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.

Although isolated reports of hard-tissue reconstruction in the cranio-maxillofacial skeleton exist, multipatient case series are lacking. This study aimed to review the experience with 13 consecutive cases of cranio-maxillofacial hard-tissue defects at four anatomically different sites, namely frontal sinus (3 cases), cranial bone (5 cases), mandible (3 cases), and nasal septum (2 cases). Autologous adipose tissue was harvested from the anterior abdominal wall, and adipose-derived stem cells were cultured, expanded, and then seeded onto resorbable scaffold materials for subsequent reimplantation into hard-tissue defects. The defects were reconstructed with either bioactive glass or β-tricalcium phosphate scaffolds seeded with adipose-derived stem cells (ASCs), and in some cases with the addition of recombinant human bone morphogenetic protein-2. Production and use of ASCs were done according to good manufacturing practice guidelines. Follow-up time ranged from 12 to 52 months. Successful integration of the construct to the surrounding skeleton was noted in 10 of the 13 cases. Two cranial defect cases in which nonrigid resorbable containment meshes were used sustained bone resorption to the point that they required the procedure to be redone. One septal perforation case failed outright at 1 year because of the postsurgical resumption of the patient's uncontrolled nasal picking habit.

There is no optimal method for reconstruction of large calvarial defects. Because of the limitations of autologous bone grafts and alloplastic materials, new methods for performing cranioplasties are needed. To create autologous bone to repair cranial defects. We performed a cranioplasty procedure with this new method in 4 patients who had large calvarial defects of different etiologies. We used autologous adipose-derived stem cells seeded in beta-tricalcium phosphate granules. For 2 patients, we used a bilaminate technique with resorbable mesh. During follow-up, there were no clinically relevant postoperative complications. The computed tomography scans revealed satisfactory outcome in ossification, and in the clinical examinations, the outcomes were good. The cranioplasty was measured in Hounsfield units from each computed tomography scan. The Hounsfield units increased gradually to equal the value of bone. The combination of scaffold material such as beta-tricalcium phosphate and autologous adipose-derived stem cells constitutes a promising model for reconstruction of human large cranial defects. The success of these clinical cases paves way for further studies and clinical applications to turn this method into a reliable treatment regimen.

Introduction: Currently, human adipose stem cells (hASCs) are differentiated towards osteogenic lineages using culture medium supplemented with L-ascorbic acid 2-phosphate (AsA2-P), dexamethasone (Dex) and beta-glycerophosphate (β-GP). Because this osteogenic medium (OM1) was initially generated for the differentiation of bone marrow-derived mesenchymal stem cells, the component concentrations may not be optimal for the differentiation of hASCs. After preliminary screening, two efficient osteogenic media (OM2 and OM3) were chosen to be compared with the commonly used osteogenic medium (OM1). To further develop the culture conditions towards clinical usage, the osteo-inductive efficiencies of OM1, OM2 and OM3 were compared using human serum (HS)-based medium and a defined, xeno-free medium (RegES), with fetal bovine serum (FBS)-based medium serving as a control. Methods: To compare the osteo-inductive efficiency of OM1, OM2 and OM3 in FBS-, HS- and RegES-based medium, the osteogenic differentiation was assessed by alkaline phosphatase (ALP) activity, mineralization, and expression of osteogenic marker genes (runx2A, DLX5, collagen type I, osteocalcin, and ALP). Results: In HS-based medium, the ALP activity increased significantly by OM3, and mineralization was enhanced by both OM2 and OM3, which have high AsA2-P and low Dex concentrations. ALP activity and mineralization of hASCs was the weakest in FBS-based medium, with no significant differences between the OM compositions due to donor variation. However, the qRT-PCR data demonstrated significant upregulation of runx2A mRNA under osteogenic differentiation in FBS- and HS-based medium, particularly by OM3 under FBS conditions. Further, the expression of DLX5 was greatly stimulated by OM1 to 3 on day 7 when compared to control. The regulation of collagen type I, ALP, and osteocalcin mRNA was modest under induction by OM1 to 3. The RegES medium was found to support the proliferation and osteogenic differentiation of hASCs, but the composition of the RegES medium hindered the comparison of OM1, OM2 and OM3. Conclusions: Serum conditions affect hASC proliferation and differentiation significantly. The ALP activity and mineralization was the weakest in FBS-based medium, although osteogenic markers were upregulated on mRNA level. When comparing the OM composition, the commonly used OM1 was least effective. Accordingly, higher concentration of AsA2-P and lower concentration of Dex, as in OM2 and OM3, should be used for the osteogenic differentiation of hASCs in vitro.