Breast cancer nipple discharge exosomal microRNAs are stable under degradative conditions.

We have previously shown that microRNAs (miRNAs) in nipple discharge are potential diagnostic biomarkers. In particular, exosomes are present in nipple discharge. Herein, we sought to elucidate the protective role of exosomes on miRNAs in nipple discharge and investigate the stability of miRNAs encapsulated in exosomes under degradative conditions. A novel TTMAAlPc-RNA complex method was used to measure the RNase concentration in colostrum and nipple discharge. Quantitative real-time polymerase chain reaction was performed to test the stability of exogenous synthetic miRNAs (cel-lin-4-5p and cel-miR-2-3p) and endogenous miRNAs (hsa-miR-4732-5p, hsa-miR-3646, hsa-miR-4484, and kshv-miR-K12-5-5p). RNase was present and functional in colostrum and nipple discharge. Endogenous miRNAs were more stably expressed compared to exogenous miRNAs at room temperature and 4°C. Triton X-100 (1%, 30 min) destroyed the exosomal membrane, causing RNA degradation in colostrum but not in nipple discharge. Therefore, we confirmed that exosomes in colostrum and nipple discharge could protect miRNAs from degradation by RNase. Exosomes in nipple discharge may be more resistant to Triton X-100 lysis compared to those in the colostrum. Exosomal miRNAs in nipple discharge in breast cancer are stable under degradative conditions. Differential Triton X-100 sensitivity of exosomes of nipple discharge and colostrum warrants further investigation.

Bacterial expression, purification, and initial characterization of a full-length Cas13b protein from Porphyromonas gingivalis.

The CRISPR-Cas13b system is a recently identified Class 2, RNA-targeting CRISPR-Cas system. The system has been repurposed to achieve robust mRNA knockdown and precise RNA-editing in mammalian cells. While the CRISPR-Cas13b system has become a powerful tool for nucleic acids manipulation, the mechanisms of the system are still not fully understood. Cas13b endonucleases from different bacterial species show poor overall sequence homologies, suggesting that structural (and probably functional) diversities may exist. It is therefore important to study CRISPR-Cas13b cases from different bacterial species. Here we report the expression, purification, and initial characterization of a Cas13b endonuclease that is associated with the 8th putative CRISPR locus from Porphyromonas gingivalis genome (Pgi8Cas13b). The full-length Pgi8Cas13b protein (1119 residues) was successfully expressed in E. Coli cells, and purified by affinity and ion-exchange chromatography methods. The purified protein is biologically active, being able to bind its cognate crRNA with high specificity and affinity. Preparation of biologically active Pgi8Cas13b protein provides the basis for further in vitro biochemical and biophysical studies of the Pgi8Cas13b CRISPR system.

TREM-1 activation modulates dsRNA induced antiviral immunity with specific enhancement of MAPK signaling and the RLRs and TLRs on macrophages.

Triggering receptor expressed on myeloid cells 1(TREM-1) is a newly identified member of the immunoglobulin superfamily and is extensively involved in the regulation of innate immunity. To determine the role of TREM-1 in innate antiviral immunity, we investigated TREM-1 expression and its downstream signaling effect in the murine bone marrow-derived macrophages or RAW264.7 macrophage-like mouse cell line by double-stranded RNA (dsRNA) stimulation. The level of TREM-1 expression was low at the baseline and could up-regulate markedly in dose- and time-dependent manners upon stimulation by dsRNA/poly IC. Inhibitor studies disclosed mitogen-activated protein kinase (MAPK) p38 and PI3K pathways were involved in dsRNA-induced up-regulation of TREM-1. Compared with lipopolysaccharide (LPS), the peak response of poly IC-induced TREM-1 expression is delayed, and cells pretreated with scrambled RNA presented higher expression of TREM-1 upon LPS challenge. After ligation with the agonist antibody, TREM-1 can potentiate type I interferon (IFN) production and antiviral inflammation induced by dsRNA, which is ralated to the enhanced phosphorylation of MAPKs and expression of RLRs and TLRs by TREM-1 ligation. This study is the first to show the regulatory role of TREM-1 in RLRs and TLRs expression, and these findings might enrich the understanding of the up-regulation mechanism and the function of TREM-1.

[Study of vascularization of hydroxyapatite/tricalcium phosphate biomaterials implanted in mice during osteoinduction].

This study aims to explore the vascularization of hydroxyapatite/tricalcium phosphate(HA/TCP)biomaterials implanted in mice during osteoinduction. The HA/TCP biomaterials were implanted in muscle of mice, and2, 4, 6, 8, 10 and 12 weeks after the implantation, the materials were harvested to prepare serial sections and hematoxylineosin(HE) staining. The process of vascularization was dynamically described, and the area percentage of neovascularization was quantitatively analyzed. The results showed that neovascularization formation was a continuous and dynamic process. The neovascularization appeared largely in the first two weeks, with a rising trend in week 4,reached peak in week 6, and gradually reduced in week 8. The results provide ideas for improving the success rate of bone tissue engineering, and indicate the mechanism of osteoinduction.

Near-infrared light-sensitive liposomes for the enhanced photothermal tumor treatment by the combination with chemotherapy.

PURPOSE: To develop a near-infrared (NIR) light-sensitive liposome, which contains hollow gold nanospheres (HAuNS) and doxorubicin (DOX), and evaluate their potential utility for enhancing antitumor activity and controlling drug release. METHODS: The liposomes (DOX&HAuNS-TSL) were designed based on a thermal sensitive liposome (TSL) formulation, and hydrophobically modified HAuNS were attached onto the membrane of the liposomes. The behavior of DOX release from the liposomes was investigated by the dialysis, diffusion in agarose gel and cellular uptake of the drug. The biodistribution of DOX&HAuNS-TSL was assessed by i.v. injection in tumor-bearing nude mice. Antitumor efficacy was evaluated both histologically using excised tissue and intuitively by measuring the tumor size and weight. RESULTS: Rapid and repetitive DOX release from the liposomes (DOX&HAuNS-TSL), could be readily achieved upon NIR laser irradiation. The treatment of tumor cells with DOX&HAuNS-TSL followed by NIR laser irradiation showed significantly greater cytotoxicity than the treatment with DOX&HAuNS-TSL alone, DOX-TSL alone (chemotherapy alone) and HAuNS-TSL plus NIR laser irradiation (Photothermal ablation, PTA, alone). In vivo antitumor study indicated that the combination of simultaneous photothermal and chemotherapeutic effect mediated by DOX&HAuNS-TSL plus NIR laser presented a significantly higher antitumor efficacy than the PTA alone mediated by HAuNS-TSL plus NIR laser irradiation. CONCLUSIONS: Our study could be as the valuable reference and direction for the clinical application of PTA in tumor therapy.

Ureteral reconstruction using autologous tubular grafts for the management of ureteral strictures and defects: an experimental study.

OBJECTIVE: To investigate whether the peritoneal cavity could function as a bioreactor to produce autologous tubular grafts for ureteral reconstruction in beagles. MATERIALS AND METHODS: 8-Fr Silastic tubes were implanted into the peritoneal cavities of 6 female beagles. At 3 weeks, the tubes were harvested and the tubular tissue covering the tubes was gently everted. A segment 3 cm in length of the right mid-ureter, involving two thirds of its diameter, was removed parallel to the ureteral axis, leaving a third of the ureteral wall. A 5-Fr double-J stent was inserted into the ureter through the created defect, and two thirds of the graft were anastomosed to both edges of the ureteral defect. One third of the graft was overlapped with the retained normal ureter and anastomosed to the external surface of the lumens. Thus, the graft was partly encapsulated by the remainder of ureteral wall. The stent was maintained for 6 weeks and removed. Excretory urography was performed at 8 (n = 3) and 12 weeks (n = 3), postoperatively. Meanwhile, the neoureter was harvested and analyzed. The left ureter served as the control and a simple intubated ureterotomy was performed. RESULTS: Histological analysis of the tubular tissue demonstrated transversely arranged myofibroblasts and an outer layer of mesothelium. The tissue was easily everted and transplanted as a ureteral graft. Eight weeks postoperatively, the neoureter demonstrated normal ureteral architecture, composed of multilayers of urothelium surrounded by smooth muscle bundles, which became increasingly organized with time. Excretory urography indicated no stenosis or hydronephrosis. CONCLUSIONS: These results show that autologous tubular tissue grown within the recipients' peritoneal cavity can be used for ureteral reconstruction in the beagle model.

PEGylated amphiphilic polymeric manganese(II) complexes as magnetic resonance angiographic agents.

Currently, the most commonly used clinical magnetic resonance imaging (MRI) contrast agents, Gd(III) chelates, have been found to be associated with nephrogenic systemic fibrosis (NSF) in renally compromised patients. Toxicity concerns related to Gd(III)-based agents prompted intensive research toward the development of safe, efficient, and long-cycle non-Gd contrast agents. Herein, three amphiphilic polymeric manganese (Mn) ligands (mPEG1k-P(L-a-HMDI)-mPEG1k, mPEG2k-P(L-a-HMDI)-mPEG2k and mPEG4k-P(L-a-HMDI)-mPEG4k) were synthesized, and then end-capped respectively with different molecular weights of polyethylene glycol monomethyl ether (mPEG 1 kD, 2 kD and 4 kD) to obtain amphiphilic polymer Mn ligands. After being chelated with Mn(II), these amphiphilic polymer Mn complexes show significantly higher T1 relaxivity than the small molecule Mn complex (MnL) at 0.5 T, 1.5 T and 3.0 T magnetic fields, respectively. Then, mPEG2k-P(MnL-a-HMDI)-mPEG2k with relatively high T1 relaxivities (23.2, 14.4 and 9.7 mM-1s-1 at 0.5 T, 1.5 T and 3.0 T, respectively), low CMC (4.7 mg L-1), reasonable size (48 nm) and excellent stability among these three polymer Mn complexes was selected for in vivo MR imaging of vascular vessels. The results suggest that mPEG2k-P(MnL-a-HMDI)-mPEG2k has an excellent and relatively long time-window vascular enhancement effect even at a low dose of 0.05 mmol Mn kg-1 BW, and could play a role in the diagnosis of vascular diseases (0.1 mmol Mn kg-1 BW). Therefore, mPEG2k-P(MnL-a-HMDI)-mPEG2k may be considered as a potential blood pool contrast agent.

Effectiveness of low-level gallium aluminium arsenide laser therapy for temporomandibular disorder with myofascial pain: A systemic review and meta-analysis.

PURPOSE: Temporomandibular disorder (TMD) causes masticatory muscle pain and mouth opening limitations and affects patients' ability to eat, practice oral health and perform other activities of daily living. Although the benefits of low-energy lasers in treating TMD have been reported, the results vary greatly depending on the equipment used and the energy output. This study systematically evaluated the efficacy of a low-level gallium aluminium arsenide (GaAlAs) laser treatment for TMD with myofascial pain and maxillary pain. METHODS: We searched the PubMed, EMBASE, Cochrane Library, Web of Science, and ClinicalTrials.gov databases for randomized controlled trials (RCTs) published since database inception to April 5, 2020, that compared low-level laser treatment to sham/placebo treatment or no intervention in patients suffering from TMD with myofascial pain. Three reviewers independently screened the literature, extracted data, and assessed the quality of the included studies according to the risk-of-bias tool recommended by the Cochrane Handbook V.5.1.0 (Cochrane Collaboration, London, UK). Then, a meta-analysis was performed using RevMan 5.3 and Stata 15.1 software. RESULTS: The data from 8 randomized controlled trials including 181 patients were analyzed. The severity of myofascial TMD pain (measured on a visual analogue scale, VAS) at the end of treatment was significantly different between the control laser therapy and the low-level GaAlAs laser therapy (weighted mean difference [WMD] = -0.76, 95% confidence interval [CI] -1.51 to 0.01, P = .046); at 3 to 4 weeks after treatment, there was no significant difference (WMD = 1.24, 95% CI -0.04 to 2.51, P = .057). In addition, there was no significant improvement in maximum mouth opening (MMO) at the end of treatment (WMD = -0.03, 95% CI -4.13 to 4.06, P = .987) or at 3 to 4 weeks after treatment (WMD = 1.22, 95% CI -2.94 to 5.39, P = .565). CONCLUSIONS: The results of this study suggest that there is insufficient evidence to indicate an efficacy of low-level GaAlAs laser therapy in improving TMD pain and maximal oral opening. These results suggest that clinicians should make appropriate recommendations to inform patient decision-making.

Co-electrospun nano-/microfibrous composite scaffolds with structural and chemical gradients for bone tissue engineering.

Recent trends in scaffold design for tissue engineering have focused on providing structural, mechanical and chemical cues for guiding cell behaviors. In this study, we presented a structural/compositional gradient nano-/microfibrous mesh by co-electrospinning, using silk fibroin-poly(ε-caprolactone) (SF-PCL) nanofibers and PCL microfibers. The pore size, porosity, and physical property of the gradient meshes were qualified. Cell proliferation of mouse osteoblast-like MC3T3-E1 cells was carried out to estimate the effect of structural and compositional gradients on biocompatibility. Furthermore, the 2-D mesh was rolled up and the compressive property of 3-D cylinder was investigated. The results suggested that the rolled-up gradient cylinder scaffold exhibited higher osteogenic differentiation compared to the pristine nanofibrous cylinder sample. By incorporating Chinese medicine ginsenoside Rg1, sustained release was achieved in composite meshes. Rg1-containing nanofibrous meshes and Rg1 gradient cylinders enhanced the cell proliferation of human umbilical vein endothelial cells (HUVECs). The developed fibrous scaffold may provide structural, compositional, and chemical gradients for bone regeneration. BRIEFS: Structural and chemical gradient fibrous scaffold fabricated by co-electrospinning.

The preparation and application of calcium phosphate biomedical composites in filling of weight-bearing bone defects.

Nowadays, artificial bone materials have been widely applied in the filling of non-weight bearing bone defects, but scarcely ever in weight-bearing bone defects. This study aims to develop an artificial bone with excellent mechanical properties and good osteogenic capability. Firstly, the collagen-thermosensitive hydrogel-calcium phosphate (CTC) composites were prepared as follows: dissolving thermosensitive hydrogel at 4 °C, then mixing with type I collagen as well as tricalcium phosphate (CaP) powder, and moulding the composites at 37 °C. Next, the CTC composites were subjected to evaluate for their chemical composition, micro morphology, pore size, Shore durometer, porosity and water absorption ability. Following this, the CTC composites were implanted into the muscle of mice while the 70% hydroxyapatite/30% ß-tricalcium phosphate (HA/TCP) biomaterials were set as the control group; 8 weeks later, the osteoinductive abilities of biomaterials were detected by histological staining. Finally, the CTC and HA/TCP biomaterials were used to fill the large segments of tibia defects in mice. The bone repairing and load-bearing abilities of materials were evaluated by histological staining, X-ray and micro-CT at week 8. Both the CTC and HA/TCP biomaterials could induce ectopic bone formation in mice; however, the CTC composites tended to produce larger areas of bone and bone marrow tissues than HA/TCP. Simultaneously, bone-repairing experiments showed that HA/TCP biomaterials were easily crushed or pushed out by new bone growth as the material has a poor hardness. In comparison, the CTC composites could be replaced gradually by newly formed bone and repair larger segments of bone defects. The CTC composites trialled in this study have better mechanical properties, osteoinductivity and weight-bearing capacity than HA/TCP. The CTC composites provide an experimental foundation for the synthesis of artificial bone and a new option for orthopedic patients.

Size and PEG Length-Controlled PEGylated Monocrystalline Superparamagnetic Iron Oxide Nanocomposite for MRI Contrast Agent.

OBJECTIVE: PEGylated superparamagnetic iron oxide (SPIO) is the most promising alternatives to gadolinium-based contrast agents (GBCAs) in MRI. This paper is to explore the imaging effects of PEGylated SPIO, which is influenced by particle sizes and surface polyethylene glycol (PEG) coating, using as MRI contrast agents at different magnetic field intensities. METHODS: Firstly, nine PEGylated monocrystalline SPIO nanoparticles with different nanocrystal sizes and different molecular weights PEG coating were prepared, and then physical and biological properties were analyzed. Finally, MRI imaging in vivo was performed to observe the imaging performance. RESULTS: Nine PEGylated monocrystalline SPIO nanoparticles have good relaxivities, serum stability, and biosecurity. At the same time, they show different imaging characteristics at different magnetic field intensities. Eight-nanometer SPIO@PEG5k is an effective T 2 contrast agent at 3.0 T (r 2/r 1 = 14.0), is an ideal T 1-T 2 dual-mode contrast agent at 1.5 T (r 2/r 1 = 6.52), and is also an effective T 1 contrast agent at 0.5 T (r 2/r 1 = 2.49), while 4-nm SPIO@PEG5k is a T 1-T 2 dual-mode contrast agent at 3.0 T (r 2/r 1 = 5.24), and is a useful T 1 contrast agent at 0.5 T (r 2/r 1 = 1.74) and 1.5 T (r 2/r 1 = 2.85). MRI studies in vivo at 3.0 T further confirm that 4-nm SPIO@PEG5k displays excellent T 1-T 2 dual-mode contrast enhancement, whereas 8-nm SPIO@PEG5k only displays T 2 contrast enhancement. CONCLUSION: PEGylated SPIOs with different nanocrystal sizes and PEG coating can be used as T 1, T 2, or T 1-T 2 dual-mode contrast agents to meet the clinical demands of MRI at specific magnetic fields.

Mussel-inspired chitosan modified superhydrophilic and underwater superoleophobic cotton fabric for efficient oil/water separation.

Superhydrophilic and underwater superoleophobic textiles exhibit excellent oil/water separation performance but are limited by the poor stability and environmental incompatibility. Inspired by strong adhesion of marine mussels, we designed and fabricated a stable and eco-friendly superhydrophilic and underwater superoleophobic cotton fabric (CF) from all renewable resources through in-situ surface deposition of polydopamine (PDA) particles followed by adsorption of hydrophilic chitosan via dip coating at room temperature. The as-prepared superhydrophilic and underwater superoleophobic CF exhibited outstanding oil/water separation performance with separation efficiency and water flux higher than 99 % and 15,000 L m-2 h-1, respectively. Moreover, it not only showed excellent resistance to mechanical abrasion and ultrasound treatment but also had outstanding superwetting stability against acid/alkali/salt erosion. We believed that the eco-friendly superhydrophilic and underwater superoleophobic CF would exhibit great potential in oil/water separation especially under harsh conditions.

[Vesselplasty for the treatment of spinal metastases complicated by posterior wall destruction of vertebral body].

OBJECTIVE: To evaluate the early clinical efficacy and safety of vesselplasty for the treatment of spinal metastases complicated by posterior wall destruction of vertebral body. METHODS: The clinical data of 19 patients(21 segments) with spinal metastases complicated by posterior wall destruction of vertebral body treated from January 2016 to January 2017 were retrospectively analyzed. There were 15 males and 4 females, aged 40 to 85 years old with a mean of (66.00±10.25) years . All patients had severe low back pain before the operation, which were diagnosed by CT as damage-type metastatic tumor of the vertebral posterior wall. All patients were treated by vesselplasty technique. Nineteen vertebrae received percutaneous unilateral pedicle puncture and two vertebrae received percutaneous bilateral pedicle puncture. VAS, ODI were recorded before operation, 1 d and 3 d after operation respectively. X-ray and CT scan were used to observe bone cement leakage and complications. RESULTS: All the operations were successful and postoperative pain was significantly relieved. Postoperative VAS score and ODI of the two groups were significantly improved (P<0.05). A small amount of bone cement leakage occurred in one vertebral body, which was a vertebral venous plexus leakage, but no clinical symptoms after operation. CONCLUSION: Vesselplasty for the treatment of spinal metastases complicated by posterior wall destruction of vertebral body can significantly reduce the symptoms of thoracolumbar back pain, improve the quality of life, reduce the incidence of bone cement leakage, and has high clinical efficacy and safety.

Construction of 2D Antimony(III) Selenide Nanosheets for Highly Efficient Photonic Cancer Theranostics.

Photonic cancer hyperthermia has been considered to be one of the most representative noninvasive cancer treatments with high therapeutic efficiency and biosafety. However, it still remains a crucial challenge to develop efficient photothermal nanoagents with satisfactory photothermal performance and biocompatibility, among which two-dimensional (2D) ultrathin nanosheets have recently been regarded as the promising multifunctional theranostic agents for photothermal tumor ablation. In this work, we report, for the first time, on the construction of a novel kind of photothermal agents based on the intriguing 2D antimony(III) selenide (Sb2Se3) nanosheets for highly efficient photoacoustic imaging-guided photonic cancer hyperthermia by near-infrared (NIR) laser activation. These Sb2Se3 nanosheets were easily fabricated by a novel but efficiently combined liquid nitrogen pretreatment and freezing-thawing approach, which were featured with high photothermal-conversion capability (extinction coefficient: 33.2 L g-1 cm-1; photothermal-conversion efficiency: 30.78%). The further surface engineering of these Sb2Se3 ultrathin nanosheets with poly(vinyl pyrrolidone) (PVP) substantially improved the biocompatibility of the nanosheets and their stability in physiological environments, guaranteeing the feasibility in photonic antitumor applications. Importantly, 2D Sb2Se3-PVP nanosheets have been certificated to efficiently eradicate the tumors by NIR-triggered photonic tumor hyperthermia. Especially, the biosafety in vitro and in vivo of these Sb2Se3 ultrathin nanosheets has been evaluated and demonstrated. This work meaningfully expands the biomedical applications of 2D bionanoplatforms with a planar topology through probing into new members (Sb2Se3 in this work) of 2D biomaterials with unique intrinsic physiochemical property and biological effect.

3-D mineralized silk fibroin/polycaprolactone composite scaffold modified with polyglutamate conjugated with BMP-2 peptide for bone tissue engineering.

In the field of bone tissue engineering, an ideal three-dimensional (3-D) scaffold should not only structurally mimic the extracellular matrix (ECM) in large tissues but also mechanically support the bone healing process and provide biochemical cues to induce osteogenesis. In this study, we investigated the feasibility of functionalisation of scaffolds by coupling polyglutamate acid conjugated with BMP-2 peptide onto silk fibroin (SF)/polycaprolactone (PCL) (SF/PCL) blend nanofibers. The morphology, composition, and mineralisation, were confirmed by FE-SEM, XRD, and FT-IR spectroscopy. The FE-SEM images revealed that wet-electrospun nanofibrous scaffolds exhibited inter-connected nano/micro-pores at different levels, and a different morphology was observed on the 3-D SF/PCL scaffold after mineralisation. Furthermore, the binding property and release behaviour of the peptide were investigated on this mineralized structure, and adipose-derived stem cells were seeded on the composite scaffolds to assay their cytocompatibility and osteogenic differentiation capacities. Results suggest that the polyglutamate motif (repetitive glutamate amino acids) exhibited markedly improved binding properties to mineralized nanofibers, and the mineralized 3-D scaffolds with the conjugated with peptide enhances the mRNA expression of osteogenic genes. The sponge-like 3-D nanofibrous scaffold mechanically and biochemically mimics the regenerative process for applications in bone tissue engineering, including the regeneration of calvarial defects.

Temperature-controlled, phase-transition ultrasound imaging-guided photothermal-chemotherapy triggered by NIR light.

Recently, nano-sized ultrasound contrast agents encapsulating drugs for cancer diagnosis and therapy have attracted much attention. However, the ultrasound signal of these agents is too weak to obtain an ideal ultrasound imaging effect. Furthermore, conventional ultrasound contrast agents with strong echo signal are not suitable for drug delivery against cancer because of their large size. To circumvent this problem, phase-transition ultrasound contrast agents are believed to be an excellent choice. Methods: Liposomes co-encapsulating doxorubicin (DOX), hollow gold nanospheres (HAuNS), and perfluorocarbon (PFC) were synthesized by film dispersion method. The morphology, particle size, and stability of these liposomes (DHPL) were investigated. The photothermal effect, drug release, particle size change, cytotoxicity, and ultrasound imaging were studied by using the near infrared (NIR) light. Furthermore, tumor accumulation of DHPL was observed by in vivo fluorescence imaging and the antitumor effect was verified in a 4T1 tumor model. Results: The nanosystem displayed a homogeneous size distribution (~200 nm) and an efficient light-to-heat conversion effect under 808 nm NIR laser irradiation. The nanometer size enabled considerable accumulation of DHPL in the tumor sites. The localized hyperthermia resulting from the photothermal effect of HAuNS could trigger the size transformation of DHPL followed by significant DOX release. Due to the gasification of PFC, a remarkably enhanced ultrasound signal was detected. DHPL also exhibited a prominent photothermally reinforced chemotherapeutic effect under the control of NIR light both in vitro and in vivo. Importantly, no systemic toxicity was observed by DHPL treatment. Conclusion: In this study, we fabricated multi-functional perfluorocarbon liposomes for ultrasound imaging-guided photothermal chemotherapy which have the potential to serve as a prospective cancer treatment approach.

The in vivo anti-fibrotic function of calcium sensitive receptor (CaSR) modulating poly(p-dioxanone-co-l-phenylalanine) prodrug.

In present study, the apoptosis induction and proliferation suppression effects of l-phenylalanine (l-Phe) on fibroblasts were confirmed. The action sites of l-Phe on fibroblasts suppression were deduced to be calcium sensitive receptor (CaSR) which could cause the release of endoplasmic reticulum (ER) Ca2+ stores; disruption of intracellular Ca2+ homeostasis triggers cell apoptosis via the ER or mitochondrial pathways. The down-regulation of CaSR were observed after the application of l-Phe, and the results those l-Phe triggered the increasing of intracellular Ca2+ concentration and calcineurin expression, and then the apoptosis and increasing G1 fraction of fibroblasts have verified our deduction. Hence, l-Phe could be seen as a kind of anti-fibrotic drugs for the crucial participation of fibroblast in the occurrence of fibrosis. And then, poly(p-dioxanone-co-l-phenylalanine) (PDPA) which could prolong the in-vivo anti-fibrotic effect of l-Phe for the sustained release of l-Phe during its degradation could be treated as anti-fibrotic polymer prodrugs. Based on the above, the in vivo anti-fibrotic function of PDPA was evaluated in rabbit ear scarring, rat peritoneum lipopolysaccharide, and rat sidewall defect/cecum abrasion models. PDPA reduced skin scarring and suppressed peritoneal fibrosis and post operation adhesion as well as secretion of transforming growth factor-ß1 in injured tissue. These results indicate that PDPA is an effective agent for preventing fibrosis following tissue injury. STATEMENT OF SIGNIFICANCE: We have previously demonstrated that poly(p-dioxanone-co-l-phenylalanine) (PDPA) could induce apoptosis to fibroblast and deduced that the inhibitory effect comes from l-phenylalanine. In present study, the inhibition mechanism of l-phenylalanine on fibroblast proliferation was demonstrated. The calcium sensitive receptor (CaSR) was found to be the action site. The CaSR was downregulated after the application of l-phenylalanine, and then the ER Ca2+ stores were released. The released Ca2+ can simultaneously activate Ca2+/calcineurin and then trigger apoptosis and G1 arrest of fibroblast. Hence, l-phenylalanine could be seen as anti-fibrosis drug and PDPA which conjugate l-phenylalanine by hydrolytic covalent bonds could be seen as l-phenylalanine polymer prodrug. Based above, the in vivo anti-fibrotic function of PDPA were verified in three different animal models.

Exercise enhance the ectopic bone formation of calcium phosphate biomaterials in muscles of mice.

OBJECTIVE: To investigate whether exercise can enhance ectopic bone formation of calcium phosphate (Ca-P) biomaterials in muscles of mice. METHODS: Firstly, ten transient receptor potential vanilloid subfamily member 1 (TRPV1) knockout mice (group KO) and ten C57BL/6 mice (group WT) were randomly chosen, 10µg Ca-P biomaterials were implanted into the thigh muscle pouch of each mouse which was far away from femur; after that, all animals were kept in open field for free exploration 5min, and the movement time and distance were automatically analyzed. Ten weeks later, the Ca-P samples were harvested for histological staining and immunochemistry. Secondly, the Ca-P biomaterials were implanted into the thigh muscle pouch of C57BL/6 mice the same as previous operation, and then randomly divided into two groups: running group and non-running group (n=10); in running group, all mice run 1h as a speed of 6m/h in a treadmill for 10weeks. Ten weeks later, the blood was collected to detect the interleukin-4 (IL-4) and IL-12 levels by enzyme linked immunosorbent assay (ELISA), and the samples were harvested for histological staining. RESULTS: In groups KO and WT, both the movement time and distance were significant higher in group KO than that in group WT (P<0.05); furthermore, the histology staining results showed that lots of new bone and bone marrow tissues were observed in group KO, but only bone matrix-like substances were observed in group WT. In running group and non-running group, the ELISA results indicated that the immunological genes, both IL-4 and IL-12 levels were significant higher in running group than that in non-running group (P<0.05); besides that, more new bone tissues were observed in running group than that in non-running group. CONCLUSION: Knockout of TRPV1 in mice could reduce algesia and induce the stronger athletic ability of mice, causing better osteoinductivity of Ca-P biomaterials both in TRPV1-/- mice and running mice; according to this, we want to offer a proposal to patients who suffer from bone defects and artificial bone transplantation: do moderate exercise, don't convalesce all the time.

In vitro and in vivo degradation of potential anti-adhesion materials: Electrospun membranes of poly(ester-amide) based on l-phenylalanine and p-(dioxanone).

Electrospun membranes of poly(p-dioxanone-co-l-phenylalanine) (PDPA) hold potential as an anti-adhesion material. Since adjustable degradation properties are important for anti-adhesion materials, in this study, the in vitro and in vivo degradation processes of PDPA electrospun membranes were investigated in detail. The morphological analysis of these membranes revealed the main degradation conditions of PDPA membranes. The weight remaining and molecular weight variation showed that the overall degradation rate of the membranes could be adjusted by modulating the molecular structure of the PDPAs. Especially, α-chymotrypsin could catalyze the degradation process of PDPAs. Based on these results, the in vitro degradation mechanism was demonstrated, and confirmed by 1 H NMR of the hydrolysis products. Finally, the in vivo degradation and biocompatibility of different PDPAs were investigated. The kinetic study showed that the in vitro and in vivo molecular weight loss of PDPAs have the first-order characteristics. The in vivo degradation rate of the most Phe-containing PDPA-3 is the slowest, and this result relates to the biocompatibilities of PDPAs. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1369-1378, 2017.

Electrospun homogeneous silk fibroin/poly (ɛ-caprolactone) nanofibrous scaffolds by addition of acetic acid for tissue engineering.

In this study, we investigated the phase separation phenomenon of silk fibroin/poly (ɛ-caprolactone) electrospinning solution to improve the performance of silk fibroin/poly (ɛ-caprolactone) electrospun nanofibers. It showed that phase separation does occur in just a few hours in the silk fibroin/poly (ɛ-caprolactone)/formic acid mixture solution. Acetic acid, small molecule nonsolvent for silk fibroin, was first introduced to silk fibroin/poly (ɛ-caprolactone)/formic acid solution, a homogeneous solution without separation for over several days was achieved after mixing for 5 h. The morphology and composition of the silk fibroin/poly (ɛ-caprolactone) and acetic acid-modified silk fibroin/poly (ɛ-caprolactone) fibrous scaffolds were examined by scanning electron microscopy, Fourier transform infrared spectroscopy and thermal gravimetric analyzer. Attachment and proliferation of mouse osteoblast MC3T3-E1 cells were tested by scanning electron microscopy and cytotoxity assay. The results indicated that the phase separation of silk fibroin/poly (ɛ-caprolactone) solution might led to inhomogeneous morphology and composition of the composite scaffolds, and the inhomogeneity of the silk fibroin/poly (ɛ-caprolactone) scaffolds with formic acid as solvent had a remarkable difference on cell adhesion and proliferation. In contrast, there was no significant difference among the silk fibroin/poly (ɛ-caprolactone) scaffolds with formic acid/acetic acid as solvent because of their good consistency in fiber morphology and composition. These obtained silk fibroin/poly (ɛ-caprolactone) nanofibers had small average diameter of 190 ± 40 nm. The obtained results proved that this study provided a facile and effective approach to achieve compositionally homogeneous silk fibroin/poly (ɛ-caprolactone) scaffolds with formic acid as solvent for effective applications.