ALPL regulates pro-angiogenic capacity of mesenchymal stem cells through ATP-P2X7 axis controlled exosomes secretion.

BACKGROUND: Early-onset bone dysplasia is a common manifestation of hypophosphatasia (HPP), an autosomal inherited disease caused by ALPL mutation. ALPL ablation induces prototypical premature bone ageing characteristics, resulting in impaired osteogenic differentiation capacity of human bone marrow mesenchymal stem cells (hBMMSCs). As angiogenesis is tightly coupled with osteogenesis, it also plays a necessary role in sustaining bone homeostasis. We have previously observed a decrease in expression of angiogenesis marker gene CD31 in the metaphysis of long bone in Alpl+/- mice. However, the role of ALPL in regulation of angiogenesis in bone has remained largely unknown. METHODS: Exosomes derived from Normal and HPP hBMMSCs were isolated and identified by ultracentrifugation, transmission electron microscopy, and nanoparticle size measurement. The effects of ALPL on the angiogenic capacity of hBMMSCs from HPP patients were assessed by immunofluorescence, tube formation, wound healing and migration assay. exo-ELISA and Western Blot were used to evaluate the exosomes secretion of hBMMSCs from HPP, and the protein expression of VEGF, PDGFBB, Angiostatin and Endostatin in exosomes respectively. RESULTS: We verified that ALPL ablation resulted in impaired pro-angiogenic capacity of hBMMSCs, accounting for reduced migration and tube formation of human umbilical vein endothelial cells, as the quantities and proteins composition of exosomes varied with ALPL expression. Mechanistically, loss of function of ALPL enhanced ATP release. Additional ATP, in turn, led to markedly elevated level of ATP receptor P2X7, which consequently promoted exosomes secretion, resulting in a decreased capacity to promote angiogenesis. Conversely, inhibition of P2X7 increased the angiogenic induction capacity by preventing excessive release of anti-angiogenic exosomes in ALPL deficient-hBMMSCs. CONCLUSION: The ALPL-ATP axis regulates the pro-angiogenic ability of hBMMSCs by controlling exosomes secretion through the P2X7 receptor. Thus, P2X7 may be proved as an effective therapeutic target for accelerating neovascularization in ALPL-deficient bone defects.

TMT-labelled quantitative proteomic analysis to predict the target promoting human odontogenic inflammatory granulation tissue transform into reparative granulation tissue.

OBJECTIVES: Odontogenic inflammatory diseases are main causes for alveolar bone breakdown and teeth loss, leaving great difficulties in denture restoration. Local inflammatory granulation tissue (IGT) is considered as pathological tissue and required to be removed. However, there are many evidences supporting that under appropriate intervention, IGT in alveolar bone maybe transformed into reparative granulation tissue (RGT), followed by ossification. Therefore, this study aimed to discover a specific target to promote this transformation. MATERIALS AND METHODS: After drawing out histological differences between IGT and RGT with haematoxylin and eosin (H&E) and immunohistochemical (IHC) assay staining, TMT-labelled quantitative proteomic analysis was applied to identify potential targets. RESULTS: The most striking histological property of RGT was found to be ECM deposition, which significantly decreased inflammatory cells, prominently increased fibroblasts as well as triggered changes of vascular types. Combined with histological findings and proteomic analysis, five KEGG pathways were associated with ECM, inflammation and angiogenesis and 49 pathways involved in differentially expressed proteins. COL1A1 was not only the most up-regulated protein, but also one of main hubs in protein-protein interaction regulatory network. Specific protease cathepsin K (CTSK) was identified. Level of CTSK in RGT was down-regulated to 69.10-76.97% (p < .05), with significantly up-regulated COL1A1, COL1A2, FN1 and TGFB1 included in focal adhesion, PI3K-Akt signalling pathways and angiogenesis. CTSK involved in transformation from IGT to RGT. CONCLUSIONS: CTSK might be a target to regulate transformation from IGT to RGT in alveolar bone through ECM, stem cells and angiogenesis mechanisms. However, further research is also clearly required.

Effect on implant drills and postoperative reactions for pre-extraction interradicular implant bed preparation during the COVID-19 pandemic and beyond.

The aim of the present study was to observe the abrasion of implant drills and postoperative reactions for the preparation of the interradicular immediate implant bed during the COVID-19 pandemic and beyond. Thirty-two implant drills were included in four groups: blank, improved surgery, traditional surgery, and control. In the improved surgery group, a dental handpiece with a surgical bur was used to decoronate the first molar and create a hole in the middle of the retained root complex, followed by the pilot drilling protocol through the hole. The remaining root complex was separated using a surgical bur and then extracted. Subsequently, the implant bed was prepared. Implant drills were used in the traditional surgery group to complete the decoronation, hole creation, and implant-drilling processes. The tooth remained intact until the implant bed was prepared. The surface roughness of the pilot drill was observed and measured. Surgery time, postoperative reactions (swelling, pain, and trismus), and fear of coronavirus disease 2019 scale (FCV-19S) were measured and recorded, respectively. Statistical analysis revealed significant difference with surface roughness among blank group (0.41 ± 0.05 µm), improved surgery group (0.37 ± 0.06 µm), traditional surgery group (0.16 ± 0.06 µm), and control group (0.26 ± 0.04 µm) (P < .001). Significant differences were revealed with surgery time between improved surgery group (5.63 ± 1.77 min) and traditional surgery group (33.63 ± 2.13 min) (P < .001). Swelling, pain, and trismus (improved group: r ≥ 0.864, P ≤ .006; traditional group: r ≥ 0.741, P ≤ .035) were positively correlated with the FCV-19S. This study proved that a new pilot drill could only be used once in traditional surgery but could be used regularly in improved surgery. Improved surgery was more effective, efficient, and economical than the traditional surgery. The higher FCV-19S, the more severe swelling, pain, and trismus.

A multifunctional drug consisting of tetracycline conjugated with odanacatib for efficient periodontitis therapy.

The treatment of periodontitis can be very challenging due to its complex etiologies. A new pharmacologic strategy entitled "host-modulation therapy," has been introduced to improve periodontal treatment outcomes. Supposedly, a multifunctional drug with the potential for bacterial infection prevention, host-response modulation and bone healing promotion would be a promising option for periodontitis therapy, but related studies remain substantially lacking. In this study, we successfully conjugated tetracycline with odanacatib (a selective inhibitor of cathepsin K) to construct a multifunctional drug (TC-ODN). We discovered that TC-ODN could promote macrophages polarizing toward anti-inflammatory phenotype and promote osteogenesis of PDLSCs under inflammatory microenvironment. In vivo, TC-ODN could be absorbed and distributed specifically to the bone after systemic administration, and accumulation of TC-ODN increased bone mineral density in ovariectomized rats. Importantly, periodontal administration of TC-ODN could successfully promote bone healing in periodontitis rats with alveolar bone loss. The findings in our study uncovered the excellent biocompatibility and multifunction of TC-ODN, including bone-targeted accumulation, immunoregulation, anti-inflammatory activity and promotion of bone healing, which might contribute to the clinical treatment of periodontitis.

Substance P-induced RAD16-I scaffold mediated hUCMSCs stereo-culture triggers production of mineralized nodules and collagen-like fibers by promoting osteogenic genes expression.

Tissue engineering has become an important therapeutic method for injuries. This study aimed to generate collagen-like matrix constructed by hUCMSCs combining self-assembled polypeptide and evaluate differentiated capacity, safety and biocompatibility. Human umbilical cord tissues were isolated and used to primarily culture hUCMSCs. hUCMSCs were identified using immunofluorescence and flow cytometry. Adipogenic- and osteogenic-differentiation of hUCMSCs were evaluated using Oil-red O and Alizarin-Red staining. Self-assembling collagen peptide RAD16-I hydrogel and substance P (SP) were prepared and combined together to form RAD16-I/SP complex. Surface morphology and ultrastructures were observed with scanning electron microscopic (SEM). hUCMSCs in simulated collagen-like matrix environment were plane-cultured and stereo-cultured. Cell viability was examined using CCK-8 and fluorescent staining assay. Osteogenic genes were detected with qRT-PCR and western blot assay. HE staining and Masson staining were used to assess production of mineralized nodules and collagen-like fibers, respectively. Collagen-like matrix complex by combining RAD16-I/SP complex with stereo-cultured hUCMSCs was successfully generated. hUCMSCs in collagen-like matrix complex demonstrated adipogenic-differentiation and osteogenic-differentiation potential. SP-induced RAD16-I mediated stereo-culture of hUCMSCs demonstrated higher cell activity and proliferation potential. SP-induced RAD16-I mediated stereo-culture of hUCMSCs promoted osteogenesis-related molecules expression. SP-induced RAD16-I mediated stereo-culture of hUCMSCs promoted production of mineralized nodules and triggered formation of collagen-like fibers. Cell-collagen-like matrix complex injection (RAD16-I/SP/hUCMSCs complex) exhibited better biocompatibility and no cytotoxicity. In conclusion, SP-induced RAD16-I mediated stereo-culture of hUCMSCs remarkably promoted osteogenesis-related gene expression, triggered production of mineralized nodules and formation of collagen-like fibers. This established cell-collagen-like matrix complex (RAD16-I/SP/hUCMSCs) injection exhibited better biocompatibility, without cytotoxicity.

Cathepsin K deficiency promotes alveolar bone regeneration by promoting jaw bone marrow mesenchymal stem cells proliferation and differentiation via glycolysis pathway.

OBJECTIVES: To clarify the possible role and mechanism of Cathepsin K (CTSK) in alveolar bone regeneration mediated by jaw bone marrow mesenchymal stem cells (JBMMSC). MATERIALS AND METHODS: Tooth extraction models of Ctsk knockout mice (Ctsk-/- ) and their wildtype (WT) littermates were used to investigate the effect of CTSK on alveolar bone regeneration. The influences of deletion or inhibition of CTSK by odanacatib (ODN) on proliferation and osteogenic differentiation of JBMMSC were assessed by CCK-8, Western blot and alizarin red staining. To explore the differently expressed genes, RNA from WT and Ctsk-/- JBMMSC was sent to RNA-seq. ECAR, glucose consumption and lactate production were measured to identify the effect of Ctsk deficiency or inhibition on glycolysis. At last, we explored whether Ctsk deficiency or inhibition promoted JBMMSC proliferation and osteogenic differentiation through glycolysis. RESULTS: We found out that Ctsk knockout could promote alveolar bone regeneration in vivo. In vitro, we confirmed that both Ctsk knockout and inhibition by ODN could promote proliferation of JBMMSC, up-regulate expression of Runx2 and ALP, and enhance matrix mineralization. RNA-seq results showed that coding genes of key enzymes in glycolysis were significantly up-regulated in Ctsk-/- JBMMSC, and Ctsk deficiency or inhibition could promote glycolysis in JBMMSC. After blocking glycolysis by 3PO, the effect of Ctsk deficiency or inhibition on JBMMSC's regeneration was blocked subsequently. CONCLUSIONS: Our findings revealed that Ctsk knockout or inhibition could promote alveolar bone regeneration by enhancing JBMMSC regeneration via glycolysis. These results shed new lights on the regulatory mechanism of CTSK on bone regeneration.

Nitrate-Functionalized poly(ε-Caprolactone) Small-Diameter Vascular Grafts Enhance Vascular Regeneration via Sustained Release of Nitric Oxide.

Artificial small-diameter vascular grafts (SDVG) fabricated from synthetic biodegradable polymers, such as poly(ε-caprolactone) (PCL), exhibit beneficial mechanical properties but are often faced with issues impacting their long-term graft success. Nitric oxide (NO) is an important physiological gasotransmitter with multiple roles in orchestrating vascular tissue function and regeneration. We fabricated a functional vascular graft by electrospinning of nitrate-functionalized poly(ε-caprolactone) that could release NO in a sustained manner via stepwise biotransformation in vivo. Nitrate-functionalized SDVG (PCL/NO) maintained patency following abdominal arterial replacement in rats. PCL/NO promoted cell infiltration at 3-months post-transplantation. In contrast, unmodified PCL SDVG showed slow cell in-growth and increased incidence of neointima formation. PCL/NO demonstrated improved endothelial cell (EC) alignment and luminal coverage, and more defined vascular smooth muscle cell (VSMC) layer, compared to unmodified PCL SDVG. In addition, release of NO stimulated Sca-1+ vascular progenitor cells (VPCs) to differentiate and contribute to rapid luminal endothelialization. Furthermore, PCL/NO inhibited the differentiation of VPCs into osteopontin-positive cells, thereby preventing vascular calcification. Overall, PCL/NO demonstrated enhanced cell ingrowth, EC monolayer formation and VSMC layer regeneration; whilst inhibiting calcified plaque formation. Our results suggested that PCL/NO could serve as promising candidates for improved and long-term success of SDVG implants.

Hyaluronan promotes the regeneration of vascular smooth muscle with potent contractile function in rapidly biodegradable vascular grafts.

The regeneration of smooth muscle with physiological functions has been a key challenge in vascular tissue engineering. Hyaluronan (HA), as a major component of the extracellular matrix, plays a vital role in regulating tissue injury and repair. In this study, a biomimetic vascular graft was prepared by co-electrospinning of synthetic degradable polymers and native ECM components including collagen type-I as well as low and high molecular weight HA (LMW HA and HMW HA). Upon implantation in the rat abdominal aorta, the grafts exhibited sustained HA release that effectively enhanced the regeneration of vascular smooth muscle. Besides, LMW HA loaded vascular grafts demonstrated rapid endothelialization compared to the other groups. More importantly, HA-loaded poly(L-lactide-co-caprolactone) grafts demonstrated an optimal vascular media layer accompanied by well-organized elastin fibers after long-term implantation (6 months), and they maintained potent physiological function up to 1/3 that of the native artery. In contrast, inadequate smooth muscle regeneration was observed in poly(ε-caprolactone) grafts due to slow degradation restricting the regeneration. The mechanism was further investigated and explained by the HA-induced migration of smooth muscle cell (SMC) via CD44-mediated signaling. Besides, low molecular weight HA can promote the migration of vascular progenitor cells that further differentiate into SMCs. These results highlight the importance of HA in the regeneration of functional vascular smooth muscle, and provide a new insight into the fabrication of tissue engineering vascular grafts (TEVGs) via combining rapidly degradable polymers and bioactive ECM components that hold great translational potential.

Correlation between genotype and phenotype in three families with Peutz-Jeghers Syndrome.

Peutz-Jeghers syndrome (PJS) is a hereditary disorder characterized by mucocutaneous pigmentations, gastrointestinal (GI) polyposis and an increased risk of certain malignancies. Little is known about the causative genes of PJS, or their association with the clinical phenotypes of PJS. The present study reports the results of clinical and genetic analysis of three Chinese families with PJS. In addition, the medical histories and clinical manifestations of these families were compared. DNA was collected from the blood samples of patients with PJS and controls. Serine/threonine kinase 11 (STK11), olfactory receptor family 4 subfamily C member 45 (OR4C45) and zonadhesin (ZAN) were amplified by polymerase chain reaction, and analyzed by sequencing and cloning. Two PJS-affected members of one family had a de novo single base deletion (NM_000455.4:c.842delC) in the STK11 gene, and their clinical presentations reflected the quantity of mutant STK11 copies in a dose-dependent manner. No pathogenic variants of OR4C45 or ZAN were found in the patients with PJS, although a new single nucleotide polymorphism (NM_003386.2:c.5768delG) of ZAN was identified. The results of the current study identified that a STK11 mutation dose-dependent genotype-phenotype relationship exists in patients with PJS. In addition, an early onset and high severity of oral pigmentations in PJS was indicative of serious GI phenotypes. These findings may aid the diagnosis and treatment of PJS.

Grading and quantification of dental fluorosis in zebrafish larva.

OBJECTIVE: The prevalence and severity of dental fluorosis in primary teeth are different from permanent teeth. Previous animal models of dental fluorosis mainly focus on juvenile rats, mice and zebrafish. Our experiment aims to set a dental fluorosis model using zebrafish larva and explore the characteristics of the first generation teeth by fluoride treatment. MATERIALS AND METHODS: After the zebrafish eggs were laid, they were exposed to excess fluoride (19ppm, 38ppm and 76ppm) for five days. The morphological characteristics of first generation teeth were examined by H&E staining, whole-mount alizarin red and alcian blue staining, and scanning electron microscope (SEM) technique. RESULTS: With whole-mount alizarin red and alcian blue staining, the tooth cusps presented red in normal control. 19ppm and 38ppmm fluoride resulted in extensive red staining from tooth cusps to the lower 1/3 of teeth. 76ppm fluoride caused malformed teeth with uneven red staining. H&E staining showed that excess fluoride caused cystic-like changes in 38ppm and 76ppm groups. SEM revealed the dose dependent pathological changes in zebrafish enameloid with fluoride treatment. Based on SEM findings, we set 0-4 dental fluorosis index (DFI) score to label the severity of dental fluorosis. CONCLUSIONS: Excess fluoride presented a dose dependent fluorosis changes in the teeth of zebrafish larva. The DFI scores in our experiment reflect dose dependent fluorosis changes in a good way and will benefit the future research of dental fluorosis.

Deficiency of ATP6V1H Causes Bone Loss by Inhibiting Bone Resorption and Bone Formation through the TGF-ß1 Pathway.

Vacuolar-type H +-ATPase (V-ATPase) is a highly conserved, ancient enzyme that couples the energy of ATP hydrolysis to proton transport across vesicular and plasma membranes of eukaryotic cells. Previously reported mutations of various V-ATPase subunits are associated with increased bone density. We now show that haploinsufficiency for the H subunit of the V1 domain (ATP6V1H) is associated with osteoporosis in humans and mice. A genome-wide SNP array analysis of 1625 Han Chinese found that 4 of 15 tag SNPs (26.7%) within ATP6V1H were significantly associated with low spine bone mineral density. Atp6v1h+/- knockout mice generated by the CRISPR/Cas9 technique had decreased bone remodeling and a net bone matrix loss. Atp6v1h+/- osteoclasts showed impaired bone formation and increased bone resorption. The increased intracellular pH of Atp6v1h+/- osteoclasts downregulated TGF-ß1 activation, thereby reducing induction of osteoblast formation but the bone mineralization was not altered. However, bone formation was reduced more than bone resorption. Our data provide evidence that partial loss of ATP6V1H function results in osteoporosis/osteopenia. We propose that defective osteoclast formation triggers impaired bone formation by altering bone remodeling. In the future, ATP6V1H might, therefore, serve as a target for the therapy of osteoporosis.