Vertical and horizontal ridge augmentation using customized three-dimensionally printed polycaprolactone mesh in atrophic posterior maxillae: a case report.

Polycaprolactone has exhibited expediency as a biomaterial for bone regenerative procedures preclinically. The present report of the two clinical cases in the posterior maxilla is the first to describe clinical application of a customized 3D printed polycaprolactone mesh for alveolar ridge augmentation. Two patients needing extensive ridge augmentation procedures for dental implant therapy were selected. Polycaprolactone meshes were virtually designed, 3D printed and applied in combination with a xenogeneic bone substitute. Cone-beam computed tomography was taken pre-operatively, immediately after the surgery, and 1.5 to 2 years after the delivery of implant prostheses. The serial cone-beam computed tomography images were superimposed to measure the augmented height and width at 1 mm increments from the implant platform to 3 mm apically. After 2 years, the mean [maximum, minimum] bone gain was 6.05 [8.64, 2.85] mm vertically and 7.77 [10.03, 6.18] mm horizontally at 1 mm below the implant platform. From immediately postoperative to 2 years, there was 14 % reduction of augmented ridged height and 24 % reduction of augmented width at 1 mm below the platform. All implants placed in augmented sites were successfully maintained until 2 years. The customized Polycaprolactone mesh might be a viable material for ridge augmentation in the atrophic posterior maxilla. This needs to be confirmed through randomized controlled clinical trials in future studies.

Human induced pluripotent stem cell line banking for the production of rare blood type erythrocytes.

BACKGROUND: The in vitro production of mature human red blood cells (RBCs) from induced pluripotent stem cells (iPSCs) has been the focus of research to meet the high demand for blood transfusions. However, limitations like high costs and technological requirements restrict the use of RBCs produced by iPSC differentiation to specific circumstances, such as for patients with rare blood types or alloimmunized patients. In this study, we developed a detailed protocol for the generation of iPSC lines derived from peripheral blood of donors with O D-positive blood and rare blood types (D-and Jr(a-)) and subsequent erythroid differentiation. METHODS: Mononuclear cells separated from the peripheral blood of O D-positive and rare blood type donors were cultured to produce and expand erythroid progenitors and reprogrammed into iPSCs. A 31-day serum-free, xeno-free erythroid differentiation protocol was used to generate reticulocytes. The stability of iPSC lines was confirmed with chromosomal analysis and RT-PCR. Morphology and cell counts were determined by microscopy observations and flow cytometry. RESULTS: Cells from all donors were successfully used to generate iPSC lines, which were differentiated into erythroid precursors without any apparent chromosomal mutations. This differentiation protocol resulted in moderate erythrocyte yield per iPSC. CONCLUSIONS: It has previously only been hypothesized that erythroid differentiation from iPSCs could be used to produce RBCs for transfusion to patients with rare blood types or who have been alloimmunized. Our results demonstrate the feasibility of producing autologous iPSC-differentiated RBCs for clinical transfusions in patients without alternative options.

Jmjd2C increases MyoD transcriptional activity through inhibiting G9a-dependent MyoD degradation.

Skeletal muscle cell differentiation requires a family of proteins called myogenic regulatory factors (MRFs) to which MyoD belongs. The activity of MyoD is under epigenetic regulation, however, the molecular mechanism by which histone KMTs and KDMs regulate MyoD transcriptional activity through methylation remains to be determined. Here we provide evidence for a unique regulatory mechanism of MyoD transcriptional activity through demethylation by Jmjd2C demethylase whose level increases during muscle differentiation. G9a decreases MyoD stability via methylation-dependent MyoD ubiquitination. Jmjd2C directly associates with MyoD in vitro and in vivo to demethylate and stabilize MyoD. The hypo-methylated MyoD due to Jmjd2C is significantly more stable than hyper-methylated MyoD by G9a. Cul4/Ddb1/Dcaf1 pathway is essential for the G9a-mediated MyoD degradation in myoblasts. By the stabilization of MyoD, Jmjd2C increases myogenic conversion of mouse embryonic fibroblasts and MyoD transcriptional activity with erasing repressive H3K9me3 level at the promoter of MyoD target genes. Collectively, Jmjd2C increases MyoD transcriptional activity to facilitate skeletal muscle differentiation by increasing MyoD stability through inhibiting G9a-dependent MyoD degradation.

In vitro erythrocyte production using human-induced pluripotent stem cells: determining the best hematopoietic stem cell sources.

BACKGROUND: Blood transfusion is an essential part of medicine. However, many countries have been facing a national blood crisis. To address this ongoing blood shortage issue, there have been efforts to generate red blood cells (RBCs) in vitro, especially from human-induced pluripotent stem cells (hiPSCs). However, the best source of hiPSCs for this purpose is yet to be determined. METHODS: In this study, hiPSCs were established from three different hematopoietic stem cell sources-peripheral blood (PB), cord blood (CB) and bone marrow (BM) aspirates (n = 3 for each source)-using episomal reprogramming vectors and differentiated into functional RBCs. Various time-course studies including immunofluorescence assay, quantitative real-time PCR, flow cytometry, karyotyping, morphological analysis, oxygen binding capacity analysis, and RNA sequencing were performed to examine and compare the characteristics of hiPSCs and hiPSC-differentiated erythroid cells. RESULTS: hiPSC lines were established from each of the three sources and were found to be pluripotent and have comparable characteristics. All hiPSCs differentiated into erythroid cells, but there were discrepancies in differentiation and maturation efficiencies: CB-derived hiPSCs matured into erythroid cells the fastest while PB-derived hiPSCs required a longer time for maturation but showed the highest degree of reproducibility. BM-derived hiPSCs gave rise to diverse types of cells and exhibited poor differentiation efficiency. Nonetheless, erythroid cells differentiated from all hiPSC lines mainly expressed fetal and/or embryonic hemoglobin, indicating that primitive erythropoiesis occurred. Their oxygen equilibrium curves were all left-shifted. CONCLUSIONS: Collectively, both PB- and CB-derived hiPSCs were favorably reliable sources for the clinical production of RBCs in vitro, despite several challenges that need to be overcome. However, owing to the limited availability and the large amount of CB required to produce hiPSCs, and the results of this study, the advantages of using PB-derived hiPSCs for RBC production in vitro may outweigh those of using CB-derived hiPSCs. We believe that our findings will facilitate the selection of optimal hiPSC lines for RBC production in vitro in the near future.

Erythroid Differentiation of Induced Pluripotent Stem Cells Co-cultured with OP9 Cells for Diagnostic Purposes.

BACKGROUND: Reagent red blood cells (RBCs) are prepared from donated whole blood, resulting in various combinations of blood group antigens. This inconsistency can be resolved by producing RBCs with uniform antigen expression. Induced pluripotent stem cells (iPSCs) generated directly from mature cells constitute an unlimited source for RBC production. We aimed to produce erythroid cells from iPSCs for diagnostic purposes. We hypothesized that cultured erythroid cells express surface antigens that can be recognized by blood group antibodies. METHODS: iPSCs were co-cultured with OP9 stromal cells to stimulate differentiation into the erythroid lineage. Cell differentiation was examined using microscopy and flow cytometry. Hemoglobin electrophoresis and oxygen-binding capacity testing were performed to verify that the cultured erythroid cells functioned normally. The agglutination reactions of the cultured erythroid cells to antibodies were investigated to confirm that the cells expressed blood group antigens. RESULTS: The generated iPSCs showed stemness characteristics and could differentiate into the erythroid lineage. As differentiation progressed, the proportion of nucleated RBCs increased. Hemoglobin electrophoresis revealed a sharp peak in the hemoglobin F region. The oxygen-binding capacity test results were similar between normal RBCs and cultured nucleated RBCs. ABO and Rh-Hr blood grouping confirmed similar antigen expression between the donor RBCs and cultured nucleated RBCs. CONCLUSIONS: We generated blood group antigen-expressing nucleated RBCs from iPSCs co-cultured with OP9 cells that can be used for diagnostic purposes. iPSCs from rare blood group donors could serve as an unlimited source for reagent production.

3D-Printed Barrier Membrane Using Mixture of Polycaprolactone and Beta-Tricalcium Phosphate for Regeneration of Rabbit Calvarial Defects.

BACKGROUND: Polycarprolactone and beta tricalcium phosphate (PCL/ß-TCP) are resorbable biomaterials that exhibit ideal mechanical properties as well as high affinity for osteogenic cells. AIM: Objective of this study was to evaluate healing and tissue reaction to the PCL/ß-TCP barrier membrane in the rabbit calvaria model for guided bone regeneration. MATERIALS AND METHODS: The PCL/ß-TCP membranes were 3D printed. Three circular defects were created in calvaria of 10 rabbits. The three groups were randomly allocated for each specimen: (i) sham control; (ii) PCL/ß-TCP membrane (PCL group); and (iii) PCL/ß-TCP membrane with synthetic bone graft (PCL-BG group). The animals were euthanized after two (n = 5) and eight weeks (n = 5) for volumetric and histomorphometric analyses. RESULTS: The greatest augmented volume was achieved by the PCL-BG group at both two and eight weeks (p < 0.01). There was a significant increase in new bone after eight weeks in the PCL group (p = 0.04). The PCL/ß-TCP membrane remained intact after eight weeks with slight degradation, and showed good tissue integration. CONCLUSIONS: PCL/ß-TCP membrane exhibited good biocompatibility, slow degradation, and ability to maintain space over eight weeks. The 3D-printed PCL/ß-TCP membrane is a promising biomaterial that could be utilized for reconstruction of critical sized defects.

Scanning Electron Microscopic Evaluation of the Internal Fit Accuracy of 3D-Printed Biphasic Calcium Phosphate Block: An Ex Vivo Pilot Study.

The aim of this study was to assess the internal fit accuracy of a three-dimensional (3D)-printed biphasic calcium phosphate (BCP) block compared with a 3D-milled poly methyl methacrylate (PMMA) block by scanning electron microscope (SEM) analysis. In a total of 20 porcine rib bones, two different types of defects having two adjacent walls and a floor were produced: a defect with a flat floor (flat defect; N = 10) and a defect with a concave floor (curved defect; N = 10). Each defect was grafted with either the 3D-printed BCP block or the 3D-milled PMMA block fabricated following the computer aided design. The defects were then cut cross-sectionally and evaluated under the SEM. The extents of internal contact and gap were measured and statistically analyzed (p < 0.05). All blocks in both BCP and PMMA groups were successfully fit to the flat and curved defects. The internal contact ratio was significantly higher in the BCP group (flat defect: 0.47 ± 0.10; curved defect: 0.29 ± 0.05) compared with the PMMA group (flat defect: 0.21 ± 0.13; curved defect: 0.17 ± 0.04; p < 0.05). The internal gap area was similar between the two groups regardless of the defect types (p > 0.05). The internal fit accuracy of the 3D-printed BCP block was reliable in both the flat and curved defects when compared with the accuracy of the 3D-milled PMMA block.

Anti-Inflammatory Potential of Complex Extracts of Ligularia stenocephala Matsum. & Koidz. and Secale cereale L. Sprout in Chronic Gingivitis: In Vitro Investigation and Randomized Clinical Trial.

Complex extracts of Ligularia stenocephala Matsum. & Koidz. (LSE) and Secale cereale L. sprout (SCSE) (TEES-10®) were prepared. The purposes of the study were to evaluate anti-inflammatory activities of TEES-10® in vitro and to observe resolution of gingivitis in human with oral administration of TEES-10®. The effects of TEES-10® on normal periodontal ligament (PDL) cell viability, lipopolysaccharide (LPS) induced PDL cell viability and the changes of inflammatory mediator expression were evaluated in vitro. In the clinical trial, 150 mg of TEES-10® powder containing capsule was administered twice daily to the test group, while the control group administered placebos in a total 100 participants with gingivitis. Probing depth (PD), bleeding on probing (BOP), clinical attachment loss, gingival index (GI) and plaque index (PI) were measured at baseline and 4 weeks. Administering TEES-10® showed significant increase in PDL cell viability compared to administering LSE or SCSE alone. In addition, treating TEES-10® to LPS induced PDL cell significantly increased PDL cell viability compared to control. TEES-10® suppressed expression of NF-κB, p-ERK, ERK, COX-2, c-Fos and p-STAT and promoted expression of PPARγ in LPS induced PDL cells. In the clinical trial, significant improvement of GI and BOP was observed in the test group at 4 weeks. In addition, the number of patients diagnosed with gingivitis was significantly reduced in the test group at 4 weeks. Salivary MMP-8 and MMP-9 was also significantly decreased compared to placebo group. Within the limitations of this study, the TEES-10® would have an anti-inflammatory potential clinically in the chronic gingivitis patients.