In Vitro Tests for Cytotoxicity: ISO 10993-5

The element may stay local – or be transported to other tissues (extravasation)

1) alter the local biological environment causing a general molecular, organelle, cellular or tissue/organ dysfunction
2) interact with a particular endogenous target molecule such as a protein, membrane component or DNA.

-
50%

80% +
IN VIVO ASSESSMENT OF TISSUE COMPATIBILITY

The material manufacture

Additives, contaminants, residues Affect the overall biological responses to a biological device
Leachable
Degradation products

Selection of tests based on intended use:

structure & function at the implantation site

Impurities, monomers,…
Inflammation / Irritation tests

High need for pharmaceuticals w/ topical application, inhaled/ingested

Fibroblasts
(epidermis)

İnitiate paracrine
response → triggers
In vivo - Draize test – ethical issues!! cytokine release from
the second layer
(dermis)

In vitro – coculture systems

Measured! Degree of
response is determined
Systemic toxicity: Acute / subacute and subchronic toxicity

Systemic toxicity: in organs/tissues away from the application site. Level is measured by fever

*Acute: 24 h
*Subacute (repeated dose): 12 – 28 days
*Subchronic: 90 days
*chronic: at least 10% of the test animal
Applications of Biomaterials

✓ Cardiovascular medical devices

- heart valves
- vascular grafts and stents
✓ Artificial cells
✓ Renal Replacement Therapy
✓ Plasmapheresis
✓ Orthopedic applications
✓ Dental implantations
✓ Adhesives & sealants
✓ Ophthalmologic applications
✓ Bioelectrolytes
✓ Cochlear prosthesis
✓ Biosensors
✓ Skin substituents & sutures
✓ Diagnostic applications
MECHANICAL AND TISSUE VALVE REPLACEMENT DEVICES

heart valves open and close with each cardiac cycle,

once per second → approximately 40 million times/ year

and 3 billion times in a 75-year lifetime.

Disorders of heart valve → stenosis (narrowing- reduces flow)

→ reverse flow across the valve

E.g. infective endocarditis (infection of a heart valve)

- may cause destruction of the infected valve
 MECHANICAL AND TISSUE VALVE REPLACEMENT DEVICES

cardiac valvular substitutes: - mechanical valves

- biological tissue valves

Tissue heart valve replacement devices. (A) Hancock porcine valve.

(B) Carpentier-Edwards bovine pericardial valve.

Mechanical prosthetic heart valves. (A). Starr-Edwards caged-ball valve. (B) Bjork- Derived from Pig valve or bovine pericardium
Shiley tilting disk valve. (C) St. Jude Medical bileaflet tilting disk heart valve Treated with glutheraldehyde
- preserves the tissue and decreases its
metallic cage (cobalt-chrome two carbon immunological reactivity, and kills the cells
or titanium alloy) hemidisks in a within the valve tissue
carbon housing - Valves can not reproduce themselves (no
lifelong anticoagulation to Pyrolytic carbon: viable cell left)
reduce the risk of high strength
thrombosis Sewing ring covered with
fatigue & wear resistance
Dacron or Teflon
Biocompatible & thromboresistence
Disks respond to changes in pressure and blood
3rd option: human cadaveric aortic or pulmonary valves
flow within the chambers of the heart.
TRANSCATHETER VALVE REPLACEMENT

The surgically implanted bioprosthetic and mechanical valves may not be used in some patients due to comorbidities

Less invasive approach : - Percutaneous balloon valvuloplasty – less efficient

- New catheter-based interventional techniques

Balloon: stainless steel, platinumiridium or

other alloys
self-expandable: nickel-titanium alloys
(e.g., Nitinol)

Percutaneous valve replacement technology. (A) The Edwards-SAPIEN™

balloon-expandable aortic valve replacement designed for percutaneous
implantation, constructed from bovine pericardium attached to a stainless
steel stent. (B) Corevalve™ aortic bioprosthesis, constructed of bovine
pericardium attached to a self-expanding nickel-titanium alloy (nitinol) stent.
VASCULAR GRAFTS AND STENTS

decreasing rate of death from cardiovascular disease; (1995-2005)

- prevention of atherosclerosis through changes in lifestyle, (reduced cigarette smoking, diet -- cholesterol and saturated
animal fats)
- improved methods of treatment of myocardial infarction and other complications of atherosclerosis-related disease
(such as bypass graft surgery)
- prevention of recurrence in patients -previously suffered serious atherosclerosis-related clinical events

clinically-important vascular disease states:

- Narrowing (stenosis) or complete obstruction of vessel lumina
- Weakening of vessel walls

**** atherosclerosis : a slowly progressive disease in which

asymmetric focal thickenings
Stents: restore the blood flow
act as a scaffold to support the vessel

deflated balloon to stenosis site (angioplasty)

Inflated

Stent (self-expanding metal mash like tube)

Reclosing prevention

Short-term failure : closure w/in hours (due to

elastic recoil of vessel, acute thrombosis at
the site)
Long-term failure: fibrous tissue formation at
the site due to excessive smooth muscle cell
proliferation
Balloons: PVC and PE (early)
nylon or polyethylene teraphthalate
stents: expandable 316L stainless steel / nitinol
fabric – larger arteries
 Stent placing – damage to endothelial lining
-- accumulation of platelets, fibrin.

Stent wires – embedded in endothelium lined fibrosis

Smooth muscle cells

??
Collagen

Thicken more & GF, chemotactic factors,

inflammatory factors..

smooth muscle cells ,

ECM , narrowing the lumen and resulting in
restenosis.

Biomaterial development: 1) bare metal stents (BMS)

2) polymer coated drug eluting stents (DES) – reduce restenosis ; polymer matrix (mixture of
poly-n-butyl methacrylate and polyethylene-vinyl acetate (EVA) or a gelatin-chondroitin sulfate coacervate film) ; drug
embedded w/in; diffusion / degradation of polymer
****increased risk of late (>1 year post-implantation) stent thrombosis causing myocardial infarction and/or sudden
death in patients who have DES ; pros & cons
3) resorbable/biodegradable stents (RBS) – clinical trial (control of degradation to limit recoil)
Vascular grafts: bypass a vessel / replace a segment of vessel

Requirements: 1) resistant to thrombosis

2) resistant to smooth muscle cell thickening, fatigue
3) similar mechanical strength to the normal site
4) suturability
A) Dacron aortic interposition graft (Terumo). (B) Dacron aorto-
iliac graft (Terumo). (C) ePTFE small vessel grafts
polyethylene terephthalate (Dacron®) ---- larger vessel applications
expanded polytetrafluoroethylene (ePTFE®) ---- small

can be made porous to enhance healing but impregnated with;

- gelatin & albumin --- clotting, reduce blood loss through pores, stimulate tissue ingrowth
- antibiotics

Porous grafts --- not impregnated -> preclotted with the patient’s own blood before implantation, minimize hemorrhage
through the graft pores.
 Implantation of graft – inner layer : plasma proteins (fibrinogen)
platelet – fibrin aggregate (not
covered with endothelium) -- pseudointima

** If covered with endothelial cells -- neointima (simulating normal)

✓ Healing with neointima is generally incomplete over its internal

surface; choices are
1) overgrowth from the host vessel across other sites
2) tissue ingrowth through sufficiently large sites (generally not
happen)
3) deposition of endothelial cells and/or stem cells from the
circulating blood

Surrounding of graft → also covered w/ connective tissue (collagen,

fibroblasts…)
That capsule extends to undisturbed body tissues

Graft interstices may also be filled w/ fibrin & cells & ECM
Stent grafts : aneurysm
Stent : stainless steel, cobalt chromium alloy
or nickel alloys (Nitinol)

Graft: polyester or polytetrafluoroethylene

PACEMAKERS
sinoatrial (SA) node (heart’s natural pacemaker)
depolarization of the cardiac myocytes
reach to atrioventricular (AV) node
impulse passes to the left&right ventricular
myocardium – depolarization and contraction
1) Pacemakers
2) Ablation of the abnormal pathway
Cardiac arrhythmias -> problem with impulse initiation
or with impulse conduction (blocks/re-entry)
May result in ventricular fibrillation (fatal).
Pacemakers: initiate contraction

(1) a pulse generator : initiate the electric stimulus and to sense cardiac electrical activity
(2) conductors leading from the pulse generator to the heart
(3) tissue interface between electrode and myocardial cells ****

nonexcitable fibrous tissue → strength of the threshold pacing stimulus required to initiate myocyte
depolarization

Strategies: - corticosteroids release

- lead design (fixation -> electrode grasp the surface of endocardial surface
-> electrodes with porous metal surfaces ; induce ingrowth

if pulse output is not set sufficiently high → loss of pacing (fatal)

maintaining output at such high levels once thresholds have stabilized greatly shortens battery life !
Ventricular Assist Devices

LVAD: replace ventricular function for extended periods

Pulsatile-flow (early devices)

inflow cannula -> left ventricle

outflow cannula -> aorta
The pump : implanted into the peritoneal cavity /
remain extracorporeal (inflow and outflow
cannulae each traversing the skin)

continuous-flow

inflow cannula -> left ventricle

outflow cannula -> aorta
The pump : smaller
continuous flow (augmentation of cardiac input)
Left Atrial Appendage Occlusion Devices

Atrial fibrillation: most common arrhythmia

Unordered contraction initiated by the SA node. So, upper

chambers fibrillate – irregular flow in the chamber

thrombosis may occur due to these flow abnormalities

at the atrial appendage

Anticoagulant usage: - varying metabolism of drug

- interaction with other drugs
- frequent blood work (monitoring)
- poor patient compliance
- life-threatening bleeding

Clog the appendage with parachute-shaped device

Cage: Nitinol
Membrane: polytetrafluoroethylene
Artificial Cells

1) can contain the same biological material as biological cells +

Drugs, enzymes can be embedded in

2) Selective permeability (size, lipoidity, affinity mechanisms)

,by choosing the correct membrane material – no cross-> allow
larger molecules_proteins_cross

3) Surface properties may vary;

- incorporation of negative/positive charge
- incorporation of albumin to increase blood compatibility
- incorporation of antigens to bind antibodies or antibodies to bind
antigen
- incorporation of polysaccharide such as heparin or polyethylene
glycol (PEG) to increase compatibility or retention time in circulation

4) Macron – nano range (ex: osteoclast size)

Hemoperfusion
5) Can be ultrathin but strong – surface area/volume blood substitutes
ex: 10 ml of 20 um artificial cells = surface area 2500 cm2 enzyme therapy
same as artificial kidney machine. molecules can potentially nanomedicine
move across 10 ml of 20 μm diameter artificial cells 100 times faster regenerative medicine
than across the artificial kidney machine
Artificial Cells in Hemoperfusion

• Removing toxins from blood

• large surface-to-volume ratio & ultrathin membrane →

bioadsorbent containing artificial cells more effective
than hemolysis

• Ex: suicidal patients; drug overdose

- encapsulating activated charcoal
- adsorbs it thus prevent from absorption from GI
Nanobiotechnology

1st generation:
Crosslinking Hg into polyhemoglobin
membrane (polyHb)

Replace blood lost in surgery;

-Keep the blood hemoglobin at an acceptable
level
Also allows lowering the
-no blood group
systemic levels of
-free from risk factors (HIV, hepatitis, bacteria..)
tyrosine needed for the
growth of melanoma

In tumors; microcirculation is different – reduction in

perfusion by oxygen carrying red blood cells = low
oxygen tension in tissues. Radiation
therapy&chemotherapty works better with oxygen
tension is higher
Hemodialysis

Waste (ΔC)
counter-current

solute transfer across a semi-permeable membrane (MW cutoff : 5000, surface area 0.5-2.1 m2)

Every 48h, for 6h

Membranes: regenerated cellulose (biocompatibility issue – complement activation)

Tubing: polyvinyl chloride, polyurethane
Improvement: remove the OH groups (modified cellulose)
- the area of these materials, and the
exposure time, is much lower than for
Synthetic membranes: polysulfone / polyamide the functional membrane surface
more biocompatible than regenerated cellulose
advantageous - range of pore sizes
Therapeutic apheresis

Apheresis: fraction of the blood (platelets, plasma, red blood cells, leukocytes) is removed, and the remaining blood is
returned to the donor

Donor apheresis: volunteers give blood fractions for the treatment of others

Therapeutic apheresis: blood fractions are selectively removed to achieve a therapeutic result
- abnormal blood proteins or cells are present in the bloodstream, and these proteins
or cells are implicated in the condition’s progression

leukemia, multiple myeloma, non-Hodgkins lymphoma

Plasmapheresis, the plasma is separated and either replaced (plasma exchange) or treated prior to recombination with the
blood (plasma treatment).

Centrifuge (batch/continuous)
Membrane filter (pore size imp)
 Plasma separation Plasma exchange Plasma treatment

Ex: by sorption
Effective; removal toxins..
Pb Pp Disadv: beneficial proteins (albumins,
Sorbent column is used.
immunoglobulins, clotting factors) will
Plasma is perfused from this column
be removed as well.
** targeted solutes can be removed
then can be recombined with
-Limits the amount of plasma to be
cell fraction and returned to the
removed
patient
-Substituation fluids – allergy/viral inf

Plasma exchange would usually be

used when the specific pathogenic
factors in the plasma are unknown or
where a specific technique to remove
these factors is not available

Pb>Pp → Mw<cutoff ; water & solutes

→ Plasma no cells

P: pressure
Orthopedic Applications

❑ Fracture fixation devices, Joint replacement, Dynamic stabilization devices

❑ metals, polymers, and ceramics

high strength
ductility
toughness
hardness
biocompatibility necessary for most loadbearing roles

Requirements
✓ material must not adversely affect its biological environment
✓ material must not be adversely affected by the surrounding host tissues and fluids

Calcified tissue: Collagen + other ECM proteins + HaP (Ca10(PO4)6(OH)2)

inter- and intrafibrillarly within the collagen

intervertebral disc replacement technology
• replace spine fusion UHMWPE sliding core two metal endplates with multiple teeth-
• Eliminate pain like projections for fixation to the vertebral endplates
• Restore structure & mobility

Early disks: cobalt alloy spheres (no fixation!)

Then: stainless steel metal spheres
Polymers did not work
titanium or cobalt–chromium cup
Cement polymethylmethacrylate (PMMA) or press fit into
place

Ceramic head

titanium or cobalt–chromium alloy femoral stem

>80 years of age, implants are cemented into place with PMMA

there are choices of surface roughness, coatings, geometry,

material composition
Fixation of the implant to the tissue is needed, many ways:

1- Morphological Fixation: cementing the device in the tissue

or by press fitting into the defect (Al2O3 implants)

2- Biological Fixation: Have to be porous so that it will be

fixed by bone ingrowth (Al2O3 or HAP coat on porous
implants)

3- Bioactive Fixation: Attachment via chemical bond (glass

ceramics and HAP)

4- Fixation of Resorbable Implants: slowly degrade and

replace with newly bone (CaP)

osteoconductive and osteoinductive growth factors such

as transforming growth factor beta (TGF beta) are being
developed for use as osteogenic surface coating
treatments to enhance orthopedic implant fixation
Tissue Adhesives

Bonding : primary _
✓ interfacial bond; biomaterial & host tissue If 2 solids → interaction energy
secondary _
✓ Bonding and/or space filling (bone cements)
Ex: tissue: calcified
tissue
Primary bond formation → resistance to tensile forc
fluid: COOH
space filling (bone cements) group
Sealing (moisture, air, biological fluid.. prevention)

Hard tissue / soft tissue adhesives 2 smooth surface – microscopically; contact “mountain peaks”

- time scale (days – years) Too small,

Can not understand the forces

Why adhesives are liquid – solidify ??

Ex: tooth enamel surface and an orthodontic bracket

fluid adhesive in between

- Initially wet both surface (low contact angle)
- conditioning with an etchant for porosity
achieving adhesive solidification (settling)

1) Phase transformation upon cooling (*necrosis); not applicable with biological tissues, 100C required)
2) Solvent evaporation
3) Polymerization of monomers (most widely used)
4) Acid-base rxn (specific ; polycarboxylate and glass–ionomer cement

These interfacial bonds can be analyzed into micro/nanostructural contributions

- micromechanical bonds (solidified adhesive extensions turns into porosity/roughness)

air voids; contaminants; and weak boundary layers disrupt these contributions – avoid by surface pretreatments
Hard Tissue Adhesives
PMMA; MMA, can be manipulated and molded

Initiator, mixed with MMA monomer → activated to forma free radicals

- heating above 60C !
- adding activator !
- propagation stage
- termination stage

In the bone – restricted place, T goes up (for 3 min, in vivo) sometimes

even higher T (90C)

Cross-section of femur and interlocking bone volatilize unreacted monomer (boiling point of MMA is 101°C).
cement showing good adaptation of the
cement to the prosthesis (now removed), and
This creates voids that can later lead to mechanical failure.
a zone of interaction with the inner surface
of the compact bone.
Rheological Factors
material at the early dough stage is forcibly injected into the femoral
space around the metallic femoral prosthesis.

viscosity decreases as shear rate is increased

Upon polymerization – shrinkage → porosity is achieved

Medical Biosensors

sensors used to transmit information into or out of the body

❑ Surface or transcutaneous electrodes- events occurs in body (monitoring or recording electrodes)

❑ stimulator electrodes - If electrodes influence specific processes that occur in the body
voltage or current waveforms are transmitted – to directly control an event
- indirect influence on the target area

Biosensor: a sensor that uses biological molecules, tissues, organisms or principles to measure chemical or biochemical
concentrations.

Physical parameters of biosensing: pressure, volume, flow, electrical potential, T etc

Chemical parameters of biosensing: concentration of a chemical species in a volume of gas, liquid or tissue
Glucose sensors

1950s: paper-based biosensors; color change in a paper when dipped into urine

Glucose oxidase & other enzymes

Rxn between, glucose, glucose oxidase and other enzymes → color change

But urine is not a good candidate –> time passes (measurement from urine – actual blood values)
→ no quantity

****enzymatic oxidation of glucose by the enzyme glucose oxidase****

Malaria Detection
Porous membrane, antibody embedded

detection of antigens produced by malarial

parasites: Plasmodium falciparum histidine
rich protein II (PfHRPII)
Sutures
Generally Absorbable ! (loose strength before losing mass)

Surgical gut sutures (enzymatic degradation)

Silk suture

Poyester/nylon sutures

UHMWPE

- bacteria sites
- tissue drag

braided vs monofilament; knots!!

Less tissue drag

But stiffness !

Coating alternative