Biocompatibility and extracellular matrix

INERT = no reaction.
BIOCOMPABILITY = perform with appropriate host response
BIODEGRADABILITY = degradation without eliciting any undesirable effect
 Long-term implantable devices = better inert and passive
 TE = support cellular activity, facilitate signaling system

TE Material =
1. Synthetic degradable polymer
2. Natural biopolymer
3. Bioactive ceramics
4. Composites
5. Tissue derived ECM

Function of TE material
1. Provide the overall shape to the construct.
2. Facilitate the delivery of signals, both molecular and mechanical to
the cells.
3. Support the cells and optimize their function within the scaffold

TE materials =
1. Scaffold  Morphology and mechanical characteristic
 3D structure
 Attachment of cell
 Nutrients can circulate

2. Matrices  resembling ECM

 Gel (hydrogel), water-based viscous matrix
 Network of structural (cross linked)

Extracellular Matrix (ECM)  good for scaffold (commercially available)

 provides structural support
 network of non-living tissue
 Often generated by cells
Main components in ECM
1. Proteins
 Collagen
 Fibronectin
 Laminin
 Glycosaminoglycans
2. Growth factors

Protein in ECM
1. Structural protein = structural properties
2. Functional protein = ‘functional’ moieties with properties (from cell
adhesion & motility to promotion or inhibition of angiogenesis)

ECM scaffold preparation

1. Decellularization

2. 3D configuration
3. Sterilization

Biologic activities of ECM scaffolds

 Antimicrobial properties
 Angiogenic properties
 Chemotactic properties
ECM Biomechanical properties is tested by Ball-burst test
ECM degrade quickly after implantation if they are not chemically crosslinked
 Need study about effect degradation – biomedical properties
 crosslinked by chemicals (but promotes foreign body response)

Constructive remodeling
Natural polymers as scaffold material

Properties are similar to those of extracellular matrix

Natural source
o Plants o Algae (seaweed)
o Animals o Microorganisms

Advantages
1. Pseudoplastic behavior (shear thinning/thickening)

2. Gelation ability
3. Water binding capacity
4. Biodegradability
5. Presence of functional groups available for chemical and/or
enzymatic modification
6. Proteins interact favorably with cells through specific recognition
domains present in their structure
7. Different natural polymers can be combined into combined materials
that are similar to ECM

Disadvantages
1. Undesirable immune response due to the presence of impurities and
endotoxins (sourcedependent)
2. Their properties may differ from batch to batch during large-scale
isolation procedures due to the inability to accurately control the
processing techniques
Polysaccharides
o Maintenance and structural integrity (e.g. cellulose, chitin)
o Energy reserve storage (e.g. starch, glycogen)
o Biological protection and adhesion (e.g. gum exudates, extracellular
microbial polysaccharides)

1. Alginate  from sea algae

Dextran  bacterial-derived polysaccharide
o Cross-linking generates some sort of gel-like behavior
o strong complexes with polycations including, synthetic polymers,
proteins and polypeptides
o for TE of bone and cartilage\

2. Chitosan
o Similar structure to GAGs
o Biorenewable, Biodegradable, Bioadhesive, Biocompatible
o Easy processability
o Most widely used for bone, cartilage, and skin but also liver, etc
o Chemical versatility and the possibility to generatebstructures
with predictable pore sizes and degradationrates, intrinsic
antibacterial activity, and ability to bind to growth

3. Cellulose
o main component of plant cell walls
o the most abundant, renewable polymer resource available today
o Limited biodegradability and difficult processing
o Successful application in bone and cardiovascular TE
o Properties of cellulose can be highly altered with chemical
modification

Proteins and other polyamides

o Hormones (Signals from one body system to another, Insulin)
o Cellular motion
o Immune system (Protect against germs)
o Enzymes (Help chemical reactions)
o Muscle tissue
Proteins (polypeptides) are made of amino acids.
Differ by the sequence & number of amino acids

Collagen
Amino acid  tropocollagen triple helix  collagen fibrils  collagen fiber

o Amino acid sequence: Glycine (Gly), Proline (Pro)

o derivative amino acids:
Hydroxyproline (Hyp), derived from proline.
Hydroxylysine (Hyl), derived from lysine (Lys)

Occur frequently

Source
1. Animal tissues (porcine and calf skin, bovine tendon, rat tail, etc.)
2. Purified from animal tissues with enzyme treatment and salt/acid
extraction.
3. Concerns over transmission of infectious agents
4. Attempts have been made to find new and safer sources of collagen,
namely from marine sources (e.g. jellyfish collagen)
TE Application
o An ideal scaffold material
o Major component of the extracellular matrix
o It can be processed into a wide variety of structures and shapes
(sponges, fibres, films, 3D gels, fleeces).
o Collagen substrates can modify the morphology, migration and in
certain cases the differentiation of cells due to the presence of cell
adhesion sequences present on its structure
o Collagen is naturally degraded by matrix metalloproteinases,
specifically collagenase and serine proteases.
o These enzymes are secreted by neutrophils during the foreign body
reaction, allowing the collagen degradation to be controlled by the
cells present at the implantation site

Collagen I: skin, tendon, vascular, ligature, organs, bone

Collagen II: cartilage
Collagen III: reticulate (reticular fibers)
Collagen IV: forms bases of cell basement membrane
Collagen V: cells surfaces, hair and placenta

Polyoxoesters (polyhydroxyalkanoic acids)

Others: elastin, starch, cellulose, soybean, and other natural polymers

Synthetic biodegradable polymers

Requirement

Polymer synthesis
1. addition polymerizations
 Double bond containing monomers such as ethylene are
polymerized by action of radicals or ionic species
 Once a reactive species is formed, successive additions of large
numbers of monomers lead to the formation of a polymer chain
 Addition polymers are not considered degradable polymers

2. step-growth polymerizations
 Characterized by the stepwise reaction of the functional groups
of the reactants.
 The size of the polymer molecule increases relatively slowly as
dimers, trimers, tetramers, pentamers, etc. are formed.
 Often a condensation reaction takes place in which a small
molecule is liberated.
 Example: Lactic acid polymerized to polyester (by remove water)
 3. ring-opening polymerizations (ROP)
 The ring-like structure of cyclic monomers is opened

Important properties of polymers that need to be optimized:

 Melting temperature (and other characteristic temperatures)
 tensile strength
 elastic modulus or stiffness
 (surface) hydrophilicity

What determines the properties?

 the monomer used
 the way in which it has been polymerized
 the molecular weight of the obtained polymer
 molecular weight distribution

Copolymer = preparation of polymers from 2 or more types of monomers

1. random- & alternating- class

intermediate between properties of different parent homopolymers
2. block- & graft- class
possess properties of both homopolymers

Biodegradation
 In polymer degradation, chain scission occurs and oligomers,
monomers and other low molecular weight species are formed.
 Due to degradation, materials erodes (loss of material by monomers
and oligomers leaving the polymer mass)
Labile (adaptable) bonds
 For biodegradabe polymeric it is necessary that the main chain of the
macromolecule contains labile bonds
 scissioned by hydrolysis or oxidation reactions to yield (soluble)
compounds of lower molecular weight

Hydrolysis factor
 the nature of the chemical bond (especially type of bond)
 pH
 the copolymer composition
 the extend of water uptake

Anhydrides and ortho-ester bonds are the most reactive ones.

In water, anhydrides are more reactive than esters and amides

 Most biodegradable polyesters degrade by a bulk erosion.

Involves hydrolysis of main chain ester bonds.
 In surface erosion, rate of hydrolysis of labile bonds is faster
 Surface-eroding polymer good for drug delivery (constant release)

In vivo vs in votro degradation

 In vivo is faster than in vitro due to tissue response, mechanical
stress, and cellular activity (except hydrogel-like polymer)
 Tissue response depends on the shape of the implant
o a higher surface area (due to fragmentation or porosity) can
affect the tissue response
o sharp angular shapes can induce a higher response than more
rounded shapes
 The solubility of the oligomers may be increased in vivo due to the
presence of lipids, resulting in a faster mass loss
Design and fabrication of scaffolds

Requirement =
1. Mechanical strength and stiffness to substitute mechanical function.
Should be sufficient to support and transmit forces to the host tissue.
2. Maintain sufficient structural integrity during the in vitroand/or in
vivo growth and remodeling process
3. Surface properties provides for cell attachment and subsequent
migration, mass transfer of nutrients and metabolites, and provision
of sufficient space for development and remodeling

Porous

Compressive stiffness (E), yield strength (σ), relative density (100-P)

Fabrication =
1. Conventional techniques
 Porogen leaching
- undefined interconnectivity of pores
- organic solvents which must be fully removed to avoid any
possible damage to the cells seededon the scaffold.
- limited thickness

 Phase-separated scaffolds
- different morphologies and characteristics can be obtained
  Gas foaming
- Gas foaming + particulate leaching = improved interconnectivity

2. Textile technologies
 Classical nonwoven textiles
- fibers are produced by extruding a polymer
- Properties depend on extrusion
- Interest for neural engineering

 Electrospinning nanofibers
  Knitting and braiding
- Individual fibers, or multifilament yarns, are woven, knitted or
braided into patterns with variable pore sizes.
- Extremely complex fabric structures can result
- The large pores between the fibers can be filled with a secondary
scaffold, such as collagen gel or electrospun fibers

3. Solid free-form fabrication (SFF)

 Systems based on laser and UV light sources
- Stereolithography
- Selective laser sintering
- Solid ground curing

(a) (b)

 Three-dimensional printing (3DP)

- The solvent drying rate is an important variable
- Very rapid drying of the solvent tends to cause warping
- warping can be eliminated by solvent with a low vapor pressure
- combine solvents to minimize warping and improve bonding.

 Systems based on extrusion/direct writing

 Indirect SFF  use negative mold
- Two-photon lithography (nanoscale precision)
Liquid resin  3D print  hardened by laser beam
Bone Tissue Engineering

What is in bone: collageen, hydroxyapatite, water, cellen

Bone achitecture: cortical bone, trabecular bone, periosteum, bone marrow

Three cell types: osteoblasts, osteocyte, osteoclast

Osteomalacia = softening of the bones, deficiency of vitamin D or calcium

too much osteoid – no mineralization

PTH = activates osteoclast

Sclerostin = synthesized by osteocytes to stop osteoblast making osteoid

Osteoconduction =
Permits bone growth on its surface or down into pores, channels or pipes

Osteoinduction =
Primitive, undifferentiated and pluripotent cells are stimulated to develop
into the bone-forming cell lineage

Treatment of bone defect (trauma or tumor) =

1. Local bone transport
2. (Vascularised) fibula autograft
3. Allograft
4. Cage reconstructions

Autologous (same individual) =

(+) Biocompatible. All required cells, Vascularized
(-) 10-30% morbidity, Limited amount

Allograph (from human) =

Demineralized bone matrix, Cancellous bone, Cortical bone

Xenogenous (another species) =

Collagen-based

Synthetic =
Calcium phosphate, Calcium sulphate, Bioglass, Polymers, Porous metals
Disadvantage allo- and xenograft
 Host-immune response
 Risk of disease transmission
 Limited resources

Calcium phosphate resoption

 Tricalcium phosphate (TCP), fast
 Hydroxyapatite (HA), slow
Can be sintered or cemented

strontium ranelate (Sr2+)\

vascular system in bone evolve by osteoclast

Bone TE technique
Bisphosphonate =
- Encourages osteoclast to undergo apoptosis
- osteoblast is allowed to work more effectively
Sclerostin =
- Protein produced by the osteocyte
- Inhibits bone formation
- Interfering canonical Wnt/β-catenin signaling

Endochondral ossification
Cartilage Tissue Engineering
Subcondral bone have a barrier for mineralization of the cartilage
 Treatment methods for cartilage lesions:
 Osteochondral transplantation
- Periosteal flap transplant

 Autologous Chondrocyte Transplantation

 tissue-engineered cartilage
  use of scaffold

Chondrocytes (cartilage) = low turn-over

Biology

Tissue engineering tools

Tissue printing
o cell and biogel together
- poor gelling control
- gelling inhomogeneity
- variable physical properties
 o hydrogel in the bottom then cell and hydrogel per layer
- better gelling control
- better homogeneity
- reproducible gel properties

VEGF (Vascular endothelial growth factor) = vascularization

BMP (bone morphogenetic proteins) = rule stem cell fate
HMW (high molecular weight) fibrin gel = as scaffold

Successful tissue engineering requires

• control of nutrient supply
• control of scaffold properties
• control of cellular differentiation
• account for dynamic interaction

Cell energy source = Glucose, Amino acids, Vitamines, Minerals

Biopolymers:
o nucleic acids (DNA / RNA - polynucleotides)
o proteins (polypeptides)
o carbohydrates (polysaccharides / “sugars”)
Type of signalling

Central cell signaling paradigm =

1. Signal initiation :
an extracellular ligand binds to a receptor on cell surface; ligand binding
changes activity of receptor, thus generating “a signal”.
- Diffusible molecules (growth factors, hormones etc.)
- Extracellular matrix proteins (Collagens, proteoglycan (PG) etc.)
- Membrane-bound ligands (Integrins, “structures”, etc)

2. Signal transduction :
activated receptor triggers signal transduction cascade in which intracellular
proteins are activated, ultimately leading to activation of a so-called
transcription factor (TF) in nucleus.
 3. Gene activation :
TF binds to regulatory sequences in target genes (i.e. promoters, enhancer
etc), resulting in gene transcription (i.e. gene activating), subsequent protein
synthesis (i.e. gene product) and ultimately cellular response (e.g. altered
physiology).

Stem cells =
o the ability to make identical copies of themselves (selfrenewal)
o the ability to form other cell types of the body (differentiation)

Morphogenesis =
Biological process that causes an organism to develop its shape

Challenge in TE
polymerase chain reaction (PCR) =
process used in molecular biology to amplify a single copy or a few copies of a
piece of DNA across several orders of magnitude, generating thousands to
millions of copies of a particular DNA sequence.

Blotting = transferring proteins, DNA or RNA, onto a carrier

Cartilage BIopsy
 cells must be dissociated from each other and from surrounding ECM
 cell release from ECM normally starts with mechanical disruption
 followed by enzymatic digestion / cell straine
Antibody-driven separation =
1. Magnetic Activated Cell Sorting (MACS, clinical use)
2. Fluorescence Activated Cell Sorting (FACS, diagnostics)
3. Cell ‘panning’ (works if antibody-coated surface has low binding
capacity for unwanted cells)

Mammalian cell culture

Usual cell growth conditions: ~37°C, 5% CO 2, ~20% O2

growth media commonly vary in:

• pH, [glucose]
• growth factors + [GF]
• nutrients + [nutrient]
• GFs, often derived from animal blood (FCS*)
Media Types =
1. Simple media
- balanced / physiological salt solutions
- usually supplemented with energy (Glc) substrates and protein (FCS)
- not supporting prolonged survival
2. Complex media
- simple media +
- amino acids, vitamines, trace elements, supplements, (FCS)
3. Chemically defined media
- essentially:= serum-free Complex media

Medium Constituent

Medium preparation
Growth factor

Selective adhesion in vitro