工程化组织的构建

生物力学检测
样品制作、保存； 实验设计：选择指标、样本量、数据处
﹡ 细胞―材料
细胞 材料
？ 有机结合体
Bioreactors for Tissue Culture
Bioreactors are purpose-designed tissue culture system；
Providing the desired biochemical and mechanical environment for cell adhesion, proliferation, differentiation and function；
工程化组织的理解
Special culture
Engineered tissue
The engineered tissue， or construct， usually consists of three-dimensional scaffolds and living cells with the desired structure and functionality.
If specific signals are not properly received or processed, then the cells dedifferentiate (i.e., become nonspecific cell types), become disorganized, and eventually die.
Preservation and storage of live engineered tissues, or bio-constructs, would be one of the major obstacles for the commercialization.
Mathematical modeling in tissue engineering
In vivo constructs after maturation in mice： Whitish with glistening； Firm in consistency.
体内、外构建的差异（关节软骨）
Articular cartilage constructs in vitro
Articular cartilage constructs in vivo
工程化组织的检测
检测对象：细胞、支架、工程化组织及植 入后新生组织;
检测指标：形态学、组织学、分子生物学 及生物力学。
形态学检测
光学显微镜； 电子显微镜； 荧光显微镜； 激光共聚焦显微镜； 原子力显微镜。
组织学检测
固定、包埋； 切片（软、硬、冰冻）； 染色（免疫组织化学：因不同组织而定）； 荧光或同位素标记； 测量（半定量、定量）。
工程化组织的构建 (Construct of engineered-tissues)
四川大学华西临床医学院 干细胞与组织工程研究室
秦廷武
Dec. 22, 2006
The concept of tissue engineering
The term ‘tissue engineering’ was officially coined at a National Science Foundation workshop in 1988 to mean the application of principles and methods of engineering and life sciences toward fundamental understanding of structure-function relationships in normal and pathological mammalian tissues and the development of biological substitutes to restore, maintain or improve tissue function.
Monitoring of the cell viability, functionality and tissue formation；
Developing continuous sampling and analytical technologies using micro- or nano-probes；
Dynamic microenvironment
Cell colonies need external cues or signals to grow into functional 3D tissues. Cells are constantly bombarded with mechanical, electrical, structural, and chemical cues that signal the cells about what they should be doing.
组织工程研究内容
组织工程研究过程
干细胞 工程
细胞 改造
细胞接种 应力与生长
生物学及 力学检测
种子细胞 支架材料
构建模式 体内 体外
工程化 组织
工程化组 织的保存
材料设计 加工检测
生物反应器、力学 植入技术 诱导与信号的转导 修复技术
植入技术 修复技术
Simple culture techniques
Purposes of tissue engineering
Medical application purpose
Research purpose
To develop biological substitutes for implantation into the body ;
To foster tissue regeneration and remodeling;
To replace, repair, maintain, or enhance function of tissues.
Testing new drugs; Simulating diseases
to develop better treatments; Reducing the use of animal tissues and organs in biological research.
皮下构建气管软骨
体内、外构建的差异
In vitro constructs formed and maintained at 8 weeks: Yellowish glistening surface； Resembling native cartilage； Stable but soft in consistency.
SEM
பைடு நூலகம்
体外构建的耳软骨
Newly formed in vitro auricular cartilage construct
Ear cartilage construct with the chondrocytes
静态、动态构建的差异（软骨）
动态构建: 有软骨陷窝; 有软骨基质分泌.
静态构建: 无软骨陷窝; 无软骨基质分泌.
体内构建
本位：功能位置； 皮下：浅表位置； 肌内：深度位置； 骨内：骨膜内。
体内构建的皮肤
Reconstructed human skin on the dorsal of the nude mice at 4 weeks after transplantation
体内构建气管软骨
皮下：浅表位置
Enabling efficient control of the tissue culture bioreactor and optimization of the construct design and bio-processing operation.
Cryopreservation of engineered tissues
Constructs are perfused using a peristaltic pump at varying flow rates to enhance matrix mineralization.
体外构建人工皮
Engineered skin constructs in vitro keratinocytes growing on a plasma polymer surface
Devising and developing new types of bioreactors, particularly addressing mass transfer and mechanical signaling.
简单
复杂
低维
多维
On-line monitoring in engineered tissue
体内、外构建的差异（皮肤）
Apligraf consists of living cells and structural proteins: Lower dermal layer combines type 1
collagen and fibroblasts (dermal cells), which produce additional matrix proteins; Upper epidermal layer is formed by
Owing to the long production cycle, for example three weeks for engineered skin grafts to eight weeks for engineered cartilage, preservation of engineered tissues is critical to ensure the off-the-shelf availability to clinicians.
静态:细胞或支架相对静止； 动态:细胞或支架相对运动； 存在的问题：
环境条件、营养条件(生物反应器) 。
生物反应器系统
静态:常规二氧化碳培养箱； 动态:旋转培养系统、拉伸/压
缩/扭转培养系统、电磁培养 系统、声学/光学培养系统。
Perfusion of Culture System
Improve mass transport throughout the scaffold by perfusing media through cell seeded scaffolds and stimulate those cells via flowinduced shear stress.
promoting human keratinocytes (epidermal cells) to multiply and to differentiate to replicate the architecture of the epidermis.
Apligraf without : • Melanocytes; • Macrophages; • Lymphocytes; • Blood vessels; • Hair follicles; • Sweat glands.
• The models can be applied both in vivo and in vitro.
工程化组织的构建方法
体外构建
（工程化椎间盘）
体内构建
（工程化耳软骨）
体外构建
在体外模拟体内细胞的动态微环境，包括 生物化学、生物物理环境。提供组织特有的生 物的、化学的和物理的刺激，产生相应的传导 信号。并提供细胞营养及代谢通路。
• Developing mathematical models for cell growth and tissue formation；
• Devising experimental techniques to determine model parameters such as oxygen consumption rate and effective diffusivity in the tissue；