Biomimetic versus sintered macroporous calcium phosphate scaffolds enhanced bone regeneration and human mesenchymal stromal cell engraftment in calvarial defects

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 (ß-TCP) constructs sharing the same interconnected macroporosity. Xeno-free expanded human bone marrow MSCs attached to the surface of the hydrophobic ß-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 ß-TCP, and conversely distributed within the pores of CDHA. Human monocyte-osteoclast differentiation was inhibited in vitro by direct culture on CDHA compared to ß-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 ß-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 ß-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.Peer ReviewedPostprint (author's final draft

Regeneration of segmental defects in metatarsus of sheep with vascularized and customized 3D-printed calcium phosphate scaffolds

Although autografts are considered to be the gold standard treatment for reconstruction of large bone defects resulting from trauma or diseases, donor site morbidity and limited availability restrict their use. Successful bone repair also depends on sufficient vascularization and to address this challenge, novel strategies focus on the development of vascularized biomaterial scaffolds. This pilot study aimed to investigate the feasibility of regenerating large bone defects in sheep using 3D-printed customized calcium phosphate scaffolds with or without surgical vascularization. Pre-operative computed tomography scans were performed to visualize the metatarsus and vasculature and to fabricate customized scaffolds and surgical guides by 3D printing. Critical-sized segmental defects created in the mid-diaphyseal region of the metatarsus were either left empty or treated with the 3D scaffold alone or in combination with an axial vascular pedicle. Bone regeneration was evaluated 1, 2 and 3 months post-implantation. After 3 months, the untreated defect remained non-bridged while the 3D scaffold guided bone regeneration. The presence of the vascular pedicle further enhanced bone formation. Histology confirmed bone growth inside the porous 3D scaffolds with or without vascular pedicle inclusion. Taken together, this pilot study demonstrated the feasibility of precised pre-surgical planning and reconstruction of large bone defects with 3D-printed personalized scaffolds.Peer ReviewedPostprint (published version

Minimal information for studies of extracellular vesicles 2018 (MISEV2018):a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points

Close to the bone: investigations into bone tissue mineralisation and mechanobiology of osteoporosis

Osteoporosis is a metabolic skeletal disease characterized by low bone mass, depleted micro-architecture and reduced strength. The public health costs of osteoporosis relate almost entirely to the fractures that are the clinical manifestation of the disease and it presents a significant cause of morbidity in today’s ageing population. Oestrogen deficiency during the menopause is the primary causative factor for postmenopausal osteoporosis and although much is known about the pathophysiology of the disease, including dysregulated bone cell function whereby more bone is digested than is formed; the underlying mechanisms involved have not yet been delineated. Recent studies have suggested that although overall strength is decreased following osteoporotic bone loss, the remaining bone tissue is stronger and stiffer, suggesting an alteration in bone tissue composition. Bisphosphonates are among drug treatments administered to tackle bone loss, however the incidence of osteoporotic fractures still remains high. Furthermore, the precise effect of drug treatment on bone tissue mineralisation is unknown.The global aim of this thesis is to discern the alterations in the quantity and distribution of bone mineral during osteoporosis. Specifically, it is sought to test the hypotheses that bone mineral distribution is altered at a tissue level following oestrogen deficiency and bisphosphonate treatment and that oestrogen depletion alters normal mineralisation and mechano-responsiveness of bone cells. Quantitative backscattered imaging (qBEI) on a scanning electron microscope was used to examine individual bone trabeculae from the proximal femur of ovariectomised sheep (oestrogen deficient state), aged matched control sheep and sheep treated with the bisphosphonate Zoledronic acid. It was found that oestrogen deficiency caused significantly higher mineral heterogeneity within trabeculae (site speficic within the femur) and along a common osteoporotic fracture line. Bone mineralisation was diminished with prolonged oestrogen deficiency and conversely was higher in older healthy sheep compared to younger control sheep. Furthermore, significantly lower mineral heterogeneity was found in OVX sheep treated with Zoledronic acid compared to untreated OVX sheep. These results indicate that changes in bone tissue mineralisation during oestrogen deficiency may be a contributing factor for reduced mechanical strength during osteoporosis, while drug induced increased homogeneity may contribute to the ability of Zoledronic acid to prevent fracture occurrence during oestrogen deficiency.The next study aimed to delineate the mechanisms responsible for such altered mineral distribution. Osteoblast and osteocyte cells were pre-treated with oestrogen and the effects of oestrogen deficiency were evaluated by subsequently withdrawing oestrogen from cells, or blocking oestrogen receptors using an oestrogen antagonist, fulvestrant. Specifically, alkaline phosphatase expression was investigated using p-nitrophenyl phosphate (pNPP), proliferation by assessing DNA content, calcium production using alizarin red assay and apoptosis by measuring for caspase 3/7 activity. Although mineral production was significantly increased by oestrogen pre-treatment, a further increase in mineral production and apoptosis were observed following oestrogen withdrawal from cells. These observations increase our understanding of the mechanisms controlling bone formation and bone cell death and may aid in the development of enhanced therapeutics for the treatment of osteoporosis.The final study of this thesis aimed to determine if the mechano-biological response of osteoblasts is impaired during oestrogen deficiency and whether changes in bone mineralisation may be related to altered bone formation in response to mechanical stimulation. Osteoblasts were pre-treated with oestrogen and subsequently oestrogen was withdrawn from cell cultures and their responses under fluid shear stress were evaluated. Firstly, daily loading cycles, using an orbital rotator, were applied to cells and mineralisation and cell viability (using alamar blue assay) were assessed after 7 and 14 days. In a separate experiment, following 2 and 7 days of oestrogen withdrawal, osteoblasts were exposed to 2 hours of shear stress in a custom designed parallel plate bioreactor. PGE2 was quantified in cell culture conditioned media using an immunoassay kit. It was found that orbital fluid flow induced shear stress significantly increased mineral production by bone cells and that under an applied shear stress, mineral production was decreased during oestrogen withdrawal. It was also observed that mechanical loading and oestrogen are required in unison to promote mineral production.PGE2 release was significantly increased with applied laminar flow, but was decreased by oestrogen withdrawal. Together, these studies provide evidence that bone cells become accustomed to levels of circulating oestrogen and that diminished oestrogen causes osteocyte apoptosis, increased osteoblast mineralisation and altered mechano-sensitivity. These changes might explain the decreased mean concentrations of mineral, together with increased mineral heterogeneity, from our earlier in vivo studies. Therefore, the results of the thesis provide a unique insight into why the tightly coupled mechanisms of matching bone’s structure and composition to the loads it experiences are disrupted when levels of circulating oestrogen are depleted

Reconstruction of Large Skeletal Defects: Current Clinical Therapeutic Strategies and Future Directions Using 3D Printing

International audienceThe 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

Advances in therapeutic applications of extracellular vesicles

International audienceExtracellular vesicles (EVs) are nanometer-sized, lipid membrane-enclosed vesicles secreted by most, if not all, cells and contain lipids, proteins, and various nucleic acid species of the source cell. EVs act as important mediators of intercellular communication that influence both physiological and pathological conditions. Given their ability to transfer bioactive components and surmount biological barriers, EVs are increasingly being explored as potential therapeutic agents. EVs can potentiate tissue regeneration, participate in immune modulation, and function as potential alternatives to stem cell therapy, and bioengineered EVs can act as delivery vehicles for therapeutic agents. Here, we cover recent approaches and advances of EV-based therapies

Mimicking the Biochemical and Mechanical Extracellular Environment of the Endochondral Ossification Process to Enhance the In Vitro Mineralization Potential of Human Mesenchymal Stem Cells

International audienceChondrogenesis and mechanical stimulation of the cartilage template are essential for bone formation through the endochondral ossification process in vivo. Recent studies have demonstrated that in vitro regeneration strategies that mimic these aspects separately, either chondrogenesis or mechanical stimulation, can promote mineralization to a certain extent both in vitro and in vivo. However, to date no study has sought to incorporate both the formation of the cartilage template and the application of mechanical stimulation simultaneously to induce osteogenesis. In this study, we test the hypothesis that mimicking both the biochemical and mechanical extracellular environment arising during endochondral ossification can enhance the in vitro mineralization potential of human mesenchymal stem cells (hMSCs). hMSC aggregates were cultured for 21 days under the following culture conditions; (1) Growth Medium-hydrostatic pressure (HP), (2) Chondrogenic Priming–HP, (3) Growth Medium + HP, and (4) Chondrogenic Priming +HP. Each group was then further cultured for another 21 days in the presence of osteogenic growth factors without HP. Biochemical (DNA, sulfate glycosaminoglycan, hydroxyproline, alkaline phosphatase activity, and calcium), histological (Alcian Blue and Alizarin Red), and immunohistological (Col I, II, and X, and BSP-2) analyses were conducted to investigate chondrogenic and osteogenic differentiation at various time points (14, 21, 35, and 42 days). Our results showed the application of HP-induced chondrogenesis similar to that of chondrogenic priming, but interestingly, there was a reduction in hypertrophy markers (collagen type X) by applying HP alone versus chon-drogenic priming alone. Moreover, the results showed that both chondrogenic priming and HP in tandem during the priming period, followed by culture in osteogenic medium, accelerated the osteogenic potential of hMSCs

Bone regeneration strategies with bone marrow stromal cells in orthopaedic surgery

International audienceBone is the most transplanted tissue human with 1 million procedures every year in Europe. Surgical interventions for bone repair are required for varied reasons such as trauma resulting non-union fractures, or diseases including osteoporosis or osteonecrosis. Autologous bone grafting is the gold standard in bone regeneration but it requires a second surgery with associated pain and complications, and is also limited by harvested bone quantity. Synthetic bone substitutes lack the osteoinductive properties to heal large bone defects. Cell therapies based on bone marrow or ex vivo expanded mesenchymal stromal stem cells (MSCs) in association with synthetic calcium phosphate (CaP) bone substitutes may be alternatives to autologous bone grafting. This manuscript reviews the different conventional biological and synthetic bone grafting procedures as well as the more recently introduced cell therapy approaches used in orthopaedic surgery for bone regeneration. Some clinical studies have demonstrated safety and efficacy of these approaches but regeneration of large bone defects remain challenging due to the absence of rapid and adequate vascularisation. Future directions in the field of bone regeneration are presented, such as testing alternative cell sources or in situ fabrication of vascularized bone grafts in patients

Pre-clinical studies of bone regeneration with human bone marrow stromal cells and biphasic calcium phosphate.

International audienceRepair of large bone defects remains a significant clinical challenge. Bone marrow stromal cells (BMSCs), a subset of which is known as bone marrow-derived mesenchymal stem cells, show therapeutic potential for bone regeneration. However, their isolation, expansion and implantation will need to be conducted under good manufacturing practices (GMP) at separate locations. An investigation which mimics this clinical scenario where large bone defects shall be regenerated is required before clinical trials can be initiated. Seven batches of 100 million human ex-vivo expanded BMSCs from five donors were transported fresh in syringes from a GMP facility in Germany to France. BMSCs were mixed with biphasic calcium phosphate (BCP) biomaterial prior to subcutaneous implantation in nude mice. The capacity of BMSCs in unison with BCP to regenerate critical sized cranial bone defects was also evaluated. BMSCs expressing luciferase were used to assess the viability and bio-distribution of implanted cells. In situ hybridization, using the human-specific repetitive Alu sequence, was performed for the identification of human cells in explants. Eight weeks after implantation of BMSCs, mineralized bone containing mature bone marrow territories was formed in ectopic sites and in calvaria defects. Significant loss of cell viability was observed by bioluminescence imaging and only 1.5 percent of the initial number of transplanted cells remained after 37 days. After eight weeks, while explants were comprised primarily of host cells, there were also human cells attached along the periphery of BCP and embedded in osteocyte lacunae dispersed throughout the newly formed bone matrix. This study demonstrates the safety and efficacy of BMSC/BCP combinations and provides crucial information for the implementation of BMSC therapy for bone regeneration