Final Report Summary - BIO-INSPIRE (BIO INSPIRED BONE REGENERATION)
Marie Curie Initial Training Network (ITN) no. 607051, BIO-INSPIRE
1 October 2013 to 30 September 2017
For a number of bone degenerative diseases the existing treatment is insufficient. Regeneration of critical bone defects requires bio-mimetic and bio-active materials to trigger the required biological events for full bone repair.
BIO-INSPIRE established a multisite network for 5 experienced researchers (ERs) and 11 early stage researchers (ESRs) at 6 partner institutes located within Europe to develop a new horizon in Orthopedic Therapy by:
1. Development of a technology platform for full tissue regeneration of a range of critical sized bone defect types and patient characteristics. This platform consists of a new recombinant collagen biomaterial scaffolds that were developed (1) as a bio-mimetic mineralized scaffold, (2) as a local delivery system for growth factors and (3) as a delivery system for (autologous) cells.
2. Training of the next generation of leading Tissue Engineering scientists in a multi-disciplinary way
The main objectives and scientific results per research track:
Biomineralization
Key scientific objective:
Development of new hybrid biomimetic mineralized recombinant collagen (RCP) based biomaterials for preclinical trials
Main scientific results:
Biomineralized scaffolds are an attractive option for bone tissue engineering, being similar to native bone.
Biomimetic mineralization processes were successfully utilized to develop various mineralized biomaterials with tailored extent of inorganic phase, obtained in the form of apatite (HA) nanoparticles heterogeneously nucleated onto assembling recombinant collagenlike polypeptide (RCP). Several geometries of biomaterials with a ‘biomimetic’ character were created, ranging from linear and isotropic structured RCP/HA freeze-dried sponges to emulsified RCP/HA microspheres in paste formulations. Biological evaluation demonstrated their potential use as bone repair materials. Injectable RCP/HA microspheres dispersed in intercalating networks were selected for further in vivo study in a rabbit osteonecrosis model. Histological images showed that treatment with a formulations comprising RCP/HA microspheres resulted in bone metabolic activity as large number of osteoblasts along trabeculae were observed. These results suggest that these pastes are very promising in future treatment of osteonecrosis.
Growth factors
Key scientific objective:
Design of a controlled release delivery system for induction of bone formation
Main scientific results:
A controlled delivery system was created by using hydrogels combined with RCP microspheres releasing Bone morphogenetic Protein 2 (BMP-2) in time. Release kinetics showed an unexpected high binding affinity of BMP-2 for RCP as the majority of the protein retained (65%-80%) in the microspheres. These BMP-2 loaded RCP microspheres spheres were able to induce ectopic bone formation in an in situ gelling system. After further optimization of the RCP microsphere-hydrogel compositions, it was possible to repair bone defects fast with a reduced BMP-2 concentration in comparison to literature values.
To further slow-down the release, 2 novel BMP2 variants were developed that could be covalent bound to RCP microspheres while maintaining the bioactivity in vitro. In vivo testing revealed that the BMP2-Plk variant did not induce bone formation, whereas the composition with BMP2-Azide gave comparable or even slightly more bone formation when compared to absorbed wild type BMP-2.
In parallel different proteins Nell1, Follistatin, HMGB1 and CCN2 were screened for their agonistic effect on bone formation. Follistatin was selected as most promising candidate based on in vitro, however, in vivo tests in a calvarial bone defect in rats did unfortunately not lead to enhanced bone formation.
Cell therapy
Key scientific objective:
Development of new injectable microcarriers for controlled cell multiplication of cells to generate substantial amounts for therapeutic purposes and assess the functionality and regenerative capacity of cell-loaded micro-carriers in new bone formation.
Main scientific results:
Microcarriers were developed that were optimized for dynamic cell culturing in terms of diameter and pore size. In addition, also the use of these microcarriers in the dynamic cultivation of cells in spinner flasks was optimized. The results showed clearly enhanced cell growth for both the C2C12 reference cell line as well as for human mesenchymal stem cells (hMSCs) on RCP particles when compared to a commercial reference. The cells could be transferred between individual microcarriers thus validating the functionality of this propagation technique mentioned before.
To improve vascularization of bone contructs, endothelial cells were also studied for cell therapy. Isolation procedures were optimized and established for micro vascular endothelial cells (mvECs) and blood outgrowth endothelial cells (BOECs). Furthermore, GMP compliant media formulations were developed for both hMSCs and for endothelial cells for future cell therapies.
In vitro and in vivo models were developed to evaluate the biocompatibility, the efficacy and the toxicity of RCP microcarriers combined or not to MSCs and BOECs. The in vivo experiment using a calvarian bone formation efficacy model showed the absence of toxicity related to the administration of the RCP beads. However, no significant bone formation efficacy could be observed probably due to low expansion of MSCs and BOECs on RCP microspheres.
Training and dissemination
A comprehensive training program was set up for the fellows in which they were being trained via i.) the secondment program, via ii.) trainings organized by the consortium, iii.) an individual selected course programme and iv.) visiting international conferences. The secondment program of exchanging knowledge between partners was one of the strongest points of the Bioinspire training program leading to >12 collaborative publications between the diverse work packages. So far, 24 peer-reviewed manuscripts were accepted for publication, 5 papers are submitted and 4 in preparation. One paper was selected on the cover of Tissue Engineering special focus issue on strategies in musculoskeletal tissue engineering. Results were presented at various international conferences (>30) with in total 33 poster contributions and 11 oral presentations. The results of BIO-INSPIRE led to the filing of 3 patents. Four fellows obtained their PhD degree in 2017 and six fellows are writing/submitting their theses and these are all expected to be defended in 2018.
Project information:
Website: www.bioinspire.eu; the Bio-inspire project logo can be found on the project website.
Contact: dr. Suzan van Dongen (suzan.van.dongen@fujifilm.com)
1 October 2013 to 30 September 2017
For a number of bone degenerative diseases the existing treatment is insufficient. Regeneration of critical bone defects requires bio-mimetic and bio-active materials to trigger the required biological events for full bone repair.
BIO-INSPIRE established a multisite network for 5 experienced researchers (ERs) and 11 early stage researchers (ESRs) at 6 partner institutes located within Europe to develop a new horizon in Orthopedic Therapy by:
1. Development of a technology platform for full tissue regeneration of a range of critical sized bone defect types and patient characteristics. This platform consists of a new recombinant collagen biomaterial scaffolds that were developed (1) as a bio-mimetic mineralized scaffold, (2) as a local delivery system for growth factors and (3) as a delivery system for (autologous) cells.
2. Training of the next generation of leading Tissue Engineering scientists in a multi-disciplinary way
The main objectives and scientific results per research track:
Biomineralization
Key scientific objective:
Development of new hybrid biomimetic mineralized recombinant collagen (RCP) based biomaterials for preclinical trials
Main scientific results:
Biomineralized scaffolds are an attractive option for bone tissue engineering, being similar to native bone.
Biomimetic mineralization processes were successfully utilized to develop various mineralized biomaterials with tailored extent of inorganic phase, obtained in the form of apatite (HA) nanoparticles heterogeneously nucleated onto assembling recombinant collagenlike polypeptide (RCP). Several geometries of biomaterials with a ‘biomimetic’ character were created, ranging from linear and isotropic structured RCP/HA freeze-dried sponges to emulsified RCP/HA microspheres in paste formulations. Biological evaluation demonstrated their potential use as bone repair materials. Injectable RCP/HA microspheres dispersed in intercalating networks were selected for further in vivo study in a rabbit osteonecrosis model. Histological images showed that treatment with a formulations comprising RCP/HA microspheres resulted in bone metabolic activity as large number of osteoblasts along trabeculae were observed. These results suggest that these pastes are very promising in future treatment of osteonecrosis.
Growth factors
Key scientific objective:
Design of a controlled release delivery system for induction of bone formation
Main scientific results:
A controlled delivery system was created by using hydrogels combined with RCP microspheres releasing Bone morphogenetic Protein 2 (BMP-2) in time. Release kinetics showed an unexpected high binding affinity of BMP-2 for RCP as the majority of the protein retained (65%-80%) in the microspheres. These BMP-2 loaded RCP microspheres spheres were able to induce ectopic bone formation in an in situ gelling system. After further optimization of the RCP microsphere-hydrogel compositions, it was possible to repair bone defects fast with a reduced BMP-2 concentration in comparison to literature values.
To further slow-down the release, 2 novel BMP2 variants were developed that could be covalent bound to RCP microspheres while maintaining the bioactivity in vitro. In vivo testing revealed that the BMP2-Plk variant did not induce bone formation, whereas the composition with BMP2-Azide gave comparable or even slightly more bone formation when compared to absorbed wild type BMP-2.
In parallel different proteins Nell1, Follistatin, HMGB1 and CCN2 were screened for their agonistic effect on bone formation. Follistatin was selected as most promising candidate based on in vitro, however, in vivo tests in a calvarial bone defect in rats did unfortunately not lead to enhanced bone formation.
Cell therapy
Key scientific objective:
Development of new injectable microcarriers for controlled cell multiplication of cells to generate substantial amounts for therapeutic purposes and assess the functionality and regenerative capacity of cell-loaded micro-carriers in new bone formation.
Main scientific results:
Microcarriers were developed that were optimized for dynamic cell culturing in terms of diameter and pore size. In addition, also the use of these microcarriers in the dynamic cultivation of cells in spinner flasks was optimized. The results showed clearly enhanced cell growth for both the C2C12 reference cell line as well as for human mesenchymal stem cells (hMSCs) on RCP particles when compared to a commercial reference. The cells could be transferred between individual microcarriers thus validating the functionality of this propagation technique mentioned before.
To improve vascularization of bone contructs, endothelial cells were also studied for cell therapy. Isolation procedures were optimized and established for micro vascular endothelial cells (mvECs) and blood outgrowth endothelial cells (BOECs). Furthermore, GMP compliant media formulations were developed for both hMSCs and for endothelial cells for future cell therapies.
In vitro and in vivo models were developed to evaluate the biocompatibility, the efficacy and the toxicity of RCP microcarriers combined or not to MSCs and BOECs. The in vivo experiment using a calvarian bone formation efficacy model showed the absence of toxicity related to the administration of the RCP beads. However, no significant bone formation efficacy could be observed probably due to low expansion of MSCs and BOECs on RCP microspheres.
Training and dissemination
A comprehensive training program was set up for the fellows in which they were being trained via i.) the secondment program, via ii.) trainings organized by the consortium, iii.) an individual selected course programme and iv.) visiting international conferences. The secondment program of exchanging knowledge between partners was one of the strongest points of the Bioinspire training program leading to >12 collaborative publications between the diverse work packages. So far, 24 peer-reviewed manuscripts were accepted for publication, 5 papers are submitted and 4 in preparation. One paper was selected on the cover of Tissue Engineering special focus issue on strategies in musculoskeletal tissue engineering. Results were presented at various international conferences (>30) with in total 33 poster contributions and 11 oral presentations. The results of BIO-INSPIRE led to the filing of 3 patents. Four fellows obtained their PhD degree in 2017 and six fellows are writing/submitting their theses and these are all expected to be defended in 2018.
Project information:
Website: www.bioinspire.eu; the Bio-inspire project logo can be found on the project website.
Contact: dr. Suzan van Dongen (suzan.van.dongen@fujifilm.com)