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Simona Cavalu
                           Professor


      Preclinical Sciences Department

Faculty of Medicine and Pharmaceutics

                 University of Oradea

                           ROMANIA
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.
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.
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.
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 details
                Ca/P= 1.80
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)
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 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.
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.
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 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
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.
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

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SIMONA CAVALU_Alternative approaches using animal model for implant biomaterials

  • 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
  • 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

Notes de l'éditeur

  1. 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.
  2. The bonebondingability of the coatings facilitates fast short-termintegration of implant with bone, but in long-term implantationand fixation, sometimes coatings are delaminated fromthe substrate or dissolved slowly and there are also concernsabout shear strength at coating/implant interface, forexample, in plasma sprayed coatings as the thickness of thecoating layer is increased.