💪Cell and Tissue Engineering Unit 3 – ECM and Cell-Matrix Interactions in Engineering

Key Concepts and Definitions

• Extracellular matrix (ECM) complex network of proteins, glycoproteins, and proteoglycans that provides structural and biochemical support to surrounding cells
• Integrins transmembrane receptors that mediate cell-ECM interactions and play a crucial role in cell adhesion, migration, and signaling
• Focal adhesions specialized structures formed at the cell-ECM interface that link the cytoskeleton to the ECM and facilitate signal transduction
• Matrix metalloproteinases (MMPs) enzymes secreted by cells that degrade ECM components and play a key role in ECM remodeling and cell migration
• Mechanotransduction process by which cells convert mechanical stimuli from the ECM into biochemical signals that regulate cell behavior and function
  • Involves activation of integrins, focal adhesion kinase (FAK), and downstream signaling pathways (Rho GTPases, MAPK)
• Tissue engineering interdisciplinary field that combines principles of engineering, biology, and materials science to develop functional tissue substitutes
  • Relies heavily on understanding and manipulating cell-ECM interactions to guide tissue formation and regeneration

ECM Components and Structure

• Collagen most abundant protein in the ECM, provides tensile strength and structural support
  • Different types of collagen (I, II, III, IV) are found in specific tissues (bone, cartilage, skin, basement membranes)
• Elastin highly elastic protein that allows tissues to stretch and recoil without damage
  • Abundant in blood vessels, skin, and lungs
• Fibronectin glycoprotein that binds to integrins and other ECM components, facilitating cell adhesion and migration
• Laminin major component of basement membranes, plays a crucial role in cell differentiation and tissue organization
• Glycosaminoglycans (GAGs) long, unbranched polysaccharide chains that form hydrated gels and provide compression resistance
  • Examples include hyaluronic acid, chondroitin sulfate, and heparan sulfate
• Proteoglycans proteins with covalently attached GAG chains, regulate cell signaling and growth factor binding
  • Aggrecan, a large proteoglycan, is a major component of cartilage ECM and provides compressive strength
• ECM organization varies depending on tissue type and function
  • Bone ECM is mineralized with hydroxyapatite crystals, providing rigidity and strength
  • Cartilage ECM is rich in collagen II and proteoglycans, allowing for compression resistance and lubrication of joints

Cell-ECM Interactions

• Cell adhesion mediated by integrins, which bind to specific ECM ligands (fibronectin, collagen, laminin)
  • Integrins are heterodimers composed of α and β subunits, with different combinations conferring specificity for different ECM components
• Formation of focal adhesions upon integrin clustering and activation
  • Focal adhesions contain proteins such as talin, vinculin, and paxillin, which link integrins to the actin cytoskeleton
• Cell migration involves dynamic remodeling of focal adhesions and the actin cytoskeleton
  • Cells extend protrusions (lamellipodia and filopodia) in the direction of migration, form new adhesions, and detach from the ECM at the trailing edge
• ECM stiffness and topography influence cell behavior and differentiation
  • Cells sense and respond to mechanical properties of the ECM through mechanotransduction pathways
  • Stem cells differentiate into specific lineages depending on ECM stiffness (soft matrices promote neurogenesis, while stiff matrices promote osteogenesis)
• Cell-ECM interactions regulate cell survival, proliferation, and differentiation
  • Anchorage-dependent cells require adhesion to the ECM to survive and proliferate
  • Loss of cell-ECM contact can lead to anoikis, a form of programmed cell death

Signaling Pathways in Cell-Matrix Communication

• Integrin activation triggers downstream signaling cascades that regulate cell behavior
  • Focal adhesion kinase (FAK) is recruited to focal adhesions and becomes phosphorylated upon integrin activation
  • FAK activates Src family kinases, which in turn activate Rho GTPases (RhoA, Rac1, Cdc42) that regulate actin cytoskeleton dynamics
• Mitogen-activated protein kinase (MAPK) pathways are activated by integrin signaling
  • Extracellular signal-regulated kinase (ERK) pathway promotes cell proliferation and survival
  • c-Jun N-terminal kinase (JNK) and p38 MAPK pathways are involved in cell stress responses and apoptosis
• PI3K/Akt pathway is activated by integrin signaling and promotes cell survival and growth
  • Akt phosphorylates and inactivates pro-apoptotic proteins (Bad, caspase-9) and activates pro-survival factors (mTOR, NF-κB)
• Crosstalk between integrin signaling and growth factor receptor signaling
  • ECM-bound growth factors (FGF, VEGF, TGF-β) can be released upon ECM degradation and activate their respective receptors
  • Integrin and growth factor receptor signaling pathways converge to regulate cell behavior and gene expression
• Wnt/β-catenin signaling pathway is modulated by cell-ECM interactions
  • β-catenin is a key component of adherens junctions and is also involved in transcriptional regulation
  • ECM composition and stiffness can influence the localization and activity of β-catenin, affecting cell fate decisions

ECM Remodeling and Dynamics

• ECM remodeling is a continuous process that involves synthesis, degradation, and reorganization of ECM components
  • Necessary for tissue development, wound healing, and adaptation to mechanical loads
• Matrix metalloproteinases (MMPs) are the main enzymes responsible for ECM degradation
  • Secreted as inactive zymogens (pro-MMPs) and activated by proteolytic cleavage
  • Specific MMPs degrade different ECM components (collagenases, gelatinases, stromelysins)
  • MMP activity is regulated by tissue inhibitors of metalloproteinases (TIMPs)
• Dysregulation of ECM remodeling is associated with various pathological conditions
  • Excessive ECM degradation occurs in cancer invasion and metastasis, allowing tumor cells to migrate through the basement membrane and invade surrounding tissues
  • Insufficient ECM degradation leads to fibrosis, characterized by excessive accumulation of collagen and other ECM components (liver cirrhosis, pulmonary fibrosis)
• ECM turnover and renewal are important for maintaining tissue homeostasis
  • Collagen synthesis and assembly are regulated by enzymes such as lysyl oxidase (LOX) and procollagen peptidases
  • Proteoglycan synthesis and GAG chain modification are controlled by specific glycosyltransferases and sulfotransferases
• Mechanical forces and cell-generated tension influence ECM remodeling
  • Cells respond to mechanical stress by altering their ECM synthesis and degradation profiles
  • Tension-induced ECM alignment and reorganization are crucial for proper tissue function (aligned collagen fibers in tendons and ligaments)

Engineering Applications of ECM

• Tissue engineering scaffolds are designed to mimic the native ECM and support cell growth and differentiation
  • Natural ECM-derived materials (collagen, fibrin, decellularized tissues) provide biocompatibility and bioactivity
  • Synthetic polymers (PLA, PGA, PLGA) offer tunable mechanical properties and degradation rates
  • Composite scaffolds combine the advantages of natural and synthetic materials
• Hydrogels are highly hydrated, cross-linked polymer networks that resemble the native ECM
  • Can be engineered to incorporate cell adhesion ligands, growth factors, and degradable cross-links
  • Injectable hydrogels allow for minimally invasive delivery and in situ gelation
• ECM-mimetic surface modifications enhance cell adhesion and differentiation on biomaterials
  • Coating with ECM proteins (fibronectin, collagen, laminin) or peptide sequences (RGD)
  • Micro- and nanoscale surface topography can guide cell alignment and migration
• Decellularized ECM scaffolds are prepared by removing cells from native tissues while preserving the ECM structure and composition
  • Retain tissue-specific biochemical cues and mechanical properties
  • Can be repopulated with patient-specific cells for personalized tissue regeneration
• 3D bioprinting enables precise control over the spatial arrangement of cells and ECM components
  • Extrusion-based bioprinting deposits cell-laden hydrogels or bioinks layer-by-layer
  • Laser-assisted bioprinting allows for high-resolution patterning of cells and ECM
• Microfluidic devices can be used to study cell-ECM interactions in controlled microenvironments
  • Gradient generators create spatial variations in ECM composition and stiffness
  • Organ-on-a-chip systems recapitulate the complex ECM organization and mechanical cues of native tissues

Challenges and Future Directions

• Achieving precise control over the spatial and temporal dynamics of ECM remodeling in engineered tissues
  • Incorporating responsive elements (MMP-cleavable peptides, light-sensitive cross-links) into scaffolds to enable on-demand remodeling
  • Developing advanced bioreactor systems that apply physiologically relevant mechanical stimuli to guide ECM alignment and maturation
• Recapitulating the complex biochemical and biophysical cues of the native ECM in vitro
  • High-throughput screening of ECM compositions and stiffness gradients to identify optimal conditions for specific cell types and applications
  • Integrating multiple ECM-mimetic cues (topography, ligand presentation, mechanical properties) into a single scaffold
• Scaling up the production of ECM-based scaffolds for clinical translation
  • Optimizing decellularization protocols to ensure complete cell removal and preservation of ECM structure and composition
  • Developing automated and reproducible manufacturing processes for large-scale scaffold fabrication
• Addressing the immunogenicity and potential for disease transmission of ECM-derived materials
  • Thorough characterization and quality control of decellularized ECM scaffolds
  • Investigating the use of genetically engineered or humanized animal tissues as ECM sources
• Advancing our understanding of cell-ECM interactions in complex, multicellular environments
  • Developing ex vivo tissue models that capture the native ECM complexity and cell-cell interactions
  • Employing advanced imaging techniques (multiphoton microscopy, super-resolution microscopy) to visualize ECM remodeling and cell behavior in real-time
• Harnessing the potential of ECM-based therapies for regenerative medicine and tissue repair
  • Combining ECM scaffolds with stem cells, growth factors, and gene delivery approaches for enhanced regenerative outcomes
  • Exploring the use of ECM-derived bioactive molecules (matricryptic peptides, cryptic growth factor binding sites) as therapeutic agents

Key Takeaways and Review

• The extracellular matrix (ECM) is a complex network of proteins, glycoproteins, and proteoglycans that provides structural and biochemical support to cells
• Key ECM components include collagen, elastin, fibronectin, laminin, glycosaminoglycans (GAGs), and proteoglycans
• Cell-ECM interactions are mediated by integrins, which cluster to form focal adhesions and link the ECM to the actin cytoskeleton
• Integrin signaling activates downstream pathways (FAK, Rho GTPases, MAPK, PI3K/Akt) that regulate cell behavior and gene expression
• ECM remodeling involves the synthesis, degradation, and reorganization of ECM components, and is regulated by matrix metalloproteinases (MMPs) and their inhibitors (TIMPs)
• Dysregulation of ECM remodeling is associated with various pathological conditions, such as cancer invasion and metastasis, and fibrosis
• Tissue engineering applications of ECM include the design of ECM-mimetic scaffolds, hydrogels, surface modifications, decellularized ECM scaffolds, and 3D bioprinting
• Future challenges and directions in ECM engineering include achieving precise control over ECM remodeling dynamics, recapitulating complex ECM cues in vitro, scaling up production for clinical translation, addressing immunogenicity and disease transmission risks, advancing our understanding of cell-ECM interactions in complex environments, and harnessing the potential of ECM-based therapies for regenerative medicine.