During the InnovaBone project TETRA developed an industrial approach to utilize the  2-photon-polymerization process.      
Two-photon polymerization (2PP) is based on the two-photon adsorption which is defined as the simultaneous absorption of two photons by a molecule. The adsorption leads to an energetically excited state of a photoinitiator resulting in cross-linking of the exposed polymer. The advantage of this effect is a very small polymerized area. In combination with a kinematic structure to move the so called voxel, 2PP is capable to write 3D structures with resolutions below one µm. Thus the 2PP process dramatically expands the possibilities of typical lithography processes used in semiconductor technology.
Within the last years 2PP-processes targeting applications like tissue engineering, photonic applications, microfluidics and MEMS have been presented. Today, 2PP process units produce structures like scaffolds, photonic crystals and single optical interconnects. Until recently, a major drawback of 2PP-processes was that only structures in small spatial dimensions and at low speed could be manufactured. For this reason, no industrial application for 2PP processes was available. Tetra, with its strong expertise in drive technology and mechatronics developed an industrial system performing 2PP processes. For the first time, a possibility for the industrialization of the 2PP process is now offered with higher speed of production resulting in higher outer dimensions of the structures.
In 2014 TETRA presented the world largest monolithic structure written by 2PP. By using biopolymers developed by the partners of the InnovaBone project TETRA supported the InnovaBone project by writing of more than 1000 scaffolds for research purposes.

The orthopedic medical device industry challenges the advanced materials science to investigate new biomaterial designs for medical devices with enhanced performance and controlled safety profiles. When the medical device regulations are not recognized during research, the industry cannot judge the feasibility of the design and does not take the risk of transferring it to the market. It will benefit the valorization process if research institutes take the medical device regulations into account early phase their research projects. For this purpose, we have introduced a systematic risk based approach to establish a ‘frozen’ design and to identify suitable design controls from a regulatory compliance point of view. This should ease the industry decision-making for accepting biomaterial designs into medical devices.

Digitalisation of R&D processes is acquiring a key role in scientific production, as it allows to share experimental procedures and data, assuring their replicability and consistency; this, in the long term, can boost achievement of reliable results.
Nevertheless, in most European research laboratories, information and data management only relies on file servers and conventional laboratory notebooks, which considerably limit the possibility to organize and share information about experimental processes.
SampleLife was designed to respond to the needs of the EU-funded InnovaBone project, and specifically:

• To trace samples and their datasheets in order to track any material and data and reenact the relation between the single parts of joint experiments;
• To manage the logistics of samples, such as information on material flows from production to testing phase: availability, location, order and shipping status;
• To harmonize datasheets through a shared and systematic framework of data structures to be customized by its users for their research needs;
• To allow replication of experimental processes and integrated data comparison.

When InnovaBone started, on the market there was no software which was flexible enough to meet such complex needs; this lead us to develop a new online tool to plan and monitor scientific experiments by managing information on materials, on their functionalization and improvement techniques, and on their in-vitro and in-vivo tests.
NetScience is also integrated with an Electronic Laboratory Notebook, which allows researchers to link sample and process data with free annotations, pictures, files and tables, as well as to comment and alert colleagues on relevant events.
NetScience also includes an online platform to manage contents, documents and project commitments towards the EC and among project partners. The tools has been developed in response to the coordination needs of InnovaBone, and is currently used by its partners to monitor the progress made in the different project tasks, as well as to upload and download project documents in an intranet where different access rights have been previously set in line with the definition of the governing bodies of the project.
Given the success obtained with the two prototypes so far, we intend to verify in LEAN mode whether it can be expanded beyond the field of biomaterials in both single laboratories and big research networks.

InnovaBone implemented an inter-disciplinary approach to foster collaborative discussions and exchanges among researches mainly from EU funded project. Moverim boosted internal training (MOOCs) and clustering actions with other projects to build up a common vision, exchange information and good practices on bone regeneration and biomaterials. Multidisciplinary fora enable to create solid new relationships and push for further collaborations. Moverim is a SME located in Brussels working on funds for R&D for many clients like European universities, Research Centre’s, SMEs, non-profit organisations, industries. The SME supports the project management, organises training activities and contributes to dissemination strategy and the results exploitation. This project enhanced Moverim competences in raising researchers skills, build up participative networking methodologies to keep research group active beyond the EU project financing.

On behalf of the InnovaBone consortium I would like to welcome you to the conference in Brussels. InnovaBone is a Large Collaborative FP7 Project with a strong multidisciplinary approach to develop smart bone biomaterials. 14 partners from 8 European countries were involved in the project.

The importance of developing novel approaches for bone repair is underscored by the heavy burden on health care costs and patient suffering caused by traumatic, osteoporotic and osteolytic metastatic bone lesions. To address these health challenges, InnovaBone is dedicated to the development of optimally performing bioinspired biomaterials mimicking the natural physiological processes underlying bone repair of non-healing bone lesions. Our ultimate aims are to ensure strong, healthy bone regeneration, reduce pain and suffering and to become a competitor in the biomaterials market of Europe. Using an extensive, state-of-the-art approach by the InnovaBone team of cellular and molecular biologists, immunologists, physicists, bioengineers, and orthopaedic surgeons, the aim is to tackle serious non-healing bone lesions. The overall approach of InnovaBone is to produce smart bioactive 3D scaffolds to fit within bone lesions, which
 will then be combined with functional, genetically-engineered self-solidifying elastin-like recombiners containing calcium phosphate nanoparticles. This combination of materials aims to encourage cells to attach within the scaffolded area, promote cell growth and ultimately start the bone regeneration process, which helps the body self repair. InnovaBone scientists established comprehensive production and testing platforms to obtain optimal products. A range of biomaterials was tested for their effects on bone cells, bone growth and healing, on immune and allergic cells with state-of-the-art in vitro and in vivo models, imaging technology, in addition to a thorough evaluation of physicochemical properties including strength, durability, elasticity, toxicity, sterilisation capacity, degradability, and more. Novel, automated 2-photon polymerization equipment and an innovative BioMEMs bioreactor system were developed during the project to upscale biomaterial production and to study the effect of the biomaterials under dynamic conditions. The InnovaBone multidisciplinary, multidimensional approach for the developments and preclinical assessment of bone biomaterials will be presented during this meeting.

Nanolayered films have been often fabricated using the layer-by-layer technology, where consecutive layers of macromolecules are assembled and stabilised by electrostatic interactions. Using adequate templates, non-flat coatings can be fabricated with tuned compositions. This enables the production of very well controlled multifunctional and structural devices using mild processing conditions that could be useful in biomedicine, including in bone tissue engineering. In such applications, where there is a direct interaction between the implant with tissues and cells, the biomaterials must exhibit adequate surface characteristics, both at the chemical and topographic points of view. Examples of structures having nano-stratified multilayered organizations as building-blocks are presented, based on the use of natural macromolecules. Functional and bio-instructive multilayers may be produced by introducing special chemical groups or bioactive agents in the assembly. Such elements may be then hierarchically organised into 3-dimensional systems for cell colonisation (e.g. capsules or scaffolds) with tuned structural and geometrical control. Adequate signals or cell sources may be used to direct the osteogenic route of the developed devices, to potentiate their bone regenerative capability.

BIOFORGE´s main task has been the development and production of a gel matrix containing bioactive molecules which could be easily injected into  the  2PP scaffolds and further solidifies at body temperature. To achieve such properties, BIOFORGE took advantage of its expertise in recombinant elastin-like thermoresponsive polymer gels and developed a biocompatible, smart and self-assembling high molecular weight polypeptide. This smart behavior arises from the elastin-like domains present in the elastin-like recombinamer (ELR) that make this material water soluble at 4ºC and to become gel-like at body temperature. Moreover, the recombinant origin of these ELRs allows BIOFORGE to incorporate bioactive domains such as cell adhesion sequence, extracellular matrix protease sensitive sequences and bone growth factors that enhance the microenvironment for regeneration and, as they are directly linked to the ELR they avoid  their elution from the gel. Following this strategy, BIOFORGE has developed several ELRs based all of them in an amphiphilic structure schematically represented in Figure 1 and with the incorporation of bioactive domains.
The final chosen bioactive domains were the αβ integrin binding domain RGD that enhances cell adhesion while migrating through the gel, the bone morphogenetic protein BMP2 (a FDA approved growth factor) and BMP7 to promote osteoblast growth and differentiation. Moreover, to facilitate cell migration through the gel and release of BMPs, elastase sensitive sequences were incorporated. The combination of bioactive domains directly linked to the ELR structure and the use of protease sensitive sequences allows the gel to constantly provide a homogenous density of bioactive domains as cells migrate through the scaffold.
Due to the limited stability of such structures for in vitro cell culture, 2 new approaches were developed in order to overcome this problem. First of all, ELRs were re-designed in order to incorporate lysines which could be further modified to incorporate chemically crosslinkable motifs. This approach was discarded due to the difficulties of handling of the resulting materials. The problem was solved by incorporating Bombix Mory silk fibroin motifs, which undergo an unreversible physical crosslink that avoids dissolution of the ELR under excess of water.
ELRs are bioproduced in E.coli under controlled conditions and further purified taking advantage of their smart behavior. Final product is checked to be pure and to match the design by different and complementary techniques such as SDS-PAGE electrofphoresis, MALDI-TOF mass spectrometry, HPLC analysis of their amino acid composition, H-NMR and FTIR spectral analysis and DSC in order to asses thermal behavior of the ELRs.
BIOFORGE has fulfilled partners’ demands providing them with highly pure ELRs during the entire project. We have bioproduced, until now, more than 27 grams of ELRs for InnovaBone partners.

Introduction
The use of nanoparticles is becoming a widely spread practice in biomedicine for many purposes ranging from imaging to cancer treatment, drug delivery and gene therapy. In any of these applications NPs should not be cytotoxic, must not degrade e.g. while carrying their cargo but should be degradable once they have had fulfilled their purpose. To find NPs sharing all these features is challenging yet, a strong candidate is hydroxyapatite (HA) NPs. HA, being the mineral phase of bone has widely been used in the bulk form for bone regeneration applications but is less investigated as NPs. One very interesting feature of HA is the fact that its crystal structure has great ability to incorporate foreign ions, a fact that is exploited in bone to concentrate traces of a wide range of ions imparting great impact on the biological performance of bone. The aim of this work is to assess if ion doping could also be used as a strategy to modulate NPs-cell interaction. For this purpose a series of different NPs doped with Mg, carbonate and a mixture of them will be made and their cytotoxity will be evaluated through cell culture testing using cancerous (MG63) and mesenchymal stem cells (rMSCs).

Materials and Methods
HA-NPs were prepared by wet chemical precipitation via neutralization of calcium hydroxide with orthophosphoric acid. Doped NPs were prepared dissolving the appropriate amount of MgCl2 or NaHCO3 into the calcium hydroxide solution before acid addition. The obtained NPs were rinsed, freeze-dried and thoroughly characterized. Cell culture studies were performed using 100 ug·ml-1 of the various NPs with 10000 cells in 96 well/plate using MG63 and rMSCs in the following conditions: in the presence/absence of 10 v/v% foetal bovine serum and in the presence/absence of the dispersant sodium citrate. Cell viability was measured through lactate dehydrogenase quantification released by alive cells. NPs internalization was assessed through TEM imaging of cells upon embedding, sectioning and staining.

Results & discussion
The graphs in Figure 1 shows that Mg-doped NPs could selectively kill MG63 cells but not rMSCs provided FBS and sodium citrate were excluded during cell culture. These results put forward two aspects: on the one hand they show that cells can sense the composition of the bare NPs and on the other hand they demonstrate that adsorption of negatively charged molecules on the NPs prevents their internalization in MG63 cells. These findings have important implications as points that through simple incorporation of non-toxic ions we could selectively kill osteosarcoma cells without the need of using current toxic drugs and at the same time proves that ion-doping can be used as a strategy to modulate cell behaviour.

The  generation of bio-degradable and porous  scaffold materials is a prerequisite for the development of the final implant architecture, as it is the supporting material for the injection of the bioactive hydrogel. For the generation of these complex structures with high µ-resolution, we focused on direct laser writing techniques in particular two-photon polymerization (2PP), to avoid the necessity of photomasks when using traditional lithography methods. Another advantage of this approach is the long wavelength of the NIR, which allows a deeper penetration of NIR radiation into the material and thus the capability of structuring in millimeter dimension was attained in the first place [1-5]. 3D scaffolds based on poly(D,L-lactide-co-ε-caprolactone) (pLC) copolymer with different compositions were manufactured by 2PP  in a variety of dimensions tailored for different biomedical applications and related testing routines. For 2PP, only optical transparent materials are suitable, since all materials show an increasing opacity with increasing caprolactone content, lactide-rich variations were explored with LA:CL ratio of 90% (LC 18:2) or 80% (LC 16:4). In addition, the number of total monomer units added at 90% LA content was halved from 20 units to 10 units  (18:2 to LC 9:1 respectively) to reduce the chain length and the resulting mesh sizes in the structure.
The reactivity of the resulting three LC variants was examined by photo-DSC at three different temperatures. The material could be classified in terms of reactivity by the time to maximal heat generation tmax, resulting in following reactivity order LC 18:2<16:4<9:1. The materials were further investigated in terms of cytotoxicity with a special focus on the initiator systems, which revealed no toxicity as a first estimation.
The 3D structure was based on a Schwarz Primitive (P) minimal surface derived unit cells, which was obtained by 2-Photon direct laser writing and validated by SEM and µ-CT. By combining the unit cells in x, y and z, a tailor-made scaffold with controlled pore size, porosity and distinct dimensions was generated. The pore sizes for all LC scaffolds were approx. 300 µm and throat sizes varied from 152 to 177 µm.  Due to the load bearing capacity of the structure it was possible to generate a material which has a high compressibility and recovers its shape even after larger stress loadings. So it is possible to handle it in clinical bone defect models, by simply pushing it into the defect site to recover there and stay in place. Overall the material has the potential for the use as a supporting material in tissue engineering applications such as bone and cartilage repair.

During the InnovaBone project TETRA developed an industrial approach to utilize the  2-photon-polymerization process.      
Two-photon polymerization (2PP) is based on the two-photon adsorption which is defined as the simultaneous absorption of two photons by a molecule. The adsorption leads to an energetically excited state of a photoinitiator resulting in cross-linking of the exposed polymer. The advantage of this effect is a very small polymerized area. In combination with a kinematic structure to move the so called voxel, 2PP is capable to write 3D structures with resolutions below one µm. Thus the 2PP process dramatically expands the possibilities of typical lithography processes used in semiconductor technology.
Within the last years 2PP-processes targeting applications like tissue engineering, photonic applications, microfluidics and MEMS have been presented. Today, 2PP process units produce structures like scaffolds, photonic crystals and single optical interconnects. Until recently, a major drawback of 2PP-processes was that only structures in small spatial dimensions and at low speed could be manufactured. For this reason, no industrial application for 2PP processes was available. Tetra, with its strong expertise in drive technology and mechatronics developed an industrial system performing 2PP processes. For the first time, a possibility for the industrialization of the 2PP process is now offered with higher speed of production resulting in higher outer dimensions of the structures.
In 2014 TETRA presented the world largest monolithic structure written by 2PP. By using biopolymers developed by the partners of the InnovaBone project TETRA supported the InnovaBone project by writing of more than 1000 scaffolds for research purposes.

Introduction
The InnovaBone project aims to develop a biomimetic product that consists of a bespoke scaffold and a bioactive self-setting gel, which will provide a microenvironment that contains active elements such as growth factors and CaP nanoparticles to promote bone repair. Scaffolds composed of different lactic acid (LA) and ɛ -caprolactone (CL) ratios were produced using a two photon polymerisation (2PP). In this study, in vitro degradation and compressive properties were conducted for the produced scaffolds in phosphate buffered saline (PBS) at 37°C. Cytocompatibility of the scaffolds was assessed using human mesenchymal stem cells (MSCs)1.
 
Materials and Methods

Scaffold production
Scaffolds were provided by IBA and TETRA and manufactured by a 2PP polymerisation technology2 in which the 3D structure was based on a Schwarz Primitive minimal surface derived unit cells. Three different LA:CL ratios (16:4, 18:2 and 9:1) were investigated and samples were coded LC16:4, 18:2 and 9:1.

In vitro degradation and mechanical testing
Degradation study of the scaffolds was performed according to the standard BS EN ISO 10993-13:2010 at 37°C using PBS buffer (pH =7.4 ± 0.2) and accelerated testing was also applied at 50 and 65°C. Compression testing was conducted using Hounsfield tester according to the standard ASTM 1621-10: 2010 at 25±1°C.

Cell culture
Human MSCs1 were seeded at a concentration of 1x106 cells per scaffold and cultured in DMEM supplemented with 10% foetal calf serum, 1% L-Glutamine, 1% non-essential amino acids, 1% penicillin/streptomycin [standard medium], supplemented with 0.1 μM dexamethasone, 50 μM ascorbic acid phosphate, and 10 mM β-glycerophosphate [Osteogenic (OS) medium)] at 37°C and 5% CO2.

Cell viability, metabolic activity, and markers of osteogenic differentiation were measured using Neutral Red, PrestoBlue, SigmaFAST & Alizarin Red assays respectively.

Results & discussion
Percentage of mass loss for LC16:4, 18:2 and 9:1 scaffolds showed a gradual increase versus degradation time as can be seen from Figure 1.
LC16:4 showed lower mass loss (ca. 20%) in comparison with LC18:2 and 9:1. This could be ascribed to the variation in ɛ-caprolactone to D,L lactide ratio (CL/LA) ratio between the scaffold materials. Compressive properties for LC18:2 and 9:1 scaffolds were also significantly higher (P<0.001) than LC16:4. Mechanical properties of these scaffolds are mainly dependent on their materials composition (i.e. CL/LA ratio) as all scaffolds have similar architecture. Accelerated degradation results show prediction of degradation rates, and the activation energies from half mass loss results were found to be 87.9, 82.7 and 94.9 kJ mol-1 for LC16:4, LC18:2 and LC9:1 respectively.
Metabolic activity and ALP activity of cells within the scaffolds are shown in Figure 2. LC18:2 promoted higher cell metabolic activity but earlier time points showed no significant difference (p>0.05) between the three compositions. LC18:2 had also the highest ALP activity in comparison with LC16:4 and 9:1 scaffolds.
LC18:2 scaffolds showed significantly higher cellular activity (ca. 20%) and mineralisation (ca. 30%) in comparison with LC16:4 and 9:1.

Conclusion
Variation of degradation and mechanical properties of LC scaffolds are related to their chemical composition and accelerated degradation was predictable. All three types of scaffold were capable of supporting cell proliferation and osteogenic differentiation of human MSCs over 21 days and LC16:4 showed the best bone cytocompatibility response. These scaffolds have a potential for use in bone repair applications.

Introduction
Biomaterials can show promise as implants both theoretically and in terms of materials characteristics, however, before a material can go into clinical trials it will have to have proved successful in in vitro and in vivo tests.
The aim of the work in Cambridge was to use primary human osteoblast and monocyte cells to investigate the osteogenic and inflammatory responses to the materials (both scaffolds and ELRs) produced by the other partners in the project. By measuring cell response to the materials it is possible to make predictions about the outcome following implantation of the InnovaBone product in the body.

Materials and Methods
The materials to be characterised were provided by other partners in the project (scaffolds and discs from IBA and ELRs by UVa). These were sterilised using
gamma irradiation.
The scaffolds or discs were pre-wetted in 70% ethanol, washed in sterile deionised water and placed in culture medium for 24 hours before being used in experiments. The ELRs were made up as a 1% solution in the culture medium used in the experiment.
The osteoblast response was measured using assays to measure cell proliferation and activity. Cells were also stained to measure alkaline phosphatase (ALP) activity and mineralisation using alizarin red. An assay to measure the increased calcium produced by the cells at day 14 was also used as an additional measure of mineralisation. The medium in all cases except the negative control had osteogenic supplements added. Positive controls included the addition of BMP 2 and BMP 7; other controls were culture medium with and without osteogenic supplements.
The inflammatory response of the materials was measured by seeding them with monocytes. The cytotoxic response was determined by measuring the amount of lactate dehydrogenase (LDH) released from the cells.  The inflammatory response was detected by measuring the release of the cytokines IL–6 and TNF α from the monocytes. Positive controls had zymosan or lipopolysaccharide added to the cells.

Results
Discs and subsequently scaffolds were found not to be toxic with low LDH release. Scaffolds of all materials were found to support osteoblast differentiation with cells showing good ALP activity. The release of inflammatory markers from cells seeded on the scaffolds was also found to be low.
Some of the ELRs were found to show an inflammatory response at a concentration of 1% but they were not toxic (Figure 1).
When the response of osteoblasts to biogels was studied it was found that the silk containing ELRs supported osteoblast proliferation and subsequent mineralisation. The presence of the ELRs prevented any staining through alizarin red and so it was only possible to see mineralisation shown using the calcium assay (Figure 2).

Conclusion
The silk ELRs were found to support the development of osteoblasts and their subsequent mineralisation.  They evoked a small inflammatory response but were not found to be toxic. The scaffolds allowed osteoblasts to differentiate and showed good ALP activity. The scaffolds were found to be non-toxic and promoted minimal inflammatory responses.

The development of predictive in vitro tests remains a major challenge for toxicology, pharmacology and also for tissue engineering.  
We have developed a simple bioreactor to host bone scaffolds and bone substitutes. It allows scaffolds filled with bone cells to be tested for a period of several weeks.
During this period the bioreactor is placed inside a standard cell culture incubator, which controls both the temperature and the partial pressure of CO2. A fluidic system guarantees the supply of nutrients and the removal of toxins from the culture.  A key feature of the bioreactor is the possibility to apply a mechanical stimulus to the scaffold during culture.  A stepper motor, spring and piston together apply a compressive force which mimics in vitro the effect of daily movement on bones and bone substitutes in vivo.  A force sensor allows accurate control of the forces applied as well as determination of the mechanical properties of the scaffold.
The bioreactor allows scaffolds to be cultured in quasi-physiological conditions.  However, new tools are needed to analyse and monitor the behaviour of cells inside the 3D scaffolds.  
While many tools are available for 2D cell culture, very few of these can be applied to 3D scaffolds.  
We are currently developing optical sensors that can be used gather information about the conditions in the centre of the scaffold. Oxygen and pH sensors using reactive dyes incorporated into porous sol-gel matrices. The combination of these matrices with optical fibres is now being explored.

The UNIVIE Bone Biology Laboratory established in vivo and in vitro platforms to evaluate biomimetic biomaterials for bone regeneration.

In vitro
To characterize biomaterials in vitro, we used mouse and human bone cells:

1 - For mouse cell assays, we cultured mouse calvaria-derived osteoblasts (OBs) with the biomaterial test samples and analyzed:

• cell viability,
• alkaline phosphatase activity,
• differentiation into mature matrix mineralizing OBs, by Alizarin S staining for Calcium.

2 - To evaluate osteoclast (OC) development and bone resorption, we used a mouse co-culture of OC precursors from bone marrow and OBs derived from calvarial bones and quantified multinucleated OC formation using a TRAP (tartrate-resistant acid phosphatase) assay and evaluated the Cathepsin K secretion, respectively.

3 -For human cell assays, we generated human OCs from peripheral blood mononucleated cell precursors, then incubated them with the test samples, and used the TRAP assay.

Results
Our results indicate that scaffolds support OB differentiation and elicit similar responses in mouse and human OC assays.

In vivo
To study bone regeneration in vivo, we used a mouse calvarial defect model, in which a critical size defect (Ø = 4 mm) was created and then filled with the test biomaterials compared with an empty defect (negative control) and a commercially available bone repair material (VitossTM; positive control). The mice were then evaluated up to 12 weeks after surgery using µComputed Tomography (CT) and histological sections of the implant area.
The materials tested included:

1. Polymer scaffolds composed of various combinations of lactide (LA), caprolactone (CL), and methacrylate (MA) produced by two-photon photopolymerisation;
2. Thermosensitive Elastin-Like-Recombinamer (ELR) biogels containing BMP2 and BMP7;
3.  Hydroxyapatite nanoparticles.

Results
Our results indicate that scaffolds support OB differentiation and elicit similar responses in mouse and human OC assays. Furthermore, scaffolds, biogels and nanoparticles in combination enhanced new bone formation in vivo. Taken together, our data suggest that mouse models may be predictive for human bone cell responses and that our biomimetic biomaterial approach for bone regeneration may have important clinical implications.

MUW focused on in vitro evaluation of ELR biogels and scaffold materials using spleen cell cultures from BALB/c and C57Bl/6 mice. We analysed in vitro cytokine responses to the scaffold disks and ELR biogels with cytokines measurement and cell proliferation assays. In vitro immune cell studies revealed that LCM3, LCM4, LCM6.1, ELR-RGD, ELR-BMP2 and ELR-BMP7 were inert. These results illustrate that these biomaterials did not induce an immune response and were not toxic to the cells. Intraperitoneal (i.p.) models in BALB/c and B6 mice were used to test the biomaterials in vivo.  Mice were implanted with scaffold, ELR biogels or a currently available biomaterial for bone repair: Vitoss Foam (OVF, Orthovita, USA). The intraperitoneal model is a 7-day protocol that provides a quick read out for early signs of foreign body reactions (FBRs). We evaluated biocompatibility by enumerating inflammatory cells and measuring cytokine levels in peritoneal lavage fluid. OVF-implanted group had a significant increase in the total number of cells with significant increases in macrophages, eosinophils and neutrophils compared to naive cells. There was no significant increase in total cell infiltration into the peritoneum in response to the scaffolds and ELR biogels demonstrating that there is no early acute inflammatory response to our biomaterial in vivo.The subcutaneous (s.c.) model was used to address longer term biocompatibility and to correlate the results from short term i.p. and longer term s.c. experiments. We sought to determine whether there were late immune responses including a foreign body response and fibrosis. We established a subcutaneous (s.c.) implantation mouse model and examined the late response by H&E and Masson’s trichrome-stained tissue sections and qPCR of the implant site at 3 and 8 weeks after s.c. implantation. OVF induced chronic inflammation with giant cells and collagen deposition compared with mild inflammation and few collagen fibers induced by scaffolds (LCM3, LCM4 and LCM6.1) and ELR biogels (ELR-RGD, ELR-BMP2, ELR-BMP7). QPCR assays revealed high upregulation of inflammation, fibrosis- and wound healing-related genes following OVF implantation compared to ELRs (ELR-RGD, ELR-BMP2, ELR-BMP7). Although the ELRs which have NP (nano-particles) induced more inflammation, collagen deposition and gene expression compared to ELR without NP and normal wound healing, but it was still less than OVF. Serum antigen-specific antibody titration showed OVF and ELRs generated antigen-specific IgG1 and IgE titres. Our data demonstrate that InnovaBone biomaterials induce minimal immune responses compared with OVF and suggest that they are safe and could be modified to increase the immune/inflammatory response in bone, if necessary by altering the ELR biogel bioactive molecues. Currently, we are profiling gene expression of calvaria defect implantation sites with the best candidate of scaffold and ELR biogels for inflammation, fibrosis or bone regeneration.

Establishing bone mineral density

Initial work by UMG-GOE included the establishment of bone mineral density (BMD) measurement with x-ray based imaging techniques, the setup of x-ray based 3D virtual histology of mouse soft-tissue stained ex-vivo with a heavy ion containing contrast agent (PTA), as well as the development of a dedicated analysis scheme to analyze bone morphologic parameters in mice in 3D utilizing the software Scry (Kuchel & Sautter GbR).
To define bone regeneration and/or osteolysis, CT studies (Quantum FX, Perkin Elmer) were performed either at low dose longitudinal in vivo or at higher resolution post-mortem in order to better characterize the only weakly mineralized new formed bone in the scaffold area. For 3D data analysis the software Scry was used. New bone formation was calculated by comparing the area of defect 12 weeks post surgery with the defect area immediately after surgery. 12 weeks after implantation of discs, scaffolds or scaffolds plus biogel and/or nanoparticles into calvarial defects of different sizes by UNIVIE, UMG-GOE received heads of mice fixed in 70% ethanol from UNIVIE. Live mice with different implants were received 1 week after surgery. CT results indicated that the scaffolds can be visualized by CT but have poor contrast against the surrounding soft-tissue. All discs and LCM6 scaffolds appeared osteolytic as shown by an increase of the area of defect 12 weeks after surgery when compared to the size of the original defect. The consortium decided that LCM3 was the most suitable scaffold, as it has the following advantages: 1) did not cause any bone resorption, 2) was more bone conductive than the other LCMs, 3) showed more regrowth of bone from the edges of the defect, 4) has some bone growth in the pores of the scaffold, and 5) was not resorbed at 12 weeks after surgery. UMG-GOE quantified the mineralisation of the regrown bone by measuring the bone mineral density (BMD) using CT in combination with a custom made density phantom. We found that the mineralisation of new bone in the mice calvaria was similar between all LCM scaffolds, but lower than normal bone.
For the evaluation of bone growth or resorption over time, longitudinal studies in live mice were performed by weekly CT as well as optical imaging in the near infrared range (NIR). For this purpose, NIR fluorescent bisphosphonate imaging agents (Osteosense) were intravenously injected at diagnostic concentrations every 4 weeks. The obtained results demonstrated a steady increase of fluorescent bisphosphonate levels over the 10 weeks of measurements for all implants. In vivo CT scans showed that the combination of LCM3 plus BMP-containing biogel plus nanoparticles is the most osteoconductive material within the scaffold. Optimization of this implant combination, in vivo scanning thereof and calculation of bone growth is ongoing.
Two steps were taken to improve the contrast of the scaffold to be able to visualise and calculate the degradation of the scaffold over time. Firstly, UMG-GOE scanned the implants using in-line free propagation phase contrast CT at the SYRMEP beamline of the synchrotron light source ‘Elettra’ (Trieste, Italy), a method which results in much higher soft-tissue contrast than classical CT, which was still not sufficient for quantitative analysis of scaffold resorption or new bone formation. Secondly, IBA incorporated 5nm or 100nm gold nanoparticles into LCM cylinders, which were scanned by UMG-GOE and showed an increased x-ray attenuation, thereby raising the contrast-to-noise ratio by a factor of 20 for the larger gold nanoparticles.

Assessing inflammation

To assess inflammation in vivo we used optical imaging in combination with near infrared fluorescent (NIRF) activatable probes (ProSense) which assess the activity of proteolytic enzymes released at the site of inflammation. ProSense was biweekly injected intravenously in mice with different implants starting from week 2 after surgery until week 12 and measured by in vivo optical imaging. The fluorescence intensities measured over the defect conclusively showed that maximum inflammation occurred at the closest time point after implantation and steadily declined over the following weeks. All implant combinations so far tested showed a considerable decrease of inflammation to near background levels by week 12 after surgery, suggesting that neither scaffold nor biogel or nanoparticles cause prolonged inflammation in the environment of the implants.

Bone is the most transplanted tissue in human with about 1 million procedures annually in Europe. Autologous bone graft is the gold standard in bone regeneration but it requires a second surgery, is limited in quantity and often associated with complications. Synthetic calcium phosphate biomaterial in association with mesenchymal stem cells is a potent alternative to autologous bone grafting. Starting from a bone marrow aspirate, several hundred millions of mesenchymal stem cells (MSC) are produced in 3 weeks in a culture medium containing human blood platelet lysate plasma. These cells are fixed on biphasic calcium phosphate (BCP) granules and then implanted in subcutis of nude mice where they produced mature bone tissue.  The mixture of human mesenchymal stem cells and biomaterial is also effective in bone healing of critical size defects in calvaria and femurs of nude rats. The procedure has also proven efficacy in regenerating diaphyseal defects in metatarsis of sheep.  In the European project REBORNE, bone regeneration was successfully achieved in 4 multi centre clinical trials. Patients suffering from non-union fractures, osteonecrosis of the femoral head, cleft palates or insufficient mandibular bone for dental implants were effectively treated with autologous stem cells and biomaterials. This presentation will give the latest pre-clinical and clinical results in bone regeneration.

The orthopedic medical device industry challenges the advanced materials science to investigate new biomaterial designs for medical devices with enhanced performance and controlled safety profiles. When the medical device regulations are not recognized during research, the industry cannot judge the feasibility of the design and does not take the risk of transferring it to the market. It will benefit the valorization process if research institutes take the medical device regulations into account early phase their research projects. For this purpose, we have introduced a systematic risk based approach to establish a ‘frozen’ design and to identify suitable design controls from a regulatory compliance point of view. This should ease the industry decision-making for accepting biomaterial designs into medical devices.