Tissue Engineering (TE) is the biotechnology that combines aspects of medicine, biology, and engineering to generate, repair, or replace human tissues. In particular, the TE approach may be used to regenerate bone by implanting a porous ceramic scaffold combined with bone marrow stromal cells (BMSC) in vivo. The scaffold plays a fundamental role since it acts as a guide and it stimulates the growth thus creating TE constructs or living biocomposites [1,2].
We develop a new methodology, based on different complementary X ray experimental techniques coupled to new analytical tools, which allow us to obtain structural information of different kind of tissue engineering from the atomic to the micrometric scale.
Our work aims to provide a full comprehension of the morphology and functionality of the mineralized biological tissue, giving the opportunity to mimic nature for the development of bio-inspired materials.
Biomineralization
Biomineralization (BM) is the widespread and fascinating process by which organisms form mineral materials, in organized crystals. In the BM process, ions in solution are converted in solid composites (biominerals) thanks to chemical-physical transformations performed by the cellular activity. The process creates sophisticated composite materials, composed of organic and inorganic compounds, with a wide range of properties depending on the many different functions they have to carry out. These include the mechanical functions of exo- and endo-skeletons and free-ions regulation or sensing. The research goals in BM are to understand the underlying mechanisms that organisms use to control mineral formation. Only the full comprehension of the morphology and functionality of the bio mineralized tissue, (i.e., shell, bone and teeth), will provide the opportunity to mimic nature for the development of bio-inspired materials.
In particular, a deeper comprehension of the BM process is at the basis of tissue engineering and regenerative medicine developments. Several in-vivo and in-vitro studies were dedicated to this purpose via the application of 2D and 3D diagnostic techniques. However, due to the complexity of the process, a complete and exhaustive explanation, still far to be reached, requires the synergy of different advanced experimental techniques.
In this framework, we have developed a new methodology, based on different complementary X ray experimental techniques coupled to new analytical tools which allowed us to obtain structural information of different kind of tissue engineering from the atomic to the micrometric scale.
High resolution X-ray Phase contrast Tomography(XrPCT), performed on a bone tissue, provided the 3D spatial distribution of the different Bone phase (Bone (B), Scaffold (SC), Soft Tissue (ST)) (figure 1a). High resolution scanning X-ray micro diffraction (uXrD) is a powerful tool to distinguish and monitor the evolution of the different phase (Collagen, Organic Matrix, HA, ACP) in the regenerated bone (figure 1c) at the organic-mineral interface within a porous scaffold.
Thanks the combination with X ray Fluorescence (XRF) (figure 1d) it is possible to verify the chemical evolution of the different growing phases, and to study the distribution of Ca in the regenerated bone.
The aim of our work is to prove a full comprehension of the morphology and functionality of the biomineralization process, which is of key importance for developing new drugs for preventing and healing bone diseases and for the development of bio-inspired materials.
Collaborators
ID 13 and 17, European Synchrotron Radiation Facility, ESRF, Grenoble, France
TOMCAT, Swiss Light Source, Paul Scherrer Institut, Villigen, & Centre d’Imagerie BioMedicale, Ecole Polytechnique Federale de Lausanne (Switzerland)
SYRMEP, Elettra – Sincrotrone Trieste S.C.p.A. (Italy)
Institute of Crystallography – CNR, Monterotondo, Rome (Italy)
Department of Experimental Medicine, University of Genova & AUO San Martino – IST Istituto Nazionale per la Ricerca sul Cancro, Genova (Italy).
Vascolarization
An ideal scaffold should present high porosity, maximum surface area for bone growth, and an interconnected pore space with pores having a sufficiently large size to allow the penetration and diffusion of the blood vessels [1]. Indeed, the efficiency of an artificially implanted construct depends on the timely delivery and exchange of nutrients (oxygen, glucose, amino acids, etc.) from surrounding blood vessels to the BMSC, and the contemporary removal of the metabolism waste products (CO2, lactate, and urea) [2-3]. Therefore, the control of the angiogenesis of the microvascular network with proper spatial organization is a key step to obtain tissue regeneration and repair [2]. Furthermore, a deeper understanding of the developmental neo-vascularization is necessary for a better treatment of many pathological conditions, including cancer, diabetes, psoriasis, and articular degeneration. In this framework, the need to detect subtle changes in tissue microvasculature requires the use of minimally invasive and bulk-sensitive experimental techniques. Currently, conventional 2D and 3D characterization techniques have limitations.
A 3D imaging spanning from a few millimeters to hundreds of nanometers, able to discriminate the smallest micro-capillaries and the volume of the scaffold with and without invasive contrast agent and without aggressive sample preparation, is extremely desirable.
In this framework, we used high-resolution in-line propagation XRPCμT for imaging the 3D vascular network in bone engineered constructs, in an ectopic bone formation mouse model using different staining preparation: (i) with MICROFIL® (Flowtech, Inc., Carver, Massachusetts); (ii) with phosphotungstic acid (PTA); (iii) without staining. The SXRPCμT was able to visualize the 3D vascularization network inside the scaffold, without any sample sectioning.
We clearly demonstrated and quantitatively analyzed in the samples where this vascularization was present (Figure 1) [4]. Moreover, our data also suggest that seeding the scaffolds with BMSC enforces the vascularization. The major difference lies in the average number of branches forming the vascular trees. The sample not seeded with BMSC was poorly ramified, while the other three samples displayed thriving trees with many branches.
In this framework, we have developed a new methodology, based on different complementary X ray experimental techniques coupled to new analytical tools which allowed us to obtain structural information of different kind of tissue engineering from the atomic to the micrometric scale.
Even though SXRPCT was able to visualize the 3D vascularization network inside the scaffold without any sample sectioning and preparation, in order to achieve a higher image quality with sub-micrometer spatial resolution, the use of a coherent, highly brilliant X-ray Synchrotron source was mandatory. This could certainly limit a possible future use of this technique in the clinical routine, but remains a highly valuable experimental approach in pre-clinical researches such as those involving investigation of different scaffold vascularization.
References
[1] Quarto R, et al. Repair of large bone defects with the use of autologous bone marrow stromal cells. Engl J Med. 344(5):385-6, (2001)
[2] Carano RA, Filvaroff EH, Angiogenesis and bone repair. Drug Discov Today. 8(21):980-9, (2003)
[3] Jain RK, Molecular regulation of vessel maturation. Nat Med. 9(6):685-93 (2003)
[4] Bukreeva I, et al. High-Resolution X-Ray Techniques as New Tool to Investigate the 3D Vascularization of Engineered-Bone Tissue. Frontiers in Bioengineering and Biotechnology 3:133. (2015)
Collaborators
TOMCAT, Swiss Light Source, Paul Scherrer Institut, Villigen, & Centre d’Imagerie BioMedicale, Ecole Polytechnique Federale de Lausanne (Switzerland)
SYRMEP, Elettra – Sincrotrone Trieste S.C.p.A. (Italy)
Institute of Crystallography – CNR, Monterotondo, Rome (Italy)
Department of Experimental Medicine, University of Genova & AUO San Martino – IST Istituto Nazionale per la Ricerca sul Cancro, Genova (Italy)