Several techniques have been developed to create 3D biomimetic scaffolds based on hydrogels, and these have recently become popular in tissue engineering. Given that the incorporation of osteogenic reagents such as VEGF and BMP2 improves bone regeneration, in this study, we evaluated a PCL-P (HEMA-NIPAAm) hydrogel consisting of polycaprolactone (PCL) blocks and poly(N-isopropylacrylamide-hydroxyethylmethacrylate) (NIPAAm-HEMA) acrylate blocks for the delivery of two growth factors, VEGF and BMP2, simultaneously. Physical characterization of PCL-P (HEMA-NIPAAm) hydrogel was evaluated by FTIR, SEM, and 1H-NMR. VEGF and BMP2 were loaded on the hydrogel and the release of both growth factors (GFs), cytotoxicity assay, and Alizarin red S staining the human dental pulp stem cells (hDPSCs) were evaluated. The initial release occurred rapidly within the first 24 hours, with 45% and 50% of both growth factors (GFs) being released. By the end of the study period, a total of 80% of BMP2 and 85% of VEGF had been released from the hydrogel scaffold.Top of Form The cell viability of samples was measured at 91%, 205%, and 470% after 24 h and on the third and fifth day, respectively. In the SEM images, hDPSCs fully adhere, colonize, and proliferate on the hybrid copolymer, which is non-toxic to cells. The produced hydrogel demonstrated no evident cytotoxicity in hDPSCs cell lines and our novel PCL-P-based hydrogel scaffold contained growth factors as a matrix for hDPSCs and cell seeding, which could be a beneficial technique used in 3D constructs with high potential for bone tissue engineering and regenerative medicine.

Objective: The stem cell theory in the endometriosis provides an advanced avenue of targeting these cells as a novel therapy to eliminate endometriosis. In this regard, studies showed that lovastatin alters the cells from a stem-like state to more differentiated condition and reduces stemness. The aim of this study was to investigate whether lovastatin treatment could influence expression and methylation patterns of genes regulating differentiation of endometrial mesenchymal stem cells (eMSCs) such asBMP2, GATA2 and RUNX2 as well as eMSCs markers.Materials and Methods: In this experimental investigation, MSCs were isolated from endometrial and endometriotic tissues and treated with lovastatin and decitabin. To investigate the osteogenic and adipogenic differentiation of eMSCs treated with the different concentration of lovastatin and decitabin, BMP2, RUNX2 and GATA2 expressions were measured by real-time polymerase chain reaction (PCR). To determine involvement of DNA methylation inBMP2 and GATA2 gene regulations of eMSCs, we used quantitative Methylation Specific PCR (qMSP) for evaluation of theBMP2 promoter status and differentially methylated region ofGATA2 exon 4.Results: In the present study, treatment with lovastatin increased expression of BMP2 andRUNX2 and induced BMP2 promoter demethylation. We also demonstrated that lovastatin treatment down-regulated GATA2 expression via inducing methylation. In addition, the results indicated that CD146 cell marker was decreased to 53% in response to lovastatin treatment compared to untreated group.Conclusion: These findings indicated that lovastatin treatment could increase the differentiation of eMSCs toward osteogenic and adiogenic lineages, while it decreased expression of eMSCs markers and subsequently reduced the stemness.

Objective(s): The loss of enteric neurons has been shown to be a major cause of slow transit constipation (STC). Gut microbiota and muscularis macrophages (MMs) are associated with the enteric nervous system (ENS) development and gastrointestinal (GI) motility. This study aimed to investigate whether Dioscin (DIO) increased GI motility and inhibited neuron loss by modulating gut microbiota profile, improving inflammation in the ENS microenvironment. Materials and Methods: The STC model was established by loperamide. The alteration of the gut microbiota was analyzed by 16S rDNA sequencing. The longitudinal muscle and myenteric plexus (LMMP) from the colon were prepared for flow cytometry, immunofluorescence, western blot, and qRT-PCR. Results: DIO increased the stool number, stool water content and shortened whole gut transit time, helped to recover the gut microbial diversity and microbiota community structure, and increased the abundance of Muribaculaceae in STC mice. Compared with the STC group, the number of MMs and the level of the iNOS, IL-6, and TNFα,genes were significantly decreased following DIO treatment. Moreover, DIO may increase the number of HuC/D+ neurons per ganglion by up-regulating the BMP2 secreted by MMs and activating the BMP2/p-Smad1/5/9 signaling pathway. Furthermore, the level of excitatory neurotransmitter AchE in colon tissues exhibited a substantial increase in the DIO group. However, the level of inhibitory neurotransmitter VIP was markedly decreased. Conclusion: Our results provide that DIO increases GI motility and inhibits neuron loss by modulating gut microbiota profile, improving inflammation in the ENS microenvironment and up-regulating the BMP2 secreted by MMs.

Endothelial cells (EC) have dynamic properties and high plasticity in response to microenvironmental change. A proinflammatory cytokine such as tumor necrotizing factor-α (TNF-α ) can induce EC phenotype shift to osteoinduction properties by releasing a potent osteogenic cytokine, namely bone morphogenetic protein 2 (BMP2). Normally BMP2 acts as an osteoblast stimulating factor in bone and cartilage tissue. BMP2 activation in vascular tissue will invite osteoblast recruitment and mineralization and generated pathological vascular stiffening and calcification. Recently, endoplasmic reticulum stress (ERS) has been emerging as a new target therapy in many vascular diseases such as vascular stiffening and calcification. Some short-chain fatty acid like 4-phenyl butyric acid has been shown had anti-ERS properties. However, the role of 4-phenyl butyric acid in BMP2 inhibition in endothelial cells is still poorly understood. Hence, we investigated the role of 4-phenyl butyric acid in inflammation-induced BMP2 expression in human vein derived endothelial cells. Endothelial cells obtained from a baby born umbilical vein were cultured and pre-treated with TNF-α (5 ng/ml) as inflammation precondition. Multiple doses of 4-phenyl butyrate acid (4-PBA) 1 nM/mL, 2 nM/mL, and 3 nM/m were used as ERS inhibitors. The expression of two ERS biomarkers, glucose-related protein-8 (GRP78) and activating transcription factor-6 (ATF6), were measured. Statistical analysis was done using one-way ANOVA and Kruskal Wallis tests, and P<0. 01 considered as significant. 4-PBA decrease luminal BMP2 at dose one nM/L, GRP78 at dose 1 nM/L, and translocated ATF6 expression at dose 1 nM/L in endothelial culture dose-dependently. Short-chain fatty acid 4-phenylbutyrate acid decreases luminal ERS marker GRP78 and translocated ATF6 expression in endothelial culture. ERS has a role in osteoinductive phenotype shifting in inflammation endothelial cells, which was the novelty of this research. Further research needs to elucidate ERS inhibition in in vivo experiment.