Community Research and Development Information Service - CORDIS

Periodic Report Summary 1 - BIO-INSPIRE (BIO INSPIRED BONE REGENERATION)

General project objectives:
For a number of bone degenerative diseases the existing treatment is insufficient. Especially in cases of larger defects in combination with age related healing impairments, diabetes or radiation therapy. Regeneration of critical bone defects requires bio-mimetic and bio-active materials to trigger the required biological events for full bone repair.
BIO-INSPIRE will develop a new horizon in Orthopaedic 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 scaffold, that will be used (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, leading them towards excellence in individual disciplines in combination with a multidisciplinary, holistic view on the (bone tissue) biological systems to be studied.

Bio-mineralization research area (1):
Objectives:
• Establishment of the procedures for scaffolds with graded mineralization
• Completion of the scaffold development for preclinical trials
Main scientific results obtained so far and expected final results:
Nano apatite phases with different compositional and crystal features were nucleated on synthetic collagen-like peptide (SCP) based matrices, to mimic the composition and structure of mineralized tissues. The ‘biomimetic’ character of the crystal phase formed in the presence of SCP was confirmed by X-ray diffraction and infrared spectroscopy.
By using emulsification technology these scaffolds were shaped in the form of injectable microspheres. These beads were subsequently dispersed in intercalating networks to obtain moldable and injectable formulations. Properties of these formulations are being optimized for in vivo experiments in terms of injectability and filling of the bone defect.
Freeze-drying was applied to obtain sponge-like biomimetic scaffolds with linear or isotropic porosity and with a well-defined pore size, interconnectivity and orientation. The mineralization of SCP with apatite phases containing magnesium and carbonate ions (replacing calcium and phosphate, respectively) was achieved and considered as optimal to exhibit high mimicry of natural mineralized tissues, thus being adequate for stimulation of cells towards new bone formation. 3D scaffolds mineralized with Mg/CO3-substituted apatites in amounts were successfully developed and will be studied in vitro and in vivo.
Bio-inspired mineralization process was also applied to nucleate apatite nanophases substituted with divalent and trivalent iron ions on SCP to impart magnetic properties. 3D scaffolds mineralized with iron-substituted apatite were successfully developed and are being optimized by means of cross-linking and mineral phase composition and content, to express adequate magnetic properties.

Growth factors research area (2):
Objectives:
• Design of a delivery system for FDA-approved growth factors/peptides derived from growth factors.
• Selection of new active peptides, growth factors or cytokines. Investigation of their effect on angiogenesis, attraction of MSC's and osteogenic differentiation.
• Assess the effects of growth factor or peptide loaded microcarriers on bone formation.
Main scientific results obtained so far and expected final results:
Synthetic Collagen Peptide (SCP) microspheres were studied for sustained release of the FDA registered Bone Morphogenic Protein 2 (BMP-2). It was shown that the majority of the protein retained on or within the microspheres and is only released after collagenase degradation of the microspheres. The released BMP2 was biologically active as demonstrated by a cell based assays using a luciferase modified cell line. To further slow-down the release, two novel BMP2 variants have been produced that contain an incorporated artificial amino-acid allowing for a site-directed, covalent binding to surface structure. Preliminary experiments proved the bioactivity of the coupled protein in cell based assays indicating that the coupling conditions do not impede the proteins structural integrity. Another strategy to increase the biological activity of endogenous BMP2 aims to block one of its natural inhibitors, Noggin. Noggin blocking peptides were selected using a phage display strategy. Next step is to synthesize peptides that will be tested for their Noggin binding capabilities.
After literature screening 4 factors were selected for attraction of MSCs and angiogenesis: Nell1, Follistatin, HMGB1 and CCN2. The effect of the candidates on osteogenesis of human bone marrow derived MSCs as well as on angiogenesis and vascularisation are currently being investigated.
An in vivo trial was started. Hydrogels were combined with BMP-2 loaded microspheres and injected subcutaneously in rats to observe ectopic bone formation. Three different in situ gelling hydrogels were optimized and prepared (two forms of alginate and a thermoreversible hyaluronic acid). The different gels were combined with two concentrations of microsphere and a fix amount of BMP2 and injected subcutaneously in immune competent rats. All constructs could be retrieved. Gel-dependent cell and blood vessel ingrowth was seen after 1 week. After 4 and 10 weeks bone formation was observed in the harvested constructs. Further analyses with semi-quantitative histology and microCT analyses are ongoing.

Cell therapy research area (3):
Objectives:
• Improvement of cell harvesting procedures of MSC's and EPC's from cell sources, including full cell characterization. Harvesting of EPC's out of patients-based sources is point of investigation.
• Controlled cell multiplication of primary cells to generate substantial amounts for therapeutic purposes, by use of new recombinant collagen-like micro-carriers.
• Improved cell survival rates by minimal invasive injections of cells on new injectable micro-carriers
Main scientific results obtained so far and expected final results:
This area is dedicated to the delivery of cells on micro-carriers to the bone defect area for neo-vascularization (EPC's) and bone regeneration (MSC's) purposes. The use of SCP based microcarriers for the dynamic cultivation of cells in spinner flasks was started. The results showed clearly enhanced cell growth of human mesenchymal stem cells (hMSCs) on SCP particles when compared to a commercial reference. The effect of crosslinking on cellular proliferation and differentiation is currently evaluated.
To improve vascularization of bone contructs, endothelial cells were also studied for cell therapy. Two types of cells were selected, micro vascular endothelial cells, mvECs and blood outgrowth endothelial cells (BOECs). For both cell types, isolation procedures were optimized and cells were characterized morphologically and immunohistologically. Additionally, cell functionality was tested by sprouting and tube formation assays induced by VEGF, FGF and combinations thereof. The isolated mvECs show a typical cobblestone morphology and express the known endothelial markers.

Orthopaedic studies:
Objective:
• Assess the functionality and regenerative capacity of biomimetic scaffolds, drug delivery carriers and cell-loaded micro-carriers, through several in vitro test systems and animal models to demonstrate biocompatibility of the material and new bone formation by the future medical implant.
Main scientific results obtained so far and expected final results:
New innovative pastes for bone regeneration were developed that can be used for the treatment of osteonecrosis of the femoral head, an important and severe condition with high incidence of rate. Preliminary ex vivo tests performed in Rizzoli showed positive results with good penetration of the paste. Based on these findings, a preclinical model will be developed.
The biocompatibility of collagen microcarriers for cell therapy was tested towards Bone Therapeutics osteoblastic cells and did not affect cell morphology or the cells phenotype. A mouse cranial defect model will be used to investigate the potential of MSC and EPC based cell therapy for bone regeneration.

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.eu)

Reported by

FUJIFILM MANUFACTURING EUROPE BV
Netherlands

Subjects

Life Sciences
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