The overall goal of VascuBone is to develop a "tool box" for bone regeneration, which includes a variation of biocompatible biomaterials and cell types, FDA approved growth factors, material modification technologies, simulation and analytical tools like molecular imaging based in vivo diagnostics (MRI and PET/CT). This tool box will be used to develop translational approaches for regenerative therapies of different types of bone defects.
The progress of the scientific work of the VascuBone project at the end of the second year is described below divided into the main research topics.
One of very important objective of the VascuBone project is the selection and modification of biomaterials which meet
requirements such as biocompatibility, volume, surface, and mechanical stability, osteoconductivity etc.
Modification of the biomaterials: Bone ceramic and composite polymer materials have been refined with different diamond particles (DP) ranging from nano- to microcrystalline sizes improving the hydrophilicity of the materials due to high surface wettability of the diamond itself.
- A reproducible fabrication chain and a tracking system of the tailored biomaterial could be realized with respect to scaling-up and regulation aspects.
- ß-TCP scaffolds, modified with hydrophilic diamond powder, as well as the co-polymer scaffolds have been determined as biocompatible regarding EN DIN ISO 10993.
- Modification of diamond-coated materials with active biomolecules to improve osteoconductive, and osteo- and angioinductive properties was theoretically modelled and realised for Angiopoietin and BMP-2.
- Degradation of three different polymer scaffolds were evaluated in vivo and in vitro. No inflammation could be seen.
Cellular components of the tool box
In bone formation the communication between mesenchymal stem cells (MSCs) and endothelial cells (ECs) is recognized as one of the most important cellular interactions. In the VascuBone project two different cell populations, (MSCs) and endothelial progenitor cells (EPCs) and subpopulations of them, will be investigated for their suitability for bone regeneration.
- Standard operating procedures (SOPs) for the isolation, expansion and immune phenotyping were established.
- MSC: Whole genome expression profiling of human MSCs (hMSCs) from differently aged donors was performed. Possible markers for involved in the regulation of stemness and differentiation revealed to be CD24, VCAM1 and CD106.
- hMSCs should be specified by:
(i) plastic-adherence when maintained in standard culture conditions,
(ii) expression of surface molecules CD90, CD73, CD105, STRO-1, CD106, CD145, CD166, CD271, CD29, CD44, CD51, CD295
(iii) absence of of CD45, CD34, CD14, HLA/DR, CD31, CD117, CD11b.
- Oxygen level has an strong influence on the expansion and differentiation of MSC (optimal levels for both are under investigation).
- MSC for the preclinical trials.
- EPC: Buffy coat was decided to be used as cell source. Cultivation and passaging conditions are in optimization. Medicyte are working on an increased expansion by upcyte® and vericyte® technology.
- A senescence test based on a reporter gene construct has been established.
The cellular cross talk between hMSCs and hECs was examined in co-cultures with different media by
immunocytochemistry, PCR and a HumanWG-6 v3.0 expression BeadChips with a one-channel Illumina platform system. The results
indicated that hECs had a significant impact on hMSCs, particularly the bidirectional gene regulation of angiogenesis and
osteogenesis. The support of hMSC on endothelial mircovascular networks growth and maturation could be enhanced under
osteogenic stimulation via media supplements.
Vericyte® factors to optimize the cell expansion of primary microvascular endothelial cells were indentified by and tested by medicyte. The cells are carefully characterized and their cell type specific characteristics, e.g. the synthesis of CD31 and vWF, could be demonstrated.
The cellular cross talk between EPC and MSC are under investigation. The analysis of the global gene expression of EPC cultured with MSC- conditioned medium or in co-culture is in progress.
Combination of biomaterial and cells
The evaluation of loading SOPs for the tailor-made implants as well as the analysis of differentiation processes and cell-matrix interactions in different cultivation models (e.g. static versus dynamical) are one important aim of the VascuBone project.
- Seeding of bone ceramic cylinders with MSC could be realized in a customized perfusion bioreactor; the comparison of dynamical and static culture is under investigation.
- After 7 and 14 days cultivating hMSCs onto the co-polymer scaffolds, the expression of osteogenic markers such as ALP, Col I, OPN and Runx2 were up-regulated, indicating that those scaffolds could support the osteogenic differentiation of hMSCs in vitro.
- Poly(LLA-co-CL) and poly(LLA-co-DXO) promoted better attachment, growth and differentiation of hMSCs than poly(LLA).
- DP coating of polymer scaffolds further enhance attachment and differentiation of MSC and bone formation.
- The strong mediator of BMSC adhesion to polymer surfaces was found to be fibronectin through the a5ß1 integrin. This suggested that polymer surfaces intended for TE can be pretreated with fibronectin or its adhesive peptide to improve stem cell adhesion efficiency.
- The prototype of a tailor-made bioreactor for the culture of BioVaSc-bone constructs was constructed at FhG. This Bioreactor allows on the hand the perfusion of the vascular system by simulation of physiological blood flow and on the other hand the stimulation of the MSC seeded on bone ceramic scaffold inside the matrix by pressure (up to 400 N and deformation up to 1 mm). Since it is known that pressure strain leads to an increased osteogenic differentiation of MSC this bioreactor should induce angiogenesis and osteogenesis in parallel. An optimized version is in production. A sub-model for glucose consumption has already been developed and will be integrated into the whole model.
- First experiments are running with mvEC seeded BioVaSc pieces in combination with bone ceramic granules to investigate vessel sprouting from the BioVaSc to the granules.
Another main task within the VascuBone project is the establishment of new non-invasive imaging techniques to e.g. visualize seeding efficiency. To reach this aim new MRI sequences and new imaging modalities have to be implemented and new tracer identified and tested.
- With a clinical 1.5 T MR scanner, imaging of the biomaterials unmodified and modified is feasible when they are filled with water.
- The visualization of MSC seeded on bone ceramic scaffolds to investigate seeding efficiency is not possible by iron oxide labelling. Fluor labelling is under investigation.
- Fluorine-based agents can be selectively taken up by monocytes resulting in the demonstration of inflammatory processes. A major advantage of 19F MR imaging is, due to the fact that fluorine is essentially absent in the human body, 19F measurements do not suffer any interference from a background signal allowing an excellent degree of specificity. Experiments to develop a fluorine-based agent for use in 19F contrast-enhanced MR inflammation imaging are ongoing. Besides efforts to develop a 19F-based agent for 19F MRI, development and optimization of contrast agents for use in 1H MRI for angiography, vessel quantification studies and inflammation imaging are underway. These 1H MRI agents include Gd-based as well as SPIO-based agents.
- The comparison of FLASE and TSE MR sequences has been adapted to visualize osseous structures as well as osseous structures plus biomaterial (MRB). The average bone volume fraction (BVF) can be calculated from the surface reconstructions. The sequences can be "calibrated" and the sequences with the better CNR or SNR can be chosen. The difference in the BVF can be corrected in the post processing. Both sequences can be used for the clinical and preclinical studies.
- µRI can be used to visualize implant ingrowth in sufficient detail (see preclinical trials).
The translational aspect of this project is the design and execution of preclinical and clinical phase I trials addressing vascularized bone regeneration in small maxillary defects and large bone defects of the facial skeleton.
- Related to the clinical trial a critical size defect model in the forehead of the sheep (desmal bone, which is comparable to the mandible) was realized, investigating different b-TCP scaffolds (Cerasorb® and ChronOs®) and a co-polymer scaffold in combination with hydrophilic diamond particles and angiogenic growth factors (angiopoetin)and bone inductive growth factors (BMP-2).(MUI)
- The animals were sacrificed after 2, 4, 12 and 24 weeks.
- The specimen will be investigated by means of histological evaluation, immunohistochemistry and RNA expression analyses.
- Only minor formation of new bone was seen at 4 weeks.
- After 12 weeks, a bony island was formed in the central of ChronOs® in addition to the peripheral new bone. The new bone formation was less for the Cerasorb® group compared with ChronOs®.
- After 24 weeks, additional bone formation was seen for both ChronOs® and Cerasorb®. Interestingly, the upper part of the defect filled with Cerasorb® was bridged by newly formed bone. Regarding the degradation of bulk bone substitutes (ChronOs® and Cerasorb®), the residual materials were present throughout all experimental time points. But it seemed that Cerasorb® degraded slightly faster which evidenced in fast replacement by new bone.
- For ChronOs®+nDP+Ang-1 group, the amount of new bone formation was similar compared to the ChronOs®+nDP and there was no significant promotion of new bone with the addition of angiogenesis factor.
- The central region of the ChronOs®+nDP+Ang-1 also showed significantly higher vessel density compared with ChronOs®scaffold.
- After 4 weeks the application of BMP-2 to Cerasorb+DP showed an enhanced bone formation in comparison to pure Cerasorb and after physisorption of Ang-1.
- Using mMRI the implant ingrowth (control vs. ChronOs®+nDP vs. ChronOs®+nDP+Ang-1) in sufficient detail could be visualized (MRB).
- Methods and protocols for the isolation and cultivation of human MSCs were adapted to sheep MSCs (FhG).
- The in vivo experiments of the femoral head necrosis started within this reporting period (UWü).
- Having the data from the preclinical trial on AVN and safety data of the MSCs from the manufacturer the outstanding investigation medicinal product dossier for the clinical trial application will rapidly finished (UWü/MUI).
- For the clinical and preclinical studies, the comparison of FLASE and TSE MR sequences has been adapted to visualize osseous structures as well as osseous structures plus biomaterial. Both sequences can be used for the clinical and preclinical studies (MRB).
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|The research leading to these results has received funding from the European Union's Seventh Framework Programme for research, technological development and demonstration under grant agreement n°242175|