We report here some pre-clinical testing of new scaffolds. To compare these second generation ceramic scaffolds more suitable for a tissue engineering approach we had to first establish animal models and analysis procedures including the use of X-ray-computed microtomography associated with X-rays synchroton radiation.

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The aim of this study was the preparation and characterization of bioactive glass-ceramic scaffolds for bone tissue engineering. For this purpose, a glass belonging to the system SiO2-P2O5-CaO-MgO-Na2O-K2O (CEL2) was used. The sponge-replication method was adopted to prepare the scaffolds; specifically, a polymeric skeleton was impregnated with a slurry containing CEL2 powder, polyvinyl alcohol (PVA) as a binding agent and distilled water. The impregnated sponge was then thermally treated to remove the polymeric phase and to sinter the inorganic one. The obtained scaffolds possessed an open and interconnected porosity, analogous to cancellous bone texture, and with a mechanical strength above 2 MPa. Moreover, the scaffolds underwent partial bioresorption due to ion-leaching phenomena. This feature was investigated by X-ray computed microcomputed tomography (micro-CT). Micro-CT is a three-dimensional (3-D) radiographic imaging technique, able to achieve a spatial resolution close to 1 microm(3). The use of synchrotron radiation allows the selected photon energy to be tuned to optimize the contrast among the different phases in the investigated samples. The 3-D scaffolds were soaked in a simulated body fluid (SBF) to study the formation of hydroxyapatite microcrystals on the scaffold struts and on the internal pore walls. The 3-D scaffolds were also soaked in a buffer solution (Tris-HCl) for different times to assess the scaffold bioresorption according to the ISO standard. A gradual resorption of the pores walls was observed during the soakings both in SBF and in Tris-HCl.

Three types of ceramic scaffolds with different composition and structure [namely synthetic 100% hydroxyapatite (HA; Engipore), synthetic calcium phosphate multiphase biomaterial containing 67% silicon stabilized tricalcium phosphate (Si-TCP; Skelite™) and natural bone mineral derived scaffolds (Bio-oss®)] were seeded with mesenchymal stem cells (MSC) and ectopically implanted for 8 and 16 weeks in immunodeficient mice. X-ray synchrotron radiation microtomography was used to derive 3D structural information on the same scaffolds both before and after implantation. Meaningful images and morphometric parameters such as scaffold and bone volume fraction, mean thickness and thickness distribution of the different phases as a function of the implantation time, were obtained. The used imaging algorithms allowed a direct comparison and registration of the 3D structure before and after implantation of the same sub-volume of a given scaffold. In this way it was possible to directly monitor the tissue engineered bone growth and the complete or partial degradation of the scaffold. Further, the detailed kinetics studies on Skelite™ scaffolds implanted for different length of times from 3 days to 24 weeks, revealed in the X-ray absorption histograms two separate peaks associated to HA and TCP. It was therefore possible to observe that the progressive degradation of the Skelite™ scaffolds was mainly due to the resorption of TCP. The different saturation times in the tissue engineered bone growth and in the TCP resorption confirmed that the bone growth was not limited the scaffold regions that were resorbed but continued in the inward direction with respect to the pore surface.

Biocompatible calcium phosphate ceramic grafts are able of supporting new bone formation in appropriate environment. The major limitation of these materials usage for medical implants is the absence of accessible methods for their patient-specific fabrication. 3D printing methodology is an excellent approach to overcome the limitation supporting effective and fast fabrication of individual complex bone substitutes. Here, we proposed a relatively simple route for 3D printing of octacalcium phosphates (OCP) in complexly shaped structures by the combination of inkjet printing with post-treatment methodology. The printed OCP blocks were further implanted in the developed cranial bone defect followed by histological evaluation. The obtained result confirmed the potential of the developed OCP bone substitutes, which allowed 2.5-time reducing of defect’s diameter at 6.5 months in a region where native bone repair is extremely inefficient.

Resorbable ceramic scaffolds based on Silicon stabilized tricalcium phosphate (Si-TCP) were seeded with bone marrow stromal cells (BMSC) and ectopically implanted for 2, 4, and 6 months in immunodeficient mice. Qualitative and quantitative evaluation of the scaffold material was performed by X-ray synchrotron radiation computed microtomography (microCT) with a spatial resolution lower than 5 mm. Unique to these experiments was that microCT data were first collected on the scaffolds before implantation and then on the same scaffolds after they were seeded with BMSC, implanted in the mice and rescued after different times. Volume fraction, mean thickness and thickness distribution were evaluated for both new bone and scaffold phases as a function of the implantation time. New bone thickness increased from week 8 to week 16. Data for the implanted scaffolds were compared with those derived from the analysis of the same scaffolds prior to implantation and with data derived from 100% hydroxyapatite (HA) scaffold treated and analyzed in the same way. At variance with findings with the 100% HA scaffolds a significant variation in the density of the different Si-TCP scaffold regions in the pre-and post-implantation samples was observed. In particular a post-implantation decrease in the density of the scaffolds, together with major changes in the scaffold phase composition, was noticeable in areas adjacent to newly formed bone. Histology confirmed a better integration between new bone and scaffold in the Si-TCP composites in comparison to 100% HA composites where new bone and scaffold phases remained well distinct.