Project context and objectives
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.
One of the very important objectives of the VascuBone project was combination of biomaterials with the cells. In this third project year, the main tasks were the optimization and validation of the different biomaterials with regard to their biocompatibility according to EN DIN ISO 10993, the control and monitoring of the cell seeding of the scaffolds, the in vivo testing and the preparation of the clinical trials.
Work performed since the beginning of the project and main results achieved so far:
One of the very important objectives of the VascuBone project is the selection and modification of biomaterials which meet
requirements such as biocompatibility, volume, surface, and mechanical stability, osteoconductivity etc.
Based on the developments of the biomaterials of the first two years the optimized material was used in year three for experimental applications at the partners and for analysis to achieve all required data for the planned clinical trials according to EN DIN ISO 10993 and 14155 as well as FDA-guidelines with respect to nano-particles:
and for nanoparticles on medical devices:
Characterisation of optimized biomaterials (bone graft substitute) - validation of the processes:The modified biomaterials focused mainly on three topics:
- Improvement of hydrophilicity,
- Increase of active surface area and
- Controlled and localized offer of growth factors within the scaffolds.
These requirements have been realized and can be provided for all bone graft substitutes. Further improvement is continuously ongoing based on the obtained in vitro and in vivo results. The processes are established and validated. Process documentation due to ISO-standards was developed and is available. In Report II results of biocompatibility tests, degradation, and inflammation in vitro and in vivo have been presented. In this reporting period the regulation aspects for the first planned clinical trial ("split mouth" based study for lateral augmentation of to narrow mandibular crests before insertion of dental implants) have been determined and the required tests and documentation were started. Additionally the polymer-materials have been improved especially with respect to mechanical properties and hydrophilicity. The capacity of binding growth factors (BMP-2, Ang.-1, VEGF) as well as the bioactivity of such bond GFs on nDPs was determined. Such functionalized nDP will provide increased active area (area in contact with bio-entities) in modified scaffolds and will simulate a cell niche via nano-structure mimicking extra cellular matrix (ECM).
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.
- Establishment of methods for standardised isolation, phenotyping, propagation and osteogenic differentiation of MSCs (readout: FACS, qPCR).
- Establishment of a novel quality assessment technology for monitoring cell cultures.
- A senescence test based on a reporter gene construct has been further characterized.
- Characterisation of the ECM production and adhesion capacity of MSC loaded on implants.
- Characterisation of cellular cross talk between EPC and MSC: Affymetrix microarray hybridization revealed gene expression of EPC cultured with MSC- conditioned medium or in co-culture (see below).
- Optimisation of MSC and EPC co-culture on the BioVaSc.
- Cultivation and passaging conditions are still in optimization
- Establishment of methods for the characterisation of growth properties of EPCs on the BioVaSc.
- Set up of bioreactor systems to cultivate cells under controlled conditions, optimisation of MSC and EPC co-culture.
- Identification of BOECs (blood outgrowth endothelial cells) as an alternative cell source for EPCs. The characterisation of the cells is in progress (Medicyte.)
- Characterisation of cellular cross talk between EPC and MSC: Affymetrix microarray hybridization revealed gene expression of EPC cultured with MSC- conditioned medium or in co-culture.
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.
- Biocompatibility of two aliphatic polyester co-polymer scaffolds (KTH) was tested by indirect and direct contact cytotoxicity assays according to DIN ISO 10993-12: 2009 and found to be noncytotoxic and biocompatible.
- Biocompatibility according to DIN ISO 10993-5 of two different scaffolds for bone reconstruction (CeraSorb® / ChronOs), which have been modified by hydrophilic diamond powder, was shown.
Evaluation of tumour promoting potential (UIB)
- To evaluate the ability of preselected functionalised scaffolds to initiate and/or enhance the invasive phenotype of premalignant cell lines derived from oral tissues in monolayer culture models, 3D organotypic models in vitro and evaluate in vivo (UIB).
- DOK and Poe9n cells were cultivated on none functionalised polymer scaffolds at various cell densities and for different length of time and trials to trypsinise them for further in vitro assay and tumorgenicity experiments.
- Protocols optimized and established for in vitro to harvest DOK and Poe9n cell lines from the scaffolds and the harvested cells were used to next optimize the tumourgenicity assays.
- A successful design established for in vivo tumourgenicity assessment model in NOG mice using the combination of cells mentioned earlier and successfully transfecting cells for in vivo imaging and in the process of optimising the scaffold implant along with cells.
- SPIO contrast agents of different particle sizes for use in 1H MRI macrophage-associated inflammation imaging of the scaffold were developed and tested in vitro with regards to their uptake by macrophages P388 (NanoPET).
- MRI measurements of the various SPIO nanoparticle contrast agents were performed and show that an increase in the relaxation rate (MR signal) is obtained with increasing particle size (MRB).
- A SPIO agent of intermediate particle size may be a suitable candidate for in vivo inflammation studies.
Evaluation of optimal cell numbers/ types for implant loading (FHG)
- The seeding process using a defined oscillary flow was the most efficient method to incorporate cells in the porous scaffold system. In combination with an oscillating pump flow (1.6 ml/min for 1 h) a good distribution of the hMSC on the CeraSorb® cylinder was revealed.
- To evaluate the influence of the dynamic culture on cell behaviour we performed live/dead staining after 7 days and analysed the expression of several genes by qRT-PCR.
- Stress markers were significantly higher in the dynamic set up on day one.
- A higher expression of osteogenic marker genes could be detected in the dynamic approach even without any further stimuli in the cell culture media.
- 5x106 hMSCs seems to be an adequate cell number for implant loading.
Establishment of techniques for proof of seeding efficiency (NanoPET, FHG, UWü, MRB)
- Synthesis of 19F crown ether nanoemulsions for use in 19F MRI was optimized by variation of the synthesis parameters. Various agents were produced with different nanoemulsion droplet sizes and different surface charge.
- Labelling protocols for the fluorine nanoemulsions were established and validated. The general feasibility of cellular labelling with the nanoemulsions was shown.
- Experiments reveal that the different droplet sizes do not have an influence on cellular uptake whereas the surface charge of the nanoemulsion plays a significant role, influencing both the cellular uptake as well as the cell viability.
Monitoring and imaging of implant ingrowth (NanoPET, MRB)
- Various contrast agents were developed, transferred to MRB and tested in vivo.
- FeraSpin XS-Type was selected as having optimal properties to quantify blood vessels in rat bones.
- The necessary steps required for the upscaling to sheep (higher volumes, higher concentrations of contrast agent) have been addressed.
- In 2013 Gd-DTPA and FeraSpin XS-Type will be provided for the large animal study in sheep.
Analytical tools: 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 tracers identified and tested.
MRI detector development (MRB)
- For the in vivo FHN sheep study in Würzburg, a dedicated MR detector (coil) has been designed and built.
MRI sequence design and validation (MRB)
- The development and testing of MR sequences has been continued in reporting period 3. Especially, the UTE sequences have been further developed:
- The implemented 3D IR-UTE MR sequence provides access to the short-T2-components in the sclerotic regions of the femur. This allows in principle to obtain additional information about the necrotic regions which is not accessible by MR sequences which are less or even not sensitive to the short-T2-components.
- The "local look technique" has been implemented and tested for TSE experiments. This technique allows reducing the field of view to the femoral head without generating aliasing artefacts.
- See also clinical trials
Regenerative therapies: 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.
Upscaling of bio materials (PPP)
- Setting-up routines to synthesize tin free polymers with reproducible qualities in a very efficient and environmentally favorable way.
- Introduction of the use of PLLA-PCL blends instead of copolymers in order to eliminate all risks for quality variations.
- Setting-up synthetic routines to fulfill the EMA and CE-requirements.
- Obtaining cytotoxicity approval of the material according to DIN ISO 10993-5.
- Setting-up quality control routines to characterize the materials both with respect to chemical composition and physical properties.
- Evaluating the material stability against gamma irradiation required for sterilization. Gamma irradiation does not influence the material. The material is not influenced by oxygen plasma either, which can be used to make the scaffold surface hydrophilic and also to bond n-DP.
- Developing a method for scaffold preparation without any influence on the material characteristics, where we can control the porosity of the scaffolds.
- Supplying scaffold samples to MRB in order to develop a method to determine the pore-size and pore-size distributions together with material distribution in the scaffolds. This allows the Vascubone group to investigate the behavior of cell growth as a function of pore sizes and pore size distributions.
- Scaffold samples have been sent to Fraunhofer for preliminary bio-reactor studies, which has shown promising results. Now, PPP has prepared 25 pieces of scaffold samples more for further evaluations.
- The non-bearing model (a critical defect in rat calvaria) has been established and represents an attractive model to study the manipulation of bone healing since the defects do not heal without intervention (UiB).
- In the non-bearing model the formation of new bone was evaluated by radiography and histology and by the expression of osteogenic markers using RT-PCR. To investigate vessel formation, histological staining was performed with EC's markers. The radiographical and histological results showed more rapid bone formation in the CO- than in the BMSC-group.
- In the bearing model (a critical defect in rat mandibula) a polymer scaffold was implanted (UiB, MUI).
- The bearing model showed an improved bone healing in BMP 2 functionalised groups.
- Both microspheres with slow released BMP-2 and diamond particles and physisorbed BMP-2 have shown a higher osteogenesis after 4 weeks than other BMP 2 coated groups.
- A more active bone remodelling which is combined inflammation and bone resorption was also detected in these two groups after 2 weeks because microspheres and diamond particles might contribute to inflammatory response.
- The μCT analysis revealed that the different immobilization techniques of BMP-2 have a time depending influence on the bone formation.
- After 4 weeks polymers with injected BMP-2, the physisorbed BMP-2 and the polymer with the BMP-2 containing microbubbles showed the highest bone formation rate. Covalently bound BMP-2, either at NCD-coated polymer or directly on the polymer was less effective. After 4 weeks most newly formed bone was found, when microbubbles, as a slow releasing system were added, whereas less bone was detected when BMP-2 was only injected or physisorbed.
Sheep model of the FHN
- In vivo experiments of the femoral head necrosis started within this reporting period (UWü).
Clinical trial protocol
- The clinical trial protocol is mostly finished; other important documents like the investigational medicinal product dossier (IMPD) and the investigator brochure are outstanding (UWü).
MR imaging of trabecular bone and avascular femoral head necrosis (MRB):
- The two MR imaging sequences FLASE and TSE were compared with a focus on the obtainable signal to noise ratio (SNR) in bone MRI:
- To minimize T2-blurring in the TSE images, low turbo factors were tested with short repetition times and optimized excitation angles. In order to keep the scan time at the same level as with the FLASE sequence, the number of readouts per unit time was kept constant, i.e. one readout per 80 ms.
- A 3D IR-prepared Ultrashort TE (UTE) sequence was programmed and implemented on the clinical 1.5 T MR scanner at the MRB. This 3D IR-UTE sequence can provide direct signal of short-T2-components in the sclerotic regions of the femoral head, which is directly related to trabecular density and characterize the sclerotic areas.
Expected final results and their potential impacts and use of the project so farAt the end of the project a "tool box" for regenerative approaches should be established which includes a variation of biocompatible approved biomaterials (knowledge-based materials) and adult stem cell types, and analytical tools (MRI and PET) which can be composed for the specific medical need. VascuBone will remove bottlenecks regarding determination of the cellular markers necessary for efficient quality control of MSC and EPC based therapies, the specification of optimal MSC subpopulation and age related adult stem cell ATMPs for bone regeneration. Important results to reach this overall aim of safe and effective therapies are available after the third year and all disseminations and milestones of the project are in time.
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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|