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Date Created: 09/23/15
Tissue Engineering Goal Replace repair or enhance biological function at the tissue or organ level Method Transplantation of cells in a suitable polymeric matrix or scaffold Implant type Dictated by the function Which the implant is designed to replace Types of Tissue Engineered Implants Type Mode of action Examples Physical Graft performs primarily Bone a biomechanical role Blood vessels Physiological Graft performs a Liver physiological role kidney Chemical Use of cells to produce Pancreatic soluble diffusible Islets components Combination Graft performs more Skin than one role Source of Cells Autologous Harvested from patient Allogenic Cells from donor Cell Lines Cells which have undergone genetic mutation Which allows adaptation to inde nite proliferation Xenogenic Cells from different species General requirements gt Bone marrow can be harvested no need to expand gt Ability to scale up important Very few cells can be expanded gt Lines which expand well keratinocytes broblasts chondrocytes myoblasts gt Lines which do not hepatic neural pancreatic islets Autologous Cells gt Simplest by patient biopsy always compatible gt Limited supply gt Takes time to harvest expand construct implant gt Very costly gt Variable patient to patient gt First application keratinocytes sheet expanded 2000 fold gt Now also chondrocytes for atricular cartilage Cells Recruited from Host gt Implant scaffold to promote cell in ltration and differentiation gt Eventual replacement of scaffold With host derived ECM gt Can include growth factors in scaffold e g BMF bone morphogenic factor gt Usually use natural scaffold e g nerves meniscous blood vessels Allogenic Cells gt No supply problems gt Can be grown up and preserved gt Reproducible gt Consistent gt More cost effective gt Can produce more complex tissues gt Need to do extensive quality control Allogenic Cell Lines gt Require in nite growth capability gt Amenable to gene transfer for speci c proteins gt Genetically modi ed cells made immortal lose some function and pose safely concerns gt Exhibit properties of neoplastically transformed cells potential of becoming tumor gt Usually encapsulate the cells gt Hepatocyte cell line use in extracorporial liver Xenogenic Cell gt Cross species therefore must be encapsulated for immune protection gt Can be genetically manipulated transgenic animals gt Many safely concerns especially Virus transmission General Safety Concerns Autologous gt Screen and validate to guard against changes in cells gt Prevent introduction of pathogens Allogenic gt Minimize transmission of infectious agent gt Prevent phenotype changes Xenogenic gt Prevent introduction of new animal virus Interesting site on TE skin manufacture httpbiomedbrowneduCoursesBI108B1108 2005 GroupsOSindexhtml Dermagraft Manufacturing Proces s Maternal Serum Donor Cells HIV 1 amp 2 Mycoplasma HTLV I EIA USP Sterility CMV and ALT Adenovirus HSV 1 amp 2 non A amp non B HIV 1 amp 2 Hepatitis Bc HTLV 1 BsAg amp C Karyology RPR Growth characteristics CMV CMV HSV I amp II Tumorigenicity HTLV I amp II Hepatitis BampC HIV 1amp2 and EBV and HHV 6 Apligraf Manufacturing Process Table 3 Cell Bank Testlnu sway Luv Myanmasma cm H mm wus Human DawHoma In vx1errns HHV 531137 Wu 1 El M MD mmangsmw Wevmsal39ansmptase chmzpwuwus w ocmn39anan MAP esHHEPonbu mm Emma name a HTL mm anmnpu yumi Hepms A 9 am won Kavymogy and meantme Caurlesyal Paremeau Organogenasrs In Can0n Muss m ndwales mama Hueom HV Nulmn nmmmadehueucy wms ran pub39msmse mm mamaquot HTLV human mu Lvm haterc WM 551 Enslavrr nrr was cmv cylamngowns HHV mama herpgswm MAP mum anybody pmduclmn m1 HER human mama ccquot Immune Reactions gt Both allografts and xenografts have reactions some are the same mechanisms gt Migration of complement mediated rejection major hurdle for xenogenic cells gt Allografts primarily T cell mediated attack graft vasculature gt Leukocytes and endothelial cells are strongly immunostimulatory need to remove passenger Leukocytes gt Keratinocytes smooth muscle cells and broblasts unable to induce generation of T helper cells and do not express HLA class 11 proteins Stem Cells 0 Stem cells cells that have the ability to divide for inde nite Periods in culture and to give rise to specialized cells 0 Pluripotent capable of giving rise to most tissues of an organism 0 Totipotent having unlimited capability Totipotent cells have the capacity to specialize into extra embryonic membranes and tissues the embryo and all postembryonic tissues and organs http stemcellsnih govinfobasics Multipotent Stem Cells The pluripotent stem cells undergo further specialization into stem cells that are committed to give rise to cells that have a particular function Examples of this include blood stem cells Which give rise to red blood cells White blood cells and platelets and skin stem cells that give rise to the various types of skin cells These more specialized stem cells are called multipotent Extracellular Matrix ECM Extarcellular gel like substance which provides 3D organization to cells and means of communication control of proliferation cell migration attachment differentiation and repair Major Macromolecular Components Glycosaminoglycans GAG s form a gel Collagen provides strength and organization Elastin provides resilience of elasticity Fibronectin adhesion of broblasts and other cells Laminin adhesion of cells to basal lamina 3D Tissue Fabrication Often just scaffold for support cells is not enough need to Mimic micro architecture of tissue Mimic microenvironment Repeating units on scale of microns Apply CADbased manufacturing technologies Fabricate vascular beds which allow for larger tissue constructs no limited by diffusion Customs build to fit defect using imaging data with CAD Drug Del Rev 61635 2004 3D Tissue Fabrication Often just scaffold for support cells is not enough need to Mimic micro architecture of tissue Mimic microenvironment Repeating units on scale of microns Apply CADbased manufacturing technologies Fabricate vascular beds which allow for larger tissue constructs no limited by diffusion Customs build to fit defect using imaging data with CAD Drug Del Rev 61635 2004 FDA Regulations 0 Ensure that donors of human cellular and tissue based products are free of communicable diseases cells and tissues are not contaminated during manufacturing 0 Maintain their integrity and function Written some regulations 0 Cover all human cell tissue and cellular and tissue based products HCTPs FDA Regulations Quality program Which would evaluate all aspects of the rm39s operations to ensure compliance With GTP Adequate organizational structure and suf cient personnel Standard operating procedures for all signi cant steps in manufacturing Facilities equipment and the environment With control and validation of manufacturing processes Adequate and appropriate storage Record keeping and management Complaint le Procedures for tracking the product from donor to recipient and from recipient to donor Time Line for Development 1993 rst regulations speci cally addressing human tissues regulations required that tissue donors be tested for certain communicable diseases such as HIV and hepatitis and screened for behavioral risk factors 1997 Proposed Approach to the Regulation of Cellular and Tissue based Products 2001 Establishment Registration and Listing for Manufacturers of Human Cellular and Tissue Based Products requires tissue facilities to register With the FDA and list their products 2004 Suitability Determination for Donors of Human Cellular and Tissue Based Products focuses on donor screening and testing requires reporting certain adverse reactions and HCTP deviations accurate and complete information in labeling and to allow FDA inspections to ensure compliance With regulations 2005 Current Good Tissue Practice for Human Cell Tissue and Cellular and Tissue Based Establishments Inspection and Enforcement requires manufacturers to recover process store label package and distribute HCTPs in a way that prevents the introduction transmission or spread of communicable diseases Tissue Engineered Constructs gt Rapidly evolving eld gt Only one FDA approved skin gtCurrent literature good source of the latest advances e g Tissue Engineering Biomaterials research Science Nature gtWill give select few examples to show What has been achieved and the complexity of the task Skin Haw Enidcmlls sum vouch What does Engineered Skin Contain mm I WWW 0 I1 Hm MI MN mlgmeuvd md mum km TN an Gum human iuxuhrmlmululuum 41mm mmm Emlyn memm lLur mum mu glnu gm 4 4 3 Dawn 1 M N iiiiiiiiinii 1 wuwux Commercial Ventures Epidermal Compon e n t Dermal Compo n e nt Apligraf Human mature Human fibroblasts in Organogenesis keratinocytes bovine collagen gel Canton MA Stratum Corneum Dermagraft N one Human fibroblasts in Smith amp Nephew PLGA scaffold London U K Orcel Immature keratinocytes Human fibroblast in bovine Ortec not confluent not collagen spong e International cornified New York NY Dermagraft was acquired in 2006 in the US by Advanced BioHealing Skin FDA approved product gt Dermagraft newborn human dermal broblasts grown on 3D degradable scaffold gt Cell source human foreskin gt Support 5 X 75 cm knitted lactic acidglycolic acid copolymer gt Medium Dulbeco s modi ed Eagle medium bovine calf serum glutamine ascorbate and non essential amino acids gt Stored frozen at 700C gt Sheet is 100 microns thick gt Achieved complete healing of diabetic foot ulcers in 12 weeks Also used for burn Victims Histological Comparisons Natura Skin Dermagraft A m Aphgra Orce Dermagraft Heals more chronic t diabetic loot ulcers Than conven onali therapy alone hnn ww 552007702715html and h p WwwdeImagraftcom De1magraft is a cryopreserved human broblastdelived dermal substitute it is composed of broblasts extracellular matIiX and a bioabsorbable scaffold DeImagraft is manufactured from human broblast cells de1ived from neWbom foreskin tissue Dun39ng the manufactuling process the human broblasts are seeded onto a bio absorbable polyglactin mesh scaffold The broblasts proliferate to ll the interstices of this scaffold and secrete human deImal collagen matIiX proteins growth factors and cytokines to create a threedimensional human dermal substitute containing metabolically active living cells Dermagraft does not contain 1 1 blood vessels or hair follicles Success Story Saver bum victim before and 6 months after heil39ruent with Dermag laft Cartilage developing gt Chondrocytes extracted from distal femoral joints of immature donors rabbit and sheep gt Seeded onto 3D porous scaffold PGA bers gtGrown in convective ow bioreactor gtImplanted in medial patellar grooves Wu et al Annals of New York Academy of Science 1999 875 405 411 Tissue Engineered Muscle Bundle myooid Approximately 3 days a er delamination of Monolayer ofmuscle cells detaching from substrate and rolling into the monolayer the a cylinder Requires Dish organized into a diameter 35 mm cylinder Robert G Dennis U Mich Representative cross section ofa myooid stained with 1 Toluidine blue scale bar is 1 pm Extracorporial Arti cial Liver gt 70 kg adult has 175 kg liver 25 body weight 12 kg of hepatocytes liver cells gt Liver failure in 1989 30000 deaths 2160 transplants gt Two types of support Passive and Bioreactors gt Passive systems remove toxins e g hemodialysis hemoperfusion plasma exchange gt No improvement still get hepatic encephalopathy HE Which leads to hepatic failure HF gt Bioreactors cell based Liver Assist Devices LAD s The Liver gt Metabolic Secretory and Synthetic functions gt Cells arranged in plates bloodhepatocyte interaction gt Two blood supplies portal vein and hepatic artery gt Vascular space known as sinusoids lined with endothelial cells with pores or fenestrations which allow free passage of solutes into the pericapillar space space of Disse gt Hepatocytes on Microvilli gt Rich in basement membrane proteins proteoglycans collagen type IV bronectin and heparin sulfate Liver gt Bile canaliculi join to form bile duct gt Highly organized unidirectional perfusion of polarized hepatocytes arrange in plates gt Hepatic acinus structure made up of 3 zones differing in blood 02 and solutes Periportal high Intermediate and pericentral low Pericentral are responsible for detoxi cation through expression of P450 cytochrome oxidase enzyme system gt This high degree of structure may be important in a tissue engineered construct Liver gt Damage often progresses in a zonal fashion gt Regeneration is possible in Vivo from hepatocytes at the portal space candidates for LAD gt Zonal hepatocyte necrosis stimulates brous scaring disrupts blood supply leads to cirrhosis gt Blood is shunted to vena cava can not pass through liver and toxins build up Functions of Replacement Liver gt Regulation of plasma protein production gt Metabolism and storage of Vitamins gt Biotransformation and gt Detoxi cation of drugs rst pass effect gt Lipoprotein and cholesterol synthesis gt Maintenance of glycogenesis and glyconolysis gt Conversion of heme to bilirubin and biliverdin Why Do Livers Fail gt Excessive alcohol consumption major cause gt Hepatitis B and C produce chronichepatitis gt Cancer gt Drugs Which give toxic metabolites With P450 enzymes gt Genetic Reasons for hope gt Rats survived after 90 hepatectomy When implanted With 10 X 107 microcarrier attached hepatocytes 1 2 of liver mass gt Human patients survive after 90 hepatectomy gt 10 of native liver function could be maintained by 120 gms or 25 X 109 cells gt Cell sources transformed cells freshly isolated hepatocytes cultured hepatocytes Cell Sources gt Transformed Cells limitless supply can screen for purity function etc concems about spontaneous changes cost tumoregenicity gt Freshly Isolated Cells Could use e g porcine cells cryopreserved have tissues similar to human inexpensive large litters gt Cultured Hepatocytes Cost and problems of culture Could use microcarriers or hollow ber reactors LAD Con gurations gt Provide maximum environment for cell Viability and function gt Good transport of nutrients oxygen toxins and metabolites gt Various designs Table 302 HepatAssistZOOOTM gt Microporous 015 micron polysulfone hollow bers gt Inoculated With rabbit hepatocytes gt Perfused With Whole blood gt Clinical evaluation ongoing of the next generation deVice LAS liver assist system Figure 303 gt Apheresis system separates plasma from blood feeds plasma to the deVice containing porcine hepatocytes gt Also have a charcoal column and an oxygenator HepatAss1 t The HepatASSTst Svstem Ts ah extracerporea bwoam cwa hver support svstem mcorporatmg Y kguyAsllgycluculy Mm p marv hepatocvtes g hv r eHs and Te 1 deSTgned to treat patTents WTth acute hver fa ure by ternporanw prowdTng essentTa hver functTons The HepatAssTst Svstem mc udes a noHoW er 2 a 3 3 m 3 m 3 3 m o X lt o m 3 a Q m 3 1 u a u a m see ngure 2 be ow The HepatAssTst Svstem T s d m c rhb h non WTth a COmmerc aHV ava ab e p asma separatTon maCHH ve thch thtuses p asma through the HepatASSTst orcuTt betore t T reconstTtuted WTth the mood es and returned to the patent Each HepatASSTst treatrnent Taste 6 hours The he atocvtes are Tso ated processe and d crvopreserv dT rhphah e WTth Good Mahutactunhg practTces CGMP Crvopreserved mm Nmnmsr momma ceH a sh pped to and stored at the chmca mgmmmm swtes where the es are thawed Just pnor to a patent treatment my www cucebm cmntechnnlagythatasa hknl MARS Molecular Adsorbent Recycling System Mechanical Design In Teraklin s MARS system blood is cleansed in an extracorporeal circuit that is a combination of both kidney and liver dialysis Established methods for kidney dialysis do not work for liver failure because kidney dialysis removes only watersoluble toxins While the liver normally removes albumin bound toxins Albumin is a protein found in the blood that carries water insoluble substances including toxins For this reason MARS uses human albumin to cleanse the blood because it can attract toxins bound to albumin in the blood that the aqueous solution in kidney dialysis cannot remove The system replaces the detoxi cation function of the liver httpbmsbrowneducurriculumblOSliverMARSpghtm Therapeutic Cloning Nucleus of a donor cell is transferred to the an enucleated oocyte to give embryo With genetic makeup identical to donor 0 Pluripotent embryonic stem cells extracted 0 Construct biological substitute for damaged tissue Current Status 0 1997 Dolly born from cloned adult somatic cell 0 Possible targets for cure Congenital abnormalities Cancer Trauma or organ failure e g kidney Infection In ammation Iatrogenic injuries caused by physician during treatment eg infection Age related Advances in Organ Transplantation Great shortage of organs First transplant 1955 kidney 1960 s rst allogenic transplant unrelated recipient Revolution in transplant surgery and immunosuppressive therapy 2001 over 23000 patients received transplants in US alone but 80000 are waiting for an organ and 6000 died awaiting an organ Time Line Cloned frogs 1962 Lambs 1996 from differentiated epithelial cells Cattle 1998 Goats 1999 Mice 1998 Pigs 2000 Therapeutic vs reproductive 0 Reproductive generation of that has identical genetic material ads cell source can be implanted in uterus 0 Therapeutic generation of early stage embryo eXplanted in Vitro Cartoon of tubule w 0 A M m 4 RUBSDRPIIOI MI I SECREHDN I Cloned Kidney Renal cells were extracted from 56 day old bovine clones expanded and seeded onto collagen coated polycarbonate membranes and renal constructs created by attaching tubes Constructs were reimplanted sub Q Unseeded control B Seeded with alogenic control cells C Seeded with cloned cells D 12 weeks after implantation Advanced Cell Technology Boston Generation of Histocompatible Tissues Using Nuclear Transplantation Nature Biotechnology 20 7 689696 2002 MARS MAmFLux DIALYZEH mmmsmnsunwnm Innuxnm m cuLuMNs ramsm sumo emcuw mnsmeMm cmcurr Dmvsnm cmcurr VitaGen Inc s Extracorporeal Liver Assist Device ELAD V1 taGeu HAD Ci rem e v r V WIND TQM neatum 573C IZI nZIJr Illne uh39alr I rcu I on a n Sunni Uses HUMAN hepatocytes httpbmsbr0wneducurriculumb108liverVitagenpghtm AlgeniX Inc39s LIVERX2000 System ttp hu grupcems umnedu Research bal ba1html The LIVERX2000 device is a hollow ber cartridge similar to that used for kidney dialysis Primary porcine hepatocytes httphugroupcemsumneduResearchbalbalhtml Fabrication Process EN I RAPMIENT 0F HEPATOCYTES mocvlm39mi cu cohmc ow mp hugaup cams umn eduResearchaJEALrhawxwmks hm Modular Extracorporeal Liver System MELS MELS Diagram PH Electrolyles Amlno ACIdS ALBUMIN RESIN CHARCOAL Thin hollow fiber membranes human hepatocytes or liver cells taken from h pcitochalitenetgojectsPROJShtml Autologous Bladder 0 Homologous Decellularised Bladder Submucosa Scaffold 0 Biodegradable Composite Scaffold Made of collagen and PGA Shaped into a bladder With polyglycolic sutures Figure 1 Construction of engineered bladd e1 Scaffold seededwith cells A 1 and engineered bladda anamamcsed to native blendi3 with running 40 perkglycol ic sutuuzs t B Implant covered with brin glue and oment um I C Atala A Bauer S Soker S Tissue engineering autologus bladders for patients needing cystoplasty Lancet 376 1241 46 2006 Anterior Cruciate Ligament ACL Major intraiarticular ligament of the knee Function 7 Joint stabilizer 7 Controls motion Ligament that connects the Femur to the Ti ia Looks like a braided rope mm mmwnmmw can heiCLl w femur Hugh bone articular cartilage ten crucxale FEET pustenar 3 cruciate ligament PCL my a I Y smn bane 4X12 3 D Braid PLAGA Fig 1 General con guration ofligament scaffold design for 3D rectangular braid JA Cooper PM Lu PK Ko JW Freeman CT Laurencin Anterior cruciate ligament regeneration using braided biodegradable scaffolds in Vitro optimization studies Biomaterials 2005 26 4805 4816 Fig 3 Electron micrographs of BALBC mouse fibroblast after 1 day in culture shows cellular spreading across the fiber left magnification 800 10 mm bar and right magnification 1500 10mm bar W i Fig 4 Electron micrographs of rabbit ACL cells after 1 day in culture shows cell migration and attachment along the fibers left and right magnification 250 and 100 mm bar Fig 5 Electron micrographs of BALBC mouse fibroblast after 8 days in culture shows large cellular networks with cells proliferating with and without the underlying scaffold left magnification 250 100 mm bar and right magnification 500 10mm bar Fig 7 Electron micrographs of rabbit ACL cells after 8 days in culture shows cell response to 3D circular braid the cells did not cover the whole scaffold but did continue to follow the underlying fibrous geometry left magnification 05 10mm bar and right magnification 1000 Desirable properties gt Biocompatible and nonImmunogenic gt Negligible toxicity locally systemically and from degradation products gt Chemically and mechanically stable gt Processable Tubes sheets foams woven etc gt Promotes cell attachment and angiogenesis gt Favorable interactionmobilization of host cells gt Ability to release bioactive compounds gt Favorable interaction with cells and ECM gt Adequate manageable pore size and distribution First Modern Biomaterials British ophthalmologist Harold Ridley late 1940s noticed that shards of canopy plastic embedded in the eye of Spit re ghter pilots from enemy machine gun re healed without ongoing reaction Used polymethyl methacrylatelenses for replacing cataracts in natural lenses First implantation of such a lens was in 1949 Current implants in over 10 million human eyes each year and have revolutionized Host Response review Response to injury Acute in ammation Formation of granulation tissue Eventual scar formation Detailed Host Response Site ooded with blood Fibrinogen cleaved to fibrin clot formation Cytokines and growth factors released White cells in ltrate mostly neutrophiles Monocytes arrive differentiate into macrophages start wound cleanup recruitment of fibroblasts and endothelial cells Fibrin clot converted to highly vascular granular tissue then ECM and usually a scar formation Foreign Body Response Review Response to biomaterial implant Nonspeci c protein adsorption Monocytes leukocytes and platelets adhere to surfaces lead to upregulation of cytokines and subsequent proin ammatory processes Chronic in ammation at the biomaterial interface macrophages unable to phagocytose the implant fuse together form multinucleated foreign body giant cells Walling off of the deVice by an avascular collagenous brous tissue 50 200ptm thick From Ann Rev Biomed Eng 2004 64175 Upon implantation in a mamma 1 mama m m mll iulznugu uuln mnh mumnux mun macmvmgI unis giant c2 luma nn a y nkina mlelsa dense annular cunagen capsule Figure l The mmng body rcnc on as illusna cd here is the nomml xencnon by Next Generation of Biomaterials Surface modi cation of biomaterials to overcome nonspeci c protein adsorption in Vivo Precision immobilization of signaling groups on surfaces Development of synthetic materials With controlled and tailored properties for drug and cell carriers Biologically inspired materials mimic natural processes Design of sophisticated 3 D architectures to produce well de ned patterns for developing bioMEMs deVices biological MicroElectroMechanical Systems and tissue engineering scaffolds Classical Approach gt Nondegradable Polydimethysiloxane polyurethanes polytetra uoroethylene polyethylene polysulphone polymethacylate poly2hydroxyethylmethacry1ate polycyanoacrylate polyamides polyvinyl chloride polyethy1enecoViny1 actetate polystyrene polyvinyl pyrrolidone polysaccharides gt Biologic Collagen GAG s glycosaminoglycans the most abundant heteropolysaccharides in the body chitosan gt Biodegradable Polyesters polyanhydrides polydioxanone polyphosphazines Polydimethysiloxane Breast penile testicular prostheses catheters PDMS drug delivery heart valves shunts oxygenators Polyurethanes PEU Arti cial hearts ventricular assist devices catheters pacemaker leads polytetra uoroethylene PTFE Heart valves vascular grafts facial prostheses hydrocephalus shunts membrane oxygenators Polyethylene PE Hip prostheses catheters Polysulphone Psu Heart valves penile prostheses Polymethyl methacylate PMMA Fracture xation intraocular lenses dentures poly2hydroxyethylmethacrylate pHEMA Contact lenses catheters polycyanoacrylate polyamidesPAN Dialysis membranes polyvinyl chloride PVC Plasmapheresis membranes blood bags polyethylenecovinyl actetate EVA Drug delivery devices polystyrene Tissue culture asks polyvinyl pyrrolidone Blood substitues Surface Modi ed Biomaterials Foreign body reaction appears to be independent of simple surface chemistry Attributed to nonspeci c protein adsorption Attempt to modify surfaces to control protein interaction Attach signaling molecules to the surface appropriately Produce nonfouling surfaces or surfaces that control protein adsorption Nonfouling Surfaces Attach Polyethylene glycol PEG also known as polyethylene oxide PEO Covalently attach to surface Effect dependent on surface chain density and can be damaged by oxidants One group rst linked PEG to mussel adhesive protein producing a sticky segment which adhered well to surfaces presenting the PEG chains at the new interface Modi ed surfaces resisted cell adhesion for up to 2 weeks in Vitro Other Surfaces 0 Radio frequency plasma deposition of tetraethyleneglycol dimethylether tetraglyme 0 Highly nonfouling crosslinked structure 0 No Chains longer than 3 tetraethyleneglycol units 0 In Vivo results not as good as in Vitro Other Surfaces cont 0 Phospholipid surfaces 0 Saccharide surfaces 0 Self Assembling Monolayers 0r SAM s Surface Speci city Binding to surfaceadsorbed proteins triggers a cascade of events inside the cell Can be used to direct cell response to the material Attach short oligopeptides that exhibit speci c binding domains Or incorporate Whole proteins Most common is arginine glycine aspartic acid or RGD from extracellular matrix proteins bronectin laminin collagen and Vitronectin Nonbiodegradable gt Long term run risk of bacterial infection chronic in ammation and development of brous capsule gt Preferred Where structural integrity is more important than function as cell scaffold gt PTFE PU Dacron used for vascular grafts gt Easily processed into 3D structures Biodegradable gt Allow time for transplant to organize in desired 3D structure gt Allow time for blood supply to develop gt No foreign residues remain gt Examples include GAGs Collagen Chitosan PLLA PGA PLGA gt Degradation rates depend on molecular weight surface area degree of crystallinity monomer Internal Structure Must be highly porous gt80 Pore size critical ZOOSOOurn optimal Many formed by salt leaching technique VVVV PreVascularization an advantage allows metabolic needs of cells to be met once vasculature is established gt Enhance prevascularization by seeding scaffold with host endothelial cells Pore Properties by Method Methods Porosit Advantages Dissadvantages Pore y size um Fiber 81 Highly porous Use solvents which are 500 Bonding scaffolds With inter poisonous to cells Unwoven connected pores immersed in them for a mesh long time Solvent 87 Structure has high Organic solvents 100 castingPartic strength or electrical used contaminate polymer ulate conductivity Very long time required leaching Gas foaming 93 Biocompatible Organic solvents 100 used contaminate polymer Gas foaming 95 Pore size and Solvents used are 13 35 porosity easily poisonous changed RGD and Whole Proteins Combine PEG and RGD diminish protein adsorption increase broblast adherance Immobilization of a cyclic RGD peptide increased human bone marrow stromal cell adhesion cf linear RGD Osteopontin potential role in mediating wound healing When immobilized on poly 2 hydroxyethyl methacrylate pHEMAsurfaces it promotes endothelial cell adhesion potential for wound healing Surface Microarchitecture 0 Nonporous implants densely packed well organized brous capsules 0 Porous implants 5 15 pm pore diameter less dense more open and disorganized brous capsule Increased vascularization adjacent to the implant enhanced diffusion of small molecules Fibrous Implants Fibers with diameters greater than 5 ptm the classic foreign body response Diameters between 1 and 5 ptm little or no capsule Fiber chemistry not surface charge appeared to have an effect on brous capsule thickness less dramatic than ber diameter Blood vessel density surrounding the bers was unaffected by ber density for ber diameters between 1 and 159 ptm Hydrogls High water content very attractive 0 Crosslinked polymer networks that are insoluble but swellable in aqueous medium 0 Mechanisms ionic e g alginate physical e g pluronics and peptide self assembling gels chemical e g brin glue and multivinyl methacrylate and acrylate derivatives hydrogen bonding Crosslinked Hydrogels 0 Cross link by photopolyrnerization radical initiated Chain polymerization 0 Degradable hydrogels based on PEG and PVA have been tuned to degrade at a rate that parallels the production of ECM by photoencapsulated Chondrocytes for cartilage The egradation behavior ofhydrogels fabricated from polyethylene glycol and polyvinyl alcohol tailored to match neotissue growth by encapsulated chondrocytes 39 aprimary r produced by chondrocytes after 6 weeks in vitro complete degradation of the gel has occurre b chondrocytes express green uorescent protein GFP after 15 days in 39 when coencapsulated with lipofectaminecomplexed DNA encoded for GFP in degradable PEG gels Images courtesy ofKristi s Anseth A New Class of Hydrogels 0 Involves a unique interaction between PVA and amino acids 0 Possibly hydrogen bonding is responsible for gel formation 0 New approach to fabricate tissue scaffolds using solid colloidal gas aphrons CGAs CGAs or microfoams contain uniform air bubbles that produce pores from 50 500 pm in size suitable for tissue scaffolds PVA and Arginine Amino Acid Hydrogel Scaffold 4 y 39 l r 7 A scanning electron microscopy image of a hydrogel foam prepared by solid colloidal gas aphrons of PVA and arginine Bar 200 pm Smart Biomaterials 0 Can respond to changes in their surrounding environment 0 Exploited to control parameters drug release cell adhesiveness mechanical properties permeability 0 Stimuli pH temperature light pH Responsive pH swellable polymethacrylic acid grafted With polyethylene glycol PMAA g PEG 0 Shrinks trapping the drug cargo at low pH 2 owing to the formation of inter polymer complexes Whereas at physiological pH 7 the gel can swell 3 25 times depending on its composition releasing its cargo in the desired compartment Thermally Responsive Two main polymers polyN isopropylacrylamide pNIPAAm and elastin like peptide ELP pNIPAAm has a lower critical solution temperature LCST at 320C LCST of ELP can be controlled by varying the length of the ELP molecule and or its amino acid composition from 300C to gt 900C ELPs used for chondrocyte encapsulation and as drug carrier for cancer therapy pNIPAAm used for tissue engineering drug deliver and biological microelectromechanical systems bioMEMs Cell Sheet Engineering 0 At 370C pNIPAAm becomes hydrophobic protein adsorption and subsequently cell adhesion 0 At T 0lt 320C change from hydrophobic to hydrophilic release of proteins and cells cultured on pNIPAAm surfaces can be lifted off of the surface 0 Technique maintains intact cell cell connections and their associated extracellular matrix Tissue engineering Application 0 Layering of cell sheets to reconstruct a homogenous tissue 0 Layering several sheets made from various types of cells to reconstruct multicellular tissues 0 The detachment of the cell sheet was accelerated When small amounts of PEG were grafted to the surface With pNIPAAm Biologically Inspired Materials 0 Self assembled biomaterials Weak noncovalent bonds typically hydrogen ionic van der Waals bonds Hydrophobic interactions 0 Biomimetic Eg Hydroxyapatite or Nacre for bone 0 Acellular materials Naturally derived tissues Self assembled Biomaterials 0 Amphiphilic molecules hydrophobic and hydrophilic segments Micelles Vesicles Tubules that are nanometers in length 0 Typical molecules Phospholipid constituent of cell membrane Polymer or oligomer of amino acids synthesized With number of different regions hydrophobic hydrophilic charged etc designed to self assemble into a macroscopic hydrogel Primary peptide sequence tuned to self assemble in situ under physiological conditions or presence of multivalent cations eg Ca Some Advantages Incorporate biological cues Within the material 0 Two peptide amphiphiles contain the biologically recognized amino acid SGqUGl lCCS RGD derived from brinogen YIGSR tyrosine isoleucine glycine serine arginine derived from laminin a o M Nquot1 viu quot xia39 3vfig f r f quot I Clmu NHAAAACKMBKVAVMH M MAM ri39r r riw gr 39ir ijf wrt iw J 2 cmuuoNHMMGGGKHGSRCONH u a Chemical structures of peptide amphiphiles a that self assemble into nano bers b at neutral pH Niece KL Hartgerink JD Donners J Stupp SI 2003 Self assembly combining two bioactive peptide amphiphile molecules into nano bers by electrostaticattraction J Am Chem SOC 1257146 47 Triggering of SelfAssembly Often use pH Salt temperature to trigger self assembly Can in uence point at which this occurs by choice of peptide sequence eg Increase hydrophobicity decreases critical salt concentration Net positive charge at neutral pH and low salt increase salt to physiological levels causes self assembly Biomimetic Biomaterials 0 Emulate the ECM and tissue architectures 0 Incorporate short oligopeptides into synthetic materials to create bioactive materials eg RGD or sequences that mediate cell adhesion cell speci c adhesion neurite extension and degradation to name few 0 Cell migration into hydrogel matrix based on PEG containing a matrix metalloproteinase MMP sensitive amino acid sequence as the crosslink and RGD tethered ligands throughout the bulk of the Threeidirnensional migration of human foreskin broblasts from a brin cluster of cells labeled into an RGDiadhesive and MMPisensitive PEG hydrogel at a day l and b day 7 PEG hydrogels containing RGD adhesive peptides were placed in a rat calvaria criticalisized defect to monitor bone healing in response to c MMPisensitive crosslinks d bone Inorphogenic protein BMP or 6 both Lutolf MR Weber FE Schmoekel HG Schense JC KohlerT et al 2003 Repair of bone defects using synthetic mimetics of collagenous extracellular matrices Nat Biotechnol 21513718 Acellular Tissues The tissue is decellularized and cell debris removed reduce host immune response Material retains many of the natural tissue s mechanical and biological properties E g glutaraldehyde xed porcine heart valve Acellular porcine small intestinal submucosa SIS naturally derived resorbable material implantation in the bladder 90 degraded Within 4 weeks Patterned Biomaterials BioMEM s micrometer or nanometer devices designed speci cally for biological applications Soft Lithography Smart Polymers 3D tissue engineered constructs Schematics illustrating several applications of PDMS stamps a Microcontact printing allows er of materials including biologically active molecules eg proteins to a surface in a wellrde ned pattern b gray scale photolithography provides a facile method with which to pattern structures of varying heights and c partial cell treatment uses laminar ow within micro uidic channels to treat part of a cell to a particular atrrx pro detachment Pane m l mm 1mm 5mm E l g e m t 1 2 Adhuml mu i i Sun Takayama S Ostuni E LeDuc P Naruse K lngber DE Whitesides GM 2003 Selective 39cal treatment of cellular m icrodomains using multiple laminar streams Chem Biol che 1 10 1237 30 polydimethylsiloxanePDMS Hydrogels containing living cells can be patterned into 3D structures by photocrosslinking each layer through a mask Two a b and three d e layered hydrogel patterns b d e all contain live cells Liu et al 2002 Journal of Biomedical Microdevices
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