H2020 MAXIBONE

In contrast to sintered calcium phosphates (CaPs) commonly employed as scaffolds to deliver mesenchymal stromal cells (MSCs) targeting bone repair, low temperature setting conditions of calcium deficient hydroxyapatite (CDHA) yield biomimetic topology with high specific surface area. In this study, the healing capacity of CDHA administering MSCs to bone defects is evaluated for the first time and compared with sintered beta-tricalcium phosphate (β-TCP) constructs sharing the same interconnected macroporosity. Xeno-free expanded human bone marrow MSCs attached to the surface of the hydrophobic β-TCP constructs, while infiltrating the pores of the hydrophilic CDHA. Implantation of MSCs on CaPs for 8 weeks in calvaria defects of nude mice exhibited complete healing, with bone formation aligned along the periphery of β-TCP, and conversely distributed within the pores of CDHA. Human monocyte-osteoclast differentiation was inhibited in vitro by direct culture on CDHA compared to β-TCP biomaterials and indirectly by administration of MSC-conditioned media generated on CDHA, while MSCs increased osteoclastogenesis in both CaPs in vivo. MSC engraftment was significantly higher in CDHA constructs, and also correlated positively with bone in-growth in scaffolds. These findings demonstrate that biomimetic CDHA are favorable carriers for MSC therapies and should be explored further towards clinical bone regeneration strategies. Statement of Significance Delivery of mesenchymal stromal cells (MSCs) on calcium phosphate (CaP) biomaterials enhances reconstruction of bone defects. Traditional CaPs are produced at high temperature, but calcium deficient hydroxyapatite (CDHA) prepared at room temperature yields a surface structure more similar to native bone mineral. The objective of this study was to compare the capacity of biomimetic CDHA scaffolds with sintered β-TCP scaffolds for bone repair mediated by MSCs for the first time. In vitro, greater cell infiltration occurred in CDHA scaffolds and following 8 weeks in vivo, MSC engraftment was higher in CDHA compared to β-TCP, as was bone in-growth. These findings demonstrate the impact of material features such as surface structure, and highlight that CDHA should be explored towards clinical bone regeneration strategies.

A wide variety of biomaterials have been developed as both stabilizing structures for the injured bone and inducers of bone neoformation. They differ in chemical composition, shape, porosity, and mechanical properties. The most extensively employed and studied subset of bioceramics are calcium phosphate materials (CaPs). These materials, when transplanted alongside mesenchymal stem cells (MSCs), lead to ectopic (intramuscular and subcutaneous) and orthotopic bone formation in preclinical studies, and effective fracture healing in clinical trials. Human MSC transplantation in pre-clinical and clinical trials reveals very low engraftment in spite of successful clinical outcomes and their therapeutic actions are thought to be primarily through paracrine mechanisms. The beneficial role of transplanted MSC could rely on their strong immunomodulatory effect since, even without long-term engraftment, they have the ability to alter both the innate and adaptive immune response which is critical to facilitate new bone formation. This study presents the current knowledge of the immune response to the implantation of CaP biomaterials alone or in combination with MSC. In particular the central role of monocyte-derived cells, both macrophages and osteoclasts, in MSC-CaP mediated bone formation is emphasized. Biomaterial properties, such as macroporosity and surface microstructure, dictate the host response, and the ultimate bone healing cascade. Understanding intercellular communications throughout the inflammation, its resolution and the bone regeneration phase, is crucial to improve the current therapeutic strategies or develop new approaches.

The healing of bone fractures is a well-orchestrated physiological process involving multiple cell types and signaling molecules interacting at the fracture site to replace and repair bone tissue without scar formation. However, when the lesion is too large, normal healing is compromised. These so-called non-union bone fractures, mostly arising due to trauma, tumor resection or disease, represent a major therapeutic challenge for orthopedic and reconstructive surgeons. In this review, we firstly present the current commonly employed surgical strategies comprising auto-, allo-, and xenograft transplantations, as well as synthetic biomaterials. Further to this, we discuss the multiple factors influencing the effectiveness of the reconstructive therapy. One essential parameter is adequate vascularization that ensures the vitality of the bone grafts thereby supporting the regeneration process, however deficient vascularization presents a frequently encountered problem in current management strategies. To address this challenge, vascularized bone grafts, including free or pedicled fibula flaps, or in situ approaches using the Masquelet induced membrane, or the patient’s body as a bioreactor, comprise feasible alternatives. Finally, we highlight future directions and novel strategies such as 3D printing and bioprinting which could overcome some of the current challenges in the field of bone defect reconstruction, with the benefit of fabricating personalized and vascularized scaffolds.

Background and aims: To review the regenerative technologies used in bone regeneration: bone grafts, barrier membranes, bioactive factors and cell therapies. Material and methods: Four background review publications served to elaborate this consensus report. Results and conclusions: Biomaterials used as bone grafts must meet specific requirements: biocompatibility, porosity, osteoconductivity, osteoinductivity, surface properties, biodegradability, mechanical properties, angiogenicity, handling and manufacturing processes. Currently used biomaterials have demonstrated advantages and limitations based on the fulfilment of these requirements. Similarly, membranes for guided bone regeneration (GBR) must fulfil specific properties and potential biological mechanisms to improve their clinical applicability. Pre-clinical and clinical studies have evaluated the added effect of bone morphogenetic proteins (mainly BMP-2) and autologous platelet concentrates (APCs) when used as bioactive agents to enhance bone regeneration. Three main approaches using cell therapies to enhance bone regeneration have been evaluated: (a) "minimally manipulated" whole tissue fractions; (b) ex vivo expanded "uncommitted" stem/progenitor cells; and (c) ex vivo expanded "committed" bone-/periosteum-derived cells. Based on the evidence from clinical trials, transplantation of cells, most commonly whole bone marrow aspirates (BMA) or bone marrow aspirate concentrations (BMAC), in combination with biomaterial scaffolds has demonstrated an additional effect in sinus augmentation and horizontal ridge augmentation, and comparable bone regeneration to autogenous bone in alveolar cleft repair.

Membranes for guided bone regeneration (GBR) were prepared from the synthetic biodegradable polymer poly-D,L-lactic/glycolic acid (PLGA). This GBR membrane has a bi-layered structure with a dense film to prevent gingival fibroblast ingrowth and ensure mechanical function, and a micro-fibrous layer to support colonization by osteogenic cells and promote bone regeneration. Hydrolysis and biodegradation were both studied in vitro through soaking in phosphate buffered saline (PBS) and in vivo by implantation in the subcutis of rats for 4, 8, 16, 26, 48 and 52 weeks. Histology revealed an excellent colonization of the micro-fibrous layer by cells with a minimal inflammatory reaction during resorption. GBR using the synthetic PLGA membrane was evaluated on critical-size calvaria defects in rats for 4 and 8 weeks. Radiographs, micro-computed tomography and histology showed bone regeneration with the PLGA membrane, while the defects covered with a collagen membrane showed a limited amount of mineralized bone, similar to that of the defect left empty. The biofunctionality of the PLGA membranes was also compared to collagen membranes in mandible defects in rabbits, associated or not with beta-tricalcium phosphate granules. This study revealed that the bi-layered synthetic membrane made of PLGA was safer, more biocompatible, and had a greater controlled resorption rate and bone regeneration capacity than collagen membranes. This new PLGA membrane could be used in pre-implantology and peri-odontology surgery.

Guided bone regeneration (GBR) is a surgical procedure used to preserve or increase bone volume, hence permitting optimal placement of dental implants. GBR requires the use of resorbable or non-resorbable membrane. Synthetic, Poly (lactic-co-glycolic acid) (PLGA) membranes have been presented as clinically efficient for periodontal/pre-implant regenerative procedures. We wanted to clinically test a newly commercialiesd PLGA membrane (Tisseos®) in a GBR procedure before implant placement.

Background: Many data are available on expansion protocols for mesenchymal stromal cells (MSCs) for both experimental settings and manufacturing for clinical trials. However, there is a lack of information on translation of established protocols for Good Manufacturing Practice (GMP) from validation to manufacturing for clinical application. We present the validation and translation of a standardized pre-clinical protocol for isolation and expansion of MSCs for a clinical trial for reconstitution of alveolar bone. Methods: Key parameters of 22 large-scale expansions of MSCs from bone marrow (BM) for validation were compared with 11 expansions manufactured for the clinical trial "Jaw bone reconstruction using a combination of autologous mesenchymal stromal cells and biomaterial prior to dental implant placement (MAXILLO1)" aimed at reconstruction of alveolar bone. Results: Despite variations of the starting material, the robust protocol led to stable performance characteristics of expanded MSCs. Manufacturing of the autologous advanced therapy medicinal product MAXILLO-1-MSC was possible, requiring 21 days for each product. Transport of BM aspirates and MSCs within 24 h was guaranteed. MSCs fulfilled quality criteria requested by the national competent authority. In one case, the delivered MSCs developed a mosaic in chromosomal finding, showing no abnormality in differentiation capacity, growth behavior or surface marker expression during long-term culture. The proportion of cells with the mosaic decreased in long-term culture and cells stopped growth after 38.4 population doublings. Conclusions: Clinical use of freshly prepared MSCs, manufactured according to a standardized and validated protocol, is feasible for bone regeneration, even if there was a long local distance between manufacturing center and clinical site. Several parameters, such as colony forming units fibroblasts (CFU-F), percentage of CD34+ cells, cell count of mononuclear cells (MNCs) and white blood cells (WBCs), of the BM may serve as a predictive tool for the yield of MSCs and may help to avoid unnecessary costs for MSC manufacturing due to insufficient cell expansion rates.

Impact statement: This work reports a new bone substitute made of precipitated apatite crystals that resemble in composition and crystallinity to the mineral phase of bone. The bone regeneration capacity of this synthetic biomimetic calcium phosphate (SBCP) was studied by using an original model of vertical bone regeneration with cups on the calvaria of rats. After 4 weeks, a significantly higher bone growth was found with SBCP compared with deproteinized bovine bone matrix and empty controls. This rapid vertical bone regeneration indicated that this new biomaterial is particularly interesting for filling bone defects in oral surgery.

Background: Autologous grafting, despite some disadvantages, is still considered the gold standard for reconstruction of maxillofacial bone defects. The aim of this study was to evaluate bone regeneration using bone marrow-derived mesenchymal stromal cells (MSCs) in a clinical trial, a less invasive approach than autologous bone grafting. This comprehensive clinical trial included subjects with severe mandibular ridge resorption. Methods: The study included 11 subjects aged 52-79 years with severe mandibular ridge resorption. Bone marrow cells were aspirated from the posterior iliac crest and plastic adherent cells were expanded in culture medium containing human platelet lysate. The MSCs and biphasic calcium phosphate granules as scaffolds were inserted subperiosteally onto the resorbed alveolar ridge. After 4-6 months of healing, new bone formation was assessed clinically and radiographically, as were safety and feasibility. Bone at the implant site was biopsied for micro-computed topography and histological analyses and dental implants were placed in the newly regenerated bone. Functional outcomes and patient satisfaction were assessed after 12 months. Results: The bone marrow cells, expanded in vitro and inserted into the defect together with biphasic calcium phosphate granules, induced significant new bone formation. The regenerated bone volume was adequate for dental implant installation. Healing was uneventful, without adverse events. The patients were satisfied with the esthetic and functional outcomes. No side effects were observed. Conclusions: The results of this comprehensive clinical trial in human subjects confirm that MSCs can successfully induce significant formation of new bone, with no untoward sequelae. Hence, this novel augmentation procedure warrants further investigation and may form the basis of a valid treatment protocol, challenging the current gold standard. Trial registration: EudraCT, 2012-003139-50. Registered on 21 August 2013. ClinicalTrials.gov, NCT 02751125 . Registered on 26 April 2016.

Regeneration of atrophied alveolar bone prior to insertion of dental implants is a major challenge for oral and maxillo- facial surgery. It has been reported that Bone Marrow (BM) derived Mesenchymal Stromal Cells (MSC) retain thera- peutic potential for bone regeneration. In the present study, a preclinical mini-pig model simulating the clinical setting was established in order to evaluate the efficacy of autologous MSC for mandible regeneration. Un- der general anaesthesia, BM aspirates were collected from tibia of mini-pigs (n = 5) and MSC were isolated, charac- terized and expanded. At the same time, a narrow alveolar ridge was simultaneously created by bilateral extraction of two premolar teeth and removal of the buccal bone in order to simulate the pathological situation in humans. After ex vivo expansion, cells were delivered fresh to the surgical operating room and seeded on Biphasic Calcium Phos- phate (BCP) granules for 1 hour followed by implantation into the simulated alveolar defects in one pig. The surgical defects were closed with sutures and left to heal for eight weeks. A bone biopsy was taken and dental implants were placed in the newly formed bone. The bone biopsy taken during the procedure showed miner- alized bone containing substantial amount of new bone with BCP granules embedded in osteoid tissues and dispersed throughout the newly formed bone matrix. The data demon- strate the osteogenic potential of autologous MSC com- bined with BCP, providing crucial pre-clinical information in a large animal aimed at the reconstruction of resorbed alveolar bone.

For the regeneration of small osseous defects, dental membranes are commonly used in oral and maxillofacial surgery. A new synthetic resorbable membrane has recently demonstrated its biocompatibility and bone regeneration capacity in preclinical studies. This membrane is made of poly-D,L-lactic/glycolic acid 85/15 (PLGA), has a bi-layered structure with a dense film to prevent gingival epithelial cell invasion and a micro-fibrous layer to support osteogenic cells and bone healing. This membrane completely degrades by hydrolysis in 4 to 6 months without signs of inflammation. On the basis of this research, a clinical study was conducted to evaluate the new membrane in guided tissue regeneration (GTR). In total, 26 patients (Age: 50.5 +/- 12.4, Min-Max 31-72 years; Male/Female 42/58 %) were operated in 7 independent private dental practices. Dental surgeons used the membrane together with various bone fillers in GTR for immediate and delayed implant placement (23 cases, 88%), and to a lesser extent socket preservation (2 cases, 8%) and alveolar crest augmentation (1 case, 4%). Surgeons reported an easy placement of the membrane (satisfaction index: 3.8/5). Fourteen days post-surgery, 15 patients had no pain while the others declared minimal pain (VRS: 2.2/10) and none had minor or serious complications related to the membrane. Exposure of the membrane without loosening of the biomaterial granules was observed in 3 cases while mucosa normally healed over time. At 4 months post-implantation, no infection or mucosal inflammation was reported and the overall dentist satisfaction with the clinical performance of the membrane was 4.5/5 on average. This clinical study demonstrated that the new synthetic resorbable membrane is safe for guided bone tissue regeneration in various dental surgery indications.