المؤلفون: Loïc Fiévet, Nicolas Espagnolle, Daniela Gerovska, David Bernard, Charlotte Syrykh, Camille Laurent, Pierre Layrolle, Julien De Lima, Arthur Justo, Nicolas Reina, Louis Casteilla, Marcos J. Araúzo-Bravo, Abderrahim Naji, Jean-Christophe Pagès, Frédéric Deschaseaux

المصدر: Stem Cell Research & Therapy, Vol 14, Iss 1, Pp 1-16 (2023)

مصطلحات موضوعية: Mesenchymal stromal cells, Single-cell RNA sequencing, Characterization, Transcriptomic, Human bone marrow, Biomarkers, Medicine (General), R5-920, Biochemistry, QD415-436

الوصف: Abstract Background Native bone marrow (BM) mesenchymal stem/stromal cells (BM-MSCs) participate in generating and shaping the skeleton and BM throughout the lifespan. Moreover, BM-MSCs regulate hematopoiesis by contributing to the hematopoietic stem cell niche in providing critical cytokines, chemokines and extracellular matrix components. However, BM-MSCs contain a heterogeneous cell population that remains ill-defined. Although studies on the taxonomy of native BM-MSCs in mice have just started to emerge, the taxonomy of native human BM-MSCs remains unelucidated. Methods By using single-cell RNA sequencing (scRNA-seq), we aimed to define a proper taxonomy for native human BM non-hematopoietic subsets including endothelial cells (ECs) and mural cells (MCs) but with a focal point on MSCs. To this end, transcriptomic scRNA-seq data were generated from 5 distinct BM donors and were analyzed together with other transcriptomic data and with computational biology analyses at different levels to identify, characterize and classify distinct native cell subsets with relevant biomarkers. Results We could ascribe novel specific biomarkers to ECs, MCs and MSCs. Unlike ECs and MCs, MSCs exhibited an adipogenic transcriptomic pattern while co-expressing genes related to hematopoiesis support and multilineage commitment potential. Furthermore, by a comparative analysis of scRNA-seq of BM cells from humans and mice, we identified core genes conserved in both species. Notably, we identified MARCKS, CXCL12, PDGFRA, and LEPR together with adipogenic factors as archetypal biomarkers of native MSCs within BM. In addition, our data suggest some complex gene nodes regulating critical biological functions of native BM-MSCs together with a preferential commitment toward an adipocyte lineage. Conclusions Overall, our taxonomy for native BM non-hematopoietic compartment provides an explicit depiction of gene expression in human ECs, MCs and MSCs at single-cell resolution. This analysis helps enhance our understanding of the phenotype and the complexity of biological functions of native human BM-MSCs.

وصف الملف: electronic resource

العلاقة: https://doaj.org/toc/1757-6512Test

الوصول الحر: https://doaj.org/article/d1b56f19350846568ce3efee6a9a7deeTest

المؤلفون: Paul Humbert, Meadhbh Á. Brennan, Julien De Lima, Régis Brion, Annie Adrait, Céline Charrier, Bénédicte Brulin, Valérie Trichet, Yohann Couté, Frédéric Blanchard, Pierre Layrolle

المصدر: Scientific Reports, Vol 11, Iss 1, Pp 1-14 (2021)

مصطلحات موضوعية: Medicine, Science

الوصف: Abstract In bone regeneration induced by the combination of mesenchymal stromal cells (MSCs) and calcium-phosphate (CaP) materials, osteoclasts emerge as a pivotal cell linking inflammation and bone formation. Favorable outcomes are observed despite short-term engraftments of implanted MSCs, highlighting their major paracrine function and the possible implication of cell death in modulating their secretions. In this work, we focused on the communication from MSCs towards osteoclasts-like cells in vitro. MSCs seeded on a CaP biomaterial or undergoing induced apoptosis produced a conditioned media favoring the development of osteoclasts from human CD14+ monocytes. On the contrary, MSCs’ apoptotic secretion inhibited the development of inflammatory multinucleated giant cells formed after IL-4 stimulation. Components of MSCs’ secretome before and after apoptotic stress were compared using mass spectrometry-based quantitative proteomics and a complementary immunoassay for major cytokines. CXCR-1 and CXCR-2 ligands, primarily IL-8/CXCL-8 but also the growth-regulated proteins CXCL-1, -2 or -3, were suggested as the major players of MSCs’ pro-osteoclastic effect. These findings support the hypothesis that osteoclasts are key players in bone regeneration and suggest that apoptosis plays an important role in MSCs’ effectiveness.

وصف الملف: electronic resource

العلاقة: https://doaj.org/toc/2045-2322Test

الوصول الحر: https://doaj.org/article/8c0605303bff4042827151db8a18481cTest

المؤلفون: Julien Herman, Benoit Le Goff, Julien De Lima, Régis Brion, Catherine Chevalier, Frédéric Blanchard, Christelle Darrieutort-Laffite

المصدر: Arthritis Research & Therapy, Vol 23, Iss 1, Pp 1-11 (2021)

مصطلحات موضوعية: Apatite, Rotator cuff tendons, Interleukin-1, Inflammasome, Air pouch model, Diseases of the musculoskeletal system, RC925-935

الوصف: Abstract Background Calcific tendonitis of the rotator cuff is due to carbonated apatite deposits in the shoulder tendons. During the evolution of the disease, an acute inflammatory episode may occur leading to the disappearance of the calcification. Although hydroxyapatite crystal-induced inflammation has been previously studied with synthetic crystals, no data are available with calcifications extracted from patients suffering from calcific tendinopathy. The objective of the study was to explore the inflammatory properties of human calcifications and the pathways involved. Methods Human calcifications and synthetic hydroxyapatite were used in vitro to stimulate human monocytes and macrophages, the human myeloid cell line THP-1, and human tenocytes. The release of IL-1β, IL-6, and IL-8 by cells was quantified by ELISA. The gene expression of pro- and anti-inflammatory cytokines was evaluated by quantitative PCR. NF-kB activation and NLRP3 involvement were assessed in THP-1 cells using a NF-kB inhibitor and a caspase-1 inhibitor. The inflammatory properties were then assessed in vivo using a mouse air pouch model. Results Human calcifications were able to induce a significant release of IL-1β when incubated with monocytes, macrophages, and THP-1 only if they were first primed with LPS (monocytes and macrophages) or PMA (THP-1). Stimulation of THP-1 by human calcifications led to similar levels of IL-1β when compared to synthetic hydroxyapatite although these levels were significantly inferior in monocytes and macrophages. The patient’s crystals enhanced mRNA expression of pro-IL-1β, as well as IL-18, NF-kB, and TGFβ when IL-6 and TNFα expression were not. IL-1β production was reduced by the inhibition of caspase-1 indicating the role of NLRP3 inflammasome. In vivo, injection of human calcifications or synthetic hydroxyapatite in the air pouch led to a significant increase in membrane thickness although significant overexpression of IL-1β was only observed for synthetic hydroxyapatite. Conclusions As synthetic hydroxyapatite, human calcifications were able to induce an inflammatory response resulting in the production of IL-1β after NF-kB activation and through NLRP3 inflammasome. In some experiments, IL-1β induction was lower with human calcifications compared to synthetic apatite. Differences in size, shape, and protein content may explain this observation.

وصف الملف: electronic resource

العلاقة: https://doaj.org/toc/1478-6362Test

الوصول الحر: https://doaj.org/article/b19c7c275c67410ba2bb12dee497d5c3Test

المؤلفون: Meadhbh Á. Brennan, Mario Barilani, Francesco Rusconi, Julien de Lima, Luciano Vidal, Cristiana Lavazza, Lorenza Lazzari, Rosaria Giordano, Pierre Layrolle

المصدر: Scientific Reports, Vol 11, Iss 1, Pp 1-15 (2021)

مصطلحات موضوعية: Medicine, Science

الوصف: Abstract Bone marrow mesenchymal stem/stromal cells (BMSCs) show great promise for bone repair, however they are isolated by an invasive bone marrow harvest and their regenerative potential decreases with age. Conversely, cord blood can be collected non-invasively after birth and contains MSCs (CBMSCs) that can be stored for future use. However, whether CBMSCs can replace BMSCs targeting bone repair is unknown. This study evaluates the in vitro osteogenic potential of unprimed, osteogenically primed, or chondrogenically primed CBMSCs and BMSCs and their in vivo bone forming capacity following ectopic implantation on biphasic calcium phosphate ceramics in nude mice. In vitro, alkaline phosphatase (intracellular, extracellular, and gene expression), and secretion of osteogenic cytokines (osteoprotegerin and osteocalcin) was significantly higher in BMSCs compared with CBMSCs, while CBMSCs demonstrated superior chondrogenic differentiation and secretion of interleukins IL-6 and IL-8. BMSCs yielded significantly more cell engraftment and ectopic bone formation compared to CBMSCs. However, priming of CBMSCs with either chondrogenic or BMP-4 supplements led to bone formation by CBMSCs. This study is the first direct quantification of the bone forming abilities of BMSCs and CBMSCs in vivo and, while revealing the innate superiority of BMSCs for bone repair, it provides avenues to induce osteogenesis by CBMSCs.

وصف الملف: electronic resource

العلاقة: https://doaj.org/toc/2045-2322Test

الوصول الحر: https://doaj.org/article/7a981bfc3e294427a1124816c78b1f10Test

المؤلفون: Paul Humbert, Julien De Lima, Meadhbh Á. Brennan, Frédéric Blanchard, Pierre Layrolle

المصدر: Bone Reports, Vol 13, Iss , Pp 100371- (2020)

مصطلحات موضوعية: Diseases of the musculoskeletal system, RC925-935

وصف الملف: electronic resource

العلاقة: http://www.sciencedirect.com/science/article/pii/S2352187220301315Test; https://doaj.org/toc/2352-1872Test

الوصول الحر: https://doaj.org/article/1cc2fc0fbb8443c6b703874399e1ea07Test

المؤلفون: Fiona E. Freeman, Meadhbh Á. Brennan, David C. Browe, Audrey Renaud, Julien De Lima, Daniel J. Kelly, Laoise M. McNamara, Pierre Layrolle

المصدر: Frontiers in Bioengineering and Biotechnology, Vol 8 (2020)

مصطلحات موضوعية: endochondral ossification, intramembranous ossification, bone tissue engineering, pre-vascularization, mesenchymal stem cells, Biotechnology, TP248.13-248.65

الوصف: There is a distinct clinical need for new therapies that provide an effective treatment for large bone defect repair. Herein we describe a developmental approach, whereby constructs are primed to mimic certain aspects of bone formation that occur during embryogenesis. Specifically, we directly compared the bone healing potential of unprimed, intramembranous, and endochondral primed MSC-laden polycaprolactone (PCL) scaffolds. To generate intramembranous constructs, MSC-seeded PCL scaffolds were exposed to osteogenic growth factors, while endochondral constructs were exposed to chondrogenic growth factors to generate a cartilage template. Eight weeks after implantation into a cranial critical sized defect in mice, there were significantly more vessels present throughout defects treated with endochondral constructs compared to intramembranous constructs. Furthermore, 33 and 50% of the animals treated with the intramembranous and endochondral constructs respectively, had full bone union along the sagittal suture line, with significantly higher levels of bone healing than the unprimed group. Having demonstrated the potential of endochondral priming but recognizing that only 50% of animals completely healed after 8 weeks, we next sought to examine if we could further accelerate the bone healing capacity of the constructs by pre-vascularizing them in vitro prior to implantation. The addition of endothelial cells alone significantly reduced the healing capacity of the constructs. The addition of a co-culture of endothelial cells and MSCs had no benefit to either the vascularization or mineralization potential of the scaffolds. Together, these results demonstrate that endochondral priming alone is enough to induce vascularization and subsequent mineralization in a critical-size defect.

وصف الملف: electronic resource

العلاقة: https://www.frontiersin.org/article/10.3389/fbioe.2020.00230/fullTest; https://doaj.org/toc/2296-4185Test

الوصول الحر: https://doaj.org/article/bf0e78b1e8b84b379f0b53a4593a193dTest

المؤلفون: Julien Herman (10727197), Benoit Le Goff (9658398), Julien De Lima (8640990), Régis Brion (530868), Catherine Chevalier (197486), Frédéric Blanchard (9511728), Christelle Darrieutort-Laffite (9658389)

مصطلحات موضوعية: Biochemistry, Medicine, Microbiology, Cell Biology, Molecular Biology, Physiology, Immunology, Developmental Biology, Cancer, Infectious Diseases, Space Science, Chemical Sciences not elsewhere classified, Apatite, Rotator cuff tendons, Interleukin-1, Inflammasome, Air pouch model

الوصف: Additional file 1: Supplementary Figure S1. IL-1β release after 6h stimulation of CD14+ monocytes, MCSF-Macrophages, GM-CSF/IFNγ-Macrophages by crystals at 250, 500 and 1000 ng/ml in presence of LPS (lipopolysaccharide) and parallel cell death rate. sHA= synthetic hydroxyapatite, CAL = human calcification CT= control group without crystal; LPS= lipopolysaccharide. n = 3. * p≤ 0,05. Supplementary Figure S2. Western Blot raw images of sHA (positive control) and three patients. Cleaved IL-1β was studied in cellular lysates (CL) and in supernatants (SN). iNF-κB= NF-κB inhibitor; iCasp1= caspase-1 inhibitor; inh= inhibitor; sHA= synthetic hydroxyapatite; CAL= human calcification; CT = control condition without crystals. Supplementary Table 1. Primers used for quantitative Polymerase Chain Reaction.

العلاقة: https://figshare.com/articles/journal_contribution/Additional_file_1_of_Pro-inflammatory_effects_of_human_apatite_crystals_extracted_from_patients_suffering_from_calcific_tendinopathy/14516698Test

الإتاحة: https://doi.org/10.6084/m9.figshare.14516698.v1Test

المؤلفون: Catherine Chevalier, Julien Herman, Frédéric Blanchard, Julien De Lima, Christelle Darrieutort-Laffite, Benoit Le Goff, Régis Brion

المصدر: Arthritis Research & Therapy, Vol 23, Iss 1, Pp 1-11 (2021)
Arthritis Research & Therapy

مصطلحات موضوعية: 0301 basic medicine, Rotator cuff tendons, Pathology, medicine.medical_specialty, Myeloid, Inflammasomes, Interleukin-1beta, Inflammation, Diseases of the musculoskeletal system, Inflammasome, 03 medical and health sciences, 0302 clinical medicine, In vivo, Apatites, NLR Family, Pyrin Domain-Containing 3 Protein, medicine, Humans, Apatite, 030203 arthritis & rheumatology, Chemistry, Caspase 1, Air pouch model, Interleukin, medicine.disease, In vitro, 030104 developmental biology, medicine.anatomical_structure, RC925-935, Tendinopathy, Tumor necrosis factor alpha, medicine.symptom, Research Article, Interleukin-1, medicine.drug, Calcification

الوصف: Background Calcific tendonitis of the rotator cuff is due to carbonated apatite deposits in the shoulder tendons. During the evolution of the disease, an acute inflammatory episode may occur leading to the disappearance of the calcification. Although hydroxyapatite crystal-induced inflammation has been previously studied with synthetic crystals, no data are available with calcifications extracted from patients suffering from calcific tendinopathy. The objective of the study was to explore the inflammatory properties of human calcifications and the pathways involved. Methods Human calcifications and synthetic hydroxyapatite were used in vitro to stimulate human monocytes and macrophages, the human myeloid cell line THP-1, and human tenocytes. The release of IL-1β, IL-6, and IL-8 by cells was quantified by ELISA. The gene expression of pro- and anti-inflammatory cytokines was evaluated by quantitative PCR. NF-kB activation and NLRP3 involvement were assessed in THP-1 cells using a NF-kB inhibitor and a caspase-1 inhibitor. The inflammatory properties were then assessed in vivo using a mouse air pouch model. Results Human calcifications were able to induce a significant release of IL-1β when incubated with monocytes, macrophages, and THP-1 only if they were first primed with LPS (monocytes and macrophages) or PMA (THP-1). Stimulation of THP-1 by human calcifications led to similar levels of IL-1β when compared to synthetic hydroxyapatite although these levels were significantly inferior in monocytes and macrophages. The patient’s crystals enhanced mRNA expression of pro-IL-1β, as well as IL-18, NF-kB, and TGFβ when IL-6 and TNFα expression were not. IL-1β production was reduced by the inhibition of caspase-1 indicating the role of NLRP3 inflammasome. In vivo, injection of human calcifications or synthetic hydroxyapatite in the air pouch led to a significant increase in membrane thickness although significant overexpression of IL-1β was only observed for synthetic hydroxyapatite. Conclusions As synthetic hydroxyapatite, human calcifications were able to induce an inflammatory response resulting in the production of IL-1β after NF-kB activation and through NLRP3 inflammasome. In some experiments, IL-1β induction was lower with human calcifications compared to synthetic apatite. Differences in size, shape, and protein content may explain this observation.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::a18bc9d94eb8b9d074ab6b6da7d601edTest
https://doi.org/10.21203/rs.3.rs-156807/v1Test

المؤلفون: Pierre Layrolle, Julien De Lima, Paul Humbert, Frédéric Blanchard, Meadhbh Á. Brennan

المصدر: Bone Reports, Vol 13, Iss, Pp 100371-(2020)

مصطلحات موضوعية: lcsh:Diseases of the musculoskeletal system, Giant cell, Chemistry, Endocrinology, Diabetes and Metabolism, Mesenchymal stem cell, Orthopedics and Sports Medicine, lcsh:RC925-935, In vitro, Cell biology

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::29a8963b1b9abb5fe698080dea57857cTest
http://www.sciencedirect.com/science/article/pii/S2352187220301315Test

المؤلفون: Dvm Borhane H. Fellah, Meadhbh Á. Brennan, Pierre Layrolle, Philippe Rosset, Stéphanie Krissian, Julien De Lima, Alain Hoornaert, Luciano Vidal

المصدر: SSRN Electronic Journal.

مصطلحات موضوعية: medicine.medical_specialty, business.industry, medicine.medical_treatment, Adipose tissue, Histology, Stromal vascular fraction, Microsurgery, Bone tissue, Surgery, Transplantation, Neovascularization, medicine.anatomical_structure, medicine, medicine.symptom, business, Bone regeneration

الوصف: Reconstructing large bone defects caused by severe trauma or resection of tumors remains a challenge for orthopedic and plastic surgeons. Free flaps comprised of a muscle flap with fibula bone and its vascularized bed can be transplanted to the reconstruction site to achieve healing. However, this transplantation technique adds morbidity, and requires extensive microsurgery and sculpting of the bone tissue to adapt the graft to both the vasculature and the anatomy of the bone defect. The aim of the current study is to evaluate an alternative approach consisting of the in situ production of a pre-vascularized synthetic bone graft and its subsequent transplantation to a critical-sized bone defect. 3D printed chambers containing biphasic calcium phosphate (BCP) granules, perfused by a local vascular pedicle, with or without the addition of stromal vascular fraction (SVF), were subcutaneously implanted into New Zealand White female rabbits. The SVF was prepared extemporaneously from autologous adipose tissue, while the vascular pedicle was isolated from the inguinal site. Chambers filled with BCP alone served as controls. After 8 weeks, the constructs containing a vascular pedicle exhibited abundant neovascularization, with significant numbers of blood vessels sprouting from the pedicle, which was further enhanced by the addition of SVF. These pre-vascularized synthetic bone grafts were then transplanted into 15 mm critical-sized segmental ulnar defects for a further 8 weeks. Micro-CT and decalcified histology revealed that pre-vascularization of synthetic bone grafts led to significantly enhanced bone regeneration. This pre-clinical study demonstrates the feasibility and efficacy of in situ production of pre-vascularized synthetic bone grafts for regenerating large bone defects, and will address an important clinical need.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::2c82d44941c876cf01a3756737f26867Test
https://doi.org/10.2139/ssrn.3559922Test