Multimodal characterization of the bone-implant interface using Raman spectroscopy and nanoindentation
Introduction
Titanium implants are widely used in orthopedic and dental surgeries, and their long-term stability relies on successful osseointegration during the healing period. Periprosthetic bone tissue forms and remodels at the bone-implant interface (BII) to achieve direct contact with the implant, adapting the tissue properties to the implant mechanical environment. The properties of newly formed bone tissue are key parameters for the implant surgical success, which clinically translates to a long-term stability [1]. Specifically, implant stability depends on the bone quantity (amount of bone) and bone quality (mechanical properties, biochemical composition, and structure of bone tissue) in a region of interest located at a distance less than about 200 µm from the implant surface [2]. Incomplete osseointegration or degradation of the bone properties (quantity and quality) at the BII during healing may ultimately lead to a lack of implant stability and implant failure, which would require a new surgery for the patient [3]. Therefore, a better understanding of the evolution of the BII properties during healing is essential for the prediction of surgical outcomes [4].
Various animal studies have been carried out to investigate the properties of newly formed bone tissue around clinical implants [2]. However, the complex geometry of commercial implants leads to multiaxial stress distribution at the BII, making it challenging to understand the determinants of osseointegration phenomena. Thus, we used an in vivo implant model with a planar interface [5], [6], [7], [8], [9] to work under more reproducible and standardized conditions. A coin-shaped implant model was designed, based on prior studies [5], [10], [11], including a gap of several hundred micrometers below the implant surface. Initially empty, the corresponding volume was filled with bone tissue during healing, thus allowing, unlike in clinical implants, to clearly distinguish between mature and newly formed bone [11], [12], [13], [14], [15], [16].
The evaluation of the BII properties is complicated due to the multiscale and composite nature of bone and its constant evolution [17]. Bone mass consists of around 70% of inorganic phase and 30% of organic phase by weight. The inorganic phase is a mineral made of hydroxyapatite (Ca5(PO4)3OH) crystals, while the organic phase contains collagen (mainly type I), non-collagenous proteins, and lipids [17], [18], [19]. The bone has a highly hierarchical structure. At the scale of tens of nanometres, the collagen molecule is a triple helix made of three polypeptides, which are chains of amino acids [20] such as phenylalanine, proline, and hydroxyproline (formed from proline). At the scale of hundreds of nanometres, the mineralized collagen molecules are grouped into bundles forming fibrils, stabilized by cross-links [20], [21]. Surrounded by extrafibrillar crystals, fibrils assemble into fibers to form the bone ultrastructure at a scale of 1–10 µm [22].
Because of the hierarchical structure of bone, a multimodal and multiphysics methodology is necessary to study biomechanical properties of bone [2], [3], [22], [23]. Nanoindentation has been used to measure the microscale elastic properties of the BII at dental implants [24], [25] to assess the effect of implant material and surface treatment [24], [26], [27], mechanical loading [28], and bone maturation and healing [27], [29]. The differences in elastic properties between the newly formed bone tissue and pre-existing mature bone have been characterized around a titanium plate [30] and around the aforementioned coin-shaped implants [13], [16]. Quantitative ultrasound techniques such as micro-Brillouin scattering [11] and echographic analyses [15] have also been used to obtain information on the biomechanical properties of the BII. Torsion tests were carried out to measure the effective adhesion energy of the BII [14]. However, to the best of our knowledge, the relationship between the microscopic mechanical properties of newly formed bone tissue around an implant and its biochemical composition, including mineral crystals and collagen structures, has not been studied.
Raman spectroscopy is an attractive technique to evaluate the local bone tissue biochemical composition at the nanoscale, which includes the mineral (hydroxyapatite) and organic (collagen components, non-collagenous proteins, and other organics) phases. Raman spectroscopy has been used to characterize the effects of metabolic disorders on bone tissue [31], [32], [33] and to investigate the remodeling process of healing bone tissues [34], [35]. Some studies have characterized bone tissue remodeling in the surroundings of composite biomaterials [36], [37] or titanium implants [38], [39]. However, these studies did not allow to distinguish between mature and newly formed bone tissues clearly.
This study aims to develop a multimodal and multiphysics approach to investigate the microscopic mechanical and compositional properties of periprosthetic bone tissue. Thus, Raman spectroscopy and nanoindentation are used in site-matched regions of interest, in mature and newly formed bone tissues, using a dedicated animal model by inserting a coin-shaped titanium implant in a rabbit femur for 10 weeks.
Section snippets
Implants
A Ti-6Al-4V coin-shaped implant (diameter of 5 mm, length of 3 mm), blasted with titanium dioxide particles (average surface roughness Ra = 1.9 µm), was surrounded by a polytetrafluoroethylene (PTFE) cap to avoid bone tissue growth around the implant and create a 750 µm gap underneath the implant, to host new bone tissue (see Fig. 1).
Surgical procedure
The implantation protocol has been detailed in previous work [11]. A New Zealand white rabbit (>3.5 kg) was anesthetized, and a 5 cm longitudinal incision was made
Results
The results obtained for the different parameters derived from Raman spectroscopy and nanoindentation are shown in Table 2, which compares the values obtained for mature and newly formed bone tissues.
For the mineral phase, the mineral crystallinity is not significantly different between mature and newly formed bone tissues, while the carbonate-to-phosphate ratio is significantly higher for newly formed bone tissue. Moreover, the standard deviation of mineral crystallinity is seven times higher
Discussion
A multimodal and multiphysics experimental approach, combining Raman spectroscopy and nanoindentation, was conducted to investigate the local compositional and mechanical properties of the newly formed bone tissue around an endosseous implant, which constitutes the originality of the present study.
Conclusions
The multimodal and multiphysics experimental approach coupled with the dedicated in vivo model represents a powerful strategy to investigate the properties of newly formed bone tissue around the BII. The composition and structure of newly formed bone show a lower mineral content, lower crystallinity, lower collagen cross-link ratio, and higher remodeling rate compared to mature bone. These characteristics explain the lower elastic properties compared to mature bone tissue. The results indicate
Declaration of Competing Interest
None of the authors has any conflicts of interest.
Acknowledgments
This project has received funding from the European Research Council (ERC) under the European Union's Horizon 2020research and innovation program (Grant agreement No. 682001, project ERC Consolidator Grant 2015 BoneImplant). This project has also received funding from the European Union's Horizon 2020research and innovation program under the Marie Sklodowska-Curie grant agreement No. 797764. This research was also partially supported by the National Science Foundation Grant DMR 15–07169 (IJ).
Ethical approval
Animal handling was approved by the ethical committee of ENVA (Ecole Nationale Vétérinaire d'Alfort) and followed the requirements of The European Guidelines for Care and Use of Laboratory Animals.
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