Biomaterials for Orthopaedic

Applications: Biocompatibility

Chaozong Liu
Professor of Orthopaedic Bioengineering
Division of Surgery & Interventional Science
University College London,
Royal National Orthopaedic Hospital

Chaozong.Liu@ucl.ac.uk
Where are UCL IOMS & RNOH
 Orthopaedic Bioengineering -
Translational in Practice at UCL
Address the translational gaps:

First translational gap Second translational gap

Basic Applied Healthcare

Research Research Policy and
Practice

Product engineering Operational models

UCL Orthopaedic Bioengineerng: Address
Unmet Needs
Musculoskeletal disorders – early diagnosis and intervention
❖ Osteoarthritis
❖ Osteoporous
❖ Bone sarcoma
❖ Avascular necrosis
Musculoskeltal tissue repair and regeneration
✓ Cartilage defects
✓ Osteochondral defects
✓ Ligament and tendon
✓ Bone defects
✓ Fracture repair
✓ Human tissue and animal models
Biomaterials and implant innovation
➢ Scaffold & Biomateirla
➢ Personalised implants
 Biomaterials for Orthopaedic Applications
Objectives:

✓ Understand the mechanical and biological behaviour of biomaterials used

as medical devices for orthopaedic and musculoskeletal applications.

✓ Understand the factors involved in the selection of a biomaterial for

orthopaedic and musculoskeletal application including mechanical,
biocompatibility, material property and fixation factors.

✓ Demonstrate a basic understanding of the major areas of current research in

both the biomaterials and biomechanics fields.

✓ Understand the challenges and difficulties in the treatment of musculoskeletal

disorders.
 Biomaterials and musculoskeletal system
What is Biomaterials?

A biomaterial is defined as a natural or synthetic material (such as a metal or

polymer) that is suitable for introduction into living tissue especially as part of
a medical device (such as an artificial joint).

• A biomaterial is any matter, surface, or construct

that interacts with biological systems.
• Used and/or adapted for a medical application:
➢ comprises whole or part of a living structure or
➢ biomedical device which performs, augments,
or replaces a natural function.
• May also be an autograft, allograft or xenograft used as
a transplant material.
 Biomaterials and musculoskeletal system
Biomaterials - Category
✓ Natural biomaterials Biomaterials must
✓ Biometals be compatible with
✓ Bioceramics the body.
✓ Biopolymers
✓ Biocomposites
➢ Host response: the reaction of a living system to the
presence of a material.
Biocompatibility is the ability of material to perform
within an appropriate host response in a specific
application. It is the quality of not having toxic or injurious
effects on biological systems.
B=f(X1,X2......Xn)
Where X: material, design, application etc
Biomaterials and musculoskeletal system

Applications:
Joint replacements
Bone plates
Bone cement
Artificial ligaments and tendons
Dental implants for tooth fixation
Blood vessel prostheses
Heart valves
Skin repair devices (artificial tissue)
Cochlear replacements
Contact lenses
Breast implants
Drug delivery mechanisms
Sustainable materials
Vascular grafts
Stents
Nerve conduits
 Biomaterials and musculoskeletal system

Metals,
alloys

Ceramics,
glasses

Biopolymers
Typical Biomaterials in Orthopaedic appliations
 Biomaterials area key components of tissue
engineering and regenerative medicine

Tissue engineering can be defined as the use of a

combination of cells, engineering materials, and
suitable biochemical factors to improve or replace
biological functions in an effort to improve clinical
procedures for the repair of damaged tissues and
organs.

Regenerative medicine - replacing, engineering or

regenerating human cells, tissues or organs to restore
or establish normal function.
When a biomaterial is placed in the body a
‘two-way’ biological interaction takes place.

Two factors involved:

1. The effect the body has on the implant material.

2. The effect the implant has on the body.

 EFFECTS THE BODY HAS ON THE
IMPLANT MATERIAL
• ENVIRONMENTAL
Body fluid is 0.9% saline, plus cells, proteins, enzymes.

• DEGRADATION
Enzymatic

• CORROSION
Mainly metals.

• PROTEIN ADSORPTION
Dependent on material properties
Biomolecular Interactions
at Medical Device Surface
Time scale
Process Length scales (Collisions
frequency)

Microns
Cells Adhesion Hours-Days
(μm)

Minutes-Hours
Nanometres-Microns
Protein Attachment (min-h)
(nm-μm)

Angstroms-
Nanoseconds
Water Adsorption Nanometres
(ns)
(Å-nm)

Physical & chemical properties

Biosurface Processes
 Plasma protein absorption to artificial ligament
Stanislawski et al (1995) J Biomat. Res 29 315-323
SDS PAGE Technique
Polylactic acid, Polyacrylamide, Polyester
,Polypropylene.
Vroman effect: The highest mobility proteins
generally arrive first and are later replaced by less mobile
proteins that have a higher affinity for the surface
Total protein

B
A

In blood on activating (clotting) surfaces

Albumin immunoglobulins fibrinogen fibronectin
Maximum adhesion of a specific protein from a multi component media
at an early time followed by a decrease in absorption with time
Protein attachment to the surface
Electrostatic Conformational change

- + + - -
- + + - -
-
- + + - -
- + + - -
- + + - -
- + + - -
-
-
- + + - -
- -
- + + - -
-
- + + - -
+
+ + + - -
+
+
+

Hydrogen bonding
Interaction of non polar groups in aqueous media hydrophobic reaction
EFFECTS THE IMPLANT HAS ON THE BODY

• Evoke an immune response

• Cell injury, inflammation

• Activation of macrophages etc.

• Upsets homeostatic equilibrium

• Scar formation
 BIOCOMPATIBILITY
Def:

Biocompatibility is the ability of a

material to perform with an appropriate
host response in a specific application.
(Williams 1987).

Definition came about when materials were developed that

were required to have specific functions as opposed to
being totally inert.
 All new materials and devices
that are to be used or placed
within the body have to be
evaluated for their
biocompatibility before their
implantation, to ensure their
safety for human use.

Biocompatibility assessment is essential

for pre-clinical testing of materials
intended for use as implantable devices.
 Biocompatibility testing

✓ in vitro testing

✓ in vivo testing
Biocompatibility involves two components:

• General aspect: ‘BIOSAFETY’

This concerns and deals with the exclusion of
deleterious effects of a biomaterial on the
organism itself.

• Specific aspect: ‘BIOFUNCTIONALITY’

This concerns and addresses the need of a
material not only to be free from damaging effects
on the host, but be able to elicit a beneficial host-
response for optimal functioning of the medical
device.
What criteria must the material fulfil to be
biocompatible?

Material must not cause:

• Chronic inflammation
• Infection
• Thrombogenesis
• Neoplasms
• Must be non-toxic in its’ primary form and must
not breakdown into any toxic substances.
 WHY IN VITRO?
• Pressure to reduce animal experimentation.
In vivo is:
- More expensive;
- Lacks control due to animal and species
differences;
- Problems with obtaining ethical permission.
- In vitro can investigate single responses in a
controlled, reproducible way.

• Considerable research in in vitro methodologies.

Testing Biomaterials: Four Phases

Raw Material
Characterization and chemical, physical, biological
Screening

cytotoxicity, sensitization,
Material irritation, systemic toxicity,
hemocompatibility,
Biocompatibility carcinogenicity

environmental, manufacturing,
Product and Process sterility, finished product
Validation qualification

periodic audit testing, release

Routine Testing
testing
• Medical Device Directive
A material used for medical devices should comply with
toxicity tests.
• The International Organisation for Standardisation
(ISO) provides guidelines.

• ISO 10993 consists of a number of parts under the general

title of ‘Biological Evaluation of Medical Devices’

• Tests for in vitro cytotoxicity and defines a series of

guidelines that can help in the selection of the most
appropriate test for biocompatibility screening.

* Policies fall in line with most worldwide bodies.

* FDA in some cases need additional work to be performed.
➢The device to be tested (the test article)
should be a representative specimen of
the mass produced device.

➢It should be finished or treated (e.g

coated and/or sterilised in the same way
as the mass-produced device.
International standards organization
ISO 10993 Biological evaluation of medical devices
• ISO 10993-2:2006 Biological evaluation of medical devices Part 2: Animal welfare
requirements
• ISO 10993-3:2003 Biological evaluation of medical devices Part 3: Tests for
genotoxicity, carcinogenicity and reproductive toxicity
• ISO 10993-4:2002/Amd 1:2006 Biological evaluation of medical devices Part 4:
Selection of tests for interactions with blood
• ISO 10993-5:2009 Biological evaluation of medical devices Part 5: Tests for in
vitro cytotoxicity
• ISO 10993-6:2007 Biological evaluation of medical devices Part 6: Tests for local
effects after implantation
• ISO 10993-7:2008 Biological evaluation of medical devices Part 7: Ethylene oxide
sterilization residuals
• ISO 10993-8:2001 Biological evaluation of medical devices Part 8: Selection of
reference materials
• ISO 10993-9:1999 Biological evaluation of medical devices Part 9: Framework for
identification and quantification of potential degradation products
• ISO 10993-10:2010 Biological evaluation of medical devices Part 10: Tests for
irritation and delayed-type hypersensitivity
• ISO 10993-11:2006 Biological evaluation of medical devices Part 11: Tests for
systemic toxicity
 ISO 10993 Biological evaluation of medical devices (contd)

• ISO 10993-12:2012 Biological evaluation of medical devices Part 12: Sample

preparation and reference materials (available in English only)
• ISO 10993-13:1998 Biological evaluation of medical devices Part 13: Identification
and quantification of degradation products from polymeric medical devices
• ISO 10993-14:2001 Biological evaluation of medical devices Part 14: Identification
and quantification of degradation products from ceramics
• ISO 10993-15:2000 Biological evaluation of medical devices Part 15: Identification
and quantification of degradation products from metals and alloys
• ISO 10993-16:1997 Biological evaluation of medical devices Part 16: Toxicokinetic
study design for degradation products and leachables
• ISO 10993-17:2002 Biological evaluation of medical devices Part 17: Establishment
of allowable limits for leachable substances
• ISO 10993-18:2005 Biological evaluation of medical devices Part 18: Chemical
characterization of materials
• ISO/TS 10993-19:2006 Biological evaluation of medical devices Part 19: Physico-
chemical, morphological and topographical characterization of materials
• ISO/TS 10993-20:2006 Biological evaluation of medical devices Part 20: Principles
and methods for immunotoxicology testing of medical devices
The ISO 10993 does not specify any single test but aims
to define a testing scheme which requires decisions to
be made in a series of steps which should lead to the
selection of the most appropriate test.

Three types of CYTOTOXICITY testing regimes are

recommended:
• Extract
• Direct contact
• Indirect contact

Choice is dependent on:

• Type of sample to be tested
• Potential site of use
• Nature of use
DIRECT

➢ Important to also evaluate reaction of cells under

normal-use conditions.
➢ Device is placed on cells growing in culture
medium.
➢ Cells are incubated and leachable chemicals
diffuse.
➢ Growth rate and cell damage monitored.
In vitro Testing: Direct Contact
• Direct contact
– monolayer, confluent cell culture,
L-929 mouse fibroblasts
– biomaterial in direct contact
– 24 hours, 37C
– cells may
• change morphology
• die
• lose adherence to dish Biomaterial

– hematoxylin blue: stains live

adherent cells
– toxicity=dead/live !
• Why L-929?
– easy to maintain
– good correlation with animals tests
– fibroblasts present in wound
healing
31
In vitro Testing: Direct Contact
A confluent monolayer (100 x magnification)
of well-defined L929 mouse fibroblast cells
exhibiting cell-to-cell contact. This
appearance is indicative of a non-cytotoxic
(negative) response
http://www.devicelink.com/mddi/archive/98/04/013.html
L929 mouse fibroblast cells (100 x
magnification) that illustrate a positive
cytotoxic reaction in the elution test method.
The cells are grainy and lack normal
cytoplasmic space; the considerable open
areas between cells indicate that extensive 32
cell lysis (disintegration) has occurred.
CELL LINES

• Primary cells optimal. Difficult to get hold of

enough human tissue especially if lots of tests
need to be done.

• Cell lines are more commonly used. Replicate

faster and have a well defined phenotype.

• Disadvantage: cells can de-differentiate after

many passages. Cell characteristics lost. Monitor
passage number.
In vitro Testing: Elution

Biomaterial
• Elution
– prepare extract of a material
– how? keep the material in oil
based or water based
solution (Why oil or water?)
– chemicals will leach into
solution
– apply solution to cell-culture
– perform similar stain based
viability tests
34
EXTRACT (eluate)
• Because the toxicity potential of materials and
devices depends to a substantial degree on the
leachability and toxicity of the soluble
components, extracts of the device are normally
used in the tests. Materials are rinsed and the
eluates tested on cell populations.

• Ideally, the extraction media should constitute a

series of media with decreasing polarity to ensure
extraction of components of widely different
solubility properties.
 EXTRACTION SOLUTIONS
• Physiological saline. Approximates the aqueous,
hydrophilic fluids of the body and permits the use of
extreme temperatures in preparing extracts – simulating
some sterilisation conditions.
• Vegetable oils are non-polar, hydrophobic solvents and
simulate the lipid fluids in the body.
• Dimethylsulfoxide and ethanol used as they
approximate solvent properties of drugs or other materials
that contact the device during normal use. At 370C for 24
-72 hours
• If medical device comprises several components
made from different materials, the ideal procedure
is to test extracts separately.

• Extraction of microbes also investigated by rinsing

device with water and testing extract. Assess the
microbiological load of raw material – also checks
efficiency of sterilisation process.
INDIRECT: AGAR DIFFUSION TEST

• A piece of test material is placed on a thin agar

layer covering a confluent monolayer of cells.

• Toxic substances leaching from the material

diffuse through the thin agar layer and kill or
disrupt adjacent cells in the monolayer.
In vitro Testing: Agar Diffusion

An agar diffusion flask containing a sample of positive control material. The discoloration that extends outward
from the material indicates that the presence of the sample has caused the cells to lyse, losing the vital stain
incorporated in the agar layer.

http://www.devicelink.com/mddi/archive/98/04/013.html
39
TYPES OF METHODS THAT CAN BE USED
TO ASSESS CYTOTOXICITY INCLUDE:

• QUALITATIVE
Assessment of cell damage by morphological means
(TEM, SEM). Light and confocal microscopy

• QUANTITATIVE
Measurement of cell damage and viability
Measurement of cell proliferation and growth
Measurement of specific aspects of cellular metabolism
Ability of cells to retain their phenotype on the test material
CELL VIABILITY - QUANTITATIVE

• VIABILITY
Trypan blue and erthromycin red. Exclusion and
Inclusion dyes. Dying non-viable cells stained blue and
pink respectively.
Coulter counters and cell sorters

Live-dead assay. Calcein am and Ethidium red.

Non-viable cells the membrane is permeated and dye bind
to DNA colouring it red under fluorescence. Calcein am
enters cells and is catalysed by viable cells and cleaved to
released green fluorescent marker.
CELL PROLIFERATION - QUANTITATIVE

• PROLIFERATION
Alamar blue and tritium-labeled thymidine uptake. MTT
assay:
The reduction of tetrazolium salts is now recognized as a
safe, accurate alternative to radiometric testing. The yellow
tetrazolium salt (MTT) is reduced in metabolically active
cells to form insoluble purple formazan crystals, which are
solubilized by the addition of a detergent. The colour can
then be quantified by spectrophotometric means.

Increase in cell growth, no effect, or a decrease in

growth due to necrosis or apoptosis.
MEMRANE INTEGRITY
Lactate dehydrogenase (LDH)

• Found in all mammalian cells.

• Normal plasma membrane impermeable to LDH.
• Damage allows leakage.

• In vitro release provides accurate measurement of

cell membrane and cell viability.
C Pendegrass 2005 DLC coating – low surface energy
C Pendegrass 2005 Smooth Ti Alloy finish
 LIVE – DEAD STAIN

Simultaneous fluorescence staining of viable and dead cells using calcein-AM

and propidium iodide (PI), which stain viable and dead cells. Nuclei staining dye PI
(red) cannot pass through a viable cell membrane. Though calcein-AM itself is not a
fluorescent molecule, the calcein generated from Calcein-AM by esterase in a viable cell
emits a strong green fluorescence). Therefore, calcein-AM only stains viable cells.
 In vivo models
• Critical for development of clinical devices
• In vitro tests cannot replace in vivo tests:
– no inflammation
– no immune response
– single cell type (usually)
– no tissue remodeling
• In vivo tests provide:
– interactions of different cell types
– effects of hormonal factors
– interactions with extracellular matrix
– interactions with blood-borne cells, proteins
and molecules
– may or may not replicate the disease process
 In Vivo Models
• Ethical Justification
– Using living sentient individuals
– Only when other methods not appropriate
– Prior to Human trials
– Appropriate numbers (power calculations)
– Species (sensitive species may not be required)
– Species variations
– Severity of procedures vs scientific and medical
benefit (Cost benefit analysis)
– Ethical review, regulatory requirements
– Regulatory requirements GLP preclinical safety
• Implant effects can be simulated in vivo:
– Interfaces can be investigated
– insoluble particulate materials released
by implants
– interaction of biological factors with the
implant
– Systemic effects
– mechanical loading experienced by
device
– Disease process?
 D
E
M G
O R
T A
I D
O A
N TI
N O
N

Factors other than material material composition that affect biocompatibility

Shape, Size, Motion, Degradation, Corrosion , Time ( Changing interface and patient
conditions),
Silicon or titanium implants with plane or porous surfaces rat abdominal wall
for evaluation of the tissue response after one, six, or 12 weeks

Titanium polished Silicon polished

Titanium anodized porous Silicon anodised porous

Wooley PH, Morren R, Andary J, Sud S, Yang SY, Mayton L, Markel D, Sieving A,
Nasser S. Inflammatory responses to orthopaedic biomaterials in the murine air pouch.
Biomaterials. 2002 Jan;23(2):517-26.

Control PMMA UHMWPE

CoCr Ti
 Macropore
Strut
Micropores
Macropore
Strut
Fused granules

Chan et al. The effects of microporosity on osteoinduction of calcium phosphate bone

graft substitute biomaterials. Acta Biomater. 2012 Jul;8(7):2788-94
 Bone response
Simplistic models of implants into bony sites

Control implants

Bone attachment

Fibrous tissue at the interface

Pull out strength

More complex models where implants are used in a

similar manner to those in humans
 Bloebaum RD, Bachus KN, Momberger NG, Hofmann AA. Mineral apposition rates of
human cancellous bone at the interface of porous coated implants. J Biomed Mater
Res. 1994 May;28(5):537-44.

19 consenting bilateral total knee arthroplasty patients. Titanium porous coated cylinders
were implanted into the medial femoral condyle of the contralateral knee during the first
of two TKAs. Retrieval was performed at the time of the second TKA (6-131 weeks
later), and fluorochrome analysis was conducted. Mean mineral apposition rates (MAR)
1.0 um/day, whereas 4 mm away, the peripheral bone had a mean MAR of 0.8 um/day.
Bone ingrowth increased up to 9 months.
Analysis showed that when bone was over 50 um from the porous coating, bone
ingrowth did not occur.
Summary

When a biomaterial is placed in the body a ‘two-way’

biological interaction:
• The effect the body has on the implant material.
• The effect the implant has on the body.

Biocompatibility: is the ability of a material to perform with

an appropriate host response in a specific application.
• In vitro test
• In vivo test

Biocompatibility test – ISO 10993 Biological evaluation of

medical devices
PhD Opportunity

UCL Institute of Orthopaedic & Musculoskeletal Science

Contact: Prof Chaozong Liu
Email: Chaozong.Liu@ucl.ac.uk