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Progress of work in 2013 and main results achieved so far:

Monitoring and quality control

Biocompatibility of CeraSorbM scaffolds modified by hydrophilic diamond powder, was determined according to DIN ISO 10993-5 as reported earlier. In addition, in vitro and in experiments were performed of this modified material and additionally the biocompatibility of two aliphatic polyester co-polymer scaffolds developed by the consortium was evaluated: Poly(L-lactide-co-e-caprolactone) and Poly(L-lactide-co-1,5-dioxepan-2-one). The surfaces of these polymer scaffolds have also been modified by nano diamond particles (nDP) and found to biocompatible. Furthermore, modifying scaffolds surfaces by nDP shown to promote cellular proliferation, differentiation and bone formation in a sheep model (Xing et al. 2013).

To develop obtain quantitative information about structural parameters for each scaffold, high resolution magnetic resolution imaging (MRI) is shown to be a valuable tool. The reproducibility of the manufacturing process of the scaffolds regarding pore size and wall thickness distributions can be verified using MRI which can be one step in the quality control for the future use as medical products. Various MRI contrast agents have also been developed by the consortium such as fluorine-containing moieties. The advantage of using fluorine-containing moieties as contrast agents is that fluorine induces a hyperintense MRI signal and can, thus, be detected unambiguously in the dark background of the scaffolds. Also, due to the fact that fluorine is essentially absent in the human body, the acquired 19F signal is background-free allowing an excellent degree of specificity. Thus, cell-specific 19F-based contrast agents were developed and demonstrated that the fluorine-based contrast agents offer an alternative for the proof of seeding efficiency by in vivo imaging methods. The emulsions were formulated for in vitro and in vivo use; i.e. the agents were made available as sterile solutions with a concentration that requires only a low injection volume.

Angiography contrast agents for in vivo MRI blood flow detection were also developed by the consortium. The novel agents included a Gd-based polymer (positive contrast) and superparamagnetic iron oxide (SPIO) nanoparticles (negative contrast) for 1H MRI, as well as 19F-based nanoemulsions for 19F MRI. These novel agents were tested in vivo in healthy rats in comparison to the standard, marketed, clinical contrast agent, Magnevist (Gd-DTPA). The results showed that from all tested agents, the optimized iron oxide-based contrast agent (FeraSpin XS-Type) induced the most significant signal changes in the bone marrow within the rat femur. Thus, this agent can be used to characterize the existence of active blood flow and, thus, angiogenesis in healing bone and remodeled biomaterials. These experiments served as a proof-of-principle to show that the SPIO contrast agent highly improves the in vivo detection of blood vessels/blood flow in bones, allowing investigation of the microvessel formation within the implant and, thus, an evaluation of the angiogenesis efficacy and implant viability. Furthermore, a substantial effort was placed during 2013 to optimize the dose of the SPIO contrast agent that can be used for evaluation of the angiogenesis efficacy in large animals as sheep. Accordingly, a preliminary and a rational dose was selected and currently tested in ongoing sheep study.

Furthermore, a novel, sensitive and non-invasive method was established for screening the tumorigenic potential of functionalized/bioactive scaffolds intended for bone regeneration. Using high potency bioluminescence (BLI) in vivo imaging of cells/scaffold construct is carried out in parallel with classical morphometric analysis. The model has been applied on testing nano diamond modified polymer scaffolds with or without BMP-2. The preliminary results of BLI showed that when the scaffolds modified with nano diamond particles the total photon intensity was reduced indicating that the tumor cells stopped proliferating. The opposite was seen when BMP-2 was added, the intensity continued to rise until the 14 weeks of imaging. It was also concluded that soluble nano diamonds do not influence tumor cell growth to a high extent although a slight increase of proliferation was observed in breast cancer cells. These results have to be considered if scaffolds coated with nano diamonds are applied in patients with bone metastases.

Design/fabrication of scaffolds

The progress can be divided in two main topics:

  • Development and optimization of biomaterial and scaffolds
  • Analysis, characterization and development of new methods for evaluation of the biomaterial

The application of the specially tailored biomaterial for in vitro and in vivo tests and their results can be found in the reports of the corresponding WPs (2, 4, 5, 6, 7, 8). The biomaterials for VascuBone are based on polymers and -TCP modified with nano diamond particles. The two different types of porous polymer-based materials are developed at KTH and PP. As ceramic based scaffold CeraSorbM of Curasan has been purchased. Modification was achieved by the developed nano diamond particle diffusion technique at DIA (described and validated during the first two years of VascuBone). The material was produced in line with the defined protocols in high reproducibility and large batches. Sterile nano diamond particles have been prepared according protocols at UW (Chem) Characterization of the novel implant material (DiaSorb) for pre-clinical studies have been realized within the consortium (UiB, FhG/IGB, IWA/UIBK) and at external accredited and authorized laboratories. New methods have been established and can be applied as standard with respect to the preparation, data evaluation and approval for the planned clinical trials. Biofunctionalization with growth factors, release measurements, evaluation and optimization for active, stable immobilization have been performed successfully at UiB and IAW. This work was strongly supported by theoretical calculations and modeling at UU and the design and development of experimental procedures.

Expected results:

The results expected and aimed in WP 3 are clearly defined by realization of the objectives in close collaboration and interaction of the VascuBone partners and support from accredited organizations. New methods for standardization and approval of nano-materials based on nano diamond particles and porous -TCP and polymer scaffolds, optimized material properties like hydrophilicity, increase of active area, biocompatibility and mechanical improvement, stable biofunctionalization and theoretical estimations to support design of experiments are demanded. Novel techniques for determination the genotoxicity, biocompatibility of nDP/scaffolds (DiaSorb) should be established. Verification of the protocols for production of sterile biomaterial in large batches achieved during the first two years of VascuBone is an additional estimated outcome.

Clearly significant results:

All expected results have been achieved successfully in year 2013 and presented in the following task-descriptions in detail. Summing up the following significant results have been achieved and have been/will be published in peer-reviewed scientific journals:

1. Quantification of nDPs and release of nDPs in/from DiaSorb

  • Release and leaching analysis techniques developed, no release observed
  • Structural analysis of DiaSorb BET surface analysis, increase of surface area up to 100 fold, pore size not influenced
  • Method development for label free detection and quantification of nDPs
  • Determination of size by NTA: no alternative to Malvern

2. Biocompatibility studies of nDPs for pre-clinical trials

  • AMES test: novel method for nano particles established
  • Single dose escalation toxicity study in rats (ongoing): nDP in glucose in higher concentration realized

3. Detachment of VEGF-165 and angiopoietin from the surfaces of nano diamond particles (nDP) by means of radiolabeling successfully realized.

4. In vivo studies determined bioactivity of bounded BMP-2 to poly(L-lactide-co-e-caprolactone) scaffolds in different binding techniques.

5. Physisorption of BMP-2 to NCD-modified CeraSorbM resulted in a higher ALP induction in comparison to non-modified materials treated with BMP-2. (n=3) The biological activity of the physisorbed BMP-2 could be verified.

6. Method to functionalize porous scaffolds and subsequently covalently bind growth factors. New technique to combine nDP and the polyester based scaffolds during the scaffold fabrication.

7. Synthesis of orthogonally functionalized nanodiamond materials with different orthogonal groups have been developed and can be used for the simultaneous grafting onto a polymer scaffold and the grafting of functional molecules, e.g. growth factors.

8. First preliminary results show that QCM can be a suitable technique for analyzing the interactions between PLA and poly(LLA-co-CL) and nDP, fibronectin and BMP-2.

Clinical observational study

One aim of the VascuBone consortium is to develop new therapeutic approaches for the regeneration of bone defects based on the application of bone marrow derived progenitor cells and biocompatible scaffolds. The translational aspect of this project is the design and executions of preclinical and clinical phase I trials addressing vascularized bone and soft tissue regeneration in:

1. Avascular necrosis of the femoral head (FHN) (FhG, UW)
2. Small maxillary defects (FhG, UiB, MUI)
3. Large bone defects of the facial skeleton (FhG, UiB, MUI)
4. Vascularized implants for the reconstruction of bronchotracheal defects (UW-UKW, FhG)

For trial 3, preclinical trials are still ongoing (WP8) to fulfil regulatory requirements. For trial 1, 2 and 4 the preclinical phase is finished. For FHN treatment , initiation of the manufacturing process for MSCs has been started at partner FhG, UKW.

Trials on small maxillary defects and large bone defects of the facial skeleton have been designed but depending on the results of the large animal studies. Visualization and monitoring of implant ingrowth and remodelling in mandibular defects was successfully established by MRI. The manufacturing process for vascularized tracheal construct was adapted to GMP. The application for the clinical trial on TraVaSc implants will be performed after approval of TraVaSc manufacturing authorization.

Cell/tissue scaffold interactions

Biomaterial as being produced and/or refined by VascuBone consortium members is being tested in order to validate and predict potential future use in clinical applications. Hence non-modified and modified CeraSorbM as well as synthetic materials are either loaded with cells or implanted in animal models in order to single out optimal scaffold modifications to enhance bone regeneration. Also using static and dynamic culture systems the response of primary human bone marrow stromal cells (MSC) to scaffolds has been tested.

Previous results indicated that

  • Both CeraSorbM and VascuBone synthetic material permit cell loading albeit under variant condition due to material specifications; showing variant cell loading attraction and capacity as well as differential proliferation of the loaded cells
  • Modification of scaffold material with ECM components allows to introduce conditions for improved cell attachment
  • Scaffolds bearing diamond nanoparticles enhance osteogenesis
  • Biomaterials refined with nDP is amenable for biofunctionalization

Evaluation of cell sources for implant loading

Main objectives were (1) understanding of EPC-MSC Interactions, (2) evaluation of age related effects on the suitability of MSC for implant loading, and (3) optimization of EPC culture conditions towards GMP production for implementation in VascuBone products.

One partner is focusing on the distinction of optimal MSC populations and conditions to efficiently induce osteogenesis. This work is based on previously established genomic and functional analyses, which were designed to determine as well as to instruct the nave cell to develop into an appropriate precursor cell type thus enabling in situ osteogenic differentiation. Applying 4-methyl umbelliferron (4MU) a fate decision in osteogenesis is enhanced mimicking the terminal stage of the osteogenic process. Notably cells cease proliferation, which is certainly of pertinent importance when employing cultivated MSC in vivo, as it is essential to warrant no further cell growth thus minimizing the risk of cancer formation. Furthermore, 4MU is capable of inducing osteocytogenesis. It is assumed that ostecytes are a robust long-living cell type, which builds bone matrix through the production of efficiently calcifying matrix. Mesenchymal cells when isolated from an environment of advanced age or otherwise when replicatively aged through excessive rounds of cell proliferation in vitro often exhibit a decreased propensity to differentiate. Treatment with a small molecule, which may also be infused at later time points during tissue engineering or presumably also in vivo post operation is a potent tool to stimulate and propel osteogenesis in a skewed situation of an elderly organism.

Endothelial precursor cells (EPCs) play an important role in postnatal vasculogenesis. Novel bone tissue engineering approaches using scaffolds seeded with MSCs plus EPCs may improve bone regeneration. Medicyte undertook efforts to find and finally establish the culture conditions of alternative cell type namely BOEC (Blood Endothelial Outgrowth Cells). Cell culture conditions have been optimized and cells have been provided to the consortium partners. To better understand cell-cell interaction of MSCs and EPCs, the changes of global gene expression patterns of human primary EPCs after having been subjected to conditioned medium of human primary MSCs or after direct cell-cell contact, and vice versa were finalized by UW in affymetrix screening procedures and are currently compared to BOEC cells. UIB has analyzed the interaction of endothelial cells and MSC in scaffolds provided by KTH. Cells have been characterized via RT-PCR, ELISA and immunohistochemical analyses. Further constructs were subcutaneously implanted into immunocompromized mice and subsequently analyzed for inflammation and angiogenesis. This will help to better understand the crosstalk of MSCs and endothelial cells, which will finally aid to the improvement of vascularized tissue engineering constructs. Although the resources for GMP production were available at FhG, the adoption of a production process for EPCs to GMP requirements has not been started, because the culture expansion and lifespan of EPC did not allow seeding of BioVaSc. Currently, BOEC cells are being used and allows for higher cell numbers. Therefore activities were focused on establishing methods to characterize the growth properties on the BioVaSc and to establish bioreactor systems to cultivate cells under controlled conditions to high density.

Development of a pre-vascularized bone implant

This work package seeks the construction of a pre-vascularized bone implant. In order to achieve this goal a range of different techniques has to be employed and refined. Equipment and materials developed and/or modified by the consortium will be combined with the findings acquired during the earlier tasks in the project to progress towards the key objective.
  • The initial step of seeding porous 3D scaffolds was optimized by the application of bioreactor systems as well as adjusting dynamic culture conditions and validated for -TCP scaffolds (CeraSorbM). Further experiments expanded the insight into the effects of these aspects on cells seeded on Polymer scaffolds (supplied by KTH) by gene expression analysis.
  • Crucial for assessing factors that induce or support angiogenesis is a method that allows quantitative measurement of such an angiogenic effect.
  • Progress towards a transfer of pre-vascularized bone implants to GMP conform manufacturing was made by additional enhancements of the current bioreactor technology.
Expectations in this work package concern the progress in techniques that ultimately allow the creation of a pre-vascularized bone implant that conforms to GMP guidelines. Progress was made in the different aspects of the WP and is presented in detail below. The following results were achieved:
  • Successful application of methods to analyze Polymer scaffolds produced by KTH qRT-PCR and revealed the increased induction of stress and osteogenic differentiation related genes in cells seeded on the polymer scaffolds under dynamic culture conditions in a bioreactor as compared to static conditions.
  • A method for quantitatively assaying angiogenic effects was developed in task 7.3. This allows the identification of optimum parameters for growth factor gradients relevant for pre-vascularizing bone implants. The method makes use of life cell imaging and subsequent trajectory analysis to quantify the effects.
  • Bioreactor technology was modified towards GMP by introducing a separation between pumps and control unit. This way, a controlled dynamic perfusion of the BioVaSc can be achieved in a GMP environment while monitoring and control of relevant parameters can be undertaken outside the GMP area.

Preclinical trials

Animal experiments in femoral hip necroses are already performed and the MRI analysis with a developed scanner is already established. The preclinical trial for lateral augmentation in the mandible is nearly finished and the preclinical evaluation of mandibular continuity defects is in progress. The preclinical model of a critical size defect in the femur in a large animal is established and the production of a prevascularized implant is standardized. The preclinical trial will start in January 2014.

To provide a sufficient lead bearing osteosynthesis in continuity defects experiments on the ideal osteosynthesis plate position are performed. Furthermore chondral ossification and host response from constructs loaded with co-cultured cells in copolymer scaffold using a rat calvarial defect model are investigated.

Dissemination and exploitation

The CA has been signed by all partners. An amendment regarding the proper handling of foreground and organizing of protection is planned, a draft amendment has been provided to the partners for commenting on 09.12.13. A draft plan for use and dissemination has been completed. Lunch symposia and EU-project session were organized in international symposia. The tools and products of VascuBone were presented at fairs, like the Bio Europe.

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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  European Union