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Digital archive of theses discussed at the University of Pisa


Thesis etd-03092015-112843

Thesis type
Tesi di dottorato di ricerca
Thesis title
Advaced Microtomographic Techniques for in vitro and in vivo evaluation of Biomaterials in Orthopedic Research
Academic discipline
Course of study
tutor Prof. Giardino, Roberto
  • Biomaterials
  • Image Analysis
  • Imaging
  • Micro-CT
  • Microstructures
Graduation session start date
Micro-CT is a very useful non-destructive technique for the 3D study of bone, biomaterials and their interactions because it is able to supply structural and densitometric information by obtaining images of the internal structure of a small object with a high spatial resolution. Besides, Micro-CT is an important tool to study the interactions between biomaterials and bone tissue from a 3D point of view. Typical uses in biomedical fields include the study of in vitro bone samples and biomaterials such as hydroxyapatite (HA), bioactive glasses, pharmaceutical granules, metals and composites (polymers + HA or calcium phosphate). Usually, the most evaluated material parameter is porosity because it plays a dominant role in the biomechanical characteristics, the initial cell attachment and thus the subsequent tissue regeneration.
Due to the linear attenuation coefficient specific for every material, is important to set X-ray source voltage and image reconstruction corrections depending on involved materials.
More specifically, bone is a connective tissue composed by an average of about 2/3 of inorganic substances and 1/3 of organic substances. The main constituents of the inorganic matrix of bone is a mixture of HA and TCP while biomaterials in orthopedics can be enclosed within two categories:- biomaterials used as replacement of bone to stimulate tissue regeneration and that can be completely absorbed and degraded (mainly ceramic and polymers);
- biomaterials for prosthetic implants that have mechanical properties that ensure stabilization of fractures or damaged joints (mainly metals, ceramics or high density polymers).
Moreover, micro-CT, deriving from clinical CT, allow studying the interactions between biomaterials and bone tissue also in a longitudinal way. The statistical power of longitudinal studies compared to cross-sectional studies has a great meaning principally because the effect of an implanted biomaterial can be evaluated over time in the same sample. In fact, an in vivo micro-CT analysis allows an assessment and quantification of the development over time in healing processes due to the application of engineered medical devices. The ability to study the time evolution of anatomical changes occurring in the course of the experiment could exclude the use of different experimental groups and, thus, has a significant ethical meaning that should not be underestimated. Limitations of the in vivo micro-CT image acquisition are: the complexity of displaying small structures that are moving dynamically due principally to breathing and heart beating; the limitation in administered X-ray dose distribution per animal due to the destructive effects on living organisms and cells; and the linear attenuation coefficient specific for every material including animal soft and hard tissues.
The evaluation of the mineral content is another important and peculiar task of micro-CT. The variations in mineral content are determinable due to the principle that the gray levels of every micro-CT section give a map of the distribution of the absorption coefficients of the X-rays related to the analyzed sample. Such coefficients depend on the material density, from the atomic number of the elements that constitute it and from the incidental energy used. In orthopedic preclinical studies, usually two different densitometric parameters are evaluated: Bone Mineral Density (BMD) and Tissue Mineral Density (TMD). Mineralization is an important aspect in several bone disease evolution such as, for example, osteoporosis or osteoarthritis.The first year of the research was dedicated to the micro-CT analysis of different kinds of biomaterials in the pre-implantation phase studying new procedures to widen the acquisition possibilities and the kinds of quantitative analytical methods.
In particular the project followed 3 different research lines:
a) the study of thixotropic carboxymethylcellulose (CMC) hydrogels added with iron magnetic-nanoparticles (CMC-NPs) examining the differences in magnetic particles distribution and using a micro-CT freezing chamber to overcome the limitation of movements during the acquisition;
b) the study of the 3D cell (MG63) distribution seeded onto a polymeric scaffold using osmium tetroxide as contrast agent and developing a new micro-CT segmentation protocol;
c) the study of dimensional metrology establishing an approach for the quantification of wear in ZrO2 head prosthesis components using micro-CT and to validate the method comparing it with the gold standard, i.e. the gravimetric analysis.
The second year was dedicated to the study of the interactions between different types of biomaterials implanted in bone tissue. The micro-CT analyses were performed as a result of in vivo preclinical studies and clinical retrieved studies. During this year, the project was divided in the following research lines:
a) the evaluation of the in vivo behaviour of ceramic custom made prosthesis in a suitable animal model (adult sheep) at 6 and 12 months from surgical cranioplasty;
b) the evaluation of the characteristics of bone quality and its microarchitecture in retrieved metal-on-metal Metal-on-metal HR;
c) the analysis of granules characteristics using a new injectable multiphasic bone substitutes based on gel-coated OsproLife® HA/TTCP.
During the third year, the non “functional”, i.e. non quantitative, information obtainable from a micro-CT analysis was deepened, testing the most important computer algorithms for 3D visualization and modelling: maximum intensity projection (MIP), shaded surface display (SSD) and volume rendering (VR).
Moreover, the Micro-CT analyses performed were divided in the following research lines:
a) the evaluation of the in vivo osteoinductive behaviour of three-dimensional interconnected porous scaffolds of gelatin with or without contents of nanocrystalline HA over time;
b) the evaluation of bone quality in terms of mineral content in a study of osteoarthritis treatment in a large animal model with engineered hyaluronic acid scaffolds.
The main objective of this PhD research was to develop innovative techniques and procedures of 3D image analysis for the characterization of polymeric, ceramic and metal biomaterials used in various fields of bone tissue preclinical research.
In detail the aims can be summarized in:
– assessing micro-CT procedures applicable to pre-implanted biomaterials through both metrological studies and in vitro studies of 3D cell scaffold colonization, with the definition of effective segmentation techniques;
– developing micro-CT techniques to evaluate different kinds of implanted biomaterials both ex vivo and in vivo establishing standard test protocols and identifying the mechanisms of material resorption and degradation in physiological environment and the mechanisms of bone remodeling;
– exploring the densitometric analysis in relation to the contribution of mineralization in healing process at peri-implant site.
These program objectives have been achieved through the development of reliable experimental procedures for the morphological and mechanical evaluation of implantation biomaterials (scaffold and prostheses); the investigation of a large number of possible applications of biomaterials in orthopedic preclinical studies through in vitro, ex-vivo and in vivo micro-CT analysis; the elaboration of new interpretative models of the bone regeneration mechanisms through 3D morphometric parameters; and the extension of the knowledge and the expertise in 3D biomaterial evaluations used in orthopedic research.