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

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly

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

Biomaterials Functionalized with MSC Secreted Extracellular Vesicles and Soluble Factors for Tissue Regeneration

International audienceAbstract The therapeutic benefits of mesenchymal stromal cell (MSC) transplantation are attributed to their secreted factors, including extracellular vesicles (EVs) and soluble factors. The potential of employing the MSC secretome as an alternative acellular approach to cell therapy is being investigated in various tissue injury indications, but EVs administered via bolus injections are rapidly sequestered and cleared. However, biomaterials offer delivery platforms to enhance EV retention rates and healing efficacy. This review highlights the mechanisms underpinning the therapeutic effects of MSC‐EVs and soluble factors as effectors of immunomodulation and tissue regeneration, conferred primarily via their nucleic acid and protein contents. Discussed is how manipulating the cell culture microenvironment or genetic modification of MSCs can further augment the potency of their secretions. The most recent advances in the development of EV‐functionalized biomaterials that mediate enhanced angiogenesis and cell survival, while attenuating inflammation and fibrosis, are presented. Finally, some technical challenges to be considered for the clinical translation of biomaterials carrying MSC‐secreted bioactive cargo are discussed

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

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

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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