture of chitosan and gelatin (CG) solutions.  Different crosslinking agents including glutaraldehyde, 1-(3-dimethylaminopropyl)-3-ethyl-carbodimide hydrochloride (EDC) and genipin were used to crosslink the scaffolds and improve their biostability.  The proliferation of human fibroblasts in the scaffolds was analyzed.  It was found that EDC crosslinked scaffolds had the greatest amount of cells after four days.  EDC crosslinked CG scaffolds had similar tensile modulus in the dry state and compressive modulus in the wet state as the commercial collagen wound dressing product.  They also showed appropriate pore sizes, high water absorption, and good dimensional stability during cell culture.  A special gelatin-based bioglue was applied on top of the CG scaffolds where keratinocytes were seeded to mimic the epidermal structure.  After 14 days, the bioglue was degraded and the keratinocytes grew to form monolayer on top of the scaffolds.  This study demonstrated that CG scaffolds crosslinked by EDC and seeded with human fibroblasts could serve as dermal constructs, while the bioglue coating seeded with keratinocytes could serve as the epidermal constructs.  Such combination may help to regenerate skin with integrated dermal and epidermal layers.  The combination may have potential use in the tissue-engineered skin.

Reference ( 169 ) 〈TOP〉

Chapter 1
連結：
[1] [1] Shevchenko RV, James SL, James SE. A review of tissue-engineered skin bioconstructs available for skin reconstruction. J R Soc Interface 2010;7:229-258.
連結：
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連結：
[5] Hendriks FM, Brokken D, Oomens CWJ, Baaijens FPT, Morales-Serrano FJ. Characterization of mechanical properties of human dermis in vivo. Philips Res. Lab., Eindhoven. Technische Universiteit Eindhoven.
連結：
[6] Carsin H, Ainaud P, Le Bever H, Rives J, Lakhel A, Stephanazzi J. Cultured epithelial autografts in extensive burn coverage of severely traumatized patients: a five year single-center experience with 30 patients. Burns 2000;26:379-387.
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