• basic
    • Morphogenesis is the developmental cascade of pattern formation, the establishment of the body plan and architecture of mirror-image bilateral symmetry of anatomical structures, culminating in the adult form.
    • 形态发生是模式形成的发育级联过程，涉及身体蓝图的建立、解剖结构镜像双侧对称性的构建，最终形成成体形态
      • The goal of tissue engineering is to locally recapitulate the developmental process of morphogenesis.
      • 组织工程的目标是在局部重现形态发生的发育过程
  • The three key ingredients for morphogenesis
    • inductive morphogenetic signal 诱导性形态发生信号
    • responding stem cells 响应性干细胞
    • extracellular matrix scaffolding ECM支架
  • gastrulation 原肠胚形成
    early morphogenesis
    • Specification of germ layers 胚层特化
    • Instruction to cells on their future position and role in the developing fetus胎儿 by morphogen signaling形态发生素信号传导
    • The ectoderm外层胚 expresses bone morphogenetic protein骨形态发生蛋白 BMP-4, which directly and supplemented with signaling via the endoderm内胚层, regulates the formation of the mesoderm中胚层
    • 外胚层表达骨形态发生蛋白 BMP-4，该蛋白直接且在內胚层信号传导的辅助下，调控中胚层的形成
    • Nodal
      • Nodal, which is like BMP4, a TGF- β family member, that is recognized as a central molecule in cell instructions during gastrulation
      • Nodal 与 BMP-4 同属转化生长因子 β（TGF-β）家族成员，是原肠胚形成过程中细胞指令传递的核心分子
      • Nodal signaling is essential for the formation of mesoderm中胚层 and definitive endoderm定型內胚层
  • Morphogen gradients
    • Morphogen gradients as positional cues 形态发生素浓度作为位置信号
    • 以Nodal为例
      • The concentration of Nodal will gradually decrease, With increasing distance away from the node (highest), establishing a so-called morphogen gradient
      • node 分泌 FGF8 Fibroblast Growth Factors 成纤维细胞生长因子
      • Cells along this gradient are exposed to different levels of Nodal, providing the cell with important instructions on its future position and role in the fetus
    • Morphogen gradients control a number of genetic mechanisms including transcriptional enhancers增强子 and repressors抑制子 of gene transcription
    • therefore are important and recurrent反复的 mechanisms by which undifferentiated未分化的 cells receive instructions on their future position and role in the body
    • Morphogen gradients
      • Spatial gradient
      • Temporal gradient
  • skeletogenesis
    Morphogenesis of musculoskeletal systems
    • Mesoderm中胚层 and neural crest神经嵴 are responsible for the formation of the skeleton during embryonic development
    • Even before the onset开始 of skeletogenesis, cells at the site of the future bone already contain information on the shape and structure of the bone they will form.
    • This information is provided by morphogen gradients, which regulate combinatorial expressions组合表达 of genes (like Hox genes同源盒基因)
    • 间充质干细胞MSC向成骨细胞、软骨细胞及脂肪细胞分化的分子调控机制
      • 通过Wnt/β-catenin 信号通路维持自我更新, 若该通路被抑制，MSC 会启动分化程序
      • 图
  • Morphogenesis in bone 骨骼的形态发生
    • DBM - Demineralized Bone Matrix 脱钙骨基质
      • basic
        • Bone grafts骨移植材料, like decalcified bone脱钙骨, have been used by orthopedic surgeons骨科医生 for nearly a century to aid bone repair.
        • Demineralized脱矿物质 segments of rabbit bone, when implanted intramuscularly在肌肉中植入, induced new bone formation.
        • The DBM is composed mainly of type I insoluble collagen, which binds fibronectin纤连蛋白
        • It was hypothesized推测 that bone might contain a substance that initiates bone growth
      • Cascading events following DBM implantation
        • The responding mesenchymal间充质 cells attached to the collagenous matrix胶原基质 and proliferated
        • Day 5, chondroblast differentiation 软骨母细胞分化
        • Days 7–8, chondrocytes 软骨细胞形成
        • Day 9, cartilage hypertrophy 软骨肥大, Vascular invasion血管入侵, osteoblast differentiation成骨细胞分化
        • Days 10–12 alkaline phosphatase was maximal碱性磷酸酶活性达到峰值
        • Day 28, Osteocalcin, bone gla protein (BGP), increase 骨钙素, 骨 γ- 羧基谷氨酸蛋白, 水平升高
        • The key steps in this cascade are chemotaxis趋化作用, mitosis, and differentiation
        • Chemotaxis is the directed migration of cells in response to a chemical gradient of signals released from the insoluble demineralized bone
          趋化作用是指细胞在脱钙骨释放的信号化学梯度引导下发生的定向迁移
      • Extracting soluble morphogens
        • Dissociative extractants解离提取剂 (guanidine HCl 盐酸胍) were used to solubilize proteins from demineralized bone matrix DBM
        • Remaining residue was mainly insoluble type I bone collagen
        • The extract alone or the residue alone was incapable of new bone induction
        • However, addition of the extract to the residue (insoluble collagen) resulted in bone induction after implantation
        • A collaboration between the soluble signal in the extract and insoluble substratum or scaffolding is required for bone formation
        • 骨形成需要提取物中的可溶性信号与不溶性基质或支架材料协同作用
        • 1 μg of active osteogenic fraction in a kilogram of bone
        • 每千克骨骼中仅含 1 微克具有成骨活性的组分
      • DBM制备以及后续应用
        • 图
    • BMP - Bone morphogenic protein 骨形态发生蛋白
      • BMPs binds to ECM
        • Bone morphogenetic proteins can bind to ECM molecules, like heparan sulfate硫酸乙酰肝素, heparin肝素 and type IV collagen
        • The binding of a soluble morphogen to extracellular matrix converts it into an insoluble matrix–bound morphogen to act locally in the solid state
        • This can establish spatial gradients of morphogens in the ECM, hence assists in morphogenesis, i.e. establishment of spatial patterns in organs and tissues
      • Functions of BMPs in morphogenesis
        • BMP is a key growth factor for bone and cartilage development.
        • BMP is also needed for the development of other organs including heart, kidney, eyes.
        • In some cases, BMP need to be inactivated by a binding protein before some tissues can develop, such as neural tissues
        • 图
  • Two ways to delivery morphogens to cells
    • Gene therapy achieves a sustained, prolonged secretion of gene products
      • The use of adenoviruses腺病毒 and tetroviruses逆转录病毒 for inserting therapeutic治疗性 genes into cell genome - safety concern over the use of viral delivery vehicle and disruption to genome 存在安全性, 基于病毒递送, 会整合到宿主基因
      • The mRNA technology - likely safer without integrating into host cell genome and based on synthetic nanoparticle delivery system 不需要整合到宿主基因, 基于纳米递送
    • Delivery of morphogens via biomaterial carriers
      • Biomimetic scaffold carrying morphogens
        • Major bone components like collagen and hydroxyapatite羟基磷灰石 bind to protein well
        • An osteoconductive骨传导性 biomimetic仿生的 biomaterial implant can progressively逐步地 function as an osteoinductive骨诱导 substance by binding to endogenous内源性 BMPs
          • Osteoconductive - the ability to act as a scaffold to which native bone cells can attach, initiating bone growth, like HA
          • 骨传导性 - 指材料能作为支架供天然骨细胞附着，进而启动骨生长的能力 (如羟基磷灰石 / HA)
          • Osteoinductive - the stimulation of osteoprogenitor cells to differentiate into osteoblasts that then begin new bone formation, like BMPs
          • 骨诱导性 - 指材料能刺激骨祖细胞分化为成骨细胞，进而启动新骨形成的能力 (如骨形态发生蛋白 / BMPs)
          • 图
  • 应用
    • Currently, two BMPs products have been approved by the Food and Drug Administration (FDA) for clinical applications (fractures of long bones, intervertebral disk regeneration)
    • by delivery in a purified collagen matrix (which is implanted in the site of the fracture)
    • products
      • Infuse BMP-2 (Medtronic)
      • OP-1 BMP-7 (Stryker)
      • 这两种产品都是促进骨生长的蛋白质
    • 脊柱融合装置
      • The device consists of three components spilt among two parts 组件分为两部分，共含三个核心成分
        • a metallic tapered spinal fusion cage金属锥形脊柱融合器 (known as the LT-CAGE™ Lumbar Tapered Fusion Device); and
        • a bone graft substitute骨移植替代物 (InFUSE™ Bone Graft) which consists of
          • a genetically-engineered human protein基因工程重组人源蛋白 (rhBMP-2)
          • a carrier/scaffold for the protein该蛋白的载体/支架 (manufactured from bovine [cow] Type I collagen)
      • 第二部分放在第一部分的融合器里面
      • 图