Final Report Summary - REBORNE (Regenerating Bone defects using New biomedical Engineering approaches)
Bone is among the most frequently transplanted tissue with about 1 million procedures annually in Europe. Bone defects may be due to trauma (non-union fractures), necrosis (osteonecrosis of the femoral head), congenital deformities (cleft palates) or ageing (mandible bone loss). Autologous bone grafting is considered the gold standard in orthopaedic and maxillo-facial surgery for regeneration of skeletal defects. However, patient’s bone stock is limited and complications are often observed at the harvesting site. The European project Reborne aims at regenerating bone by using mesenchymal stem cells and advanced biomaterials.
For the first time at the European level, we have demonstrated safety and efficacy of cells and biomaterials to regenerate bone in clinical trials (Rosset et al, Orthop Traumatol Surg Res. 2014). We have shown that mesenchymal stem cells (MSC) in combination with biphasic calcium phosphate (BCP) biomaterials have the capacity to regenerate bone tissue. On the basis of our pre-clinical research demonstrating safety and efficacy (Brennan et al, Stem Cell Res Ther 2014), 5 clinical trials using Advanced Therapy Medicinal products (ATMP) have been authorized by the National Competent Authorities in several European countries. In these clinical studies, MSC were isolated from bone marrow and amplified in culture with human platelet lysate plasma (PLP) in GMP facilities prior to re-implantation in patients requiring bone regeneration.
In the first clinical trial, non-union fractures were successfully treated in 30 patients using this cell therapy approach leading to bone healing at 12 months in 93% of cases (EudraCT 2011-005441-13). In a second multicenter study, autologous MSCs were used for the treatment of osteonecrosis of the femoral head involving 25 patients (EudraCT 2012-002010-39). In the same indication, a third pilot trial using allogeneic MSC has also been approved (EudraCT 2014-000516-34). Alveolar cleft palates were successfully reconstructed in 20 children using osteoinductive calcium phosphate (CaP) granules. Lateral and vertical bone augmentations were achieved in 10 patients using autologous MSC and CaP granules (EudraCT 2012-003139-50).
In addition to achieving 5 clinical trials with ATMP, Reborne has contributed to a large dissemination of knowledge with 34 publications in high impact journals, organization of 8 workshops and 190 communications in major scientific conferences and to the general public (e.g. interviews on Arte TV, newspapers and magazines). In the field of biomaterials, 3 new products have been CE-marked by medical devices companies and a spin-off of the University has been created. The production and quality controls of cell therapy products has been successfully standardized in Germany, France, Italy and Spain. Transportation of fresh patient’s cells over long distances through Europe was also demonstrated. Different cell sources such as bone marrow, adipose tissue and umbilical cord blood were investigated for bone regeneration. Several animal models were developed to demonstrate safety and efficacy of cells and biomaterials. Ethical and regulatory expertise was gained over the years as illustrated by the effective approvals of the 5 multi-centre clinical trials in orthopaedic and maxillo-facial surgeries.
The European project Reborne has therefore produced significant knowledge and expertise in bone tissue engineering from basic research to clinical translation.
Project Context and Objectives:
With more than 1 million procedures every year in Europe, bone is the most transplanted tissue. The standard of care uses autologous bone grafting but it has drawbacks such as 2 surgical sites, limited bone stock, post-operative pain and limited efficacy. The European project Reborne proposes alternatives to bone grafts by using cultured mesenchymal stem cells and biomaterials.
To achieve these objectives, the project was structured in five main themes:
- Research and development of biphasic calcium phosphate ceramics as scaffolds for bone tissue engineering including specific devices for association with mesenchymal stem cells (MSCs)
- Research and development of injectable bone substitutes having osteoconductive, osteoinductive properties and supporting bone healing and vascularization, using CaP ceramic particles and hydrogels for injection, encapsulation, proliferation and differentiation of mesenchymal stem cells (MSCs)
- Research and development of composites based on calcium phosphate cements having intrinsic osteoinductive properties, allowing for the incorporation of vascular and/or bone growth factors, supporting cell growth, having high strength and being resorbable
2. Cell production and quality control
- Development of specific protocols for GMP production of mesenchymal stromal cells (MSC) to be used in the consortium clinical trials
- Defining GMP culture conditions that allow optimal synergy between cells and the biomaterials to be used in bone repair
- Evaluating the potential of different sources of MSC regarding easiness of culture, cell yield, osteoblastic differentiation
- Definition and standardization of cryopreservation protocols of the MSC from different sources
- Establish, validate and perform quality controls for MSC alone or in combination with suitable bioscaffolds for bone repair
- Establish and validate safety controls including: genetic stability and immuno-reactivity for cryopreserved/thawed MSC
- Establish rapid potency assays including: immunophenotypic and gene profile cytokine secretion assays
3. Preclinical validation
- Providing standardized and novel potency assays combining human mesenchymal stem cells (MSC) and biomaterials to be translated into GMP settings of REBORNE
- Standardize optimal in vitro culture conditions capable to rescue and maintain selected human and animal MSC with robust osteogenic potential when combined with biomaterials
- Establish biomaterial and MSC combinations focusing on both angiogeneic and osteogenetic potentials to obtain a stronger bone regeneration
- Explore, in vitro and in vivo, the impact of immune system on matched and mismatched, differentiated and undifferentiated MSC. The use of immunosuppressive drugs will be additionally assessed considering their influence on bone differentiation
- Combine biomaterials with optimal source/s of MSC, as single entity or in associations, for an optimal bone regeneration in animal models mimicking selected human diseases
4. Clinical trials
- Evaluation of efficacy of autologous MSCs combined to biomaterial to obtain bone healing in patients with closed, comminuted tibia or femur or humerus diaphyseal fracture (Trafton B and C closed tibial fractures, Winquist and Hansen type III or IV closed femoral fractures, treated by locked intramedullary nailing, and A.O. type B and C closed humeral dyaphyseal fractures after intramedullay nailing or osteosynthesis) presenting with delayed consolidation (no consolidation after 3 months) requiring standard supply of bone graft, in patients aged 18 to 65. A second objective in this indication will be the evaluation of efficacy of MSCs combined to injectable biomaterial for percutaneous administration, when made available by the Consortium
- Evaluation of efficacy of autologous MSCs combined to biomaterial to obtain bone healing enhancement in patients (age 18-65 yr) with avascular necrosis of the femoral head (Ficat-Arlet I or II, MRI confirmation)
- Bone healing enhancement in patients (age 12-18 yr) with avascular necrosis of the femoral condyle or head (Ficat-Arlet I or II, MRI confirmation) with previously treated hematologic malignancy with immunosuppression
- Evaluation of safety and efficacy of calcium phosphate granules associated with membranes and autologos MSC in mandible region behind the canine teeth prior to dental implants
- Evaluation of efficacy of a bone substitute with intrinsic osteoinductive property for regenerating bone in cleft palates in children
5. Ethics and dissemination
- Establishing the general ethical perspectives and legal framework in preclinical and clinical R&D of new biomedical engineered products based on allogenic and autologus adult human MSC and biomaterials
- Disseminating and exploiting the foreground generated within REBORNE
On the basis of pre-clinical studies, the consortium has achieved major milestones with the approval of 4 clinical trials in different European countries. These important achievements have received considerable attention from the media across Europe and also abroad. These clinical studies are among the few in Europe with Advanced Therapy Medicinal Products. It is remarkable that the clinical studies are driven by non-profit governmental organizations under the support of the FP7 program. In addition, partners of the REBORNE consortium have published several excellent scientific papers and communicated in numerous international conferences. Based on the obtained results, a new standard in bone regeneration is born.
The project, structured in differents workpackadge, generated specifics sciences and technological results.
About biomaterials, REBORNE:
- Performed a preclinical demonstration of equivalent performance between TCP composite and autograft (Xpand#9).
- Devlopped a own brand labelling (CE mark) by a commercial partner for osteoinductive TCP (Xpand#9)
- Made a demonstration of volume preservation and complete bioresorption of osteoinductive TCP composite (Putty) in 6 month in vivo (Xpand#9)
- Developpe a novel macroporous CDHA and TCP scaffolds, either biomimetic or high temperature, respectively
- Revealed some osteoconductive properties of biomaterials.
- Optimize the production process of chitosan succinamide
- Developpe analytical methods for chitosan succinamide
- Identify the parameters for optimal and reproducible hydrogel properties
- Optimize of the hydrogel formulation process
- Initiate the manufacturing development of gel alone with Fill & Finish CMO’s
- Establish the biocompatibility package needed for CE mark as class III implantable medical device
One of the most significant foreground of the project is the edition and the validation of a the protocol for expansion of MSC for clinical trials. This is especially based on the manufacturing and characterization of a supplement containing platelet derived growth factors for ex-vivo expansion of MSC. During the project, all cell production centers performed 69 cell expansion for 3 clinical trials:
- Investigational medicinal product, i.e. GMP-grade bone marrow-derived MSC has been manufactured for 29 patients in the clinical trial ORTHO-1. These clinical scale ex-vivo expansions fully reproduced results which were previously obtained during validation runs. Starting material, i.e. bone marrow aspirate, was quite heterogeneous with regard to cell count, overall cell number, frequency and overall number of MSC progenitors (CFU-F). Despite this heterogeneity, the parameters describing the efficacy of MSC expansion itself were quite stable.
This demonstrates the feasibility of large scale expansion of bone marrow-derived MSC with a target cell dose > 200 x 106 cells. Furthermore we could demonstrate that it is possible to produce an ATMP in different manufacturing sites without any obvious differences in the efficacy of expansion. This is important for future multicentre clinical trials with several manufacturing sites which might be located distant from the clinical sites.
- Investigational medicinal product, i.e. GMP-grade bone marrow-derived MSC has been manufactured for 21 patients in the clinical trial ORTHO-2. These clinical scale ex-vivo expansions fully reproduced the results previously obtained during validation runs and the first clinical trial ORTHO-1 (see above). Starting material, i.e. bone marrow aspirate, was quite heterogeneous with regard to cell count, overall cell number, frequency and overall number of MSC progenitors (CFU-F). Despite this heterogeneity, the parameters describing the efficacy of MSC expansion itself (population doublings and doubling time) were quite stable.
Thus, we could demonstrate the feasibility of large scale expansion of bone marrow-derived MSC with a target cell dose of 140 - 200 x 106 cells in ORTHO-2. Furthermore we could demonstrate by ORTHO-2 clinical trial (as already shown for the clinical trial ORTHO-1) that it is possible to produce an ATMP in different manufacturing sites without any obvious differences in the efficacy of expansion.
- Investigational medicinal product, i.e. GMP-grade bone marrow-derived MSC has been manufactured for 11 patients in the clinical trial MAXILLO-1. These clinical scale ex-vivo expansions fully reproduced the results previously obtained during validation runs and the first clinical trial ORTHO-1 and ORTHO-2 (see above). Starting material, i.e. bone marrow aspirate, was quite heterogeneous with regard to cell count, overall cell number, frequency and overall number of MSC progenitors (CFU-F). Despite this heterogeneity, the parameters describing the efficacy of MSC expansion itself (population doublings and doubling time) were quite stable.
Thus, we could demonstrate the feasibility of large scale expansion of bone marrow-derived MSC with a target cell dose of at least 100 x 106 cells in MAXILLO-1. A specific feature of the clinical trial Maxillo-1 was the substantial distance between manufacturing site (Ulm) and the clinical trial site (Bergen). This implied bidirectional transport (bone marrow aspirate from Bergen to Ulm and return of expanded cells to Bergen with a transportation time between 18 and 24 hours). MAXILLO-1 trial demonstrated feasibility of a trial with autologous MSC including distant sites. (see Deliverable report D.2.9 for more details on transport validation).
Also, the quality control has been an important topic of the project. We demonstrate:
- The efficiently performing standardized quality controls (QC) during MSC production for ORTHO-1, ORTHO-2 and MAXILLO-1 trials;
- The standardiziation of the protocols for QC characterization of immune regulatory properties of BM, AD and CB –MSC with and without biomaterials.
Specific fonctunal controls were performed with the following significant results:
- We report a test to quantify three-dimensional biomineralisation ex-vivo as a rapid, cost-effective potency assay for bone formation by cGMP amplified MSC;
- Our data on shipping fresh cells in normal saline open a new platform of tissue regeneration aiming to the further development of cellular therapies based on non-frozen cells;
- Our data on shipping fresh cells in normal saline may be linked to a novel business model in the cellular therapy arena;
- We were able to dramatically reduce MSC differentiation time and establish a faster bone potency assay that was aligned to the context of cGMP facilities: it was possible to measure gene expression changes significantly associated with osteogenic differentiation within 7 days, thus reducing the duration of this assay, which usually lasts between 2 and 3 weeks;
- Differentiation assays were successfully tested on MSC and biomaterial combinations in three dimensional (3D) cultures using MBCP+ granules that are going to be introduced in the ORTHO1 trial of the WP-5;
- More interestingly, these assays could be reproduced in testing other materials The standardization of this differentiation assay in 3D is now allowing the in vitro screening of distinct biomaterial and MSC combinations avoiding the introduction of massive screening based on animal models, in the full respect of the 3R rules on animal experimentation. This is a relevant benefit for the companies that are belonging to the consortium allowing them to rapidly synthetize and screen different type of biomaterials that can be used in a close future into the clinic. In vitro screeing will again be used to limit the animal testing only on materials with proven osteogenic properties in vitro;
- Specific markers could be used to detect native non-plastic adherence MSC;
- We were able to reveal the existence of human non-adherent cells (possibly MSC precursors) that are persisting for long-time in vitro and that are generally discarded after 4-5 days from the BM-MSC isolation in standard protocols.
On clinical trials, encouraging results have been observed on Ortho-1 (29 patients), Ortho-2 (25 patients), Maxillo-1 and Maxillo-2. Definitive conclusion will allow to give more details about the pertinence of MSCs and biomaterials on treatment of bone defects.
In addition, a last clinical trials has been approved (ORTHO-3) but not performed during the project.
During the project, we demonstate first the ability to perform a multicentric and multicountry clinical trial on regenerative medicine, using cell therapy. Reborne validate the possibility to expand, transport and use MSCs for treatment thanks to a standardisation of the protocol and strict quality controls.
Moreover, we have developed expertise in regulatory procedures for acceptance of innovative clinical trials via Voluntary Harmonisation Procedure for ORTHO-2 and ORTHO-3. Also, ae obtained authorisation for the multicountries clinical trial Ortho-3 in 60 days.
Bone is among the most frequently transplanted tissue with about 1 million procedures annually in Europe. The worldwide market of bone replacement materials is currently estimated at 5 billion € with a 10% annual growth. Despite their considerable disadvantages, including the risk of disease transfer and immunologic rejection, limited supply of bone, costs and complications, allografts and autografts account for more than 80% of total graft volume. Significant growth opportunities exist for synthetic bone grafts in association with mesenchymal stem cells from autologous or allogenic sources as alternatives to biological bone grafts in orthopaedic and maxillofacial surgery. The objectives of REBORNE was to perform clinical trials using advanced biomaterials and cells triggering bone healing in patients. In order to reach this goal, 4 phase II clinical studies have been performed. Two orthopaedic trials concerning the treatment of long bone fractures and osteonecrosis of the femoral head in adults or children have been conducted using bioceramics, hydrogel for percutaneous injection and stem cells from autologous sources. Clinical research have also concerned maxillofacial surgery with bone augmentation prior to dental implants and the reconstruction of cleft palates in children. The safety and efficacy of the new therapies have been assessed clinically using X-rays, CT scans and MRI as well as histology of biopsies. These ambitious clinical targets required research and development efforts from a large consortium of top world class laboratories, SMEs manufacturing biomaterials, GMP-cell producing facilities and surgeons in hospitals as well as the consideration of ethical and regulatory issues. REBORNE expanded the competitiveness of Europe through new CE-marked bioproducts in the field of regenerative medicine and giving proof of concept of effectiveness of cell therapy on several bone issues.
Reflecting the excellent work done during the project, 34 publications were published in international peer-reviewed journal since 2010. Other publications are planned in the coming months, including the results of clinical trials. During the project, the coordinator and the partners have been fully involved in the communication of project results to the scientific community in order to promote progress in regenerative medicine. This was reflected by 190 communications (oral presentations, posters, organisation of workshops, press release...) in scientific events such as congress or conferences. The excellence of the work has been exhibited both at global, European and local level. Specific sessions were dedicated to the project in international conferences such as in the International Symposium on Apatites and correlative Bioceramics in Nantes on 5-8 June 2013 or in the European Orthopaedic Research Society Conference.
In addition, the coordinator and the partners have been fully involved in the communication of project results to the general public. This was reflected by 27 communications conducted through interviews or press releases to promote the project as widest as possible in Europe.
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