Final Report Summary - RESTORATION (Resorbable Ceramic Biocomposites for Orthopaedic and Maxillofacial Applications)
Executive Summary:
Ceramic composite materials have for many years been considered to show great promise in the repair of musculoskeletal defects. The materials can mimic the structure of bone, and devices made from the materials can be structured to closely match the mechanical requirements of implant sites. In addition, wide ranges of bioactivity are possible, from inert to fully resorbable.
Bioceramics have most commonly been used to date in dentistry, and in some orthopaedic applications, e.g. as an injectable paste for vertebroplasty, or as a coating material for metal orthopaedic implants. However, advances in cellular medicine bring great opportunity for significant growth in the bioceramics industry – bioceramics and bioceramic composites offer levels of bioactivity which far exceed those available from metal implants, together with combinations of strength and modulus which exceed anything which can be offered by bioactive polymers on their own. Working in tandem with cells, proteins and other biologically active agents (both from the host and introduced) bioceramic composites have the potential to revolutionise many treatments and therapies, giving new, highly effective early stage clinical interventions for conditions where no approach has existed to date.
In order to deliver on the potential shown by bioceramic composites the combination of mechanical design, materials, processing, clinical delivery and subsequent biological interaction all have to be understood in an integrated and systematic way. The RESTORATION research project is addressing these underlying research and technological challenges in order to develop new bioceramic products for five SME partner companies.
The RESTORATION project has focussed on the development of new materials and medical devices for the treatment of osteoarthritis, maxillofacial fractures and vertebral fractures. New material processing routes, new materials and new material structures have been created, and assessed for their biocompatibility and mechanical properties.
The partners will now seek exploit the project results and bring these new technologies to the market in the coming years. Specific outcomes in terms of new products and their progress are:
• New bioceramic plugs as early stage treatments for osteoarthritis has been developed to the stage where they have been tested in a large animal model, and their development will be taken forward by Glass Technology Services, JRI Orthopaedics and Newcastle University.
• A new bioactive cement for use in vertebroplasty procedures has also been evaluated in a large animal model and will be further developed by COREP.
• A new particulate bone filler has been evaluated in a small animal model and will be taken forward by Glass Technology Services and JRI Orthopaedics.
• A new biocomposite paste containing platelet rich plama for use in vertebroplasty procedures has been evaluated in a small animal model and will be further developed by Sagetis-Biotech and the Institut Quimic de Sarria (IQS).
• A new maxillfacial plate design and material have been developed and will be taken forward by Newcastle University.
In addition, all the materials and technologies developed within the project have been assessed in terms of environmental impact to idtentify issues to take into account for industrial scale-up.
The project has delivered an integrated pre-clinical assessment of new bioceramic composites in order to underpin the development of a range of new musculoskeletal medical devices.
Project Context and Objectives:
Ceramic composite materials have for many years been considered to show great promise in the repair of musculoskeletal defects. The materials can mimic the structure of bone, and devices made from the materials can be structured to closely match the mechanical requirements of implant sites. In addition, wide ranges of bioactivity are possible, from inert to fully resorbable.
Bioceramics have most commonly been used to date in dentistry, and in some orthopaedic applications, e.g. as an injectable paste for vertebroplasty, or as a coating material for metal orthopaedic implants. However, advances in cellular medicine bring great opportunity for significant growth in the bioceramics industry – bioceramics and bioceramic composites offer levels of bioactivity which far exceed those available from metal implants, together with combinations of strength and modulus which exceed anything which can be offered by bioactive polymers on their own. Working in tandem with cells, proteins and other biologically active agents (both from the host and introduced) bioceramic composites have the potential to revolutionise many treatments and therapies, giving new, highly effective early stage clinical interventions for conditions where no approach has existed to date.
In order to deliver on the potential shown by bioceramic composites the combination of materials science, mechanical response, processing, clinical delivery and subsequent biological interaction all have to be understood in an integrated and systematic way. The RESTORATION consortium have collaboratively address this underlying research and technological challenge in order to develop new bioceramic products for five research led SME partner companies.
The project involved:
- The selection of a limited number of advanced bioceramic materials (apatite containing bioceramics, calcium phosphate and calcium sulphate based materials, mesoporous bioactive glasses and borosilicate glasses) that have potential to create high added value biomedical products for the SME partners.
- The development of advanced high added value medical devices that offer improved and controlled biological interaction, and become part of the biological system.
- Research to modify and adapt the advanced bioceramic materials for new production processes, suitable for future in-hospital or in-clinic manufacture of personalised devices.
- Full assessment of the new bioceramic materials and devices, on the grounds of potential clinical effectiveness, economics, and environmental impact.
The clinical targets with related objectives were:
- Osteoarthritis (OA). OA is a degenerative joint disease, typified by a loss of quality of cartilage and bone at the interface of a joint, resulting in pain, stiffness and reduced mobility. The process is not confined to the joint but can involve regional muscles and ligaments. Treatment can be considered non-surgical and surgical. Non-surgical treatment includes exercise, weight loss, physical therapy and medication (e.g. pain relief). For severe disease, when the non-surgical measures cannot control the symptoms, there is currently no other widely recognised treatment other than joint replacement (including newer designs such as joint resurfacing). OA is the most frequent form of arthritis in humans, and is significantly disabling. Survey data indicates that it affects over 11% of females and 7% of males by the time they reach their mid-fifties, rising to over 33% of females and 19% of males by the time they reach their mid-70’s. The current global market in joint replacements is estimated to be in excess of €7 billion, with in excess of €2 billion within the EU and the bulk of this is hip and knee procedures for OA. Within the project we have developed early stage interventions for OA, capable of being applied to local defects within the joint, through minimally invasive surgical techniques. OA puts significant pressure on healthcare systems and developing procedures which are minimally invasive and “day case” will reduce this pressure, significantly delay or completely remove the need for a total joint replacement, and significantly enhance quality of life.
- Vertebral Compression Fractures (VCF). Increased life expectancy is leading to a rise in age related pathologies which often involve the decay of bone quality (for example from osteoporosis) causing vertebral compression fractures. Approximately 35-50% of women and 20-30% of men develop vertebral compression fractures, and approximately 50% of those affected develop multiple fractures throughout their life . In addition, trauma, bone diseases, and cancers can lead to vertebral fractures and bone loss in younger patients. Vertebroplasty (VP) and kyphoplasty (KP) are two surgical minimally invasive procedures that are now considered the “gold standard” for the medical treatment of VCF. They both aim to augment and stabilize the weakened vertebral body and KP also aims to restore as much of the height and functional state as possible. VP consists of the percutaneous injection of bone cement directly into the fractured vertebral body. KP injects bone cement into a cavity created in the vertebral body by the inflation of a balloon. The global market in products for VCF is in excess of €400 million, and is growing significantly as a result of the ageing population. Within the project we have developed new injectable bioactive ceramic composites able to stimulate bone regeneration and to deliver specific drugs to the target site.
- Maxillofacial Bone Fracture Alignment. Maxillofacial bone fractures occur either as a result of trauma or as part of a surgical procedure designed to correct a developmental fault. Fractures of the mandible account for around 6% of trauma cases, with an increasing incidence of cases seen in the elderly over the last 30 years, and the global market for maxillofacial bone fracture alignment devices is estimated at €100 million. The limited anatomical space within the maxillofacial region means that fractures of the bones are usually aligned using internal fixation plates, with correct alignment of the bones to allow correct eating, speaking and the quality of life of the patient post-operatively. However, there are a significant numbers of revisions required each year due to post-operative problems associated with current plate designs. The development of new materials that will reduce the need for these revisions will greatly reduce the economic burden on the health systems and improve the quality of life of the patient. Within the project we have started the development of new bioceramic polymer composites, which can be manufactured to conform to the patient’s anatomy, possess sufficient stiffness and strength to protect and support the broken bone, and which will resorb non-toxically within 24 months of placement.
Through developing new treatments for these three conditions the project:
- Has developed commercial opportunities for the SME partners.
- Has developed internationally leading science on the development of bioactive ceramic composites, their processing and their behaviour as part of a biological system.
- Will make significant contributions to improving the quality of life for sufferers, initially in the EU and later throughout the world.
Project Results:
The project has been carried out in four stages:
- Selection of a limited number of advanced bioceramic materials that have potential to create high added value biomedical products for the SME partners
- The development of advanced high added value medical devices that offer improved and controlled biological interaction, and become part of the biological system
- Research to modify and adapt the advanced bioceramic materials for new production processes, suitable for future in-hospital or in-clinic manufacture of personalised devices
- Full assessment of the new bioceramic materials and devices, on the grounds of potential clinical effectiveness, economics, and environmental impact
In overview the main achievements were:
- The first RTD workpackage (WP) was WP2, Clinical Processes and Specification. The main aim of this short WP was to explicitly capture all the requirements for the products that the project will deliver. This WP is complete.
- WPs 3, 4 and 5 were materials development WPs for the vertebroplasty, osteoarthritis and maxillofacial fracture fixation application areas respectively. These WP’s developed and evaluated the new materials which are the foundation of the project.
- WP6 was a major integrating WP, and also a significant product development stagegate. Within this WP the materials developed in WPs 3, 4, and 5 are being evaluated in terms of their bioactivity, using human and animal primary cells and both a non-load bearing and load bearing small animal model. This WP has been extended (without resource or timing implications) to further support WPs 7, 8 and 9.
- WPs 7, 8 and 9 were product development WPs, again for the vertebroplasty, osteoarthritis and maxillofacial fracture fixation application areas respectively. These WPs took input from WPs 2-6 in order to specify, design and evaluate mechanically and in vitro full scale products, and to develop variants of those products to be evaluated in WP11.
- WP10 is took input from WPs 7-9 to develop life cycle cost models, to understand the environmental impacts of the products, and to examine ways in which both the life cycle costs and environmental impact can be minimised to ensure the best long term value for healthcare systems.
- WP11 was the final RTD WP, and is using a large animal model in order to evaluate the performance of the products in vivo. WP11 assessed the products over implantation times of six months in order to provide high quality information on the behaviour in vivo, so that at the end of the project the products are ready to progress to clinical trial.
Further details for each WP (where commercial confidentiality allows) are described below.
In WP2 device specifications were created and distributed, and these documents were used throughout the project to steer the research activity.
In WP3 Bionica Tech managed to produce mesoporous bioactive glass particles (MBGs), both through an optimized sol-gel synthesis and through a rapid-kinetic process, i.e. the spray-drying technique. Both synthesis methods allowed to obtain glasses characterised by a satisfactory mesoporosity and a remarkable bioactivity. The composition of the sol-gel glass was also modified adding zirconia to the glass network, in order to gain radiopacity, while maintaining a mesoporous structure and a good bioactivity. The transition from the traditional sol-gel synthesis to the spray-drying technique allows to obtain spherical particles through a remarkable less time-consuming process and then represents an appealing option for an industrial transfer of the mesoporous glass production. Moreover, a new synthesis based on the use of water as solvent was tested with the spray-drying technique and as a preliminary but solid result, mesoporous spherical silica has been obtained.
Traditional MBGs or the sprayed particles (SC-SD) were used as dispersed phases in a calcium sulphate matrix in order to obtain a resorbable osteoinductive cement. The first matrix chosen was a calcium sulphate hemihydrate bought from Sigma-Aldrich; this cement, named Spine-Ghost (with the addition of a third glass-ceramic reinforcing phase, SCNZgc) resulted to be sufficiently radiopaque, well-injectable and released a highly amount of bioactive ions (in particular silicon and calcium). However, Spine-Ghost showed inappropriate mechanical properties and an inadequate setting time.
Due to these reasons, Bionica Tech sought a new matrix, in order to obtain optimised properties for its composite cement. After a series of evaluation, the selected solution consisted in using a different typology of calcium sulphate hemihydrate: in fact, while the Sigma-Aldrich hemihydrate is composed of β-calcium sulphate (as confirmed by the DSC analysis), Bionica Tech sought an α-calcium sulphate, like the one widely used in dentistry, which gives a stronger dihydrate after a faster precipitation mechanism. This difference is mainly based by a larger crystal size. Bionica Tech chose an alpha type-III dental gypsum and carried out using this matrix the optimisation of the different composite parameter as well as a complete characterization of the new cement. At first, a new liquid to powder ratio has been tuned, in order to obtain a paste that was sufficiently injectable through a vertebroplasty needle (15 gauge). The new cement (Spine-Ghost III) showed a shorter setting-time and thus traditional MBG particles have been substituted by spray-dryer ones in order to facilitate the technological transfer at the industrial scale. Spine-Ghost III-SD cement was then prepared: it is easily injectable, bioactive, has good mechanical compressive strength and shows more satisfactory working and setting times.
The main tasks developed by the combined Sagetis and IQS team in WP3 can be summarized as follows:
• Developing different combinations of polymeric biodegradable paste
Both the design of the polymer and the combination of polymer/bioceramic have been optimized for the polymeric paste.
The polymer synthesis was also optimized in order to allow the future GMP synthesis of the polymer. The original microwave-assisted polymerization was changed to a traditional wet chemistry synthesis. Three formulations were sent for in vitro testing to Karolinska and one of them was tested ex vivo at Evora.
A protocol of the injectability of the paste was established, heating the paste at 50ºC before injection. These injectability conditions were also tested ex vivo at Evora with sheep vertebrae. A syringe-heating device has been purchased to be used in future in vivo experiments.
Finally a protocol to increase the radiopacity of the paste has been developed using ZrO2 as radiopaque material. Also a protocol of sterilisation using gamma radiation is under development.
• Developing of new hydroxiapatite ceramic materials with controlled crystallinity
A new bioceramic material have been developed, based in a sol-gel process that incorpores SiO2 to the hydroxiapatite structure. The presence of SiO2 keeps the crystallinity level of HA low, expecting a better bioactivity of the bioceramic developed.
• Establishing the protocol to isolate Rich Platelet Fibrin (RPF) from our own blood samples.
The protocol is established. The yield of isolation was increased using a rew designed improved centrifuge tubes. Different blood samples were used (human, pig and rat). A key issue was identified regarding the growth factors concentration in the different animal’s blood.
The inactivation of the growth factors due to the sample preparation was tested. It has been demonstrated that the growth factors remain active even after the paste heating at 50ºC for injection.
Optimizing the polymer synthesis and the formulation with the PRP
Paste with different PRP has been prepared and send to Karolinska for evaluation. The results confirmed that the Paste is not toxic and the presence of PRP promotes fibroblasts viability.
In WP4, task T4.1 an in-situ precipitation of nHAp method has successfully developed. The precipitation of nanoHAp crystallites is based on the reaction of PO43- and Ca2+ from phosphate and calcium solutions. Precipitation parameters, including concentration, temperature, pH value and titration rate, have been studied. Through control of these parameters the potential size, morphology, chemistry and lattice parameters of the HA crystallites have been defined.
Within task T4.2 there were issues with particle agglomeration upon drying and mixing to form composites. The process was modified a number of times to reduce the degree of particulate aggregation, with consistent and good results finally achieved and reported in D4.55.
Task T4.3 was carried out by UNEW and GTS, and as a result of extensive research into the areas of glass chemistry and glass formulation provided a scientific basis for new and innovative compositions that will yield new bioceramics. The glass compositions developed cover a range of compositions that can be considered to be soda lime silica, borosilicate and phosphate based compositions. Work to date can be summarised as:
• The glasses were then made and melted to produce the new trial materials.
• Biological evaluation has been conducted on the seven new bioactive glass compositions and this has included the cytotoxicity of the innovative glass powders.
• The evaluation of the ions released from each composition in normal physiological environments over different time periods.
• The pH variation in deionised water has been investigated for the full range of the melted glasses.
• Scaffold fabrication depends also on the sintering ability of the glass powders, so thermal analyses were used to evaluate the new bioceramics sintering conditions
• Bioceramic pellets were prepared and characterised by X-ray diffraction (XRD) and scanning electron microscopy (SEM)
• The cellular MTT assays have shown no detrimental effects on the cell viability, indicating that the new materials are not cytotoxic
From task T4.4 both ceramic and ceramic polymer-structures were created using low temperature moulding and 3D printing techniques which were then used to create baseline PLA, PLGA and AW scaffolds which have been supplied to the Karolinska Institute for evaluation in WP6, and which will be used to create prototype medical devices for WP8.
Task T4.5 identified fused filament fabrication and the Z-corp 3D printing process as the most relevant and appropriate processes to create the structures which were required for device manufacture in WP8.
In WP5 Task T5.1 a novel resorbable copolymer of polylactic acid and polyglycolic acid was developed with vinyl functionalisation of end groups. Early experiments concentrated on increasing the molecular weight of polymer to maximise the polymer strength. Composites were then created with the addition of hydroxyapatite powder as filler. The concentration of filler to resorbable polymer optimised in terms of flexural strength of the resultant composite. Composite were shown to maintain their flexural strength over 6 weeks storage in an aqueous environment, exhibiting better performance than commercially available resorbable polymers.
Within task T5.2 two approached were taken. The first involved using methacrylate-based monomers, while the second involved developing an adhesive based around DOPA. The methacrylate-based adhesives exhibited the highest bond strengths and so were taken forward for further study, involving aging in an aqueous environment. Bond strength testing revealed that the bond strength between the resorbable composite and porcine mandibular specimens reduced significantly after 7 days storage in an aqueous environment. Careful analysis revealed that this was due to the hydrophilic monomers in the adhesive formulation adsorbing water during storage and swelling.
During task T5.3 micro-CT images were obtained from the Radiology Department of Newcastle Dental Hospital and software routines developed so that these images could be simply transformed into a suitable format for use in 3D printing devices. Prototype devices were produced from polylactic acid and feedback from oral surgeons allowed the geometry of the final devices to be optimised.
In WP 6 the viability of the cells seeded on the biomaterials was assessed by MTT assay (Roche Diagnostics, Germany) or LDH assay (Cytotoxicity Assay, Promega Co., USA) according to the provided protocols.
The NCL2, NCL7, PLA and AW/PLA discs were cyto-compatible after 24h and 48h culture. However, their cell viability values were lower than AW alone.
The “Spine-Ghost III”biocement and the polymeric paste were tested using the indirect-contact culture. The biocement and the polymeric paste were well tolerated by the BMSCs after 24h culture (Figure 1). It can be concluded that both paste and biocement are cyto-compatible in indirect-contact culture.
PLGA discs were evaluated without preconditioning. The fresh culture media containing rat BMSCs were added onto the discs (10000 cells/disc/well; 48-well plate). The cells were cultured for 24h or 72h in a humidified atmosphere of 5% CO2 at 37C. All PLGA discs were cyto-compatible after 24h and 72h culture. The HT-PLGA cell viability values were lowest.
The MSGP was cytocompatible only in indirect cell culture. The cells phagocytized the particles in direct culture (Figure 2). With the increased MSGP concentration the cell viability decreased (Table 5).
The AW microparticles were evaluated starting with preconditioning in NM for 24h. The AW viability data were compared with the beta-TCP microparticles (chronOS, SYNTHES) viability data or culture plastic. The AW and beta-TCP microparticles were cyto-compatible after 24h and 48h culture.
The in vitro osteogenic properties were assessed by measuring alkaline phosphates (ALP) activity of the rat BMSCs.
The ALP activity was lower for the BMSCs indirect-contact cultured in the presence of the polymeric paste or “SG-III” biocement compared to the culture plastic.
The MSGP increased the ALP activity after 14 days indirect-contact culture.
The ALP activity of the BMSCs seeded directly onto the NCL2 and NCL7 discs, or plastic pre-coated with MSGP was undetectable.
The ALP activity of the BMSCs seeded directly onto the PLA or AW/PLA was much lower than for the AW discs. It can be concluded that the AW disc alone has been the best candidate to support the BMSCs osteogenic differentiation in vitro.
The lowest ALP activity of the BMSCs was in the presence of the PLGA high Mw both after 7 days and 14 days in culture. The highest ALP activity was for PLGA low Mw. However the ALP activity of the BMSCs seeded on the plastic was always the highest.
The ALP activity of the BMSCs seeded directly onto the AW microparticles was better than for the beta-TCP microparticles, however lower than for the culture plastic, except for the cells seeded into 48-well plates after 14 days in culture.
The ALP activity was significantly enhanced by culturing the cells with the MSGP conditioned media, prepared by overnight incubation at +4 C, compared to osteogenic media.
The in vitro chondrogenic properties of the new (open-wall structure) PLA and AW/PLA discs were assessed in vitro using the BMSCs micromass culture or pellet-type culture methods. The AW and AW/PLA discs have not supported the chondrogenic differentiation of the rat BMSCs in micromass-type culture, however some fibrocartilage-like structures were formed in pellet-type culture (Figure 5). Based on the previous reports, it appears that PLA discs have better chondrogenic properties than AW or AW/PLA discs.
The cell-compatibility and osteogenic properties of the several ORLA protein coatings of the AW- and PLA-discs were assessed in vitro. All protein-coated samples (PLA- and AW-ORLA) were cyto-compatible after 24h culture in normal media. The ORLA165+178 (Col 4 + OPN) had highest MTT value for PLA, and ORLA178 (OPN) for AW.
The ORLA protein coatings of the AW discs induced a minor increase in the ALP activity compared to the uncoated AW. All ORLA-PLA coated samples induced some increase in the ALP activity, mostly higher after 14 days culture.
For in vivo assessment a total number of 116 rats were operated.
The histological assessment of the calvarial defects treated with the PLA discs proved that the large part of the PLA remained at the implantation site. However the PLA scaffolds were well integrated into the host tissues already at 2 weeks after surgery. The new designed scaffolds were well tolerated by the host tissues. No new bone formation was observed in the defects treated with the PLA.
The new designed AW/PLA presented quite impressive new bone formation observed at 12 weeks after surgery on the AW side of the discs. The new bone was penetrating the AW and mostly from the PLA upper-site of the scaffolds. The fibrous connective tissue ingrowth was present at the interface between the AW and PLA parts of the discs already after 2 weeks. The new design AW/PLA discs were biocompatible and osteoconductive; however the AW alone could to be more effective. Moreover the new AW seems to be better than the older AW where the new bone was found only on the surface.
The AW microparticles and the beta-TCP microparticles were biocompatible. The beta-TCP microparticles appeared to biodegrade faster than the AW microparticles. However more new bone seems to form around implanted AW microparticles at 12 weeks after surgery. More in vivo data are necessary to confirm those findings.
In WP7 the final composition of the vertebroplasty materials was defined – details of this work are commercially sensitive.
In WP8 over 1,000 scaffolds have been produced for mechanical and in vitro testing, and in vivo testing. In Task 8.1 UNEW, LEITAT, GTS, IQS, and JRI collaborated to develop three device designs: a biocramic plug based on apatite-wollastonite (AW); a biocomposite plug based on AW together with a PLA or PLGA cartilage facing surface; and an injectable nanoHA-PLGA system.
In task T8.2 LEITAT led work which focussed on modelling of the implants, and developed a model of bone ingrowth, and stiffness development which is reported in D7.71 and D8.72. These models indicated that, for the geometries of defect which the project was developing implants for, that bone growth and development would take place over a period of approximately two months, which was well beyond the dissolution rate for the scaffolds and so there was little risk of the implant dissolving before new bone had the opportunity to develop and mature.
In task 8.3 devices were evaluated. The potential for the AW and AW-PLA plugs was evaluated through mechanical and in vitro testing, and through small animal in vivo models in WP6, and was evaluated through evaluating porous AW alone, porous PLA alone, and AW-PLA composites. Mechanical property testing indicated that the porous AW structure had mechancial properties at the lower end of those expected for cortical bone, and that the porous PLA structure had mechanical properties at the level associated with cancellous bone, and that these were maintained over extended periods in phosphate buffered saline or simulated body fluid. Bioactivity testing indicated that the porous AW material alone was the most osteoinductive, followed by the PLA structure alone, followed by the composite, and so on the basis of those results two final device designs were developed based on AW alone and PLA alone, as shown in Figure 1 below. Batches of 10 of these plugs were manufactured by UNEW and supplied to UE for evaluation in the large animal model in WP11.
In WP9, for task 9.1 UNEW and JRI developed detailed specifications for the fracture fixation device based around the methacrylate-terminated PLGA and adhesive designed in WP5. In task T9.2 LEITAT led work which focussed on modelling the mechanical properties of intact, fractured and repairing mandibles. They then considered the mechanical environment when a fracture plate was attached to the modelled mandible. In task T9.3 the manufacturing process designed to allow the production of fracture plates, tailored to the patient anatomy was developed. Using the procedures developed in WP5 routines for converting clinical images into CAD CAM files enabled the production of proto-type devices. Consultation with maxillofacial surgeons allowed for the establishment of the critical dimensions of the devices to be produced. Full details were presented in the deliverable report D9.73.
In task T9.4 methods of applying the adhesive to bone were developed, using ex vivo porcine mandibles are test substrates. Using procedures similar to those used to apply similar adhesives in dentistry a simple brush application technique was developed. In task T9.5 specimens of the resorbable polymer were attached to ex vivo porcine mandible specimens using the developed adhesive to establish the bond strength both at time of placement and after storage in an aqueous environment, to simulate the in vivo situation. The first adhesive considered, while exhibiting high initial bond strengths, did not show sufficient long term high bond strength. Careful analysis of the constituents of the first adhesive allowed the development of a second adhesive, which proved to be more stable over the whole 6 week storage period.
The tasks of WP10 consist in performing an environmental and economic assessment of the medical devices developed in RESTORATION project and proposing alternatives or recommendations to reduce the life cycle cost and environmental impact related to production and distribution stages. The work has been lead by LEITAT, with the collaboration of the rest of the partners of the RESTORATION Consortium.
A methodology was defined for the environmental and economic assessment for the four types of devices: Vertebroplasty (VP)/Kyphoplasty (KP), Osteochondral (AO) and Maxillofacial applications. The goal and the scope of the studies were defined according to the goals of the project and the guidelines of the sector.
The inventory of the data has been done. Datasheets with the detail of data needed from the studied processes (inputs, outputs and economic information) were elaborated and sent to each partner responsible for the different processes. This deliverable includes the inventory data for materials, manufacturing processes, pre-use and use. For some stages (pre-clinical, clinical assays, surgical and post-surgical stages) generic data from the healthcare sector have been collected. An approach of the regulatory framework has been done and the key parameters to be assessed have been defined and requested to the partners involved. Despite this, some data could not be gathered. In this sense, it was sought reference data to model all these stages, with the collaboration of the partners of the Consortium. It is worth mentioning that further analysis about economic data could be done in the future to obtain a more realistic Life Cycle Cost Assessment.
The environmental and economic assessment, with the inventory data gathered has been performed. The analyses have been done according to the Life Cycle Methodology which is based on the ISO 14040, ISO 14044 and the ILCD Handbook. The environmental and economic Life Cycle Analysis (LCA) have been developed in accordance with the four stages stables by the ISO reference: goal and scope, life cycle inventory, life cycle assessment and interpretation.
The interpretation of the results indicates that in reference to VP and KP surgeries the device with minor Carbon Footprint is the Bioactive composite containing Rich Platelet Fibrin (0.331 kg CO2 eq.). The Mesoporous Bioactive Glass Cement (MBGs), the other device developed for the VP/KP interventions, has the highest Carbon footprint (2,438 kg CO2 eq.). For the AO applications the best suited device is the AW device (0.309 kg CO2 eq.), that it has also, the lowest Carbon Footprint. It is followed by NCL7 (0.312 kg CO2 eq.) and NCL2 (0.322 kg CO2 eq.). Finally, for the Maxillofacial applications only one device has been developed and its Carbon Footprint is 0.936 kg CO2 eq.
Main environmental impact contributions are focused on the material synthesis or manufacturing of the device stages, due to high energy consumption related to this phases. In this sense, for the recommendations for industrial scale up, the energy systems and efficiencies could be optimised (furnace, heating and cooling systems, mixing and aging processes, etc.). In terms of economic costs, savings from lower complications and shorter recovery time and therapy (foreseen for innovative RESTORATION devices procedures) can significantly reduce the total cost of the devices.
Using sheep as a large animal model, WP11 assessed the bone response to the developed materials for distinct applications: injectable bioceramic for vertebroplasty, and devices for osteochondral repair in load-bearing conditions.
Potential Impact:
The project has the potential to make a significant impact to future healthcare. The RESTORATION project has focussed on the development of new materials and medical devices for the treatment of osteoarthritis, maxillofacial fractures and vertebral fractures. New material processing routes, new materials and new material structures have been created, and assessed for their biocompatibility and mechanical properties.
The partners will now seek exploit the project results and bring these new technologies to the market in the coming years. Specific outcomes in terms of new products and their progress are:
• New bioceramic plugs as early stage treatments for osteoarthritis has been developed to the stage where they have been tested in a large animal model, and their development will be taken forward by Glass Technology Services, JRI Orthopaedics and Newcastle University.
• A new bioactive cement for use in vertebroplasty procedures has also been evaluated in a large animal model and will be further developed by COREP.
• A new particulate bone filler has been evaluated in a small animal model and will be taken forward by Glass Technology Services and JRI Orthopaedics.
• A new biocomposite paste containing platelet rich plama for use in vertebroplasty procedures has been evaluated in a small animal model and will be further developed by Sagetis-Biotech and the Institut Quimic de Sarria (IQS).
• A new maxillfacial plate design and material have been developed and will be taken forward by Newcastle University.
In terms of dissemination, the project has:
- Contributed to two special sessions at European Biomaterials conferences (ESB2013; eMRS2014).
- Led to over 50 conference papers and presentations.
- Will lead to over 10 journal papers, which are in preparation.
- Produced communication materials which were distributed at conferences and events.
- Produced a video describing the project objectives.
List of Websites:
www.restoration-project.eu
Ceramic composite materials have for many years been considered to show great promise in the repair of musculoskeletal defects. The materials can mimic the structure of bone, and devices made from the materials can be structured to closely match the mechanical requirements of implant sites. In addition, wide ranges of bioactivity are possible, from inert to fully resorbable.
Bioceramics have most commonly been used to date in dentistry, and in some orthopaedic applications, e.g. as an injectable paste for vertebroplasty, or as a coating material for metal orthopaedic implants. However, advances in cellular medicine bring great opportunity for significant growth in the bioceramics industry – bioceramics and bioceramic composites offer levels of bioactivity which far exceed those available from metal implants, together with combinations of strength and modulus which exceed anything which can be offered by bioactive polymers on their own. Working in tandem with cells, proteins and other biologically active agents (both from the host and introduced) bioceramic composites have the potential to revolutionise many treatments and therapies, giving new, highly effective early stage clinical interventions for conditions where no approach has existed to date.
In order to deliver on the potential shown by bioceramic composites the combination of mechanical design, materials, processing, clinical delivery and subsequent biological interaction all have to be understood in an integrated and systematic way. The RESTORATION research project is addressing these underlying research and technological challenges in order to develop new bioceramic products for five SME partner companies.
The RESTORATION project has focussed on the development of new materials and medical devices for the treatment of osteoarthritis, maxillofacial fractures and vertebral fractures. New material processing routes, new materials and new material structures have been created, and assessed for their biocompatibility and mechanical properties.
The partners will now seek exploit the project results and bring these new technologies to the market in the coming years. Specific outcomes in terms of new products and their progress are:
• New bioceramic plugs as early stage treatments for osteoarthritis has been developed to the stage where they have been tested in a large animal model, and their development will be taken forward by Glass Technology Services, JRI Orthopaedics and Newcastle University.
• A new bioactive cement for use in vertebroplasty procedures has also been evaluated in a large animal model and will be further developed by COREP.
• A new particulate bone filler has been evaluated in a small animal model and will be taken forward by Glass Technology Services and JRI Orthopaedics.
• A new biocomposite paste containing platelet rich plama for use in vertebroplasty procedures has been evaluated in a small animal model and will be further developed by Sagetis-Biotech and the Institut Quimic de Sarria (IQS).
• A new maxillfacial plate design and material have been developed and will be taken forward by Newcastle University.
In addition, all the materials and technologies developed within the project have been assessed in terms of environmental impact to idtentify issues to take into account for industrial scale-up.
The project has delivered an integrated pre-clinical assessment of new bioceramic composites in order to underpin the development of a range of new musculoskeletal medical devices.
Project Context and Objectives:
Ceramic composite materials have for many years been considered to show great promise in the repair of musculoskeletal defects. The materials can mimic the structure of bone, and devices made from the materials can be structured to closely match the mechanical requirements of implant sites. In addition, wide ranges of bioactivity are possible, from inert to fully resorbable.
Bioceramics have most commonly been used to date in dentistry, and in some orthopaedic applications, e.g. as an injectable paste for vertebroplasty, or as a coating material for metal orthopaedic implants. However, advances in cellular medicine bring great opportunity for significant growth in the bioceramics industry – bioceramics and bioceramic composites offer levels of bioactivity which far exceed those available from metal implants, together with combinations of strength and modulus which exceed anything which can be offered by bioactive polymers on their own. Working in tandem with cells, proteins and other biologically active agents (both from the host and introduced) bioceramic composites have the potential to revolutionise many treatments and therapies, giving new, highly effective early stage clinical interventions for conditions where no approach has existed to date.
In order to deliver on the potential shown by bioceramic composites the combination of materials science, mechanical response, processing, clinical delivery and subsequent biological interaction all have to be understood in an integrated and systematic way. The RESTORATION consortium have collaboratively address this underlying research and technological challenge in order to develop new bioceramic products for five research led SME partner companies.
The project involved:
- The selection of a limited number of advanced bioceramic materials (apatite containing bioceramics, calcium phosphate and calcium sulphate based materials, mesoporous bioactive glasses and borosilicate glasses) that have potential to create high added value biomedical products for the SME partners.
- The development of advanced high added value medical devices that offer improved and controlled biological interaction, and become part of the biological system.
- Research to modify and adapt the advanced bioceramic materials for new production processes, suitable for future in-hospital or in-clinic manufacture of personalised devices.
- Full assessment of the new bioceramic materials and devices, on the grounds of potential clinical effectiveness, economics, and environmental impact.
The clinical targets with related objectives were:
- Osteoarthritis (OA). OA is a degenerative joint disease, typified by a loss of quality of cartilage and bone at the interface of a joint, resulting in pain, stiffness and reduced mobility. The process is not confined to the joint but can involve regional muscles and ligaments. Treatment can be considered non-surgical and surgical. Non-surgical treatment includes exercise, weight loss, physical therapy and medication (e.g. pain relief). For severe disease, when the non-surgical measures cannot control the symptoms, there is currently no other widely recognised treatment other than joint replacement (including newer designs such as joint resurfacing). OA is the most frequent form of arthritis in humans, and is significantly disabling. Survey data indicates that it affects over 11% of females and 7% of males by the time they reach their mid-fifties, rising to over 33% of females and 19% of males by the time they reach their mid-70’s. The current global market in joint replacements is estimated to be in excess of €7 billion, with in excess of €2 billion within the EU and the bulk of this is hip and knee procedures for OA. Within the project we have developed early stage interventions for OA, capable of being applied to local defects within the joint, through minimally invasive surgical techniques. OA puts significant pressure on healthcare systems and developing procedures which are minimally invasive and “day case” will reduce this pressure, significantly delay or completely remove the need for a total joint replacement, and significantly enhance quality of life.
- Vertebral Compression Fractures (VCF). Increased life expectancy is leading to a rise in age related pathologies which often involve the decay of bone quality (for example from osteoporosis) causing vertebral compression fractures. Approximately 35-50% of women and 20-30% of men develop vertebral compression fractures, and approximately 50% of those affected develop multiple fractures throughout their life . In addition, trauma, bone diseases, and cancers can lead to vertebral fractures and bone loss in younger patients. Vertebroplasty (VP) and kyphoplasty (KP) are two surgical minimally invasive procedures that are now considered the “gold standard” for the medical treatment of VCF. They both aim to augment and stabilize the weakened vertebral body and KP also aims to restore as much of the height and functional state as possible. VP consists of the percutaneous injection of bone cement directly into the fractured vertebral body. KP injects bone cement into a cavity created in the vertebral body by the inflation of a balloon. The global market in products for VCF is in excess of €400 million, and is growing significantly as a result of the ageing population. Within the project we have developed new injectable bioactive ceramic composites able to stimulate bone regeneration and to deliver specific drugs to the target site.
- Maxillofacial Bone Fracture Alignment. Maxillofacial bone fractures occur either as a result of trauma or as part of a surgical procedure designed to correct a developmental fault. Fractures of the mandible account for around 6% of trauma cases, with an increasing incidence of cases seen in the elderly over the last 30 years, and the global market for maxillofacial bone fracture alignment devices is estimated at €100 million. The limited anatomical space within the maxillofacial region means that fractures of the bones are usually aligned using internal fixation plates, with correct alignment of the bones to allow correct eating, speaking and the quality of life of the patient post-operatively. However, there are a significant numbers of revisions required each year due to post-operative problems associated with current plate designs. The development of new materials that will reduce the need for these revisions will greatly reduce the economic burden on the health systems and improve the quality of life of the patient. Within the project we have started the development of new bioceramic polymer composites, which can be manufactured to conform to the patient’s anatomy, possess sufficient stiffness and strength to protect and support the broken bone, and which will resorb non-toxically within 24 months of placement.
Through developing new treatments for these three conditions the project:
- Has developed commercial opportunities for the SME partners.
- Has developed internationally leading science on the development of bioactive ceramic composites, their processing and their behaviour as part of a biological system.
- Will make significant contributions to improving the quality of life for sufferers, initially in the EU and later throughout the world.
Project Results:
The project has been carried out in four stages:
- Selection of a limited number of advanced bioceramic materials that have potential to create high added value biomedical products for the SME partners
- The development of advanced high added value medical devices that offer improved and controlled biological interaction, and become part of the biological system
- Research to modify and adapt the advanced bioceramic materials for new production processes, suitable for future in-hospital or in-clinic manufacture of personalised devices
- Full assessment of the new bioceramic materials and devices, on the grounds of potential clinical effectiveness, economics, and environmental impact
In overview the main achievements were:
- The first RTD workpackage (WP) was WP2, Clinical Processes and Specification. The main aim of this short WP was to explicitly capture all the requirements for the products that the project will deliver. This WP is complete.
- WPs 3, 4 and 5 were materials development WPs for the vertebroplasty, osteoarthritis and maxillofacial fracture fixation application areas respectively. These WP’s developed and evaluated the new materials which are the foundation of the project.
- WP6 was a major integrating WP, and also a significant product development stagegate. Within this WP the materials developed in WPs 3, 4, and 5 are being evaluated in terms of their bioactivity, using human and animal primary cells and both a non-load bearing and load bearing small animal model. This WP has been extended (without resource or timing implications) to further support WPs 7, 8 and 9.
- WPs 7, 8 and 9 were product development WPs, again for the vertebroplasty, osteoarthritis and maxillofacial fracture fixation application areas respectively. These WPs took input from WPs 2-6 in order to specify, design and evaluate mechanically and in vitro full scale products, and to develop variants of those products to be evaluated in WP11.
- WP10 is took input from WPs 7-9 to develop life cycle cost models, to understand the environmental impacts of the products, and to examine ways in which both the life cycle costs and environmental impact can be minimised to ensure the best long term value for healthcare systems.
- WP11 was the final RTD WP, and is using a large animal model in order to evaluate the performance of the products in vivo. WP11 assessed the products over implantation times of six months in order to provide high quality information on the behaviour in vivo, so that at the end of the project the products are ready to progress to clinical trial.
Further details for each WP (where commercial confidentiality allows) are described below.
In WP2 device specifications were created and distributed, and these documents were used throughout the project to steer the research activity.
In WP3 Bionica Tech managed to produce mesoporous bioactive glass particles (MBGs), both through an optimized sol-gel synthesis and through a rapid-kinetic process, i.e. the spray-drying technique. Both synthesis methods allowed to obtain glasses characterised by a satisfactory mesoporosity and a remarkable bioactivity. The composition of the sol-gel glass was also modified adding zirconia to the glass network, in order to gain radiopacity, while maintaining a mesoporous structure and a good bioactivity. The transition from the traditional sol-gel synthesis to the spray-drying technique allows to obtain spherical particles through a remarkable less time-consuming process and then represents an appealing option for an industrial transfer of the mesoporous glass production. Moreover, a new synthesis based on the use of water as solvent was tested with the spray-drying technique and as a preliminary but solid result, mesoporous spherical silica has been obtained.
Traditional MBGs or the sprayed particles (SC-SD) were used as dispersed phases in a calcium sulphate matrix in order to obtain a resorbable osteoinductive cement. The first matrix chosen was a calcium sulphate hemihydrate bought from Sigma-Aldrich; this cement, named Spine-Ghost (with the addition of a third glass-ceramic reinforcing phase, SCNZgc) resulted to be sufficiently radiopaque, well-injectable and released a highly amount of bioactive ions (in particular silicon and calcium). However, Spine-Ghost showed inappropriate mechanical properties and an inadequate setting time.
Due to these reasons, Bionica Tech sought a new matrix, in order to obtain optimised properties for its composite cement. After a series of evaluation, the selected solution consisted in using a different typology of calcium sulphate hemihydrate: in fact, while the Sigma-Aldrich hemihydrate is composed of β-calcium sulphate (as confirmed by the DSC analysis), Bionica Tech sought an α-calcium sulphate, like the one widely used in dentistry, which gives a stronger dihydrate after a faster precipitation mechanism. This difference is mainly based by a larger crystal size. Bionica Tech chose an alpha type-III dental gypsum and carried out using this matrix the optimisation of the different composite parameter as well as a complete characterization of the new cement. At first, a new liquid to powder ratio has been tuned, in order to obtain a paste that was sufficiently injectable through a vertebroplasty needle (15 gauge). The new cement (Spine-Ghost III) showed a shorter setting-time and thus traditional MBG particles have been substituted by spray-dryer ones in order to facilitate the technological transfer at the industrial scale. Spine-Ghost III-SD cement was then prepared: it is easily injectable, bioactive, has good mechanical compressive strength and shows more satisfactory working and setting times.
The main tasks developed by the combined Sagetis and IQS team in WP3 can be summarized as follows:
• Developing different combinations of polymeric biodegradable paste
Both the design of the polymer and the combination of polymer/bioceramic have been optimized for the polymeric paste.
The polymer synthesis was also optimized in order to allow the future GMP synthesis of the polymer. The original microwave-assisted polymerization was changed to a traditional wet chemistry synthesis. Three formulations were sent for in vitro testing to Karolinska and one of them was tested ex vivo at Evora.
A protocol of the injectability of the paste was established, heating the paste at 50ºC before injection. These injectability conditions were also tested ex vivo at Evora with sheep vertebrae. A syringe-heating device has been purchased to be used in future in vivo experiments.
Finally a protocol to increase the radiopacity of the paste has been developed using ZrO2 as radiopaque material. Also a protocol of sterilisation using gamma radiation is under development.
• Developing of new hydroxiapatite ceramic materials with controlled crystallinity
A new bioceramic material have been developed, based in a sol-gel process that incorpores SiO2 to the hydroxiapatite structure. The presence of SiO2 keeps the crystallinity level of HA low, expecting a better bioactivity of the bioceramic developed.
• Establishing the protocol to isolate Rich Platelet Fibrin (RPF) from our own blood samples.
The protocol is established. The yield of isolation was increased using a rew designed improved centrifuge tubes. Different blood samples were used (human, pig and rat). A key issue was identified regarding the growth factors concentration in the different animal’s blood.
The inactivation of the growth factors due to the sample preparation was tested. It has been demonstrated that the growth factors remain active even after the paste heating at 50ºC for injection.
Optimizing the polymer synthesis and the formulation with the PRP
Paste with different PRP has been prepared and send to Karolinska for evaluation. The results confirmed that the Paste is not toxic and the presence of PRP promotes fibroblasts viability.
In WP4, task T4.1 an in-situ precipitation of nHAp method has successfully developed. The precipitation of nanoHAp crystallites is based on the reaction of PO43- and Ca2+ from phosphate and calcium solutions. Precipitation parameters, including concentration, temperature, pH value and titration rate, have been studied. Through control of these parameters the potential size, morphology, chemistry and lattice parameters of the HA crystallites have been defined.
Within task T4.2 there were issues with particle agglomeration upon drying and mixing to form composites. The process was modified a number of times to reduce the degree of particulate aggregation, with consistent and good results finally achieved and reported in D4.55.
Task T4.3 was carried out by UNEW and GTS, and as a result of extensive research into the areas of glass chemistry and glass formulation provided a scientific basis for new and innovative compositions that will yield new bioceramics. The glass compositions developed cover a range of compositions that can be considered to be soda lime silica, borosilicate and phosphate based compositions. Work to date can be summarised as:
• The glasses were then made and melted to produce the new trial materials.
• Biological evaluation has been conducted on the seven new bioactive glass compositions and this has included the cytotoxicity of the innovative glass powders.
• The evaluation of the ions released from each composition in normal physiological environments over different time periods.
• The pH variation in deionised water has been investigated for the full range of the melted glasses.
• Scaffold fabrication depends also on the sintering ability of the glass powders, so thermal analyses were used to evaluate the new bioceramics sintering conditions
• Bioceramic pellets were prepared and characterised by X-ray diffraction (XRD) and scanning electron microscopy (SEM)
• The cellular MTT assays have shown no detrimental effects on the cell viability, indicating that the new materials are not cytotoxic
From task T4.4 both ceramic and ceramic polymer-structures were created using low temperature moulding and 3D printing techniques which were then used to create baseline PLA, PLGA and AW scaffolds which have been supplied to the Karolinska Institute for evaluation in WP6, and which will be used to create prototype medical devices for WP8.
Task T4.5 identified fused filament fabrication and the Z-corp 3D printing process as the most relevant and appropriate processes to create the structures which were required for device manufacture in WP8.
In WP5 Task T5.1 a novel resorbable copolymer of polylactic acid and polyglycolic acid was developed with vinyl functionalisation of end groups. Early experiments concentrated on increasing the molecular weight of polymer to maximise the polymer strength. Composites were then created with the addition of hydroxyapatite powder as filler. The concentration of filler to resorbable polymer optimised in terms of flexural strength of the resultant composite. Composite were shown to maintain their flexural strength over 6 weeks storage in an aqueous environment, exhibiting better performance than commercially available resorbable polymers.
Within task T5.2 two approached were taken. The first involved using methacrylate-based monomers, while the second involved developing an adhesive based around DOPA. The methacrylate-based adhesives exhibited the highest bond strengths and so were taken forward for further study, involving aging in an aqueous environment. Bond strength testing revealed that the bond strength between the resorbable composite and porcine mandibular specimens reduced significantly after 7 days storage in an aqueous environment. Careful analysis revealed that this was due to the hydrophilic monomers in the adhesive formulation adsorbing water during storage and swelling.
During task T5.3 micro-CT images were obtained from the Radiology Department of Newcastle Dental Hospital and software routines developed so that these images could be simply transformed into a suitable format for use in 3D printing devices. Prototype devices were produced from polylactic acid and feedback from oral surgeons allowed the geometry of the final devices to be optimised.
In WP 6 the viability of the cells seeded on the biomaterials was assessed by MTT assay (Roche Diagnostics, Germany) or LDH assay (Cytotoxicity Assay, Promega Co., USA) according to the provided protocols.
The NCL2, NCL7, PLA and AW/PLA discs were cyto-compatible after 24h and 48h culture. However, their cell viability values were lower than AW alone.
The “Spine-Ghost III”biocement and the polymeric paste were tested using the indirect-contact culture. The biocement and the polymeric paste were well tolerated by the BMSCs after 24h culture (Figure 1). It can be concluded that both paste and biocement are cyto-compatible in indirect-contact culture.
PLGA discs were evaluated without preconditioning. The fresh culture media containing rat BMSCs were added onto the discs (10000 cells/disc/well; 48-well plate). The cells were cultured for 24h or 72h in a humidified atmosphere of 5% CO2 at 37C. All PLGA discs were cyto-compatible after 24h and 72h culture. The HT-PLGA cell viability values were lowest.
The MSGP was cytocompatible only in indirect cell culture. The cells phagocytized the particles in direct culture (Figure 2). With the increased MSGP concentration the cell viability decreased (Table 5).
The AW microparticles were evaluated starting with preconditioning in NM for 24h. The AW viability data were compared with the beta-TCP microparticles (chronOS, SYNTHES) viability data or culture plastic. The AW and beta-TCP microparticles were cyto-compatible after 24h and 48h culture.
The in vitro osteogenic properties were assessed by measuring alkaline phosphates (ALP) activity of the rat BMSCs.
The ALP activity was lower for the BMSCs indirect-contact cultured in the presence of the polymeric paste or “SG-III” biocement compared to the culture plastic.
The MSGP increased the ALP activity after 14 days indirect-contact culture.
The ALP activity of the BMSCs seeded directly onto the NCL2 and NCL7 discs, or plastic pre-coated with MSGP was undetectable.
The ALP activity of the BMSCs seeded directly onto the PLA or AW/PLA was much lower than for the AW discs. It can be concluded that the AW disc alone has been the best candidate to support the BMSCs osteogenic differentiation in vitro.
The lowest ALP activity of the BMSCs was in the presence of the PLGA high Mw both after 7 days and 14 days in culture. The highest ALP activity was for PLGA low Mw. However the ALP activity of the BMSCs seeded on the plastic was always the highest.
The ALP activity of the BMSCs seeded directly onto the AW microparticles was better than for the beta-TCP microparticles, however lower than for the culture plastic, except for the cells seeded into 48-well plates after 14 days in culture.
The ALP activity was significantly enhanced by culturing the cells with the MSGP conditioned media, prepared by overnight incubation at +4 C, compared to osteogenic media.
The in vitro chondrogenic properties of the new (open-wall structure) PLA and AW/PLA discs were assessed in vitro using the BMSCs micromass culture or pellet-type culture methods. The AW and AW/PLA discs have not supported the chondrogenic differentiation of the rat BMSCs in micromass-type culture, however some fibrocartilage-like structures were formed in pellet-type culture (Figure 5). Based on the previous reports, it appears that PLA discs have better chondrogenic properties than AW or AW/PLA discs.
The cell-compatibility and osteogenic properties of the several ORLA protein coatings of the AW- and PLA-discs were assessed in vitro. All protein-coated samples (PLA- and AW-ORLA) were cyto-compatible after 24h culture in normal media. The ORLA165+178 (Col 4 + OPN) had highest MTT value for PLA, and ORLA178 (OPN) for AW.
The ORLA protein coatings of the AW discs induced a minor increase in the ALP activity compared to the uncoated AW. All ORLA-PLA coated samples induced some increase in the ALP activity, mostly higher after 14 days culture.
For in vivo assessment a total number of 116 rats were operated.
The histological assessment of the calvarial defects treated with the PLA discs proved that the large part of the PLA remained at the implantation site. However the PLA scaffolds were well integrated into the host tissues already at 2 weeks after surgery. The new designed scaffolds were well tolerated by the host tissues. No new bone formation was observed in the defects treated with the PLA.
The new designed AW/PLA presented quite impressive new bone formation observed at 12 weeks after surgery on the AW side of the discs. The new bone was penetrating the AW and mostly from the PLA upper-site of the scaffolds. The fibrous connective tissue ingrowth was present at the interface between the AW and PLA parts of the discs already after 2 weeks. The new design AW/PLA discs were biocompatible and osteoconductive; however the AW alone could to be more effective. Moreover the new AW seems to be better than the older AW where the new bone was found only on the surface.
The AW microparticles and the beta-TCP microparticles were biocompatible. The beta-TCP microparticles appeared to biodegrade faster than the AW microparticles. However more new bone seems to form around implanted AW microparticles at 12 weeks after surgery. More in vivo data are necessary to confirm those findings.
In WP7 the final composition of the vertebroplasty materials was defined – details of this work are commercially sensitive.
In WP8 over 1,000 scaffolds have been produced for mechanical and in vitro testing, and in vivo testing. In Task 8.1 UNEW, LEITAT, GTS, IQS, and JRI collaborated to develop three device designs: a biocramic plug based on apatite-wollastonite (AW); a biocomposite plug based on AW together with a PLA or PLGA cartilage facing surface; and an injectable nanoHA-PLGA system.
In task T8.2 LEITAT led work which focussed on modelling of the implants, and developed a model of bone ingrowth, and stiffness development which is reported in D7.71 and D8.72. These models indicated that, for the geometries of defect which the project was developing implants for, that bone growth and development would take place over a period of approximately two months, which was well beyond the dissolution rate for the scaffolds and so there was little risk of the implant dissolving before new bone had the opportunity to develop and mature.
In task 8.3 devices were evaluated. The potential for the AW and AW-PLA plugs was evaluated through mechanical and in vitro testing, and through small animal in vivo models in WP6, and was evaluated through evaluating porous AW alone, porous PLA alone, and AW-PLA composites. Mechanical property testing indicated that the porous AW structure had mechancial properties at the lower end of those expected for cortical bone, and that the porous PLA structure had mechanical properties at the level associated with cancellous bone, and that these were maintained over extended periods in phosphate buffered saline or simulated body fluid. Bioactivity testing indicated that the porous AW material alone was the most osteoinductive, followed by the PLA structure alone, followed by the composite, and so on the basis of those results two final device designs were developed based on AW alone and PLA alone, as shown in Figure 1 below. Batches of 10 of these plugs were manufactured by UNEW and supplied to UE for evaluation in the large animal model in WP11.
In WP9, for task 9.1 UNEW and JRI developed detailed specifications for the fracture fixation device based around the methacrylate-terminated PLGA and adhesive designed in WP5. In task T9.2 LEITAT led work which focussed on modelling the mechanical properties of intact, fractured and repairing mandibles. They then considered the mechanical environment when a fracture plate was attached to the modelled mandible. In task T9.3 the manufacturing process designed to allow the production of fracture plates, tailored to the patient anatomy was developed. Using the procedures developed in WP5 routines for converting clinical images into CAD CAM files enabled the production of proto-type devices. Consultation with maxillofacial surgeons allowed for the establishment of the critical dimensions of the devices to be produced. Full details were presented in the deliverable report D9.73.
In task T9.4 methods of applying the adhesive to bone were developed, using ex vivo porcine mandibles are test substrates. Using procedures similar to those used to apply similar adhesives in dentistry a simple brush application technique was developed. In task T9.5 specimens of the resorbable polymer were attached to ex vivo porcine mandible specimens using the developed adhesive to establish the bond strength both at time of placement and after storage in an aqueous environment, to simulate the in vivo situation. The first adhesive considered, while exhibiting high initial bond strengths, did not show sufficient long term high bond strength. Careful analysis of the constituents of the first adhesive allowed the development of a second adhesive, which proved to be more stable over the whole 6 week storage period.
The tasks of WP10 consist in performing an environmental and economic assessment of the medical devices developed in RESTORATION project and proposing alternatives or recommendations to reduce the life cycle cost and environmental impact related to production and distribution stages. The work has been lead by LEITAT, with the collaboration of the rest of the partners of the RESTORATION Consortium.
A methodology was defined for the environmental and economic assessment for the four types of devices: Vertebroplasty (VP)/Kyphoplasty (KP), Osteochondral (AO) and Maxillofacial applications. The goal and the scope of the studies were defined according to the goals of the project and the guidelines of the sector.
The inventory of the data has been done. Datasheets with the detail of data needed from the studied processes (inputs, outputs and economic information) were elaborated and sent to each partner responsible for the different processes. This deliverable includes the inventory data for materials, manufacturing processes, pre-use and use. For some stages (pre-clinical, clinical assays, surgical and post-surgical stages) generic data from the healthcare sector have been collected. An approach of the regulatory framework has been done and the key parameters to be assessed have been defined and requested to the partners involved. Despite this, some data could not be gathered. In this sense, it was sought reference data to model all these stages, with the collaboration of the partners of the Consortium. It is worth mentioning that further analysis about economic data could be done in the future to obtain a more realistic Life Cycle Cost Assessment.
The environmental and economic assessment, with the inventory data gathered has been performed. The analyses have been done according to the Life Cycle Methodology which is based on the ISO 14040, ISO 14044 and the ILCD Handbook. The environmental and economic Life Cycle Analysis (LCA) have been developed in accordance with the four stages stables by the ISO reference: goal and scope, life cycle inventory, life cycle assessment and interpretation.
The interpretation of the results indicates that in reference to VP and KP surgeries the device with minor Carbon Footprint is the Bioactive composite containing Rich Platelet Fibrin (0.331 kg CO2 eq.). The Mesoporous Bioactive Glass Cement (MBGs), the other device developed for the VP/KP interventions, has the highest Carbon footprint (2,438 kg CO2 eq.). For the AO applications the best suited device is the AW device (0.309 kg CO2 eq.), that it has also, the lowest Carbon Footprint. It is followed by NCL7 (0.312 kg CO2 eq.) and NCL2 (0.322 kg CO2 eq.). Finally, for the Maxillofacial applications only one device has been developed and its Carbon Footprint is 0.936 kg CO2 eq.
Main environmental impact contributions are focused on the material synthesis or manufacturing of the device stages, due to high energy consumption related to this phases. In this sense, for the recommendations for industrial scale up, the energy systems and efficiencies could be optimised (furnace, heating and cooling systems, mixing and aging processes, etc.). In terms of economic costs, savings from lower complications and shorter recovery time and therapy (foreseen for innovative RESTORATION devices procedures) can significantly reduce the total cost of the devices.
Using sheep as a large animal model, WP11 assessed the bone response to the developed materials for distinct applications: injectable bioceramic for vertebroplasty, and devices for osteochondral repair in load-bearing conditions.
Potential Impact:
The project has the potential to make a significant impact to future healthcare. The RESTORATION project has focussed on the development of new materials and medical devices for the treatment of osteoarthritis, maxillofacial fractures and vertebral fractures. New material processing routes, new materials and new material structures have been created, and assessed for their biocompatibility and mechanical properties.
The partners will now seek exploit the project results and bring these new technologies to the market in the coming years. Specific outcomes in terms of new products and their progress are:
• New bioceramic plugs as early stage treatments for osteoarthritis has been developed to the stage where they have been tested in a large animal model, and their development will be taken forward by Glass Technology Services, JRI Orthopaedics and Newcastle University.
• A new bioactive cement for use in vertebroplasty procedures has also been evaluated in a large animal model and will be further developed by COREP.
• A new particulate bone filler has been evaluated in a small animal model and will be taken forward by Glass Technology Services and JRI Orthopaedics.
• A new biocomposite paste containing platelet rich plama for use in vertebroplasty procedures has been evaluated in a small animal model and will be further developed by Sagetis-Biotech and the Institut Quimic de Sarria (IQS).
• A new maxillfacial plate design and material have been developed and will be taken forward by Newcastle University.
In terms of dissemination, the project has:
- Contributed to two special sessions at European Biomaterials conferences (ESB2013; eMRS2014).
- Led to over 50 conference papers and presentations.
- Will lead to over 10 journal papers, which are in preparation.
- Produced communication materials which were distributed at conferences and events.
- Produced a video describing the project objectives.
List of Websites:
www.restoration-project.eu