Excellence in Research: Skin Tissue Regeneration using Smart Scaffolds

卓越的研究：使用智能支架进行皮肤组织再生

• 批准号: 1831282
• 负责人: Komal Vig
• 金额: $ 50万
• 依托单位: Alabama State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-10-01 至 2024-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1831282&HistoricalAwards=false
• 关键词: Excellence; Research; Skin; Tissue; Regeneration

The need is great for specialized skin tissues that can be used for wound healing and for replacement of skin damaged due to disease (e.g. diabetes caused ulcers) or injury (e.g. burns.) Though bioengineered skin research has been very active for over 40 years, available commercial products are still subject to rejection, infection and scarring, lack vascularization (blood vessels) and do not match the texture or color of the surrounding skin. The goal of this project is to address these limitations by developing a fully functional skin with minimal or no graft rejection, with wound healing capabilities, and that is individualized to match the texture, color and vascularization of the host tissue. Structure for the bioengineered skin will be provided by electrospun or 3D printed smart scaffolds consisting of polymers or composite hydrogels and bioinks (fluids that contain materials that provide the environment needed to support cell adhesion, proliferation and differentiation) that have been optimized for tissue generation and tested for antibacterial and antiviral activities. The optimized cell scaffolds, 3D printing methods, strategies and other technologies can be further refined to regenerate other organs (e.g. livers), thus establishing an exciting new research direction. The multidisciplinary expertise in nanobiotechnology, bioengineering, cell biology and immunology required for this project will provide invaluable opportunities for students involved in the research. Students will also benefit from the unique community of Alabama State University, a historically black university (HBCU) located in Central Alabama, and collaborative Universities and industry scientists and engineers. This setting, the Principal Investigator's strong commitment to undergraduate education and the project's appeal is expected to result in an increase in the number of minorities and women participating in the workforce in the region, state, and nation in STEM disciplines.The goal of this project is to bioengineer fully functional skin tissues that can be used for wound healing and replacing skin damaged by disease or injury. Meritorious attributes include: no or minimal graft rejection, wound healing capabilities, antimicrobial protection, vascularization, and individualized texture and pigmentation that matches the host tissue. Key to the project's success is the development of smart scaffolds and bioinks into which growth factors, antimicrobials, nanoencapsulated DNA/cells, proangiogenic materials, decellularized ECM, etc. can be incorporated and skin models in which texture and pigmentation can be controlled. The Research Plan is organized under seven objectives. OBJECTIVE 1 is to develop novel biocompatible, cell and tissue specific, hydrogel materials and bioinks. Components to be designed and optimized include: synthetic polymer (PLA and PEG) scaffolds, composite hydrogels (collagen-chitosan and chitosan-alginate) scaffolds, electrospun composite (chilosan-pCL with PLGA-PGA nanofibers) scaffolds, gelatin-sodium alginate bioink, gelatin-fibrinogen-collagen bioink and decellularized ECM bioink (porcine cartilage and heart tissues). 3D scaffolds will be printed from the described hydrogels and bioinks, with further incorporation of various growth factors and ECM proteins. OBJECTIVE 2 is to produce smart infection resistant and genetically modified scaffolds and bioinks. Antibacterial/antiviral enhancement will be achieved by incorporation of encapsulated coated (silver PVP, gold, and silver) carbon nanotubes into the scaffolds and bioinks. A non-viral gene delivery system to enhance tissue generation will also be developed. OBJECTIVE 3 is to characterize the physical and chemical characteristics of the scaffolds and bioinks. Scaffold morphology will be analyzed using Scanning Electron, Transmission Electron and Atomic Force Microscopy (SEM, TEM and AFM). Functional groups in macromolecules will be determined using Fourier Transform Infrared Spectroscopy (FT-IR). The compression modulus will be determined using an INSTRON machine. The equilibrium swelling ratio will be measured using the conventional gravimeter method. Porosity will be measured using a displacement liquid that easily enters pores. OBJECTIVE 4 is optimization, development and characterization of skin tissues. Two layer skin tissues will be grown using fibroblasts (dermis) and keratinocytes (epidermis) and stem cells that can be differentiated into fibroblasts and keratinocytes as the skin grows to full thickness. Optimization of cell scaffolds will be assessed by determining seeding efficiency, cell proliferation, cell viability and cell attachment. Scaffolds and skin tissues will be analyzed histologically using SEM, TEM and AFM analyses, and mRNA expression levels of different skin compartment markers will be investigated using real-time PCR. OBJECTIVE 5 is to develop skin tissues with vascularization. Two approaches will be used: a) ex vivo development of vascularized skin by incorporating proangiogenic molecules in cell scaffolds prior to skin tissue development, and b) an in vivo approach that relies on dermal microvasculature cells to achieve vascularization post-skin tissue development. OBJECTIVE 6 is to develop skin tissues with melanotic features. Primary epidermal melanocytes will be cultured and used with fibroblasts and keratinocytes for 3D bioprinting of full thickness skin. Melanocyte morphology, proliferation, viability, melanin production and interactions with the fibroblasts and keratinocytes will be determined using technologies similar to those used in previous objectives. OBJECTIVE 7 is to assess the antimicrobial effects of skin tissues. The smart scaffolds with antimicrobial properties developed under Objective 2 will be used to develop human and mouse 3D skins that will be infected with skin bacteria, e.g. Staphylococcus aureus or Pseudomonas aeruginosa. Antimicrobial efficacy will be assessed by quantifying the post infection bacterial load (plate counting) and structural evaluation (ultrasound) of the infected skin.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

对于可用于伤口愈合和替换因疾病（如糖尿病引起的溃疡）或损伤（如烧伤）而受损的皮肤的专门皮肤组织的需求很大。尽管生物工程皮肤研究已经非常活跃了40多年，但现有的商业产品仍然容易受到排斥，感染和疤痕，缺乏血管（血管），并且与周围皮肤的质地或颜色不匹配。该项目的目标是通过开发一种功能齐全的皮肤来解决这些限制，这种皮肤具有最小或没有移植排斥反应，具有伤口愈合能力，并且可以个性化地与宿主组织的纹理，颜色和血管化相匹配。生物工程皮肤的结构将由静电纺或3D打印智能支架提供，该支架由聚合物或复合水凝胶和生物墨水（含有提供支持细胞粘附、增殖和分化所需环境的材料的液体）组成，这些材料已经过组织生成优化并经过抗菌和抗病毒活性测试。优化后的细胞支架、3D打印方法、策略等技术可以进一步细化到其他器官（如肝脏）的再生，从而建立一个令人兴奋的新研究方向。该项目所需的纳米生物技术、生物工程、细胞生物学和免疫学等多学科专业知识将为参与研究的学生提供宝贵的机会。学生还将受益于阿拉巴马州立大学的独特社区，这是一所位于阿拉巴马州中部的历史悠久的黑人大学（HBCU），以及合作大学和行业科学家和工程师。在这种情况下，首席研究员对本科教育的坚定承诺和项目的吸引力预计将导致少数民族和妇女参与该地区、州和国家STEM学科劳动力的数量增加。该项目的目标是通过生物工程制造功能齐全的皮肤组织，用于伤口愈合和替换因疾病或损伤而受损的皮肤。值得表扬的属性包括：没有或最小的移植物排斥反应，伤口愈合能力，抗菌保护，血管化，以及与宿主组织匹配的个性化纹理和色素沉着。该项目成功的关键是开发智能支架和生物墨水，其中可以纳入生长因子，抗菌剂，纳米封装DNA/细胞，促血管生成材料，脱细胞ECM等，以及可以控制纹理和色素沉着的皮肤模型。研究计划有七个目标。目的1：开发新型生物相容性、细胞和组织特异性的水凝胶材料和生物墨水。设计和优化的组件包括：合成聚合物（聚乳酸和聚乙二醇）支架，复合水凝胶（胶原-壳聚糖和壳聚糖-海藻酸盐）支架，静电纺丝复合材料（chilosan-pCL与PLGA-PGA纳米纤维）支架，明胶-海藻酸钠生物链接，明胶-纤维蛋白原-胶原生物链接和脱细胞ECM生物链接（猪软骨和心脏组织）。3D支架将由所描述的水凝胶和生物墨水打印，并进一步掺入各种生长因子和ECM蛋白。目标2是生产智能抗感染和转基因支架和生物墨水。通过将包裹的（银PVP，金和银）碳纳米管掺入支架和生物墨水中，可以实现抗菌/抗病毒增强。还将开发一种非病毒基因传递系统，以增强组织生成。目的3是表征支架和生物墨水的物理和化学特性。将使用扫描电子、透射电子和原子力显微镜（SEM、TEM和AFM）分析支架形态。利用傅里叶变换红外光谱（FT-IR）测定大分子中的官能团。压缩模量将使用INSTRON机器确定。平衡膨胀比将用传统的重力仪方法测量。孔隙度将使用易于进入孔隙的置换液体来测量。目的4：优化、开发和表征皮肤组织。将利用成纤维细胞（真皮）和角质形成细胞（表皮），以及在皮肤发育到全厚时分化为成纤维细胞和角质形成细胞的干细胞，培育出两层皮肤组织。细胞支架的优化将通过测定播种效率、细胞增殖、细胞活力和细胞附着来评估。采用扫描电镜（SEM）、透射电镜（TEM）和原子力显微镜（AFM）对支架和皮肤组织进行组织学分析，采用实时荧光定量PCR （real-time PCR）检测不同皮肤隔室标志物的mRNA表达水平。目的5：培养具有血管化的皮肤组织。将使用两种方法：a)在皮肤组织发育之前，通过在细胞支架中加入促血管生成分子来实现血管化皮肤的体外发育；b)在体内方法，依靠皮肤微血管细胞来实现皮肤组织发育后的血管化。目的6：培养具有黑色素特征的皮肤组织。原代表皮黑色素细胞将被培养并与成纤维细胞和角质形成细胞一起用于全层皮肤的3D生物打印。黑素细胞的形态、增殖、活力、黑色素的产生以及与成纤维细胞和角化细胞的相互作用将使用类似于先前目标中使用的技术来确定。目的7：评估皮肤组织的抗菌作用。在目标2下开发的具有抗菌特性的智能支架将用于开发人类和小鼠的3D皮肤，这些皮肤将被皮肤细菌感染，例如金黄色葡萄球菌或铜绿假单胞菌。抗菌效果将通过量化感染后细菌负荷（平板计数）和感染皮肤的结构评估（超声）来评估。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。