Phthalate Esters Released from Plastics Promote Biofilm Formation and Chlorine Resistance.

Phthalate esters (PAEs) are commonly released from plastic pipes in some water distribution systems. Here, we show that exposure to a low concentration (1-10 µg/L) of three PAEs (dimethyl phthalate (DMP), di-n-hexyl phthalate (DnHP), and di-(2-ethylhexyl) phthalate (DEHP)) promotes Pseudomonas biofilm formation and resistance to free chlorine. At PAE concentrations ranging from 1 to 5 µg/L, genes coding for quorum sensing, extracellular polymeric substances excretion, and oxidative stress resistance were upregulated by 2.7- to 16.8-fold, 2.1- to 18.9-fold, and 1.6- to 9.9-fold, respectively. Accordingly, more biofilm matrix was produced and the polysaccharide and eDNA contents increased by 30.3-82.3 and 10.3-39.3%, respectively, relative to the unexposed controls. Confocal laser scanning microscopy showed that PAE exposure stimulated biofilm densification (volumetric fraction increased from 27.1 to 38.0-50.6%), which would hinder disinfectant diffusion. Biofilm densification was verified by atomic force microscopy, which measured an increase of elastic modulus by 2.0- to 3.2-fold. PAE exposure also stimulated the antioxidative system, with cell-normalized superoxide dismutase, catalase, and glutathione activities increasing by 1.8- to 3.0-fold, 1.0- to 2.0-fold, and 1.2- to 1.6-fold, respectively. This likely protected cells against oxidative damage by chlorine. Overall, we demonstrate that biofilm exposure to environmentally relevant levels of PAEs can upregulate molecular processes and physiologic changes that promote biofilm densification and antioxidative system expression, which enhance biofilm resistance to disinfectants.

Polydopamine-coated UiO-66 nanoparticles loaded with perfluorotributylamine/tirapazamine for hypoxia-activated osteosarcoma therapy.

BACKGROUND: Hypoxia is a characteristic of solid tumors that can lead to tumor angiogenesis and early metastasis, and addressing hypoxia presents tremendous challenges. In this work, a nanomedicine based on oxygen-absorbing perfluorotributylamine (PFA) and the bioreductive prodrug tirapazamine (TPZ) was prepared by using a polydopamine (PDA)-coated UiO-66 metal organic framework (MOF) as the drug carrier. RESULTS: The results showed that TPZ/PFA@UiO-66@PDA nanoparticles significantly enhanced hypoxia, induced cell apoptosis in vitro through the oxygen-dependent HIF-1α pathway and decreased oxygen levels in vivo after intratumoral injection. In addition, our study demonstrated that TPZ/PFA@UiO-66@PDA nanoparticles can accumulate in the tumor region after tail vein injection and effectively inhibit tumor growth when combined with photothermal therapy (PTT). TPZ/PFA@UiO-66@PDA nanoparticles increased HIF-1α expression while did not promote the expression of CD31 in vivo during the experiment. CONCLUSIONS: By using TPZ and PFA and the enhanced permeability and retention effect of nanoparticles, TPZ/PFA@UiO-66@PDA can target tumor tissues, enhance hypoxia in the tumor microenvironment, and activate TPZ. Combined with PTT, the growth of osteosarcoma xenografts can be effectively inhibited.

Improving the Solubility of Artificial Ligands of Streptavidin to Enable More Practical Reversible Switching of Protein Localization in Cells.

Chemical inducers that can control target-protein localization in living cells are powerful tools to investigate dynamic biological systems. We recently reported the retention using selective hook or "RUSH" system for reversible localization change of proteins of interest by addition/washout of small-molecule artificial ligands of streptavidin (ALiS). However, the utility of previously developed ALiS was restricted by limited solubility in water. Here, we overcame this problem by X-ray crystal structure-guided design of a more soluble ALiS derivative (ALiS-3), which retains sufficient streptavidin-binding affinity for use in the RUSH system. The ALiS-3-streptavidin interaction was characterized in detail. ALiS-3 is a convenient and effective tool for dynamic control of α-mannosidase II localization between ER and Golgi in living cells.

Polyethylene glycol modified PAMAM dendrimer delivery of kartogenin to induce chondrogenic differentiation of mesenchymal stem cells.

Partly PEGylated polyamidoamine (PAMAM) dendrimer was used as the nanocarrier for the cytoplasmic delivery of kartogenin (KGN) to induce chondrogenic differentiation of mesenchymal stem cells (MSCs). Here, KGN was conjugated to the surface of PAMAM and the end group of polyethylene glycol (PEG) to obtain PEG-PAMAM-KGN (PPK) and KGN-PEG-PAMAM (KPP) conjugate, respectively. The effects of PPK and KPP on the in vitro chondrogenic differentiation of MSCs were evaluated. KPP induced higher expression of chondrogenic markers than PPK and free KGN. In particular, after treatment of KPP, CBF ß nuclear localization intensity was significantly increased, indicating enhanced efficacy of chondrogenesis. The fluorescein labeled PEG-PAMAM was capable to persist in the joint cavity for a prolonged time of both healthy and osteoarthritis (OA) rats. Thus, PEG-PAMAM could be a useful nanocarrier for intra-articular (IA) delivery of drug to treat OA.

Injectable microspheres adhering to the cartilage matrix promote rapid reconstruction of partial-thickness cartilage defects.

An effective treatment for the irregular partial-thickness cartilage defect in the early stages of osteoarthritis (OA) is lacking. Cartilage tissue engineering is effective for treating full-thickness cartilage defects with limited area. In this study, we designed an injectable multifunctional poly(lactic-co-glycolic acid) (PLGA) microsphere to repair partial-thickness cartilage defects. The microsphere was grafted with an E7 peptide after loading the microsphere with kartogenin (KGN) and modifying the outer layer through dopamine self-polymerization. The microsphere could adhere to the cartilage defect, recruit synovial mesenchymal stem cells (SMSCs) in situ, and stimulate their differentiation into chondrocytes after injection into the articular cavity. Through in vivo and in vitro experiments, we demonstrated the ability of multifunctional microspheres to adhere to cartilage matrix, recruit SMSCs, and promote their differentiation into cartilage. Following treatment, the cartilage surface of the model group with partial-thickness cartilage defect showed smooth recovery, and the glycosaminoglycan content remained normal; the untreated control group showed significant progression of OA. The microsphere, a framework for cartilage tissue engineering, promoted the expression of SMSCs involved in cartilage repair while adapting to cell migration and growth. Thus, for treating partial-thickness cartilage defects in OA, this innovative carrier system based on stem cell therapy can potentially improve therapeutic outcomes. STATEMENT OF SIGNIFICANCE: Mesenchymal stem cells (MSCs) therapy is effective in the repair of cartilage injury. However, because of the particularity of partial-thickness cartilage injury, it is difficult to recruit enough seed cells in situ, and there is a lack of suitable scaffolds for cell migration and growth. Here, we developed polydopamine surface-modified PLGA microspheres (PMS) containing KGN and E7 peptides. The adhesion ability of the microspheres is facilitated by the polydopamine layer wrapped in them; thus, the microspheres can adhere to the injured cartilage and recruit MSCs, thereby promoting their differentiation into chondrocytes and accomplishing cartilage repair. The multifunctional microspheres can be used as a safe and potential method to treat partial-thickness cartilage defects in OA.

Intra-Articular Injection of PLGA/Polydopamine Core-Shell Nanoparticle Attenuates Osteoarthritis Progression.

Osteoarthritis (OA) is a common joint disease characterized by progressive cartilage degeneration. Unfortunately, currently available clinical drugs are mainly analgesics and cannot alleviate the development of OA. Kartogenin (KGN) has been found to promote the differentiation of bone marrow mesenchymal stem cells (BMSCs) into chondrocytes for the treatment of cartilage damage in early OA. However, KGN, as a small hydrophobic molecule, is rapidly cleared from the synovial fluid after intra-articular injection. This study synthesized a KGN-loaded nanocarrier based on PLGA/polydopamine core/shell structure to treat OA. The fluorescence signal of KGN@PLGA/PDA-PEG-E7 nanoparticles lasted for 4 weeks, ensuring long-term sustained release of KGN from a single intra-articular injection. In addition, the polyphenolic structure of PDA enables it to effectively scavenge reactive oxygen species, and the BMSC-targeting peptide E7 (EPLQLKM) endows KGN@PLGA/PDA-PEG-E7 NPs with an effective affinity for BMSCs. As a result, the KGN@PLGA/PDA-PEG-E7 nanoparticles could effectively induce cartilage in vitro and protect the cartilage and subchondral bone in a rat ACLT model. This therapeutic strategy could also be extended to the delivery of other drugs, targeting other tissues to treat joint diseases.

Chitosan/polycaprolactone multilayer hydrogel: A sustained Kartogenin delivery model for cartilage regeneration.

Cartilage regeneration using biomaterial-guided delivery systems presents improved therapeutic efficacy of the biomolecules while minimizing side effects. Here, our hypothesis was to design a multilayer scaffold of chitosan (CS) hydrogel and polycaprolactone (PCL) mat to enhance the mechanical properties, integrity and stability of CS, especially for subsequent in vivo transplantation. After conjugation of the Kartogenin (KGN) into this structure, its gradual release can promote chondrogenesis of mesenchymal stem cells (MSCs). Initially, a thin electrospun PCL layer was sandwiched between two CS hydrogels. Subsequently, KGN was superficially immobilized onto the CS matrix. The successful conjugation was confirmed by scanning electron microscopy (SEM) and infrared spectroscopy. These novel KGN-conjugated scaffolds possessed lower swelling and higher compressive modulus and showed gradual release of KGN in longer retention times. Immunofluorescent and histological staining represented more cells located in lacunae as well as more Coll2 and Sox9 positive cells on KGN-conjugated scaffolds. Gene expression analysis also revealed that SOX9, COLL2 and ACAN expression levels were higher in the presence of KGN, while COLLX expression was down-regulated, indicating a hypertrophy phenomenon with synergistic effect of TGF-ß. This multilayer structure not only facilitates the effective treatment, but also provides a proper mechanical structure for cartilage engineering.

Alginate sulfate-based hydrogel/nanofiber composite scaffold with controlled Kartogenin delivery for tissue engineering.

In this study, we fabricated two different arrangements of laminated composite scaffolds based on Alginate:Alginate sulfate hydrogel, PCL:Gelatin electrospun mat, and Kartogenin-PLGA nanoparticles (KGN-NPs). The optimized composite scaffold revealed a range of advantages such as improved mechanical features as well as less potential of damage (less dissipated energy), interconnected pores of hydrogel and fiber with adequate pore size, excellent swelling ratio, and controlled biodegradability. Furthermore, the synthesized KGN-NPs with spherical morphology were incorporated into the composite scaffold and exhibited a linear and sustained release of KGN within 30 days with desirable initial burst reduction (12% vs. 20%). Additionally, the cytotoxicity impact of the composite was evaluated. Resazurin assay and Live/Dead staining revealed that the optimized composite scaffold has no cytotoxic effect and could improve cell growth. Overall, according to the enhanced mechanical features, suitable environment for cellular growth, and sustained drug release, the optimized scaffold would be a good candidate for tissue regeneration.

Kartogenin Enhances Chondrogenic Differentiation of MSCs in 3D Tri-Copolymer Scaffolds and the Self-Designed Bioreactor System.

Human cartilage has relatively slow metabolism compared to other normal tissues. Cartilage damage is of great clinical consequence since cartilage has limited intrinsic healing potential. Cartilage tissue engineering is a rapidly emerging field that holds great promise for tissue function repair and artificial/engineered tissue substitutes. However, current clinical therapies for cartilage repair are less than satisfactory and rarely recover full function or return the diseased tissue to its native healthy state. Kartogenin (KGN), a small molecule, can promote chondrocyte differentiation both in vitro and in vivo. The purpose of this research is to optimize the chondrogenic process in mesenchymal stem cell (MSC)-based chondrogenic constructs with KGN for potential use in cartilage tissue engineering. In this study, we demonstrate that KGN treatment can promote MSC condensation and cell cluster formation within a tri-copolymer scaffold. Expression of Acan, Sox9, and Col2a1 was significantly up-regulated in three-dimensional (3D) culture conditions. The lacuna-like structure showed active deposition of type II collagen and aggrecan deposition. We expect these results will open new avenues for the use of small molecules in chondrogenic differentiation protocols in combination with scaffolds, which may yield better strategies for cartilage tissue engineering.

Scale-up of non-toxic poly(butylene adipate-co-terephthalate)-Chitin based nanocomposite articles by injection moulding and 3D printing.

Poly(butylene adipate-co-terephthalate) (PBAT), a compostable polymer, filled with different weight percentage of unbleached nano chitin (NC; 10%, 30% and 50%), a biodegradable filler from crustacean waste, were prepared from the extruded blends by injection moulding and 3D printing. The nanochitin required was prepared from chitin isolated from prawn shells (Fenneropenaeus indicus). The nanochitin crystals were observed to contain carboxylic acid surface functional groups as assessed by FT-IR, 13C solid state NMR (SS NMR) spectroscopy, zeta potential measurements and the extent of the same was estimated by potentiometric titration. The PBAT-NC nanocomposites were characterized SS NMR spectroscopy, FT-IR spectroscopy, wide angle X-ray diffraction, dynamic mechanical analysis, DSC and TGA. Thermal and mechanical properties of the nanocomposites were determined. The moulded nanocomposites changed more and more rigid with increasing weight percentage of NC without significant change in the tensile strength. The TGA indicated that the thermal stability of PBAT could be improved but not significantly by the addition of NC. Wound healing was enhanced in the presence of the nanocomposite while in vivo toxicity was significant at high concentration. The PBAT-NC nanocomposites could be moulded in to useful articles such as laptop charger cover, rat cover for washing machine, planters and key holders under conditions similar to that used in the processing of LDPE.

Kartogenin-loaded coaxial PGS/PCL aligned nanofibers for cartilage tissue engineering.

Electrospinning is a valuable technology for cartilage tissue engineering (CTE) due to its ability to produce fibrous scaffolds mimicking the nanoscale and alignment of collagen fibers present within the superficial zone of articular cartilage. Coaxial electrospinning allows the fabrication of core-shell fibers able to incorporate and release bioactive molecules (e.g., drugs or growth factors) in a controlled manner. Herein, we used coaxial electrospinning to produce coaxial poly(glycerol sebacate) (PGS)/poly(caprolactone) (PCL) aligned nanofibers (core:PGS/shell:PCL). The obtained scaffolds were characterized in terms of their structure, chemical composition, thermal properties, mechanical performance and in vitro degradation kinetics, in comparison to monoaxial PCL aligned fibers and respective non-aligned controls. All the electrospun scaffolds produced presented average fiber diameters within the nanometer-scale and the core-shell structure of the composite fibers was clearly confirmed by TEM. Additionally, fiber alignment significantly increased (>2-fold) the elastic modulus of both coaxial and monoxial scaffolds. Kartogenin (KGN), a small molecule known to promote mesenchymal stem/stromal cells (MSC) chondrogenesis, was loaded into the core PGS solution to generate coaxial PGS-KGN/PCL nanofibers. The KGN release kinetics and scaffold biological performance were evaluated in comparison to KGN-loaded monoaxial fibers and respective non-loaded controls. Coaxial PGS-KGN/PCL nanofibers showed a more controlled and sustained KGN release over 21 days than monoaxial PCL-KGN nanofibers. When cultured with human bone marrow MSC in incomplete chondrogenic medium (without TGF-ß3), KGN-loaded scaffolds enhanced significantly cell proliferation and chondrogenic differentiation, as suggested by the increased sGAG amounts and chondrogenic markers gene expression levels. Overall, these findings highlight the potential of using coaxial PGS-KGN/PCL aligned nanofibers as a bioactive scaffold for CTE applications.

Chitosan modified Fe3O4/KGN self-assembled nanoprobes for osteochondral MR diagnose and regeneration.

Chondral and osteochondral defects caused by trauma or pathological changes, commonly progress into total joint degradation, even resulting in disability. The cartilage restoration is a great challenge because of its avascularity and limited proliferative ability. Additionally, precise diagnosis using non-invasive detection techniques is challenging, which increases problems associated with chondral disease treatment. Methods: To achieve a theranostic goal, we used an integrated strategy that relies on exploiting a multifunctional nanoprobe based on chitosan-modified Fe3O4 nanoparticles, which spontaneously self-assemble with the oppositely charged small molecule growth factor, kartogenin (KGN). This nanoprobe was used to obtain distinctively brighter T2-weighted magnetic resonance (MR) imaging, allowing its use as a positive contrast agent, and could be applied to obtain accurate diagnosis and osteochondral regeneration therapy. Results: This nanoprobe was first investigated using adipose tissue-derived stem cells (ADSCs), and was found to be a novel positive contrast agent that also plays a significant role in stimulating ADSCs differentiation into chondrocytes. This self-assembled probe was not only biocompatible both in vitro and in vivo, contributing to cellular internalization, but was also used to successfully make distinction of normal/damaged tissue in T2-weighted MR imaging. This novel combination was systematically shown to be biosafe via the decrement of apparent MR signals and elimination of ferroferric oxide over a 12-week regeneration period. Conclusion: Here, we established a novel method for osteochondral disease diagnosis and reconstruction. Using the Fe3O4-CS/KGN nanoprobe, it is easy to distinguish the defect position, and it could act as a tool for dynamic observation as well as a stem cell-based therapy for directionally chondral differentiation.

Biofunctionalized chondrogenic shape-memory ternary scaffolds for efficient cell-free cartilage regeneration.

Cartilage defect repair remains a great clinical challenge due to the limited self-regeneration capacity of cartilage tissue. Surgical treatment of injured cartilage is rather difficult due to the narrow space in the articular cavity and irregular defect area. Herein, we designed and fabricated chondrogenic and physiological-temperature-triggered shape-memory ternary scaffolds for cell-free cartilage repair, where the poly (glycerol sebacate) (PGS) networks ensured elasticity and shape recovery, crystallized poly (1,3-propylene sebacate) (PPS) acted as switchable phase, and immobilized bioactive kartogenin (KGN) endowed the scaffolds with chondrogenic capacity. The resultant scaffolds exhibited shape-memory properties with shape-memory fixed ratio of 98% and recovered ratio of 97% at 37°C for PPS/PGS/KGN-100, indicating a good potential for minimally invasive implantation. The scaffolds gradually degraded in Dulbecco's phosphate-buffered saline and released KGN up to 12 weeks in vitro. In addition, the scaffolds promoted chondrogenic differentiation while inhibiting osteogenic differentiation of bone marrow-derived mesenchymal stem cells in a concentration-dependent manner and cartilage regeneration in full-thickness defects of rat femoropatellar groove for 12 weeks. Consequently, the PPS/PGS/KGN-100 scaffolds stimulated the formation of an overlying layer of neocartilage mimicking the characteristic architecture of native articular cartilage even in the absence of exogenous growth factors and seeded cells. This study provides much inspiration for future research on cartilage tissue engineering. STATEMENT OF SIGNIFICANCE: There are two crucial challenges for cartilage defect repair: the lack of self-regeneration capacity of cartilage tissue and difficult scaffold implantation via traditional open surgery due to space-limited joints. Herein, bioactive body-temperature-responsive shape memory scaffolds are designed to simultaneously address the challenges. The scaffolds can be readily implanted by minimally invasive approach and recover by body-temperature of patient. The integration of kartogenin endows scaffolds the bioactivity, leading to the first example of bulk shape-memory scaffolds for cell-free cartilage repair. These characteristics make the scaffolds advantageous for clinical translation. Moreover, our developed material is easy to be functionalized due to the presence of extensive free hydroxyl groups and provides a versatile platform to design diverse functional shape memory biomaterials.

Injectable double-crosslinked hydrogels with kartogenin-conjugated polyurethane nano-particles and transforming growth factor ß3 for in-situ cartilage regeneration.

Articular cartilage has a limited ability for self-repair after injury. Implantation of scaffolds functionalized with bioactive molecules that could induce the migration and chondrogenesis of endogenous mesenchymal stem cells (MSCs) provides a convenient alternative for in-situ cartilage regeneration. In this study, we found the synergistic effects of kartogenin (KGN) and transforming growth factor ß3 (TGF-ß3) on chondrogenesis of MSCs in vitro, indicating that KGN and TGF-ß3 are a good match for cartilage regeneration. Furthermore, we confirmed that KGN promoted the chondrogenesis of MSCs through attenuating the degradation of Runx1, which physically interacted with p-Smad3 in nuclei of MSCs. Meanwhile, we designed an injectable double-crosslinked hydrogel with superior mechanical property and longer support for cartilage regeneration by modifying sodium alginate and gelatin. When loaded with KGN conjugated polyurethane nanoparticles (PN-KGN) and TGF-ß3, this hydrogel showed biological functions by the release of KGN and TGF-ß3, which promoted the MSC migration and cartilage regeneration in one system. In conclusion, the cell-free hydrogel, along with PN-KGN and TGF-ß3, provides a promising strategy for cartilage repair by attracting endogenous MSCs and inducing chondrogenesis of recruited cells in a single-step procedure.

Preparation and characterization of the collagen/cellulose nanocrystals/USPIO scaffolds loaded kartogenin for cartilage regeneration.

The regeneration of cartilage is a challenging problem for lack of innate abilities to mount a sufficient healing response. Kartogenin (KGN), an emerging chondroinductive non-protein small molecule, bound to the surface of the ultrasmall super-paramagnetic iron-oxide (USPIO) by innovational one-step technology, followed by being incorporated into the cross-linking collagen/cellulose nanocrystals (Col/CNC) bioactive scaffolds to stimulate an appropriate microenvironment for the growth and differentiation of bone marrow-derived mesenchymal stem cells (BMSCs), thus facilitating the formation of chondrocyte. Herein, USPIO not only served as a carrier for small molecule drugs, but also as MRI contrast agents, which can non-invasively monitor the degradation of the scaffolds and the self-repair capacity of cartilage. In vitro studies showed that the KGN could release from the composite scaffolds in a sustained and stable manner and promote the chondrogenic differentiation of BMSCs based on UV spectrophotometry test, and specific markers analysis. Of note, USPIO labeled composite scaffolds retained their stability without loss of relaxation rate the composite scaffolds can be a promising biomaterials for cartilage repair, with the function of noninvasive visualization and semiquantitative analysis of scaffolds degradation and neocartilage.

Bioactivity of Marine Streptomyces sp. VITJS4: Interactions of Cytotoxic Phthalate Derivatives with Human Topoisomerase II α: An In Silico Molecular Docking Analysis.

Despite clinical advances in antimicrobial and anticancer therapy, there is an urge for the search of new bioactive compounds. In the present study, previously isolated Streptomyces sp. VITJS4 strain (NCIM No. 5574) (ACC No: JQ234978.1) crude extract tested for antibacterial activity showed a broad spectrum at the concentration of 20 mg/mL against pathogens. The antioxidant potential tested at 0.5 mg/mL concentration exhibited reducing power activity with a maximum of 90 % inhibition. The anticancer property by MTT assay on HeLa and HepG2 cells showed cytotoxic effect with IC50 of 50 µg/mL each. The DNA fragmentation pattern observed in both HeLa and HepG2 cell indicated laddering pattern at 40 µg/mL concentration. GC-MS analysis revealed that the significant peak corresponding at m/z 149 (M+) was identified as phthalate derivatives. The extract was further separated by HPLC with their retention times (t r) at 6.294 min. The above-obtained results were also supported by molecular docking studies which provide an insight into ligand binding to the active site of the receptor. The in silico docking studies revealed better binding affinity with a binding energy of -5.87 kJ mol-1 of the ligand toward topoisomerase II α.

(E)-2-Methoxy-4-(3-(4-methoxyphenyl) prop-1-en-1-yl) phenol attenuates PMA-induced inflammatory responses in human monocytic cells through PKCδ/JNK/AP-1 pathways.

(E)-2-Methoxy-4-(3-(4-methoxyphenyl) prop-1-en-1-yl) phenol (MMPP), a new (E)-2,4-bis(p-hydroxyphenyl)-2 - butenal derivative, reportedly has therapeutic effects such as anti-arthritic properties. Although previous studies showed that MMPP has anti-arthritic effects on rheumatoid arthritis (RA), the anti-inflammation mechanism of MMPP remains unclear. In this study, phorbol-12-myristate 13-acetate (PMA) was used as an inflammatory stimulus to evaluate the detailed mechanism of the MMPP-mediated anti-inflammatory effect in human monocytic THP-1 cells. We investigated the effects of MMPP on inflammation-related pathways including protein kinase Cδ (PKCδ), mitogen-activated protein kinase, and activator protein-1 (AP-1). PMA induced the translocation of PKCs from the cytosol to the membrane and phosphorylated JNK. MMPP inhibited PMA-induced membrane translocation of PKCδ, phosphorylation of JNK, and nuclear translocation of AP-1, resulting in downregulation of cyclooxygenase-2 and chemokine ligand 5 production. These findings indicate that MMPP inhibits inflammatory responses in THP-1 cells by mitigating PMA-induced activation of PKCδ and JNK and nuclear translocation of AP-1. Therefore, MMPP may be useful as an anti-inflammatory drug.

Development of kartogenin-conjugated chitosan-hyaluronic acid hydrogel for nucleus pulposus regeneration.

Injectable constructs for in vivo gelation have many advantages in the regeneration of degenerated nucleus pulposus. In this study, an injectable hydrogel consisting of chitosan (CS) and hyaluronic acid (HA) crosslinked with glycerol phosphate (GP) at different proportions (CS : GP : HA, 6 : 3 : 1, 5 : 3 : 2, 4 : 3 : 3, 3 : 3 : 4, 2 : 3 : 5, 1 : 3 : 6, V : V : V) was developed and employed as a delivery system for kartogenin (KGN), a biocompound that can activate chondrocytes. In vitro gelation time, morphologies, swelling, weight loss, compressive modulus and cumulative release of KGN in hydrogels were studied. For biocompatibility assessments, human adipose-derived stem cells (ADSCs) were encapsulated in these hydrogels. The effects of KGN on stem cell proliferation and differentiation into nucleus pulposus-like cells were examined. The hydrogels with higher concentrations of HA showed a slightly shorter gelation time, higher water uptake, faster weight loss and faster KGN release compared to the hydrogels with lower concentrations of HA. As the KGN-conjugated hydrogel prepared with the proportions 5 : 3 : 2 displayed good mechanical properties, it was chosen as the optimal gel to promote cell proliferation and differentiation. No significant difference was seen in the expression levels of nucleus pulposus markers induced by KGN or TGF-ß. Additionally, inclusion of KGN and TGF-ß together did not produce a synergistic effect in inducing nucleus pulposus properties. In conclusion, we have developed a KGN-conjugated CS/HA hydrogel (5 : 3 : 2) with sustained release of KGN in hydrogel that can promote ADSC proliferation and nucleus pulposus differentiation. This kind of hydrogel may be a simple and effective candidate for the repair of degenerative NP tissue after minimally invasive surgery.

Evaluation of the potential of kartogenin encapsulated poly(L-lactic acid-co-caprolactone)/collagen nanofibers for tracheal cartilage regeneration.

Tracheal stenosis is one of major challenging issues in clinical medicine because of the poor intrinsic ability of tracheal cartilage for repair. Tissue engineering provides an alternative method for the treatment of tracheal defects by generating replacement tracheal structures. In this study, we fabricated coaxial electrospun fibers using poly(L-lactic acid-co-caprolactone) and collagen solution as shell fluid and kartogenin solution as core fluid. Scanning electron microscope and transmission electron microscope images demonstrated that nanofibers had uniform and smooth structure. The kartogenin released from the scaffolds in a sustained and stable manner for about 2 months. The bioactivity of released kartogenin was evaluated by its effect on maintain the synthesis of type II collagen and glycosaminoglycans by chondrocytes. The proliferation and morphology analyses of mesenchymal stems cells derived from bone marrow of rabbits indicated the good biocompatibility of the fabricated nanofibrous scaffold. Meanwhile, the chondrogenic differentiation of bone marrow mesenchymal stem cells cultured on core-shell nanofibrous scaffold was evaluated by real-time polymerase chain reaction. The results suggested that the core-shell nanofibrous scaffold with kartogenin could promote the chondrogenic differentiation ability of bone marrow mesenchymal stem cells. Overall, the core-shell nanofibrous scaffold could be an effective delivery system for kartogenin and served as a promising tissue engineered scaffold for tracheal cartilage regeneration.

Comparison of P75 NTR-positive and -negative etcomesenchymal stem cell odontogenic differentiation through epithelial-mesenchymal interaction.

OBJECTIVES: The aim of this study was to investigate differences of odonto-differentiation between P75 -neurotrophin receptor (P75 -NTR)-positive ectomesenchymal stem cells (P75+EMSCs) and P75 -NTR-negative ectomesenchymal stem cells (P75-EMSCs), and their underlying mechanisms. MATERIALS AND METHODS: Primary cranial neural crest-derived cells (CNC) were isolated from the first branchial arches, and P75+EMSCs and P75-EMSCs were sorted by fluorescence-activated cell sorting. Differentiation of P75+EMSCs or P75-EMSCs into odontoblast-like cells was induced by dental epithelial cells in vitro or in vivo. Differential gene expression profiles between P75+EMSCs and P75-EMSCs were analysed by microarray assay. Smad4-specific small interfering RNA and activator kartogenin were used to treat the cells, to evaluate effects of Smad4 in odonto-differentiation of P75+EMSCs or P75-EMSCs. RESULTS: Under induction of dental epithelium conditioned medium, P75+EMSCs had more mineralized node formation and higher expression of Dmp1 and Dspp compared to P75-EMSCs. In our in vivo study, graft of P75+EMSCs recombination with dental epithelium showed higher expression of DMP1 and DSP. Knock-down of Smad4 in P75+EMSCs significantly downregulated expression of DMP1 and DSP, while activation of Smad4 in P75-EMSCs by the activator kartogenin, significantly increased DSP and DMP1 expression. CONCLUSIONS: P75+EMSCs showed more odonto-differentiation potential than P75-EMSCs both in vivo and in vitro. Smad4 played a critical role in determination of odonto-differentiation potential of CNC-derived EMSCs.