Alternative approaches using animal model for implant biomaterials:

advantages and disadvantages

Simona Cavalu Professor

Preclinical Sciences Department

Faculty of Medicine and Pharmaceutics

University of Oradea

ROMANIA

MotivationDevelopment 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.

Factors for consideration when choosing an animal model

Implant design and surface textureAnimal selection: macrostructure,

microstructure, bone composition, bone remodeling - similarities when comparing to the human bone

Costs to aquire and care for animalsAvailability, acceptability to societyTolerance to captivity and ease of housing.International standard (ISO 10993-6) and

Animal Protection Act.

Materials to be tested in this studyDense 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.

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

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.

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

Granules with irregular shape - small animal model protocol

Small animal model: Wistar rat

Collagen film

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.

Monitoring the osseointegration process at different time intervals (3, 6 weeks). Radiographic images

Post surgery fracture failure may occur

SEM images of the sheared implant surfaces

3 weeks after surgery

Ca/P= 1.60

6 weeks after surgery

Haversian canal detailsCa/P= 1.80

Histological images to detect any immunological or inflammatory responses

osteoblasts

A network of woven bony trabecular architecture with cellular infiltration was observed (H&E stain)

Bone marrow cells

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.

Rabbit modelThe 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.

Machined alumina/zirconia ceramics - cylindrical shape, suitable for rabbit model

Biomedical coatingsThe use of surface covering layers (i.e. coatings) provides methods to control the biological response to materials and material devices including implants and prostheses (Figure 1). Depending on implant location and function, implants require specific biological responses. For instance, bone implants require fast integration with native bone tissue. On the other hand, soft tissue implant functionality is improved by the absence of a contractile fibrous tissue capsule. The aim of our research on biomaterial coatings is to optimize the biological response for specific applications of biomedical implants.Organic coatingsSeveral types of organic materials can be used to generate a coating with specific modulatory effects on the biological response. Examples include proteins, DNA, sugars, etc. Specific biological responses that can be controlled are cell attachement and behavior. Organic coatings consisting of proteins are generally based on the presence of these proteins at the implant location. Members of the extracellular matrix (ECM) are the most commonly used proteins. DNA is interesting as a structural molecule, as it is homogeous within all vertebrate species. Consequently, as an implant coating, it masks the implant from being recognized as a foreign body.

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.

Protein (fibrinogen) coating on alumina/zirconia ceramics

Rabbit model: surgical procedure

Radiographic images at 6 weeks

Histology: implant- bone marrow cells interaction

Histology: implant- host bone interaction

Goldner’s Trichrome stain

SEM/EDX bone-implant interface

Ca/P= 1.62

Ca/P= 1.77

Histological and SEM observationsThe 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

ConclusionsNo 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.

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 .

Wild rabbit in red wine sauce