In vitro and in vivo assessment of a 3D printable gelatin methacrylate hydrogel for bone regeneration applications.

Bone tissue engineering (BTE) has made significant progress in developing and assessing different types of bio-substitutes. However, scaffolds production through standardized methods, as required for good manufacturing process (GMP), and post-transplant in vivo monitoring still limit their translation into the clinic. 3D printed 5% GelMA scaffolds have been prepared through an optimized and reproducible process in this work. Mesenchymal stem cells (MSC) were encapsulated in the 3D printable GelMA ink, and their biological properties were assessed in vitro to evaluate their potential for cell delivery application. Moreover, in vivo implantation of the pristine 3D printed GelMA has been performed in a rat condyle defect model. Whereas optimal tissue integration was observed via histology, no signs of fibrotic encapsulation or inhibited bone formation were attained. A multimodal imaging workflow based on computed tomography (CT) and magnetic resonance imaging (MRI) allowed the simultaneous monitoring of both new bone formation and scaffold degradation. These outcomes point out the direction to undertake in developing 3D printed-based hydrogels for BTE that can allow a faster transition into clinical use.

Pharmacokinetics of Single Domain Antibodies and Conjugated Nanoparticles Using a Hybrid near Infrared Method.

Iron oxide nanoparticles and single domain antibodies from camelids (VHHs) have been increasingly recognized for their potential uses for medical diagnosis and treatment. However, there have been relatively few detailed characterizations of their pharmacokinetics (PK). The aim of this study was to develop imaging methods and pharmacokinetic models to aid the future development of a novel family of brain MRI molecular contrast agents. An efficient near-infrared (NIR) imaging method was established to monitor VHH and VHH conjugated nanoparticle kinetics in mice using a hybrid approach: kinetics in blood were assessed by direct sampling, and kinetics in kidney, liver, and brain were assessed by serial in vivo NIR imaging. These studies were performed under "basal" circumstances in which the VHH constructs and VHH-conjugated nanoparticles do not substantially interact with targets nor cross the blood brain barrier. Using this approach, we constructed a five-compartment PK model that fits the data well for single VHHs, engineered VHH trimers, and iron oxide nanoparticles conjugated to VHH trimers. The establishment of the feasibility of these methods lays a foundation for future PK studies of candidate brain MRI molecular contrast agents.

Sensitive detection of extremely small iron oxide nanoparticles in living mice using MP2RAGE with advanced image co-registration.

Magnetic resonance imaging (MRI) is a widely used non-invasive methodology for both preclinical and clinical studies. However, MRI lacks molecular specificity. Molecular contrast agents for MRI would be highly beneficial for detecting specific pathological lesions and quantitatively evaluating therapeutic efficacy in vivo. In this study, an optimized Magnetization Prepared-RApid Gradient Echo (MP-RAGE) with 2 inversion times called MP2RAGE combined with advanced image co-registration is presented as an effective non-invasive methodology to quantitatively detect T1 MR contrast agents. The optimized MP2RAGE produced high quality in vivo mouse brain T1 (or R1 = 1/T1) map with high spatial resolution, 160 × 160 × 160 µm3 voxel at 9.4 T. Test-retest signal to noise was > 20 for most voxels. Extremely small iron oxide nanoparticles (ESIONPs) having 3 nm core size and 11 nm hydrodynamic radius after polyethylene glycol (PEG) coating were intracranially injected into mouse brain and detected as a proof-of-concept. Two independent MP2RAGE MR scans were performed pre- and post-injection of ESIONPs followed by advanced image co-registration. The comparison of two T1 (or R1) maps after image co-registration provided precise and quantitative assessment of the effects of the injected ESIONPs at each voxel. The proposed MR protocol has potential for future use in the detection of T1 molecular contrast agents.

Application of BMP-Bone Cement and FGF-Gel on Periodontal Tissue Regeneration in Nonhuman Primates.

The ultimate challenge of tissue engineering research is the translation of experimental knowledge into clinical application. In the preclinical testing phase of any new therapy, animal models remain the gold standard. Therefore, the methodological choice of a suitable model is critical to meet the requirements for a safe clinical application of the developed treatment. For instance, we have shown in rats that the application of calcium phosphate cement (CPC)/propylene glycol alginate (PGA) with bone morphogenetic protein (BMP)-2 or fibroblast growth factor (FGF)-2 resulted in the regeneration of periodontal defects. However, it is debated whether using small models form a predictive method for translation to larger species. At the same time, the 3R framework is encouraged as guiding principles of the ethical use of animal testing. Therefore, based on the successful rat study, the objective of this study was to further investigate the periodontal regenerative efficacy of the CPC/BMP and PGA/FGF system in a periodontal defect model with a low number of nonhuman primates (NHPs). Three Macaca fascicularis-overstocked from breeding for other purposes-were used (reuse of animals and appropriateness of the experimental animal species according to 3R framework). Three-wall periodontal defects were surgically created in the mandible. In total, 10 defects were created and distributed over two groups: (1) control group: PGA+CPC (n = 5) and (2) experimental group: PGA/FGF+CPC/BMP (n = 5). After 3 months, tissue regeneration was evaluated by histomorphometry and radiographic measurements. Data showed that epithelial downgrowth, cementum, and ligament regeneration were significantly enhanced in the experimental group compared with the control group (n = 5; p = 0.013, p = 0.028, and p = 0.018, respectively). However, the amount of newly formed bone did not differ (p = 0.146). Overall, as a translational proof-of-principle study, the hybrid periodontal regenerative method of CPC/BMP+PGA/FGF promoted periodontal regeneration in NHPs. This study warrants the application of CPC/BMP/PGA/FGF in clinical trials. Impact Statement This study validated an earlier successful periodontal regeneration strategy from a rat model into a few spare nonhuman primates (NHPs). The hybrid periodontal regenerative method of calcium phosphate cement (CPC)/bone morphogenetic protein (BMP)-2/propylene glycol alginate (PGA)/fibroblast growth factor (FGF)-2 promoted periodontal regeneration in NHPs, which corroborated the previous rat results. This translational approach was a very practical option and thus reduced the number and species of experimental animals in translational research. These results found in NHPs indicate a consistent conclusion with the earlier findings in the rat model. It further warrants the application of CPC/BMP-2+PGA/FGF-2 in human clinical trials.

Enhancing X-ray Attenuation of 3D Printed Gelatin Methacrylate (GelMA) Hydrogels Utilizing Gold Nanoparticles for Bone Tissue Engineering Applications.

Bone tissue engineering is a rapidly growing field which is currently progressing toward clinical applications. Effective imaging methods for longitudinal studies are critical to evaluating the new bone formation and the fate of the scaffolds. Computed tomography (CT) is a prevailing technique employed to investigate hard tissue scaffolds; however, the CT signal becomes weak in mainly-water containing materials, which hinders the use of CT for hydrogels-based materials. Nevertheless, hydrogels such as gelatin methacrylate (GelMA) are widely used for tissue regeneration due to their optimal biological properties and their ability to induce extracellular matrix formation. To date, gold nanoparticles (AuNPs) have been suggested as promising contrast agents, due to their high X-ray attenuation, biocompatibility, and low toxicity. In this study, the effects of different sizes and concentrations of AuNPs on the mechanical properties and the cytocompatibility of the bulk GelMA-AuNPs scaffolds were evaluated. Furthermore, the enhancement of CT contrast with the cytocompatible size and concentration of AuNPs were investigated. 3D printed GelMA and GelMA-AuNPs scaffolds were obtained and assessed for the osteogenic differentiation of mesenchymal stem cells (MSC). Lastly, 3D printed GelMA and GelMA-AuNPs scaffolds were scanned in a bone defect utilizing µCT as the proof of concept that the GelMA-AuNPs are good candidates for bone tissue engineering with enhanced visibility for µCT imaging.

Magnetic Resonance Imaging of Hard Tissues and Hard Tissue Engineered Bio-substitutes.

Magnetic resonance imaging (MRI) is a non-invasive diagnostic imaging tool based on the detection of protons into the tissues. This imaging technique is remarkable because of high spatial resolution, strong soft tissue contrast and specificity, and good depth penetration. However, MR imaging of hard tissues, such as bone and teeth, remains challenging due to low proton content in such tissues as well as to very short transverse relaxation times (T2). To overcome these issues, new MRI techniques, such as sweep imaging with Fourier transformation (SWIFT), ultrashort echo time (UTE) imaging, and zero echo time (ZTE) imaging, have been developed for hard tissues imaging with promising results reported. Within this article, MRI techniques developed for the detection of hard tissues, such as bone and dental tissues, have been reviewed. The main goal was thus to give a comprehensive overview on the corresponding (pre-) clinical applications and on the potential future directions with such techniques applied. In addition, a section dedicated to MR imaging of novel biomaterials developed for hard tissue applications was given as well.

Bisphosphonate Functionalized Gadolinium Oxide Nanoparticles Allow Long-Term MRI/CT Multimodal Imaging of Calcium Phosphate Bone Cement.

Direct in vivo monitoring of bioconstructs using noninvasive imaging modalities such as magnetic resonance imaging (MRI) or computed tomography (CT) is not possible for many materials. Calcium phosphate-based composites (CPCs) that are applicable to bone regeneration are an example where the materials have poor MRI and CT contrast; hence, they are challenging to detect in vivo. In this study, a CPC construct is designed with gadolinium-oxide nanoparticles incorporated to act as an MRI/CT multimodal contrast agent. The gadolinium(III) oxide nanoparticles are synthesized via the polyol method and surface functionalized with a bisphosphonate (BP) derivative to give a construct (gadolinium-based contrast agents (GBCAs)-BP) with strong affinity toward calcium phosphate. The CPC-GBCAs-BP functional material is longitudinally monitored after in vivo implantation in a condyle defect rat model. The synthetic method developed produces nanoparticles that are stable in aqueous solution (hydrodynamic diameter 70 nm) with significant T1 and T2 relaxivity demonstrated in both clinical 3 T and preclinical 11.7 T MRI systems. The combination of GBCAs-BP nanoparticles with CPC gives an injectable material with handling properties that are suitable for clinical applications. The BP functionalization prolongs the residence of the contrast agent within the CPC to allow long-term follow-up imaging studies. The useful contrast agent properties combined with biological compatibility indicate further investigation of the novel bone substitute hybrid material toward clinical application.

Gelatin methacrylate scaffold for bone tissue engineering: The influence of polymer concentration.

Gelatin methacrylate (GelMA) is an inexpensive, photocrosslinkable, cell-responsive hydrogel which has drawn attention for a wide range of tissue engineering applications. The potential of GelMA scaffolds was demonstrated to be tunable for different tissue engineering (TE) applications through modifying the polymer concentration, methacrylation degree, or UV light intensity. Despite the promising results of GelMA hydrogels in tissue engineering, the influence of polymer concentration for bone tissue engineering (BTE) scaffolds was not established yet. Thus, in this study, we have demonstrated the effect of polymer concentration in GelMA scaffolds on osteogenic differentiation. We prepared GelMA scaffolds with 5 and 10% polymer concentrations and characterized the scaffolds in terms of porosity, pore size, swelling characteristics, and mechanical properties. Subsequent to the scaffolds characterization, the scaffolds were seeded with bone marrow derived rat mesenchymal stem cells and cultured in osteogenic media to evaluate the possible osteogenic differentiation effect exerted by the polymer concentration. After 7, 14, 21, and 28 days, DNA content, calcium deposition, and alkaline phosphatase (ALP) activity of scaffolds were evaluated quantitatively by colorimetric bioassays. Furthermore, the distribution of the calcium deposition within the scaffolds was attained qualitatively and quantitatively by microcomputer tomography (µCT). Our data suggest that GelMA hydrogels prepared with 5% polymer concentration has promoted homogeneous extracellular matrix calcification and it is a great candidate for BTE applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 201-209, 2018.

Visualization of calcium phosphate cement in teeth by zero echo time 1 H MRI at high field.

1 H magnetic resonance imaging (MRI) by a zero echo time (ZTE) sequence is an excellent method to image teeth. Calcium phosphate cement (CPC) materials are applied in the restoration of tooth lesions, but it has not yet been investigated whether they can be detected by computed tomography (CT) or MRI. The aim of this study was to optimize high-field ZTE imaging to enable the visualization of a new CPC formulation implanted in teeth and to apply this in the assessment of its decomposition in vivo. CPC was implanted in three human and three goat teeth ex vivo and in three goat teeth in vivo. An ultrashort echo time (UTE) sequence with multiple flip angles and echo times was applied at 11.7 T to measure T1 and T2 * values of CPC, enamel and dentin. Teeth with CPC were imaged with an optimized ZTE sequence. Goat teeth implanted with CPC in vivo were imaged after 7 weeks ex vivo. T2 * relaxation of implanted CPC, dentin and enamel was better fitted by a model assuming a Gaussian rather than a Lorentzian distribution. For CPC and human enamel and dentin, the average T2 * values were 273 ± 19, 562 ± 221 and 476 ± 147 µs, respectively, the average T2 values were 1234 ± 27, 963 ± 151 and 577 ± 41 µs, respectively, and the average T1 values were 1065 ± 45, 972 ± 40 and 903 ± 7 ms, respectively. In ZTE images, CPC had a higher signal-to-noise-ratio than dentin and enamel because of the higher water content. Seven weeks after in vivo implantation, the CPC-filled lesions showed less homogeneous structures, a lower T1 value and T2 * separated into two components. MRI by ZTE provides excellent contrast for CPC in teeth and allows its decomposition to be followed.

Perfluorocarbon/Gold Loading for Noninvasive in Vivo Assessment of Bone Fillers Using 19F Magnetic Resonance Imaging and Computed Tomography.

Calcium phosphate cement (CPC) is used in bone repair because of its biocompatibility. However, high similarity between CPC and the natural osseous phase results in poor image contrast in most of the available in vivo imaging techniques such as computed tomography (CT) and magnetic resonance imaging (MRI). For accurate identification and localization during and after implantation in vivo, a composition with enhanced image contrast is needed. In this study, we labeled CPC with perfluoro-15-crown-5-ether-loaded (PFCE) poly(latic-co-glycolic acid) nanoparticles (hydrodynamic radius 100 nm) and gold nanoparticles (diameter 40 nm), as 19F MRI and CT contrast agents, respectively. The resulting CPC/PFCE/gold composite is implanted in a rat model for in vivo longitudinal imaging. Our findings show that the incorporation of the two types of different nanoparticles did result in adequate handling properties of the cement. Qualitative and quantitative long-term assessment of CPC/PFCE/gold degradation was achieved in vivo and correlated to the new bone formation. Finally, no adverse biological effects on the bone tissue are observed via histology. In conclusion, an easy and efficient strategy for following CPC implantation and degradation in vivo is developed. As all materials used are biocompatible, this CPC/PFCE/gold composite is clinically applicable.