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Module 1: Material Characterization

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Video 1: Biocompatibility
Good morning everyone.So, in the previous classes we have learnt about what are surface properties and bulkproperties and how to characterize the surface properties and Mechanical Characterizationof the materials.So, we have learnt about that.So, coming to the final characterization, which is biological characterization.So, this is the end game which determines whether your material is biocompatible ornot; which is basically whether, whatever implant or devices you are producing withwhich will be taken by the host system or will be rejected by the host system.So, in biological characterization, there are three major categories.One is in vitro biomaterial characterization and another is in vivo biomaterial characterizationand apart from this there is a separate section, which is bacteria biomaterial interaction.So, in vitro is where you will do all the biological related experiments on a lab scaleenvironment.In vivo where you will experiment the material in animal models like rat, pigs, canine, speciesand all.So, bacterial interaction is mainly needed when you are looking for infection on thematerial surfaces and all.So, when you are implanting a material into the body, so within a nanosecond the proteinswill get adsorbed onto the material surface.So, based on the protein adsorption, it determines how the cells are adhering on to the materialsurface which will lead to the host material; host cells and material interaction.If the cell attachment it continues and if there is a proper cell attachment, it willlead to spreading of the cells and then which will lead to the differentiation and migration.So, all these steps will lead to complete integration of a material into the body.So, based on the application you have to decide what type of interaction you need.Like, if you need an interaction between the cells and the material for the applicationsuch as orthopedic implants, wound dressing and all, so you need your material to be integratedwith the host cell.But, if you are looking into cardiovascular devices and all where, you do not need anycells to be adhered onto the surfaces and all.You should avoid the cell attachment on the material surfaces and all.So, coming into what are the aspects you have to look, for the material to be a biocompatible.So, there are a lot of aspects based on again application.So, until now whatever the surface property and the bulk property you have analyzed orcharacterized, those can be changed based on the results you are getting.But for this biocompatibility, it is entirely dependent on the how the material is interactingwith the cells.So, what type of cells you are using and what type of experiment you are doing, based onthat it will interact with your material ok.So, whatever material you have characterized and suitable, made suitable for your applicationand all, it should be attached to the cells based on how effective it can be integratedinto the system.So, coming into aspects, so first is sensitization, irritation and intracutaneous, reactivity.So, this is mainly applicable for wound dressing materials.So, what happens is when you are putting a wound dressing on a particular wound, it shouldnot cause irritation or it should not produce any allergic reaction on the surfaces andall.So, we have to test for that so that is that aspect, then systemic toxicity.So, systemic toxicity involves, what duration of toxicity you are going to observe overthe period of time.So, toxicity which is a commonly used term for studying the biocompatibility of any scaffolds,hydrogels or materials; so, what you are basically going to do is, whether the material you haveproduced is leaching out any compound or it is interacting with the cells and killingthe cells.So, we have to check that using systemic toxicity.Genotoxicity which is the material, which can cause damage to the genes; so, which canlead to mutation and all.So, implantation mainly observed when you are the using a material, which can affectthe nearby tissues and organs also.If you are using orthopedic implants and all it can have a effect on nearby bones and nearbytissues, muscular tissues also.So, based on the where you are implanting, you have to decide, you have to find out thefactors affecting that.Haemocompatibility it is primarily involved for cardiovascular applications, where youneed to your material to be fully blood compatible.And, carcinogenicity it is again linked to genotoxicity or the mutagenicity where, ifthere is a mutation it can lead to causing cancer causing effects and all which willlead to further rejection of your implants.Reproduction and development it is not primarily used for all the materials.But if there is a possibility, if there is a possibility that your material leachateor your nanoparticles or the molecules, which you are using can affect the reproductionsystem or the embryonic development stage it has to be checked for that.Biodegradation it is used for a lot of application such as vascular sutures and staples whereyou are using that material and it should be degraded over the period of time, so youhave to check those things and all.How the cells will degrade those materials and all you will check in that biodegradationand all.Immune response is basically involving the total immune system, whether the in introducedmaterial is having any antigenic effects, so that it will trigger a immunogenic cascade.So, that it will lead to inflammation of the site of implants.So, if you are introducing a metal orthopedic implants and, if it is causing leach out immunogenicresponses and all, that place will have a inflammation occurring.So, these are the major aspects.So, in this one commonly used test for assessing the biocompatibility are cytotoxicity, whichis the initial test we have to do for all the materials.Whether that material is killing the cells or it is having a inert effect or it is promotingthe cell growth and all.Then, haemocompatibility where you have a blood related application and all.Mutagenicity and carcinogenicity, where you will check for any change in mutation occurredin the system.Then bone remodeling, it is a separate topic where you can estimate how much of the materialis affecting the bone integration or bone growth and all, so that is bone remodeling.So, in cytotoxicity, I will explain in detail all the assays.It has direct and indirect contact and couple of other experiments MTT assay and the XTTassay, which is used for calculating the how much your material is toxic to the cells.Haemocompatibility where, these experiments will tell you whether your material is notcausing any damage to the blood cells or it is leading to any platelet aggregation orblood clot formation.So, mutagenicity and carcinogenicity Ames test and chromosomal aberration and mouselymphoma assay.So, in Ames test we can do it in a bacterial study, where a bacteria usually a salmonellaspecies will be there.So, in that one, we will have a mutant which is having a histidine, lack of histidine genein that one.So, if there is no histidine supplied in the media, the bacteria cannot grow ok.So, you will introduce your compound, so if it is causing mutagenic reaction on the gene,it will convert the histidine and it will grow; so, that is the basics of Ames test.So, by this you can observe that whether the Ames test is, whether the compound you areusing is causing mutagenic response or not.So, for all these tests that would be a control where you will have a normal bacterial gene,which has a histidine utilizing gene also.So, if it is not enhancing your compound is not enhancing that, it can suggest that yourcompound is only changing the gene of the histidine lacking species.So, in chromosomal aberration, we will check how the chromosome is after mitotic divisionand all, we will check with the compound and without compound.So, if there is a chromosomal abbreviation, there would be either a insertion, deletionor translocation observed on the chromosome chromosomal structures, so that can be observedin chromosomal abbreviation.So, mouse lymphoma assay, similar to Ames assay, but we will do it in a mouse lymphomacells.So, in the cells which will have a thymidine kinase enzyme so, that would be knocked outin a knocked out mouse.So, if you are compound is, and they will supply thymidine, so if your compound canmutate the cells then it can use that thymidine.So, by that you will understood that your compound is causing any mutagenic responsesor not.So, in bone remodeling, so the main factors we will look up are alkaline phosphatase assay.So, in bone remodeling when you are using a dental implant or orthopedic implants, whereyou have a bone cements and all, that will lead to integration of the hosts cells, osteoblastcells and all.Lead to formation of increased in calcium content, phosphate content and osteocalcinis the major molecule for the bone cells.So, if there is increase in these factors and all, you can actually estimate them usingthis assays and all.And found that that whatever the bone scaffold or whatever you are the material is introducingit has a effect on the improving the bone cell integration within your scaffold andall.So, systemic toxicity, yeah.What is the role of ALP enzyme in bone growth?.So, ALP enzyme what it does is, it converts phosphates to inorganic phosphates and all.So, other phosphate derivatives into inorganic phosphates, those phosphates are major contentof bone.So, they are the basic components calcium phosphates, so there are a lot of phosphates,so, based on that you will understood that, if there is a higher activity then there isa faster growth of bone tissue and all ok.So, systemic toxicity it determines how long the implant you are going to use it in a system.So, acute system where you will use it less than 24 hours and all, which is people usecatheters and all to do angioplasty and other treatments and sub-acute which is 24 hoursto 28 days.Then, sub-chronic 28 days to 90 days urinary catheters and all it would be within 2 monthsor 3 months they will remove it.Then chronic which is above 90 days.So, you have to test whether the material is not having any effect on the cells surroundingit, so those chronic will involve all the bone implants and cardiovascular implantsand all.So, based on this time duration, we have to check the material, it is exactly, up tillthat time it is not having any adverse effect on the cells.
Video 2: In Vitro Cytotoxicity and Cell Adhesion
So, coming into toxicity so, this is a primary test we will do for all the material, eitherit is a hydrogel, scaffold or nanoparticles, whatever the material.So, we have to check whether it is accepted by the cells or not.So, that can be done based on three different methods; direct contact, indirect contact,elution or extract assay.So, all these variation is based on what is your sample and what type of sample you arehaving.Direct contact is where you have see, so you can see the figure.The first one where you have grown a cells on the tissue culture plate and you put yoursample on the top of it; and check whether it is affecting any of the cells underneathit.So, that can be done for your nanoparticles or not having too much load samples.So, if you are having too much load it can destroy your cells automatically.So, indirect test, which is done using, there would be agar layer on the top of the cellsand your sample would be there.In that, which if your sample is leaching out some compounds or some erosion or someother drugs are releasing from the material, then that can leach out and affect the cellsunderneath it so, that is indirect contact.So, indirect contact can be done based on, how it is interacting with the cells also.So, as you can see in the right hand side top figures, where first you grow the cellson a filter membrane, so then after the cells are adhered on to the surfaces and all thenyou invert it and then you will put the sample on the top.So, your sample is not exactly in contact with the cells, but the leachate of your samplewould be going into the cells and whether if it has any effect, it will kill the cellsor if it promotes the cells, it will have that effect.So, extraction or elusion assays are used for soft materials, where you can keep thatmaterial in a media for 24 hours or 48 hours based on your application, then you have toextract the media.If there is a leach out from these compounds, you can take that media and culture it inthat with the cells or with the indirect test method.So, you have the cells, you have the extracts extracted media that, whether that leachatehas any effect and all you can test it.So, all these things are based on the sample size and how effective the leachate is comingout of the sample and all.So, if there is no leachate coming out of the sample, it will not have any effect andall.So, for that you can move on to the next one of test which is involved, which is whichinvolves a cell adhesion and proliferation.So, all these steps, all these experiments can be done for those assays also where, youkeep your sample at first then you seed the cells on top of them.So, at this one, toxicity measures how much of your material is toxic and all.So, if you found out that material is not toxic, not causing any effect, for all theseassays and all then you can go for cell adhesion.So, whether your material is promoting cell adhesion and all because, cell adhesion isvery important when you are integrating your material with the host system and all.So, for that you will have the sample at the bottom and the cells would be seeded at thetop of the material.And if over the period of time, if the cells are having adhered, adhesion property basedon your material surface and all that can be estimated also.So, for all the assays while involving biological characterization, control is very important;you should have a positive control and negative control.Negative control as such there would be a standard material.So, if you are testing orthopedic implants and all there would be a standard material,stainless steel, stainless steel would be there.So, you have to test that material for cytotoxicity.So, the negative control will not have any effect on the cells and all, there would bea positive control also.So, the positive control will have a maximum effect, maximum killing effect on their cellsand all.So, based on those only, you can within that range only you can decide whether your materialis cytotoxic or not.So, usually according to literature, if your material causes below, below 70 percentagedecrease in cell, cell viability then that material is not suitable for the biologicalapplication and all.So, now we saw how the experiment to identify your material is toxic or not.So, how exactly you identify that thing?So, that can be done by qualitative and quantitative.Qualitative involves you observe them visually using microscope, so you can use a normaldye like trypan blue or calcein-AM and propidium iodide.So, calcein-AM will go inside the cells and it will bind with the cyto cytoskeleton andit will give a green fluorescence; and propidium iodide which will go and bind to the nucleusand it will give a red colour.So, if there is a cell death and all, you can use these stains and identify whetherthe cells are alive or dead, you can observe them under the fluorescence microscope andall.so that is a qualitative test.And quantitative test, there are a lot of assays are there.So, these are the commonly used one.So, MTT, XTT and MTS so, all these are tetrazolium salts.So, what it does is, so using electron receptor it will convert into formazan.So, so the formazan will give a different colour, in MTT what happens is, it will goinside the cells and mitochondrial dehydrogenase, it will reduce the MTT and it will form aformazan crystals.So, in MTT it is a final step of the reaction, where you will kill off all the cells andall.But in XTT, MTS and alamar blue and all cells would be alive because, in MTT the formazanformed is, the formazan crystal formed is inside the cells.So, you have to estimate how much of the quantity of formazan has been formed and all.So, you have to rupture the cells and dissolve them using a solubilizing agent like DMSOand all to check the absorbance of the formazan crystals formed.Whereas, in XTT and MTS, which are water soluble tetrazolium salts.So it happens outside that cells itself with the help of phenazine methosulfate, PMS, itinvolves a plasma membrane electron transport, which converts these tetrazolium salts intoformazan dye.And without rupturing the cells, you can directly dissolve it in a culture media or the bufferand check the formazan, amount of formazan crystals formed and all.That formazan formed is not insoluble.In MTT.No in XTT and MTS.No it is water soluble.So, can you use the same cells again for some other experiment?Yeah.You can wash those cells and you can observe it for, but if you keep on repeating that,the cells will obviously die.So, you can immediately takeout and you can use that.So, next to that is alamar blue assay which involves resazurin dye.So, this dye involves a cytoplasmic redox reaction, so it goes into the cells and dueto redox reaction it will convert into resorufin which will give a pink colour.So, compared to these above three methods, this is a highly sensitive method where youcan estimate even a very small amount of cells also.And it has another advantage that it has a fluorescent, fluorescence property also.So, you can using fluorescence spectrophotometer, you can calculate even a minor change in thecell quantity also.So, for all these things there would be a control.So, where you do not treat your cells with any of your samples and all and there wouldbe another control where there will not be any cells.But, whatever the material you are using will, will be in the media also because, those controlsare very important, because all this involved colorimetric assays.So, if you are material is changing the media pH or any other color adding colorimetricvalue to the system then, the results obtained can be of a false positive.So, to avoid that, we should always have the control in all of the assays and all.So, these are the methods for cytotoxicity.So, after cytotoxicity, if your material is not toxic you can go for the cell adhesionand all.So, cell adhesion its involves the same list of procedures itself, where you can put yoursample underneath the cells before seeding.So, in cytotoxicity what you do is, you seed the cells, you grow them, you make them forall these animal cell lines it has to have a substrate to attach on to the attach attachmentsshould be there.So, you have a tissue culture plate, your cells would be properly spread on that tissueculture plate and then you put on your material, if it is causing toxicity that would be observed.In cell viability or the cell adhesion assay what you do is, you put your material andthen you seed the cells on to the material surface, material means any flat surface materialor scaffold and all.So, if based on your surface properties whether it is a hydrophilic, or it has any receptor,ligand for the cell receptors and all based on that, the cells will attach and you canestimate the amount of cells attached on to the material you are having.So, coming into cell motility and cell migration assay; so, these are next stage of characterizationwhere, it is predominantly used in wound healing application.So, in wound healing application, what you have is there would be a wound separatingtwo different tissues, so you same tissue so, you have to join them.So, that can be done using this scratch assay.So, you have a material you seed the cells on to the material and you scratch them andwhatever your compound which will enhance the healing property and all.So, you put that compound and check whether how the wound is getting closed.So, as you can see in the figures, so the left side one is control and the right sideone is treated samples.So, they first grown cultures then, they using a small tip or spatula they create the wound.So, in the left hand side there will not be any treatment at all.In the right hand side they have used the Annona squamosa extract, which is a extractof custard apple leaves.So, they have used it and, as you can see the wound has been closed within two daysand all.So, it has a wound healing property.So, by this you can identify how much of, how fast your wound can be healed using adifferent concentration of compound and, different composition of the compound and all.What are those HDF?So, HDF is Human Dermal Fibroblasts.So, it is a skin cells, so dermal fibroblasts.So, whatever the material you are using, if it is non-toxic to one type of cell does notmean it is a non-toxic to all types of cells.So, each cell has a different property and different mechanism.So, based on the application, if you are going for the bone application and all you haveto check with the osteoblast cells and if you are going for the vascular application,you have to check with the endothelial cells.And if you are going for a wound healing application, you have to check with fibroblasts cells andall.So, each cells has a different attachment property and different effect on materialsurfaces and all, so that you have to always think.
Video 3: Hemocompatibility and Biofilm
So, another important segment in biocompatibility is haemocompatibility.So, haemocompatibility it is specific to blood related application.So, if the if a material is haemocompatible, it has a good blood compatibility.For example, if you consider a heart valves, vascular grafts and all, there you have acontinuous contact with the blood the throughout the system, throughout the time; so, at thetime of implantation and at the time of removal so, there would be a continuous contact ofthe blood cells with the material.So, if there is any thrombus, so what is thrombus formation?So, thrombus is a blood clot.So, which is formed by adhesion of platelets and fibrin mesh formed on to the surfaces.So, first platelet will adhere on to the material surface then aggregation happens.So, if one platelet get adheres, it will send out extracellular signals which will activatethe surrounding platelets and all, it will act as a homing beacon for making the otherplatelets to come into its nearby area; and it will form an aggregate that is called plateletaggregation.So, when that stable platelet aggregate has been formed, there would be a parallel reaction,coagulation cascade happens which forms a fibrin mesh.So, that fibrin mesh acts as a network, which will trap this platelet aggregates and forma blood clot.So, if you are having a vascular graft and if a blood clot is formed on to the on itssurfaces and all it will lead to stroke and other complications and all.So, in the right hand side it is a example of how surface modification can improve thehaemocompatibility.So, the top one they have used the CTI coated catheter.So, CTI is corn trypsin inhibitor, so which will inhibit the clot formation and bottomone is without any, without any coating.So, as you can see based on this study, you can identify a material can be avoid clotformation or not.So, what are the experiments you can do for haemocompatibility the main experiment isplatelet adhesion because, that is the initial step of thrombus formation.So, that can be observed using SEM, so, you incubate your sample with platelets, you extracthealthy platelets from human volunteers, then you can check how the platelets is adheringonto the material surface and you can observe it under SEM.Or you can quantitative using previous whatever cytotoxicity experiments you can MTT assayalso you can quantify.There is an assay called lactate dehydrogenase activity assay, which all involves the liveplatelets using the reductase enzyme, it will using a reductase enzyme and forming a colorimetricproduct which will lead to quantification of this platelets.So, all these things, you should have a standard, so you have to count the number of cells andwhat is the number of cells having that effect, like in MTT for the particular absorbancewhat is the number of cells?So, you have to make a standard and all, so from that only you can calculate the actualnumber of cells on your material surfaces or how much of has been reduced or increasedfrom the control.So, standard is very important for all these biological aspects.Other than, platelet adhesion there is a commonly used assay which is called hemolysis.So, hemolysis as the name suggests the lysis of the hemoglo, erythrocytes.So, whenever your RBCs are broken down it will release hemoglobin that can be estimatedusing UV visible spectroscopy.So, based on the standard international organization for standardization, hemolysis index shouldbe below 2 for a non-hemolytic material, it should not have any clot formation on thematerial surfaces and all.If it is slightly hemolytic, it is the range is 2 to 5 and if it is highly hemolytic itis above 5, which is not at all recommended for any applications.So, hemolysis is calculated based on absorbance of the sample minus absorbance of the negativecontrol by posh absorbance of positive control minus absorbance of negative control.So, what are the negative and positive controls are negative control is a normal saline solutionwhich does not have any effect on the RBCs.Positive control can be either water or triton X, where it will lies the cells immediatelyon contact with RBCs.And sample is where whatever sample you are having.So, whether that sample will have any effect on RBCs that is the sample.So, based on this you will find out the hemolytic index and identify a material is blood compatibleor not.So, other than these methods there are the partial thromboplastin time, which, whichis to estimate the amount of estimate the time required for the thrombus formation.So, you have your control material and you have your sample material; and in controlmaterial you put on your blood sample in both of them and you add a coagulation factor inboth of them.So, coagulation factor, which will lead to thrombus formation.So, if your material is non haemolytic, which is avoiding thrombus formation, there wouldbe a delay in thrombus formation.Whereas, in this reference material it would be immediately formed so, based on that youfind out the partial thromboplastin time.And you can look into markers of coagulation cascade like C3a, C5a.So, all these are there is a series of pathway involved for involved in the coagulation cascade.So, you can check for the particular markers, whether it is an inactivated or activatedstate and all.From that, you can identify that the material which you are inserting is activating theblood cells and causing this coagulation cascade and all.Because, it is a serious issue that if a small clot also forms, if it gets detached fromthe surfaces, it can go and block somewhere in the arteries where which is called emboli;that movable clot is called emboli.So, that can block the arteries and it can lead to serious issues.So, when you are looking for a blood contacting application, you should always do the haemocompatiblestudies and all.So, coming into bacterial interaction with the biomaterials, one of the major complicationsimmediately arises when you are introducing a material into the system is infection.So, if there is a infection there would be an immediate rejection; because, if thereis a bacterial infection on the material surface it can lead to immune responses and all othertoxic to the cells and all so, it would be immediately rejected by the cells.So, we have to make the material as much as possible to avoid any bacterial adhesion andforming a bacterial adhesion on the surface and all.So, this is majorly observed and it is majorly observed in dental plagues, where you havea dental implants where you have lot amount of bacteria, exposed to the material and all.And infection on urinary catheters, so urinary catheters, there would be a lot of bacteriaexcreted and all.So, there is another place and orthopedic prosthesis, there also if there is a littlebit of bacterial interaction is there, that can lead to infection serious issues.In normal condition the bacterial would be as a planktonic state, which is suspensionin the liquid solution.But when this bacteria is adhering on to the material surface, it becomes a sessile statewhich will lead to the formation of biofilm.So, what are biofilm?So, as a normal bacterial adhesion, it is reversible.So, due to shear force and all it can be easily the removed off from the surfaces and all.But, when a bacteria adheres and it forms aggregates and all it then changes its metabolismand secretes exopolysaccharides.So, these exopolysaccharides what they do is, they cover this bacterial aggregates andform a thin biofilm and all.So, these exopolysaccharides will protect them from the immunogenic cells and all, thereby it will be there for over a long period of time.And whenever the host immune system is low, these can come out of the exopolysaccharidesand it can infect the nearby tissues and cells and all.So, biofilm is a major problem and your material should be avoiding the biofilm formation asmuch as possible.For biofilm estimation, you can there are two basic methods; one is turbidity measurementand another one is a plate count measurement.So, in turbidity measurement, you measure the number of bacterial cells based on theturbidity observed at, at O.D of 600 nanometers, it is a standard for observing bacteria, youcan estimate the amount of bacteria.So, you have a control you have your sample and all, you inoculate the control with thebacteria and you have your sample and inoculate with the bacteriaSo, all those things you have to again, how much of cells are adhered onto the surfacesand all, whether it has caused any immunogenic responses and all.So, all those things you have to do for that.