Alternative approaches using animal model for implant biomaterials: advantages and disadvantages

1. Simona Cavalu
Professor
Preclinical Sciences Department
Faculty of Medicine and Pharmaceutics
University of Oradea
ROMANIA

2. Motivation
 Development of an optimal interface between bone
and orthopedic or dental implant.
 Determining whether a newly developed implant
material confirms to the requirements of
biocompatibility, mechanical stability and safety.
 The use of animal models is an essential step in testing
orthopedic and dental implants prior to clinical use
in humans.
 Results from in vitro studies (cells culture) can be
difficult to extrapolate to the in vivo situations.

3. Factors for consideration when
choosing an animal model
 Implant design and surface texture
 Animal selection:
macrostructure, microstructure, bone
composition, bone remodeling - similarities when
comparing to the human bone
 Costs to aquire and care for animals
 Availability, acceptability to society
 Tolerance to captivity and ease of housing.
 International standard (ISO 10993-6) and Animal
Protection Act.

4. Materials to be tested in this study
 Dense Al2O3-YSZ composites prepared by Spark Plasma Sintering
 High performance biocomposite that combines the excellent
material properties of alumina in terms of chemical stability and
low wear and of zirconia with its superior mechanical strength and
fracture toughness.
 Alumina/zirconia ceramics were successfully used in total hip/knee
arthroplasty in the last decades as alternative to metal devices.
 For dental application: root canal posts, orthodontic brackets,
implant abutments and all- ceramic restaurations.

5. Al2O3/ ZrO2 have been considered as bioinert
ceramics since they cannot induce apatite formation
in SBF. They do however support bone cell
attachment, proliferation and differentiation.
A. Gonzalez, 2010, Osteoblasts on
bioceramic, Copyright Univ. of
Aberdeen

6. Materials characterization by
SEM, XRD, FTIR, mechanical tests
 Effect of CeO2 addition on densification and
microstructure of Al2O3–YSZ composites, I. Akin, E.
Yilmaz, F. Sahin, O.Yucel, G.Goller, Ceramics
International 37 (2011) 3273–3280.
 Correlation between structural properties and in vivo
biocompatibility of alumina/zirconia bioceramics, S.
Cavalu & all, Key Eng. Mater. 493-494 (2012) 1-6.
 XRD and FTIR investigation of zirconia-taughened
alumina composites, V. Simon, S. Cavalu, I. Akin, O.
Yucel, G. Goller, Studia UBB Physica, LVI, 1(2011)67-72.

7. SEM details
of Al2O3 and Al2O3-YSZ
composites (G. Göller &
all, Istanbul Technical
University)

8. Granules with irregular shape - small
animal model protocol

9. Small animal model: Wistar rat
Collagen film

10. Collagen membrane
• S. Cavalu & all, In vitro study of collagen coating by electrodeposition on acrylic
bone cement, Digest J. Nanomater. Biostruct (2011 ) 1, 89-97.
• S. Cavalu & all, Bioactivity and antimicrobial properties of PMMA/Ag2O bone
cement collagen coated, Digest J. Nanomater. Biostruct (2011 ) 2, 779-790.
• S. Cavalu & all, Improving the bioactivity and biocompatibility of acrylic bone
cement by collagen coating, Key Eng. Mater (2012) 493-494, 391-396.

11. Monitoring the osseointegration process at different
time intervals (3, 6 weeks). Radiographic images
Post surgery fracture failure
may occur

12. SEM images of the sheared implant surfaces
3 weeks after surgery
Ca/P= 1.60

13. 6 weeks after
surgery
Haversian canal details
Ca/P= 1.80

14. Histological images to detect any
immunological or inflammatory responses
osteoblasts Bone marrow cells
A network of woven bony trabecular architecture with cellular infiltration was observed
(H&E stain)

15. SEM and Histological analysis revealed:
 Fibrinous and collagenous matrix with three-dimensional interconnected porous
structure after first 3 weeks.
 Distinct gaps between the implant and the bone were observed in a few locations.
 After 6 weeks, the matrix around the surface implanted area appeared more
densely, well covered and integrated into a mixture of mineralized tissue, osteoid
and dense matrix.
 From the EDAX spectra, calcium/phosphate ratio is an indicative of the surface
implant coverage for a successful osseointegration, varying from 1.7 (after 3 weeks)
to 1.8 (after 6 weeks).
 No clinical signs of inflammation or mobility were present. Newly
formed bone surrounded the implant surfaces, and many osteoblasts secreting
osteoid matrix were observed.

16. Rabbit model
 The most commonly used model for medical research
(aprox. 35% of the musculoskeletal research studies –Al.
Pearce, Eur Cells Mater 13 ,2007).
 Histologically, rabbit long bones have a very different
microstructure from humans.
 In comparison with other species, the rabbit has faster
skeletal change and bone turnover (significant
intracortical, Haversian remodelling). This make it
difficult to extrapolate results from studies performed in
rabbits onto the likely human clinical response.

17. Machined alumina/zirconia ceramics -
cylindrical shape, suitable for rabbit model
Alumina/zirconia ceramics are
bioinert materials: once placed in
the natural tissue, it has a minimal
interaction with the surrounding
tissue, generally a fibrous capsule
might form around the implants.
Surface properties control the amount and quality of cells adhered on the
implant and consequently, the tissue growth. Surface treatment techniques:
sandblasting, acid-etched, organic (protein) or inorganic (Ca/P) coating.

18. Protein (fibrinogen) coating on
alumina/zirconia ceramics

19. Rabbit model: surgical procedure

20. Radiographic images at 6 weeks

21. Histology: implant- bone
marrow cells interaction

22. Histology: implant- host
bone interaction
Goldner’s Trichrome stain

23. SEM/EDX bone-implant interface
Ca/P= 1.62
Ca/P= 1.77

24. Histological and SEM observations
 The presence of young, compact lamellar bone and
osteocytes near the implant surface indicated good
biocompatibility, and certainly the presence of the
implant did not disturb the processes of bone formation
at the interface.
 Slight vascular congestion in contact with the bone
marrow, but no giant cells
 Microstructure details (including Haversian canals) of
bone and bone marrow tissue and elemental composition
at the interface indicated Ca/P =1.62 - 1.77

25. Conclusions
 No animal species fulfils all of the requirements of an ideal
model, but an understanding of the differences in bone
architecture and remodeling may assist in the selection of a
suitable model for a defined research question.
 Organic (proteic) film as surface conditioning might be an
alternative approach to induce the bioactivity and improve
the biocompatibility of dense bioceramics designed to
load bearing bone replacement (hip joint, dental abutments)
and to optimize the biological response for specific
applications of biomedical implants.

26. The team:
* Prof. dr. Viorica Simon and dr. Oana Ponta
Babes-Bolyai University, Faculty of Physics &
Institute of Interdisciplinary Research in Bio-Nano-
Sciences, Cluj-Napoca, Romania.
*Prof. dr. Gultekin Goller and assist. prof. Ipek
Akin, Istanbul Technical University, Materials
Science Department.
*Assist. prof. Cristian Ratiu , assist dr. Silviu Vlad
and dr. Ioan Oswald University of Oradea, Faculty
of Medicine and Pharmaceutics, Oradea, Romania.
Romania-Turkey Bilateral Cooperation 2011-2012 and
CNCS-UEFISCDI project PNII-ID-PCE 2011-3-0441
contract nr. 237/2011 .

27. Wild rabbit in red wine sauce