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Rapid Prototyping of Custom-Made Bone-Forming Tissue Engineering Constructs.

Final Report Summary - RAPIDOS (Rapid Prototyping of Custom-Made Bone-Forming Tissue Engineering Constructs.)

Executive Summary:
In this RAPIDOS European and Chinese consortium, RP technologies were applied to create custom-made biomaterial constructs by integrating 1) imaging and information technologies, 2) biomaterials and process engineering, and 3) biological and biomedical engineering for novel and truly translational bone repair solutions. The main objective of this project was to apply precise and rapid prototyping technologies for custom-made bone implants with optimised macro-architecture, osteoinduction via the inclusion of calcium phosphate and a Chinese medicine phytomolecule (icaritin), and bactericidal properties.
First, the partners have developed a clinical CT imaging process technology workflow for development of anatomically relevant and precise custom-made macro-structured designed scaffolds. The goal of this patented workflow is to allow the surgeons to design and self-assess patient specific implants taking into account the constraints of the biomaterial and fabrication process.
Secondly, biodegradable composite materials were developed and their printability by low-temperature rapid prototyping and stereolithography established. Functionalisation with bactericidal macromolecules and loading with icaritin were performed. The optimisation of composite formulations; poly(trimethylcarbonate) (PTMC)/calcium phosphate and poly(lactic-co-glycolic)/tricalcium phosphate/magnesium (PLGA/TCP/Mg) respectively, for stereolithography and low temperature rapid manufacturing has been performed and already implant scaffolds were fabricated by both stereolithography and low temperature rapid prototyping. Biodegradable polymeric nanofibers and microspheres loaded with icaritin, a Chinese medicine phytomolecule as potential drug delivery vehicle have been prepared and incorporated into the photo-polymerisable resin formulation for stereolithography and assessed in vitro. In vitro studies have also shown the osteopromotive effect of the hydroxyapatite nanoparticles loaded into PTMC scaffolds. In vivo studies in rabbit calvarial defects showed the enhance bone ingrowth in Calcium phosphate nanoparticles loaded PTMC Stereolithography scaffolds in comparison to PTMC scaffolds. The osteopromotive effect of icaritin in PLGA/TCP/Mg and hydroxyapatite/PTMC scaffolds was assessed too in vivo and in vitro. Quaternised chitosan and magnesium were shown to decrease biofilm formation onto the surface of PLGA/TCP/Mg scaffolds in vitro and in vivo without being detrimental to mesenchymal stem cells osteogenic differentiation.
Finally, a preclinical proof of concept showed the robustness of the process and the patient specific osteoinductive orbital floor implant produced by stereolithography.
The European and Chinese RAPIDOS project activities have already led to 22 peer-review manuscripts and the filling of 2 patents. Four workshops were organised and attended by European and Chinese partners with several exchange missions between partners. Finally, the RAPIDOS results have contributed to the creation of one Spin-off company for production of new biomaterials and facilitated the merging of European industry partner.

Project Context and Objectives:
Road traffic injuries kill nearly 1.3 million people annually while 50 million sustain non-fatal injuries. If current trends continue, road crashes are predicted to rise from 9th leading cause of death to 5th by 2030. Worldwide, the number of bone grafts used in surgical procedures has been estimated at over 24 million in 2010. Indeed, bone is the most often transplanted tissue after blood and the need for bone graft substitute materials is enormous. About one third represents the European market, while the total Asian markets (China and India) for bone graft substitutes and other biomaterials increased by 53.2% from 2009 to 2010. The bone graft sales are forecasted to reach a total of $3.3 billion worldwide in 2017. Therefore, the global increase of needs for bone graft substitutes and emergence of large healthcare providers in Asia support the necessities for better bone repair solutions based on biomaterial scaffolds.
Among pertinent non-healing bone fractures occurring in road traffic accidents, the region of the head is a major target for development of precise custom-made bone constructs. In cranio-maxillofacial surgery, large blow-out orbital floor fractures have still mitigated outcomes and improved scaffold solutions are needed. The reconstruction of large bone defects in proximal femur or proximal tibia is also an enormous challenge for biomaterial devices due to the requirements for both complex shape and partial load bearing ability, but also due to the risk of incidence of steroid-associated osteonecrosis or infection which may exceed 30% for large open fractures. The technical issues for the engineering of scaffold for bone tissue engineering therapies are: (i) fabrication of biomaterial scaffolds for anatomical fit of complex three-dimensional large bone defect, (ii) fabrication of biomaterials with adequate mechanical and structural stability/degradation kinetics and (iii) fabrication of biomaterials with optimised macro-architecture for improved mass transport and perfusion for delivery of biological effectors.
Advanced solid free form fabrication also called rapid prototyping (RP) could provide the necessary control to create such innovative medical devices. For example, stereolithography offers the high resolution that is necessary to create controlled architecture and anatomically fitting device while low temperature rapid prototyping is a unique technique in its ability to incorporate into the scaffolds temperature sensitive active compounds.
Thus, the goal of this European and Chinese consortium is to apply RP technologies to create custom-made tissue engineered biomaterial constructs by integrating 1) imaging and information technologies, 2) biomaterials and process engineering, and 3) biological and biomedical engineering for novel and truly translational bone repair solutions. The main objective of this project is to apply precise and rapid prototyping technologies for custom-made bone tissue engineering with optimised macro-architecture, osteoinduction via the inclusion of calcium phosphate and a Chinese medicine phytomolecule (icaritin), and bactericidal properties.

For this 4 years period, the general goal was to apply Rapid Prototyping technologies to create custom-made tissue engineered biomaterial constructs by integrating 1) imaging and information technologies, 2) biomaterials and process engineering, and 3) biological and biomedical engineering for novel and truly translational bone repair solutions.

The main objective of this project is to apply precise and rapid prototyping technologies for custom-made bone tissue engineering with optimised macro-architecture and bio-functionality.
The RAPIDOS project concept is to be realised by 1) Establishing an imaging process, morphing patient specific implant shape and optimized internal macro-structure; 2) Developing precise and rapid prototyping technologies and advance biomaterials incorporating optimised macro-architecture and biological effectors; 3) Validating and optimizing architecture design and bioactivity of scaffolds for bone Tissue Engineering in vitro and in vivo; and 4) Consolidating EU-China biomaterials research through close collaboration between partners.

RAPIDOS Project objectives for the 4 years period

WP 3- Imaging process morphing patient specific implant shape and optimized internal
• Mathematical modelling for the choice of the most suitable implant scaffold in relation to bone defect size and location;
• Analyse and process retrospective clinical CT cases affected by unilateral orbital floor fractures to provide computer templates matching the shape and size of the orbital defects;
• Development of model scaffold architectures for improved cells seeding efficiency and mass transport;
• Morphing of clinical image data and design internal architectures for rapid prototyping technologies.

WP 4- Rapid prototyping technologies and advance biomaterials incorporating optimized macro-architecture and exogenous factors
• Prepare and characterize calcium phosphate particles;
• Prepare and characterize PTMC resin, PTMC/CaP composite, PLGA microspheres/nanofibers loaded with Icaritin and their combination thereof;
• Develop and optimize SLA technology for preparation of custom-made scaffolds with controlled and precise macro-architecture;
• Develop and characterize SLA scaffolds releasing biological effectors (e.g. icaritin).

WP 5- In Vitro Bone Tissue Engineering
• To define optimal scaffold properties in combination or not with hydrogel carrier for the seeding and differentiation of hMSCs into bone in vitro.
• Within this WP, the influence of the following parameters will be examined; (1) PTMC/CaP ratio, (2) scaffold architecture, (3) quantity and released icaritin from loaded microspheres.

WP 6- In Vivo Bone Tissue Engineering
• In this WP, small and large animal models are proposed to assess orbital floor fracture. As WPs 1-3 was successful in developing a blue print of custom-made scaffold for bone tissue engineering and identifying in vitro, the best conditions to repair bone. The different PTMC/nano-hydroxyapatite (HA) scaffolds were also implanted in a small animal model (rabbit calvarial defect) in order to select the best candidate allowing for bone defect repair. Finally, we validated the PTMC/HA patient-specific implant (PSI) using orbital floor defect in a larger pre-clinical animal model (sheep model).

WP 7- Consolidate EU-China biomaterials research and further enhance collaboration
• Disseminate information on the research performed to the consortium, the scientific community, the AO orthopedic surgeons network, the industry throughout Europe, and China;
• Ensure proper exploitation of RAPIDOS Project.

Project Results:
RAPIDOS main S & T results/foregrounds
Work package 3- Imaging process morphing patient specific implant shape and optimized internal macro-structure
Summary of progress towards objectives:
The main objective of the WP3 for this reporting period was to create tools to analyse clinical imaging data like computed tomography (CT) images and exploit this original data set to create, for example a 3D stereolithographic model of an implant that can be transferred as a set of coordinates for the printing system to create a custom-made scaffold implant. A mathematical (engineering) model has been prepared that estimates the stress-strain behaviour of orbital floor implants under loading (Task 3.1 completed). A computer workflow has been developed and patented for design of an orbital floor implant based on clinical computed tomography data (Task 3.5 completed). The method is based on a semi-automatic procedure allowing for a PSI to be generated based on the intact side. It is envisioned that the surgeons will have a tool to decide which scaffold design is suitable for reconstruction of the defect in the orbital floor based on an interactive system, addition of landmarks for fixation and selection of size and thickness of the implant. Five retrospective clinical CT cases affected by unilateral orbital floor fractures were provided by Chinese partner (301 partner) after ethical approval, and process workflow and analysis performed. Similarly, the methodology was applied on clinical CT scan of sheep for the pilot study to be conducted in the following period. (Tasks 3.2 completed and 3.3 completed). Furthermore, the virtual defined implant shapes have been morphed with macro-architecture designs and adapted to the rapid and precise fabrication techniques considered (SLA and LT-RP). STL files have been provided to the UT partner for additive manufacturing processing (Tasks 3.4 completed).

WP 4- Rapid prototyping technologies and advance biomaterials incorporating optimized macro-architecture and exogeneous factors
Summary of progress towards objectives:
The work performed in the work package 4 represented the core biomaterial activities and objectives of the RAPIDOS project. The preparation, characterization and optimization of calcium phosphate ceramic for stereolithography process were performed by XPAND who provided partners with calcium phosphate particles (Task 4.1 completed). The preparation of drug loaded PLA microspheres was investigated by QMUL, AOF and UT, using icaritin and dexamethasone as drug models (Task 4.2 completed). Icaritin loading in electrospun fibers has been achieved and the release of the icaritin upon degradation of the fibers was performed. In addition, QMUL and AOF have prepared and characterized composite polymer-polymer structures loaded with dexamethasone and investigated its activity in vitro on MSCs. Preparation and optimization of poly(trimethylcarbonate) (PTMC) resin for stereolithography process in combination with CaP particles and nanofibers was performed. The influences of CaP particles and shopped nanofibers mixing on the curing of the PTMC resin and the mechanical properties of photo-cured composite materials have been assessed (Task 4.3 completed). Additionally, CaP particles from XPAND were introduced in the electrospun nanofibers in order to ease the process of cutting continuous electrospun fibers into discreet fibers that could be more easily combined to the PTMC resin. Stereolithography manufacturing of PTMC/CaP formulation has successfully been performed (Task 4.4 completed).
The preparation, characterization and optimization of calcium phosphate ceramic and magnesium for blending with PLGA and low temperature rapid prototyping fabrication has been performed (Task 4.5 completed). PLGA/TCP/Mg compositions preparation and LT-RP optimization have been performed demonstrating the versatility of the additive manufacturing employed (Task 4.6 completed). The in vitro degradation of the PLGA/TCP/Mg scaffolds in dynamic conditions was performed showing the fast release of Mg2+ ion is solution (Task 4.7 completed). Materials; PTMC resin, icaritin, dexamethasone, antimicrobial agent and electrospun fibers were exchanged between RAPIDOS partners (Task 4.8 completed) with notably the distribution of icaritin from SIAT to all EU partners. Also the Chinese partners have completed their project, exchanges of materials and preparation of manuscripts were actively performed.

WP 5 In Vitro Bone Tissue Engineering
Summary of progress towards objectives:
The objectives of this work package 5 was to assess the influence of the biomaterials and scaffolds prepared in work package 4 on human mesenchymal stromal cells (hMSCs) behaviour in vitro. It was expected that the CaP, Mg and icaritin loaded in the biomaterials will have an osteoconductive and potentially inductive effect and will enhance the differentiation of hMSCs toward an osteogenic phenotype. These works will allow the selection of a most bone promoter composition, but also assess the potential of a tissue engineering approach combining material and stem cells. PTMC/CaP scaffold compositions have been assessed in vitro by XPAND and UT partners demonstrating the safety and biocompatibility of the materials (Task 5.1 completed). The screening of the biological influence icaritin release in vitro has been performed indicating that icaritin is an osteopromotive molecule which biological effect on hMSCs should be assessed in osteogenic conditions in vitro (Basal media +100 nM dexamethasone, 50 μg/ml ascorbic acid, 5 mM β-glycerophosphate). However, icaritin used in conjunction with PTMC/CaP did not enhance further the osteopromotive effect of the CaP.
The bacteria colonization and biofilm of bacteria on the PLGA composite scaffolds in vitro was performed showing the beneficial effect of the Magnesium on reducing colonization and biofilm formation at short time point (Task 5.4 completed). The proliferation and osteogenic differentiation of hMSCs in the PLGA/TCP scaffold combined icaritin/Mg in vitro has been performed and published in the literature. Additional work in understanding the mechanistic effect of icaritin was pursued by SIAT. The suppression of adipogenesis by icaritin has notably been observed and could suggest that in turn this could promote osteogenesis in hMSCs (Task 5.5 completed).

WP 6- In Vivo Bone Tissue Engineering
Summary of progress towards objectives:
Isolation and expansion protocols of the equivalent bone rabbit and sheep mesenchymal stromal cells have been made available by AOF, SIAT and SJTU partners to all consortium partners (Task 6.1 completed). The objective of this work was to ensure that partners of the consortium would be able to compare cells work performed in vivo. Selection of animal a small animal model has been performed and the model performed (Task 6.2 completed).
The tasks 6.3 6.4 regarding small animal model were completed. A calvarial model in rabbit was employed by the European partners to screen PTMC/CaP stereolithography scaffolds with two contents of CaP. The addition of CaP in the PTMC scaffolds showed to enhance significantly the bone formation and healing. It was decided based on the in vitro and the first in vivo results that the cell-free tissue engineering approach will be pursued as more suitable for translation into the clinics.. An animal segmental bone defects study associated with infection was performed by SJTU partner demonstrating the potential of quaternized chitosan loaded biomaterial for the treatment of local infection in open fracture (Task 6.5 completed).
An in vivo study using PLGA/TCP/Mg composite scaffolds in bone tunnel after repair core decompression in steroid-associated osteonecrosis (SAON) was performed by SIAT demonstrating the ability of the LT-RP scaffold developed in the RAPIDOS project to improve bone healing in a compromise bone situation. (Task 6.6 completed). An animal model of rabbit ulna bone segmental defect was also used and performed by SIAT to assess the PLGA/TCP composite scaffolds loaded with icaritin. The beneficial effect of icaritin in the bone formation was demonstrated on the PLGA/TCP, but interestingly, addition of icaritin at two concentrations into the optimized PTMC/CaP did not further enhance the bone formation and healing in the calvarial bone rabbit model (Task 6.7 completed). This could be due to the animal model selected or specific interactions of the icaritin with the materials. Finally, an orbital floor model in large animal, sheep, was developed and used to assess the patient specific orbital floor implant process developed in the RAPIDOS project (WP3) and the selected PTMC/CaP stereolithography technology optimized (WP4). The RAPIDOS printed implants performed similarly to pre-shaped commercial titanium implants used as positive control with no failure or negative signs of inflammation or influence of the animals' eyesight. However, the osteoinductivity of the PTMC/HA was demonstrated, indicating the ability to produce, for the first time, osteoinductive patient specific implants for complex fractures of the orbital floor (Task 6.8 completed).

Potential Impact:
RAPIDOS potential impact
The RAPIDOS project addressed the key topics of the call [NMP-2013-EU-China]. The objectives of the RAPIDOS project have been achieved and reported (see deliverables and final scientific report).

Main advantages of the RAPIDOS concept strategy by the consortium industrial partner were
1. High market demand for bone substitutes
2. Breakthrough concept with pre-clinically validated prototypes for treatment of none healing blow out fracture in orbital floor.
3. The new validated concept will be implemented to tackle other disease conditions and need for bone non-healing.
The pre-clinical studies performed during the RAPIDOS Project have validated the safety of the biomaterials and the additive manufacturing technologies. Importantly, for the first time the osteoinductive effect of the composite stereolithography scaffold was demonstrated in a large animal study. In addition, the ability to reproduce the technological concept and surgical procedure in a clinical-like setting for a bioactive patient specific orbital floor implant product was demonstrated. Therefore, the CaP material developed by XPAND provide a solid basis for bone substitutes products. The RAPIDOS project validated prototypes for treatment of none healing blow out fracture in orbital floor. This work can provide the basis for implementation to other disease conditions and need for bone non-healing.

Main envisaged obstacles to the RAPIDOS concept exploitation
1. Still long time to market from preclinical validated prototypes to human validated products
2. Competition with products already in market
3. Price for this therapeutic approach
The biological free approach developed by the RAPIDOS partners was chosen to avoid lengthy regulatory pathway and hurdles. To date, competition exists but is scarce. Among potential treat, Tissue Regeneration Systems, Inc. (TRS) recently acquired by DePuy Synthes Products, Inc., part of the Johnson & Johnson Family of Companies, is creating patient-specific, bioresorbable implants with a unique mineral coating intended to support bone healing in patients with orthopaedic and craniomaxillofacial deformities and injuries. The technology developed by TRS is a classical fuse deposition modelling additive manufacturing with a post process with bioactive coating. There are not yet a product from TRS, but this suggests that the RAPIDOS approach could be of huge interest for large players in the field of additive manufacturing for health.

The consortium strategy to further materialize the RAPIDOS project outputs has been:
• The partners have produced IP within the project by following up and evaluated existing patents in their portfolio and research relevant fields. The RAPIDOS partners feel that no other patents would be required to protect their freedom to operate.
• Further in vivo studies and Phase I/II clinical studies has not yet been planned, but the new GLP accredited facility and clinical investigation department of AOF could provide readily available technical support for further investigations. The investments and funds requested for these studies have not yet been detailed.
• It was identified that with the newly investment fund available through the AO foundation, a collaborative effort to translate the RAPIDOS findings could be achieved and is being assessed.
• The scientific and technology demonstration of RAPIDOS project has facilitated the merger of Xpand Biotechnology BV with KUROS Bioscience AG in 2017.
The innovative approach within RAPIDOS has contributed to an increased competitiveness of the SME involve in the RAPIDOS project, but also in general to the European biomaterial and biomedical industry by giving rise to patented innovations to be exploited by the existing networks of the SME and academic Partners.

Strategy advance for 2013-2017
Commercial exploitation
The project has generated exploitable knowledge (see periodic reports).
Therefore, the following provisions or activities have been put in place:
1. The work programme as defined in Annex I of the Contract with the European Commission. The work programme defines deliverables that have been reported.
2. The consortium agreement. The CA defines in detail IPR, handling IPR and exploitation rights, including provisions for cases where knowledge originating from the project is owned by more than one partner.
3. IPR issues were handled by Technology Transfer Units (TTU) or departments or persons assigned to manage IPR issues at every partner institution.
4. Every Partner has informed all other partners of the names and contact details of the persons responsible for IPR management in their respective institutions.

To date, AOF (AO Technology AG) has submitted a patent application on "Method for manufacturing an auxiliary device suitable for the manufacture of a patient customized implant" L. Kamer, D. Eglin. PCT/CH 2015/000001, 12 January 2015.
The SIAT has deposed a patent "A porous acellular tissue engineering cartilage and preparation methods" CN104399130A.
Additional patents applications may be expected.

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