Work performed since the beginning of the project and main results achieved so far:
The VascuBone consortium decided to focus on two types of resorbable synthetic implant materials:
- A commercially available beta-tricalcium phosphate (ß-TCP),
- A biocompatible hybrid polymer scaffolds, which are developed within the consortium.
Modification of the biomaterials: All 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.
- Within the first project year a reproducible fabrication chain of modified, 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.
The osteoconductive, and osteo- and angioinductive properties of the biomaterial should be improved by coating with growth factors (GF) or components of the ECM. For an efficient and stable grafting of these molecules on the DP-coated implant material first theoretical modelling using example substances like chitosan and fibronectin were performed.
- Both of these molecules were found to bind strongly to the diamond surfaces, with a clear preference for chitosan.
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, although the underlying mechanisms are not well understood so far. 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 and expansion were established in the consortium in the first project year.
- MSC: Whole genome expression profiling of human MSCs (hMSCs) from differently aged donors resulted in the identification of first candidates (e.g. VCAM1/CD106) for valid cell surface marker involved in the regulation of stemness and differentiation.
- hMSCs should be specified by:
- Plastic-adherence when maintained in standard culture conditions,
- Expression of surface molecules CD90, CD73, CD105, STRO-1, CD106, CD145, CD166, CD271, CD29, CD44, CD51, CD295
- Absence of of CD45, CD34, CD14, HLA/DR, CD31, CD117, CD11b.
- EPC: A protocol to use buffy coat as cell source was established. Passaging conditions have been optimized resulting in up to 10-fold higher cells numbers.
The cellular cross talk between hMSCs and hECs during a 5 and 15 days co-culture was examined by 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. More detailed study of the microarray data is warranted to explore further possible cellular and molecular interactions of importance in bone tissue engineering.
Additionally, to optimize the cell expansion of primary cells, a novel and innovative upcyte® technology was implemented. Different gene combinations were screened and genes capable of pushing microvascular Endotheliacells (mvECs) into extended proliferation were identified. The cells are carefully characterized and their cell type specific characteristics, e.g. the synthesis of CD31 and vWF, could be demonstrated.
Combination of biomaterial and cells
Another important aim in the first year of the project was the definition of implant loading conditions and the construction of a bioreactor system for dynamical 3D culture.
- First in vitro experiments under static conditions, seeding hMSCs on ß-TCP scaffolds or human osteoblast-like cells on co-polymer scaffolds, demonstrated the adhesion and proliferation of these cells on the materials.
- 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).
- Computational modelling was set up in order to design and construct a complex bioreactor for dynamic bone culture conditions. Beside mechanical aspects also biochemical processes were integrated in order to predict the consumption of nutrition inside the porous scaffold.
- A sub-model for glucose consumption has already been developed and will be integrated into the whole model.
- The results of the computational model were then used to design a first bioreactor generation. The bioreactor was
constructed with two main functions: to provide sufficient amount of nutrition and to exert mechanical stimulation up to 400
N and deformation up to 1 mm.
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.
- With a clinical 1.5 T MR scanner, imaging of the biomaterials alone is feasible when they are filled with water.
- The diamond coating has no influence on the MR measurements.
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.
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 planned, investigating different ß-TCP scaffolds and a co-polymer scaffold in combination with hydrophilic diamond particles and angiogenic growth factors (angiopoetin) and bone inductive growth factors (BMP-2).
- The animals will be sacrificed after 2, 4, 12 and 24 weeks.
- The specimen will be investigated by means of histological evaluation, immunohistochemistry and RNA expression analyses.
- The in vivo experiments started within this reporting period.
- We also applied for an in vivo experiment on the treatment of the femoral head necrosis (FHN). Aim of the investigation is the evaluation of the efficiency of MSCs in the treatment of this pathological entity. Different concentrations of the MSCs will be investigated and compared to standard decompression technique.
The first clinical phase I studies for small maxillary defects and the FHN were designed.
- Clinical observational studies will be conducted as randomized split-mouth controlled clinical trials.
- With respect to the clinical trial a scientific advice was received by the local authorizing authority (PEI, Paul-Ehrlich-Institute) in Germany.
- Recommendations and modifications have been adapted to the study protocol.
- Safety and tolerability of the clinical study will be evaluated by recording adverse and serious adverse events, vital signs, safety laboratory assessment, physical exam, incidence of infection, and concomitant (non-investigational) medication throughout.
Expected final results and their potential impacts and use of the project so far
At 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. First important results to reach this overall aim of safe and effective therapies are available after the first year and all disseminations and milestones of the project are in time.Back to top
|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|