Lecture 20: Engineering Tissues & Organs PDF

🥽 Lecture 20: Engineering tissues & organs do notes according to summary points at the end of lecture slide instead of your normal LOs (where applicable) What are hydrogels? Definition: Hydrogels are an insol...

🥽 Lecture 20: Engineering tissues & organs do notes according to summary points at the end of lecture slide instead of your normal LOs (where applicable) What are hydrogels? Definition: Hydrogels are an insoluble network of polymer chains that swell in water (3D swollen network). Composition: Natural or synthetic polymeric networks. Water content: Ranges from at least 30% to over 90%. Properties: A major class of biomaterials. Possess tissue-like structural properties. Hydrogels are generated by crosslinking: 1. Chemical Crosslinking (Covalent Crosslinks): Lecture 20: Engineering tissues & organs 1  Involves a chemical reaction. 2. Physical Crosslinking (Non-Covalent Crosslinks): Includes hydrogen bonds, ionic bonds, and self-assembly. Application: Hydrogels can be pipetted or 3D-printed. Cells can be embedded in hydrogel networks. Types of hydrogels and biomaterials There are three different types of hydrogels depending on the origin of the biomaterial used (natural, semi-synthetic, synthetic). Lecture 20: Engineering tissues & organs 2  Natural hydrogel: derived from naturally occurring biomaterials. Synthetic hydrogel: engineered from synthetic polymers. Natural Hydrogels: Matrigel: Composition: Mix of matrix proteins and growth factors. Usage: Widely used matrix for organoid cultures. Limitations: Chemically undefined. High batch-to-batch variation. Collagen Hydrogels: Composition: Primary constituent of the extracellular matrix. Preparation: Reconstituted collagen in acidic solution. Limitations: Lack of control over matrix composition, architecture, and collagen type. Synthetic Hydrogels: PEG (Polyethylene Glycol) Hydrogels: Lecture 20: Engineering tissues & organs 3  Common Usage: Widely used as a hydrogel component in biomedical applications. Properties: Chemically Crosslinked: Can be crosslinked in various ways to form stable hydrogels. Non-Adhesive: Non-adhesive to cells and proteins, making it ideal for specific biomedical uses. Eliminability: Can be eliminated from the body, enhancing its biocompatibility. Applications: Coating Biomaterials: Used to coat biomaterials to induce ‘stealth’ properties, making them non-thrombogenic (reducing clot formation). Functionalization: Functionalized with peptides for cancer models and other tissue engineering applications. Design Flexibility: Allows precise tuning of hydrogel architecture and stiffness. Lecture 20: Engineering tissues & organs 4  Incorporates matrix-binding and degradative motifs for targeted applications. Crosslinking and Structure: Typically involves calcium ions and enzymes like transglutaminase for forming a non-fibrous, elastic network suitable for 3D cell cultures Peptide Amphiphiles (PAs): Properties: High design flexibility, customizable with specific motifs. Usage: Cancer models and other tissue engineering applications. Limitations: Low mechanical properties, similar to Matrigel. Self-assembling peptide amphiphiles Definition: Self-Assembly: Spontaneous organization of single components into an ordered structure without human intervention. Commonly found in nature (e.g., micelles, protein complexes, membranes). Lecture 20: Engineering tissues & organs 5  Examples in Nature: Micelles Protein complexes Membranes (e.g., lipid bilayers) Application in Research: Tumour Models: Developed a tumour model using self-assembling peptide amphiphiles. Schematic Overview: Peptide amphiphile fibers act as a scaffold. Fibers can include various proteins (represented by blue fibers). Different cell types are incorporated, including tumour cells and supporting cells. Functionalisation: Includes proteins or peptides such as integrin-binding peptides or interactive sequences. Allows high control over hydrogel properties (molecular composition, matrix stiffness, degradation). Methodology: Components (peptides, cells) are mixed in a test tube. After a reaction time of 30 minutes, the hydrogel forms. Hydrogels are free-floating and can be grown in 48-well plates for up to four weeks. Lecture 20: Engineering tissues & organs 6  Applications: Drug Screening: Used for testing tumour cell reactions to drugs. Matrix Analysis: Studied how matrix components assemble at tumour sites. Hydrogen swelling Starts with the dry and soluble hydrogel material placed in water Water enters the material and hydrates the most hydrophilic groups (primary bound water) Network swells and exposes the hydrophobic groups (secondary bound water) Osmotic pressure drives additional water uptake until it is opposed by the network chain extension and reaches an equilibrium swelling (free water) Lecture 20: Engineering tissues & organs 7  Important because it controls solute diffusion, optical and mechanical properties: Solute Diffusion: Swelling affects how molecules (solutes) move through the hydrogel. A more swollen hydrogel has larger pores, allowing solutes to diffuse more easily. Conversely, less swelling restricts solute movement. Optical Properties: The transparency and refractive index of the hydrogel can change with swelling. Swollen hydrogels often become more transparent as the water content increases, affecting how light passes through the material. Mechanical Properties: Swelling influences the stiffness, elasticity, and strength of the hydrogel. A more swollen hydrogel is typically softer and less rigid, whereas a less swollen hydrogel retains more mechanical strength Hydrogel swelling and pH pH Sensitivity: Most pH-sensitive polymers swell at high pH and collapse at low pH. Drug Delivery Mechanism: This property is utilized for triggered drug delivery systems. Lecture 20: Engineering tissues & organs 8  Stomach (Low pH): The drug is not released due to hydrogel collapse. Small Intestine (High pH): The hydrogel swells, triggering drug release. Colon (Low pH): The hydrogel is degraded, ensuring targeted drug release. Schematic Overview: The process involves the hydrogel absorbing water, swelling, and releasing the drug in specific pH conditions, providing a controlled release mechanism through the gastrointestinal tract. Lecture 20: Engineering tissues & organs 9  Hydrogel Degradation Degradation mechanisms include different components that may degrade via cleavage of: Polymer / material backbone Crosslinker Side group Degradation rate depends on: Mechanism Chemistry of degradable link Catalyst Lecture 20: Engineering tissues & organs 10  In vitro and in vivo degradation rates are different Triggered Degradation: Can be induced by light or mechanical force, providing spatial and temporal control. Control over hydrogel degradation Lecture 20: Engineering tissues & organs 11  Chemically crosslinked hydrogels can be engineered to degrade at a programmed, cell-induced or user-defined rate with varying degrees of spatiotemporal control. Chemically crosslinked hydrogels can be designed to break down in a controlled manner, either on their own or in response to external factors. This degradation can be: Programmed Rate: The hydrogel is engineered to degrade at a predetermined speed based on its chemical composition and environmental conditions, such as pH or temperature. Cell-Induced Degradation: The degradation is triggered by biological processes, such as the action of enzymes produced by cells, allowing the hydrogel to degrade as cells interact with it. User-Defined Rate: The degradation can be adjusted by external inputs, such as applying a specific chemical or physical stimulus (e.g., light, heat, or mechanical force). Lecture 20: Engineering tissues & organs 12  Spatiotemporal Control: This refers to controlling both the location and timing of degradation, enabling precise manipulation of the hydrogel’s structure and function for applications like targeted drug delivery or tissue engineering. Stimulus-responsive hydrogels Swelling and Shrinking: Hydrogels can transition between a swollen, highly interconnected network and a dry, shrunken state. External Stimuli: Environmental factors can induce, inhibit, or reverse swelling/shrinking processes. Examples of stimuli include: pH: As previously discussed, changes in pH can trigger swelling or collapse. Temperature: Thermal changes can influence the hydrogel's state. Electricity: Electrical stimuli can modify hydrogel properties. Magnetic Fields: Magnetic stimuli can also induce changes. Light: Exposure to light can trigger responses. Humidity: Changes in humidity affect swelling. Chemical Reactions: Reactions such as redox processes can be utilized. Lecture 20: Engineering tissues & organs 13  Conclusion: Hydrogels respond to changes in external conditions or stimuli (pH, temperature, light, mechanical forces etc). Responsive biomaterials: 4D printing Plant-Based Stimulus Example: Utilizes a plant-based stimulus (e.g., a flower) introduced into medical modeling. The material is printed using a 3D printer. Upon exposure to water pressure or osmotic pressure, the material swells, creating a dynamic "fourth dimension" in the structure. The 3D structure evolves over time, swelling to form a complex, dynamic structure, as shown in the example image. Lecture 20: Engineering tissues & organs 14  Temperature-Dependent Biowork Example: Focuses on bio-work with robots using temperature-sensitive materials. At lower temperatures, the material keeps flower petals closed. As temperature increases (up to 50°C), the petals open, mimicking the blooming process of a flower. Utilizes shape memory polymers to achieve this effect. Lecture 20: Engineering tissues & organs 15  Application in Bioprinting: The shape memory polymers enable the creation of temperature- responsive structures. High-resolution projection stability tomography is used for precise printing. These materials allow control over thermal and mechanical behavior. Bio-Robotic Gripper Example: Multi-material grippers are designed with various structures and sizes. Process Overview: A: Different designs of multi-material grippers. B: Transition between the printed shape and the temporary shape, triggered by temperature changes. C: Snapshots of the gripper grabbing an object, controlled solely by temperature changes. Highlights the potential for engineering tissues, organoids, and organ functions through dynamic material design Lecture 20: Engineering tissues & organs 16  Hydrogel applications Soft contact lenses: Hydrogels provide a soft, oxygen-permeable material that holds water, keeping lenses moist and comfortable. Personal care (nappies/diapers): Hydrogels absorb and lock in moisture, keeping skin dry and preventing leaks. Agriculture (water-storing granules): Hydrogels absorb water and release it gradually, helping plants stay hydrated during dry periods. Food (thickening agents): Hydrogels enhance texture and stability by retaining water and thickening food products. Cell encapsulation (organoids): Hydrogels create a supportive 3D matrix for cells, simulating natural tissue environments. Drug delivery: Hydrogels control the timed release of medications, improving precision and efficacy. Lecture 20: Engineering tissues & organs 17  Tissue engineering (cartilage, bone): Hydrogels provide a scaffold that supports cell growth and guides tissue regeneration. Regenerative medicine (organ functions): Hydrogels support cell growth and tissue repair, aiding in organ regeneration. Cancer research (organoids, 3D cancer models): Hydrogels mimic the tumor microenvironment, enabling realistic cancer modeling and drug testing. Many hydrogels are non-adhesive to cells. They have a similar surface tension to native tissue so have reduced levels of protein adsorption, therefore minimal foreign body reaction. Hydrogels in Tissue and Organ Development Early Use of Matrigel: Initially used for simple tissue and organ studies. Applied to organoids and later to cancer cell studies. Advancements with 3D Bioprinting: Allows printing of tissue analogues layer by layer. Key difference from traditional 3D printing: Inclusion of cells, growth factors, or drugs in the biomaterial. Scaffolds can mature over time, e.g., in bioreactors. Specific Applications Tissue Replacement: Hydrogels used to create scaffolds for damaged tissue. Scaffolds mature and respond before being implanted into patients. Lecture 20: Engineering tissues & organs 18  Example: 3D Bioprinted Prosthetic Ovary Development: Uses gelatin-based scaffolds. 3D printing at different angles (30°, 60°, 90°) to create fiber structures. Cells (e.g., green fluorescent protein-labeled follicles) seeded onto the scaffold. Observations: Larger angles (60°, 90°) favored cell attachment. Scaffold implanted in mice after ovaries were removed. Results: Implanted scaffolds led to successful reproduction in mice. Offspring produced across multiple generations, demonstrating functional organ replacement. Melt Electrospinning Writing (MEW) for Scaffold Regeneration Process Overview: Lecture 20: Engineering tissues & organs 19  A technique used for regenerating scaffolds from biomaterials, specifically polycaprolactone (PCL). Can produce various scaffold shapes: Large sheets. Tubular structures for bone tissue engineering. Printing Method: Polymer is heated to high temperatures, generating a jet of tiny fibers. These fibers are deposited on a collector and gradually form scaffold structures. Fiber deposition can be controlled at different angles (90°, smaller angles) for patterning. Lecture 20: Engineering tissues & organs 20  Sterilization and Use: Post-printing scaffolds need to be sterilized before use. Used in lab settings to culture human osteoclasts and produce bone scaffolds. Advantages of MEW: Electrospun fibers mimic the size and structure of native extracellulr matrix fibers (0.5–3 microns in diameter). Provides a tissue-specific microenvironment for cellular growth. Lecture 20: Engineering tissues & organs 21  Example: Human Osteoclast Culturing: Human osteoclasts were seeded onto 3D printed scaffolds. Cells attached to the fibers, filling pores and depositing their own extracellular matrix over time. Visualized through microscopy using specific markers for extracellular matrix components (e.g., collagen). PEG-Based Hydrogels for Cancer Research Hydrogel Application: Used to create models for ovarian cancer. Patient-derived ovarian cancer cells grown in PEG-based hydrogels. Lecture 20: Engineering tissues & organs 22  Properties of PEG-Based Hydrogels: Customizable mechanical properties (soft vs stiff). Functionalized with peptide sequences, such as RGB peptides, to modify binding sites and mechanical characteristics. Findings with Ovarian Cancer Cells: Soft vs Stiff Scaffolds: Cancer cells preferred soft scaffolds over stiff ones. Increased stiffness led to changes in cell proliferation. MMP-Sensitive Hydrogels: Incorporating matrix metalloproteinase (MMP) sensitive peptides increased cancer cell proliferation. Hydrogels without MMP-sensitive peptides showed reduced proliferation. Lecture 20: Engineering tissues & organs 23  Drug Screening: Hydrogel systems enable screening of cancer drug inhibitors (e.g., MMP inhibitors). Identified that MMP inhibitors reduced cancer cell proliferation significantly. Patient-Specific Bioengineering for Cancer Research Initial Process: Research starts with patient-specific tumor tissue (e.g., pancreatic cancer). Fresh tumor tissues undergo mechanical testing to measure tissue stiffness or softness, which informs the biomaterials used in the 3D printing process. Biomaterial Use in 3D Printing: The mechanical properties of the tissue guide the selection of fibers (e.g., cleaning fibers with specific stiffness). Post-printing, patient-derived cells are added to the biomaterial, and the tissue is observed for how the cells grow and interact with the material. Patient-Derived Cells: Lecture 20: Engineering tissues & organs 24  When enough tissue is available, patient-derived cells (e.g., cancer- associated fibroblasts, myeloid cells) are isolated and used in the bioengineering process. These cells can be mixed into a biomaterial for printing, creating multicellular models or "hydrogel chocolates," where different cell types are encapsulated. Multi-Cellular Models: The goal is to create complex models that mimic the tumor microenvironment, including various cell types. These models can then be used for drug testing to understand patient- specific responses to treatments. Research Aim: Personalized treatment strategies for cancer, allowing for better targeting of therapies based on individual patient needs. Research tools – biomaterials 1. PEG-Based Hydrogels (Synthetic Hydrogels): Purpose: Used in the lab to grow cells and mimic their extracellular matrix (ECM). Lecture 20: Engineering tissues & organs 25  Function: Cells form their own ECM fibers within the PEG hydrogel. Key Feature: The absence of functionalized ECM molecules allows researchers to study cell interactions in a simpler synthetic matrix. Comparison: Scanning electron microscopy (SEM) can be used to study how cells interact with the PEG matrix. 2. GelMA Hydrogels (Gelatin Methacryloyl): Purpose: Another commonly used hydrogel for creating tissue scaffolds. Applications: Frequently used in tissue engineering and cell culture models. Properties: Derived from gelatin, GelMA provides a natural, biocompatible scaffold for cell growth. 3. Peptide-Functionalized Hydrogels (Peptide Amphiphiles): Purpose: These hydrogels are functionalized with peptides, offering enhanced control over the fibrous network structure. Function: Peptides allow for the creation of ECM-like fibers, such as collagen fibers, which are found in native tissues. Comparison to Synthetic Hydrogels: These hydrogels have a softer structure, more akin to natural ECM compared to other synthetic hydrogels. Applications: Used in bioengineering to create scaffolds that mimic native tissue environments more closely. 4. Matrigel/Collagen Gels: Purpose: These are naturally derived hydrogels that are used for cell culture and tissue engineering. Applications: Provide a natural ECM environment for cell growth. Comparison: Scanning electron microscopy can be used to compare peptide-functionalized hydrogels with naturally occurring ECM structures like matrigel and collagen to ensure the best mimicry of native tissue architecture. 5. Melt Electrospinning Writing (PCL-Based Electrospun Scaffolds): Lecture 20: Engineering tissues & organs 26  Purpose: PCL (polycaprolactone) fibers are electrospun to create scaffolds. Advantages: This method offers high control over fiber placement and structure, crucial for applications where precision is key. Applications: Used in bone tissue engineering and other fields that require precise control of the scaffold's microstructure Tissue Engineering – 1993 and today Early Tissue Engineering (1993): Key components: Cells Biodegradable Polymer Scaffold Matrix for Tissue Construction Used to regenerate tissues such as bone, cartilage, and liver. Tissue Engineering Over Time: Stem Cells: The use of stem cells (e.g., iPS cells) for tissue engineering, a major advancement in the field. Biomaterial Engineering: Hydrogels, with a focus on biodegradable and bioactive materials. Lecture 20: Engineering tissues & organs 27  The integration of hydrogels with biomaterials for more efficient tissue engineering. Technology: The development of bioprinting and 3D printing technologies for creating tissue constructs. Biomechanics: Importance of considering the stiffness and biomechanics of the engineered tissues to match the native tissue characteristics. Current Applications and Technologies: Self-Assembly: Using self-assembling materials (e.g., peptide hydrogels) for tissue engineering. Bioprinting/3D Printing: Key methods for creating tissue constructs with precision. Organ Decellularization: Example: Using leftover tissue from liver transplantations or other organs, which is decellularized to preserve the native extracellular matrix. This matrix is then repopulated with patient-derived cells to regenerate functional organs. Future Considerations: Patient-Centric Approach: Emphasizing that tissue engineering should start with patient-specific considerations, such as the biomechanics and stiffness of tissues Lecture 20: Engineering tissues & organs 28  Applications of 3D Platforms and Approaches in Bioengineering Disease Modeling: Human Disease: Used to study diseases such as cancer, modeling both early disease steps and progression. Metastatic Processes: Helps in studying cancer metastasis and progression. Incorporating Microfluidics: Organ-on-a-Chip or Cancer-on-a-Chip Devices: Allows integration of circulatory systems and microfluidics to better mimic human tissues and processes. Tumor Vascularization: Tumorigenesis Models: These platforms can model tumor vascular networks and tumorigenesis, including macrophage interactions with tumors. Mechanical and Matrix Interactions: Matrix Stiffness: Studies the mechanical plasticity and interactions between tumor cells and the extracellular matrix (ECM). Screening and Monitoring: Lecture 20: Engineering tissues & organs 29  Circulating Tumor Cells (CTCs): Can be used for screening and monitoring cancer therapies or circulating tumor cells. Podocyte Activity: Research on substrates, particularly in the context of podiatrists, using engineered tissues. Preclinical Drug Screening: Drug Discovery: Tumoroids, organoids, and spheroids are crucial for preclinical drug screening of biologics and new therapeutics such as antibodies, cell therapies, and biological therapeutics Benefits and limitations of tumour tissue engineering Benefits of Tumor Tissue Engineering: 1. Control and Reproducibility: Defined biomaterial-based hydrogel models offer high control over composition and are reproducible. These models do not have the batch-to-batch variation seen in tissue matrices. 2. Personalization: Can model patient-specific tumor features and responses, enabling personalized treatment models. 3. Controlled Analysis: Lecture 20: Engineering tissues & organs 30  Offers the ability to perform very controlled analyses on disease models. Limitations of Tumor Tissue Engineering: 1. Labor-Intensive: Creating and using these models can be labor-intensive, requiring a lot of effort in handling and experimentation. 2. Cost: Expensive due to the materials and technologies (e.g., 3D printing, bioink, and equipment) involved. In-house recipe development for some components adds further costs. 3. Multifaceted Model Limitations: Incorporation of Multiple Cell Types: Challenges remain in incorporating all necessary cell types (e.g., malignant and non- malignant) in these models. Multifactorial Nature: Models may require multiple biomaterials, each with different physical and chemical properties. 4. Optimization for Specific Tissue Models: Each tissue or organ model requires optimization of bioprinting parameters (e.g., biomaterial selection, culture conditions, growth factors) to support cell maturation. 5. Access to Patient-Derived Materials: Patient tissue availability remains a challenge in accessing materials needed for accurate and personalized modeling. 6. Interdisciplinary Nature: Requires a multidisciplinary approach, as these bioengineering techniques often combine expertise from fields like serology, bioengineering, and biomedical sciences Lecture 20: Engineering tissues & organs 31