Solvent-Free Preparation of Porous Poly(L-Lactide) Microcarriers for Cell Culture and Bone Tissue Engineering

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3D-printed scaffold combined to 2D osteoinductive coatings to repair a critical-size mandibular bone defect

International audienceThe reconstruction of large bone defects (12 cm3) remains a challenge for clinicians. We developed a new critical-size mandibular bone defect model on a minipig, close to human clinical issues. We analyzed the bone reconstruction obtained by a 3D-printed scaffold made of clinical-grade polylactic acid (PLA), coated with a polyelectrolyte film delivering an osteogenic bioactive molecule (BMP-2). We compared the results (computed tomography scans, microcomputed tomography scans, histology) to the gold standard solution, bone autograft. We demonstrated that the dose of BMP-2 delivered from the scaffold significantly influenced the amount of regenerated bone and the repair kinetics, with a clear BMP-2 dose-dependence. Bone was homogeneously formed inside the scaffold without ectopic bone formation. The bone repair was as good as for the bone autograft. The BMP-2 doses applied in our study were reduced 20- to 75-fold compared to the commercial collagen sponges used in the current clinical applications, without any adverse effects. Three-dimensional printed PLA scaffolds loaded with reduced doses of BMP-2 may be a safe and simple solution for large bone defects faced in the clinic

Heparan sulfate co-immobilized with cRGD ligands and BMP2 on biomimetic platforms promotes BMP2-mediated osteogenic differentiation

The chemical and physical properties of the extracellular matrix (ECM) are known to be fundamental for regulating growth factor bioactivity. The role of heparan sulfate (HS), a glycosaminoglycan, and of cell adhesion proteins (containing the cyclic RGD (cRGD) ligands) on bone morphogenetic protein 2 (BMP2)-mediated osteogenic differentiation has not been fully explored. In particular, it is not known whether and how their effects can be potentiated when they are presented in controlled close proximity, as in the ECM. Here, we developed streptavidin platforms to mimic selective aspects of the in vivo presentation of cRGD, HS and BMP2, with a nanoscale-control of their surface density and orientation to study cell adhesion and osteogenic differentiation. We showed that whereas a controlled increase in cRGD surface concentration upregulated BMP2 signaling due to β3 integrin recruitment, silencing either β1 or β3 integrins negatively affected BMP2-mediated phosphorylation of SMAD1/5/9 and alkaline phosphatase expression. Furthermore, the presence of adsorbed BMP2 promoted cellular adhesion at very low cRGD concentrations. Finally, we proved that HS co-immobilized with cRGD both sustained BMP2 signaling and enhanced osteogenic differentiation compared to BMP2 directly immobilized on streptavidin, even with a low cRGD surface concentration. Altogether, our results show that HS facilitated and sustained the synergy between BMP2 and integrin pathways and that the co-immobilization of HS and cRGD peptides optimised BMP2-mediated osteogenic differentiation

Impact of heterozygous ALK1 mutations on the transcriptomic response to BMP9 and BMP10 in endothelial cells from hereditary hemorrhagic telangiectasia and pulmonary arterial hypertension donors

International audienceAbstract Heterozygous activin receptor-like kinase 1 ( ALK1 ) mutations are associated with two vascular diseases: hereditary hemorrhagic telangiectasia (HHT) and more rarely pulmonary arterial hypertension (PAH). Here, we aimed to understand the impact of ALK1 mutations on BMP9 and BMP10 transcriptomic responses in endothelial cells. Endothelial colony-forming cells (ECFCs) and microvascular endothelial cells (HMVECs) carrying loss of function ALK1 mutations were isolated from newborn HHT and adult PAH donors, respectively. RNA-sequencing was performed on each type of cells compared to controls following an 18 h stimulation with BMP9 or BMP10. In control ECFCs, BMP9 and BMP10 stimulations induced similar transcriptomic responses with around 800 differentially expressed genes (DEGs). ALK1 -mutated ECFCs unexpectedly revealed highly similar transcriptomic profiles to controls, both at the baseline and upon stimulation, and normal activation of Smad1/5 that could not be explained by a compensation in cell-surface ALK1 level. Conversely, PAH HMVECs revealed strong transcriptional dysregulations compared to controls with > 1200 DEGs at the baseline. Consequently, because our study involved two variables, ALK1 genotype and BMP stimulation, we performed two-factor differential expression analysis and identified 44 BMP9-dysregulated genes in mutated HMVECs, but none in ECFCs. Yet, the impaired regulation of at least one hit, namely lunatic fringe ( LFNG ), was validated by RT-qPCR in three different ALK1 -mutated endothelial models. In conclusion, ALK1 heterozygosity only modified the BMP9/BMP10 regulation of few genes, including LFNG involved in NOTCH signaling. Future studies will uncover whether dysregulations in such hits are enough to promote HHT/PAH pathogenesis, making them potential therapeutic targets, or if second hits are necessary