Bioeng II zk PDF

BIOENGINEERING field combining biological and...

BIOENGINEERING field combining biological and engineering disciplines to = various + gain products high value services ↳ chemical eug of biochem processes , biomedical biomechanical eng / genetic eng , eng... 1 UPSTREAM TYPICAL BIOENG PROCESS preparation and propagation :. - ↳ preparation tMicrobiacutea dia.. 1. CULTIVATION- cultivation in the bioreactor ↳ monitoring and controlling of the process. DOWNSTREAM PROCESSING 3 separation of cells - ↳ cultivation medium and cell disruption (intracell. process ↳ concentration, isolation and purification of the product ↳ finalization CULTIVATION STRATEGIES : 1.BATCH CULTIVATION - closed system - no nutrients added, G no products removed growth is described by GROWTH CURVE time is money we want to maximize ju - ↳ optimalization of cultivation conditions composition of cultivation media,temperature, oc - CONTROL STRATEGIES acration... to maximal X · when the product is BIOMASS itself - we want to prolong exponentional phase gain metabolite production also we want to prolong exp phase for primary -. · metabolite shorten exp phase and prolongation of stationary phase for production of 20 -. · E - E - when we want primary product we want - to prolong exponentional phase (also when the product is biomass itself) - When we want secondary product- we want to shorten exp phase and. prolong stationary phase  (but nothing is removed) L. FED-BATCH CULTIVATION - we fed onelmore nutrients > - increasing the cultivation volume of GFEEDING the bioreactor STRATEGIES-control of the process G slow constant flow of medium-linear of the biomass growth exponentional inflow of medium exponentional = growth of biomass feedback regulation direct measurement of the substrate cons. measurement of other parameters- pHiO , GO.... CONTINUOUS CULTIVATION 3 - Open system continuous - supply of untients and d continuous removal of medium - INFLOW DATE = OUTFLOW RATE constant volume of the working - TURES : CHEMOSTAT bioreactor inflow outflow -dilution rated growth under optimal conditions · = - usually problem-contamination F - = -D by adjusting D we can eliminate contamination... TURMIDISTAT constant turbidity · AUXOSTAT · constant parameter associated with grow SEMl-CONTINUOUS CULTIVATION ↳ batch repeated , fee-batch - a part of the culture (10-30% ) is left in the reactor after the end of the cultivation and "fresh" medium is added ↳ massive contamination repeating until there are physiological changes or ↳ of the culture  Aso inenesa A= 0 stationary ↑ state ACCUMULATION the increment = of the balanced quantity g INPUT + SOURCE-OUTCOME + ACCUMUATION EQUATION = source-the ↑ balanced amount of system quantity that arises in the during the balance period (positive (zero Inegative value ( CELL GROWTH must follow the law of constant energy and mass STIRRING wanttoreach ve concentrationandtemperaturehomoi a -> ↓ to make the batch homogenous - conc.temp the importance economics , energy demands , regulation, - - monitoring of the processaeration... 1 SPONTANEOUS STIRRING.. FORCED STIRRING 2 A) MECHANICAL STIRRING classification of stirrers by movement (notationvibration - - by RPM (slow /fast rotation on flow directionen racial  - types of stirrers : d 6 ↓ for AEROBIC PROCESSES with high compromise between large pumping capacity · · oxygen acmands turbine and propeller ↳ they homogenize well · efficient in mass transfer and gas disportion · high speed · high speed · radial stirring RADIAL : AXAL · AXIAL flow affordable high price · high-speed · · · 6 blacks dividing disc · BPNEUMOTICAL - by gas D HYDRAULICAL-by pump RATION - bubbles size smallerate d 20mm ,5-6mm 0 & da 36mm sokoi ? Piscositdspeeden & " surface tension velocity deco jaw speed, asi michlostri gradient - - - = -  & < uniform bubbles o interactions -actions varied bubble sizes bubbles large in layers - - - - Suzitikyslike - - - - - - ACCUMULATION = INPUT-OUTPUT + TRANSPORT-CONSUMPTION - - d - for growth products non-cellular reactions viaccie efectivita premos lyslike a plynne & > fate (vzduchulplymel do Cultival media I C - wirenefektivity acrace a michaini v biorealtorech = &Nisamimahsememnona - -  resistant ↳ from somenews for example fermenteda various mixtures↑ separate individual components in membranes to using Bioengineering II ↑ most common-separation based on the particle/molecule size &Membrane Bioreactors Fundamental Membrane-Based Processes in Biotechnology Use special membranes enabling the separation of individual components in various mixtures (e.g., fermented culture media). The most common separation mechanism is based on particle/molecule size. Modern technologies utilize nanofibrous membranes made from resistant polymers. semipermeablemembranealowionsandsmall modules to larger holea go through ; retains * Dialysis and particles A separation membrane process driven by a di]erence in chemical potentials. (DNA , peptides , Employs a semi-permeable membrane: cells ,polysacavides o Allows ions and small molecules through. o Retains larger molecules and particles (DNA, proteins, peptides, ↳ polysaccharides, cells). for removing Common application: Removing salts from protein samples and exchanging salts ,exchanging bu]ers. buffers... - Factors AJecting Dialysis EJiciency: &Sample volume - Bu]er volume -- Number of bu]er changes Temperature - C Time Pore size pomernofiber usingmembranespreszenmdriven by PRESST & Ultrafiltration separation Separation using membranes with pore sizes approx. 10–50 nm. proteins DIFFERENCE Used for separation, concentration, or partial purification of proteins/biomolecules. r Driving force: Pressure di]erence (1–10 × 10⁵ Pa). retetate PERMEATE Membranes for Ultrafiltration: 1.-Polymer Membranes: o Created by phase inversion (polymer dissolved in organic solvent, cast as thin layer, coagulation bath solidifies porous film). 2.-Nanofiber Membranes: Modern alternatives. - Process Details: Separates molecules by size, shape, and charge. Retained molecules (retentate) vs. molecules passing through e(permeate). - "Cut-o]" defines the molecular size retained with 90% e]iciency, typically in the range of 10³–10⁶ Da. - e Advantages: Gentle - on labile molecules (e.g., enzymes). - Maintains pH and ionic strength. - No organic solvents required. Independent - - of temperature. Possible Arrangements:  Plate, spiral, tube, capillary, or hollow fiber modules. e Driving Force: Pressure-driven. -Trans-membrane potential (TMP): o Defined as pressure di]erence across the membrane. - o Higher TMP accelerates ultrafiltration but risks membrane damage at critical values. e Applications: 1. Sample pre-concentration. - 2. Bu]er exchange and salt removal (diafiltration, ultradialysis). - 3. Fractionation and purification by molecular size. -- separation systems e Membrane Bioreactors (MBR) combines bioreactions with > - membrane Bioreactors containing special membranes for separation of fractions or e compartmentalization for simultaneous processes. back toe goes - Types of MBRs: ratenate Preparates - media 1. Bioreactors with Built-In Membranes: all ↓ Good process control. o - o Limited - e]ective membrane area. - 2. Bioreactors with Attached Membrane Modules: elimination o Separate cells from fermented broth and return them to the process. Fundamental Configuration of MBR permeate Separates cells and media; returns cells to the reactor. E Common for water-soluble metabolites (e.g., ethanol, lactic acid). Eliminates inhibitory product e]ects, improving process yield. - Challenges: & o Maintaining sterile conditions. - o Preventing cell fouling via high-velocity flow (shear stress risks cell - damage). iMBR Design: Membrane inside the reactor. Overpressure system drives mass transfer. Lower operating costs and reduced cell shear stress. Aeration prevents fouling. Common in wastewater treatment. dMBR Design: ZA↓ Di]usion-driven mass transfer (can be slow, limiting e]iciency). Allows functional separation of processes. E Fouling of Membranes Major issue reducing membrane area and mass transfer rate. Fouling Mechanisms: ↓ 1. Adsorption of macromolecules or colloidal particles. 2. Cell adhesion and biofilm formation. 3. Precipitation of dissolved substances forming crusts. eFouling Types: adsorption of macromolecules or it creates particles - colloid biofilm  can remove the macromolecules , colloid particles... from the membrane -we & Reversible: Removed by physical/mechanical means (e.g., backwashing). Irremovable: Requires chemical treatment. - Irreversible: Cannot be removed by any method. - - Factors Influencing Fouling: 1. Extracellular Polymeric Substances (EPS): - o Organic macromolecules (polysaccharides, proteins, nucleic acids, lipids). 2. Soluble Microbial Products (SMP): o Metabolic byproducts - (organic acids, c peptides). o Risk of precipitation clogging pores. 3. Precipitating Components: o Culture media substances (e.g., phosphates, carbonates). - 4. Membrane Characteristics: o Material (e.g., PE fouls faster than PVDF). 87 o Geometry (planar modules more prone to fouling). o Hydrophobic membranes foul faster than hydrophilic ones. - 5. Ionic Strength: o Impacts flocculation and fouling. - o High cation concentration increases fouling. --Applications of MBR 1. Biotechnological Processes: o Example: Human AAT production. -2. Wastewater Treatment: o Common usage for compartmentalized bioreactors. -  Bioreactors with Immobilized Cells or Enzymes -utically components active Immobilized Systems livinglataucalm active cellsorganel... Definition: Modern concept where catalytically active components (e.g., living or metabolically active cells, organelles, enzymes) are immobilized on a solid I carrier in a bioreactor. catalytically active & Advantages: components can be reused o Catalyst is retained in the bioreactor for reuse, reducing costs. - reducing costs ; > - o Enables continuous processes. enables continuous processes - o Eliminates need for cell separation steps. & Challenges: - o Intracellular products and fast-growing cells are problematic. oC = Clogging, mass and energy transfer, and homogenization issues arise, especially for living cells requiring gas transfer. - Methods of Immobilization on a Solid Carrier - > non-covalent (vanderwatsioninis , 1. Non-Covalent Adsorption: Simple and inexpensive. mobilizationthroughwa interactions d Preserves high activity through weak non-bonding interactions (van der Waals, ionic, hydrophobic, hydrogen bonds). ↓ simple , inexpensive ISSUE catalyst can wash out of : Sensitivity to temperature, pH, and ionic strength. the carrier ↓ sensitivity to pH) Common carriers: silicates, MOF, PCP, clay materials, synthetic polymers. temperature,ionic Issue: Catalyst can wash out of the carrier. -catalyst & 2. Covalent Adsorption: is bonded to the carrier through covalent bond, strength d Process: Covalent binding of catalyst surface structures to carrier (must have functional groups for gentle bond formation).must have functional groups ADVANTAGE-strong bond Advantages: Strong bond. toogentle bond formation & e & Challenges: Conformational changes may reduce activity or availability of active sites. FUNCTIONAL GROUPS : e Common materials: anhydrides ,aldehydes, - o Chitosan with glutaraldehyde. epoxides... e o Eupegrit® C (macroporous methacrylate spheres). - o Inorganic supports (e.g.,-silicates) modified with functional groups. Functional groups include: anhydrides, aldehydes, epoxides, isothiocyanates, etc. & 3. Entrapment (into the Matrix): physical entrapment into the polymer matrix -> Physical entrapment within a polymer matrix. ↳ common carriers Preserves catalyst structure. : alginatagarosees a PA Common carriers: gel-forming biopolymers (alginate, agarose, cellulose derivatives) or synthetic polymers (PAA, PVA). & Example: Sol-gel immobilization (transforming colloidal suspension into a gel). 4. Encapsulation (into Particles): catast is enclosed in particles separated by semipermeable membrane Catalyst enclosed in particles separated by a semipermeable membrane. - Common materials: alginate (forms gel with polyvalent cations like Ca²⁺) or common materials d : Sa D synthetic polymers (PVA, PAA). LENTILLY men Example: LentiKats® technology. alginate , PUA ,PAA. - alginatea ene - seaweed from mat.  NHL is also carrier-linked via catalyst a & 5. Cross-Linking: Catalyst serves as its own carrier, linked via NH₂ using bifunctional reagents (e.g., glutaraldehyde). Techniques: o CLE (cross-linked enzymes). o CLEAs (cross-linked enzyme aggregates). o CLECs (cross-linked enzyme crystals). CLECs: o Microporous, highly active materials. o Useful in undiluted organic solvents. o Stabilized through crystallization and cross-linking. CLEAs: o Aggregates cross-linked after precipitation (e.g., ammonium sulfate, PEG). o Allows simultaneous precipitation of multiple enzymes (combi-CLEA). & Immobilization of Enzymes vs. Cells = Cell immobilization is more -- complex due to structural and functional demands. Common methods: encapsulation or entrapment. & Challenges with viable cells: o Potential damage during immobilization. - o Growth within the matrix causing release or clogging. Solution: Use inactivated cells maintaining metabolic activity. Bioreactors for Immobilized Systems Key Designs: 1. Ideally Stirred Reactor: Standard design. 2. Packed Fixed-Bed Reactor: o Catalyst layer remains constant. o Issues with mass/energy transfer and removal of waste gases. 3. Fluidized-Bed Reactor: o Particles suspended by high gas flow rates. o Improved mass and energy transfer, aeration. o Shape modifications enhance mixing. 4. Reversed Fluidized-Bed Reactor: Specialized variation of fluidized beds. 5. Gas-Stirred Bioreactor: Geometry ensures e^icient mixing through gas. Factors Complicating Mass Transfer Damköhler Number (Da): Ratio of maximal reaction rate to maximal mass transfer rate. o Influenced by e^ective di^usivity (De), layer thickness (l), and substrate concentration (Sb).  Es Cultivation of Cell Cultures cultured under controlled conditions - often outsid Definition standard parameters Cell Cultures: Cells cultured under controlled conditions, often outside standard parameters. Typically refers to eukaryotic cells (animal, human, plant) ↓ isolated from tissues by specialized procedures. typically eukaryotic cells Applications of Cell Cultures Research on cancer and diseases. Janimal ,human, Biotechnological production of vaccines, antibodies, hormones, proteins, interferons. plant Stem cell development. APPLICATION OF CELL CULTURES Drug and treatment procedure development. Gresearch on cancer and diseases Toxicological testing of substances and materials. production of vaccines, biotechnological - Advantages over simpler systems: antibodies, normones , proteins-- substances Complex post-translational modifications like glycosylation. toxicological and materials testing of - Genetic engineering: Expands technological potential significantly. treatment procedures -- drug and and development - Types of Cell Cultures - 1. Primary Cell Cultures: Derived from tissue via enzymatic digestion (e.g., trypsin). o o Cultured in flasks with media. - o Diploid chromosome set; mixture of cell types. - 2. Secondary Cell Cultures: o Passaged primary cultures with a single cell type. - o -Diploid chromosome set. o Lifespan of ~30–50 passages. -- 3. Cell Lines: o Derived via mutagenesis or tumors. - o Heteroploid and immortal. - o Example: HeLa cells from Henrietta Lacks. -- - Plant Cell Cultures: Totipotent, capable of regenerating into full plants. - Used for compound extraction or whole-plant regeneration. - Adherent vs. Suspension Cultures -Adherent Cultures: o Require adhesion and cell contact. - o Form a monolayer. - Suspension Cultures: o Grow freely in suspension. o Adaptable from adherent types. - Cell Morphology E 1. Fibroblast-like: Elongated, bipolar. 2. Epithelial-like: Polygonal, adherent. 3. Lymphoblast-like: Spherical, suspension growth. - Cultivation Media Basal Media: Basic nutrients; supplemented with fetal bovine serum (FBS). E Serum-Reduced Media: Minimized serum, enhanced with nutrients. Serum-Free Media: Specific cocktails replace serum. Cultivation Conditions - Narrow Optimal Range: - Compared to microbes. pH ~7.4 (varies with species). - Temperature: Usually 36–37°C for human cells. CO₂ Atmosphere: Regulates pH via bicarbonate bu^ering. - Challenges: - o High cost. - o Sensitivity to contamination. o Susceptibility to shear forces. - - Contamination in Cell Cultures 1.-Bacteria: Most common; rapid growth- and metabolic impact. Treated with antibiotics. - 2.OYeast: E^icient sugar metabolism; una^ected pH. - c 3. Fungi: Resistant spores and toxins. 4. Viruses: High specificity; detection by PCR/ELISA. 5. Mycoplasma: Smallest bacteria, undetectable until severe damage. - Cell Cultures in Biotechnology 1.CCHO Cells: o Industry standard for therapeutic proteins. - o High yield, FDA-approved. C 2. Insect Cell Lines: o Easy to culture, high tolerance. - o Challenges with lysis and glycosylation. E Equipment for Cell Culture 1.-Laboratory Scale: o Disposable plastics for adherent cells (microplates, flasks). - 2. Mechanically Stirred Bioreactors: o Similar to microbial systems, adapted for shear sensitivity. o Types: Batch, fed-batch, continuous. - o Industrial scale: 10,000–20,000 L. 3.-Alternative Bioreactors: o Fluid air-lift, membrane, or immobilized cell designs for gentle mixing. 4.-Disposable Bioreactors: o Lower sterilization costs, shorter delays. o Limited scalability and transfer e^iciency. - - - Utilization for Vaccines Critical application area with advanced techniques. -  before purification we need to know as much & detail as stability possible to about the target proteinstress temperature , pot , osmotic , - - activators , pl Isolation and Purification of Proteins detergents ,inhibitors ,... ↳ from literature,experiments... T Importance of Protein Purification - - Research Motivation: Proteins play diverse roles in living systems; understanding their structure, function, and properties requires high purity and concentration. - - Application Motivation: Proteins are essential in industries such as pharmaceuticals, cosmetics, food, and chemicals. Applications demand - specific levels of purity. - & Industrial Use of Proteins 1.- - Single Cell Protein (SCP): Animal feed; minimal purification. 2.- - - Food Proteins: Extracts from whey or cereals; high safety and purity requirements. - 3. Industrial Catalysts: Microbial enzymes; range from crude to highly pure. 4. Pharmaceutical Preparations: Enzymes, antibodies, interferons, vaccines; very high purity standards. - - Challenges in Protein Purification Multiple steps with partial purification at each stage. = Significant loss of target protein during the process. Purity requirements depend on the application. - Key Steps Before Purification - 1. Determine Requirements: Define purity and quantity. 2.- Understand Target Protein: o Stability under various conditions (pH, temperature, osmotic stress). o Physical properties: pI, Mw, hydrophobicity. o Intracellular, extracellular, or membrane-bound location. - 3. Analytical Tests: Reliable assays for activity and concentration (e.g., Bradford or Lowry tests). - Sample Preparation & Intracellular Proteins: - inside the cell-cutoplasma , organes, quotation d Cell disruption (ultrasound, enzymes, detergents). peroxidasefrench presen Removal of debris by centrifugation or filtration. Extracellular Proteins: of balat proteins Lower contaminant levels compared to intracellular. - Concentration through precipitation or ultrafiltration. - Membrane-Bound Proteins: - Isolate membrane fractions via centrifugation and sucrose gradients. - Detergents remove residual membranes. - ⑭ Techniques for Purification 1. Protein Precipitation: E^ective for concentrating proteins but may cause denaturation. Methods: o Salt addition (e.g., ammonium sulfate). - o pH adjustment to pI. o Organic solvents (acetone, methanol). - - o Polymers - or heavy metals. 2. Ultrafiltration: - small molecules goes through Separates based on size through semi-permeable membranes. Retains proteins in the retentate while smaller molecules pass through. - ⑳ - Maintains pH and ionic strength. 3. Dialysis: Removes salts and bu^ers using semi-permeable membranes. E^iciency influenced by sample volume, bu^er changes, temperature, and pore - size. E Protein Purification by Chromatography Overview: Emphasis on preparative function (purity) rather than analytical function. - Uses low-pressure liquid chromatography (LPLC). - & Instrumentation: 1.- Peristaltic Pump: Wide flow range; suitable for low-pressure applications. E - - 2. Gradient Former: Gradually changes bu^er composition. - 3. Columns: Proper preparation and maintenance are crucial. @ - 4. Detectors: UV and conductivity detectors monitor elution. The 5. Fraction Collector: Collects fractions based on time or volume. Chromatography Techniques: the pH the protein depends on 1. Ion Exchange Chromatography (IEX): charge of - o Separates based on charge. (pF) 1- - o Reversible binding between proteins and oppositely charged stationary phases. o Elution via pH or ionic strength gradients. - 2. Hydrophobic Interaction Chromatography (HIC): o Binding via hydrophobic interactions. o High salt concentration enhances binding; elution reduces salt or changes - polarity. - - 3. A^inity Chromatography (AC): o Targets specific interactions (e.g., antigen-antibody). - o High resolution and capacity. o Ligand-based separation; elution by competitive or non-specific methods. - - 4. Size Exclusion Chromatography (SEC): o Separates - based on molecular size using gel matrices. o Large molecules elute faster; small molecules di^use into the gel and - elute slower. - o Applications include molecular weight determination and salt removal. - IEX > - HIU - AC > - GF- RPC - Applications of Purification Techniques Protein sequencing and structural studies. Determining enzyme kinetics. Investigating protein interactions (protein-protein, protein-DNA, etc.). =Biotechnological production and pharmaceutical applications.  Downstream Processing (DSP) & Common DSP Techniques need to disrupt es to Cell Disruption: Required for intracellular products;- 1. - access intracelular components performed gently to protect labile components (proteins, antioxidants). - o - - Mechanical methods: Ultrasonic homogenizer, French press, cavitation. o - Chemical/Enzymatic methods: Detergents, lytic enzymes (e.g., lysozyme, proteinase K), osmotic lysis, freezing/thawing. musical 2. Separation of Solid Particles: ⑳paration o Centrifugation: Fixed angle vs. swinging rotor, chilled vs. non-refrigerated. o Filtration: Removes cellular debris. - 3. Crystallization/Precipitation: Converts dissolved substances into solids for ↓ easier separation. Sinitial purification pegupan ⑭ o Methods include supersaturation, pH adjustment, temperature change, or use of antisolvents and precipitating agents (e.g., ammonium sulfate). - 4. Membrane Techniques: o Ultrafiltration, dialysis for protein concentration, and buOer exchange. - 5. Extraction: Separates substances into organic phases or via techniques like countercurrent extraction, ionic liquids, or supercritical fluids. - 6. Preparative Chromatography: Used for purifying specific biomolecules. Cell Disruption A critical step for accessing intracellular components. Gentle disruption is necessary to preserve product integrity. Common methods: o Mechanical: Ball mills, French press, ultrasonic devices. o Enzymatic: Lysozyme, proteolytic enzymes. o Physical: Freezing and thawing, osmotic shock. Centrifugation Separates solid particles from liquid based on density. Types: o Industrial centrifuges: Hydrocyclones, tubular centrifuges. o Lab-scale centrifuges: Fixed angle, swinging rotor. Key parameter: Relative Centrifugal Force (RCF). Crystallization and Precipitation Used to isolate and purify products. Precipitation: Suitable for initial purification; high impurity content. Crystallization: Produces high-purity products; often used in final steps. Examples: o Citric acid: Precipitated via pH adjustment. o Sophorolipids: Precipitation induced by lowering pH.  Extraction Transfers target substances into a diOerent phase (e.g., aqueous to organic). Types: 1. Physical extraction: Using immiscible liquids. 2. Ion pair extraction: Via reverse micelles. 3. Membrane extractions. 4. Supercritical fluid extraction (e.g., CO2). Example: Penicillin isolation via pH adjustments and solvent extractions. Electrodialysis Uses an electric field to migrate ions through selective membranes (cationic or anionic). Suitable for charged compounds like organic acids. DSP Applications Industrial examples: o Polyhydroxyalkanoate (PHA) isolation. o Purification of microbial metabolites. Benefits: EOicient recovery and purification of target compounds. Delivery Systems in Bioengineering - Bioengineering Overview Definition: Field combining biological and engineering disciplines to gain various - products and high-value services. - Scope: Crozsah : 1. Chemical engineering of biochemical processes. - 2. Biomedical engineering - application of engineering in healthcare. - 3. Genetic engineering, systems biology, bioinformatics. - 4. Biomechanical engineering, bionics, bioprinting, bio-robotics. - Interaction of Living Organisms with Materials Biocompatibility: o Tolerance of materials in biological environments (e.g., human organism). o Judged by interactions with the environment, considering undesirable eOects: § Toxicity § Allergic reactions § Carcinogenesis, mutagenesis § Initiation of infection § Coagulation, inflammation § Stability vs. (bio)degradation Key Factors in Biocompatibility: o Williams’ definition: Ability of material to perform a function without causing undesirable reactions.  o Application-dependent; influenced by conditions, location, and price/performance ratio. Biodegradability vs. Biocompatibility: o Abiotic or biological degradation of materials in physiological environments. o Applications: § Absorbable implants and sutures. § Controlled transport of biologically active substances. o Considerations: § Degradation rate and identification of degradation products. § Impact on tissues and cells (e.g., inflammatory responses). o Advantageous if degradation products are endogenous (e.g., lactic acid polymers). o Certain degradation products may exhibit biological activity (e.g., P4HB degrades into 4-hydroxybutyrate, a neurotransmitter). Risks: Mutagenicity and Carcinogenicity Historical Observation: o Carcinogenic potential of plastics observed in invasive injections (1940s). Mechanisms: 1. Release of chemical substances (organic or inorganic) from materials, leading to genetic mutations and carcinogenesis. 2. Inflammatory reactions producing radicals and peroxides, causing genetic damage and solid-state carcinogenicity. Uses of Biomaterials in Biomedicine Implants (soft tissues, bones, dental implants, organs, blood vessels). ScaOolds for tissue therapy. Wound covering and healing. Delivery systems for biologically active substances (BAS). Delivery Systems Tools and formulations ensuring: 1. Gradual release of BAS. 2. Targeted transport of BAS to specific cells or tissues. Advantages for Patients: o Reduction of therapeutic doses (e.g., cytostatics). o Decreased frequency of drug administration. o Stability for unstable drugs (e.g., peptides, enzymes). Gradual Release Systems 1. DiTusion-Controlled: o BAS released through defined pores of a membrane. 2. Water Penetration-Controlled: o Water pushes BAS out of the system. 3. Osmosis-Controlled: o Water passes through a semi-permeable membrane, gradually releasing BAS. 4. Chemically Controlled: o Biodegradable polymers release BAS as they degrade. 5. Responsive Systems: o Combine drug delivery with biosensors monitoring drug levels. Polymer-Based Delivery Systems Common Forms: Nano-/micro-particles and fibers. Applications: Transport of BAS, including peptides, enzymes, and DNA (gene therapy). Key Parameters: Particle size, surface charge, morphology, and release kinetics. Limitations: o DiOicult scaling of production. o Low encapsulation capacity. o Wide particle size distribution. Categories: 1. Protein-Based Nanoparticles: o Biodegradable, non-toxic, stable. o Proteins include: § Silk proteins. § Collagen and gelatin (require chemical cross-linking). § β-casein: Stabilizes hydrophobic drugs. § Albumin: High binding capacity for BAS. 2. Polysaccharide-Based Systems: o Biodegradable and biocompatible. o Polysaccharides include: § Chitosan: Degraded by enzymes. § Alginate: Forms gels with Ca2+. § Hyaluronic acid: Cross-linking enables particle formation. 3. Polyester-Based Nanoparticles: o Materials include PLGA, PLA, PHA, PMLA. Dendrimers and Dendrons Branched, highly symmetric molecular systems. Properties depend on functional surface groups. Applications: Drug transport and gene therapy. Liposomes Nano-/micro-particles formed by amphiphilic molecules (e.g., phospholipids). Basic Structure: Phospholipid bilayer. Formation: Occurs above phase transition temperature (Tm). Types: o Multilamellar (MLV). o Unilamellar (SUV, LUV, GUV). Applications: Transport systems in medicine, cosmetics, and functional foods. Specialized Liposomes: 1. Stealth Liposomes: Coated with polyethylene glycol (PEG) for prolonged circulation. 2. Cationic Liposomes: Transport DNA fragments for gene therapy. 3. Targeted Liposomes: Use antibodies for targeting specific cells (e.g., tumors). Applications of Delivery Systems Pharmaceutical Uses: o Cancer treatment: Dose reduction and targeting. o Gene therapy: Delivery of DNA/RNA. o Encapsulation of labile substances (peptides, enzymes). Specific Examples: o Liposomal drugs (e.g., Doxil for cancer). o Controlled and prolonged drug release. Tissue Engineering Overview Definition: Tissue engineering is an interdisciplinary field combining medicine, tissue culture techniques, and material sciences to repair damaged tissues or create new tissues entirely. Scope: o Includes transplant medicine and regenerative medicine in some definitions. ScaTolds in Tissue Engineering Purpose: o Provide structural support for cell growth. o Allow cells to grow in vivo on artificial supports. Materials: o Biodegradable polymers preferred; they degrade over time, eliminating the need for surgical removal. Design Considerations: o Chemical, mechanical, and morphological/topographical properties of materials. o Biocompatibility and cell adhesion are essential. Customization: o ScaOolds can be modeled to mimic specific tissue sizes and shapes. Demands on ScaTolds: 1. Biocompatibility: Non-toxic and supportive of cell growth. 2. Surface Properties: Promote cell adhesion and proliferation. 3. Mechanical Properties: SuOicient strength and flexibility. 4. Porosity: Fully open, interconnected pores for nutrient transport and cell communication. 5. Pore Size: Must allow cell penetration and vascularization. 6. Biodegradability: Rate must align with cell growth rates. Non-Fibrous ScaTold Preparation 1. Solvent Casting and Particulate Leaching (SCPL): o Dissolve material in solvent; pour into mold with porogen (e.g., salt, sucrose); evaporate solvent and leach out porogen. o Result: Porous structure. 2. Gas Foaming: o Expose polymer disks to CO2 overpressure to create a porous structure. o Limitations: Interconnected pores are not guaranteed. 3. Lyophilization: o Freeze polymer emulsion and remove solvent/water via lyophilization. o Challenge: Small, poorly connected pores. Advantages: Easy to prepare. Disadvantages: Reproducibility, use of toxic solvents, insuOicient porosity. Fiber ScaTold Preparation 1. Melt Spinning: o Polymer melted and fibers formed via cooling. o No organic solvents required. 2. Force Spinning: o Fibers spun from melt or solution using centrifugal force. o Similar to cotton candy production. 3. Electrospinning: o High voltage applied to polymer solution; fibers form as solvent evaporates. o Features: Highly porous, precise control of geometry and properties. Other ScaTold Concepts Spider Silk Proteins: o Recombinant spider silk proteins form thin films suitable as scaOolds. Injectable ScaTolds: o Cells and materials injected as a mixture; material hardens in situ. Sources of Cells 1. Autologous Cells: o From the patient; highest acceptance and lowest immune response risk. o Requires healthy tissue and time for culture. 2. Mesenchymal Stem Cells: o Multipotent; diOerentiate into various cell types. o Found in bone marrow, umbilical cord blood, and fat tissue. 3. Allogeneic Cells: o From other individuals of the same species; ethical concerns but useful in some applications. 4. Xenogenic Cells: o From other species. 5. Syngeneic/Isogenic Cells: o From genetically identical individuals (e.g., twins). Cultivation of Animal Tissue (In Vitro Meat) Concept: Culturing animal tissues without raising or slaughtering animals. Procedure: o Stem cells (embryonic or induced pluripotent) diOerentiated into tissues. o Grown in basal media; cost is a major limitation. o ScaOolds (e.g., cellulose, chitin, collagen) often used for structured products. Applications: o Bioreactors for large-scale cultivation. 3D Printing in Tissue Engineering Revolutionary Approach: o Creation of tissues and organs via 3D printing of cell cultures.