Patient-Specific Bioimplants and Reconstruction Plates for Mandibular Defects: Production Workflow and In Vivo Large Animal Model Study.

A major challenge with extensive craniomaxillofacial bone reconstruction is the limited donor-site availability to reconstruct defects predictably and accurately according to the anatomical shape of the patient. Here, patient-specific composite bioimplants, consisting of cross-linked poly(trimethylene carbonate) (PTMC) networks and ß-tricalcium phosphate (ß-TCP), are tested in vivo in twelve Göttingen minipigs in a large mandibular continuity defect model. The 25 mm defects are supported by patient-specific titanium reconstruction plates and receive either osteoconductive composite bioimplants (PTMC+TCP), neat polymer network bioimplants (PTMC), autologous bone segments (positive control), or are left empty (negative control). Postoperatively, defects treated with bioimplants show evident ossification at 24 weeks. Histopathologic evaluation reveals that neat PTMC bioimplant surfaces are largely covered with fibrous tissue, while in the PTMC+TCP bioimplants, bone attached directly to the implant surface shows good osteoconduction and histological signs of osteoinductivity. However, PTMC+TCP bioimplants are associated with high incidence of necrosis and infection, possibly due to rapid resorption and/or particle size of the used ß-TCP. The study highlights the importance of testing bone regeneration implants in a clinically relevant large animal model and at the in situ reconstruction site, since results on small animal models and studies in nonloadbearing areas do not translate directly.

Epigenetic alterations in mesenchymal stem cells by osteosarcoma-derived extracellular vesicles.

Extracellular vesicles (EVs) are central to intercellular communication and play an important role in cancer progression and development. Osteosarcoma (OS) is an aggressive bone tumour, characterized by the presence of malignant mesenchymal cells. The specific tumour-driving genetic alterations that are associated with OS development are currently poorly understood. Mesenchymal stem cells (MSCs) of osteogenic lineage have been postulated as likely candidates as the cells of origin for OS, thus indicating that MSCs and OS stroma cells may be related cell types. Therefore, this study set out to examine the EV-mediated intercellular crosstalk of MSCs and OS. MSCs and pre-osteoblasts were treated with OS-EVs at different time points, and the epigenetic signature of OS-EVs was assessed by methylation analysis of LINE-1 (long interspersed element) and tumour suppressor genes. In addition, surface markers and expression of specific genes were also evaluated. Our data indicated that OS-EVs mediated LINE-1 hypomethylation in MSCs, whereas an opposite effect was seen in pre-osteoblasts, indicating that MSCs but not pre-osteoblasts were susceptible to epigenetic transformation. Thus, OS-EVs modulated the fate of MSCs by modulating the epigenetic status, and also influenced the expression of genes related to bone microenvironment remodelling. Overall, this study provided evidence that epigenetic regulation appears to be an early event in the transformation of MSCs during the development of OS. Elucidating the mechanisms of EV-mediated communication may lead to new avenues for therapeutic exploitation.

Monocyte-derived extracellular vesicles stimulate cytokine secretion and gene expression of matrix metalloproteinases by mesenchymal stem/stromal cells.

Intercellular communication is essential in bone remodelling to ensure that new bone is formed with only temporary bone loss. Monocytes (MCs) and osteoclasts actively take part in controlling bone remodelling by providing signals that promote osteogenic differentiation of mesenchymal stem/stromal cells (MSCs). Extracellular vesicles (EVs) have attracted attention as regulators of bone remodelling. EVs facilitate intercellular communication by transferring a complex cargo of biologically active molecules to target cells. In the present study, we evaluated the potency of EVs from MCs and osteoclasts to induce a lineage-specific response in MSCs. We analysed gene expression and protein secretion by both adipose tissue-derived MSCs and bone marrow-derived MSCs after stimulation with EVs from lipopolysaccharide-activated primary human MCs and (mineral-resorbing) osteoclasts. Isolated EVs were enriched in exosomes (EVs of endosomal origin) and were free of cell debris. MC- and osteoclast-derived EVs were taken up by adipose tissue-derived MSCs. EVs from activated MCs promoted the secretion of cytokines by MSCs, which may represent an immunomodulatory mechanism. MC-derived EVs also upregulated the expression of genes encoding for matrix metalloproteinases. Therefore, we hypothesize that MCs facilitate tissue remodelling through EV-mediated signalling. We did not observe a significant effect of osteoclast-derived EVs on gene expression or protein secretion in MSCs. EV-mediated signalling might represent an additional mode of cell-cell signalling during the transition from injury and inflammation to bone regeneration and play an important role in the coupling between bone resorption and bone formation. DATABASE: Gene expression data are available in the GEO database under the accession number GSE102401.

Efficient ultrafiltration-based protocol to deplete extracellular vesicles from fetal bovine serum.

Fetal bovine serum (FBS) is the most commonly used supplement in studies involving cell-culture experiments. However, FBS contains large numbers of bovine extracellular vesicles (EVs), which hamper the analyses of secreted EVs from the cell type of preference and, thus, also the downstream analyses. Therefore, a prior elimination of EVs from FBS is crucial. However, the current methods of EV depletion by ultracentrifugation are cumbersome and the commercial alternatives expensive. In this study, our aim was to develop a protocol to completely deplete EVs from FBS, which may have wide applicability in cell-culture applications. We investigated different EV-depleted FBS prepared by our novel ultrafiltration-based protocol, by conventionally used overnight ultracentrifugation, or commercially available depleted FBS, and compared them with regular FBS. All sera were characterized by nanoparticle tracking analysis, electron microscopy, Western blotting and RNA quantification. Next, adipose-tissue mesenchymal stem cells (AT-MSCs) and cancer cells were grown in the media supplemented with the three different EV-depleted FBS and compared with cells grown in regular FBS media to assess the effects on cell proliferation, stress, differentiation and EV production. The novel ultrafiltration-based protocol depleted EVs from FBS clearly more efficiently than ultracentrifugation and commercial methods. Cell proliferation, stress, differentiation and EV production of AT-MSCs and cancer cell lines were similarly maintained in all three EV-depleted FBS media up to 96 h. In summary, our ultrafiltration protocol efficiently depletes EVs, is easy to use and maintains cell growth and metabolism. Since the method is also cost-effective and easy to standardize, it could be used in a wide range of cell-culture applications helping to increase comparability of EV research results between laboratories.