p75NTR antibody-conjugated microspheres: an approach to guided tissue regeneration by selective recruitment of endogenous periodontal ligament cells.

Repairing defects in alveolar bone is essential for regenerating periodontal tissue, but it is a formidable challenge. One promising therapeutic approach involves using a strategy that specifically recruits periodontal ligament cells (PDLCs) with high regenerative potential to achieve in situ regeneration of alveolar bone. In this study, we have created a new type of microsphere conjugated with an antibody to target p75 neurotrophin receptor (p75NTR), which is made of nano-hydroxyapatite (nHA) and chitosan (CS). The goal of this design is to attract p75NTR+hPDLCs selectively and promote osteogenesis. In vitro experiments demonstrated that the antibody-conjugated microspheres attracted significantly more PDLCs compared to non-conjugated microspheres. Incorporating nHA not only enhances cell adhesion and proliferation on the surface of the microsphere but also augments its osteoinductive properties. Microspheres effectively recruited p75NTR+ cells at bone defect sites in SD rats, as observed through immunofluorescent staining of p75NTR antibodies. This p75NTR antibody-conjugated nHA/CS microsphere presents a promising approach for selectively recruiting cells and repairing bone defects.

p75NTR promotes tooth rhythmic mineralization via upregulation of BMAL1/CLOCK.

The circadian clock plays a critical role in dentomaxillofacial development. Tooth biomineralization is characterized by the circadian clock; however, the mechanisms underlying the coordination of circadian rhythms with tooth development and biomineralization remain unclear. The p75 neurotrophin receptor (p75NTR) is a clock factor that regulates the oscillatory components of the circadian rhythm. This study aims to investigate the impact of p75NTR on the rhythmic mineralization of teeth and elucidate its underlying molecular mechanisms. We generated p75NTR knockout mice to examine the effects of p75NTR deficiency on tooth mineralization. Ectomesenchymal stem cells (EMSCs), derived from mouse tooth germs, were used for in vitro experiments. Results showed a reduction in tooth mineral density and daily mineralization rate in p75NTR knockout mice. Deletion of p75NTR decreased the expression of DMP1, DSPP, RUNX2, and ALP in tooth germ. Odontogenic differentiation and mineralization of EMSCs were activated by p75NTR. Histological results demonstrated predominant detection of p75NTR protein in odontoblasts and stratum intermedium cells during rapid formation phases of dental hard tissue. The mRNA expression of p75NTR exhibited circadian variations in tooth germs and EMSCs, consistent with the expression patterns of the core clock genes Bmal1 and Clock. The upregulation of BMAL1/CLOCK expression by p75NTR positively regulated the mineralization ability of EMSCs, whereas BMAL1 and CLOCK exerted a negative feedback regulation on p75NTR by inhibiting its promoter activity. Our findings suggest that p75NTR is necessary to maintain normal tooth biomineralization. Odontogenic differentiation and mineralization of EMSCs is regulated by the p75NTR-BMAL1/CLOCK signaling axis. These findings offer valuable insights into the associations between circadian rhythms, tooth development, and biomineralization.

A potential role of p75NTR in the regulation of circadian rhythm and incremental growth lines during tooth development.

Objective: Tooth morphogenesis and the formation of hard tissues have been reported to be closely related to circadian rhythms. This study investigates the spatiotemporal expression and relationship of p75NTR with core clock genes, mineralization-related or odontogenesis-related genes, and aims to derive the potential role of p75NTR in regulating circadian rhythm and incrementality growth line formation during tooth development. Materials and methods: The dynamic morphology of the rat dental germ was observed at seven stages (E14.5 d, E16.5 d, E18.5 d, P.N. 4 d, P.N. 7 d, P.N. 10 d, and P.N. 15 d). Next, the expressions of p75NTR and other target factors were traced. The ectomesenchymal stem cells (EMSCs) were isolated from the E18.5d rat dental germs and synchronized using 50% of fetal bovine serum. Then, they were cultured in light/light (L.L.), dark/dark (D.D.), and light/dark (L.D.) conditions for 48 h. The total RNA was collected every 4 h, and the circadian rhythm dynamics of target factors were observed. To reveal the mechanism further, p75NTR was down-regulated in p75NTR ExIII-/- mice and up-regulated in immortalized mouse dental apical papilla progenitor cells. The change tendencies of other target factors were also detected. Results: The clock genes Bmal1, Clock, Per1, and Per2 were all expressed in tooth germs before the formation of dental hard tissues and demonstrated a regular oscillating expression pattern in EMSCs from dental germs. Their expression was affected by the L.D. stimulus, and most of them were promoted by D.D. conditions. p75NTR presented a similar expression pattern and a positive or negative relationship with most clock genes, mineralization-related and odontogenesis-related factors, such as brain and muscle ARNT-like protein-1 (Bmal1), runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), MSH-like 1 (MSX1), dentin matrix acidic phosphoprotein 1 (Dmp1), and dentin sialophosphoprotein (Dspp). Moreover, the arrangement, morphology, and even boundary in pre-odontoblast/pre-ameloblast layers were disordered in the p75NTR ExIII-/- mice. Conclusion: Circadian rhythm was found to affect tooth development. p75NTR might play a crucial role in regulating clock genes in the mineralization and formation of the dental hard tissues. p75NTR is actively involved in the odontoblast-ameloblast junction and cell polarity establishment during tooth morphogenesis.

Phage-free peptide ELISA for ochratoxin A detection based on biotinylated mimotope as a competing antigen.

To perform the biopanning of a mimotope peptide with reduced affinity to anti-ochratoxin A (OTA) monoclonal antibodies (mAbs), we executed two improved biopanning approaches with a commercial 7-mer peptide library. In the first approach, anti-mouse IgG antibodies were used to erect the anti-OTA mAbs; in the second approach, an ultralow OTA concentration (0.1 ng/mL) was used to perform the competitive elution of phage particles. After the fourth round of biopanning was completed, 30 identified clones were positive phage particles; of these phage particles, 16 exhibited strong competitive inhibition with a low OTA concentration of 0.1 ng/mL. DNA sequencing results revealed that the 16 phage particles represented six different peptide sequences. Among these particles, the phage particle with a peptide sequence of "GMVQTIF" showed the highest sensitivity to OTA detection. The biotinylated 12-mer peptide "GMVQTIF-GGGSK-biotin" was designed as a competing antigen to develop a competitive peptide ELISA. Under the optimal parameters, the proposed peptide ELISA with the biotinylated 12-mer peptide as a competing antigen exhibited good dynamic linear detection for OTA in the range of 0.005 ng/mL-0.2 ng/mL with a detection limit of 0.001 ng/mL. The median inhibition concentration of OTA was 0.024 ng/mL (n=6), which is approximately fivefold more efficient as a competing antigen than the OTA-HRP conjugates. Reaction kinetics revealed that the biotinylated 12-mer peptide exhibited lower affinity to anti-OTA mAbs than the conventional chemical OTA antigen. The practicality of the proposed peptide ELISA was compared with a conventional ELISA method. In summary, this study demonstrated a novel concept of the development of phage-free peptide ELISA for the detection of OTA by using a biotinylated mimotope peptide as a competing antigen. This novel strategy can be applied to sensitively detect other toxic small molecules during food safety monitoring.