Circulating peptide is a potential source of biomarkers for cancer detection. However, the existence of large molecular weight proteins in plasma have a disastrous effect on circulating peptides isolating and detecting. Herein, nanotrap fractionation following by mass spectrometry have been applied to quantify the levels of bradykinin (BK) and hydroxylated bradykinin (Hyp-BK) as a relative measure of KRAS-regulated Prolyl-4-hydroxylase alpha-1 (P4HA1) which may serve as early diagnosis marker for pancreatic ductal adenocarcinoma (PDAC). We found that P4HA1 can be upregulated by KRASG12V, which is a PDAC driver mutation, using HPNE/KRAS and HPNE cells. And we revealed that P4HA1 is overexpressed in PDAC tumors, compared to normal and inflamed pancreatic tissues. RNA interference revealed that P4HA1 activity was primarily responsible for Hyp-BK production. Mass spectrometry analysis revealed that plasma Hyp-BK/BK ratio was higher in PDAC than pancreatitis patients and healthy controls, while the area under the receiver operating characteristic (ROC) curve (AUC) is 0.8209 (95%CI, 0.7269–0.9149). The Hyp-BK/BK association with PDAC was reproduced in another cohort, where this ratio was found to increase with advancing tumor stage. These novel findings paved the way for wider applications of Nanotrap coupled mass spectrometry as a powerful tool for revealing biosignatures from plasma.
Hydroxyapatite (HA) is a bioceramic material that shares similar crystal and chemical structures with inorganic components of the bone. However, HA lacks osteoinductive activity and has a brittle nature, making it challenging to apply for direct load-bearing bone applications. In this study, we used a wet chemical method to synthesize zinc-doped hydroxyapatite powders with different Zn/(Zn+Ca) molar ratios of 0, 0.025, 0.05, and 0.1. The corresponding Zn-HA was designated as HA, Zn2.5-HA, Zn5-HA, and Zn10-HA. The Zn-HA powders at 30 wt% were used to fabricate poly(propylene fumarate) (PPF)-based nanocomposite scaffolds (HA/PPF, Zn2.5-HA/PPF, Zn5-HA/PPF, and Zn10-HA/PPF). The physical properties of obtained scaffolds were examined by scanning electron microscopy, energy-dispersive X-ray spectroscopy (EDS), transmission electron microscopy (TEM), and atomic force microscopy (AFM). Live/dead cell viability assay showed that these scaffolds were biocompatible and supported excellent adhesion of MC3T3-E1 preosteoblast cells. Additionally, the proliferation of cells was detected at 1, 4, and 7 days on these scaffolds. Alkaline phosphatase (ALP) activity measurement and alizarin red staining showed good osteogenic differentiation and matrix mineralization for MC3T3-E1 cells growing on these scaffolds. Taken together, the results here indicate that Zn5-HA/PPF nanocomposite scaffolds are promising scaffold material for bone tissue engineering.
Synovial inflammation mainly resulting from interleukin-1 beta (IL-1β) plays a crucial role in the early and late stage of osteoarthritis. Recent progress in therapeutic gene delivery systems has led to promising strategies for local sustained target gene expression. The aim of this study was to design a nanoparticle made of chitosan (CS)/hyaluronic acid (HA)/plasmid-DNA (pDNA) encoding IL-1 receptor antagonist gene (pIL-1Ra) and furtherly use it to transfect the primary synoviocytes, and then investigate whether CS/HA/pIL-1Ra nanoparticles could make the synoviocytes overexpress functional IL-1Ra to attenuate inflammation induced by IL-1β. In this study, CS was modified with HA to generate CS/HA nanoparticles and then combined with pIL-1Ra to form CS/HA/pIL-1Ra nanoparticles. The physicochemical characteristics results showed that CS/HA nanoparticles exhibited an appropriate particle size (144.9?±?2.8?nm) and positive zeta potential (?+?28?mV). The gel retardation assay revealed that pDNA was effectively protected and released in a sustained manner more than 15 days. Cytotoxicity results showed that CS/HA/pIL-1Ra nanoparticles had a safe range (0-80?μg/ml) for the application to synoviocytes. RT-qPCR and western blot analysis demonstrated that CS/HA/pIL-1Ra nanoparticles were able to increase IL-1Ra expression in primary synoviocytes, and reduce the mRNA and protein levels of matrix metalloproteinase-3 (MMP-3), matrix metalloproteinase-13 (MMP-13), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in IL-1β-induced synoviocytes. Our findings indicated that CS/HA/pIL-1Ra nanoparticles efficiently transfected synoviocytes and attenuated synovitis induced by IL-1β, which will provide a potential strategy for OA synovitis.
The target of the recent study is to achieve a significant inexpensive and eco-friendly way for getting ZTA/HA composites, based on the nano-HA derived from the eggshell biogenic source. Combining simultaneously the porous structure; which is considered as a bone formation key, with developed mechanical properties and adequate biocompatibility, is another purpose of this study. Furthermore, the impact of ZTA addition from 10–30 mass-%, fabricated by uniaxial pressing and sintering at 1200–1300?°C for 2?h, on the physical and mechanical properties, microstructure and phase composition of ZTA/HA composite bodies was investigated. The results demonstrated that the increasing of ZTA content increases the bodies’ apparent porosity and decreases the bulk density due to the decomposition of HA into β-TCP. Where the formation of β-TCP possessed the predominant impact on the mechanical properties of the sintered ZTA/HA composites. ICP, SEM, EDX and thin film XRD results of composites containing 20 mass-% ZTA affirmed the excellent bioactivity of the bodies.
This paper reports the corrosion resistant and cytocompatible properties of the hyaluronic acid-silane coating on AZ31 Mg alloy. In this study, the osteoinductive properties of high molecular weight hyaluronic acid (HA, 1–4?MDa) and the corrosion protection of silane coatings were incorporated as a composite coating on biodegradable AZ31 Mg alloy for orthopaedic applications. The multi-step fabrication of coatings first involved dip coating of a passivated AZ31 Mg alloy with a methyltriethoxysilane-tetraethoxysilane sol-gel to deposit a dense, cross-linked and corrosion resistant silane coating (AZ31-MT). The second step was to create an amine-functionalised surface by treating coated alloy with 3-aminopropyl-triethoxy silane (AZ31-MT-A) which facilitated the immobilisation of HA via EDC-NHS coupling reactions at two different concentrations i.e 1?mg.ml?1 (AZ31-MT-A-HA1) and 2?mg.ml?1 (AZ31-MT-A-HA2). These coatings were characterised by Fourier transform infrared spectroscopy, atomic force microscopy and static contact angle measurements which confirmed the successful assembly of the full coatings onto AZ31 Mg alloy. The influence of HA-silane coating on the corrosion of Mg alloy was investigated by electrical impedance spectroscopy and long-term immersion studies measurements in HEPES buffered DMEM. The results showed an enhanced corrosion resistance of HA functionalised silane coated AZ31 substrate over the uncoated equivalent alloy. Furthermore, the cytocompatibility of MC3T3-E1 osteoblasts was evaluated on HA-coated AZ31-MT-A substrates by live-dead staining, quantification of total cellular DNA content, scanning electron microscope and alkaline phosphatase activity. The results showed HA concentration-dependent improvement of osteoblast cellular response in terms of enhanced cell adhesion, proliferation and differentiation. These findings hold great promise in employing such biomimetic multifunctional coatings to improve the corrosion resistance and cytocompatibility of biodegradable Mg-based alloy for orthopaedic applications.
The in vitro and in vivo osteoblastic differentiation of rat bone marrow stromal cells (MSCs) was assessed on hydroxyapatite disks with 3 different
porosities: 30%, 50%, and 70% (HA30, HA50, and HA70, respectively). MSCs obtained by 10-day culture of fresh bone marrow cells
were subcultured for 2 weeks on 3 kinds of porous HA disks in the presence and absence of dexamethasone (Dex). After 2 weeks
of subculture, alkaline phosphatase (ALP) activity and osteocalcin production of MSCs/HA composites with Dex were higher than
those without, and increased with increasing porosity. The resultant bone tissue grafts “cultured-bone/HA constructs” were
implanted subcutaneously into the backs of syngeneic rats, and harvested 1, 2, and 4 weeks after implantation. At 1 week,
only cultured-bone/HA70 constructs exhibited expanded bone formation. At 2 and 4 weeks, active osteoblasts and progressive
bone formation were observed morphologically in both cultured-bone/HA50 and HA70 constructs. At 4 weeks, bone tissue was observed
even in cultured-bone/HA30 constructs. ALP activity and osteocalcin production also increased with increasing porosity and
time after implantation. In this in vivo model, different scaffold porosity with similar crystal morphology of the apatite phase demonstrated marked differences in
ability to support osteogenesis by implanted rat MSCs. 相似文献
