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Executive Summary:
The main objective of OPHIS is to develop new engineered biomaterials for the repair and regeneration of both osteochondral regions and vertebral bodies in patients affected by severe osteoarthritis and osteoporosis. Both these bioactive and biomimetic substitutes are designed to be implanted under minimally invasive surgery. These will take the form of malleable constructs in the case of osteoarthritis-related lesions and injectable paste formulations in the case of vertebroplasty. Biochemical functionalization are designed to be linked to the scaffold to instruct cells toward specific regenerative activity.
The biomaterials developed in OPHIS for the healing of osteoarthritis are based on the unique combination of bio-polymeric scaffolds with a macroporous architecture presenting nanostructured functionalization able to mimic the extracellular matrices (ECM) of either bone or cartilage, thus being able to direct, control and preserve the phenotypes of both osteoblasts and chondrocytes in their related histological compartments. The functionalization are based on semi-dendrimers linking relevant peptide sequences mimicking glycosaminoglycans and proteoglycans of the extra-cellular matrix, relevant for cartilage regeneration, or exposing carboxybetaine, an amino-acid derivative suitable for binding growth factors. These novel, nano-structured, bio-functional devices are obtained by biologically-inspired self-assembling/mineralization process reproducing in vitro the natural phenomena occurring during formation of bone tissue. The process was applied on blended or interpenetrating composite polymer networks to achieve improved mechanical strength and stability. These devices were synthesized in a graded fashion to reproduce the composition and structure of multi-functional human tissues thus being able to promote regeneration of trabecular bone, mineralised cartilage, hyaline cartilage when implanted in joint regions.
The biomaterials addressed to the healing of vertebral bodies affected by osteoporosis-induced weakening are based on self-setting ceramic pastes made of Sr-substituted α -Ca3(PO4)2 added with bio-polymers to achieve adequate rheological properties and enhanced osteoconductivity in vivo. A unique synthesis process enabled the direct crystallization of Sr-substituted calcium phosphates precursors in the form of powders that, by suitable processing, gained the ability of self-hardening and transformation into Sr-substituted hydroxyapatite within times considered adequate for vertebroplasty procedures and with compressive strength suitable for early physical stabilization of vertebral bodies.
Upon preliminary evaluation in small and large animal model, new hybrid devices based on collagen/cellulose blends with graded mineralization demonstrated ability in regenerating critical-sized defects in bone and osteochondral regions. On the other hand, the new cements demonstrated easiness of handling and use in the operatory arena, and when injected in small animal, showed new bone formation and osteoconduction in the whole defect, in a greater extent when compared to a commercial apatite cement of proven effectiveness in vertebroplasty procedures. Therefore, OPHIS project succeeded in the development of new malleable and injectable bio-devices for regeneration of critical bone and osteochondral defects, that can be further exploited towards new therapies for hard tissues diseased by Osteoarthritis and Osteoporosis.

Project Context and Objectives:
Osteoarthritis (OA) is the most common joint disease in older adults and is characterized by progressive deterioration and sclerosis of articular cartilage and sub-chondral bone, as well as changes in the synovial membrane as a result of mechanical and biological processes that modify cartilage homeostasis. Several factors, including biochemical and genetic ones, contribute to the progress of OA. Moreover, a correlation exists between increasing age and the prevalence of osteoarthritis, thus suggesting that there are some changes in chondrocyte function. The processes that lead to osteoarthritis are characterized by an imbalance between anabolic and catabolic activities of the resident chondrocytes that result in a loss of matrix elements and deterioration in the functional and structural properties of the cartilage. So far no medical intervention has been shown to halt disease progression or reverse joint damaging; moreover, many of these drugs are beset with serious side effects.
The main challenge in the treatment of OA by regenerative medicine approaches is represented by the need to regenerate two different and adjacent tissues: the cartilage and the underlying trabecular bone. These two tissues are closely connected within the osteochondral unit despite their distinct physical, chemical and biological characteristics and different healing potential. Thus, aided regeneration of the osteochondral compartment by biomaterials or tissue engineering constructs requires tailored technological solutions able to fulfil the different histological and physiological features of both the tissue types.
Osteoporosis (OP) is the most prevalent skeletal disorder characterized by decreased bone mass and bone mineral density. In addition, deterioration of the overall bone structure leads to bone fragility and to increased risk of fracture. In OP the bone micro-architecture is disrupted, and the amount and variety of non-collagenous proteins in bone is altered. Furthermore, fractures are significantly exacerbated and healing is often impaired in osteoporotic patients. Post-menopausal OP is the most common and significant form of OP in which oestrogen deficiency gives rise to a high turnover rate in bone metabolism, indeed accelerated bone resorption by osteoclasts has been established as a principal mechanism in OP. At the same time, the reduced bone formation is an effect of a decrease in cell recruitment, precursor cell proliferation, and/or differentiation. Fracture incidence increases exponentially with age and can be over 20-fold greater in an elderly population compared to a young healthy population. Any efficient treatment of osteoporosis should target two main issues: (i) fracture risk should be strongly reduced and (ii) the treatment should have a sustained effect to reduce or eliminate the needs for daily medication for the remainder of the patient’s life. However, this is not the case for the currently available pharmacological treatments. Thus, it is vital to identify a treatment regimen that creates a permanent reduction in fracture risk even after the therapy is discontinued. This might be achieved with a combination of biomaterials and drugs or with a completely new approach.
Tissue replacement and augmentation by implants is adopted in cases where pharmacologic treatment proves ineffective and OA and OP conditions cause persistent pain and disability. In particular, implants such as artificial knee prostheses and vertebroplasty and/or kyphoplasty are adopted when the tissue structure and functionality is irreversibly compromised. However, the prosthetic solution is generally reserved for older patients, due to the fact that the life span of prosthetic materials is limited to 10-15 years. For that reason the treatment of early arthritis in relatively young and active patients is even more problematic as it leads to revision surgery. In recent years, innovative bioengineering approaches have been proposed for the early treatment of arthritis. However, despite the emergence of encouraging laboratory data, the clinical application of tissue engineering products have not produced the expected clinical benefits.
Vertebroplasty and balloon kyphoplasty (injection of acrylic bone cement into a fractured vertebra) are widely used to relieve pain related to pressure on the spinal nerves caused by the deformation and/or fracture of the vertebral bodies, however they do not provide adequate tissue regeneration. Although the systemic nature of OP will always require a degree of pharmaceutical treatment, new biomimetic, minimally invasive biodegradable biomaterials and tissue engineering constructs, will provide a jump-start for bone regeneration and will enhance the chance of successful clinical outcome. These new regenerative materials will eliminate the need for more radical surgical intervention.
In response to the above reported issues OPHIS aimed to provide suitable bases for new therapies for the repair and regeneration of diseased bone and osteochondral regions, through the designed synthesis and engineering of:
(i) tailored bio-hybrid composites and hydrogels in form of 3D- blended or interpenetrating polymer scaffolds for the regeneration of osteochondral tissues damaged by OA. Specifically designed bi- or tri-compartmental materials will be used to repair large defects;
(ii) injectable composite pastes/cements tuned for their optimum viscosity and porosity and with appropriate biomimetic and biomechanical characteristics for spinal regions weakened by OP.

The above mentioned biomaterials are intended to be:
⇒ functionalized and/or doped with chemical (e.g. strontium ions, oxygen scavengers) and biochemical (e.g. bioactive/bio-docking peptides, genes) agents able to control cell phenotype and activity;
⇒ tuned for an efficient bone or cartilage formation through the controlled delivery of bioactive agents using a pH-sensitive controlled release from nano-carriers and through the activation of cells modified by signal transduction and gene transfection.

The new engineered biomaterials are intended to act as triggers for cell phenotype regulation in the form of:
1) micro-environments mimicking the ECM composition;
2) biochemical stimuli mimicking the growth factor pathways of healing tissues.
In particular, composite hydrogel scaffolds, prepared to reproduce the intrinsic features of ECM, were developed and used as a framework on which to assemble specific bio-cues not inherently present in the base materials that are selected for the regeneration of the different tissue types (trabecular bone, mineralized cartilage and hyaline cartilage). More specifically:
- for cartilage regeneration, the collagen-like construct will be integrated and/or interpenetrated with an alginate matrix;
- for bone regeneration, the structural integrity of the 3D bio-hybrid composites will be provided by bio-engineered cellulose scaffolds co-integrated with a mineralized collagen matrix known to possess biomechanical properties comparable to trabecular bone.

The mineralization of assembling polymeric matrices will be performed through bio-inspired process and graded to create natural osteochondral tissue features which induce the required cellular responses.
These hydrogel scaffolds will be complemented by the integration of bio-triggers in form of nanostructured functionalization that will be achieved by grafting semi-dendrimeric macromolecules and nanobeads. The semi-dendrimers will provide a nano-structured exposure of bio-ligands key to cell/substrate recognition processes, while the nanobeads will represent reservoirs for the controlled delivery of growth factor analogues. In addition, specific semi-dendrimers will be synthesized and grafted on the bio-polymeric matrices to obtain a control of oxygen tension leading to the expression of hypoxic phenotypes in cells. These nano-structured components will be specifically designed for each type of target tissue.
For cartilage repair, OPHIS will develop semi-dendrimeric structures able to mimic the glycosaminoglycans and proteoglycans of the articular cartilage. Semi-dendrimers will be coupled with the candidate biopolymers that will be engineered as gels of different mesh size. These biomaterials will regulate the oxygen tension within the hydrogel by reducing it to levels similar to those of the articular cartilage.
For the purpose of bone repair semi-dendrimers will be functionalized with bioactive peptides with a role in stem cell homing, and osteoblast migration. The gel formulations for bone repair will also be combined with two essential components favoring their angiogenesis and mineralization (i.e. BMP-2 and VEGF analogue peptides). The second component will be biomimetic nano-size strontium-carbonate-hydroxyapatite as nucleating agents for biomineralization, able to release strontium ions to stimulate osteoblast activity in vivo.
The bioactive systems described above will also be included in new formulations of injectable pastes/cements. Classical cement setting mechanisms will be investigated and employed to mechanically stabilize the construct. Induced changes of pH will control the precipitation of phosphates and the nucleation of the new mineral phase in the gel network triggering the setting of the composite cement into the vertebra.
The semi-dendrimeric carriers will also be designed to introduce the human Lim Mineralization Protein (LMP) gene which is spliced to produce LMP-1 and LMP-3 isoforms and induce bone formation. This will be a feasibility study with the potential to be extended to future genetic modification of human cells. A 3D bioreactor system will be used to assess the potential of these biomaterials to induce tissue regeneration in vitro. The main features of this bioreactor will be to mimic the biomechanical stimuli and oxygen tensions of the tissues. Such a clinically reflective model will reduce the need for animal tests. The animal tests will be performed on critical size defects of bone and cartilage as well as osteo-chondral damage generated to reproduce OA and OP conditions in small / medium animals. Later, clinically reflective osteoporotic sheep models will be employed to mimic both the surgical procedures and clinical outcome of vertebroplasty and osteochondral defect treatment. Atomic Force Microscopy (AFM) will be used as a molecular scale analytical method to measure directly and in a quantitative way the interactions between the engineered constructs and native tissue components.

Project Results:
Main S&T results
The workplan of OPHIS is subdivided into 7 R&D work packages as highlighted in the following scheme:
The project activities started with WP1 and WP2 simultaneously. In WP1 the 3D scaffolds made of natural polymers composite (ISTEC, FSUJ, TUD) and the injectable pastes (ISTEC, FSUJ) were designed and synthesized; in WP2 the initial dendrimer structures with oxygen-regulating ability (UOB) were designed, synthesized and characterised. The preliminary steps of the whole work were based on the partners experience in combination with a literature study, and considering the properties, based on literature and clinical experience, required from the final products for clinical and surgical applications (FIN-CER, IOR, UCSC). A strategy for selection and development of relevant peptides and bioactive molecules maintaining the cell phenotype and able to favor the recruitment of cells involved in the natural cascade of specific tissue regeneration was adopted in WP3 (UOB, TUD, ISTEC, FSUJ). The functionalization of dendrimers linked to the scaffolds and nanobeads was performed by identification of suitable pH-sensitive switching agents (WP3: ISTEC, UOB). Moreover, a transgenic approach mediated by dendrimers was also investigated (WP3: UCSC). The final assembly of the various components prepared under WP1, WP2 and WP3 was performed within WP4 which is mainly devoted to the scaling up of the synthetic procedures to produce the final samples (FIN-CER) in compliance with the existing regulatory issues. For the development of composite phenotypic triggers and its intended use for bone and cartilage repair, risk management process was also carried out to identify the health hazards associated with the innovative component.
The optimal phenotypic triggers were developed through an iterative procedure and a final performance evaluation carried out in vitro under dynamic conditions in an established bioreactor system in WP5 (UHBS). In vitro evaluation in static condition was carried out in WP6 (LEMI), while in vivo pre-clinical assessment on small and big size animal model was carried out in WP7 (UCSC, IOR).

Work progress and achievements during the project
All the milestones foreseen in OPHIS were reached in time. The main results and achievements of the project OPHIS are summarized as follows:
- New bio-polymeric matrices and blends suitable for guided assembling/mineralization and generation of 3D scaffolds for bone and osteochondral regeneration with designed composition and microstructure.
- New hybrid bone and osteochondral scaffolds presenting dendrimer-mediated functionalization triggering bone and vascular regeneration.
- Apatite nano-phases with multiple bio-competent ion-substitution.
- New αTCP phases presenting tailored substitution with strontium.
- New injectable, self-setting pastes based on Sr- αTCP with tailored viscosity, setting time and mechanical strength.
- New injectable self-setting cements based on Sr-substituted apatites with enhanced osteogenic and osteoconductive ability.
- New alginate-based osteochondral scaffolds.
- New bone scaffolds based on bacterial nanocellulose.
- New semi-dendrimers exposing O2 scavengers as triggers of chondrocytic differentiation.
- New semi-dendrimers mediating gene transfection.
- New peptide sequences mimicking bone (BMP-2), cartilage (IGF-1) and vascular (VEGF) growth factors.
- New polymeric nanobeads with controlled size and ability of controlled delivery.
- New pH-sensitive bio-polymeric constructs with ability of controlled delivery.
- Preliminary assessment of a AFM nanoindentation-based method to predict, in a quantitatively and spatially resolved way, the behaviour of grafts for osteochondral regeneration upon implantation.

The most relevant results achieved by OPHIS including their application impact are here listed.

1) New hybrid scaffolds

• Hybrid graded scaffolds for osteo-chondral tissue regeneration obtained by bio-inspired assembling/mineralization of blended biopolymer matrices
Among the evaluated blends formed by a mixture of: collagen (Coll), bacterial nano-cellulose (BNC), chitosan (Chit) and alginate (Alg), two stable blends were selected: Coll-BNC (70/30 wt%) and Coll-Chit (70/30 wt%). Stable and homogeneous blends were successfully prepared through a pH controlled assembling process, polymers ratio and cross-linking reactions were optimized.
Bio-inspired nucleation of magnesium doped hydroxyapatite (MgHA) on the polymeric blends was performed by a biomimetic synthesis process of contemporarily assembling and mineralization (ISTEC) to obtain highly biomimetic and bioactive mineralized composites.
The Coll-BNC blend and the respective mineralized composite MgHA/Coll-BNC, because of the high hydrophilicity of BNC demonstrate suitable properties in terms of swellability, on the other hand the low biodegradability of BNC resulted very effective in terms of enzymatic resistance.
The bi-layer Coll-BNC and MgHA/Coll-BNC, mimicking bone and cartilage, was freeze-dried generating 3D highly flexible constructs they showed also proper characteristics to be perfused in a bioreactor (UHBS) where an optimal level of cell colonization was reached.
In vivo evaluations performed in an osteochondral lesion on femoral condyle of sheep model (IOR) assessed the absence of any significant toxicity and inflammatory reactions in the surrounding tissues. Was observed the formation of few new bone growth inside the defect and partial integration of the scaffold, both at bone and cartilage compartments.
In conclusion the material can be considered suitable for the regeneration of osteochondral lesions but, due to the high hydrophilicity and the huge swelling, the 3D micro-CT evaluation showed a huge variability and heterogeneity between the cases and a high percentage of empty voids reducing the total amount of newly formed bone, thus further improvements are required to guarantee a longer permanence of the scaffold in vivo.

As a second choice Coll/Chit and MgHA/Coll-Chit materials were developed, since they showed best mechanical performances in respect to MgHA/Coll used as reference, in fact the stress-strain behavior indicate lower deformation. Because of the high mechanical performances of the 3D constructs made of Coll/Chit and MgHA/Coll-Chit they were considered for the final in vivo evaluations: in most of the samples, the cartilage defect was filled with fibrous tissue resembling fibrocartilage, with proteoglycan synthesis as evidenced by intense red staining with Safranin-O. As far as the bony compartment, none of the samples evidenced bone healing. Additionally, in the majority of the samples cysts within trabecular tissue, delimited by a thick connective membrane, were also recognized.
In conclusion the chitosan affected negatively the osteoconductivity of collagen and this is particularly true when the mineral component is present on the surface of collagen hindering the collagenic functional groups.

Application impact: in OPHIS the bio-inspired process already assessed for the development of high regenerative scaffolds based on type I collagen has been implemented for application on composite biopolymeric matrices, including bacterial nano-cellulose and chitosan. This approach enhance from one side the flexibility and wettability and from the other side the stiffness of the 3D bio-inspired constructs, thus being adequate for implantation in larger tissue defects.
However further improvement are still necessary to identify the final features to make the new constructs suitable for development in clinics.

• Multi-layered scaffolds mimicking bony and cartilaginous regions obtained by ionotropic gelation
A bi-layered but monolithic alginate-based scaffold for osteochondral regeneration was obtained by ionotropic gelation of alginate (TUD). The synthesis method generated constructs associating a bone-mimicking mineralized layer with channel-like pores, and a non-mineralized chondral-like layer. The synthesis method enable direct cell seeding during synthesis that result embedded into the chondral-like layer.
Since the number of alive cells was too low in the cellularized constructs, the cell-free formulation was selected for the final in vivo evaluations in osteochondral lesion on femoral condyle of sheep model (IOR).
In conclusion the micro-CT and the histological evaluation demonstrate an uncompleted bone regeneration highlighted by the presence of empty areas and some areas the presence of newly formed repair tissue and the integration of the scaffolds with the host cartilage. It seems reasonable to assess that the reabsorption rate of alginate is too high and that the extent of cross-linking exerted by calcium is too low for application in vivo.

Application impact: the new alginate-based osteochondral scaffold is an easy-to-handle implantable material which can be loaded with autologous cells for stimulation of the healing of osteochondral defects. In OPHIS the safety of the cell-free biomaterial was proven, while the efficacy evaluated in a large animal model (IOR) resulted still inadequate for further development towards a clinical product.

• Hybrid nanocomposite scaffold based on Bacterial Nanocellulose

Pure BNC fibers were prepared by a dynamic cultivation of gluconacetobacter xylinus in a starch modified Hestrin-Schramm-Medium. Those fibers demonstrate to be suitable for the development of 3D polymeric porous scaffold and hybrid bone scaffolds mineralized with HA nanoparticles. The nanoscaled BNC fibers were successfully mineralized by hydrothermal treatment upon adequate oxidation of BNC fibers enabling linking of Ca2+ ions (FSUJ). After the assessment of mechanical and in vitro performances (LEMI), both the materials, pure and mineralized BNC were selected as suitable material for in vivo evaluation in rabbit condyle mode.
In conclusion few new bone growth inside the defect was observed and it was localized especially close to the material surface. The sagittal sections details showed the partial integration of the material.

Application impact: The engineering of the mineralized with non-mineralized BNC fibers enables the production of new bone and osteochondral scaffolds which deserves further investigation for future application.

2) Functionalization

• pH-sensitive bio-polymeric mineralized scaffolds for bone and osteochondral regeneration
Novel, nano-structured pH-sensitive hybrid scaffolds based on type I collagen mineralized with MgHA phase and engineered with chitosan matrices and polyvinylpyrrolydone (PVP) were developed (ISTEC). The chitosan-based matrix was integrated in the mineralized collagen matrix by both blending and interpenetration in the wet state. The final constructs are biocompatible (LEMI) and exhibit ability of swelling/collapsing depending on the pH of their surrounding environment.
In vivo test performed on rabbit condyle showed that the direct contact between bone tissue and scaffold was absent in most of the investigated cases because of the presence of connective tissue around the scaffold, characterized in some specimens, by the presence of an inflammatory cell infiltrates.
In conclusion the presence of pH-sensitive function (made of chitosan based polymer) on the scaffold increased hydrophobicity and induced encapsulation by means of the fibrous tissue.

Application impact: the new scaffold, exhibiting ability of reacting to specific pH values, can be exploited as a new implantable drug delivery system associating the high regenerative ability of collagen/MgHA scaffolds with the stimuli-responsive features of modified chitosan that enable controlled release of anti-inflammatory or antibiotic molecules, thus potentially improving the process of tissue regeneration.

• Hybrid beads to be loaded with bioactive molecules
Hollow micro/nano spheres based on PLLA and hydroxyapatite were developed by following the Pickering theory (ISTEC). These devices may enable multiple functionalization, by taking advantage of the active surface sites of the polymer and the apatite phase; moreover drugs or bioactive molecules can be loaded in the cavity of the spheres. The chemical-physical features of the spheres such as size, polymer crystallinity and surface charge can be tailored in view of the specific application.
In vitro test showed good biocompatibility of the micro-nano-spheres.

Application Impact: The developed hybrid nano/micro-beads have potential uses as building blocks for the functionalization of 3D scaffolds or injectable paste for bone regeneration or smart drug delivery system.

• Oxygen sequestrators
Semi-dendrimers with oxygen consuming properties are a highly innovative progress far beyond the state-of-the-art that was achieved during OPHIS (UoB/TUD).
The final results have shown the highly reproducible and scaled up synthesis of these macromolecules, their ease of grafting on biomaterial surfaces and their ability to generate hypoxia micro-environment stimulating cell proliferation and extracellular matrix production.

Application Impact: the application of dendrimer-based functionalization to medical devices represents a powerful tool to improve the regenerative properties of bone and cartilage tissues

• Functionalized hybrid scaffolds with semi-dendrimeric structures able to mimic the cartilage micro-environment.
The synthesis of semi-dendrimers based on hyperbranched poly-L-lysine and the functionalisation of their uppermost branching generation with molecules of carboxybetaine has been achieved at suitable scaled-up quantities (UOB). For the first time, these macromolecules have been used to functionalize hybrid scaffolds for osteochondral regeneration (ISTEC) to create new bio-functionalities capable of specifically docking endogenous or loaded cells into scaffold porosity. In vitro studies (UoB, LEMI) have shown that this covalent functionalisation increases the efficiency of cell loading (or recruitment) within scaffolds and offer a control of progenitor cells and chondrocytes phenotype in a manner superior to non-functionalised devices.
Moreover in vivo studies (IOR) report the repair of the osteochondral defect with formation of new bone in both trabecular and cortical compartments. The presence of newly formed hyaline-like tissue, with evidence of viable chondrocytes uniformly distributed in all cartilage layers, with no evident cell clusters, and good integration of the scaffolds were detected.
Concluding in consequence of these promising results, a specification of the final product, osteochondral scaffold functionalized with carboxybetaine dendron (R-G3K(CB)16) has been drafted to identify the most appropriate analytical methods and protocol (FINCER/UoB), in the view of product standardization and commercialization.

Application Impact: This novel bio-functionalisation method can be applied to any biomaterial scaffold to confer enhanced tissue regeneration properties. Hence, they can be applied to scaffolds for the regeneration of osteo-articular defects as well as to other types of tissues where the recruitment of endogenous cells or the loading of cells prior to implantation needs to be maximized and controlled. All the tests and improvements of the functionalized collagen-based scaffold allowed the scale-up of the process in terms of efficiency and reproducibility. All these requirements are necessary to lead the product to actual exploitation and to the clinical application.

3) Injectable bioactive cements for bone regeneration
A new injectable bone cement based on calcium-deficient apatite partially doped with strontium ions was developed and optimized for use in vertebroplasty procedures (ISTEC). The ionic substitution with strontium was targeted to specific anti-osteoporotic effect, i.e. enhanced osteogenesis. The new cement also contains small amount of alginate, a bio-erodible polymer that can chemically interact with calcium thus providing enhanced physical stability to the cement and improve the setting behavior, as also observed during 1 month of ageing in physiological-like conditions. Moreover the bio-dissolution of alginate in vivo enables the penetration of new bone into the injected mass, thus favoring osteointegration and establishment of improved mechanical properties in the bone/cement construct. By preliminary in vitro and vivo investigation (UCSC), the new cement demonstrated improved cell behavior and bone penetration when compared with a commercial cement widely used in vertebroplasty procedures. Moreover, signs of resorption due to microfragmentation were observed. Further in vivo studies on rats (UCSC) assessed the absence of any significant toxicity in soft and peri-implanted tissues. Besides, tissue engineering approaches were performed by implanting the cement with living fibroblast cells thus enabling tissue engineering approach and gene therapies.

In conclusion the new cement has been patented and is now object of further investigation in more clinically-relevant animal models, as well as of preliminary activities of standardization, quality assurance and addressing of the relevant regulatory issues (e.g. concerning scaled up synthesis, sterilization, packaging, ageing and storage) which are being developed positively thus making suitable the commercial exploitation of the new cement (FINCER).

Application Impact: The unique properties of the new Sr-substituted cement make it adequate and very promising for mini-invasive surgery such as vertebroplasty/kyphoplasty, percutaneous treatment of fragility methaphyseal fractures (femur, tibia humerus, wrist), and hip, shoulder and knee prosthetic revision surgery. The mechanical strength and cohesion of the new cement, which are stable over 1 month of ageing in simulated body fluid at 37 °C, as well as the preliminary results of in vivo tests, prove that early physical stabilization of diseased vertebrae without sudden fracture is feasible, thus opening the way to new therapies against fractures of vertebral and load-bearing bones.


Scaffolds for osteoarthritis
3D biomimetic hybrid scaffolds for the healing of osteochondral regions diseased by osteoarthritis were developed, based on different macromolecular matrices (i.e. collagen, bacterial and regenerated nanocellulose, alginate, chitosan) mineralized with biomimetic hydroxyapatite nanoparticles to mimic bone and osteochondral tissues. The biomimicry of the different compartments of osteochondral regions was obtained by developing a nano-technological biomineralization process mimicking the nucleation of calcium phosphates mediated by an organic matrix (collagen). The obtained constructs were intended to be functionalized in order to expose bio-cues triggering regeneration of relevant healthy tissues (see Figure 1).
In particular, biomineralization was achieved by pH-driven self-assembling of bio-polymeric fibres and simultaneous heterogeneous nucleation of biomimetic hydroxyapatite nanoparticles, thus mimicking the natural process of new bone formation in vivo (see Figure 2).
Natural polymers were assembled into blends in aqueous media at body temperature (Figure 3); the process conditions were directed and controlled to enhance chemical and physical interaction between the bio-polymeric components and form 3D composite matrices enabling biomineralization. 3D hybrid constructs with tailored properties were developed with different mineralization extent mimicking different histological compartments of articular regions.
Hybrid tissue-mimicking layers were engineered and assembled in wet state to produce hydrogels with graded morphology and composition. Directed freeze-drying processes were applied to obtain the final osteochondral scaffolds; controlled cooling and heating cycles enabled to create structures with wide open porosity (see Figure 4). The scaffold were then cross-linked to adjust porosity and stiffness, as well as to confer suitable resorption kinetics in vivo. Cross-linking procedures were thoroughly investigated by means of different chemical approaches, i.e. using BDDGE or Genipin after freeze drying in suitable amounts to achieve adequate properties. The obtained bio-devices were characterize by suitable permeability to cells and optimal shape-memory properties for implantation by minimally-invasive single step procedures.
Multi-layered scaffolds mimicking bony and cartilaginous regions were also obtained by ice-templating processes and ionotropic gelation. Ice-templating processes were applied to obtain scaffolds based on cellulose; in this respect bacterial cellulose samples originated from different bacterial strands as well as regenerated cellulose were investigated and subjected to oxidation processes to create new surface functional groups that enable the binding of Ca2+ ions, in turn promoting bio-inspired mineralization with hydroxyapatite. Successful mineralization was achieved by hydrothermal process applied to oxidised cellulose matrices. Bi-layered constructs simulating mineralized and non-mineralized tissues were achieved by incorporation of Sr-substituted hydroxyapatite nanoparticles. From this basis, composite matrices were developed through blending of different biopolymers with collagen, with the purpose to achieve improved functionality, such as mechanical properties. Particularly, mineralized blends were achieved with chitosan and bacterial nano-cellulose (BNC) (see Figure 5).
Multi-layered scaffolds for osteochondral regeneration were also produced by methods based on ionotropic gelation applied on alginate-based matrices. The method allowed to obtain mono-, bi- e tri-phasic osteochondral scaffolds with parallel aligned pore channels (Figure 6). The embedding of cells into the scaffold during its synthesis was carried out in sterile conditions, thus enabling the production of cellularized constructs.
Scaffolds for osteochondral regeneration were synthesized by a similar approach where hyaluronic acid was introduced in the chondral-like part. Alginate/collagen and alginate/cellulose blends were developed to function as drug delivery systems.
Several samples were prepared and tested in vivo. Figure 7 shows some of these final scaffolds.

Scaffold functionalization
Bio-competent semi-dendrimers / new polymers were synthesized, as ligands for specific growth factors and chemical/biochemical cues, to be grafted to hybrid osteochondral scaffolds (Figure 8). In particular, the novel semi-dendrimeric structures presented functional groups of the cartilage and bone extracellular matrix. The hyperbranched and nanospaced structure of these polymers were designed to increase the exposure of functional groups to the cells as well as controlling their spacing around the cell. The functionalization aimed at enabling the exposure of specific carbohydrate units mimicking the glycosaminoglycans and proteoglycans of the articular cartilage extracellular matrix. Separate batches of semi-dendrimers were developed to present bioligands specific for stem cell and osteoblast receptors to encourage the colonization of the bone regenerating scaffolds. For both cartilage and bone regeneration a class of semi-dendrimer exposed a specific amino acid derivative, the carboxybetaine which has previously been shown to bind growth factors (RG3K(CB)16). In this respect, successful functionalization occurred with all the types of bio-polymers used in OPHIS, i.e. type I collagen, oxidized cellulose and alginate. Thorough characterization of the new dendrimers aimed at assessing the purity of the product and the effective grafting on the different bio-polymers.
The functionalization of scaffolds for cartilage regeneration aimed to reproduce the main features of the chondron, the unit in which chondrocytes are encapsulated. These features are (i) a glycosaminoglycan environment and (ii) a collagen type II-mimicking environment capable of binding growth factors such as IGF-1.
New polymers with ability of oxygen regulation were also developed and grafted on dendrimers. In particular, the presence of quinone derivatives with oxygen-chelating groups can generate localized hypoxia domains within the osteochondral substitute scaffolds. The so obtained constructs (RG3K(HGA)16) were then linked to 3D collagen and alginate-based matrices, to achieve functionalized scaffolds with ability to favour cell differentiation in chondrocytes. The physical, chemical and mechanical features of the functionalized scaffolds were assessed. The perspective to endow scaffold for osteochondral regeneration with specific triggers for selective cell differentiation towards the reconstruction of healthy cartilage can be considered as far beyond the state of the art existing at the beginning of project OPHIS.
Polymeric nano- and micro-beads made of BNC, alginate or chitosan were developed in different sizes and by different methods, including spray drying and emulsification methods (see Figure 9). Multi-layer beads were also developed by Pickering emulsion. The new beads are intended to be functionalized with osteogenic and angiogenic growth factors and anti-osteoporotic agents (e.g. Sr-hydroxyapatite nanoparticles) and incorporated in calcium phosphate-based pastes to obtain functionalized cements for regenerative vertebroplasty.
Linear peptide sequences mimicking the activity of BMP-2, VEGF and IGF-1 growth factors were synthesized at scaled up batches, with the aim to functionalize dendrimers and nanobeads and enable enhanced osteogenesis, chondrogenesis and vasculogenesis. The loading of the new scaffolds with such analogues was performed by means of both grafting to semi-dendrimers for suitable exposure to the physiological environment and incorporation in nano-/micro-beads with the purpose to achieve a controlled delivery of such relevant bio-molecules. In this respect the release of BMP-2 analogues from chitosan beads was monitored over several days, revealing the ability of the carriers to ensure an early release of therapeutically-significant doses of the bioactive peptide followed by a sustained release. Moreover semi-dendrimers functionalized with moieties enabling oxygen sequestration were linked to hybrid scaffolds to enhance new cartilage formation. Also, collagen and alginate-based nanobeads were engineered and made available for peptide entrapping. In particular, the ability of chitosan nanobeads to uptake and release growth factors was assessed. To facilitate the release of growth factor mimics under the reduced pH environment associated with the inflammatory conditions of osteoarthritis, pH-sensitive polymers based on chitosan were developed and their ability to release drugs at inflammatory conditions was assessed.
These new polymers were also integrated into the formulations of collagen-based constructs to create smart scaffolds able to respond and to deliver therapeutic agents in the case of inflammation.

Bone cements for osteoporosis
New bioactive and osteoconductive bone cements based on calcium phosphates with different compositions were developed for regeneration of collapsed vertebral bodies due to osteoporosis. The new cements were conceived to exhibit high osteogenic character, osteoconductivity and progressive resorption in vivo, thus enabling complete bone regeneration. The concept for the development of the new solutions against bone weakening by osteoporosis bases on the functionalization of new injectable formulations to enhance osteogenic, osteoconductive and bio-resorption ability (see Figure 10).
In one approach Sr-substituted calcium phosphate precursors were synthesized and processed to undergo phenomena of dissolution and recrystallization of apatite phases when in contact with aqueous media and yield self-setting of the cement. The precursor powders were obtained by a new synthesis process enabling crystallization of -TCP phase ionically substituted with strontium, introduced as specific anti-osteoporotic medium. The precursor powder was processed by milling methods to reduce specific surface area, to achieve early transformation into strontium-substituted hydroxyapatite nanoparticles (Sr-HA) that yield the setting of the cement by physical entanglement. The cement was added with alginate to improve rheological behaviour under injection and to improve the cement cohesion and mechanical strength after setting. Besides, the introduction of alginate aimed at providing enhanced bio-dissolution by progressive digestion (Figure 11). The setting kinetics of the new Sr-HA cement was optimized to reach values suitable for clinical practice (i.e. < 15 min). The dissolution of Sr-TCP and subsequent recrystallization gave rise to formation of elongated nanosized particles of Sr-HA (Figure 12) thus enabling physical entanglement and hardening of the injected body.
Brushitic cements were also developed by a different approach yielding formation of a calcium phosphate cement with adequate mechanical strength. The cement formulation was added with sugars acting as porogens upon progressive leaching in vivo. Radio-opaque phases were also added to enable monitoring of the cement after injection in vivo.
Both cement formulations were thoroughly investigated and optimized to achieve suitable injectability, cohesion and setting times suitable for the clinical practice. Also, repeatable procedures of mixing, handling and injection were established and optimized by making use of commercial mixing devices.
Preliminary investigation in vivo determined that the best-in-class cement was Sr-HA, which was subsequently subjected to further optimization in the view of its scaling up. In consideration of the cement features exhibited so far, suitable additional clinical applications are now identified in the treatment of long bone fractures, particularly in the case of tibia and humerus.

Cement functionalization
Strontium is recognized as an efficient anti-osteoporotic agent, since it stimulates the activity of osteoblast cells, and prevents bone resorption. The incorporation of strontium ions in the crystal of hydroxyapatite was carried out, to trigger specific anti-osteoporotic effect. Also, the introduction of tailored amounts of alginate yielded injectable pastes with tailored viscosity and setting time. After the first studies in vivo, that assessed the good ability of the new cement to induce new bone formation and cement fragmentation improving bone penetration and osseointegration, further analysis are currently in progress to assess the specific effect of strontium on bone regeneration.

Cell transfection by semi-dendrimers
New methods for gene transfection were investigated, in particular OPHIS focused on the development of an efficient non-viral carrier system based on semi-dendrimers with an optimized molecular design to efficiently bind the target plasmid and to transport it into the fibroblast nucleus.
In particular, fibroblast cells were transfected with LMP3FL-eGFP plasmid, by using FFG3K, CG3K and CG3K(CAB)16 semi-dendrimers, synthesized to ensure suitable penetration of the cell membrane and to enable the binding to DNA. The efficiency of transfection was assessed and monitored by grafting of fluorescent probes (FITC). The transfection efficiency of these dendrons relied on their ability to form stable complexes with plasmid DNA through either positively charged primary amine groups or specific bioligands (i.e. carboxybetaine) which were well-exposed on their uppermost branching generation.
The in vitro proof of concept was obtained by comparing the new dendrimers with a commercial one in the transfection of dermal fibroblasts using conventional tissue culture conditions. After examination, the results indicated that the considered dendron was capable of binding the plasmid and carry the new gene expression vector inside cells thus achieving a transfection efficiency of ~80% after 24 hours. In respect to these experiments, the correct handling and use of dendrimers and transfected cells was also investigated, in order to maintain adequate efficacy. The experiments clearly showed that the considered dendrons are capable of binding to the plasmid thus opening possibility of cell transfection without using adenoviral carriers.

In vitro static and dynamic assessment in bioreactor
Static in vitro tests were carried out to assess the cytocompatibility of biomaterials and devices for OA and OP. In particular bio-polymers, calcium phosphates, hybrid composites and self-setting pastes were tested for cytotoxicity prior further evaluation. This enabled to screen the various developed materials towards optimization and final assessment in vivo. Further evaluation was performed by analysis of cell-material interaction by using Human Osteogenic Cells, Human Articular Chondrocytes, as well as evaluation of the inflammatory potential by monocyte/macrophage activation.
Long-term in vitro tests on disks of Sr-HA cements were carried out; in 3 months of cell culture the Sr-HA cement showed a continuous cell proliferation and progressively increasing expression of osteogenic character, so that in vitro osteoinductivity was demonstrated. These tests are a suitable complement to in vivo tests to assess the biologic behavior of the new cements.
3D clinically-reflective models of osteochondral repair in bioreactor were developed, for the assessment of scaffold functionality in terms of oxygen regulation and bio-cue delivery. Several types of scaffolds, among the new devices developed in OPHIS, were tested, particularly bi-layered osteochondral scaffolds based on collagen, collagen-BNC blends, collagen-alginate blends that presented also a bone-like layer made of collagen mineralized with Mg-substituted HA. In this respect, with the goal to establish a 3D bioreactor-based culture system, different perfusion bioreactor flow regimes were applied for cell seeding on the scaffolds. The advantage of using a 3D model rather than 2D culture in petri dishes was demonstrated. In particular 3D cultures gave rise to formation of stromal-like tissue structures, characterized by heterogeneous cell population and morphologies in physical contact. 3D-perfusion expanded cells maintained a higher clonogenicity and superior multi-lineage differentiation ability by means of osteogenicity, chondrogenicity and adipogenicity. Also, the influence of defined hypoxia levels on cell differentiation was investigated, by testing scaffolds functionalized with oxygen-chelating peptides. As a result, the functionalization of the scaffold resulted in a higher cellularity. Besides, the effect of the various bioactive components on the synthesis of pro-inflammatory mediators was investigated. In particular, the effect of inflammatory cells on the cartilage-forming ability of mesenchymal stem cells was investigated. Preliminary results reported that factors released by macrophages do not modulate MSC chondrogenesis and that the tissue repair macrophages induce higher and more reproducible chondro-inductive effects, in comparison with inflammatory macrophages. It was also found that cell co-cultures are more suitable to ensure cell viability and proliferation; also, instructed cells in co-cultures were able to express higher amounts of glycosaminoglycans, a base component of cartilage.

Therefore, an optimized bioreactor culture conditions for the generation of an osteochondral implant was achieved. Using defined conditions, scaffolds could be seeded with chondrocytes throughout the cartilage layer, while minimizing chondrocyte attachment to bone layer. During in vitro culture, cells could proliferate and generate a cartilaginous extracellular matrix.
During the project, new methods to assess the specific functional properties of tissue engineered cartilage were settled by using atomic force microscopy (AFM)
In particular it was assessed: (i) whether AFM measurements can be employed to obtain both quantitative and spatially resolved data on the elastic response of tissue engineered cartilage to short exposure to IL-1 β (i.e. a condition mimicking the initially inflammatory implantation site) and how these measurements correlate with standard biochemical or histological assays. In this study it was demonstrated that AFM nanoindentation allows to monitor, in a quantitative and spatially resolved way, the IL-1 β mediated reduction of tissue elastic modulus. AFM-based quantification of elasticity in advanced culture systems may be used in conjunction with biochemical and histological data to predict the graft behavior upon implantation, in order to estimate a suitable degree of cartilage maturation for functional tissue engineering and ultimately to define “how good is good enough” for clinical use. In this perspective, this result can be considered as far beyond the state of the art existing at the beginning of project OPHIS. In this respect, in order to use AFM-based measurements as non-invasive quality control for tissue engineered cartilage, future work will have to be undertaken to render the assessment compatible with tissue-specific culture constraints (e.g. fixation without glue and maintenance of sterility).

In vivo assessment
Ethical issues
The beneficiaries involved in the management of biological tissues and in vivo tests, i.e. IOR, UCSC, LEMI and UHBS, carried out their activity in compliance with ethics issues described in D10.4 and D10.5.
Briefly, animal experimentation was limited as much as possible and replaced with alternative in vitro models. All research activities conducted within OPHIS were carried out in compliance with fundamental ethical principles, including animal welfare requirements, in conformity with Community Laws and Directives.
The choice of the animal models and the number of animal involved in each study was made to ensure clinically relevant results and adequate statistical significance; on the other hand the overall number of animals was kept the lowest, and the follow up times was kept the shortest. The treatment of all the animals, including anaesthesia as well as the pre- and post-operative stages was strictly regulated by Ethical Committee of each specific institute, to which the detailed protocol of the studies including scientific objectives was preliminarily submitted for approval and authorization.

New scaffolds for osteochondral regions diseased by Osteoarthritis
During OPHIS new implantable scaffolds for regeneration of bone and osteochondral tissues were developed and tested in vivo on small (rabbit) and large (sheep) animal model.
In particular, the scaffolds tested on rabbit were:
I GROUP: RegenOss-like scaffold – this group was used as a control
II GROUP: Mineralized alginate scaffold
III GROUP: Collagen + Chitosan + HA
IV GROUP: BNC (half treatment group with an unmineralized and half with a mineralized scaffold)

The follow-up time of these experiments was 6 months.

The scaffolds tested in sheep were:
I GROUP: 4 sheep (MaioRegen-like bi-layer scaffold)
II GROUP: 4 sheep (bilayered BNC-based scaffold)
III GROUP: 4 sheep (alginate-based scaffold)
IV GROUP: 4 sheep (MaioRegen functionalized)
V GROUP: Control - 2 sheep (untreated defects)
VI GROUP: 4 sheep (Collagen/Chitosan bilayer)

The in vivo tests highlighted that the best-in-class among the new materials for osteochondral regeneration was the bi-layered HA/Collagen produced by ISTEC, functionalized with semi-dendrimers produced by UoB (IV Group). In this respect this material will be further investigated also after OPHIS, to assess whether surface functionalization can actually provide enhanced tissue regeneration, particularly regarding the process of cell recruitment and homing in the scaffold (i.e. in vivo tissue engineering).
Among the other tested scaffolds, some were considered interesting and with good handling; in this respect, several bio-polymeric blends were considered in OPHIS in order to provide improved mechanical performance, thus extending the field of application in bone and osteochondral surgery. Although in these animal tests the results were worse than the control (a collagen-based osteochondral scaffolds obtained by biomineralization), it is considered that surface functionalization by dendrimers may improve the biologic response, thus improving the results of in vivo tests.
With more detail, BNC and alginate are worthy to be further investigated; contrary, chitosan resulted the worst among the tested polymers, likely due to its hydrophobicity that prevented cell adhesion, penetration and resorption, as also highlighted by the histologic results.

New cements for vertebral bodies diseased by Osteoporosis
The two cements developed in OPHIS were assessed by preliminary in vivo tests in small animal, in comparison with a commercial cement of proven effectiveness in kyphoplasty procedures. The Sr-HA cement developed by ISTEC resulted the best in class after one month in respect to new bone formation and penetration inside the bone defect. Then, deeper in vivo studies were carried out at follow up times up to 3 months in rabbit, osteoinduction tests were carried out in osteoporotic animal model (rats). The Sr-HA cement was also tested in large animal (goat) at times up to 3 months.
The new Sr-HA cement for the reconstruction of vertebral bodies affected by osteoporosis gave very good results in the small animal tests, thus resulting more bioactive than a commercial product widely used in procedures of vertebroplasty/kyphoplasty, taken as a reference (KyphOS FS, Medtronic). In particular, the early effect of Sr-HA cement in inducing new bone penetration inside the boned defect was particularly promising in the view of early physical stabilization of vertebral bodies weakened by osteoporosis. This is an issue of paramount importance for the further development of the new cement; in fact, calcium phosphate cements have the drawback of a reduced mechanical strength that still limit their use in favor of acrylic cements. Thus, the possibility to enhance osseointegration in the earliest stages of bone regeneration is very promising for increasing the strength of the bone/cement construct and paves the way for the use of regenerative bone cements in vertebroplasty, as well as in the healing of other bone districts such as tibial plateau, wrist and ankle. Besides, the possibility of tailoring the viscosity and setting behavior of the Sr-HA cement open interesting perspectives for its use in therapies against different pathologies affecting bone parts where the application of solid 3D scaffolds is difficult or not feasible. In this respect, it was observed that Sr-HA cement can be used in the healing of osteonecrosis affecting femoral heads.
The new Sr-HA cement was also evaluated by means of in vivo toxicity, biocompatibility, and osteoinductive/conductive activity in an experimental model of osteoporosis in order to well simulate pathological conditions in which the biomaterial will be used in humans (osteoporotic vertebral collapse) and ensure therapeutic efficacy. In this respect, osteoporotic rats were examined and no toxic effects were detected on blood analysis as well as on liver, spleen and peri-implanted tissues carried out after explant.
In vivo tests were also carried out by implanting Sr-HA in large animal (goats). For application in vertebral surgery, the actual radio-opacity of the cement has still to be definitively assessed. In fact, good radio-opacity was detected during implantation of the cement in rabbit. However since the goat bones are quite thicker than those of rabbits and close to the human ones, it has still to be elucidated whether the flowing of the cement in a bone defect can be controlled in real time by radiographic control, during clinical vertebroplasty procedures. The final evaluation of these last tests will be completed at the end of 2014.

Scaling-up and further product development
The activities of scaling up of the final devices were carried out in compliance with the directives relevant to the Regulatory Issues for EC mark and the assessment of Quality Assurance. To do this, preliminary activities to define and validate the formulation of the product, the procedure of manufacturing and the conditions of sterilization, storage and transportation were carried out. The final devices consdiereed for the healing of bone and osteochondral tissues diseased by osteoarthritis are based on type I collagen (i.e. obtained by bio-inspired assembling and mineralization of collagen/BNC matrices), bacterial cellulose (i.e. obtained by hydrothermal treatment of oxidized BNC matrices and subsequent ice-templating process) and alginate (i.e. monolithic and biphasic constructs obtained by ionotropic gelation). The scaling up of the new products focused on four general aspects: Clinical need; Compliance with regulation; Product development; Industrialization.
All the information collected from the four point above will be used to develop a “SWOT analysis” (i.e. Strengths, Weaknesses, Opportunity and Threats) useful to define the internal and external factors that are favorable and unfavorable to achieve the commercial exploitation of devices foreseen in the project.
For the scaling up of new Sr-HA bone cement, clinical need part deals with an epidemiologic study of osteoporotic fractures with a focus for vertebral compression fractures and a deep analysis of main competitors useful to complete the second part of the SWOT analysis. This thing has to be done in three different areas:
- Traumatology and osteoporotic fractures in wrist
- Stable vertebral fractures (osteoporotic or traumatic)
- Not stable osteoporotic vertebral fractures

The compliance with regulation regards a classification and identification of essential requirements and standard to be applied for device characterization according to ISO.

This passage will be carried out according to the following directives of the European Community:
• Directive 93/42/EEC; Directive 2000/70/EC; Directive 2007/47/EC
In particular what we need to identify all the essential requirements is a short description of the product, an intended use of the product and a checklist of the essential requirements to guarantee the product safety during its lifetime.
Product development and Industrialization slowly approach the industrial exploitation considering a defined development of the final formulation, wondering if the product could be patentable and other technical aspects as the development of easy to use packaging and a sterilization method.
In the industrialization phase there is an identification of quality control parameters, a verification of pilot batch reproducibility and the study of requirements for process scale-up.
Regarding the analysis of possible competitors, for over 60 years old patients the main solution used to treat damaged or weakened vertebral bodies is PMMA cement, but with younger patients it’s possible to treat the problem with bioactive cements thanks to a higher response from the host tissues, in spite of their limited initial mechanical strength, compared to acrylic cements.
It has been defined the intended use of the new devices developed in the project. In particular the new functionalized osteochondral scaffolds will be used as “Medical device indicated for treatment of lesions caused by OA in relatively young (<60-65 years) and/or severe patients. Reconstruction and regeneration of osteochondral defects”.

For all these materials the classification of production area is still missing; this parameter will be defined after selection of the material for commercial exploitation.
A future relevant action will be to clarify whether or not the new dendrons could give to the device an activation through pharmacological, immunological or metabolic means once implanted in the body.

Similarly, for the new cements the intended use is identified as: “Bioactive, radio-opaque and injectable paste/cement for regenerative vertebroplasty”. Further info are still necessary for the classification of the productive area and the selection of the sterilization method.
On this basis, the product specifications for both the new osteochondral scaffolds and the new bone cements were defined.
The hyperbranched poly (ε-lysine) dendrons (R-G3K) synthesis developed and studied by the UoB has been transferred to FINCER in order to start the process of industrialization and validation of the technology toward the clinical application.
The equipment required for the dendron synthesis and scaffold functionalization have been set up and validated in FINCER facilities.

In particular, for the new cements the following critical issues for the process validation and scale-up:
- Sterilization of the liquid component
- Mechanical compression test at different time point in the vivo simulated condition
- Stability test
- Scale-up of the powder synthesis
In this respect the preparation and handling of the cement were standardized by using commercial mixing systems (MedMix).
Tests of stability of the alginate-containing liquid solution also started to assess the suitable storage conditions by applying ASTM F1980-07 “Standard guide for accelerated aging of sterile barrier system for medical devices”. The preliminary results are encouraging since the solutions remained stable so that it is expected that the material can be stored at room temperature.
Mechanical testing in controlled, accelerated conditions in SBF were also carried out to assess the stability of the injected paste with time. It resulted that compressive strength remained unchanged over 1 month. The tests are still in progress.
Finally the scaling up of the components of the new cements is as well in progress. In particular it is being investigated the repeatable synthesis of Sr-TCP, the liquid solution, as well as the procedures of sterilization and storage of both components (see also above). The preliminary results show that no critical issues are raised so far for these activities.

Regulatory issues
The purpose of this activity is to properly define the regulatory issues for the new developed scaffolds; in this respect the EC directives to the results of the project will be applied.
The regulatory process consists of the following steps towards certification and CE mark:
• Results of the research
• Interest (Commitment) of industrial companies
• Regulatory pathway for classification
• CE mark
To achieve adequate classification of the new products, the Intended Use and the Principle of Action serve to define the use for which the new product was designed and the scientific evidence supporting the statement. This information is relevant to classify the product as medical device or medicinal product. In case of doubts the product is classified as borderline.
Functionalized semi-dendrimers can direct cell behavior and phenotype, so that the classification of functionalized scaffolds is still uncertain. Hence, future steps in the scaling up of such new devices, the notified body will be contacted; in this respect a possible future action may be a further in vivo assessment at much longer follow up times to assess the category to which the product belongs.

Potential Impact:
The most relevant achievements obtained during OPHIS can generate a relevant impact as concerns:
1) regeneration of bone and osteochondral tissues diseased by severe Osteoarthritis
2) regeneration of bone fractures affecting vertebral bodies or other bony regions affected by weakening or fracture due to trauma or Osteoporosis
In particular, two research products developed in OPHIS resulted very promising for commercial exploitation:
1) New osteochondral scaffolds made of type I collagen mineralized with Mg,CO3-substituted apatite functionalized with dendrimers (carboxybetain) that enhance the specific docking of endogenous or loaded cells into the scaffold porosity, as well as specific differentiation. The in vivo tests on large animal report the repair of the osteochondral defect with formation of new bone in both trabecular and cortical compartments. The presence of newly formed hyaline-like tissue, with evidence of viable chondrocytes uniformly distributed in all cartilage layers, with no evident cell clusters, and good integration of the scaffolds were detected. Due to these promising results the new scaffolds can be indicated for the treatment of bone and osteochondral lesions caused by Osteoarthritis in relatively young (<60-65 years) and/or severe patients.
The new device has been validated in large animal model, which is a relevant environment in the view of pilot clinical studies, thus reaching a TRL of 5.

2) New strontium-substituted apatite bone cements obtained by transformation of Sr-doped β-tricalcium phosphate added with sodium alginate. The new cement has been subjected to a number of in vivo validation procedures, i.e. small and clinically-reflective vertebroplasty in large animal, as well as toxicity in rats, demonstrating enhanced osteogenic, osteoconductive and bio-resorption ability in comparison with a commercial apatite cement widely used in vertebroplasty. Due to these results, the new cement can be indicated for the treatment of trauma or osteoporosis-related weakening or fracture by regenerative vertebroplasty/kyphoplasty, percutaneous treatment of fragility methaphyseal fractures (femur, tibia humerus, wrist), and hip, shoulder and knee prosthetic revision surgery.
The new device has been validated in small and large animal models, including toxicity, which is a relevant environment in the view of pilot clinical studies, thus reaching a TRL of 5.

More specifically, in OPHIS several foreground results can be relevant as milestones for the development of new therapies enhancing the healing of diseased bone and osteochondral tissues:
1) Hybrid graded scaffolds for osteo-chondral tissue regeneration obtained by bio-inspired assembling/mineralization of blended biopolymer matrices: the bio-inspired process already assessed for the development of high regenerative scaffolds based on type I collagen has been implemented for application on composite biopolymeric matrices, including bacterial nano-cellulose and chitosan. This approach enhances from one side the flexibility and wettability, and from the other side the stiffness of the 3D bio-inspired constructs, thus being adequate for implantation in larger tissue defects.
2) Multi-layered scaffolds mimicking bony and cartilaginous regions obtained by ionotropic gelation: the new alginate-based osteochondral scaffold is an easy-to-handle implantable material which can be loaded with autologous cells for stimulation of the healing of osteochondral defects. In OPHIS the safety of the cell-free biomaterial was proven, while the efficacy evaluated in a large animal model (IOR) resulted still inadequate for further development towards a clinical product.
3) Hybrid nanocomposite scaffold based on Bacterial Nanocellulose: The engineering of the mineralized with non-mineralized BNC fibers enables the production of new bone and osteochondral scaffolds which deserves further investigation for future application.

1) pH-sensitive bio-polymeric mineralized scaffolds for bone and osteochondral regeneration: the new scaffold, exhibiting ability of reacting to specific pH values, can be exploited as a new implantable drug delivery system associating the high regenerative ability of collagen/MgHA scaffolds with the stimuli-responsive features of modified chitosan that enable controlled release of anti-inflammatory or antibiotic molecules, thus potentially improving the process of tissue regeneration.
2) Oxygen sequestrators: the application of dendrimer-based functionalization to medical devices represents a powerful tool to improve the regenerative properties of bone and cartilage tissues
3) Functionalized hybrid scaffolds with semi-dendrimeric structures able to mimic the cartilage micro-environment: This novel bio-functionalisation method can be applied to any biomaterial scaffold to confer enhanced tissue regeneration properties. Hence, they can be applied to scaffolds for the regeneration of osteo-articular defects as well as to other types of tissues where the recruitment of endogenous cells or the loading of cells prior to implantation needs to be maximized and controlled. All the tests and improvements of the functionalized collagen-based scaffold allowed the scale-up of the process in terms of efficiency and reproducibility. All these requirements are necessary to lead the product to actual exploitation and to the clinical application.
Due to these promising results the new scaffolds can be indicated for the treatment of bone and osteochondral lesions caused by Osteoarthritis in relatively young (<60-65 years) and/or severe patients.
The new device has been validated in large animal model, which is a relevant environment in the view of pilot clinical studies, thus reaching a TRL of 5.

Injectable bioactive cements for regenerative vertebroplasty: the unique properties of the new Sr-substituted cement make it adequate and very promising for mini-invasive surgery such as vertebroplasty/kyphoplasty, percutaneous treatment of fragility methaphyseal fractures (femur, tibia humerus, wrist), and hip, shoulder and knee prosthetic revision surgery. The mechanical strength and cohesion of the new cement, which are stable over 1 month of ageing in simulated body fluid at 37 °C, as well as the preliminary results of in vivo tests, prove that early physical stabilization of diseased vertebrae without sudden fracture is feasible, thus opening the way to new therapies against fractures of vertebral and load-bearing bones.
Due to these results, the new cement can be indicated for the treatment of trauma or osteoporosis-related weakening or fracture by regenerative vertebroplasty/kyphoplasty, percutaneous treatment of fragility methaphyseal fractures (femur, tibia humerus, wrist), and hip, shoulder and knee prosthetic revision surgery.
The new device has been validated in small and large animal models, including toxicity, which is a relevant environment in the view of pilot clinical studies, thus reaching a TRL of 5.

The results gathered in the project OPHIS will offer a unique opportunity to improve competitiveness of the European industry. Indeed, despite the relatively high investment in tissue engineering made by non-EU companies, no clear product has emerged that is able to lead the market in the field of OA and OP treatments. The development of the new tissue substitutes in OPHIS were underpinned by a robust and systematic research activity leading to a strong knowledge platform in the field of tissue regeneration of complex tissues following pathological conditions. The impact that OPHIS will have on the knowledge-based competitiveness of industry is tangible when framed in the context of the benefits for the two industrial partners of the consortium.
FINCER will be able to exploit products where nano-sized components confer a bioactive/biocompetent character to composites for biomedical applications. The company will be able to integrate a novel class of biomaterials in their portfolio and targeting a specific market so far dominated by the pharmaceutical industry and medical implant companies. FINCER will have the opportunity to draw a flexible strategy where either a-cellular products or tissue engineering constructs can be exploited depending on the clear clinical advantage and commercial value.
Likewise, the objective of optimising clinically-reflective in vitro models to test the biocompatibility of the substitutes, their fine regulation on cell activity and, ultimately, their clinical potential will offer a clear commercial advantage to LEMI, the industrial partner of OPHIS with a mission in the provision of biocompatibility test services. Indeed, according to the EC requirements, significant progress should be made in the field of material characterisation to gain knowledge and well define their properties, “in particular the structure–property relationships at different scales, to improve materials assessment, reliability”.

Uniquely, the objective of developing bio-analytical AFM testing of the interactions occurring between implanted materials and natural tissue will offer to the company a potential added value. Indeed, in the view of the new EC regulatory framework of the tissue engineering products as “Advanced Biological Products”, this could represent a clear commercial advantage for the company at global scale.
For both FINCER and LEMI and/or any other third commercial partner, the OPHIS technology represents an avenue towards the manufacture of “products and related processes and technologies able to meet customer requirements as well as growth and public health”. Although the future products deriving from the OPHIS research activity are expected to be significantly more expensive that conventional biomaterials and medical implants, the commercial sustainability imparted by these tissue substitutes is balanced by the expected impact consisting in reduced hospitalisation time, reduced cost burn associated with pharmaceutical intervention and reduced postoperative morbidity and absenteeism. These advantages clearly address the EC policy driven by concerns on balance in economic growth and social well-being.
A quantification of the economic framework in which OPHIS is expected to provide its impact is reported below in the sections dedicated to the economic and societal impact and to the strategic impact. The nature of the OPHIS tissue substitutes in terms of manufacturing capability and knowledge-based resources is naturally oriented towards SMEs who can more easily adapt their production processes to assemble the innovative multi-component OPHIS tissue substitutes that are based on “a multidisciplinary and integrative RTD approach” in the area of nano-sciences and nanotechnologies. This will address the specific EC requirement for “families of new materials with high application potentials in sectors such as … health” and in particular for “biomimetic gels and polymers… and composite materials”. More specifically, the bottom-up approach to achieve bio-functionality in specific clinical applications is reached by assembling nanoscale and biocompetent biomaterials with macroporous scaffolds and composites. Uniquely, according to the EC vision, the OPHIS tissue substitutes will combine the “novel properties” of the nano-scale functionalities with the “pre-defined properties” of the biopolymer and respective composites.
The composition of the partnership of OPHIS clearly responds to the EC requirement for improving “interdisciplinary approaches in a collaborative research (environment) that may include several fields of sciences or disciplines such as: biological sciences, physics, chemistry”. In fact, in OPHIS the synergy between different fields of knowledge enabled the development of new products by introducing multi-functionality at biochemical, cellular and histological levels thus obtaining highly performing bio-inspired materials. More specifically, graded hybrid scaffolds for regeneration of complex osteo-cartilaginous regions were developed by imparting compositional and morphological mimesis of the different native tissues; besides, the bio-competence of the different layers was further enhanced by exposure of nanosized moieties able to trigger specific, tissue-relevant cell phenotypes. This completely new devices can enable new therapies for local treatment of articular regions diseased by osteoarthritis.
Similarly, the new apatite cements developed in OPHIS provided breakthrough results in the actual scenario of injectable cements for vertebroplasty. In this respect, specific anti-osteoporotic agents, i.e. strontium ions, were incorporated in the structure of the cement by a novel chemical approach enabling progressive and predictable release in vivo, thus opening new perspective for local treatment of bone weakening due to osteoporosis.
In conclusion, the new approach of OPHIS can represent a first basis for new local therapies against degenerative diseases that can boost tissue regeneration even in the case of patients with reduced endogenous regenerative potential. Besides, the optimization and validation of local, personalized therapies can reduce the recourse to systemic approaches that often result affected by concerns for undesired and serious side effects, as also illustrated in more details in the next sections.

Contribution to community societal objectives
The increase in average age in the EU, together with expectation of an active life among the elderly, has led to a corresponding increase in the societal and economic impacts of degenerative diseases including OA and OP. In response to these pressures, modern medicine tries to offer solutions that can improve the quality-of-life throughout aging. Unfortunately, the current clinical solutions, namely pharmacological treatments and tissue replacement and augmentation by implants, suffer from significant limitations and they are not able to restore completely the patient’s mobility and quality of life. Currently available pharmacological solutions, including topical agents used in patients as adjuncts or as systemic medications, suffer from limitations and they can only lead to the short-term reduction of mild-to-moderate pain in non-severe chondropathies or osteoporosis. For example, studies have suggested that intra-articular injections of corticosteroids are of short-term benefit (1 or 2 weeks only) for pain and function. Moreover, some evidence suggests drug-based interventions are not able to alter the progression of the disease and may have detrimental consequences on joint structures. In addition, anti-inflammatory drugs present potential side-effects and require close patient monitoring due to toxicity (including renal disease, hepatotoxicity, cardiovascular and gastrointestinal toxicity). Injectable visco-supplements that mimic healthy synovial fluid are also of limited efficiency and can provoke side-effects when abused or misused.
In summary, there are currently no available therapeutic solutions proven to slow down the progression of OA and OP. For this reason, a significant amount of effort is being dedicated worldwide to the development of innovative, engineered and “intelligent” biomaterials. It is envisaged that such materials can be obtained through their functionalization with biological cues normally involved in tissue regeneration. It is anticipated that significant progress in the treatment of these pathologies can only be achieved through a regenerative medicine approach.
OPHIS project pursued this strategy that offers a systematic and intelligent approach towards the resolution of complex tissue damage by coordinated and targeted material design. As previously stated, this project adopts a bottom-up approach where the requirements for tissue regeneration will be addressed from the biochemical to the cellular to the histological level. Therefore, the characteristics of the tissue substitutes developed during the project will fulfil the main objectives of the EC strategies as defined in the call NMP-2009- 2.3-1 to which OPHIS refers as well as the expected impact of this call that is:
“Development of such biomaterials will result in biomedical implants having characteristics close to those of natural tissues. The designed structures and approaches should provide remedies and improved strategies to combat musculoskeletal disorders and arthritis…..”

In respect to these issues, the achievements of the OPHIS project will impact on:
- the clinical methods used to treat severe OA and OP based on the restoration of the natural functionality of the bone, cartilage and osteochondral tissue by controlling the phenotypes of both osteoblasts and chondrocytes in their respective histological compartments. These alternative methods will reduce the need for late treatment of the damaged tissues by conventional medical implants bearing limitations in terms of clinical outcomes and costs. Therefore, the long-term impact of the OPHIS approach on health and social costs is clearly evident;
- the number of invasive surgical procedures for the treatments of OA and OP resulting in (i) a reduced hospitalisation time, (ii) reduced post-surgical pain and (iii) fewer potential complications;
- the dependence upon systemic pharmacological approaches to the treatment of OA and OP that in turn impact on the number of drug-related complications. The improved accuracy in the delivery of bioactive molecules to the targeted anatomical sites will offer highly specific, localised activation of the tissue regeneration mechanisms;
- the availability of clinically performing biomaterials based on biologically active components that can be implanted through minimally-invasive procedures. Indeed, the new tissue substitutes will provide the clinicians with a pioneering solution towards the restoration of tissue functionality in patients with OA and OP. Particularly, in the case of vertebroplasty where acrylic-based cements still play a major role, the new cements developed in OPHIS demonstrated fast bone regeneration and osseointegration which may promote early physical stabilization and complete bone healing. Therefore, the effect of the new cements can be much more beneficial than the ones provided by the currently available solutions, even though hospitalization time may be a bit longer. The advantages are much more evident if considering the numerous drawbacks affecting the use of acrylic cements in vertebroplasty.

Economic and societal impact
The outcomes of the OPHIS project will have enormous economic and societal impacts on degenerative diseases such as OA and OP which involve millions of people each year in the EU. These diseases result not only in progressive disability but may also impact at the psycho-social level with approximately 40% of adults with knee OA reporting their health as “poor” or “fair”. Moreover, one third of the European population will be at least 60 years old by 2050 and the increased physical activity and modified lifestyle provoke a high incidence of these diseases even in the young population. Thus, unless more efficient therapeutic solutions are found, the economic impact of OA and OP is destined to increase enormously in the coming decades. Therefore, the impact of the results of OPHIS is expected to be relevant to future clinical practice and to lead to a significant reduction of the costs impinging on the healthcare system.
OA is the most common form of arthritis, with a major impact on patient mobility, productivity and independence and ranks among the top ten causes of disability worldwide. Symptoms and disability increase in prevalence with increasing age and people with OA use health-care services at a higher rate than a representative group of all adults. With the population aging, the prevalence of osteoarthritis is increasing and its consequences are impacting significantly on society. In fact, OA is a leading cause of chronic disability in the USA and EU. Numbers are impressive, accounting for over 20 million diseased people in the USA and over 50 million in EU, registering as much as 25% of the total visits to primary care physicians, and half of all Non-Steroidal Anti-Inflammatory Drugs (NSAID) prescriptions. In the USA, hospitalisations for OA soared from about 322,000 in 1993 to 735,000 in 2006.
The number of people with OA-related disability is expected to double by the year 2020, thereby increasing the already significant economic burden resulting from the condition. Costs of OA-related illnesses have risen over recent decades accounting for up to 1–2.5% of the gross national product in developed countries. Moreover, focus on the direct costs to the health-care system or on the direct, out-of-pocket expenditures by patients for items such as medications, assistive devices, transport and home adaptations, may lead to an underestimation of costs associated with the condition. Failure to consider informal care costs may also lead to an underestimation of the true financial burden related to OA. In fact, among OA-related costs, indirect costs account for, on average, 81% of their total economic burden. Indirect patient costs of OA are attributable to the cost of informal care (e.g. help with personal care, household and yard chores provided by unpaid caregivers) necessitated by OA-related disability. Informal care plays a major role in the total care provided to people with chronic diseases like OA. Moreover, with increasing efforts by governments to contain health-care expenditures by minimising lengths of in-hospital stay for joint replacement surgery and shifting postoperative rehabilitation from an in-patient setting to home, the need for informal care is expected to increase. Arthritis has a significant impact on psychosocial and physical function and is known to be the leading cause of disability in later life. Approximately 40% of adults older than the age of 70 suffer from osteoarthritis of the knee. Among these, about 80% of patients have some degree of movement limitation and 25% cannot perform major activities of daily living (ADL’s), 11% of adults with knee OA need help with personal care and 14% require help with routine need. Therefore, there are also significant out-of-pocket costs and loss of earnings due to changes in occupation and roles in domestic duties. Costs of OA are substantial and are due mainly to informal costs, including indirect costs due to lost wages and reduced productivity, which have also to be considered. Approximately 200 million women are affected by osteoporosis worldwide, with a consequent high incidence of bone fractures representing a serious clinical and economic problem. In the United States more than 250,000 hip, 700,000 vertebral and 250,000 wrist fractures are annually attributable to OP, with an increasing incidence related to age. The yearly cost to heal the vertebral fractures and related pain is in excess of 2 billion Euro and for all the fractures related to OP, over 20 billion. Over 400,000 hip fractures occur every year in the EU member states. A hip fracture almost always requires hospitalisation and major surgery and almost invariably results in chronic pain, reduced mobility, disability, and loss of independence. Hospital costs for hip fractures alone amounted to over 3.5 billion Euro in the EU in 1999, with half a million beds occupied, foreseen to double in the next 50 years. Additional costs, up to 2.5 times greater than the direct hospital costs, are due to rehabilitation and nursing assistance for immobilized patients. Indeed, osteoporosis accounts for more days spent in hospital than other diseases, including diabetes, myocardial infarction and breast cancer for women over 45 years of age. The social and economic cost may be further increased, because of the associated long-term morbidity. For example patients with symptomatic vertebral fracture consult their GPs 14 times more often than do controls in the year following fracture, so are likely to continue to use health and social service resources at an increased rate. Osteoporosis is a major health problem, comparable to other diseases. Osteoporosis in Europe results in more “lost years of healthy life” (DALYs) than most of the cancers. One in three women (more than for breast cancer) and one in five men (more than for prostate cancer) will get osteoporosis, and every 30 seconds someone in the European Union has a fracture as a result of osteoporosis. As a consequence, the quality of life in osteoporotic patients is significantly affected with pain and disability often impacting on their psychological wellbeing, leading to anxiety, depression, fear for the future, and altered perception of their social role. Among the long term complications caused by vertebral fracture, it is worth mentioning the loss of lung functionality and the increased risks of further vertebral fractures. Women with one pre-existing vertebral deformity have a five-fold greater risk of further vertebral fracture; 20% of postmenopausal osteoporotic women suffer a further vertebral fracture within one year of an incident vertebral fracture. Moreover, the risk of fractures in other anatomical areas is also increased. Women with a previous vertebral fracture have a 3.8-fold increased risk of hip fracture, compared with the background female population.
This extended damage reduces life expectancy with 24% of women and 33% of men dying within one year after a hip fracture. Various studies have shown that loss of function and independence among survivors is profound, with ca. 40% unable to walk independently and ca. 60% requiring assistance a year later. Because of these losses, around a third of the patients is totally dependent and resides in nursing homes within a year following a hip fracture. Therefore, unless solved, future economic and logistic impact on health care services will be enormous due to the need for more hospital beds, more rehabilitation, greater demand on outpatient services and long-term nursing care. In the past decade the USA has carried out a multidisciplinary strategy of investments on nanotechnology and fundamental research on nano-biosystems, health issues and bio-nano devices. The European reaction with the EC 7th Framework programme has emphasized the importance of this area dedicating funding of over 3 billion Euros to nanotechnology and new materials including repair and regenerative functions in the health sector. Therefore this project proposal aims to achieve scientific, technical and economic competitiveness through a highly innovative approach in the field of tissue regeneration. Every year in the EU several hundred thousand surgical procedures required the use either of bone graft or other devices to resurface or reconstruct joins impaired by OA. Regrettably, the majority of those devices are developed, produced and marketed by non-European companies, resulting in a progressive increase of the related costs for the import of the materials and consequently in a high economic impact on the European Community. The development of advanced products in regenerative medicine will surely enhance the competitiveness of EU-based industry. SME and large companies will have an opportunity to strengthen their IP and product portfolios by penetrating and extending their shares in the orthopaedic and spinal market. Just to provide some self-explanatory numbers on the European joint prosthesis market, please note that in 2006, as much as 908 M€ of hip implant were sold in the five major EU countries (UK, DE, FR, IT and ES), as well as 957 M€ on knee implants and 54 M€ on shoulder. Moreover, it is calculated that the bone substitutes market for orthopaedic and spinal applications generated, in the same year and same countries, was more than 70 M€. The number of regenerative and reconstructive surgeries is estimated to increase at an annual rate of approximately 15% for at least the next 10 years. Furthermore, there is a great demand for the European health services to reduce the costs related to patient treatment. A key approach is to develop new strategies for tissue regeneration with the goal of reducing the costs of patient care whilst improving the quality of care of the patient and, consequently, their quality of life. This goal will be achieved by reducing hospitalisation time and costs, patient pain, risk and costs related with bone grafts. Patient healing, time for return to work and time taken to resume normal activity will be accelerated with a better psychological and clinical outcome.
Economic growth in Europe will also depend on the development of new products with high added value. The bone graft market and the joint resurfacing market offers to this new generation of products high potential to expand treatment options, improve patient quality and reduce healthcare costs. Therefore, any new approach to bone and cartilage regeneration harnessing the body’s own regenerative potential is not only a very attractive to physicians but also economically very attractive to patients, the end users. The development of such an approach is the overall goal of the project and will enable the industrial partners to maintain and sustain a competitive edge.

Working conditions, Employment
The establishment of new and advanced technologies can create new highly skilled jobs. EU industry is under pressure from imports from other parts of the world, especially Asia. This also applies for low-tech medical devices. Regenerative medicine is a key emerging technology driven by biotechnology and advanced materials research. The products and services arising from these technologies will surely be advanced, sophisticated, high tech products. Those products will require a high level of expertise, skills and accuracy, customised and extremely organised production lines, state-of-the-art risk management and quality control competence, and will clearly be subjected to strict regulation and standards. Industrial production of such high-tech products, with attractive return on investment, will be carried out in the Euro-zone (where a network of research centres and specialized service is already available) hence ensuring long term benefit to growth and employment in Europe. In other words, the project will lead to research and manufacturing businesses located in Europe and employing predominantly skilled and well-educated work forces.

In recent years the increasing application of nanotechnology to regenerative medicine and the constant progress in research has led to the development of advanced biomaterials for regenerating connective hard tissues. Nowadays, regenerative medicine requires the close integration of various fields of knowledge: materials science, biology, chemistry, engineering, and clinical practice. The participation of six universities in OPHIS provided a direct opportunity for the research students and post-doctoral staff in the project to develop a knowledge based on a multi-faceted trans-disciplinary approach, with the supervision of senior scientists, with a high level of experience in the different field of this complex and critical arena. This occurred through interactions between the institutions and by visits to laboratories. The field of nano-technology as applied to regenerative medicine has already highlighted the need for highly specialised training of human resources. The involvement of the universities has contributed to providing new courses and training through undergraduate, graduate and short courses in this field. The transfer of new knowledge from a multidisciplinary context of specialists to young researchers has taught them how to develop a concrete and pragmatic approach to problem analysis and problem solving as well as effective research management skills.

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