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Components of Bone Tissue Engineering

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Video 1: Bone Tissue Engineering
Good afternoon everyone, I welcome you all for my second session of Bone Tissue Engineering.In our first session, we discussed about the function of bone, brief introduction aboutwhat is bone and its function.And, we studied its anatomy and physiology in detail and also the formation of bone aswell.So, today in this session we will be discussing about bone tissue engineering strategies andwhat are the key components present in tissue engineering application.So, in the last 3 decades, bone tissue engineering has been a very new concept and a useful conceptfor orthopedic surgeons as well as is the biomedical community in order to overcomethe limitations for the enhancement and regeneration of bone defects.So, to start with, we should know what is tissue engineering, I think by this time youall would know what is the definition of tissue engineering which is nothing, but where itencompasses the knowledge of life sciences and engineering together.For the development of a biomaterial in order to replace, restore or regenerate a diseasedtissue or organ.Or, in general it has been defined as the application of scientific principles to thedesign, construction, modification and growth of living tissues.There are three major elements present in tissue engineering which composes the tissueengineering triad.The first element is biological scaffold which is biomaterial and the second one is cellsand the third one is growth factors.So, scaffold, cells and growth factors all together makes a tissue engineering triad.So, this is just a brief introduction about tissue engineering and tissue engineeringtriad.So, what is the need for bone tissue engineering, I think we have discussed that in our firstsession as well.So, to overcome the limitations like which we are facing in current clinical operationslike in grafting procedures, like autograft where we graft tissues from the same individual,but due to limited availability, donor site morbidity.And, the second option is allograft where it as the chances of immune rejection andpathogen transfer.So, in order to overcome all these limitation we need a substitute, bone graft substituteto enhance the bone regeneration or to repair the bone defect.So, the field of tissue engineering, bone tissue engineering focuses on the alternativeoption that will completely eliminate the above said limitations which are facing inthe current grafting, bone grafting procedures.As I said like over 2 million procedures, surgical procedures as the performed in everyyear, bone grafting procedure where bone is considered to be the second mostly transplantedtissue after blood.So, this diagram explains the classic bone tissue engineering paradigm which highlightsthe several key players.The key players are the three major components of tissue engineering triad the cells.The first one is biocompatible scaffold that closely mimics; the scaffolds are nothing,but the 3D structure which mimics the extracellular matrix, scaffolds.And, the osteogenic cells to lay down the bone tissue matrix.When I was explaining about the cells, types of cells present in the bone, we have fourdifferent types of cells were osteoblast cells, osteocytes, osteogenic cells and osteoclastcells.So, osteoblasts is for responsible for the formation of bone, osteoclast is responsiblefor bone resorption and osteogenic cells is the only bone cells that can divide, differentiateinto osteoblast cells.So, we need osteoblast cells sorry, osteogenic cells to lay down the bone tissue matrix,the second essential component.And, the third one is the growth factors which is the morphogenetic signals that can helpto direct the cells to the phenotypically desired desirable type and also for the growth;enhancement of the growth that is a growth factors, that the third major component ofbone tissue engineering.So, scaffold cells and biological factors, all three together to composes the tissueengineering triad, bone tissue engineering triad.So, in this session we will be dealing about in detail about the each component, key componentof bone tissue engineering.So, bone tissue engineering involve the uses of porous 3D scaffolds.The 3D scaffolds as I said take, it mimics the extracellular matrix or in general itacts as the template, it acts as the structural template for the cell to enter, adhere, proliferateand differentiate and thereby it enhances the formation of new tissue and it has todegrade.So, it should be degradable and it is the temporary implant, it cannot be assigned toa permanent implant.And so, it involves the uses of porous 3D scaffolds that along with cells and bioactivefactors that can provide support for cells to spread, migrate, differentiate for newtissue formation; sorry three components are biomaterials, cells and growth factors.So, we will be seeing the strategies for biomaterials, cells based approaches and growth factor basedapproaches in the later presentation.So, to start with biomaterials-based approach, we should know what is a biomaterial.What is a biomaterial?Biomaterial is a natural or synthetic substance that has been engineered to construct to performin a construct in a, to perform a biological function with a medical purpose ok.It can be either medical purpose can be either therapeutic or diagnostic one.Evolution of biomaterials, in 1960s or 1970s the first 70s first generation biomaterialshas been developed.So, it was developed in a idea just to mimic the tissue which got damaged due to physicalreasons or where it has to mimic the physically damage tissue because of fracture or diseaseor any other traumas.So, they are just a bioinert materials and they do not interact with the biology of thehost organism.So, first generation materials are called as bioinert materials for example, stainlesssteel and its alloys comes under bioinert materials.And, then they switch the gears from the passive materials to the active materials; the second-generationmaterials are called bioactive materials.Bioactive materials where it can interact with the interact with the host biology, hostorganism biology; there where there will be an interaction between the graft as well asthe cellular level interaction between the host organisms.And, in 2000s third generation of biomaterials has been developed, where it is the bioresorbablematerials; where it helps in the formation of new tissue.It combines the properties of bioactive as well as the bioresorbable where it interactswith the cellular level, cellular level in the host organism, as well as it gives a specificresponse.And, also it develops the new tissue formation; it helps in the development of new tissueformation.And, in 2020 where the develop biomimetic materials where the develop a material whichmimic the nature of the natural material.So, in this biomaterials-based approach will be focused mainly on third generation materialsfor example, polymer and this composites.So, again they are classified into osteoinductive materials, hybrid materials, advanced hydrogels,immuno modulatory materials.The first category is osteoinductive material.The word osteoinduction, the word osteoinduction which means it instructs the surrounding cellsfor the formation of bone, surroundings stem cells for the formation of bone.So, those material should have the ability, if I say this is an osteoinductive material;that means, that material has the ability to induce the bone formation by instructingthe surrounding cells in vivo environment to form bone.For example, hydroxyapatite and calcium phosphate or ceramic based materials are prone to haveosteoinductive property.The second generation class of biomaterials is hybrid materials.They are nothing but the combination of two or more biomaterials with enhanced functionalitiesin the, either in the form of copolymers or polymer-polymer blend or polymer ceramic composites.Copolymers are nothing but it is a substance where it developed from two or more monomericspecies.For example, PLGA where it developed from the monomeric units of polylactide and polyglycolide.So, we can tune the properties of both the monomers where PLA has the glass transitiontemperature above room temperature and it has very long degradation rate whereas, PGApolyglycolide has glass transition temperature below room temperature and it has shorterdegradation rate.So, we can combine and tune the properties of PLA and PGA which are FDA approved polymers.And, we can develop polymer which is copolymer, hybrid material, PLGA with tailorable tailoredproperty in the end.And polymer-polymer blends.So, again the same example I will tell you about PLGA, the degradation product of PLGAwill be acidic in nature.So, prolong exposure of tissue to acidic product will lead to tissue necrosis and eventuallythe implant will lead to failure of implants.So, what we researchers will do is they combine with the polymer like for example, phosphazenes,phosphazenes where the degradation product of phosphazenes are in neutral pH.So, they combine PLGA as well as phosphazenes to develop a biomaterial to give a non-toxicdegradation products.The final one is polymer ceramic composites which are very useful in bone tissue engineeringand this we can call it as biomimetic scaffolds.It has all the desirable properties for bone tissue engineering.Then third class of biomaterial is advanced hydrogels.Hydrogels with because of its physical properties and its structure it has it has been widelyused in tissue engineering application.And, the recent research have showed that self assembling peptides have gained recentenormous attention.So, forming scaffolds as they are completely biological, biocompatible and biodegradable.For example: RADI-16, it is an self assembling peptide where it aims to mimic natural extracellularmatrix and it can be readily synthesized.And, it is injected in the form of nanofibers; it is injected in the form of nanofibers,once it enters the physiological fluids and it becomes the gel and it serves as the templatefor the starting material; template as in starting material.The final one is immuno modulatory biomaterial.So, in order to suppress the immune reaction, we need, we can develop a biomaterial thatcan modulate or manipulate the immune system in a favourable manner for the enhancementof bone regeneration.So, these are about the biomaterials-based approach where I will focus mainly on scaffolds.

Video 2: Scaffolds and Cellular Based Approaches
The scaffolds, scaffolds are the masterpiece of bone tissue engineering.A bone scaffold is the 3D matrix that allows and stimulates the attachment, proliferationof osteoinducible cells on its surfaces.And, the first one was developed by Green in early 1970, where they injected, wherethey seeded cartilage cells into the scaffold that is how it has developed seeding cellson scaffolds.And, the image which shows there is a long bone where there is a defect and we have toplaces the scaffold in the defect and check for the enhancement or regeneration of thelong bone.So, scaffold since the scaffold it is in a masterpiece of the bone tissue engineering,we should be more careful in selecting scaffold and its properties, the materials and forthe everything for the preparation of scaffold.So, the first one the scaffolds can be categorized into four classes; polymeric, ceramic, compositeor metallic scaffolds.Polymers again polymerics scaffold can be derived from either natural polymers or syntheticpolymers.And, composites maybe the combination of polymers and ceramics, and, the scaffolds requirementof the ideals scaffold.And, any ideals scaffold for bone tissue engineering, it should be biocompatible.And, it should be biodegradable and it should not be a permanent implant, it is an temporaryimplant.And, it is non-toxic and highly interconnected porosity, this porosity plays a very importantrole in bone tissue engineering, because, the pore size.The pore size is very important for the cells to enter and proliferate, differentiate andalso for the diffusion in and out of the nutrients and wastage.The pore diameter or pore size, it should be greater than 100 micrometer and it shouldbe mechanically strong.Since, we are aiming for bone tissue engineering, the scaffold should be ideally strong andit should mimic the either cortical, if in case for a cortical bone tissue engineering;it should have the mechanical properties similar to cortical bone.Whereas, the spongy bone, the cancellous bone it should mimic the properties of this spongybone.Also, it should enhances vascularization, vascularization is the formation of new bloodvessels.So, any ideals scaffold for bone tissue engineering it should be biocompatible, it is it shouldnot be toxic.It should be biodegradable, it should have highly interconnected porosity.It should be mechanically strong and it should enhances vascularization.Those the three properties of scaffold which clearly shows the ideal scaffold properties.The osseointegration which is nothing but the biocompatibility, it should integratewell with the host tissue.And, osteoconductivity where it helps in the formation of new bone on the surface of thegraft, and osteoinductivity it should direct the cells, surrounding cells for the formationof bone.So, these are the main properties of the ideal scaffold.And, micro and macro structures are the nothing but the porosity structures, mechanical properties,it should be mechanically strong.So, these are the main properties of a scaffold to be used in bio bone tissue engineering.So, this picture where it shows the treatment of bone defects with the presence of scaffold.The first there is a bone defect in the bone gap, defect gap in the long bone where weplaces scaffold in that defect gap, defective gap.And, this scaffold first it should, it should be osteointegrated.It should be osteointegrated, then it should be osteoconductive, osteoinductive and thenthe healing process.This is nothing but the vascularization, the formation of new vessels.So, from this, there will be some few graphical images just for teaching purposes, shouldbe aware of this graphical images which are taken from the literature; for knowing aboutthe information what they have done for scaffold based strategies.So, in these are called as first generation scaffolds which are nothing but the groupin National University of Singapore collaborated with Temasek polytechnic where they optimizethe parameters for PCL, polycaprolactone and its composites for the development of scaffoldby FDM method.FDM method is Fusion Deposition Modelling which is nothing but the 3D method, 3D printingmethod for the development of biomimetic scaffold.They considered this as a first generation scaffolds, where they perform clinical trialsfor clinical studies for 5 years.After 5 years they said that the outcome was positive.And, again the another research group use this kind of burr hole plugs for the treatmentof cranioplasty, the burr hole plugs for the treatment of cranioplasty.The another group was tested with another clinical trial, another set of experiments,where they used this PCL burr hole plugs and they studied in rats.And, after after 12 months, 12 months they got an positive outcome and this CT imagesshows the perfect alignment.The CT images shows the perfect alignment of PCL sheets to the 3D orbital cavity.The first image where the use this PCL sheets in the autologous iliac crest, where theyremoved a skin from there, where they place this sheet and tested for the regenerationof bone and they found it successful.The second where they did for skull defects, the second image where they place this PCLburr hole sheets on the skull defects.And, then after 12 months of the study they found that they have perfectly aligned andthey heal the bone defect.So, then they have move to the second generation of scaffolds where they wanted to study withPCL and composites ok, where they have done they prepared scaffold using PCL tricalciumphosphate and collagen.And, they created defects around 5 mm diameter in rat parietal bone, in rat parietal boneand then they tested for healing of the bone defects.And, in the C and D you can see the micro CT scanning image where the skull defect,after 14 months there is the complete closure of the bone.There is the closure of the bone with the presence of the scaffold, when it is comparedwith the blank where there is no scaffold at all and this was done in rat parietal bone.Using PCL sheets with and its composites, this scaffolds which, these are the biomimeticscaffolds.And, this one is the pigs spine fusion model and the below pictures are the rabbit NewZealand white rabbit skull defects.They have tested for cranioplasty, again analysis of tissue engineering the constructs showsthat the formation throughout the entire scaffold and no fibrous tissue formation is seen.And, micro CT analysis revealed that the 30 percent of PCL-TCP is resorbed and replacedby bone.And, this is for the treatment of load bearing osteochondral defects, again load bearingsince bone we need to test for load bearing defects.Again, with the PCL and calcium phosphate where they have drilled hole in a load bearingosteochondral defect; they created load bearing osteochondral defect where they have seededmesenchymal stem cells along with the scaffold PCL CaP scaffold.And, then they inserted on the defect and after after 12 months or I am not sure aboutthe month or the time; after a certain time there is a closure of these bone defect inthe presence of scaffold.So, this all about the scaffold, the scaffold we have seen about its properties of the scaffoldrequired for the ideal bone graft.And, few examples about biomimetic scaffold where they developed for the first time firstengineer, first generation scaffold and the second generations scaffold.And, now we move on to the strategies for bone regeneration cellular based approach.First we seen about scaffold based approach and now it is the cellular based approach.The cellular based approach we have seen that there are four types of cells: osteoblastcells which are responsible for the formation of bone.Osteocytes which are the primary cell for a matured bone and which are responsible forto maintaining the concentration of matrix, mineral concentration of the matrix.And, osteogenic cells which are responsible for the differentiation into osteoblast andosteoclast cells which are responsible for the bone resorption.And so, cellular; so, though the volume of cells present in the bone is less, but itsfunction is crucial.So, we need to have bone cells in order to repair bone defects, there are four basedthese are the first proposed approach.Firstly, proposed approach is, cellular based approach is implantation of unfractionatedfresh bone marrow is the first approach.And, the second one is the purified culture expansion of mesenchymal stem cells and thethird one differentiated osteoblasts and chondrocytes.And, the fourth one is the cells that have been modified genetically to have a recombinantbone morphogenetic protein.So, we will look into first approach.So, these approaches mainly based on the primitive mechanism primarily targets the, see cellularbased approach in bone tissue engineering primarily targets the early stages of bonerepair when the skeletal progenitors becomes impaired.So, this can be mainly due to either trauma or any disease conditions or maybe can bedue to aging as well.These proposed mechanism by which implanted cells enhance bone regeneration in bone tissueengineering involve; first is early release of key osteogenic and vasculogenic moleculesand growth factors.And, the second one is it forms the template to recruit the osteogenic cells and vasculogeniccells.And, the third one is actively laying down the bone matrix and vascularizing the boneconstruct.These are the steps that follows in the cellular based approaches.And, the first approach is the unfractionated fresh bone marrow approach; in the first picturewhere the patient the first is they are anesthetized.And, the second one is the defect and where they remove, directly remove the fresh bonemarrow and inject the cells into the defective site.So, they remove the bone marrow; so, we all know cells present in the bone marrow region.So, they remove the cells from the bone marrow and they inject into the defect site directlyas well as along with the some matrix.So, they inject bone marrow cells along with the matrix.So, after few days or a few months we can see from the radiograph images, where thereis a complete healing of the bone defect.We can see the difference in the picture B and C where there is complete healing of bonedefect, due to the injection of bone marrow cells.Osteogenic cells directly from iliac crest, the posterior wing of iliac crest and we injectinto the defect side.But, the disadvantages of this is availability again it is autologous as well as it increasespain and it require two surgeries where we need to remove and we need to inject intothe defect sites.So, it requires lot of pain and time consuming and everything.And, the next approach is differentiated osteoblasts and chondrocytes approach.We all know the bone mechanism, formation of bone mechanism.As I explain in my first section session formation of bone occurs in two mechanism via intramembranousand endochondral ossification.So, what people researches have thought like why do not we inject osteoblast cell differentiatedosteoblast cells or chondrocytes directly into the defect or along with the mesenchymalstem cells, ok.So, first they have tried, they inject differentiated osteoblast cells, they achieved in that.They achieved increase in enhancement of bone regeneration whereas, in the second picture,this picture where what they have tried is they tried to inject chondrocytes.So, because they thought that endochondral ossification, first the formation of cartilaginoustemplate thereby laying down bone matrix, right.So, what they thought why do not we inject chondrocytes directly into the defect sites.So, Vacanti and Vacanti and his group they did this research; what they did it is likethey injected chondrocytes into the bone defect and they compared with the periosteal cells.What they found is there was a formation of cartilage layer in both the defects, theycompared with periosteal cells and chondrocytes cells.And, but after cartilage formation the defect where they inject only chondrocytes, theydid not find any formation of vascularization or angiogenesis nothing, it was like that.In turn they found that the chondrocytes produces the precursor cells which has the cues, whichinhibit the vascularization.So, the other defect where it they are found the cartilaginous template as well as theformation of bone and everything it was there.And whereas, in the defect where they injected only chondrocytes, they did not they couldnot find the vascularization network or neoangiogenesis which is the primary formation of blood vessels.So, they found that injecting chondrocytes alone will not support in cell based approach.Then the, this approach the mesenchymal stem cells approach is effective approach wherethey can this mesenchymal stem cells can undergo replication without differentiation.It can go and we can passage 30 times of this mesenchymal stem cells on which increasesto up to 1 billion-fold of cells without differentiation, it has to differentiate only in the presenceof implantation.So, this is one of the research where the recently produced where in the segmental defectthey created a bone defect with the inserted collagen matrix.What happened was the endogenous mesenchymal stem cells slightly invaded into the matrixthen, then this research group with the help of ultrasound they in they injected BMP, BoneMorphogenetic Protein in order to start the bone repair process with a microbubbles.So, they suspended in microbubbles and injected into the mesenchymal stem cell populated scaffold.And, with ultrasound what happens, this gene enter into the cells with the external stimuli.After few days and this segment after few months this segment got healed completely.So, this they have done it with all the all the rat animals which are treated with thiskind of approach has been completely healed with this mesenchymal stem cells approach.So, first there is the creation of segmental defects, they implanted the collagens scaffold,placed this collagens scaffold, this is the scaffold and bone marrow.After 2 week this mesenchymal stem cells slowly it is invading these scaffolds.And, then they inject DNA and microbubbles which is which has bone morphogenetic protein.And, they apply ultrasound, the external stimuli.Now, all the DNA will get into the cells; now it will start bone repairing process.See how beautiful is this approach.So, where they have scaffold, cells and growth factors as well.All the three combined together and used for the repair and regeneration of that, thatbig bone defect.In the next session we will be dealing about the growth factor based approach and the commerciallyavailable bone grafts and what are the current trends and future direction of bone tissue engineering.