Influence of nanoscopic bone composition and structure on microscopic elastic properties at the BII

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Bone components and hierarchical structure

Bone tissue is a composite material composed of around 70 % of an inorganic phase and 30 % of an organic phase. The inorganic phase is a mineral constituted of hydroxyapatite Ca5(P O4)3OH crystals. The organic phase is mainly composed of type I collagen, of non-collagenous proteins, proteoglycans (GAGs) and lipids [163, 172]. Both phases contain water as well. At the scale of tens of nanometres, the collagen molecule is a triple helix made of three polypeptides which are chains of amino acids [113] such as phenylalanine, proline, and hydroxyproline. At the scale of hundreds of nanometres, bone crystals, which are thin plate-shape crystals, gather into crosslinks between collagen molecules forming bundles, grouped into mineralised collagen fibrils (Fig. 1.1) [113, 209]. Surrounded by extrafibrillar crystals, fibrils finally gather into lamella at the scale of 2–9 m forming lamellar bone microstructure [189].
Composed of the same nano- and microscale hierarchical structures previously de-scribed, lamellar bone tissue assembles into two distinctive structures at the mesoscale. Cortical bone, the outer layer of bone (Fig. 1.1 and Fig. 1.2), is a dense and stiff tissue made of osteons at the scale of 100 m. Under the cortical layer, trabecular bone (Fig. 1.1 and Fig. 1.2) is a more porous and softer tissue, made of plates and rods.

Bone remodelling and healing

Bone is a dynamic, vascular, living tissue that changes throughout life [45] as its matrix is formed, repaired and restructured in a continuous process called bone remodelling. Every year, an adult remodels 25 % of his trabecular bone and 4 % of his cortical bone [59].
Several bone cells are involved in the bone remodelling process. First, osteoclasts remove the old bone tissue. After bone resorption, osteoblasts form new bone tissue by producing bone organic matrix and depositing hydroxyapatite enabling then bone mineralisation. Some osteoblasts are trapped during the bone formation process and become osteocytes. The precise role of those cells is still investigated, but osteocytes, in association with bone lining cells on the bone surface, appear to be part of the signalling pathway triggering the bone remodelling process in response to mechanical stress [107].
This basic multi-cellular unit ensures bone formation in four different situations: i) ini-tial formation of bone in embryos and fetus ; ii) bone growth during youth until reaching an adult size ; iii) bone remodelling during all life ; and iv) bone healing. During adult life, in addition to the continuous bone remodelling, bone formation is triggered when bone is affected by microcracks, fracture or bone damages due to a disease or surgery. To reconstitute the tissue continuity, a healing process will then start creating first an irregular woven bone which will later undergo remodelling to form lamellar and structured bone [45]. The case of healing around an implant after surgery, studied in this thesis, is presented in the next section.

Bone-implant interface (BII)

Implants

Nowadays, trauma injuries provoked by road traffic, sports and work accidents often occur. Moreover with population ageing, bone disorders, like osteoporosis weakening bone, become more and more common. In these clinical situations, inserting implants characterised by their design and their biomaterials within bone tissue has become a common practice in orthopaedic and dental surgery [221]. Replacing a joint or a tooth, the main functions of implants are to provide mechanical and structural support, restore the functionality of the treated organ, integrate with the damaged tissue and promote healing [3].
Implant material is chosen to be biocompatible and to have mechanical properties adapted to the implantation site [221]. More than 70 % of implants (and up to 95 % in or-thopaedics) are made of metal [79]. Metal implants are used for their mechanical support. They aim to strongly bind to bone, limiting movements between implant and host tissue, and providing physiological load bearing functionality to the implantation site [3]. With their biocompatibility, low elastic modulus and high resistance to corrosion [180], titanium alloys are often used to design orthopaedic and dental implants, especially Ti6Al4V (the material studied in this thesis) composed of 90 % of titanium (Ti), 6 % of aluminium (Al) and 4 % of vanadium (V).

Implant stability and osseointegration

Implant stability is essential as it determines the long-term surgical success. Imme-diately after surgery, primary stability mostly depends on the surgical procedure, the implant design and bone properties at the implantation site [76]. Primary stability of the implant is a necessary condition to obtain a long-term stability resulting from osseointe-gration phenomena.
During the weeks following surgery, peri-implant healing process takes place at the implantation site and bone tissue develops around the implant, and in particular in direct contact with the implant surface. Initially defined by Branemark et al. in 1977 [27] and by Albrektsson et al. in 1981 [7], osseointegration is described as a direct contact between the living bone and implant on the microscopic level [7]. It creates a bone-implant interface (BII), which allows the long-term implant stability defined as secondary stability. Secondary stability relies on the implant’s design, material and surface properties, on the frictional properties between the implant and surrounding bone, as well as on the geometry and quality of bone tissue at the implantation site and its remodelling activity [76].
During osseointegration, osteoinduction occurs [6] as osteogenic cells differentiate into osteoblasts which will synthesise new bone through a woven collagenous matrix, later mineralised into bone and the whole process will spatially progress. Contact osteogenesis corresponds to de novo bone formation on the implant surface which progresses towards the host bone. In parallel, distance osteogenesis corresponds to bone formation from existing host bone following surgery and progresses towards the implant surface [45, 112]. When both contact and distance osteogenesis occur, bone tissues formed in the two di-rections merge at late osseointegration stage representing the end of the healing process with a tight BII [77].

Osseointegration performance

Despite their routine clinical use, failures of implant surgery still occur and improve-ment is needed [3]. One reason of osseointegration failure is the amplitude of micromo-tions at the BII. During bone healing, low-amplitude micromotions (lower than 40-70 m) stimulate bone remodelling, whereas fibrous tissues may develop instead of an osseointe-grated interface in the case of excessive interfacial micromotions (above 150 m) following surgery [26]. The effect of micromotions explains why primary stability (subsection 1.2.2) should not be too low to avoid excessive interfacial micromotions [53, 197] causing implant failure. It should also not be too high since an excessive level of stresses may lead to bone necrosis [44, 54].
To improve osseointegration outcomes, various factors have been identified to affect the amplitude of micromotions and bone properties at the implantation site, such as the surgical procedure, as well as the implant geometry and material. Among implant prop-erties, surface roughness influences osseointegration. Surface roughness can be modified with sandblasting and/or chemical treatments [121, 186]. It is usually evaluated using the Sa value, the average surface roughness, measured by mechanical contact profilometres, optical profiling devices or scanning probe microscopes [218]. Rough surfaces increase the friction coefficient at the BII, thus reducing micromotions and increasing primary stabil-ity. Moreover, rough implants present a higher specific surface area on which bone cells can interact, also stimulating bone tissue repair [67]. However, a compromise should be found with a high roughness to sufficiently stimulate bone remodelling without creating stress concentration and debris, which could damage bone tissue and thus hamper the osseointegration process. In particular, surface roughness between 3.6 and 3.9 m seem to be optimal for osseointegration [179].

Bone content and tissue quality at the BII

As explained in subsection 1.2.2, the BII formed during healing will condition long-term stability. Therefore, to evaluate osseointegration performances, the BII needs to be fully characterised to identify properties ensuring long-term implant stability. Resulting in the formation of the BII, osseointegration phenomena lead to an increase in the amount of bone tissue in direct contact with the implant surface. The obtained bone-implant contact (BIC) at the BII, defined as the first tens micrometre-thick layer of bone tissue at the implant surface, enables implant anchorage and is besides an important determinant of implant stability [76]. Another factor is the amount of bone tissue in the surroundings, further from the implant surface, which also develops during healing. This bone content beyond the direct contact layer around the implant surface is defined as bone quantity [76]. Within bone ingrowth, bone properties evolve during mineralisation as bone adapts to its mechanical environment at every scales from its composition to its nano-, micro-and mesostructure, inducing changes in its mechanical properties [67]. The combination of bone structural, compositional and mechanical properties define bone quality [50]. Therefore, bone-implant contact, bone quantity and quality within a region of interest located at a distance up to 200 m from the implant surface determine surgical success [76] and should then be investigated.

In vivo models of the BII

The study of osseointegration to understand implant stability requires to be able to replicate the conditions of surgical implantations. Different strategies can be adopted. In vitro studies consider bone-implant contact outside any living being using synthetic materials to mimic bone tissue [216]. Conversely, when the implant is inserted within a living being to monitor osseointegration evolution during healing, this is an in vivo study [212, 215]. In vivo studies in the human body are called clinical studies [111]. At the end of an animal in vivo study, samples can be harvested to conduct ex vivo analyses on the resulting osseointegrated implants [212]. However, due to the interdependency of all bone parameters and properties, bone experimental characterisation may not give clear explanations, justifying in silico approaches to characterise the BII at various scales using computer simulation [85, 134, 214].

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Animal models

In vivo animal studies are interesting ways to produce osseointegrated samples for ex vivo analyses as in vitro studies cannot replicate many of bone features. Even if bone tissue has many common points throughout species, every animal model is specific. Common animal models include mice, rabbits, sheeps, pigs, dogs or primates [167, 205]. The choice of the animal model depends on the topic of the study (such as bone disease or characteristic bone structure) and the animal convenience (such as size, housing or treatment).
Rabbit (often New Zealand white rabbit) is a common animal model to study bone ingrowth and the BII around an implant [205], on the distal part of the femur or the bilateral tibiae [122]. Despite different size, shape and loading, rabbit and human bones have similar composition with comparable bone mineral density and fracture toughness of mid-diaphyseal bone [122, 167]. Rabbit bone microstructure is made of vascularised osteons parallel to the long axis of the bone [167]. Furthermore, rabbits quickly reach skeletal maturity at only 6 months and have a faster bone turnover than other species (primates or some rodents, for instance) [122, 143, 167, 205]. This small animal model is also often chosen for its availability and convenience in housing and treatment compared to other species such as sheeps or pigs [122, 167].

Implant models

Different implant models have been studied in the literature, from clinical implants to adapted models to isolate phenomena and consider standardised configurations aiming at a better understanding of the osseointegration process.
Clinical dental implants have often been inserted into in vivo animal models to eval-uate bone composition and structure [89] as well as mechanical properties of the tissue developed around the implant [36] or within the thread [228], in order to understand im-plant stability [212]. To isolate the implant surface from its biological environment and study the dependency between contact and distance osteogenesis (subsection 1.2.2), the implantation setup was modified by inserting a titanium tube between the implant and neighbouring bone (Fig. 1.3A) [38].
C) Hollow dental screw with a bone chamber inside [17]. D) Implant design with an inner hollow canal (adapted from [114]). E) Titanium pin inserted in rat tibia [46]. F) Cut view of the subperiosteal titanium plate. The bone chamber filled with new bone is surrounded by red [42]. G) Hydraulic bone chamber [109].
Around clinical implants, newly formed and mature bone tissues are intertwined, which does not allow precise characterisation of new bone tissue. To study more specifically the bone ingrowth resulting from healing, bone chamber compartments isolating newly formed bone were created within threads (Fig. 1.3B) by modifying the shape of cylindrical titanium implants [30] and of commercial titanium screw implants [19]. A bone chamber was also designed inside a hollow titanium dental screw (Fig. 1.3C), where new bone could develop thanks to ingrowth openings and bone graft could be introduced [17]. Other implant designs were used to place bone graft [49, 114] or bone substitute [49] in an inner canal to evaluate the impact on bone growth located in the bone chamber in the inner hollow canal and/or outside in the gap between the implant surface and cortical bone (Fig. 1.3D) [49, 100, 114].
Complex implant geometry generates multi-axial stress distribution at the BII, making the process inducing the observed bone properties difficult to understand. Thus, simplified implant geometry have been used such as non-weight bearing titanium rods (Fig. 1.3E) avoiding any mechanical effect of the threading to characterise more specifically bone microstructure at the BII [46, 124] and its strength [46]. Implant geometry has also been adapted to study a plane BII limiting complex stress distribution with, for example, a subperiosteal titanium plate inserted with a trough to create a bone chamber (Fig. 1.3F) [42]. Cylindrical coin-shaped implants also allow a plane BII and such implants were used with the hydraulic bone chamber (Fig. 1.3G) where cyclic compressive loading through an hydraulic pressure was applied to assess the effect on bone microstructure within a new bone chamber [109].

Experimental characterisation methods

Similarly, based on the implant model described in [175] (Fig. 1.4A), a coin-shaped implant model presented in Fig. 1.4B has been designed our group [136, 139] and is used in this thesis. Its simple geometry with a planar interface allows reproducible and standardised biomechanical conditions. The coin-shaped Ti6Al4V implant is surrounded by a polytetrafluoroethylene (PTFE) cap in order to i) limit bone growth and attachment on its lateral sides and ii) create a bone chamber between the cortical bone surface and the implant. The bone chamber is initially empty and will be filled with new bone tissue during healing, allowing a clear distinction between newly formed and mature bone (see Fig. 1.4B).

Table of contents :

Chapter 1. Bone and osseointegration 
1.1 Bone tissue
1.1.1 Bone components and hierarchical structure
1.1.2 Bone remodelling and healing
1.2 Bone-implant interface (BII)
1.2.1 Implants
1.2.2 Implant stability and osseointegration
1.2.3 Osseointegration performance
1.2.4 Bone content and tissue quality at the BII
1.3 In vivo models of the BII
1.3.1 Animal models
1.3.2 Implant models
1.4 Experimental characterisation methods
1.4.1 Bone-implant contact, bone quantity and structure
1.4.1.1 Quantitative ultrasound (QUS) technique
1.4.1.2 Histology
1.4.1.3 Micro-computed tomography (CT)
1.4.1.4 X-ray scattering techniques
1.4.1.5 Other techniques evaluating bone structure
1.4.2 Bone composition
1.4.2.1 Raman spectroscopy
1.4.2.2 Other techniques evaluating bone composition
1.4.3 Bone mechanical properties
1.4.3.1 Nanoindentation
1.4.3.2 Micro-Brillouin scattering
1.4.3.3 Macroscopic tests
1.4.3.4 Other techniques evaluating bone mechanical properties
1.5 Numerical models
1.5.1 Mechanical models
1.5.1.1 Modelling the BII
1.5.1.2 Complementing the experimental characterisation methods
1.5.1.3 Preventing stress shielding
1.5.2 Acoustical models
1.6 Key points
Chapter 2. Bone-implant contact at the BII
2.1 Introduction
2.2 Methods
2.2.1 Implants
2.2.2 Topographical analysis
2.2.3 Surgical procedure
2.2.4 Quantitative ultrasonic (QUS) device
2.2.5 Signal processing
2.2.6 Histological analysis
2.2.7 Measurement errors and statistical analysis
2.3 Results
2.3.1 Implant surface characterisation
2.3.2 QUS analysis
2.3.3 Histological analysis
2.3.4 Comparison of QUS and histological measurements
2.4 Discussion
2.4.1 Bone-implant contact increases with healing time
2.4.2 Heterogeneity of bone-implant contact during osseointegration
2.4.3 Implant surface roughness influences osseointegration
2.4.4 Validation with the numerical microscale BII model
2.4.5 A higher sensitivity of the QUS technique compared to histology
2.4.6 Influence of bone quality
2.4.7 Limitations
2.5 Conclusion
2.6 Key points
Chapter 3. Influence of nanoscopic bone composition and structure on microscopic elastic properties at the BII
3.1 Introduction
3.2 Methods
3.2.1 Sample preparation
3.2.2 Raman spectroscopy
3.2.3 Nanoindentation measurements
3.2.4 Statistical analysis
3.3 Results
3.4 Discussion
3.4.1 Composition and mechanical properties within newly formed and mature bone
3.4.2 Bone mineral phase of newly formed tissue under remodelling
3.4.3 Immature organic phase within newly formed bone
3.4.4 A lower mineral content in newly formed bone
3.4.5 Effects of compositional and structural changes on bone elastic properties
3.4.6 Limitations
3.5 Conclusion
3.6 Key points
Chapter 4. Spatio-temporal variations of bone elastic properties at the BII 
4.1 Introduction
4.2 Methods
4.2.1 Sample preparation
4.2.2 Histological analysis
4.2.3 Nanoindentation measurements
4.2.4 Micro-Brillouin scattering measurements
4.2.5 Statistical analysis
4.2.6 Local density estimation
4.3 Results
4.3.1 Histological analysis
4.3.2 Nanoindentation measurements
4.3.3 Micro-Brillouin scattering measurements
4.3.4 Local relative variations and density estimation
4.4 Discussion
4.4.1 Bone elastic parameters
4.4.2 Effect of healing time on bone elastic properties
4.4.3 Spatial variations of bone elastic properties at the BII
4.4.4 Contact osteogenesis within the bone chamber
4.4.5 Limitations
4.5 Conclusion
4.6 Key points
Chapter 5. Perspectives 
5.1 Further characterisation of the BII
5.1.1 Towards a better quantification of bone growth at the BII
5.1.2 Further biomechanical characterisation of the BII
5.1.2.1 Taking into account bone anisotropy and viscoelasticity
5.1.2.2 Investigating the rupture of the BII under mechanical loading
5.1.2.3 Characterisation of bone-implant friction
5.1.3 Effect of mechanical loading during osseointegration
5.1.3.1 Bone adaptation
5.1.3.2 Mechanotransduction
5.2 Clinical applications and medical devices
5.2.1 Optimising clinical implants and treatments
5.2.1.1 Optimisation of the implant surface properties
5.2.1.2 Optimisation of medical treatments
5.2.2 Developing medical devices for stability assessment
5.3 Key points

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