Biomaterials for Bioengineers Lecture Notes - 2020/2021 PDF

Biomaterials for Bioengineers Lecture Notes Mestre, Miguel 2021/2022 Contents Introduction to Biomaterials................................................................................................................... 2 Early Biomedical Materials................................................................................................................. 2 The Need for Biomaterials.................................................................................................................. 2 Implants, Transplants and Prosthesis................................................................................................. 2 Problems with current implants......................................................................................................... 3 Bioactive Spectrum............................................................................................................................. 4 Molecular and Cellular Biology: Overview.............................................................................................. 5 Cells..................................................................................................................................................... 5 The Central Dogma of Molecular Biology........................................................................................... 9 Life and Death of Cells...................................................................................................................... 10 Tissues and Organs........................................................................................................................... 10 Cells of the Blood and Immune System............................................................................................ 14 Metals and Ceramics............................................................................................................................. 17 Metals............................................................................................................................................... 18 Ceramics............................................................................................................................................ 20 Polymers and Composites..................................................................................................................... 24 Bioresorbable Polymers........................................................................................................................ 32 Hydrogels.............................................................................................................................................. 41 Tissue Engineering: Introduction and cell source................................................................................. 49 Cartilage & Cartilage Repair.................................................................................................................. 56 Cartilage – Structure and Content.................................................................................................... 56 Disease and Damage......................................................................................................................... 60 Treatment Options............................................................................................................................ 61 Bone: Structure and Mechanical Properties......................................................................................... 64 Joint Replacements and Bone Cement................................................................................................. 73 Tissue Response to Biomaterials.......................................................................................................... 82 Bioinert Polymer Devices...................................................................................................................... 88 Bioactive Ceramics................................................................................................................................ 94 Bioactive Glass.................................................................................................................................... 100 Scaffold Materials............................................................................................................................... 104 Tissue Adhesives................................................................................................................................. 108 Teeth and Dental Biomaterials........................................................................................................... 112 Hemocompatibility and Stents........................................................................................................... 116 1 Introduction to Biomaterials Early Biomedical Materials Materials aren’t a new field as there are very early examples of biomedical materials that date back to the iron ages (300 BC) or the ancient Egypt (1500 BC). In the iron ages were used iron pins as dental implants and in the ancient Egypt was common the use of gold in order to hold the teeth together, almost like a container does nowadays. Gold is characterised for being biocompatible, soft (ductile), even though it is quite expensive. Dating back to 1815 (Battle of Waterloo) it was common to use prosthesis, which were strapped on to a soldier leg (amputee), were made of wood, being very heavy quite cumbersome and also relatively strong. However, this prosthesis could be easily damage, despite their strength. These prostheses were a good solution in case of amputation, which was the typical historical treatment for infected limbs. Note that this type of prosthesis don’t allow integration of the host tissue, being just an artificial limb. The Need for Biomaterials One of the main reasons to the development of biomaterials is due to the fact that we have an aging population. This will lead to a greater amount of medical issues requiring care, creating stress sunder the health systems. This leads to shortage of resources as for example there will be shortage of organs for transplantation as there will be less donors than people requiring them. The solution for this problem is materials that can help regenerate tissues. Note that populations are getting older globally due to many factors such as good nutrition, health care, being the number one contributing factor water cleanliness. As we get older tissues begin to fail and there are no donors of most of those tissues and they are difficult to graft so there is the emerging need to find therapies and materials that can help do that. Tissue Engineering is a complementary field to biomaterials that could potentially reduce the organ waiting list, as the longstanding goal of Tissue Engineering is to grow new organs. A good example of an aging tissue is the bone. Osteoporosis is the phenomenon of loss of bone density with age. The loss of bone density will make the bones weaker and more brittle, making them more prone to fracture. This disease is a chronic condition. The reasons behind the origin of osteoporosis is the balance between two different cells involved in the remodelling process of bone – osteoblasts, which are responsible of producing more bone, and osteoclasts, which resort bone. These cells act continuously as the bone is a living tissue and is continuously being remodelled/ refreshes. However, in osteoporosis there is an enhanced action of osteoclasts leading to a greater bone resorption than production, ultimately leading to a decrease in bone density. This phenomenon doesn’t happen only to elderly people but also to astronauts. These space travellers when they return from space usually present enhanced osteoporosis even though they might be young. This is because bones require mechanical loading (forces) to regulate the osteoblast-osteoclast activity, reacting to these external stimuli. In a space shuttle due to zero gravity, this mechanical loading does not exist, ultimately leading to a mismatch in osteoblast-osteoclast activity. Therefore, bones require acceleration to keep their natural process of remodelling. Note that in women osteoporosis can be enhanced by menopause. Implants, Transplants and Prosthesis  Implant is an object, or something inserted or embedded surgically in the body. This could be a stitch or a mess or even a heart valve. 2  Transplant is a tissue or organ moved from one body or body part to another. o Autograph – transplant from another site within the patient’s body. This process can be detrimental to the donor site and can be painful and require a long recovery. o Allograft/ Homograft – transplant from another human being. Usually there is a lack of donor availably, there is a risk of disease transmission and immunorejection. o Xenograft – transplant from another species. This is very rare as this leads to a huge immune response by the body. So, if a patient was to have a xenograft it would require to be on immunosuppressants for the rest of their life. This type of transplant has a high risk of disease transmission and rejection. One of the main downsides of all of these procedures is infection.  Prosthesis is a manmade device used inside the body to replace or augment a diseased or damaged part of the body. Examples of Medical Devices Implants: 1. Currently in terms of the replacement of tissues there is a huge amount of operations (~500.000 spinal cord operations alone). One of the most common operations is a total hip replacement, where it is used a medical device made of ceramic ball, which fits into the hip joint, and a metal femoral stem onto which the ceramic will sit on. These replacements are used when cartilages wear out. Note that cartilages wear out and cannot be replaced as cartilage is a complex tissue with very low vascularization (avascular), being therefore very difficult to regenerate. Therefore, often is removed/debrided away and a device is used to replace it. 2. Nowadays are also used Intraocular Lens. These devices are surgically implanted into the eye, by removing the lens in patients with cataracts when the lens starts clouding up. This an example of an implanted material, which is something that doesn’t elicit an immune response. This specific material is made of PMMA (Poly (methyl methacrylate)), which is a synthetic polymer used in various areas of the body such as for bone cement. Note that when we insert an inert material in the skin the body will scar when the tissue repairs, being rejected by the body. Therefore, an inert material doesn’t involve regeneration and will lead to scaring. This does not happen in the eye when we insert the intraocular lens due to the fact that there is no immune system (no blood supply), being immune privileged. 3. Use of artificial organs such as a pacemaker, which supports the heart to beat within more regular intervals. This device is inserted by a lead, going to the right atrium and into the right ventricle. The pacemaker itself it has multiple components, requiring therefore specific characteristics such as being anti-thrombotic. This shows that the different organs have different environments and components which ultimately lead to different demands to be fulfilled by the materials, requiring therefore different design criteria. Problems with current implants.  Mots of the manmade materials is that these materials don’t interact with the body, or adapt and change to it, remodelling. Therefore, there is no manmade material available that can completely match the biomechanical characteristics of living tissues. Nowadays, we can capture some elements of it but not all.  There is also no manmade material capable of repairing itself.  Manmade materials are not capable of adjusting their structure and properties due to changes in environment or mechanical loads that it encounters. It is possible to pre-program things like degradation, or that can alter pH or Temperature, but this is a very limited realm still. 3 Currently life expectancy is increasing, and we are outliving our body parts, and we need to be able to help our body regenerate diseased or damaged tissue. Health systems are struggling due to the pressure they are under being required novel materials that can help in fulfilling these needs. The Future entails that we need Regeneration of Tissues. Tissue Engineering as a field requires 3 different things: scaffolds, tissues, and cells. There are two aspects to growing tissues within biomaterials: we can get in situ tissue regeneration (Biomaterial instructs cells inside the body and replace cells as it degrades away, and all the biology of regeneration is provided by the body), or, alternatively, we can use tissue engineering scaffolds, combine it with cells and signals such as proteins and growth factors to then carry an implantation, containing the scaffold the information to regenerate the tissue. Note that in this context of tissue engineering scaffold provide the structural support for cell attachment. However, there are still a lot of limitations to what can be done with tissue engineering as:  Nobody can grow tissue outside the body to 100% perfection.  Engineered tissues can take too long to grow. The difficulty of this task is due to the complexity of Biology, which has a Hierarchical Structure well defined:  Molecules – Nucleic Acids, Amino acids, Sugars, Lipids, Minerals  Macromolecules – DNA, Polysaccharides, proteins, Bilipid Layer, HCA  Organelles – Nucleus, Mitochondria, Membrane, …  Cells – very complex unit of living organisms  Extracellular Matrix, Fibres  Tissue  Organs  Life form (mm-m) Bioactive Spectrum This shows the relative bioactivity or percentage of interfacial bone tissue as a function of the implementation time. This spectrum allows to divide the materials into 4 distinct categories:  Type 1 – Nearly inert – Somewhat permanent, they last for a long period of time, usually not degraded, body quickly encapsulates them. If made more porous we can reduce the encapsulation by the body allowing ingrowth.  Type 2 – Porous ingrowth – materials that have high porosity and allow the tissue to in grow forming a scaffold  Type 3 – Bioactive material – They stay for a prolonged amount of time, but they degrade away, allowing the body to repair. They are not encapsulated as they interact with the body, simulating a body response which is not scaring or fibrosis.  Type 4 – Resorbable material – Material that quickly degrades away, as the body can resort it. 4 Molecular and Cellular Biology: Overview The body is made of different structure, being organised in a series of different structures. Looking at the different scales, the nucleus contains all the information necessary to make up an organism, having all that information tightly packed. Cells Cells are the building blocks of all living organisms. There are 2 main types of cells: Prokaryotic and Eukaryotic. Prokaryotic Cells (‘Pro’ – before + ‘Karyotic’ – Nucleus) are cells that don’t have a nucleus as well as all membrane bound organelles. All components of the cell are dispersed within its inside, being only bound by a cell membrane and a cell wall. This type of cell is very common in bacteria, and they are vert small cells, smaller than eukaryotic ones. This is as there is no membrane bound organelles and DNA and proteins are all mixed together, the production of proteins (Transcription + Translation) are coupled much faster as the molecules don’t have to travel between compartments, leading to faster division. Thereby why bacteria go much faster than other organisms and cells. What’s the importance of Bacterial in biomaterial science? One of the biggest problems today are infection related deaths in hospital mostly due to the formation of biofilms. Therefore, we want to prevent the bacteria from adhering to the surface of materials and create this structure that will promote continuous bacterial growth leading to the progression of the infection. To stop their formation there are different ways of doing it: 1. Antifouling – create a surface of materials that prevents the biofilm formation. This very common in catheters and it can be done with different polymers, preventing adhesion. 2. Antibacterial – Use of antibiotics, which will either have a contact killing (just by touching the surface the bacteria will die), or there can be some sort of trigger release through enzymatic cleavage reactions Eukaryotic Cells are more interesting as they are present in higher order organisms. These have a nucleus and a lot of membrane bound organelles. They are also much bigger than prokaryotic cells. Since the DNA is inside the nucleus and proteins are produced outside, within the cytoplasm, there is 5 no coupling between transcription and translation, leading for the process to be much slower as will also be cell division. Cell Membrane This is a lipid bilayer composed of phospholipids, which are amphipathic molecules, modelled as a fluid mosaic (Singer and Nicholson 1972), which contains embedded trans-membrane proteins bound to sugars (glycoproteins) and cholesterol. This structure is semi-permeable allowing transport through it  Free diffusion – Hydrophobic, non-polar and small polar.  Protein Mediated Transport- larger polar and ions  Endocytosis -fluids and large particles). This structure has several functions such as:  Insulates and isolates cell internals from external elements – barrier  Encapsulates all functional components of the cell and the cytoplasm  Controls traffic of molecules in and out  Plays key role in signaling and signal recognition. Protein-mediated transport takes care of the larger polar molecules and ions, being there 3 main types:  Uniporter – Protein only allows one single molecule from one side of the cell, and it is regulated by either voltage, stress, or ligands. These use ATP and are mainly found in mitochondria and neurons.  Synporter – Protein allows two different molecules in the same direction. What happens is that, for instance, it is pumped one ion, and it is used ATP to pump it against the gradient. Eventually when we get a difference in concentration the transporter opens allowing the second molecule in – regulated by electrochemical gradients. Eg.: SGLT1  Antiporter – These proteins allow two different molecules to move in opposite directions. Similarly, to Synporters, they are regulated by electrochemical gradients. Eg.: Na+/Ca2+ exchanger. Endocytosis and Exocytosis is an extremely important mechanism of transporting substances inside of the cell for biomaterials specially in the realm of nano biomaterials. Endocytosis is usually receptor mediated, so there are ligands or small particles that connect to receptors on the surface of the cell (Eg.: Clathrin-mediated or Caveolae-mediated). These receptors, when a nanoparticle comes in contact with the surface of the cell, they will assemble and pull the membrane in to form a vesicle like structure, allowing the particle to enter the cell. The process is called Pinocytosis for liquids and Phagocytosis for larger particles (dust, cell debris, etc.). Exocytosis is the opposite mechanism to endocytosis, where the cell takes things into vesicles and empties them out to the extracellular environment. This is a way to get rid of waste or to release neurotransmitters and hormones. The most common event that happens after endocytosis is the fusion of the vesicle into a lysosome, which is an organelle bound compartment that has a lot of digestive enzymes. So, the early endosome 6 will lower the pH gradually going towards alate endosome stage, fusing, finally into a lysosome. The Lysosome will be responsible for breaking down all molecules (polyssacharides, proteins, lipids). Nanoparticles use endocytosis to go into the cell. Therefore, we try to deliver a chemotherapeutic agent, which need to reach the nucleus, the last thing we want is for the particles to end up in a lysosome, being degraded, and not completing its goal. Additionally, one possible downside of interaction of lysosomes with nanoparticles is that by their dissolution they might release toxic chemicals to the cytoplasm. Therefore, in the last couple of years the biomaterials field has found a lot of strategies, which allow molecules to escape the endosome, being able to deliver the content of the nanoparticles in the respective organelle desired. Endosomal Escape Strategies: a) Membrane Fusion – Very common if we use liposomes to deliver the drugs as the surface of a liposome is very similar to the membrane of the cell and of the vesicles, so it can very easily fuse with the membrane of the endosome and release its content onto the cytoplasm. b) Osmotic Rupture – Also known as the Proton Sponge Effect. These vesicles have proton pumps which pump protons to the inside of the cell, lowering the pH of its microenvironment. If there is a polymer capable of absorbing those protons, the pH will never lower, and these pumps will continue its action constitutively. Since for every proton that comes in an anion has to come in to compensate for the charge, the solute concentration will increase leading to a rush of water from the outside into the vesicle, eventually leading to rupture of the vesicle. c) Particle Swelling – Some particles, during the process of pH lowering carried by the proton pumps in the endosome will not absorb the proton, but will swell, breaking the endosome around it. d) Membrane Destabilization – The change in pH of the endosome will break down the polymeric content into enzymes which can interact with the membrane, disrupting it. Movement of substances in the cell is performed by the cytoskeleton. This is a structure composed of fibres that assemble and disassemble very quickly, in order bring things from one point to another or to move a cell from one place to another. The fibres provide the rails of transport within the cell. It also plays a vital role in cell adhesion and migration, and generation of contractile force in places such as muscle or for the closure of wounds. Two of the most important fibres are actin and tubulin Note that everything else in the cell that is not enclosed in the nucleus is in cytoplasm. The cytoplasm contains the cytosol, which is an aqueous fluid that is composed of water, dissolved ions, small molecules, and large water-soluble molecules such as proteins, and organelles. Normal cytosolic pH is 7, i.e., that if some other pH is required for cells to perform some catalytic function, they will be enclosed in membrane bound organelles. The organelles are membrane bound intracellular compartments, distinct and highly organized, which contain specific chemicals for carrying their assigned functions. They are suspended in the cytoplasm or anchored to the cytoplasm and only exist in eukaryotic cells. Organelles are structures such as the Rough Endoplasmic Reticulum, the Golgi Apparatus, the Ribosomes, Lysosomes, Mitochondria, etc. 7 The Mitochondria is the powerhouse of the cell, producing energy in the form of ATP which is the energy currency of the cell. Mitochondria produces energy via its two membranes. It is the only organelle with two membranes, which are needed to create a gradient of protons that, when go back into the matrix will allow the production of ATP. These structures contain their own DNA (circular), which is believed to be evidence of evolution, being proposed that mitochondria were actually prokaryotic cells that somehow were suffered endocytosis and survived. Nucleus  The nucleus is argued to be the most important organelle of the self. This structure is surrounded by an envelope, which is called the nuclear envelope. This is a lipid bilayer with large pores which allow molecules to travel back and forth from the cytosol, controlling this transport.  The nucleus contains the DNA which is tightly packed and wrapped around proteins (histones), which then coil into chromatin and further condense into chromosomes. One chromosome which is about 4-5 microns in length contains about 4 centimetres of DNA if uncoiled.  The nucleus contains 23 pairs of chromosomes, which carry all the genetic information of the person.  DNA is formed by 4 nucleotides: Adenine (A), Guanine (G), Cytosine (C) and Thymine (T). Table 1- Summary of Eukaryotic and Prokaryotic Cells Prokaryotic Cells Eukaryotic Cells Eukaryotic cell parts: 1. Plasma membrane 2. Extracellular matrix 3. Nucleus Prokaryotic cell parts: 4. Nuclear envelope 1. Plasma membrane 5. Nuclear pores 2. Cell wall 6. Nucleolus 3. Capsule 7. Endoplasmic reticulum (ER) 4. Ribosomes 8. Ribosomes 5. Cytoplasm 9. Golgi apparatus 6. Circular DNA 10. Cytoplasm 7. Nucleoid region 11. Mitochondria 8. Flagella 12. Microfilaments 9. Pili 13. Vacuole 14. Microtubules 15. Centrioles 16. Lysosomes 8 Key characteristics: Key characteristics: Larger (10-100 μm) Small (0.5-5 μm) Transcription & Translation separately Transcription & Translation coupled – – slower protein synthesis faster protein synthesis Separate nucleus and membrane- Faster cell division – binary fission bound organelles Slower cell division – mitosis The Central Dogma of Molecular Biology This is the basis of all Molecular Biology, and it describes the flow of information between 3 main types of molecules: DNA, RNA, and proteins. DNA is transcribed into RNA in the nucleus. RNA is ten in turn translated into proteins via ribosomes in the cytoplasm. How does this process actually occur inside the cells? The relaying of information is done via code, which is literally what DNA molecules have engraved inside them. Inside the molecule of DNA, the nucleotide bases are organised in particular sequences, and they are transcribed into RNA bases (Adenine (A), Guanine (G), Cytosine (C) and Uracil (U) – this is the equivalent to thymine in DNA). T  A; A  U; C  G; G  C The RNA that travels to the outside of the nucleus is translated into a protein via codons, which are three nucleotide sequences that encode for one amino acid. The relationship between the codon and the respective amino acid is described within the genetic code. This is universal as all known living organisms use the same genetic code, unambiguous, since codon codes for just one amino acid (or start or stop) and redundant as most amino acids are encoded by more than one codon. The redundancy of the genetic code is an important characteristic, being known as an evolutionary checkpoint as to minimise the effect of mutations in the protein produced. Note that all codons with U in the middle code for hydrophobic amino acids, while those with A in the middle encode for hydrophilic large amino acids. Note that the translation of the transcribed and processed RNA molecule (mature mRNA) is carried by the ribosome, which slides through each sequence and transfer RNA molecules which are responsible of detecting each codon and attaching the correspondent amino acid, catalysing the formation of the peptide bond and ultimately leading to the growth of the polypeptide chain. The ribosomes are not floating freely in the nucleus, they are attached to the Rough Endoplasmic Reticulum (RER). After the synthesis of the protein this can either 9 go to the RER to have a function or they can go to the Golgi Apparatus for post-transcription modifications (phosphorylation, glycosylation, etc.) and ultimately to vesicles and exocytosis from the cell as hormones or neurotransmitters. The production of proteins results from the expression of genes. This expression can be controlled at all series of different levels being there factors responsible for this. In fact, even though, all cells have the exact same genetic sequences (DNA) not all genes are expressed equally across all cells and expressed at once. Factors that affect gene expression:  Transcription Factors – alter start of transcription; modulate chromatin opening; turn genes on and off.  Spliceosomes – rearrangement of exons after splicing (removal of introns – non coding regions of the DNA).  Epigenetics – effects of environment (such as UV light) through mechanisms such as DNA methylation, histone modifications and non-coding RNA. Phenotype = Genotype + Environment + Disease + Molecular Mechanisms + Chance Note that the simplicity of the Central dogma of biology is ideal to explain all the mechanisms of how we go from DNA to proteins, but it is not enough to explain the many variations of gene expression that are possible. Ultimately, the phenotype of an organism is the mixture of all the factors that can influence the expression of genes and it’s what ultimately controls protein production. Life and Death of Cells  The life span of different cells vary greatly.  All cells are continuously growing, such that one fertilized egg can generate 1 trillion (10 12) cells! This means that controlled cell division is a key of a part of development of an organism. At the end of a life span of a cell a cell, the cell die, being the two most common mechanism through which this happens necrosis and apoptosis.  Necrosis is the result of a cell dying unexpectedly due to starvation, poisoning, after heart attach or stroke for example. These cells literally explode releasing all their content and attract the immune system, resulting in an inflammatory process.  Apoptosis is a programmed cell death due to DNA damage, external signalling from the environment or other cell, or viral infection. These effects activate mechanisms where the cells shrink and get into small vesicles, which and then be recycled or degraded the action of the immune system Tissues and Organs Cells specialise to perform functions. One single zygote differentiates into multiple cells, each one of them with different functions. Examples: 10 a) macrophages have microvilli for transport, as by increasing the surface of the cell membrane there can be a lot more transport through the membrane b) There are cells which have greater numbers of mitochondria due to the high demand of energy such as cardiomyocytes. c) Cells such as neurons require a greater Endoplasmic Reticulum due to the production of neurotransmitters. All these differences arise from stem cells. Stem cells are cells that are self- renewing, being able to keep dividing and have all the information available as they are undifferentiated. These cells can differentiate as a response to stimuli from the environment. Note that there are different types of stem cells:  Totipotent Stem Cells – cells that can differentiate into any other cell and placental cells (These cells are present in the early stages of development).  Pluripotent Stem Cells – cell that can differentiate into any cell but not placental ones.  Multipotent Stem Cells – cell that can give rise to a few cell types. (Hematopoietic stem cells for example).  Unipotent Stem Cells – cell that can origin one fully differentiated cell type. (Fibroblast - Progenitor for skin and tendon for example). Nowadays, research wants to understand how we can move from one state to the other, i.e., how can we go from multipotent stem cells to pluripotent stem cells. This is crucial as working with embryos gives rise to a series of ethical issues. Note that is already possible to go from multipotent to pluripotent. These cells are called iPSCs (induced Pluripotent Stem Cells). There is a very interesting type of multipotent stem cells Mesenchymal Stem Cells. This is as they are present in adults, being possible to take them from a patient and they can differentiate into a multitude of tissues. They are present in areas bone marrow, umbilical cord, and fat tissue. These characteristics make them of great interest for Biomaterials. These cells can be taken isolated, grown, put into a material and implant them back in the body. The interesting part is that the material composition (structural and physical properties) play a role in differentiating the cells into one type or another (such as Adipocyte cells - Skin, Osteocyte, Muscle Cells, Chondrocytes – cartilage, and Neurons). This characteristic allows us to use MSCs in Medicine 11 for things such as heart attack, brain stroke, spinal damage, Parkinson’s disease, osteoarthritis, and diabetes (beta cells). How are cells glued together to perform a function? Cells are glued together by the Extracellular Membrane or Tight Junctions. The Extracellular Membrane (ECM) is a non-cellular component in all tissues and organs, it provides scaffolding of cells. It is also the basis for all the biochemical and biomechanical cues that keep those cells of the tissue in homeostasis. Genetic abnormalities that affect the ECM can lead from mild (loose joints for example) to severe syndromes, such as aortic aneurysm. The ECM is made of water and mostly of fibrous proteins and what are called proteoglycans (proteins and sugars bound together). The proteins are unique for each typology of tissue, which define the mechanical properties of organs and directs organisation and physiological functions. The ECM interacts in the cell having an important role in signalling through the cell receptors:  Mediate cell anchorage  Trigger cellular signalling, regulating cellular pathways, either through cell surface receptors or mechanotransductors.  Involved in the growth and cell cycle by presenting the cell cycle. 12 The ECM is subject to constant remodelling due to the action of enzymes, which can come from the cells or outside the ECM. The action of enzymes can lead to the release of factors that are crucial for the cell homeostasis. Therefore, the ECM is not purely scaffolding. As said the ECM varies from tissue to tissue, being very specific to the body. For instances, things like hydroxyapatite and osteocalcin are only seen in bone and not other tissues. Also, while in cartilage the collagen fibres are surrounding the chondrocytes, collagen fibres in connective tissue or in the cornea are aligned. This defines mechanical properties. Therefore, if we want maximum tensile strength, we will put all the fibres aligned (parallel) with each other. In cartilage we care about compression so by putting fibres in an orthogonal disposition we get an increase in compressive force rather than tensile one. If we want to do tissue engineering, we need to keep in mind what are the mechanical force that each tissue experience and develop material that have the right mechanical properties (either soft or stiff for instance). Also, nowadays, we are evolving to have materials that have lifelike changes in time and space. This is where the field is at the moment and the importance of ECM for the development of the right materials for tissue regeneration. The ECM is highly dynamic, and it is constantly renovating itself, but it is also very sensitive to changes in the environment. Time for example can induce changes to the ECM (aging) as well as tumours. Tumours will lead to the orientations of the proteins and the growth of proteins to change completely. All these factors change the ECM composition, amounts and the cell topography. For instance, in a tumour there will be more fibrous proteins with greater cross-linking levels leading to a stiffer matrix with reduced elasticity when compared to healthy tissue. To mimic the ECM, we can use either synthetic or natural materials. In the skin we have the ECM with embedded cells in deeper layers and the outer layer is composed of epithelial cells which align everything together and connect to each other through cell junctions. These two structures are glue together by the basement membrane, which is also responsible for strengthening the integrity of the ECM. The epithelium is a continuous protective layer of cells, which are connected to each other through cell junctions. 13 Cell Junctions  Tight Junctions – seal the two neighbouring cell membranes together, acting as a wall within the organ, preventing molecules from leaking through the epithelium into the organ.  Anchoring Junctions – They connect cells with each other through the actin cytoskeleton (adherens junctions) or through the intermediate filaments (desmosomes). The cells can also be linked to the basement membrane through actin filaments (focal adhesions) or intermediate filaments (hemidesmosomes).  Gap Junctions - They are communicating junctions which link the cells and allow the transport of nutrients, molecules, ions, and electrical impulses in some cases. Epithelial Structures: Epithelial Structures vary strongly form organ to organ. The most thing about this type of cells is that they are polarised, which means that they have directionality, and they don’t look the same at the bottom and at the top. This makes sense as the top is exposed to different environments, while the bottom is connected to the ECM Example: Lung Epithelium (Interaction with air); Gut Epithelium (absorption of nutrients); Hair Cells (Mechanotransduction of sound). Cells of the Blood and Immune System The cells of the blood and immune system derive from (multipotent) haematopoietic stem cells. These cells are not part of a tissue they are circulating in the blood and in the lymphatic system. They don’t have an ECM, but they are of extreme relevance to the biomaterials field. This multipotent stem cell can differentiate into two main stem cells:  Common Myeloid Progenitor o It is related to the bone marrow, and it is associated with the formation of Red Blood Cells and Granulocytes). o Red Blood Cells are important as they are responsible for delivering oxygen in the body o Granulocytes patrol where the infections might arise, act quickly to fight the infection and alert lymphocytes (adaptive immune system). This is the common name to all the cells of the immune system that can kill through phagocytosis.  Common Lymphoid Progenitor o Adaptive immune system – T and B cells. o Natural Killer Cells 14 o This is related to the lymphatic system. o These cells act much slower and are more specific (cell- or antibody-mediated immunity). Immune response is very complex - Immunology There are different types of cells  Innate Immune system – second line of defence when the skin is breached and you’re exposed to the external world. If anything comes in, such as bacteria or virus, this group of cells will create a very rapid response, which lacks memory. The cells won’t remember if it was exposed to these pathogens or agents before. It will fire ‘from scratch’ every time. o NK Cells – Kill anything without self-markers o their surface. o Complement – Plasma proteins activated by microbes and trigger phagocytic cells. o Mast cells and basophils release molecules that create an inflammatory response. o Phagocytes (dendritic cells, macrophages, eosinophils, neutrophils) – engulf the microbes or cells that are infected, damaged, or dying.  Adaptative Immune System – This is the Third line of defence. If after the action of the innate immune system there is the need to fight pathogens or external agents, then the T and B cells will come in. These cells provide the body with long-lasting protection (having a memory). o B Cells – produce antibodies very specific to antigens of pathogens. o T Cells – express cell-surface receptors that recognise antigens. Biomaterials and the Immune System The most important thing in terms of biomaterials is that there is this one process that is one most undesired process carried by the immune system called the Foreign Body Reaction (FBR). This is an inflammatory process that can take the duration from weeks to months after a material is implanted. When we implant the material the tissue around it is hurt, generating an immune reaction, where the proteins bind to the surface of the material (Innate Immune System). Due to the big proportions of the material the macrophages (these cells drive the FBR) cannot phagocytose the material so they start fusing with each other around it leading to the formation of foreign body giant cells, which release all sorts of signals, which will attract fibroblasts. The fibroblasts will then be responsible of releasing collagen, producing a fibrotic response, and encapsulating the material, isolating it to minimise interactions to the body. This response ultimately compromises the function and integrity of the material. So, if it was supposed to deliver a drug it will no longer be able to do it. There are strategies to minimise the FBR.  Physical properties (size, shape, roughness, mechanical strength)  Biochemical properties: coating with ECM-derived proteins (collagen, CS, HA, etc.) – this trick the body into thinking that the material is collagen and there is no immune response required. 15  Controlled release of drugs to modulate inflammation – to keep the immune system in check and trick the body to not think that there is no inflammatory response needed.  New materials: zwitterionic polymers, modified alginates, polypeptide-based materials – poorly understood still. 16 Metals and Ceramics Materials are composed of atoms which are held together by strong interatomic forces. The strength of these interactions will determine whether a material is a liquid, gas or a solid. The physical properties of solids depend on the nature and strength of the interatomic bonds. This can lead to different types of solids. There are 3 main types of interatomic bonds:  Ionic Bonds o This involves an electron donor atom (usually metallic), which transfers one or more electrons to an electron acceptor (usually non-metallic). As a result of this electron transfer the atoms become a cation and an ion, respectively, being strongly attracted by electrostatic effect – Example: Table Salt NaCl. o Ionic solids are poor electrical conductors as the bound electrons are not available to serve as charge carriers.  Covalent Bonds o This occurs in elements that fall along a boundary between metals and non-metals (such as Carbon and Silica). o These elements tendency to donate and accept electrons is the same, sharing their valence electrons locally. o The localisation of the valence electrons also makes these materials very poor electrical conductors.  Metallic Bonds o This type of bond is characterised by a regular atomic arrangement with the electrons being able to travel freely (free electrons) around the metallic ions, forming an electron cloud. This leads to good heat and electrical conduction as well as plastic deformation due to the non-localise nature of the bonds, there can be movement within the atomic structure. o Note that conductivity requires the capacity of electrons to freely move, which is why it is very hard to get conductance in other materials. Note that the first two type of bonds are present in ceramics and glasses, while the second is reserved only for metals. There will also be other interactions aside from interatomic forces (non-covalent), such as hydrogen bonds, Van der Waals forces. These weak interactions are of role relevance for this topic as they don’t have a dominant role on the resultant material properties of ceramics, glasses, and metals. Atomic Structure and microstructure A bulk material (macro structure) is also organised in different organizational structures:  Microstructure – array of crystals or grains, which are separated by boundaries. Within each crystal grain there is an arrangement of atoms. Note that the key point about microstructures is that the size of the grain determines how the material behaves and within each grain there is a collection of atoms.  Atomic Structure – 3D array of atoms, which are arranged constituting a crystal. o Crystalline structure – the atoms or ions are arranged in an orderly repeating pattern in 3D. The smallest repeating unit of the structure is called the unit cell. o Amorphous structure – random orientation of atoms – present in glasses. 17 The microstructure defines behaviour of the material having plenty of different features: grain size, grain shape, grain orientation, grain boundaries, porosity, microcracks (The last two are big deal for ceramic based materials as this is where brittle lies, being capable of leading to failure of the material). Grains can move over each other slightly. Small grains move more easily. Smaller grains lead to stronger and more ductile materials. Metals Principal Characteristics  Ductile – exhibit plastic deformation under loads  Good conductors of heat and electricity as they have free electrons which can travel all across the metal leading to conduction Why do Metals bend? Looking at the unit cell on the atomic structure, i.e., the metallic bonds between the metallic atoms, there is a crystalline structure. However, there are irregularities within the crystal structure (called dislocations or slip lane), which ultimately leads to the ductility of the metal. When applied sheer stress to the material at the slip lane/ dislocation there will be a reorientation of the metallic bonding within the material, leading to the absorption of energy. This dislocation would move through the grain until it reaches the grain boundary, being fully removed to the grain boundary and forming a so-called unit step of slip. So, the removal of these dislocations and the energy required to do so is what determines and gives the metal its toughness and ductility.  Metals, in general, corrode and oxidise very easily – To solve this problem are developed alloys, which are a mixture or solution of different metals, which contribute with different properties to the material. For instance, stainless steel is an alloy composed of iron, chromium, and carbon. Alloys are a good way of improving the mechanical properties and corrosion resistance of a metal. Alloys are formed by a mixture of atoms (solid solutions), by dissolving one material into another. Ultimately, there are 2 types of Solid Solutions: 1. Substitutional Solid Solution – Both types of mixed atoms have approximately the same size and can just substitute into the metallic structure – Aluminium + Copper. 2. Interstitial Solid Solution – atoms that are substituted are much smaller and can fit within the crystalline structure of a metal – Iron + Carbon (Carbon is much smaller than Iron). Looking back at the microstructures of alloys, their elementary structure has 2+ types of atoms within the metallic structure, leading to the optimisation of the metal properties.  A solid solution leads to stopping dislocations as they are not able to move as easily through the material, ultimately requiring more force to move the slip lane to the grain boundary – improving mechanical properties. o A stainless spoon is very ductile and malleable but can be broken and just before breaking it heats up as a result of the energy put into it through work to move the dislocations, plastically deforming the material. This process is called Work Hardening. – Note that dislocations can move within grains but not across grain boundaries. Mechanical Properties of Metals: 18 Tensile Test – It can be easily made from the material to be tested a dumbbell/ dog bone shaped specimen, which can be gripped to either end so that is put under tension – pulling the material apart. This test allows us to plot the stress and strain of the material by measuring the force and extension of the material. During this test the material will undergo through different regions during the test: 1. Elastic Region – where the stress and strain are linear relatively to one another. 2. Plastic Region – where the material starts to fail this region ends up in the fracture of the material, such that it cannot withstand any more deformation. 3. Fracture These regions as well as the stress vs strain plot allow us to characterise the mechanical properties of the material through different basic properties:  Yield Strength – where the material leaves the elastic region and enters the plastic regime, i.e., the stress required to plastically deform the material.  Tensile Strength – the maximum stress that the material can undergo.  Fracture Point.  Young’s Modulus – this is a measure of stiffness described within the elastic regime of the plot, corresponding to the gradient of the linear curve that describes this area. o The highest the value of the Young’s Modulus the stiffer the material is. o The young’s modulus can be compared between different materials using a tensile test. It is expected that alloys (blend of different materials/ metals), the strength and stiffness will increase (i.e., higher young’s modulus).  Toughness – the material resistance to crack propagation. How tough and ductile a material can be measured as the area under the curve of the stress strain plot. o The smaller the area under the curve the less tough the material is, i.e., the more brittle it is. The opposite will lead to an increase in ductility. Fatigue is a property of metals which tells us that a material may fracture at service stresses below the yield stress. If the material was to be repeatedly subject to cyclic loads, the material could be subject to stress and ultimately leading to failure below the yield stress. This happens as repetitive loading can produce microcracks, which can lead to the weakening of the material. This is an important property in any biomedical material as materials in the body will be subject to repetitive cycles – relevant for things such as heart valves and prosthetic joints which are biomedical applications in regions of the body where these repetitive loads will be a certain factor. Microstructure and Properties:  The grain size has a large effect on the mechanical properties. The smaller the grain size, the greater the strength. This is described by the Hall-Petch relationship: 𝜎 = 𝜎 + 𝑘𝑑 Where d is the grain size, σy is the yield stress, m ~ 0.5 and k is a constant. As grain size decreases there is an increase in the mechanical properties of the material. So, smaller grains tend to make the material tougher because the grains can slide more over each other and also due to the fact that there will be more dislocations moving to the grain boundary. 19 Smaller grains have greater ratios of surface area to volume, which means a greater ratio of grain boundary dislocations. Metals in Medicine  Metals are used in a series of different applications in the body such as orthopaedics, oral and maxillofacial surgery, cardiovascular surgery, dentistry, etc.  Most commonly alloys are used to increasing the strength and reducing the fatigue of these implants. Some of the most common alloys used in medicine are: o Stainless Steel (316L = alloy of Fe, Cr, Ni, low C) – most commonly used. o Cobalt-based alloy (Co-Cr) – used for hip prosthesis. o Titanium and Ti-based alloys – Titanium is used due to its lightweight and great biocompatibility.  Example: Hip Prosthesis o Charnley Prosthesis – standard low friction type prosthesis due to the presence of a polymer cup made of Ultra-high molecular weight polyethylene (UHMWPE) with metal backing, which articulates with a cemented femoral component. This type of prosthesis is characterised by its reduced friction, which constitutes a benefit in this type of medical devices as they will undergo high loads very often, and therefore it will allow the materials to last for longer periods of time. If the material had high friction the material would degrade much faster as more stress would go through it. o Metals used For Total Joint Replacements: Note: The use of porous integration is something desired to promote osseointegration, to encourage bone growth. Ceramics Ceramics are solid inorganic compounds which have various combinations of ionic and covalent bonding. They have more complex crystal structures than metals. They are more brittle than metals as they result from a combination of ionic and covalent bonding. In ionic bonds, atoms are charged, so shearing will make ions repel each other. Covalent bonds are highly directional, so shearing will lead to their breakage. Principal characteristics  Hard – very high young’s modulus (steep curve)  Brittle – low ductility and toughness (small area under the curve).  Incombustible 20  Corrosion resistant  Poor conductors of heat and electricity  Light materials, weight much less than metals  These materials have high melting points; good resistance to heat, not deforming; good chemical stability at high temperatures (not oxidising). Ceramics Microstructure – aggregate of randomly oriented crystallites (small single crystals of less than 100um diameter) intimately bonded together to form a solid. Inspecting the structure under the microscope it is possible to visualize porosity (empty space between grains) and grain boundaries, which constitute a defect in the structure of the material and can therefore be a source of cracks, making the materials therefore brittle. Types of Ceramic Materials:  Oxide Ceramics: Silicates (also inorganic glasses and glass-ceramics), Calcium Phosphates, Al2O3, ZrO2 and TiO2.  Non-Oxide Ceramics: Carbides, Nitrides (and Carbon). How are Ceramics Processed? They are usually processed from a powder and then heated (sintering). Therefore, the solid powders can be fused together under ear and extreme temperatures in a furnace. There is no perfect fusion, which leads to the formation of defects such as porosity, which is unwanted. This defects in the microstructure can be the source of the cracks in the structure that can lead to brittleness and low toughness/ductility. Bioceramics in Medicine:  Used in dentistry as implants – restorative materials. Bioceramics can be used as a replace for skeletal hard connective tissue o Eg. Alumina, Lucite.  These materials have to be processed very carefully as we, for example, don’t want brittle teeth. Inorganic Glasses:  The atomic structure of glasses has a quite distinct orientation of atoms as they are not crystalline. These constitute amorphous structures. The structure of glass is similar to that of a liquid, i.e., that an orderly repeating arrangement of atoms is not maintained over long distances.  Glasses can be form readily from a series of oxide molecules such as SiO2, GeO2, B2O3 and P2O5. o Silicon-oxygen assemble themselves in a tetrahedron structure which can be extended in a very long network without border.  Glasses are favourable for several biomedical applications because they are more widely available, they are low cost and easy to fabricate. For instance, they don’t need to be processed in such high temperatures as for Bioceramics.  Inorganic glasses are also very interesting as they can be made to degrade (biodegradability). Glass-Ceramics Materials Glass-Ceramic materials are crystalline materials obtained by controlled crystallisation of a previously amorphous powder and glass. 21 This controlled crystallization process requires very specific concentrate compositions, and it is usually a two-stage heat treatment to get crystallization to occur. The result of this process is a glassy alloy (glass-ceramic) which has better thermomechanical properties than their parent glasses. Glasses and Glass-ceramics in Medicine:  Eyeglasses  Diagnostic instruments  Chemical ware  Thermometers, tissue culture flasks.  Restorative materials in dentistry  Implants – replace skeletal hard connective tissue – mineralized tissue rather than soft tissues. Ceramics, Glass and Glass Ceramics VS Metals Mechanical properties wise, if we compare the metals to ceramics, it can be seen that in the elastic region, the curve is much steeper for ceramics than for metals. They have a higher young’s modulus, being stiffer than metals. However, the ceramics are brittle not having a plastic region. They have an elastic region and then they fracture, being also characterised by a low area of the plot that is translated into a low fracture toughness when comparing to metals. This is due to the grain boundaries and porosity:  Grain boundaries can be a site for collection of impurities, glassy phases, stress concentration and microcracking source.  Porous can be open or close, can decrease the Young’s Modulus, strength hardness and thermal conductivity of the materials. o In fact, it can be seen on the picture on the side the relation between porosity and the bending strength of the material. As the porosity (%) increases the material loses its capacity to bend without fracturing. An interesting property of glasses is that as they become more flexible the thinner. Therefore, they allow us to produce materials such as fibre optic cables and bone filler materials to encourage mineralization, which are materials that require a great deal of flexibility to handle and process. Classification of Bioceramics:  Bioinert – no toxic response from the body on implantation. Usually results in fibrous encapsulation (scar tissue formation) as there is no such thing as a truly inert material. Thereby why biodegradability is such an important characteristic. o E.g.: Alumina, Zirconia. 22  Bioresorbable – undergoes degradation in the body. Dissolution produces harmless and can be secreted naturally. o E.g.: Tricalcium Phosphate (TCP).  Bioactive - Produces a biological response from the body that results in a bond between the material and the host tissue. Specially important to reduce fibrous encapsulation. o E.g.: Hydroxyapatite, bioactive glass. However, for several or most or many biomedical applications we want some degree of porosity to encourage tissue in growth as well as increase the degradation of the material. These are very difficult properties for ceramics, restricting the way they can be used. Usually, these Bioceramics are restricted to non-loading implant application as they are too brittle when made porous, or used for scaffolds for tissue engineering, where they are used in vitro and there is a degree of tissue growth within the material and the scaffold serves its purpose before it is implanted. Therefore, porosity is a key parameter that we wont to import into ceramics without leading to too much degradation. For instance, bone will grow within interconnecting pore channels near the surface maintaining its vascularity and long-term viability. 23 Polymers and Composites Polymers are a material widely used in surgery. Some examples are  Endoscope (Polyethylene -PE; Polyurethane - PU) – this endoscope would be desired to be flexible, processed to be smooth.  Sutures (Polypropylene – PP; Poly (glycolic acid) - PGA) – these sutures can be degradable (PGA) or non-degradable (PP). PGA is degradable as it contains ester bonds in its backbone and esters are prone to hydrolysis.  Angioplasty balloon catheters (Polyurethane – PU) – material is required to be flexible and capable of being inflated under high pressure so that it can resolve any structures in the blood vessel wall, anti-clotting. – Angioplasty is a procedure require to treat atherosclerosis, which is the build-up of fats, cholesterol, and other substances in and on the artery wall, making it harder for blood to flow.  Artificial Arteries (Polyethylene terephthalate -PET) – this polymer should be very flexible so that it can dilate and contract easily and to be anti-thrombotic as the blood, which contains, platelets is in contact with it and therefore we want to avoid a thrombotic event to happen, which could lead to a disastrous effect.  Artificial Hip – Cup can be made of UHMWPE and the bone cement can be made of Poly (methyl methacrylate)- PMMA.  Intraocular lenses (PMMA).  Brest Implants (Silicone) – Inorganic Polymer – very flexible and biocompatible. Generally, polymers are lightweight, flexible, and strong in tension. This is due to the chemical nature and the orientation of the bonds in the polymer. These are very long molecules that are randomly orientated and that are usually highly coiled and intertwined. They can therefore be stretched until its aligned the polymer chains, being very strong in tension. Polymers – a polymer is a material made of many units, where each individual unit is called a monomer. Note that the length of the polymer chain governs the properties of the material. There are two types of polymers:  Natural o E.g.: Collagen – This polymer is a very abundant polymer in the body. This polymer is composed of a single alpha chain, which self assembles to form a triple stranded helical rod. The alpha chain is composed of x and y amino acids, which are commonly proline and hydroxyproline.  Synthetic o Thermoplastic – these are long thin string like molecules that can easily entangle with themselves. The longer these polymers are the greater the degree of entanglement, which is what gives the polymer strength. Greater molecular weight means there will be a greater length of the polymer chain and more entanglements, which will hold the polymer together. These polymers can be easily manipulated after synthesis (vacuum forming, injection moulding). – E.g.: Teflon, Polypropylene. o Thermosetting – These are cross-linked polymer chains. Different chains are connected to each other via cross-links. The toughness and fracture properties of these materials depend on the cross-linking density. In fact, if there is a greater 24 degree of cross-linking the polymer will be much harder to deform, leading to stronger properties and more brittle materials. Note that both PMMA and Silicone can be thermoplastic or thermoset materials, depending on the application. They are thermoplastics when all intermolecular interactions are based on entanglement. If the polymer chains are covalently-bonded between themselves, then they are considered thermoset materials, as these bonds are irreversible. Methods of Polymer Synthesis:  Chain-grown (Addition) Polymers – this consists of building a chain on monomer at a time (‘stringing pearls into a necklace’). Polymers that are synthesised through this method are produced by free radical addition reactions from unsaturated monomers, i.e., monomers containing C+C bonds. o E.g.: polyethylene and poly(methyl methacrylate). Initiation Reaction - other initiators can be used to create the monomer free radical that will be the initiator of the polymer chain. Elongation Reaction of the polymer chain where new molecules will react with the monomer free radical ultimately leading to the formation of the polymer chain. The reaction ends when all resources are exhausted and there are no more monomers or when the radicals get quenched. The radicals can get quenched by two radicals coming together or due to exposure to the air where they can react with oxygen radicals.  Step-grown (Condensation) Polymers – these polymers are formed by reacting two monomers together in a reaction in which a small molecule is eliminated (water). Condensation polymers may be hydrolysed in the body as they have esters in the backbone of the polymer. This is not formed one monomer at a time. Different polymers react with each other eventually coming together to form a single polymer chain. Note that water is produced during this process, but water can also hydrolyse substances, so this is sometimes a limitation of this type of molecules. Therefore, it is needed to be removed the water as best as possible so that the equilibrium isn’t reached, and the water doesn’t hydrolyse the polymer allowing us to grow the polymer. If this doesn’t happen large molecular weights won’t be achieved as there will be a competition between hydrolysis and condensation reactions. Most natural polymers (polysaccharides, proteins) are made by condensation polymerization. 25 o E.g.: polyamides and polyesters (formed by an amine or an alcohol reacting with a carboxylic acid, being secreted a water molecule per bond formed) Polymer Properties:  Degree of polymerisation (N) is the number of monomer units in the polymer chain (molecule). N is generally 100-1000. Anything below 100 is no considered to be a polymer as there needs to be a critical polymer weight for the chains to be able to entangle to give it some strength. Therefore, below 100 they are called oligomers and they don’t have very good properties lying in the boundary between waxes and oils.  Molar Mass – the molecular weight (length of the chain) of a polymer molecule. Most polymers have a distribution of chain lengths, i.e., they are polydisperse. The molecular weight of the polymer is the molecular weight of the monomer times the degree of polymerisation. The polymers are polydisperse as when we grow a polymer through a growth reaction (addition or condensation) the polymers will not all grow at the same rate, being there a distribution. Therefore, the molecular weight in this context is only representative as an average as we cannot make all polymers to have the same value. Structural types of Polymers:  Homopolymer – a polymer consisting of one type of monomer unit.  Copolymer – a polymer consisting of two monomer types, which can be brought in different types. o Random (ABABBAAABABBAABABA) o Alternating (ABABABABA) – this will be a condensation polymer normally an acids can only react with amines. o Block (AAAABBBBAAAA) – These can be done by bonding two types of polymers or doing reactions of the two types of polymers separately, i.e., polymerise A and then B and then A again, and so on. Therefore, this type of polymer has regions of monomer rich domains (typical in biodegradable sutures). When we look at copolymers, we can start to impart some degree crystalline within its structure. Polymers are usually very amorphous, very flexible and there is no crystallinity. Copolymers, however, can have crystalline regions being characterised as semi-crystalline polymers. So, when we have monomer rich domains, where the same repeating unit is highly concentrated, it can associate with itself, forming some degree of order, called small crystalline domains. These are only small regions within the polymer. 26 Note that polymers don’t have grains or grain boundaries, but polymer chains can fold on each other because the polymer chain is quite simple or has some type of similarity. Note that the more complicated the polymer the harder it is to get these crystalline domains because the chains won’t associate, and won’t be able to pack with each other. Therefore, a simple block copolymer will be where we start to get some degree of crystallinity. Note that a functional group is more prone to obstruct crystalline regions formation the bigger and chunkiest it is, as it will be harder to pack it.  Amorphous – where there is no crystallinity and glass like behaviour.  Semi crystalline – contain amorphous and crystalline domains. Along one single polymer chains there can be amorphous parts and crystalline parts. o The crystalline regions act as pseudo-crosslink sites as they are more associated with each other. This improves the strength of these materials. These ‘cross-links’ resist flow, creep, and plastic deformation, therefore, being necessary to apply more strength for these domains to be broken apart. o The degree of crystallinity will also influence the mechanical properties (moduli, strength, etc.). More crystalline polymers are usually stiffer and stronger than their amorphous counter parts. o The degree of crystallinity will also influence gas permeability and water uptake. When we get crystallinity the diffusion behaviour across the polymer sample will also be changed. Mechanical Properties of Polymers: There is an elastic region, a yield point, and a very long plastic region. The materials are therefore very flexible (in tension). The graph of stress vs Strain on the side corresponds to a semicrystalline polymer with a relatively low Glass Temperature such as a high-density polyethylene. Thermoplastic Polymers: Thermoplastic polymers are long thin ‘string like’ molecules. As such like string they become entangled, and the entanglements are what hold the polymer together. Note that there are no physical knots. The fracture is essential the result of the completion of the disentanglement process of the polymer. Note that the fracture properties will depend on the molar mass. We need the molecular weight to be above a critical point otherwise there won’t be any entanglements. To get a good strength and toughness of the polymer we need its molar mass to be greater than 100,000. Note that thermoplastic polymers can be easily manipulated after synthesis:  Vacuum Forming – Take a polymer sample and dissolve I and then apply the polymer into the mould, and using suction (vacuum) the polymer will be hold to the mould desired conformation.  Compression Moulding – Mechanical type of moulding where the polymer is compressed to a specific mould conformation. In this situation is usually used a polymer powder, which is 27 compressed into a mould to hold it in place. It can also be applied some heat. When the mould is released, we yield our compressed polymer sample.  Injection Moulding – there is a hopper where we have our polymer sample usually in the form of powders or beads. The polymer goes through the machine, and it is melted by heat and it is then forced into the mould where it will adhere to the specific desired conformation.  Electrospinning – commonly used for scaffolds for tissue engineering. The polymer solution is placed into a nozzle, and it is applied a potential difference between the nozzle and the collection plate. This can be done with a polyester solution for instance dissolved in organic solvent. They are extruded by the nozzle (positively charged) onto a collection plate (negatively charged). This leads to the production of very thin strands. It produces a cotton wool like material, which is very similar to many tissues in our body such as the ECM. This can be collected on the plate, which can be rotated on a mandrill. Afterwards the fibre network can be retrieved and processed and possibly seeded with cells or cross-linked even to increase the rigidity of the scaffold, for example.  3D printing or Bioprinting – similar to some of the industrial processes but it gives us much more control and accuracy over the moulding of the material. It is possible to melt polymer samples, print them onto a stage where they can solidify to create a 3D geometry.  Extrusion – polymer is forced (extruded) through a die so it has a specific cross-sectional area. The polymer material in form of pellets is fed into an extruder through a hopper. Configuration and conformation The configuration of the polymer is essentially the chemical structure. To convert from one configuration to another it is necessary bond breakage. This corresponds to the idea of monomer units joining head-to-head or head to tail. Etc. The conformation of a polymer involves its 3D structure and only bond rotation is required to convert one conformation to another.  Chiral Centres: A carbon atom with 4 different groups attached is termed as a chiral centre (chirality). There are left-handed (L) and right-handed (D) forms of a chiral centre. Through different bond rotations we can get L and D chirality of the same individual configuration of atoms. Chiral centres are ubiquitous in some polymer samples used for biomedical materials, notably polyesters. These chiral centres have significant implications as they can change the way the material pack and the arrangement of the polymer chains within the centre. A polymer chain with chiral centre can polymerise in 3 different ways: o Atactic – The chiral centres (LH and RH) are linked randomly. o Syndiotactic – The chiral centres (LH and RH) are linked in an alternating fashion. o Isotactic – The chiral centres are linked together using one arrangement (All right- handed or all left handed). Note that D and L stand in Latin for dexter and laevus which translate to left and right. These nomenclatures allow us to distinguish two molecules that are different in terms of reflection, being a mirrored version of one another Properties of a Polymer The properties of a polymer depend on two different thermal characteristics:  Melting Temperature (Tm) 28 o Melting corresponds to the flow by chain movement. Therefore, the melting temperature corresponds to the energy required to encourage the chain movement by flow. o As Molecular weight decreases, Tm will decrease. o As branching increases Tm will decrease, if the molecular weight remains constant. o As number of free chain ends increases Tm will decrease. o As degree of chain rotation increases Tm will decrease. o As degree of cross-linking increases Tm will increase. o As crystallinity increases Tm will increase.  Glass Temperature (Tg) o The glass transition Temperature corresponds to the transition from a glass to a rubbery state. It coincides with the segmental motion of the polymer chains and a 4- 5-fold reduction in stiffness (Young’s Modulus) o Very flexible materials are characterised for having a very low glass transition temperature. o The glass transition temperature is very important in determining the characteristics of the polymer such as how rubbery it is. o The Diffusion processes increase on passing through Tg, i.e., that the higher the glass transition Temperature, the harder it is for gases to diffuse through the material o The Tg is affected by the same factors as the Tm in the same fashion. o Polymers behave like amorphous glass below the Tg.  Time Dependence of the Polymers o Polymers have time dependent mechanical properties. They have both elastic and viscous character which changes through time scale of the test. This behaviour of the material is known as “viscoelasticity”. They are elastic because after a strain due to the application of a stress the material is capable of recover. On the other hand, the material is viscous due to its capability to creep (extend) after the strain – ‘fading memory’ behaviour. o A viscoelastic polymer will see an increase in strain with time if stress is held constant. o Properties are most viscoelastic close to their Tg. o Polymers have elastic and viscous behaviour. That depends on the amount of stress that they undergo in terms of what strength we measure. o The viscoelastic response (combination of elastic and viscous behaviour where applied stress results in an instantaneous elastic strain followed by a viscous, time- dependent strain) is reduced by the presence of crystalline regions. o Note that viscoelasticity has very profound effects on cell behaviour, because a lot of tissues in the body are viscoelastic.  Factors affecting the mechanical properties: o Molecular Weight (Chain length) – chain entanglement o Degree of freedom for bond rotation and the degree of crystallinity (resistance to creep and flow) o Structural type (homo, alternating, block or random polymer) – amorphous vs semi- crystalline. o Cross-link density – tells us whether we have thermoset or thermoplastic o Temperature, depending on whether we are above or below the glass transition temperature or the melting temperature. o Time of loading in terms of viscoelasticity. 29 Polymers can also be classified into two main classes two main classes:  Bioinert – polymers are not completely inert as they can be fibrously encapsulated in the body.  Bioresorbable by dissolution/hydrolysis or by cells/enzymes (polymer is recognized by the cells and starts to be degraded by enzymatic methods). Polymer Composites – result from combining materials together to yield synergistic properties. Therefore, a composite is a material consisting of two or more phases. E.g.: glass fibre reinforced plastic – polymer matrix provides flexibility and stops the cracks from running through the brittle component, which is the glass. Bone is a polymer composite as it has hydroxyapatite crystals and collagen which are bond together, giving a strong composite material that is very strong and resists to load. Very hard to capture synthetically. Most natural structural biological properties are polymeric composites e.g. bone is a composite of collage (protein) and apatite (ceramic). By using two phases we can get, adjust, and manipulate the properties of the composite. Composites may be isotropic (have the same properties in all directions) or anisotropic (different properties in different directions, being there some degree of order). Lots of structures in the body are anisotropic. They have some degree of order, not being completely random. Mechanical properties of Polymer Composites: If we analyse the Young’s Modulus against the classic example of a polymer composite (HAPEX – hydroxyapatite reinforced polyethylene) It is possible to see that as the volume fraction within the material of hydroxyapatite is increased, the Young’s Modulus increases and the strain of failure decreases. So, increasing the ceramic content means that the stiffness of the material goes up and the toughness and flexibility goes down. There will be a sweet spot depending on what the material is going to be used for as it can be controlled the volume fraction and weight fraction of the polymer and ceramic components. It can be predicted somewhat the results and properties of the Young’s Modulus of these composites by using the rule of mixtures for continuous fibre composites: 𝑬𝑪 = 𝑬𝒇 𝑽𝒇 + 𝑬𝒎 𝑽𝒎 This is looking at the Young’s Modulus of the filler and the respective volume fraction and the Young’s Modulus of the matrix of the material and the respective volume fraction. This usually gives a good estimate. However, this rule doesn’t always hold. In polymer composites there is no bond between the polymer matrix and the glass fibre material, for example, which can be a source of crack and reduce the resistance and toughness of these materials. Therefore, it is not only necessary to do the right blend of materials to yield the right stiffness, but it 30 Bioresorbable Polymers Introduction to bioresorbable polymers Bioresorbable polymers are polymers which degrade safely within the body. Examples of such are sutures, temporary implants (PLG), drug delivery platforms. However, this type of polymers have some disadvantages such as the biocompatibility of the by-products of the polymer. Sometimes a polymer per say is not toxic, but when it breaks down it produces toxic agents that will have undesired interactions with the body. Another disadvantage is uncontrolled degradation. In fact, the material can be put in the body and some sudden event not predicted happens leading to a degradation of the material at a non-desired rate, leading to loss of mechanical properties, higher toxicity, and others. Therefore, there is a need for careful consideration of the design the function and the chemistry of the bioresorbable polymers, in order to have precise and relevant degradation with no toxic by- products. Key Definitions:  Biodegradation is a chemical process by which bonds are cleaved (i.e., hydrolysis of PLA, enzymatic degradation of poly(L-Lactide) by proteinase K. Note that biodegradation and dissolution are not the same. Dissolving results from the interaction of water with the polymer, which will lead to the polymer chains to be pulled apart. Biodegradation requires a cleavage/breakage of bonds.  Erosion – Process that results in the mass of loss of a material that may result in a change of size, shape, or mass. Note that erosion is a type of degradation, but relates more with the event of after bond cleavage the material degraded leaves the bulk. o Surface Erosion – restricted to the surface of a material (polyanhydrides). Material retains more a less the same shape but decreases in mass, being there a constant release of mass over time. o Bulk Erosion – throughout the sample, causing the whole material to degrade (PGA). Things are being degraded but they get entangled in the structure. There is no visible decrease in mass straight away. A decrease in mass will only be visible once the resulting species from bond cleavage are small enough that they offset the results of entanglement. Note that usually, even though it depends on the application, we want to keep degradation profile to surface erosion. For instance, when releasing a drug if we do it with a material that is subject to surface erosion that process will be linear. The same won’t happen if the material is subject to bulk erosion.  Resorption – process of eliminating the degradation products. Obviously, bond gets cleaved, and the mass decreases and the body needs to ‘clean up’. This can happen in multiple ways: the degradation products can be collected by the blood and then expelled from the body, or some degradation products might be able to partake in specific pathways of the body. For 32 instance, Poly(glycolic acid), when broken down into glycolic acid it can be used in the Krab’s Cycle. How can we control degradation/erosion? 1. Polymer Backbone Functionality  Most common functionalities are hydrolytically susceptible groups – they will degrade in the presence of water.  Different groups lead to different degradation kinetics.  Degradation rate can be affected by other factors (i.e., the presence of hydrophobic groups, changes in local pH, polymer architecture, etc.)  The most used for bioresorbable polymers are polyanhydrides, polycarbonates and polyesters as they have a good degradation time on a relevant time scale, applicable to medical processes. A) Polyanhydrides  These polymers are the least hydrolytically stable bond, degrading at very high rates. The hydrophobicity of the polymer can limit water penetration and significantly reduce the hydrolysis rate in the presence of water. This can be done by making the R group hydrophobic. o Example – Gliadel is a bioresorbable polymer with an R hydrophobic General structure of Polyanhydrides group, which contains a chemotherapy drug incorporated/ trapped in its structure. This polymer is used for the treatment of glioblastoma (brain tumour). These wafers are incorporated into the brain, and they degrade at lower rates than if they were just polyanhydrides.  These polymers have mostly surface erosion, mostly as these polymers are very tightly packed.  The release kinetics is defined by the shape of biomaterials – This statement will always be true whenever we have surface erosion. B) Polyesters  Most popular class of degradable polymers.  These polymers hydrolyse at the ester bond in the backbone. The hydrolysis is governed by the accessibility of water. o Adding methyl groups or hydro carbonated chains the polymer becomes more hydrophobic leading to a slower degradation. Poly(glycolic acid) and Poly(lactic acid) are a good example. Poly(lactic acid) degrades slower as it has one methyl group in its chain that contributes to its hydrophobicity and to make it harder for water to go in. 33  The degradation products of polyesters leads to carboxylic acid formation, lowering the pH. Since they their degradation is accelerated by acidic pH, there could be a point where we lose control over the degradation rate.  Most commonly used Poly esters o Poly(glycolic acid) or PGA. o Poly(lactic acid) or PLA. o Copolymers of these, or PLGA. o Polycaprolactone or PCL C) Poly-p-dioxanone  The presence of less ester groups by substituting them for an ether group, we will lead to less places of bond breakage within the chain, while maintaining the same level of hydrophobicity/ hydrophilicity.  This polymer is more flexible than PGA (PGA has a glass transition temperature of 37°C while PPD has a glass temperature of between -10 and 0°C).  Lower density of ester units leads to slower degradation rate.  This polymer has a semicrystalline structure (~55%). D) Polycaprolactone (PLC)  PCL has a high solubility and low melting temperature (59 - 64°C).  It is semi-crystalline with low Tg.  Not as used as PGA or PLA as it has lower mechanical properties, i.e., it cannot be used to produce structures such as sutures.  This polymer is used for drug delivery in implantable contraceptive device (Capronor). 2. Polymer Architecture There are different ways of controlling polymer architecture: A) Route of polymerisation  By controlling the route of polymerisation, we can yield different shapes.  Yield precise molecular weights.  Controlled synthesis of copolymers with various compositions and topologies.  By playing around with characteristics such as hydrophilicity and hydrophobicity we can generate structures such as vesicles, where the hydrophobic structures align themselves on the extremes of the membrane. Other structures possible are micelles which are composed of one single layer of the molecules, which align their hydrophobic part in the inside of the structure, being therefore good to encapsulate hydrophobic drugs. 34 B) Molecular Weight  An increase in molecular weight will lead to a slower degradation rate as an increase in molecular weight will allow polymers to be more tightly packed.  Therefore, a higher molecular weight will lead to the production of harder, more rigid materials, making it more difficult for enzymes and water to access the backbone of the structure. C) Glass Transition Temperature (Tg)  The glass transition temperature is the temperature at which a polymer goes from a glassy to a rubbery state. This corresponds to the temperature over which a polymer has enough thermal energy to have coordinated movement.  There will be more rapid degradation above Tg of the polymer as there is more energy for the polymer to move around.  For example, PGA has a Tg of about 37°C, while PLLA has a Tg around 66°C. This means that at room temperature (25°C) the chains of PLLA are much more packed than PGA. Being there less movement in the first polymer as the polymer is more tightly packed. Therefore, enzymes and water can act more efficiently and quickly in PGA, leading to its greater degradation rate when compared to PLLA. D) Crystallinity  A structure can be crystalline (orderly organized) or amorphous (randomly and loosely packed organization of polymer chains). Note that crystalline regions are areas where there is an interaction, usually non-covalent, such as hydrophobic, hydrogen bonds or ionic bonds for example.  If a polymer has a lot of chance to generate crystalline regions, when the polymer gets packed together it will be harder for water to enter its structure and hydrolyse bonds, leading to a slower degradation. Therefore, crystalline domains resist to infiltration of water and enzymes, leading to slower surface erosion, while amorphous domains tend to have much faster degradation processes.  One way of controlling the crystallinity of the polymer is by creating copolymers. If we have a polymer composed only by PLA or PGA the structures will be able to align themselves in a quite orderly fashion. However, if we have PLGA by mixing PLA and PGA, since they have different chemical structures, they won’t be able to be 35 so orderly packed, creating more amorphous region and ultimately leading to faster degradation rates. E) Stereoisomers (Enantiomers)  One carbon group connected to 4 different residues, we have a chiral centre, which can be D (clockwise) or L (anti-clockwise).  Lactic acid for example has chiral centres such as D-lactide, L-lactide and DL-lactide.  Therefore, we can get PLLA and PDLLA which will have different chiral centres. PLLA has only L-lactide packed in very orderly fashion and forming crystalline regions. However, PDLLA has a mixture of D and L lactide chiral centres leading to a more amorphous structure and greater difficulty in forming crystalline regions. Higher tensile strength, lower elongation, and slower PLLA Semicrystalline Tg = 66°C degradation. Lower tensile strength, higher elongation, and faster PDLLA Amorphous Tg= 55°C degradation.  Note that with PDLLA we have less level of control as we cannot dictate what position, and in which fashion the D and L lactides will be in the polymer chain. F) Relative

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