Xiangrong Cheng

Porphyromonas gingivalis may play an important role in the pathogenesis of periodontitis-associated rheumatoid arthritis.

Rheumatoid arthritis (RA) is a common, systemic autoimmune disease which leads to destruction of the joint architecture and consequent disability. Although the aetiology of RA remains unknown, accumulating studies have established a strong association between RA and periodontitis (PD). Recently, anti-cyclic citrullinated peptide (anti-CCP) autoantibody and citrullinated peptide have been realized to be involved in the breaking of self-tolerance and development of autoimmune in RA. The citrullinated peptide is generated by post-translational modification (citrullination) of protein-bound arginine by peptidylarginine deiminase (PAD). Porphyromonas gingivalis(P. gingivalis), the major aetiological agent of PD and the only bacterium known to express a PAD enzyme, has been reported to be significantly associated with RA. The antibody titers to P. gingivalis are significantly increased in patients with RA and P. gingivalis antibody titers are significantly correlated with anti-CCP antibody isotypes that are specific to RA. Recent study indicates that the major synovial targets of the RA-specific anti-CCP autoantibodies are deiminated forms of the alpha- and beta- chains of fibrin. Meanwhile, it is also confirmed that bacterial PAD produced by P. gingivalis has the capacity of deiminating arginine in fibrin found in the periodontal lesion. Whats more, it has been demonstrated that citrullination of HLA binding peptide causes a 100-fold increase in peptide-MHC affinity and leads to the activation CD4(+)T cells in HLA DRB1 0401 transgenic mice. Therefore, we postulate that P. gingivalis may play a crucial role in the pathogenesis of periodontitis-associated RA. P. gingivalis, which colonizes in the oral cavity, produces PAD enzyme continuously that leads to the citrullination of RA autoantigen such as fibrin in synovium joint. These PAD engendered antigens, presented in association with major histocompatibility complex (MHC) molecules by antigen-presenting cells (APC), ultimately lead to production of the anti-CCP antibody. The anti-CCP antibodies form immune complexes with citrullinated proteins, which can be bound by inflammatory cells via their Fc receptors. The roles of these immune complexes and inflammatory cells are mediated by a complex cascade involving complement activation. These mechanisms result in a release of mediators of inflammation and joint destruction ultimately leading to the onset of RA. This hypothesis reveals that oral bacterial infection may play a role in peptide citrullination which might be involved in loss of self-tolerance and development of autoimmune in RA.

Enhanced bone regeneration around dental implant with bone morphogenetic protein 2 gene and vascular endothelial growth factor protein delivery

OBJECTIVE To evaluate the synergistic effect of bone morphogenetic protein 2 (BMP-2) and vascular endothelial growth factor (VEGF) on the repair of bone defects around dental implants. MATERIAL AND METHODS Five groups of scaffold were fabricated by a freeze-drying method, including pure chitosan/collagen scaffold; scaffold loaded with adenoviruses expressing BMP-2, adenoviruses expressing VEGF, both adenoviruses expressing BMP-2 and VEGF, VEGF protein and adenovirus expressing BMP-2. In vitro studies examined whether bone marrow stromal cells were responsive to these scaffolds over time. Bone formation capacity, bone-to-implant contact, as well as removal torque values were investigated in vivo. Differences between the various groups were statistically analyzed using the one-way analysis of variance test. RESULTS The in vitro study revealed a burst and rapid release of VEGF with a sustained high-level expression of BMP-2 in scaffold combined with VEGF protein and adenoviruses expressing BMP-2. Histomorphometry demonstrated that scaffolds expressing BMP-2 enhanced more bone formation compared with other groups; VEGF alone is insufficient to promote bone formation. New bone formation in the bone defects around dental implants, bone-to-implant contact and mean peak removal torque showed statistically significant difference for the adenoviral vector encoding human bone morphogenetic protein 2 (Ad-BMP-2) and VEGF protein and adenovirus expressing BMP-2 groups. Furthermore, scaffold combined with VEGF protein and Ad-BMP-2 represented the best outcomes in this model. CONCLUSIONS A combination of BMP-2 gene and VEGF protein could have a synergistic effect in promoting bone healing.

The synergetic bone-forming effects of combinations of growth factors expressed by adenovirus vectors on chitosan/collagen scaffolds

Supporting and enhancing the regeneration and stability of alveolar bone at dental implant sites is necessary to improve implant integration and stability. In this study chitosan/collagen scaffolds combined with adenoviruses expressing either bone morphogenetic protein 7 (BMP-7), platelet-derived growth factor B (PDGF-B), or a combination of both BMP-7 and PDGF-B, were prepared by a freeze-drying method. The cytotoxicity of the resulting biomaterials, and their potential effects on cell proliferation and differentiation towards an osteoblast phenotype, were studied in an initial in vitro study using human periodontal ligament cells (HPLCs). These in vitro results indicate that the scaffold materials are not cytotoxic, and moreover that scaffolds expressing PDGF-B show a higher proliferation rate, while scaffolds expressing BMP-7 show a stronger differentiation towards the osteoblast phenotype. In a subsequent in vivo study, scaffolds were implanted into surgically induced defects on both sides of the dog mandible. Bone formation, which was evaluated histomorphometrically at 4 and 8 weeks and after sacrificing the animals at 12 weeks, revealed a significant increase in bone formation in scaffolds expressing only BMP-7 and also the combined growth factors, with the highest increase occurring for the scaffold expressing both BMP-7 and PDGF-B. This study demonstrates the promising potential of biomaterial expression of combinations of growth factors such as BMP-7 and PDGF-B for bone regeneration in tissue engineering applications.

Novel gene-activated matrix with embedded chitosan/plasmid DNA nanoparticles encoding PDGF for periodontal tissue engineering.

Recently, much attention has been paid to tissue engineering and local gene delivery system in periodontal tissue regeneration. Gene-activated matrix (GAM) blends these two strategies, serving as a local bioreactor with therapeutic gene expression and providing a structural template to fill the lesion defects for cell adhesion, proliferation and synthesis of extracellular matrix (ECM). In this study, we designed a novel GAM with embedded chitosan/plasmid nanoparticles encoding platelet derived growth factor (PDGF) based on porous chitosan/collagen composite scaffold. The chitosan/collagen scaffold acted as three-dimensional carrier and chitosan nanoparticles condensed plasmid DNA. The plasmid DNA entrapped in the scaffolds showed a sustained and steady release over 6 weeks and could be effectively protected by chitosan nanoparticles. MTT assay demonstrated that periodontal ligament cells (PDLCs) cultured into the novel GAM achieved high proliferation. Luciferase reporter gene assay displayed that the novel GAM could express 1.07 x 10(4) LU/mg protein after 1 week and 8.97 x 10(3) LU/mg protein after 2 weeks. The histological results confirmed that PDLCs maintained a fibroblast figure and the periodontal connective tissue-like structure formed in the scaffolds after 2 weeks. Semi-quantitative immunohistochemical results suggested that PDGF protein expressed at a relatively high level after 2 weeks. From this study, it can be concluded that the novel GAM had potential in the application of periodontal tissue engineering.

Three-dimensional Nanohydroxyapatite/Chitosan Scaffolds as Potential Tissue Engineered Periodontal Tissue

The development of suitable three-dimensional scaffold for the maintenance of cellular viability and differentiation is critical for applications in periodontal tissue engineering. In this work, different ratios of porous nanohydroxyapatite/chitosan (HA/chitosan) scaffolds are prepared through a freeze-drying process. These scaffolds are evaluated in vitro by the analysis of microscopic structure, porosity, and cytocompatibility. The expression of type I collagen and alkaline phosphatase (ALP) activity are detected with real-time polymerase chain reaction (RT-PCR). Human periodontal ligament cells (HPLCs) transfected with enhanced green fluorescence protein (EGFP) are seeded onto the scaffolds, and then these scaffolds are implanted subcutaneously into athymic mice. The results indicated that the porosity and pore diameter of the HA/chitosan scaffolds are lower than those of pure chitosan scaffold. The HA/chitosan scaffold containing 1% HA exhibited better cytocompatibility than the pure chitosan scaffold. The expression of type I collagen and ALP are up-regulated in 1% HA/chitosan scaffold. After implanted in vivo, EGFP-transfected HPLCs not only proliferate but also recruit surrounding tissue to grow in the scaffold. The degradation of the scaffold significantly decreased in the presence of HA. This study demonstrated the potential of HA/ chitosan scaffold as a good substrate candidate in periodontal tissue engineering

Synthesis and inflammatory response of a novel silk fibroin scaffold containing BMP7 adenovirus for bone regeneration

Gene therapy has garnished tremendous awareness for the repair of osseous defects. It exhibits high efficiency gene transfer and osteogenic differentiation potential making it well suitable for the sustained delivery of growth factors to local tissues. In the present study a simplified solution-based in situ biomimetic synthesis method is demonstrated for bone morphogenetic protein 7 (BMP7) adenovirus combined with silk fibroin scaffolds. This scaffold not only provides the three dimensional space for bone ingrowth, but also releases the BMP7 adenovirus which targets its secretion by host cells in vivo. Scaffolds were tested both in vitro for their osteogenic potential as well as in vivo in a critical-size calvarial defect in mice. Scaffolds loaded with bone morphogenetic protein 7 adenovirus (adBMP7) were able to sustain release of adBMP7 for up to 21 days and support cell proliferation and differentiation to bone forming osteoblasts. Calvarial defects treated with scaffolds containing adBMP7 significantly induced new bone formation in vivo. To demonstrate immuno-compatibility with host tissues, IL-2, IL-6 and TNF-α were measured up to 4 weeks post-implantation. Although these scaffolds demonstrated an initial pro-inflammatory response, levels of IL-2, IL-6 and TNF-α returned to baseline control values at either 2 or 4 weeks post-implantation demonstrating long term compatibility for growth factor delivery via gene therapy. The results from the present study indicate the promise of gene delivery scaffold systems for robust, low cost, and high quality bone tissue engineering applications.

Cellulose nanowhiskers: Preparation, characterization and cytotoxicity evaluation

OBJECTIVE To characterize the structure and to evaluate the cytotoxicity of cellulose nanowhiskers (CNW) prepared from cotton linters. METHODS A series of CNW dispersions was prepared by hydrolyzing cotton linters with sulfuric acid. The structure of CNW was characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR) and X-ray diffraction (XRD). The cytotoxicity of CNW dispersions to L929 cells was preliminarily investigated by cell culture and MTT assay. RESULTS SEM and TEM observation showed that the original cotton litters have been successfully acid-hydrolyzed into cellulose nanowhiskers having needle- or short rod-like structure with an average length of 250 nm and diameter of 10 nm. XRD patterns suggested that degree of crystalline in the CNW was much higher than that in the cotton linters as a result that the acid-hydrolysis process has removed the amorphous domains in the cotton linters. The results from MTT assay and cell morphology observation indicated that CNW with concentration from 0.01% to 0.2% had low toxicity to L929 cells, while cytotoxicity showed an increase tendency with an increase in the concentration of CNW. CONCLUSIONS Cellulose nanowhiskers dispersion could be prepared from cotton linters by acid-hydrolysis process. CNW showed potential applications as nanobiomaterials due to its low cytotoxicity.

RhBMP-2 Microspheres-Loaded Chitosan/Collagen Scaffold Enhanced Osseointegration : An Experiment in Dog

The purpose of this study is to develop a novel recombinant human bone morphogenetic protein-2 (rhBMP-2) sustained release scaffold for dental implant osseointegration, and to evaluate the effect of this scaffold on promoting bone formation. RhBMP-2 was encapsulated in the poly-D,L-lactideco-glycolide (PLGA) biodegradable microspheres, which were subsequently dispersed in a chitosan/collagen composite scaffold. This rhBMP-2 microspheres-loaded scaffold (S-MB) was compared with a chitosan/collagen scaffold without microspheres that directly encapsulated rhBMP-2 (S-B) in vitro and in vivo. The microstructure of the new scaffold was examined with scanning electron microscopy. The release profile of rhBMP-2 in vitro was measured at interval periods. The effect of rhBMP-2 encapsulated scaffolds on enhancing bone formation through implantation in dogs’ mandibles was identified by histological examination of the regenerated bone after 4 weeks of implantation. Due to PLGA microspheres being loaded, the S-MB exhibited lower values at porosity and swelling rate, as well as a higher effective release dose than that of the S-B. Bone density, bone-implant contact, and bone-fill values measured from dog experiments demonstrated that the S-MB induced bone regeneration more quickly and was timely substituted by new bone. It was concluded that this sustained carrier scaffold based on microspheres was more effective to induce implant osseointegration. *Author to whom correspondence should be addressed. E-mail: xiangrongcheng@hotmail.com Figures 1, 2, 6 and 7 appear in color online: http://jba.sagepub.com JOURNAL OF BIOMATERIALS APPLICATIONS Volume 23 — January 2009 331 0885-3282/09/04 0331–16

Novel MesoPorous BioGlass/silk scaffold containing adPDGF‐B and adBMP7 for the repair of periodontal defects in beagle dogs

10.00/0 DOI: 10.1177/0885328208090013 SAGE Publications 2009 Los Angeles, London, New Delhi and Singapore

Platelet-derived growth factor BB gene-released scaffolds: biosynthesis and characterization.

AIM The local delivery of growth factors via gene therapy has gained tremendous awareness in recent years due to their sustained growth factor delivery to target tissues. The aim of this study was to fabricate and investigate a scaffold able to release growth factors via gene therapy for the repair of periodontal tissues. MATERIALS AND METHODS Novel mesoporous bioglass (MBG)/silk fibrin scaffold combined with BMP7 and/or PDGF-B adenovirus was fabricated and tested in vitro for cell migration, proliferation and differentiation. Furthermore, acute-type buccal dehiscence periodontal defects (mesiodistal width × depth: 5 × 5 mm) were created on the buccal portion of the maxillary premolars in five normal male beagle dogs (12 months old, 15.0 ± 2.0 kg) and histologically examined for periodontal regeneration following implantation of the following five groups: (1) no scaffold, (2) MBG/silk scaffold alone, (3) scaffold + adPDGF-B, (4) scaffold + adBMP7, (5) scaffold + adPDGF-b + adBMP7. RESULTS In vitro findings demonstrated that adPDGF-B was able to rapidly recruit periodontal ligament (PDL) cells over sixfold more effectively than adBMP7, whereas adBMP7 was more able to induce osteoblast differentiation of PDL cells. In vivo findings demonstrate that scaffolds loaded with adPDGF-B were able to partially regenerate the periodontal ligament while adBMP7 scaffolds primarily improved new bone formation. The combination of both adPDGF-B and adBMP7 synergistically promoted periodontal regeneration by allowing up to two times greater regeneration of the periodontal ligament, alveolar bone and cementum when compared to each adenovirus used alone. CONCLUSIONS Although both PDGF-B and BMP7 are individually capable of promoting periodontal regeneration to some degree, their combination synergistically promotes wound healing in acute-type buccal dehiscence periodontal defects when delivered simultaneously. This study demonstrates the promise for successful delivery of low-cost, effective growth factor delivery via gene therapy for the treatment of periodontal defects.