© 2011 France Lambert Department of Periodontology and Oral Surgery Service de Médecine Dentaire Domaine du Sart Tilman, Bat B35 B-4000 Liège +32 4 366 82 90 [email protected] All right reserved. No part of this book may be reproduced or transmitted in any form or any mean, electronic or mechanical, including photocopy, recording, or any information storage and retrieval sys- tem, without permission in writing from the copyright owner.
Department of Periodontology and Oral Surgery Faculty of Medicine University of Liège, Belgium
Pr E.Rompen, University of Liège, Belgium Pr V.Bours, University of Liège, Belgium Pr J.Martial, University of Liège, Belgium Pr M.Lamy, University of Liège, Belgium Pr A.Vanheusden, University of Liège, Belgium Dr M-C De Pauw, University of Liège, Belgium Dr M.Muller, University of Liège, Belgium Pr P.De Coster, University of Ghent, Belgium Promotor:
President:
Jury Members:
« The man who moves a mountain begins by carrying away small stones. »
Confucius, Antique Chinese philosopher
Acknowledgements:
This work would not have been possible without the support of numerous people.
I would first like to thank my promoter, Prof. Eric Rompen, Head of the Department of Periodontology and Oral Surgery, University of Liège. He is the very first person who gave me the taste for periodontology and implantology, he imparted to me his clinical and scientific know-how over the last 8 years and he utterly trusted me in the completion of this PhD Thesis.
This work allowed me to meet amazing people, and gave me the opportunity to build up a strong network of scientific professionals; I hope we will be able to further collaborate as efficiently in the future. I would like to express my gratitude to:
Prof. Angélique Léonard and Laurent Fraikin from the Department of Applied Chemistry, University of Liège.Merci Angélique de m’avoir consacré du temps même pendant les moments où tu expérimentais tes qualités de jeune maman.
Prof. Pierre Drion and Luc Duwez, from the Central Animals Facility, Giga-R, University of Liège.Pierre merci pour ton soutient, j’espère que ce projet est le départ d’une longue collaboration et amitié.
Prof. Pierre Layrolle, and Sophie Sourice from the Laboratory of Physiopathology of Bone Resorption and the Laboratory for Osteo-articular and Dental Tissue Engineering, University of Nantes. This work could not have been completed without the amazing histological expertise of the group of Nantes.Merci Pierre pour cette collaboration et pour avoir partagé ta connaissance des biomatériaux.
Prof. Adelin Albert, Laurence Seidel and Sophie Vanbelle from the Department of Informatics and Biostatistic, University of Liège.Vraiment, merci pour la volonté que vous avez mise ces derniers mois dans ce travail. Prof. Albert, j’admire votre dévouement et la passion que vous avez pour votre métier. Laurence, je ne sais pas comment vous avez fait pour ne pas devenir folle avec mes données !
Prof. Peter De Coster from the Department of Oral Biology, University of Ghent for his availability and expertise.
Dr. Geoffrey Lecloux from the Department of Periodontology and Oral Surgery, University of Liège for his surgical involvement in several studies of this Thesis.Geoffrey, en plus d’avoir contribué à ce que je suis devenue professionnellement aujourd’hui, tu sais que tu es un ami…
Dr. Bart Vandenberghe from the Department of Prosthodontics, University of Leuven. Bart,
« the IT guy » un grand merci pour ton énergie et ton dynamisme, j’espère que l’on va pouvoir
I kindly thank all the members of my Thesis committee: Prof. V. Bours, Prof. J. Martial, Dr. M-C De Pauw, Dr M. Muller for their wise advises over the last 4 years. I would also like to sincerely thank the jury members of this PhD Thesis who were not in my committee: Prof.
M. Lamy and Prof. A. Vanheusden, you contributed to my education and it is a pleasure today to treat patients together with you as a team, Prof. De Coster, and finally I address a very special acknowledgment to Prof. Ronald Jung: We met for the first time when I had just graduated from Dental School, it is today a great honor to have you among the jury of my PhD Thesis.
Although they were not directly involved in the present PhD Thesis, Dr. H-P Weber and Dr. Gallucci have undoubtedly contributed to the development of my scientific knowledge during my stay in Harvard School of Dental Medicine, Boston, MA, USA, and I strongly thank them for sharing their expertise. This thesis could not have been properly conducted without the financial support of the University of Liège (Classical funds: C-08/77) and of Geistlich Pharma AG (Wolhusen, Switzerland).
Of course, I also want to express my gratefulness to my dear colleagues:Laurie, Kim, Vanessa, Laurence, Leila, Elise and Charlotte vous savez que vous avez aussi apporté votre pierre à ce travail, merci pour votre aide. Francine, Bertrand, vous m’avez vu grandir et c’est un plaisir de travailler à vos cotés. Je souhaite également remercier l’équipe de nursing et le personnel administratif du Sart-Tilman et du Brull, pour beaucoup de choses mais surtout pour leur grande patience ! Tommie, tu as probablement été mon meilleur coach pour la réalisation de cette thèse, ton expérience et tes conseils m’ont été vraiment très précieux.
Je remercie aussi infiniment mes parents et ma famille qui m’ont toujours supportée (dans les 2 sens du terme !). Papa, maman, depuis le début, vous avez toujours cru en moi et m’avez encouragée. Mickey (et Harmony), Carine, Fabienne, Pat, Olivier, Aline, Marine, Sybille, vous savez à quel point je vous adore, merci d’avoir toujours été là pour me remonter le moral et aussi pour avoir partagé nos plus beaux moments.
J’ai aussi une série de vraiment très bons et vieux amis que je dois remercier pour avoir toujours été si fidèles ! L’équipe du Sartay, 15 ans après, on partage encore tellement, les filles…
(dentistes), pas besoin de grand discours pour exprimer notre amitié, et tous les autres, vous avez été témoins de l’énergie que j’ai mise dans mon boulot et dans cette thèse… Je suis sûre que vous vous demandez, what is next ?
Je dois aussi chaleureusement remercier les personnes qui m’ont aidées et permises de réaliser ce manuscrit : Gérard Scrève, Géraldine Troisfontaine, Guillaume Giraud.
Finalement, je veux aussi remercier mais surtout dédier cette thèse à ma grand-mère paternelle, le Dr. Louise-Marie Lambert et à mon grand-père maternel, le Dr. Raymond Toussaint.
Bonne Mamanlou, tu t’es battue pour étudier la médecine alors qu’à cette époque c’était un métier d’homme, tu as consacré ta vie aux autres et à tes patients, avant d’aller rejoindre ton mari adoré. C’est avec une immense fierté que j’ai toujours apprécié les comparaisons que
Table of content
Introduction and aims
Chapter 1 :Biomaterials Properties
Physico-chemical and morphological properties of biomaterials used in alveolar bone augmentation and preservation
Chapter 2 : Biological concept of sinus lift procedures Influence of space-filling materials in sub-sinusal bone augmentation:
Blood clot vs autogenous bone chips vs covine hydroxyapatite Chapter 3 : Calcium phosphate based Biomaterials
Influence of calcium phosphate based space-fillers in sub-sinusal bone augmentation: A comparison of four different types of biomaterials Chapter 4 : Titanium biomaterials
Bone regeneration using porous titanium particles vs. bovine hydroxyapatite: A sinus lift study in rabbits
Chapter 5 : Collagenated biomaterials
Effect of collagenated biomaterials in sinusal bone augmentation:
A study in rabbits
Chapter 6 : Sinus lift clinical trial
One-step approach for implant placement and sub-sinusal bone regeneration using bovine hydroxyapatite: A 2- to 6-year follow-up study
Chapter 7 : Socket preservation : Hard tissue changes Description of the bone-remodeling patterns after socket preservation procedures in human: A methodological study
Conclusion and guidelines Summary / Résumé Curriculum Vitæ
12 14
44
66
90
108
126
146
166 168 173
General introduction and objectives:
Partial or complete edentulism have a significant impact on the quality of life by reducing the ability to eat and by altering social relationships. It was clearly demonstrated that dental prosthesis retained with implants can significantly improve mastication, aesthetics, and even intimate relationships.
Dental implants are nowadays a reliable solution to replace missing teeth and have been widely documented. However, they require a minimal bone quantity (in height and thickness).
But alveolar bone defects are very frequent, for instance due to periodontitis, traumatism or acute dental infection. Moreover, a simple tooth extraction leads to significant bone resorption. Therefore, alveolar bone regeneration is often necessary in order to place implants and to restore the patient's dentition with removable or fixed prosthesis.
The residual bone height in the posterior edentulous maxilla is frequently limited by the presence of the maxillary sinuses. Indeed, the absence of molars and/or premolars in the maxilla allows the sinus to pneumatize, reducing the height of the alveolar ridge. Although there is a tendency to use short and wide implants in those cases of reduced bone height, at a certain point, the sinusal pneumatization might impair implant placement. To overcome those limits, it is possible to perform sub-sinusal bone regeneration, more frequently called
“sinus lift”; in order to augment the bone quantity and consequently place implants. This procedure to increase the residual bone height of the posterior edentulous maxilla, as initially described by Tatum and Boyne, was relatively heavy and traumatic (Boyne and James, 1980). Today, the protocol has much evolved, not only technically, but also with the introduction of biomaterials.
Furthermore, dimensional changes of the alveolar ridge occur after tooth extraction. The resulting available bone quantity might compromise implant placement and/or aesthetic outcomes of conventional or implant-supported prosthodontic rehabilitation. However, bone collapse after extraction seems to be minimized by socket preservation procedures, consisting in the placement of biomaterialsinto the void.
The number of commercially available biomaterialsfor bone regeneration is growing every day and some materials are not supported by strong scientific data in the professional literature. And the heterogeneity of this literature makes it difficult to compare the influences of those biomaterials on osteogenesis and to elaborate on the advantages of one biomaterial over another, which might also depend on clinical indications.
The overall objective of this thesis is to contribute to the understanding of the biological
To develop a data sheetintegrating the physico-chemical and morphological properties of biomaterials often used in dentistry, as a tool for clinicians. Therefore, a literature review on the impact of physico-chemical characteristicsof several biomaterials on osteogenesis was performed.
To understand the physiology and the biological model of sub-sinusal bone augmentation by using either a simple blood clot, autogenous bone chips or biomaterials as space fillers under the lifted membrane, in a sinus lift model in rabbits.Hypothesis: Osteogenesis can occur with whatever space filler; however, non-resorbable biomaterials might better withstand sinusal repneumatization.
To compare the performances, in terms of bone formation, resorption rate and 3-D stability, of four calcium phosphate-based biomaterialsoften used for alveolar ridge augmentation, in a sinus lift model in rabbits.Hypothesis: Resorbable biomaterials might not withstand sinusal repneumatization over a long period of time.
To assess, in a rabbit model, the bone formation process and the 3-D volume stability of sub-sinusal bone regeneration using porous titanium granulesor bovine hydroxyapatite granules, and to evaluate the effect of BHA particle hydration with a therapeutic concentration of doxycycline solution.Hypothesis: 1) Porous titanium particles offer identical osteo- conductive properties as BHA. 2) In addition to its antibiotic effect, doxycycline might enhance osteogenesis and decrease biomaterial resorption rate.
To investigate the effect of collagenated biomaterialsat different stages of the osteogenesis process, still in rabbit sinuses.Hypothesis: Collagen might slow down the bone healing process.
To assess the clinical outcome of a minimalized sub-sinusal bone augmentation procedure in 40 patients using only biomaterials, simultaneously with the placement of non-submerged implants. Implant and prosthodontic survival rates as well as complications were evaluated after a follow-up period of 2 to 6 years.Hypothesis: Based on adequate primary stability, similar clinical outcomes might be found with a single-stage and less traumatic sinus lift surgical technique compared to conventional staged protocols.
To develop a new method in order to objectively evaluate in humans the 3-D volume variation of alveolar ridge preservation (socket preservation) over time using CTscan.
1
2
3
4
5
6
7
Abstract
The use of biomaterials for alveolar bone regeneration procedures has been repeatedly shown to be effective. Although an ever growing number of biomaterials are available, the origins, material characteristics, and properties of these products are not always sufficiently known by the clinician, which often hampers the correct product selection.
The purpose of this review is to provide an overview of eight types of osteoconductive biomaterials that are frequently used in dentistry and to summarize their physico-chemical and morphological characteristics at the macro- and micro-scales. Furthermore, the influence of these properties on the in vivobehavior of these biomaterials is discussed.
The results of this review are presented as data sheets, which may prove to be helpful for clinicians during the design of treatment strategies.
Introduction
Alveolar bone engineering techniques are frequently used in periodontal therapies, such as oral and maxillofacial surgical procedures, with effective clinical outcomes [1-3].
Although autogenous bone has long been regarded as the golde standard for bone augmentation procedures because it supplies osteoinductive growth factors, osteogenic cells, and a structural scaffold [4], its use may present considerable drawbacks, such as a limited availability and a risk of morbidity at the donor site. For these reasons, biomaterials have been developed as promising alternatives for autogenous bone [5-7], and both the natural and synthetic materials have been used with increasing frequency to fill bone defects [8-16]. Although natural osteoconductive biomaterials, such as porcine and bovine xenografts, are currently used on a large scale because of their similarity to human bone in terms of chemical composition and structure [17-19], the interest in synthetic grafting materials has increased for ethical reasons. These synthetic products are being developed at a growing pace with the objective of fabricating biomaterials that mimic the extracellular matrix of bone, not only with respect to the chemical composition, but also to its structural and functional properties [20]. Calcium phosphate-based particulate scaffold materials are currently produced in varying calcium phosphate (Ca/P) ratios, with different solubility rates under physiological conditions. Calcium tetraphosphate (Ca4P2O9) has the greatest solubility rate, followed in decreasing order by anhydrous dicalcium phosphate (Ca2(PO4)), tricalcium phosphate (Ca3(PO4)2), and hydroxyapatite (Ca10(PO4)6(OH)2). Previous studies have shown that materials with a low Ca/P ratio resorb more rapidly, resulting in loss of mechanical strength, and those with greater Ca/P ratios, such as hydroxyapatite (HA), are more stable and degrade more slowly [21-23].
However, the chemical composition of the scaffold is not the only factor that determines the nature and extent of biodegradation. Physical material characteristics, such as crystallinity, crystal size, particle size, porosity and surface roughness have been reported to influence the biological performance of biomaterials. It is known that surface area and topography (mainly pore size) exert a significant influence over the interaction of osteogenic cells with the biomaterial surface [24, 25]. The particle size, for example, appears to affect not only the contact area but also the packing characteristics of the material, ultimately determining the interconnecting macro-porosity of the particle ensemble, which is crucial for bone regeneration. Additionally, recent studies have demonstrated that micro-porosities in the biomaterial significantly accelerate osseointegration [21, 24, 26-29].
The biomaterials that are used in bone regeneration procedures are available in different forms, including particles, powders, blocks, cements or hydrated pastes. However, because the macrostructural properties may influence the handling during clinical procedures, the physical form in which a material is delivered to the clinician may also have an important impact on osteogenesis, in particular on the cell and blood vessel colonization of the substrate.
The purpose of this chapter is to provide an overview of the physico-chemical properties of a variety of commercially available mineral-based biomaterials that are frequently used as bone fillers in periodontal and oral surgeries. Each product was studied with respect to the three following aspects: macromorphology, micro-morphology and chemical composition. To summarize the information, a data sheet for each material of interest was created to be used by the dental practitioner.
Materials and methods
A data sheet was created to summarize the physico-chemical characteristics of the eight biomaterials, on three scales: the macro-scale, (millimeters, 10-3m); the micro-scale, the size of a bone cell (micrometers, 10-6m); and the pico-scale, at the molecular level (10-12m) (Fig. 1.1).
Fig. 1.1:
Representation of the
The physicochemical characteristics were reviewed from the literature, and additional macroscopic and microscopic analyses using photography and scanning electronic microscopy (SEM), respectively, were performed. All samples were obtained directly from the manufacturers in sealed vials and were used without further treatment.
Macroscopic morphology analysis
High-magnification pictures were taken using a digital Nikon 200D camera with a 100-mm lens at the highest magnification. Additionally, higher magnification pictures (20X) were obtained using binocular stereozoom microscope (Zeiss, Germany).
Microscopic analysis
Samples were placed on carbon conductive tape on cupper tabs and sputtered with platinum for 45 s. SEM (JEOL 6400) was performed at the Institut des Matériaux de Nantes. The photos were taken using the secondary electron mode at 7 keV under the following magnifications: 200x, 500x, 1000x, 2000x, 5000x.
Additional characteristics of the materials, including macro- and micro-porosity, as well as total porosity, density, and specific surface area were extracted from the literature, if available.
The micro-porosityof the material describes the voids within the particle, which are also defined as micro-pores or the intra-particle space. These pores have diameters on the order of 10 µm. The macroporosity of the material corresponds to larger pores, ranging from 100 to 500 µm in diameter, which allow the invasion of cells, tissue and blood vessels.
The macro-porosityresults from the empty spaces between the particles, commonly designated as the inter-particle spaces. The size of these voids, which is related to the way the particles are packed, depends on the particle size and shape, as well as the particle size distribution. These macro- and micro-porosities are usually measured using mercury intrusion with an increasing pressure. The porosity is calculated as the ratio of the intruded volume to the total sample volume [30].
The total porosityof the particles is calculated from the sum of the intra-particle micro- porosity and the inter-particle interstitial spaces (macro-porosities).
The particle density(sample mass/volume of the solid) is determined using gas pycnometry [30]. This method excludes the granule interstices and most pores because the small gas molecules (helium) are able to penetrate almost all of the empty spaces.
The specific surface area (m2/g) is the total surface that is in contact with the bodily fluids and is measured using gas (nitrogen) adsorption. This value depends directly on the micro-topography and micro-porosity of the material [30].
Chemical analysis
To identify the crystalline phases of the biomaterials, high-resolution x-ray powder diffractometry (XRD) was usually used in the different reviewed studies. The XRD results indicate the chemical composition (presence of crystalline phases) and the amount of impurities, which can increase the risk of foreign body reactions [30, 31]. The American Society for Testing and Materials (ASTM) requires a phase purity of at least 95% (ASTM F 1088-04).
To precisely determine the water, organic material (such as collagen), and mineral (calcium phosphate) contents, thermogravimetric analysis (TGA) was used. During this analysis, incorporated water is lost at temperatures ranging from room temperature to approximately 200°C. Above temperatures of approximately 300°C, organic materials such as collagen, fat tissue, and proteins burn. At temperatures of approximately 400°C, only the mineral phase (calcium phosphate) remains. If the mineral contains some carbonate in the form of carbonated apatite, there is an additional mass loss between temperatures of approximately 400°C and 900°C [16,20].
Results
The results acquired from the literature review, as well as the images obtained using the magnifying device and SEM, have been summarized for each biomaterial in the form of a data sheetincluding :
1) the manufacturer’s description including the fabrication method and handling information, 2) the macroscopic morphology,
3) the microscopic morphology, and 4) the chemical composition.
Manufacturer’s description:
Bio-Oss® spongiosa granules, produced by Geistlich Pharma AG (Wolhusen, Switzerland), are reported to be a natural bone mineral, derived from bovine bone and containing carbonate apatite. The particle size of the granulate ranges from 0.25 to 2 mm. It is used primarily in dental surgery.
Fabrication method:
All organic material is removed by a stepwise annealing process (up to 300°C), followed by a chemical treatment (NaOH) that leaves a porous hydroxyapatite bone chip material.
Handling:
The hydrated particles can be handled with a spatula.
Fig. 1.2:
a) High-magnification photography shows that Bio-Oss® is a particulate biomaterial,
Trade name: Bio-Oss®
Company: Geistlich (www.geistlich.com) Country: Switzerland
Macroscopic morphology
The macroscopic morphology is shown in Fig. 1.2. a, b.
datasheet 1
datasheet 2
datasheet 3
datasheet 4
datasheet 5
datasheet 6
datasheet 7
datasheet 8
Microscopic morphology
The microscopic morphology is shown in Fig. 1.3. a, b.
Fig. 1.3:
Scanning electron micrograph of Bio-Oss®
particles at magnifications of a) 500x and b) 2000x. Higher magnification shows the granule surface and the presence of micro-porosities.
Chemical composition
Natural hydroxyapatite derived from bovine bone (Fig. 1.4, Table 1.1).
Total porosity: 63.5%
Intra-particule porosity: 51% (micro-pore size: 0.03 µm) Inter-particle porosity: 12.5% (macro-pore size: 400 µm)
Density: 3.21 g/cm3 Specific surface area: 59.7 m2/g
Fig. 1.4:
a) Morphology of the hydroxyapatite crystal (including the Miller
indices).
b) Hydroxyapatite (x,y) projection of the plane (4 unit cells).
[30]
a b
a b
Manufacturer’s description:
Bio-Oss® Collagen is a mixture of Bio-Oss® granules with identical properties con- taining 10% highly
purified porcine collagen fiber, provided in block form.
Handling:
The block is deformable and can be directly inserted into the defect.
Fig. 1.5:
(a)High-magnification photography shows that Bio-Oss® collagen is in the form of a soft block.
(b) Higher magnification image (2.5x) of Bio-Oss®
Trade name: Bio-Oss® Collagen
Company: Geistlich(www.geistlich.com) Country: Switzerland
Macroscopic morphology
The macroscopic morphology is shown in Fig. 1.5 a, b.
datasheet 1 datasheet 2
datasheet 3
datasheet 4
datasheet 5
datasheet 6
datasheet 7
datasheet 8
Microscopic morphology
The microscopic morphology is shown in Fig. 1.6 a, b.
Fig. 1.6:
Scanning electron micrographs of Bio-Oss® Collagen at magnifications of a) 500x and b)2000x.
Note the presence of collagen particles.
Chemical composition
Natural hydroxyapatite derived from bovine bone (90%) and natural collagen derived from porcine (10%) (Fig 1.7 and Table 1.2).
Total porosity: unavailable Intra-particule porosity: unavailable Inter-particle porosity: unavailable Density: unavailable Specific surface area: unavailable
Fig. 1.7:
a) Collagen fibril.
b) Morphology of the hydroxyapatite crystal (including Miller indices).
a b
a b
Manufacturer’s description
Cerasorb® M is a synthetic, porous, biocompatible, ceramic material made of pure-phase betatricalcium phosphates in granular form. Cerasorb® M has an interconnecting total porosity ranging from 60 to 70%.
Fabrication method:
The material is obtained using high-temperature calcination (ceramic sintering) processes according to the reaction:
2 CaHPO4+ CaCO3=> Ca3(PO4)2+ CO2 + H2O
Because the material is heated to more than 1000°C, microorganisms and pyro- gens are completely excluded. The production of the granules occurs by the com- pacting of the primary particles with a porogen. After the porogen is burned out, the compact body is broken and classified by granule sizes. This technique produces the porosity and the tamponade-like effect of the granules. After a sterilization step, the material is decanted and sterilized with Á-radiation (25 kGy).
Hanling:
The hydrated particles can be handled with a spatula.
Fig. 1.8:
a) High-magnification pho- tography shows that Cerasorb® M is a particulate biomaterial.
Trade name: Cerasorb M
Company: Curasan (www.curasan.de) Country: Germany
Macroscopic morphology
The macroscopic morphology is shown in Fig. 1.8a, b.
datasheet 1 datasheet 2 datasheet 3
datasheet 4
datasheet 5
datasheet 6
datasheet 7
datasheet 8
Microscopic morphology
The microscopic morphology is shown in Fig. 1.9a, b.
Fig. 1.9: Scanning electron micrograph of Cerasorb® M particles at magnifications of a) 200x and b) 1000x . The broken form of the granule
surface and the high porosity are visible.
Chemical composition
Synthetic beta-tricalcium phosphate (Fig. 1.10 and Table 1.3).
Total porosity: 65 +/- 5 %
Intra-particule porosity: 51 vol% ( micro-pore size: 15 - 50 µm) Inter-particle porosity: 14% ( macro-pore size: 100-500 µm)
Density: 3.066 ± 0.002 g/cm3µm) Specific surface area: unavailable
Fig. 1.10:
Beta- tricalcium phosphates chemical formula.
Data received from the manufacturer.
a b
Manufacturer’s description
Bone Ceramic® is a fully synthetic bone graft substitute of medical-grade purity in particulate form that is composed of biphasic calcium phosphate, a mixture of 60%
hydroxyapatite, which is 100% crystalline, and 40% beta-tricalcium phosphate.
Bone Ceramic® is 90% porous with interconnected pores with diameters ranging from 100 to 500 µm.
Fabrication method:
Unavailable.
Handling:
The hydrated particles can be handled with a spatula.
Fig 1.11:
a) High-magnification photography shows that Bone Ceramic® is a particulate biomaterial. b)
Higher magnification (2.5x) of the Bone
Trade name: Bone Ceramic®
Company: Straumann (www.straumann.com) Country: Switzerland
Macroscopic morphology
The macroscopic morphology is shown in Fig. 1.11a, b.
datasheet 1 datasheet 2 datasheet 3 datasheet 4
datasheet 5
datasheet 6
datasheet 7
datasheet 8
Microscopic morphology
The microscopic morphology is shown in Fig. 1.12a, b.
Fig. 1.12.
a) Scanning electron micrograph of Bone Ceramic® particles at magnification of a) 500x and b) 2000x. Note the absence of micro-porosities.
Chemical composition
Synthetic beta-tricalcium phosphate: 40%.
Synthetic hydroxyapatite: 60% (Fig. 1.13 and Table 1.4).
Total porosity: 90%
Intra-particule porosity: unavailable.
Inter-particle porosity: unavailable.
Density: unavailable.
Specific surface area: unavailable
Fig. 1.13:
a) Morphology of the hydroxyapatite crystal (including the Miller indices).
b) Beta- tricalcium phosphates chemical formula..
Data received from the manufacturer.
a b
a b
Manufacturer’s description
Natix® is manufactured using commercially-pure Grade 1 titanium and is available as irregularly shaped particles ranging in size from 0.7 to 1 mm in size, with a porosity of approximately 80%. Natix® is sterilized by heat and Á-radiation.
Fabrication method:
Unavailable.
Handling:
The hydrated particles can be handled with a spatula.
Fig. 1.14:
a) High-magnification photography shows that Natix® is a particulate biomaterial. b) Higher
Trade name: Natix®
Company: Tigran (www.tigran.se) Country: Sweden
Macroscopic morphology
The macroscopic morphology is shown in Fig. 1.14a, b.
datasheet 1 datasheet 2 datasheet 3 datasheet 4 datasheet 5
datasheet 6
datasheet 7
datasheet 8
Microscopic morphology
The microscopic morphology is shown in Fig. 1.15a, b.
Fig. 1.15:
Scanning electron micrograph of Natix®
particles at magnification of a) 200x and b) 2000x.
Note the presence of micro-porosities.
Chemical composition
Grade 1 titanium (Fig. 1.16 and Table 1.5).
Total porosity: 55.8%
Intra-particule porosity: unavailable
Inter-particle porosity: unavailable (micro-pore size: 241 µm).
Density: unavailable Specific surface area: unavailable
Fig. 1.16.
Titanium crystal.
[ ??]
a b
Manufacturer’s description
Genos® is a xenograft of material made of porcine cortico-cancellous particles and collagen. The granule size ranges from 600 to 1000 µm.
Fabrication method:
The xenogenic bone particles are not calcinated at high temperatures (the maximum process temperature is below 200 °C) to preserve the collagen of the porcine bone.
Handling:
The hydrated particles can be handled with a spatula.
Fig. 1.17:
a) High-magnification photography shows that Genos® is a particulate biomaterial. b) Higher
Trade name: Genos®
Company: Tecnoss Dental (www.osteobiol.com) Country: Italy
Macroscopic morphology
The macroscopic morphology is shown in Fig. 1.17a, b.
datasheet 1
datasheet 2
datasheet 3
datasheet 4
datasheet 5
datasheet 6
datasheet 7
datasheet 8
Microscopic morphology
The microscopic morphology is shown in Fig. 1.18 a, b.
Fig. 1.18:
Scanning electronic micrograph at magnification of (a) 500x and (b) 2000x of a Genos ® particle. Note the collagen fibers.
Chemical composition
Porcine hydroxyapatite and collagen (Fig. 1.19 and Table 1.6).
Total porosity: 33.1%
Intra-particule porosity: 12.1% (micro-pore size: 0.02 µm) Inter-particle porosity: 21% (macro-pore size: 400 µm)
Density: unavailable Specific surface area: 42.4 m2/g
Fig. 1.19:
a) Collagen fibril.
b) Morphology of the hydroxyapatite crystal (including Miller indices).
[30]
a b
a b
Manufacturer’s description
MP3® is a mix of cortico-cancellous particles of porcine origin (90%) that is collagenated and prehydrated in a porcine collagen gel. The granule size ranges from 600 to 1000 µm. The xenogenic bone particles are not calcinated at high temperatures (the maximum process temperature is below 200 C°) to preserve the collagen of the porcine bone. The 10% collagen gel added to the granules has the primary function of making the biomaterial sticky, stable and easy to handle.
Handling:
The material is injectable.
Fig. 1.20:
a) High-magnification photography shows that MP3® is a heterogen
Trade name: MP3®
Company: Tecnoss Dental (www.osteobiol.com) Country: Italy
Macroscopic morphology
The macroscopic morphology is shown in Fig. 1.20a, b.
datasheet 1
datasheet 2
datasheet 3
datasheet 4
datasheet 5
datasheet 6
datasheet 7
datasheet 8
Microscopic morphology
SEM of a hydrated material is not feasible.
Fig 1.11
Scanning electronic micrograph of Cerasorb® M
particles. The broken form of the granule
and the high porosity are visible. (x 200) Fig 1.12
Higher magnification of the granule surface, note the presence of microporosities.
Chemical composition
Porcine hydroxyapatite and collagen (Fig. 1.21).
Total porosity: unavailable Intra-particule porosity: unavailable Inter-particle porosity: unavailable Density: unavailable Specific surface area: unavailable
a b
Fig. 1.21:
a) Collagen fibril. (b) Morphology of the hydroxyapatite crystal (including Miller indices).
a b
Manufacturer’s description
Ostim® is a nanocrystalline, precipitated hydroxyapatite that contains approximately 40% water. It has a viscous, fluid-like consistency and can therefore be directly injected into a defect or mixed previously. This material is quite different than self-hardening bone cements as it is a fluid dispersion. It can be used in dental and orthopedic surgery.
Fabrication method:
Unavailable.
Handling:
The material is injectable.
Fig. 1.22:
a) High-magnification photography shows
Trade name: Ostim®
Company: Heraeus Kulser (www.heraeus-dental.com) Country: Germany
Macroscopic morphology
The macroscopic morphology is shown in Fig. 1.22 a, b.
datasheet 1
datasheet 2
datasheet 3
datasheet 4
datasheet 5
datasheet 6
datasheet 7
datasheet 8
Fig. 1.23.
Pure nanocrystalline hydroxyapatite (Ostim®) forms aggregates of needle-shaped crystals in transmission electron microscopy a) 7500x;
b)150 000x. [54]
Chemical composition
Synthetic hydroxyapatite nanocrystals (Fig. 1.24 and Table 1.VII).
Total porosity: unavailable Intra-particule porosity: unavailable Inter-particle porosity: unavailable Density: 1.000 g/cm3. Specific surface area: unavailable
Fig. 1.23.
a) Morphology of the hydroxyapatite crystal (including the Miller indices). b) Hydroxyapatite (x,y) projection of the plane (4 unit cells)
a b
Microscopic morphology
SEM of a hydrated material is not feasible. However, transmission electron microscopy reveals the needle-like shape of the hydroxyapatite nanocrystals (Fig. 1.23).
a b
Discussion and conclusion
Influence of biomaterial characteristics on osteogenesis at different scales.
The characteristics of a biomaterial can be evaluated at different scales, each of which appears to have an influence on osteogenesis (Figure 1.1).
Macroscopic morphology (milli-scale, 10-3m)
Calcium-based biomaterials used in the dental field to promote bone preservation/regeneration exist in different forms, such as particles, powders, blocks, cements or hydrated pastes. Some of these products contain particulate materials mixed with collagen, primarily to facilitate handling during surgery.
This aspect is further discussed in Chapter 5.
The particle sizeaffects not only the contact area but also the packing characteristics of the materials, ultimately determining the macro-porosityof the particle ensemble [26, 28]. Although the ultimate biological response cannot be predicted, smaller particles (approximately 300 µm) appear to result in a better performance [26, 28, 32]. However, controversy exists regarding the effect of porosity on bone regeneration. Induction of bone growth requires a mechanically stable surface that avoids micro-motion caused by debris and loose particles [30, 33].
Microscopic morphology (micro- to nano-scales; 10-6and 10-9m, respectively) Previous studies confirm that the surface area and scaffold architecture of the materials, particularly the pore size and pore interconnectivity, exert a major influence on the interaction of osteogenic cells with the biomaterial surface [24, 25, 34]. Although it is generally recognized that large pores (greater than 100 µm) enhance new bone formation [34], it has been reported that micro-porosity can be used to accelerate osteointegra- tion [24, 27, 29, 35]. Moreover, it has been shown that nano-porous structures improve cell adhesion, proliferation, and differentiation [26]. On the other hand, a trade-off exists between the porosity and mechanical performance of the scaffolds, with greater porosity resulting in reduced mechanical strength [34, 36].
Recent investigations support the hypothesis that surface reactivityalso plays a role in osteoinduction on the surface of the material; a dissolution-precipitation process takes place along with the coprecipitation of relevant endogenous factors, such as cytokines or proteins [33, 37]. This specific property was previously found to be associ- ated with specific material geometries [38, 39], favoring the binding of an optimal amount of endogenous bone morphogenetic proteins (BMPs), allowing the material to become osteoinductive. Researchers have specified that bone is not induced on mate- rials with a specific surface area below a certain low level; on the other hand, bone for-
high level of because of a fast resorption process. Although these findings imply that the optimal surface area that leads to maximal osteoinduction is a material-specific property, the minimal pore size requirement for this process has been shown to be approximately 100 µm [33] because of cell transport, migration requirements and nutrient transport.
Pore sizes between 300 and 400 ?m are recommended because of the formation of capillaries [30, 33, 40].
Chemical composition (pico-scale; 10-12m)
The rationale for the development of calcium phosphate-based biomaterials is their similarity to bone mineral in composition and in some physico-mechanical properties.
The natural bone mineral is essentially a carbonate hydroxyapatite rather than a calcium hydroxyapatite, approximated by the formula (Ca,X)10(PO4,HPO4,CO3)6(OH,Y)2, where X is one of the cations (Mg2+, Na+or Sr2+) that can substitute for the calcium ions, and Y is one of the anions (Cl-or F-) that can substitute for the hydroxyl groups [41]. For comparison, the formula of calcium hydroxyapatite is Ca10(PO4)6(OH)2. Dissolution is a first and necessary step in biomaterial-induced osteogenesis, providing saturation of the extracellular environment with calcium and phosphate ions as a prerequisite to matrix mineralization (Fig. 1.25). The composition (chemical properties) and also the microstructure and topography of the biomaterials strongly determine both the dissolution or resorption rate of the material and the ability of the material to facilitate cell proliferation, cell attachment and phenotypic expression [42-46].
The dissolutionof biomaterials primarily depends on the composition (i.e., the proportion of slowresorbing hydroxyapatite relative to fast-dissolving beta-tricalcium phosphate, with higher carbonate content leading to increased substrate resorption and osteoclastogenesis) [47]. For materials of similar composition, the extent of dissolution will depend on the method of preparation, which determines the particle size, porosity (both micro- and macro-porosity), specific surface area, and crystallinity [42, 48-50].
Fig.1.25:
Dissolution/precipitation schema of the process of bone formation in presence of CaP biomaterials. The acidic environment resulting from cellular activity produces a partial dissolution of Ca/P, leading to an increased
In bovine-derived apatite, the dissolution was shown to be the greatest from non-sintered bone without organic matrix, followed by non-sintered bone with organic matrix, and then sintered bone. This dissolution behavior can be explained by the crystal size, which is much greater in sintered (1000°C) bone than in non-sintered bone; CO3content;
and the protective effect of the organic matrix phase in acidic buffered conditions [51].
Recent studies have additionally shown that sintering temperatures and times have a great effect on the density of different apatites [52, 53], allowing the tailoring of both the mechanical properties and phase content of apatite.
For synthetic Ca/P materials, the extent of dissolution is the greatest for amorphous CaP, followed by alpha-tricalcium phosphate, and then hydroxyapatite [49], depending strongly on the origin of the material and the preparation method. Previous studies showed that incorporation of different ions causes changes in the morphological features, such as crystal size and shape, as well as in the dissolution properties of the apatite [50]. Several reports sustain the postulation that controlling carbonate substitution in hydroxyapatite may be a route to controlling biomaterial resorption. However, because the resorbing cells have not always clearly been characterized, it remains difficult to ascertain the biological effect of carbonate substitution on osteoclasts.
Conclusion
Although the described physicochemical and morphological characteristics influence the in vivobehavior of the biomaterial, they are not often taken into consideration when the biological performance of a material is evaluated, thereby producing conflicting results in the literature.
Furthermore, there are a limited number of studies that compared the physico-chemical properties and preclinical or clinical performances of commercially available bone substitutes. As shown in this study, these synthetic materials show significant differences depending on the manufacturing process. Some are sintered at high temperature, have low micro-porosity and thus low bioactivity, and others are able to induce ectopic bone formation when solely implanted in the muscles of animals. Comparative studies using different biomaterials implanted in standardized animal models are of great importance to provide proof-of-principle for the selection of the most appropriate bone substitute for a clinical application. Nevertheless, these types of studies are rarely found in the literature. Moreover, clinical studies comparing two or more groups of bone substitutes under standardized conditions are rarer. In conclusion, a detailed understanding of the influence of the material characteristics on the in vivobehavior of a biomaterial is crucial for the clinician to decide which grafting material to use for preservation or regeneration
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Abstract
Aim:The first objective of the present study was to compare the short-term and the continuing 3D volume stability of sub-sinusal bone regeneration in rabbits using different space fillers. The second objective was to assess qualitatively and quantitatively the early bone formation process and the behavior over a longer period of time of the regenerated bone.
Materials and methods:Rabbits underwent a double sinus lift procedure using: blood clot (Clot), autogenous bone chips (Auto) and bovine hydroxyapatite (BHA). Animals were sacrificed at 1 week, 5 weeks and 6 months. Samples were subjected to x-ray microtomography and histology. Variations in the volume of bone augmentations were calculated at different time points. Qualitative analysis was performed using 7-µm sections and quantitative histomorphometric analyses were carried out using SEM.
Results:From baseline (100%) to 5 weeks, the augmented volumes dropped to 17.3%
(Clot), 57.6% (Auto) and 90.6% (BHA). Afterwards, until 6 months, only 19,4 % (Clot) and 31,4 % (Auto) of initial volumes were found, while it remained more stable in the BHA group (84%). At 1 week, an initial osteogenesis process could be observed in the 3 groups along the bone walls. At 5 weeks, despite the volume dropped significantly, newly formed bone density was higher with Clot and Auto than with BHA.
At 6 months, bone densities were statistically similar in the 3 groups. However, after 6 months, the surface invaded by newly formed bone (Regenerated area) was signifi- cantly higher when BHA was used as space filler. In the BHA group, the space filer area remained quite stable and the density of the composite regenerated tissue (bone + BHA) reached more than 50% at 6 months.
Conclusions and clinical implications:The 3 space fillers allowed bone formation to occur. Nevertheless, augmented volumes dropped in the Clot and Auto groups, while they remained stable with BHA. Therefore, a slowly resorbable biomaterial might be suitable in sub-sinusal bone augmentation for preventing the re-expansion process and for augmenting the density of the regenerated tissues.
Introduction
The presence of pneumatized sinuses impairing implant placement in the posterior maxilla is a frequent anatomical situation [1]. To overcome this difficulty, sub-sinusal bone regeneration, more frequently called a “sinus lift”, has proven to be successful with various biomaterials used as space fillers [2]. Which biomaterial is the ideal choice for this purpose remains an open question.
Although autogenous bone is still often regarded as the gold standard in bone augmentation procedures, morbidity at the donor site as well as reports of significant levels of resorption whether intraoral, extra-oral, block or particulated bone were used, necessitate the consideration of alternative biomaterials [3-6].
Some authors have even reported successful sub-sinusal bone augmentation and implant outcomes after 1 and 3 years with only a blood clot and no grafting material [7-9].
It has been shown that, after tooth extraction in the posterior maxilla, the alveolar bone will be subjected to an internal resorption due to sinus expansion in an inferior direction [10]. Therefore, a re-expansion of the sinus can also occur after a regeneration procedure.
For instance, in one study [11], sinus reexpansion was observed on panoramic x-rays 3 to 10 years after sub-sinusal augmentation procedures using a 2:1 autogenous bone / xenograft mixture: at the end of the observation period, the majority of implants were found to protrude into the sinus. Moreover, it has been shown that, when re-expansion occurs below the apex of an implant, the sinusal membrane is in close contact with the implant [12, 13]. From those studies, it appears that implants do not interact with their neighboring bone in the same way as the periodontium of natural teeth. Therefore, the long-term tridimensional stability of the regenerated volume appears to be of major importance, as a re-expansion diminishes the total bone to implant contact.
The biological concept of sub-sinusal augmentations has been well described in animal models [14-18]. These studies showed that this concept corresponds to the biological concept of Guided Bone Regeneration (GBR): complete bone regeneration in a created space can occur after the placement of a sole blood clot. Nevertheless, the regenerated bone will be progressively resorbed by a re-expansion of the sinusal cavity due to the positive air pressure inside the sinus associated with nasal breathing. Some of these publications also showed that re-pneumatization can be counteracted by the placement of bovinehydroxyapatite (BHA) in combination with the blot clot.
Although the ability of bone to regenerate in a sub-sinusal model is well documented both in vivoand clinically, the early steps of bone formation and bone dynamics over time remain poorly explored.
The first objective of the present study was to compare the 3D volume stability of sub-sinusal bone regeneration in rabbits when a sole blood clot, autogenous bone or bovine hydroxyapatite are used as space fillers. The second objective was to qualitatively and quantitatively assess the early bone formation process as well as the continuing behavior of the regenerated bone with these three different types of filling material.
Material and methods
Animals
New Zealand White rabbits (adult, males, average body weight of 3.0 kg) were used in the study. All experimental procedures and protocols used in this investigation were reviewed and approved by the Institutional Animal Care and Use Ethics Committee of the University of Liège, Belgium. The "Guide for the Care and Use of Laboratory Animals", prepared by the Institute of Laboratory Animal Resources, National Research Council, and published by the National Academy Press, was followed carefully [19].
Study design
This study is part of an overall project where 96 sinus-lift procedures performed on 48 rabbits using 10 different types of space fillers were assessed at 3 distinct time points, 1 week, 5 weeks and 6 months, respectively. Specifically, the space fillers were randomly allocated to the sinuses and 16 rabbits were sacrificed at each time point, so that at least 3 sinuses were available for each space filler at each time point, yielding a two-factor experimental design (space filler and time) with repeated measurements. In the present study, 3 space fillers were compared: a coagulated blood clot (Clot), autogenous bone chips (Auto) or bovine hydroxyapatite (Bio-Oss®, Geistlich®, Wolhusen, Switzerland)(BHA). A total of 28 sinus-lift procedures were analyzed from 22 different rabbits.
Surgical procedure
Anesthesia of the rabbits was induced by administration of a ketamine/xylazine bolus (respectively 65/4 mg/kg, IM). 20 min after a fentanyl/dehydrobenzperidol premedication (0.22 ml/kg of a bolus 25µg/1.25 mg/ml IM). 2 hours before surgery, animals also received buprenorphin at a dose of 0.05 mg/kg. This was administered twice a day for
2 days. Surgical interventions were performed under strict sterile conditions. The surgical area was shaved and disinfected with iodine, and a straight incision was made to expose the nasal bone and the naso-incisal suture lines. The soft tissues were reflected with the periosteum in order to access to the upper bone wall of the sinus. Two ovoid windows (approximately 6x4 mm) were created bilaterally using a round diamond bur (Fig. 2.1a).
The membrane was carefully raised from the floor and lateral walls and the space-filling material was inserted into the created compartment (Fig. 2.1b). The volume of filling material was standardized to 0.4 ml per sinus using insulin syringes. Particulated autogenous bone was harvested from the skull using a scraper (Biomet 3i, Warsaw, Indiana, USA). Blood was collected from the ear vein and transferred into an insulin syringe to clot naturally before insertion into the sinus. The bony windows were covered with a resorbable membrane (Biogide, Geistlich, Wolhusen, Switzerland) and the wounds were sutured with 4/0 polyester thread (Permasharp, Hu Friedy, Rotterdam, NL). (Fig. 2.1c). Animals were sacrificed by injection of pentobarbital (200 mg/kg, IV, after the same premedication as for surgeries). Samples were dissected (Fig. 2.2) and soaked in fixative (6 % formol).
a b c
Fig. 2.1: Surgical procedure.
a) The maxillary sinus was opened and the membrane was pushed inwards.
b) The volume of space filler was standardized and it was inserted into the
created cavity using a customized syringe.
c) Windows were covered with collagen membranes before suturing.
Fig. 2.2:
Samples of rabbit augmented maxillary sinuses at 5 weeks:
a) BHA.
b) Autogenous bone chips.
