The ERα/KDM6B regulatory axis modulates osteogenic differentiation in human mesenchymal stem cells

Osteoporosis is a highly prevalent public health burden associated with an increased risk of bone fracture, particularly in aging women. Estrogen, an important medicinal component for the preventative and therapeutic treatment of postmenopausal osteoporosis, induces osteogenesis by activating the estrogen receptor signaling pathway and upregulating the expression of osteogenic genes, such as bone morphogenetic proteins (BMPs). The epigenetic regulation of estrogen-mediated osteogenesis, however, is still unclear. In this report, we found that estrogen significantly induced the expression of lysine-specific demethylase 6B (KDM6B) and that KDM6B depletion by shRNAs led to a significant reduction in the osteogenic potential of DMSCs. Mechanistically, upon estrogen stimulation, estrogen receptor-α (ERα) was recruited to the KDM6B promoter, directly enhancing KDM6B expression. Subsequently, KDM6B was recruited to the BMP2 and HOXC6 promoters, resulting in the removal of H3K27me3 marks and activating the transcription of BMP2 and HOXC6, the master genes of osteogenic differentiation. Furthermore, we found that estrogen enhanced DMSC osteogenesis during calvarial bone regeneration and that estrogen’s pro-osteogenic effect was dependent on KDM6B in vivo. Taken together, our results demonstrate the vital role of the ERα/KDM6B regulatory axis in the epigenetic regulation of the estrogen-dependent osteogenic response.

A Narrative Review of Cell-Based Approaches for Cranial Bone Regeneration

Current cranial repair techniques combine the use of autologous bone grafts and biomaterials. In addition to their association with harvesting morbidity, autografts are often limited by insufficient quantity of bone stock. Biomaterials lead to better outcomes, but their effectiveness is often compromised by the unpredictable lack of integration and structural failure. Bone tissue engineering offers the promising alternative of generating constructs composed of instructive biomaterials including cells or cell-secreted products, which could enhance the outcome of reconstructive treatments. This review focuses on cell-based approaches with potential to regenerate calvarial bone defects, including human studies and preclinical research. Further, we discuss strategies to deliver extracellular matrix, conditioned media and extracellular vesicles derived from cell cultures. Recent advances in 3D printing and bioprinting techniques that appear to be promising for cranial reconstruction are also discussed. Finally, we review cell-based gene therapy approaches, covering both unregulated and regulated gene switches that can create spatiotemporal patterns of transgenic therapeutic molecules. In summary, this review provides an overview of the current developments in cell-based strategies with potential to enhance the surgical armamentarium for regenerating cranial vault defects.

Single-cell analysis identifies a key role for Hhip in murine coronal suture development

Craniofacial development depends on formation and maintenance of sutures between bones of the skull. In sutures, growth occurs at osteogenic fronts along the edge of each bone, and suture mesenchyme separates adjacent bones. Here, we perform single-cell RNA-seq analysis of the embryonic, wild type murine coronal suture to define its population structure. Seven populations at E16.5 and nine at E18.5 comprise the suture mesenchyme, osteogenic cells, and associated populations. Expression of Hhip, an inhibitor of hedgehog signaling, marks a mesenchymal population distinct from those of other neurocranial sutures. Tracing of the neonatal Hhip-expressing population shows that descendant cells persist in the coronal suture and contribute to calvarial bone growth. In Hhip−/− coronal sutures at E18.5, the osteogenic fronts are closely apposed and the suture mesenchyme is depleted with increased hedgehog signaling compared to those of the wild type. Collectively, these data demonstrate that Hhip is required for normal coronal suture development.

Bioclickable Mussel-Derived Peptides With Immunoregulation for Osseointegration of PEEK

Polyether ether ketone (PEEK)–based biomaterials have been widely used in the field of spine and joint surgery. However, lack of biological activity limits their further clinical application. In this study, we synthesized a bioclickable mussel-derived peptide Azide-DOPA4 as a PEEK surface coating modifier and further combined bone morphogenetic protein 2 functional peptides (BMP2p) with a dibenzylcyclooctyne (DBCO) motif through bio-orthogonal reactions to obtain DOPA4@BMP2p-PEEK. As expected, more BMP2p can be conjugated on PEEK after Azide-DOPA4 coating. The surface roughness and hydrophilicity of DOPA4@BMP2p-PEEK were obviously increased. Then, we optimized the osteogenic capacity of PEEK substrates. In vitro, compared with the BMP2p-coating PEEK material, DOPA4@BMP2p-PEEK showed significantly higher osteogenic induction capability of rat bone marrow mesenchymal stem cells. In vivo, we constructed a rat calvarial bone defect model and implanted PEEK materials with a differently modified surface. Micro-computed tomography scanning displayed that the DOPA4@BMP2p-PEEK implant group had significantly higher new bone volume and bone mineral density than the BMP2p-PEEK group. Histological staining of hard tissue further confirmed that the DOPA4@BMP2p-PEEK group revealed a better osseointegrative effect than the BMP2p-PEEK group. More importantly, we also found that DOPA4@BMP2p coating has a synergistic effect with induced Foxp3+ regulatory T (iTreg) cells to promote osteogenesis. In summary, with an easy-to-perform, two-step surface bioengineering approach, the DOPA4@BMP2p-PEEK material reported here displayed excellent biocompatibility and osteogenic functions. It will, moreover, offer insights to engineering surfaces of orthopedic implants.

Human Sex Matters: Y-linked lysine demethylase 5D drives accelerated male osteogenic differentiation

Female sex is increasingly associated to a loss of bone mass during aging and an increased risk for fractures developing nonunion. Hormonal factors and cell-intrinsic mechanisms are suggested to drive these sexual dimorphisms, although underlying molecular mechanisms are still a matter of debate. Here, we observed a decreased capacity of calvarial bone recovery in female rats and a profound sexually dimorphic osteogenic differentiation human adult neural crest-derived stem cells (NCSCs). Next to an elevated expression of pro-osteogenic regulators, global trancriptomics revealed Lysine Demethylase 5D (KDM5D) to be highly upregulated in differentiating male NCSCs. Loss of function by siRNA or pharmacological inhibition of KDM5D significantly reduced the osteogenic differentiation capacity of male NCSCs. In summary, we demonstrate craniofacial osteogenic differentiation to be sexually dimorphic with the expression of KDM5D as a prerequisite for accelerated male osteogenic differentiation, emphasizing the analysis of sex-specific differences as a crucial parameter for treating bone defects.

miR-196a-5p Promoted the Osteogenic Differentiation and Calvarial Bone Defect Repair of Wharton’s Jelly Umbilical Cord Stem Cells

Background: To investigate the effect of miR‐196a-5p on the osteogenic differentiation and defected bone repair of Wharton’s jelly umbilical cord stem cells (WJCMSCs). Methods: miR‐196a-5p mimic or inhibitor was applied to overexpress or silence miR‐196a-5p expression in WJCMSCs. The alkaline phosphatase (ALP) activity, mineralization ability, and osteogenic markers expression were used to test WJCMSCs osteogenic potential in vitro. Calvarial bone defect model of rat was used to evaluate WJCMSCs bone regeneration ability in vivo. mRNA microarray was used to reveal the underling mechanisms that miR‐196a-5p regulated bone repair.Results: miR-196a-5p inhibition reduced the ALP activity, mineralization ability, and level of osteogenic markers OCN, DSPP, DMP1 and BSP, while miR-196a-5p overexpression enhanced the ALP activity, mineralization ability, and level of OCN, DSPP, DMP1 and BSP of WJCMSCs in vitro. Next, the micro-CT and histopathology results showed miR-196a-5p-overexpressed-WJCMSCs obviously promoted the new bone tissue regeneration and calvarial bone defect repair after MSCs transplanted for 12 weeks. Further, mRNA microarray of miR-196a-5p-overexpressed-WJCMSCs revealed totally 959 significantly differentially expressed genes (DEGs), among which 34 upregulated and 925 downregulated. Also, 241 miR-196a-5p targeted genes were predicted by using miRNA targeted websites and only 19 predicted genes were consistent with microarray results. On this basis, one significantly downregulated gene SERPINB2 was selected and revealed that SERPINB2 deletion obviously enhanced the ALP activity and mineralization ability of WJCMSCs in vitro.Conclusions: miR-196a-5p promoted the osteogenic differentiation potential and calvarial bone defect repair ability of WJCMSCs. And SERPINB2 acted as one key downstream gene to participate in the miR-196a-5p promoted MSCs osteogenic differentiation.