Definitive proof is lacking on mesenchymal stem cell (MSCs) cellular therapy to regenerate bone if biological potential is insufficient. High number of MSCs after GMP expansion may solve the progenitor insufficiency at the injury but clinical trials are pending. A prospective, multicenter, multinational Phase I/IIa interventional clinical trial was designed under the EU-FP7 REBORNE Project to evaluate safety and early efficacy of autologous expanded MSCs loaded on biomaterial at the fracture site in diaphyseal and/or metaphysodiaphyseal fractures (femur, tibia, humerus) nonunions. The trial included 30 recruited patients among 5 European centres in France, Spain, Germany, and Italy. Safety endpoints (local and general complication rate) and secondary endpoints for early efficacy (number of patients with clinically and radiologically proven bone healing at 12 and 24 weeks) were established. Cultured MSCs from autologous bone marrow, expanded under GMP protocol was the Investigational Medicinal Product, standardised in the participating countries confirming equivalent cell production in all the contributing GMP facilities. Cells were mixed with CE-marked biphasic calcium phosphate biomaterial in the surgical setting, at an implanted dose of 20−106 cells per cc of biomaterial (total 10cc per case) in a single administration, after debridement of the nonunion.Background
Methods
Delayed bone healing and nonunion are complications of long bone fractures, with prolonged pain and disability. Regenerative therapies employing mesenchymal stromal cells (MSC) and/or bone substitutes are increasingly applied to enhance bone consolidation. The REBORNE project entailed a multi-center orthopaedic clinical trial focused on the evaluation of efficacy of expanded autologous bone marrow (BM) derived MSC combined with a CaP-biomaterial, to enhance bone healing in patients with nonunion of diaphyseal fractures. To complement the clinical and radiological examination of patients, bone turnover markers (BTM) were assayed as potential predictors of bone healing or non-union. Peripheral blood was collected from patients at fixed time-endpoints, that is at 6,12 and 24 weeks post-surgery for implantation of expanded autologus MSC and bone-like particles. Bone-specific alkaline phosphatase (BAP), C-terminal-propeptide type I-procollagen (PICP), osteocalcin (OC), β-Cross-Laps Collagen (CTX), soluble receptor activator of NFkB (RANKL), osteoprotegerin (OPG) were measured by ELISA assays in blood samples of 22 patients at BM collection and at follow-up visits. A significant relationship with age was found only at 6 months, with an inverse correlation for CTX, RANKL and OC, and positive for OPG. BTM levels were not related to gender. As an effect of local regenerative process, some BTM showed significant changes in comparison to the baseline value. In particular, the time course of BAP, PICP and RANKL was different in patients with a successful healing in comparison to patients with a negative outcome. The BTM profile apparently indicated a remarkable bone formation activity 12 weeks after surgery. However, the paucity of failed patients in our case series did not allow to prove statistically the role of BTM as predictors of the final outcome. Blood markers related to bone cell function are useful to measure the efficacy of a expanded MSC-regenerative approach applied to long bone non-unions. Changes of the markers may provide a support to radiological assessment of bone healing.
Properties of human amniotic membrane are particularly interesting. To use it as an Advanced Therapeutic Medicinal Product in bone surgery, we are evaluating its association with a potentially osteoinductive scaffold. The human Amniotic Membrane (hAM) is known to have a good potential to help the regeneration of tissues. It has been used for 100 years in many medical disciplines because of its properties: a membrane containing stem cells and growth factors, with low immunogenicity and anti-microbial, anti-inflammatory, anti-fibrotic and analgesic properties. Moreover, previous published data showed the possibility of We aim to use hAM as an Advanced Therapeutic Medicinal Product for bone repair to treat large defects or pseudarthrosis. So we are studying the association of hAM with nanofiber jet sprayed polycaprolactone (PCL) scaffolds and the possibility to induce its osteodifferenciation.Summary
Introduction
Human amniotic membrane has interesting properties for regenerative medicine. To use it as an Advanced Therapeutic Medicinal Product in bone surgery, we are evaluating: the necessity of its osteodifferentiation and the impact on immunogenicity; its optimal condition for storage. The human Amniotic Membrane (hAM) is known to have a good potential to help the regeneration of tissues. It has been used for 100 years in many medical disciplines because of its properties: a flexible scaffold containing stem cells and growth factors, with low immunogenicity and anti-microbial, anti-inflammatory, anti-fibrotic and analgesic properties. Previous published data showed the possibility of in vitro osteodifferentiation of the whole tissue. We aim to use this «boosted membrane» as an Advanced Therapeutic Medicinal Product for bone repair to treat large defects or pseudarthrosis, so, we are studying: The necessity to osteodifferentiate the tissue and its consequence on the immunogenicity; Its in vivo osteogenic potential; The effects of the cryopreservation on cell viability and function.Summary
Introduction
