Characterization and cytocompatibility of thermosensitive hydrogel embedded with chitosan nanoparticles for delivery of bone morphogenetic protein-2 plasmid DNA.

A novel injectable chitosan thermosensitive hydrogel was designed as a target multi-effect scaffold for endogenous repair of the periodontium. The hydrogel complex was designed by embedding chitosan nanoparticles (CSn) loaded with bone morphogenetic protein-2 plasmid DNA (pDNA-BMP2) into a chitosan (CS)-based hydrogel with α,ß-glycerophosphate (α,ß-GP), termed CS/CSn(pDNA-BMP2)-GP. Characterization, the in vitro release profile for pDNA-BMP2, and cytocompatibility to human periodontal ligament cells (HPDLCs), were then conducted. The average diameter of the CSn(pDNA-BMP2) was 270.1 nm with a polydispersity index (PDI) of 0.486 and zeta potential of +27.0 mv. A DNase I protection assay showed that CSn could protect the pDNA-BMP2 from nuclease degradation. Encapsulation efficiency and loading capacity of CSn(pDNA-BMP2) were more than 80 and 30 %, respectively. The sol-gel transition time was only 3 min when CSn(pDNA-BMP2) was added into the CS/α,ß-GP system. Scanning electron microscopy showed that CSn(pDNA-BMP2) was randomly dispersed in a network with regular holes and a porous structure. Weighting method showed the swelling ratio and degradation was faster in medium of pH 4.0 than pH 6.8. An in vitro pDNA-BMP2 release test showed that the cumulative release rate of pDNA-BMP2 was much slower from CS/CSn-GP than from CSn in identical release media. In release media with different pH, pDNA-BMP2 release was much slower at pH 6.8 than at pH 4.0. Three-dimensional culture with HPDLCs showed good cell proliferation and the Cell-Counting Kit-8 assay indicated improved cell growth with the addition of CSn(pDNA-BMP2) to CS/α,ß-GP. In summary, the CS/CSn(pDNA-BMP2)-GP complex system exhibited excellent biological properties and cytocompatibility, indicating great potential as a gene delivery carrier and tissue regeneration scaffold for endogenous repair of the periodontium.

Systemically transplanted human gingiva-derived mesenchymal stem cells contributing to bone tissue regeneration.

As novel postnatal stem cells, gingiva-derived mesenchymal stem cells (GMSCs) have been considered as an ideal candidate cell resource for tissue engineering and cell-based therapies. GMSCs implanted into sites of injury have been confirmed to promote the injury repair. However, no studies have demonstrated whether systemically transplanted GMSCs can home to the bone injuries and contribute to the new bone formation in vivo. In this study, we transplanted human GMSCs into C57BL/6J mice with defects in mandibular bone via the tail vein to explore the capacity of transplanted GMSCs to promote bone regeneration. Results showed that the transplanted GMSCs were detected in the bone defects and employed in new bone formation. And the newly formed bone area in mice with GMSCs transplantation was significantly higher than that in control mice. Our findings indicate that systemically transplanted GMSCs can not only home to the mandibular defect but also promote bone regeneration.

[Cytocompatibility of chitosan-based thermosensitive hydrogel to human periodontal ligament cell].

PURPOSE: The aim of this investigation was to evaluate the cytocompatibility of an in situ chitosan-quaternized chitosan/α, ß-glycerophosphate (CS-HTCC/GP) thermosensitive hydrogel in vitro. METHODS: The primary cells were isolated from human periodontal ligament and cultured. The role of different concentrations of CS-HTCC/GP extract to HPDLCs was evaluated by MTT assay and alkaline phosphatase (ALP) activity. Also, the ultra-architecture of HPDLCs was determined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) respectively. SPSS13.0 software package was used for statistical analysis. RESULTS: By immunocytochemical method, the cells were stained positively to antibodies against vimentin, and negatively to antibodies against cytokeratin, which indicated that they were external embryo mesenchymal cell without epithelial cell mixure. CS-HTCC/GP thermosensitive hydrogel promoted proliferation of HPDLCs,especially at 3d and 5d, the results was significantly different (P<0.001). ALP activity was significantly greater in group 2 and 3 than in group 4 after 5d (P<0.001). Also, no negative influence to ultrastructure of HPDLCs was found through SEM and TEM. CONCLUSIONS: The results indicate that CS-HTCC/GP thermosensitive hydrogel exhibits excellent cytocompatibility and has potential to be used as an in situ injectable local periodontal drug delivery vehicle and a tissue-engineering scaffold for periodontal disease therapy.

In vitro evaluation of mucoadhesion and permeation enhancement of polymeric amphiphilic nanoparticles.

Oleoyl-carboxymethy-chitosan (OCMCS) nanoparticles based on chitosan with various molecular weights were prepared using coacervation process, which demonstrated particle size of 150-350 nm, zeta potential of 10-20 mV, and high encapsulation efficiency of fluorescein isothiocyanate dextran (FD4). OCMCS nanoparticles were found to be adsorbed onto the excised carp intestinal mucosa, the extent of adsorption increased with increasing chitosan molecular weight. In comparison to FD4 solution, OCMCS nanoparticles promoted FD4 transport through excised carp intestinal mucosa by 3.26-6.52 folds, which were observed via fluorescence microscope. The OCMCS nanoparticulate systems that interacted with the Caco-2 cells decreased the transepithelial electric resistance (TEER) and induced increasing the apparent permeability coefficient (Papp) of FD4 by 3.61-6.32 folds. Cytotoxicity studies in Caco-2 monolayers verified the safety of the delivery system. The improvement of mucoadhesive ability and permeability enable the OCMCS nanosystems suitable carriers for the intestinal absorption of protein drugs.

A novel injectable chlorhexidine thermosensitive hydrogel for periodontal application: preparation, antibacterial activity and toxicity evaluation.

The aim of this paper was to evaluate the application potential of CS-HTCC/GP-0.1%Chx thermosensitive hydrogel which was synthesized using chitosan (CS), quaternized CS, and alpha,beta-glycerophosphate (alpha,beta-GP) loading with 0.1% chlorhexidine (Chx) (w/v) for periodontal treatment. An aqueous solution of CS-HTCC/GP-0.1%Chx was transformed into hydrogel at 6 min when the temperature was increased to 37 degrees C. The scan electron microscopy (SEM) image of the gel was a porous, loose and crosslinked network. In vitro, Chx released over 18 h from the CS-HTCC/GP thermosensitive hydrogel in artificial saliva pH 6.8. Release rate could be controlled through adjustment of alpha,beta-GP or Chx concentration. CS-HTCC/GP-0.1%Chx thermosensitive hydrogel exhibited excellent inhibitory activity against primary periodontal pathogens. CS-HTCC/GP-0.1%Chx thermosensitive hydrogel had no acute toxicity; the maximum tolerated dose in rats was 400 mg/ml. All results indicated that CS-HTCC/GP-0.1%Chx thermosensitive hydrogel is a strong candidate as a local drug delivery system for periodontal treatment.

[An in vitro evaluation of the antibacterial activity of chitosan-based thermosensitive hydrogel against periodontal pathogens].

PURPOSE: To evaluate the in vitro antibacterial activity of chitosan - quaternized chitosan/alpha, beta-glycerophosphate (CS-HTCC/GP) thermosensitive hydrogel against three periodontal pathogens- P. gingivalis, P. intermedia, and A. actinomycetemcomitans. METHODS: An agar diffusion method was used to assess the antimicrobial property of CS-HTCC/GP thermosensitive hydrogel with minimum inhibitory concentration (MIC) and inhibitory zone measurement. SPSS13.0 software package was used for Student's t test. RESULTS: Three periodontal pathogens strains were all susceptible to CS-HTCC/GP thermosensitive hydrogel. Both matrix of thermosensitive hydrogel and antibiotic exhibited stronger antibacterial activity especially when they were combined. CONCLUSIONS: CS-HTCC/GP thermosensitive hydrogel is not only as the vehicle of antibiotics which joins the local drug delivery system but as an activator which takes part in the antibacterial process.

In vitro evaluation of the biomedical properties of chitosan and quaternized chitosan for dental applications.

The aim of this study was to evaluate the potential dental applications of chitosan (CS) and N-[1-hydroxy-3-(trimethylammonium)propyl]chitosan chloride (HTCC). HTCC was prepared by reacting CS with glycidyltrimethylammonium chloride (GTMAC). CS and HTCC were characterized by infrared (FITR) and (1)H NMR spectroscopy. The antibacterial activity of CS and HTCC against oral pathogens, their proliferation activity and effects on the ultrastructure of human periodontal ligament cells (HPDLCs) were investigated. The results indicated that four oral strains were susceptible to CS and HTCC with minimum inhibitory concentrations (MICs) ranging from 0.25 to 2.5mg/mL. The in vitro 3-(4,5-dimethyl-2-thizolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay determined that CS at 2000, 1000, 100, and 50microg/mL could stimulate the proliferation of HPDLCs. Instead, HTCC inhibited the proliferation at the same concentrations but accelerated the proliferation of HPDLCs at relatively low concentrations (10, 3, 1.5, 1, and 0.3microg/mL). Transmission electron microscopy (TEM) observations revealed that the ultra-architecture of HPDLC was seriously destroyed by HTCC treatment at 1000microg/mL. Taken together, these results contribute information necessary to enhance our understanding of CS and HTCC in the dental field.

Injectable thermosensitive hydrogel based on chitosan and quaternized chitosan and the biomedical properties.

A novel injectable thermosensitive hydrogel (CS-HTCC/alpha beta-GP) was successfully designed and prepared using chitosan (CS), quaternized chitosan (HTCC) and alpha,beta-glycerophosphate (alpha,beta-GP) without any additional chemical stimulus. The gelation point of CS-HTCC/alpha beta-GP can be set at a temperature close to normal body temperature or other temperature above 25 degrees C. The transition process can be controlled by adjusting the weight ratio of CS to HTCC, or different final concentration of alpha,beta-GP. The optimum formulation is (CS + HTCC) (2% w/v), CS/HTCC (5/1 w/w) and alpha,beta-GP 8.33% or 9.09% (w/v), where the sol-gel transition time was 3 min at 37 degrees C. The drug released over 3 h from the CS-HTCC/alpha,beta-GP thermosensitive hydrogel in artificial saliva pH 6.8. In addition, CS-HTCC/alpha,beta-GP thermosensitive hydrogel exhibited stronger antibacterial activity towards two periodontal pathogens (Porphyromonas gingivalis, P.g and Prevotella intermedia, P.i). CS-HTCC/alpha, beta-GP thermosensitive hydrogel was a considerable candidate as a local drug delivery system for periodontal treatment.

Biological evaluation of a novel chitosan-PVA-based local delivery system for treatment of periodontitis.

In the study, we intend to design a suitable localized drug delivery system (LDDS) with chitosan and poly vinyl alcohol (PVA) for treating serve periodontitis. For that, a novel formulation based on the incorporation of chitosan-based microspheres into PVA film was prepared. As the core parts of the novel formulation, chitosan-based microspheres were prepared form chitosan and/or carboxymethyl-chitosan (CM-chitosan) by using water-in-oil emulsification method. Then basic in vitro and in vivo experiments focusing on biocompatibility and biodegradability of the two chitosan-based microspheres were carried out to evaluate the feasibility of the novel LDDS. In vitro tests, besides having no hemolysis, chitosan microsphere (Cs1-Ms), and CM-chitosan microsphere (Cs2-Ms) have adsorbed little proteins on their surfaces. Moreover, plasma proteins adsorbed on Cs2-Ms, most of which can easily desorbed, are much less than that adsorbed on Cs1-Ms. This indicates that Cs2-Ms perhaps has better biocompatibility than Cs1-Ms. In vivo tests, Cs1-Ms and Cs2-Ms were subcutaneously implanted in rat to investigate the host tissue inflammatory response. Implantations of Cs1-Ms and Cs2-Ms induced a little more severe inflammation when compared with the implantation of PVA film. However, the difference on in vivo biocompatibility between Cs1-Ms and Cs2-Ms could not be confirmed by the implantation model of our experiments. Both Cs1-Ms and Cs2-Ms had suffered bioerosion when they were subcutaneously implanted. The hard and compact matrixes of Cs1-Ms were degraded very slowly, and only some trifling degradation had been found until 4 weeks of implantation. In contrast, Cs2-Ms is soft and more hydrophilic, and can be quickly degraded in a form of diffluence by the physiological circumstance. All these results suggested that Cs2-Ms had better potentials used as core parts of the novel designed LDDS in the future developments.

Preparation and function of composite asymmetric chitosan/CM-chitosan membrane.

A novel composite asymmetric chitosan/CM-chitosan membrane (C-P-C) was prepared, the top-layer was chitosan (CS), the intermediate was PVA, and the substrate was carboxymethyl chitosan (CM-CS). C-P-C membrane had capability in mechanical strength, light transparence, vapor permeability, and wound skin joining. The CS and CM-CS in C-P-C membrane were selected by series independent experiments, respectively. CS (MW 90,000 Da) had the highest antibacterial activity for E.coli. CM-CS had biocompatibility, no cytotoxicity, and had the activity of promoting growth of human skin fibroblast and inhibiting the growth of keloid fibroblast. The normal skin fibroblast can growth on the CM-CS surface of C-P-C, and have no conglomeration in higher cell density, and the keloid fibroblast could not growth on CM-CS surface of C-P-C. The animal experiment demonstrated that wound, covered with the C-P-C membrane, was hemostatic, healing quickly and had histocompatibility. The results indicated that the C-P-C membrane could be used as dressing of skin repair, and had the potential in promoting wound healing and inhibiting the keloid formation.