The Effect of Polydeoxyribonucleotide Extracted from Salmon Sperm on the Restoration of Bisphosphonate-Related Osteonecrosis of the Jaw.

Bisphosphonates (BPs) used for treating skeletal diseases can induce bisphosphonate-related osteonecrosis of the jaw (BRONJ). Despite much effort, effective remedies are yet to be established. In the present study, we investigated the feasibility of polydeoxyribonucleotide (PDRN) extracted from salmon sperm for the treatment of BRONJ, in a BRONJ-induced rat model. Compared with BRONJ-induced samples, PDRN-treated samples exhibited lower necrotic bone percentages and increased numbers of blood vessels and attached osteoclast production. Moreover, local administration of PDRN at a high concentration (8 mg/kg) remarkably resolved the osteonecrosis. Findings from this study suggest that local administration of PDRN at a specific concentration may be considered clinically for the management of BRONJ.

Chitosan for Tissue Engineering.

Chitosan, a deacetylated chitin, is one of the few natural polymers similar to glycosaminoglycans (GAGs) widely distributed throughout connective tissues. It has been believed that the excellent biocompatibility of chitosan is largely attributed to this structural similarity. Chitosan is also known to possess biodegradability, antimicrobial activity and low toxicity and immunogenicity which are essential for scaffolds. In addition, the existence of free amine groups in its backbone chain enables further chemical modifications to create the additional biomedical functionality. For these reasons, chitosan has found a tremendous variety of biomedical applications in recent years. This chapter introduces the basic contents of chitosan and discusses its applications to artificial skin, artificial bone, and artificial cartilage in tissue engineering purpose.

The Cocktail Effect of BMP-2 and TGF-ß1 Loaded in Visible Light-Cured Glycol Chitosan Hydrogels for the Enhancement of Bone Formation in a Rat Tibial Defect Model.

Bone tissue engineering scaffolds offer the merits of minimal invasion as well as localized and controlled biomolecule release to targeted sites. In this study, we prepared injectable hydrogel systems based on visible light-cured glycol chitosan (GC) hydrogels containing bone morphogenetic protein-2 (BMP-2) and/or transforming growth factor-beta1 (TGF-ß1) as scaffolds for bone formation in vitro and in vivo. The hydrogels were characterized by storage modulus, scanning electron microscopy (SEM) and swelling ratio analyses. The developed hydrogel systems showed controlled releases of growth factors in a sustained manner for 30 days. In vitro and in vivo studies revealed that growth factor-loaded GC hydrogels have no cytotoxicity against MC3T3-E1 osteoblast cell line, improved mRNA expressions of alkaline phosphatase (ALP), type I collagen (COL 1) and osteocalcin (OCN), and increased bone volume (BV) and bone mineral density (BMD) in tibia defect sites. Moreover, GC hydrogel containing BMP-2 (10 ng) and TGF-ß1 (10 ng) (GC/BMP-2/TGF-ß1-10 ng) showed greater bone formation abilities than that containing BMP-2 (5 ng) and TGF-ß1 (5 ng) (GC/BMP-2/TGF-ß1-5 ng) in vitro and in vivo. Consequently, the injectable GC/BMP-2/TGF-ß1-10 ng hydrogel may have clinical potential for dental or orthopedic applications.

Preparation and Evaluation of Dexamethasone (DEX)/Growth and Differentiation Factor-5 (GDF-5) Surface-Modified Titanium Using ß-Cyclodextrin-Conjugated Heparin (CD-Hep) for Enhanced Osteogenic Activity In Vitro and In Vivo.

The most ideal implant models in the dental and orthopedic fields to minimize the failure rate of implantation involve the improvement of osseointegration with host bone. Therefore, a focus of this study is the preparation of surface-modified titanium (Ti) samples of disc and screw types using dexamethasone (DEX) and/or growth and differentiation factor-5 (GDF-5), as well as the evaluation of their efficacies on bone formation in vitro and in vivo. X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and contact angle measurement were used to evaluate the surface chemical composition, surface morphology and wettability, respectively. The results showed that implant surfaces were successfully modified with DEX and/or GDF-5, and had rough surfaces along with hydrophilicity. DEX, GDF-5 or DEX/GDF-5 on the surface-modified samples were rapidly released within one day and released for 28 days in a sustained manner. The proliferation and bone formation of MC3T3-E1 cells cultured on pristine and surface-modified implants in vitro were examined by cell counting kit-8 (CCK-8) assay, as well as the measurements of alkaline phosphatase (ALP) activity and calcium deposition, respectively. MC3T3-E1 cells cultured on DEX/GDF-5-Ti showed noticeable ALP activity and calcium deposition in vitro. Active bone formation and strong osseointegration occurred at the interface between DEX/GDF-5-Ti and host bone, as evaluated by micro computed-tomography (micro CT) analysis. Surface modification using DEX/GDF-5 could be a good method for advanced implants for orthopaedic and dental applications.

Surface Modification of Titanium with BMP-2/GDF-5 by a Heparin Linker and Its Efficacy as a Dental Implant.

In this study, we prepared human bone morphogenetic protein-2 (hBMP-2)/human growth and differentiation factor-5 (hGDF-5)-coated titanium (Ti) disc and screw types for controlled release of the growth factors (GFs). The two growth factors were coated onto Ti with a smooth surface using their specific interaction with heparin, because they have heparin binding sites in their molecular structures. Efficacy of the two growth factor-coated Ti for enhancement of bone formation and osseointegration was compared to pristine Ti, and hBMP-2- and hGDF-5-coated Ti in vivo. The surface chemical composition, surface morphology, and wettability characteristics of the metal samples were determined by X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), and contact angle measurement, respectively. The initial burst of hBMP-2, hGDF-5, and their combination, occurred within one day of the release study, resulting in 12.5%, 4.5%, and 13.5%/3.2%, and then there was a sustained, even release of these two growth factors from the coated metal for 30 days. In vitro tests revealed that MC3T3-E1 cells cultured on the two growth factor-coated Ti had a higher proliferation rate and a higher activity for alkaline phosphatase (ALP), which led to a larger amount of calcium deposition and larger expressions of type I collagen (COL 1), ALP, and osteocalcin (OCN) mRNAs. In vivo animal tests using ten white New Zealand rabbits showed that the two growth factor-coated Ti enhanced bone formation and osseointegration at the interface between the implants and host bone. In addition, histological evaluation showed that bone remodeling, including bone formation by osteoblasts and bone resorption by osteoclasts, actively occurred between the two growth factor-coated Ti and host bone. Consequently, it is suggested that Ti surface modification with the combination of hBMP-2 and hGDF-5 for the two growth factor-coated Ti implants can improve the clinical properties of implants for orthopedic and dental applications.

Effects of Titanium Mesh Surfaces-Coated with Hydroxyapatite/ß-Tricalcium Phosphate Nanotubes on Acetabular Bone Defects in Rabbits.

The management of severe acetabular bone defects in revision reconstructive orthopedic surgery is challenging. In this study, cyclic precalcification (CP) treatment was used on both nanotube-surface Ti-mesh and a bone graft substitute for the acetabular defect model, and its effects were assessed in vitro and in vivo. Nanotube-Ti mesh coated with hydroxyapatite/ß-tricalcium phosphate (HA/ß-TCP) was manufactured by an anodizing and a sintering method, respectively. An 8 mm diameter defect was created on each acetabulum of eight rabbits, then treated by grafting materials and covered by Ti meshes. At four and eight weeks, postoperatively, biopsies were performed for histomorphometric analyses. The newly-formed bone layers under cyclic precalcified anodized Ti (CP-AT) meshes were superior with regard to the mineralized area at both four and eight weeks, as compared with that under untreated Ti meshes. Active bone regeneration at 2-4 weeks was stronger than at 6-8 weeks, particularly with treated biphasic ceramic (p < 0.05). CP improved the bioactivity of Ti meshes and biphasic grafting materials. Moreover, the precalcified nanotubular Ti meshes could enhance early contact bone formation on the mesh and, therefore, may reduce the collapse of Ti meshes into the defect, increasing the sufficiency of acetabular reconstruction. Finally, cyclic precalcification did not affect bone regeneration by biphasic grafting materials in vivo.

In Vitro Osteogenic Differentiation Enhanced by Zirconia Coated with Nano-Layered Growth and Differentiation Factor-5.

Zirconia (Zr) is also known as a biocompatible material with favorable mechanical properties as well as low plaque adhesion. In this study, we examined the efficacy of Zr coated with growth and differentiation factor-5 (GDF-5) bonded via click reaction as a substrate to support osteogenic differentiation of MC3T3-E1 cells. Pristine and surface-modified Zr surfaces were characterized by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS), resulting that GDF-5 was successfully coated to the pristine Zr surface. GDF-5 coated to Zr surfaces was released for 28 days in a sustained manner. New bone formation onto GDF-5 coated Zr (Zr/GDF-5) surface was confirmed by in vitro test including cell proliferation, alkaline phosphatase activity and calcium deposition assays, and in vivo test including real-time polymerase chain reaction (qPCR) assay including osterix (OSX), runt-related transcription factor 2 (Runx 2), COL 1 (type I collagen) and osteocalcin (OC). Cell proliferation, alkaline phosphatase activity, and calcium deposition of MC3T3- E1 cells were significantly enhanced when the cells were cultured on Zr/GDF-5. Additionally, the results of qPCR revealed that genes related with osteogenic differentiation were up regulated when the cells were cultured on Zr/GDF-5. Our findings demonstrate that Zr/GDF-5 could be used as a material for enhancing the efficacy of osteogenic differentiation.

Theranostic potential of biodegradable polymeric nanoparticles with paclitaxel and curcumin against breast carcinoma.

In this study, integrin-mediated targeting and near-infrared fluorescence (NIRF) traceable polyethylene glycol-b-poly(lactic-co-glycolic acid) (PEG-PLGA)-based polymeric nanoparticles (NPs) were prepared to investigate the effects of paclitaxel (PTX) and curcumin (CUR) combination therapy on breast cancer. Cyclic (arginine-glycine-aspartic acid-phenylalanine-lysine) (cRGDfK) was selected as a ligand for breast cancer and conjugated to the end of NPs (cRGDfK-NPs). For fluorescence imaging, sulfo-cyanine 5.5 (Cy5.5) was incorporated into NPs (Cy5.5-NPs). A series of hybrid NPs consisting of NPs, cRGDfK-NPs, and Cy5.5-NPs with drugs encapsulated inside the core (Cy5.5-cRGDfK-NPs/PTX + CUR) were prepared by self-assembly. The efficacy of PTX and CUR combination and the ability of the integrin-mediated targeting of NPs were systemically investigated using a 4T1 mouse breast cancer cell line and a nude mouse xenograft model. We suggested that Cy5.5-cRGDfK-NPs/PTX + CUR has superior theranostic potential against breast carcinoma.

Injectable hydrogels based on MPEG-PCL-RGD and BMSCs for bone tissue engineering.

The aim of this study was to investigate the osteogenic potential of bone marrow-derived mesenchymal stem cells (BMSCs) seeded on novel thermosensitive in situ forming hydrogel systems comprising methoxy polyethylene glycol-polycaprolactone (MP) and RGD-conjugated MP (MP-RGD) in vitro and in vivo. Real-time polymerase chain reaction (PCR) together with immunofluorescence staining revealed the strong expression of osteogenic markers (collagen 1 and osteocalcin) of BMSCs in MP/MP-RGD samples compared to MP samples. PCR array testing also showed the upregulation of the interconnected signaling networks regulating cell proliferation and differentiation, which was further verified through the Kyoto Encyclopedia of Genes and Genomes pathway analysis. Histological findings and computed tomographic analysis demonstrated that the MP/MP-RGD hydrogel dramatically promoted new bone formation in a rabbit calvarial defect model. In conclusion, this hydrogel appears to elicit cellular behaviors desired for bone tissue regeneration.

Preparation of poly-l-lysine-based nanoparticles with pH-sensitive release of curcumin for targeted imaging and therapy of liver cancer in vitro and in vivo.

Poly-l-lysine (PLL) nanoparticle (NP) system was prepared for the controlled release of curcumin (CUR) by pH stimuli, and its theranostic efficacy on cancer was compared to that of CUR solution in vitro and in vivo. Deoxycholic acid (DOCA), methoxy polyethylene glycol (MPEG) and cyanine 5.5 (cy5.5) were conjugated to the amine group of PLL through condensation reaction (PLL-DOCA-MPEG-cy5.5), followed by encapsulation of CUR by dialysis method (PLL-DOCA-MPEG-cy5.5/CUR NPs). The composition, morphology and size distribution of PLL-DOCA-MPEG-cy5.5 NPs were characterized by proton nuclear magnetic resonance (1H NMR), transmission electron microscopy (TEM) and dynamic light scattering (DLS), respectively. In vitro tests exhibited that changes in the charge and size of the NPs at low pH led to the improved cellular uptake of CUR into human hepatoma Hep3B cell line by electrostatically absorptive endocytosis. PEGylation with MPEG was turn out to be very effective to have a prolonged blood circulation time, in turn increased the EPR effect. In addition, the incorporation of Cy5.5 into NPs provided successful biodistribution images in vivo and ex vivo. Our findings suggest that PLL-DOCA-MPEG-cy5.5/CUR NPs may have promising applications in cancer theranosis.

Cellular behaviour of hepatocyte-like cells from nude mouse bone marrow-derived mesenchymal stem cells on galactosylated poly(D,L-lactic-co-glycolic acid).

Previously, the galactosylation of poly(d,l-lactic-co-glycolic acid) (PLGA) surface was accomplished by grafting allylamine (AA), using inductively coupled plasma-assisted chemical vapour deposition (ICP-CVD) and conjugating lactobionic acid (LA) with AA via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (EDC/NHS) activation for hepatic tissue-engineering purposes. As a continuation study, the cellular behaviour of hepatocyte-like cells (HLCs) on the surface of the galactosylated PLGA were investigated. Nude mouse bone marrow-derived mesenchymal stem cells (MSCs) were cultured under hepatogenic conditions and the differentiated cells were characterized by reverse-transcription polymerase chain reaction (RT-PCR), immunofluorescence and periodic acid-Schiff (PAS) staining. Galactosylated PLGA enhanced the proliferation rate of HLCs compared to the control; HLCs on the surface of the sample became aggregated and formed spheroids after 3 days of culture. A large number of cells on the surface of the sample exhibited increased liver-specific functional activities, such as albumin and urea secretions. In addition, multicellular spheroids in the sample strongly expressed phospholyated focal adhesion kinase (pFAK) (cell-matrix interactions), E-cadherin (cell-cell interactions) and connexin 32 (Cox32; gap junction).

A Titanium Surface-Modified with Nano-Sized Hydroxyapatite and Simvastatin Enhances Bone Formation and Osseintegration.

The aim of the present study was to evaluate whether coating pristine titanium (Ti) with nano-sized hydroxyapatite (HAp) and simvastatin could enhance bone formation and osseointegration in vitro and in vivo because both HAp and simvastatin have the characteristic of osteogenetic induction. Pristine Ti was sequentially surface-treated with NaOH, 1,1-carbonyldiimidazole (CDI), beta-cyclodextrin-immobilized HAp powders (ß-CD/HAp), and simvastatin before analysis using scanning electron microscopy (SEM), X-ray photoelectron microscopy (XPS), and static contact angle measurement. This revealed that simvastatin was released continually for up 28 days. Modification of the Ti surface with nano-sized HAp and simvastatin (Ti/ß-CD/HAp/Sim) discs enhanced the osteogenic differentiation of MC3T3-E1 cells in vitro. Furthermore, Ti/ß-CD/HAp/Sim of screw type enhanced bone formation between the screw and the host bone when the screw implanted to the proximal tibia and femoral head of rabbits. These results suggest that surface modification of nano-sized HAp and simvastatin are effective tools for developing attractive dental implants.

Surface modification of titanium with hydroxyapatite-heparin-BMP-2 enhances the efficacy of bone formation and osseointegration in vitro and in vivo.

Surface-modified titanium (Ti) samples with hydroxyapatite (HAp) and heparin (Hep)-bone morphogenetic protein-2 (BMP-2) complex (Ti/HAp/Hep/BMP-2) were prepared, and their efficacies on the enhancements of bone formation and osseointegration in vitro and in vivo were examined as compared to Ti/HAp and Ti/Hep/BMP-2. The modified surfaces were characterized by X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and contact angle goniometry. In vitro studies revealed that MG-63 human osteosarcoma cell lines grown on Ti/HAp/Hep/BMP-2 increased the amounts of alkaline phosphatase (ALP) activity, calcium deposition and the levels of OCN mRNA gene expression as compared to those grown on Ti/HAp, Ti/Hep/BMP-2 or pristine Ti. Moreover, Ti/HAp/Hep/BMP-2 exhibited higher bone volume (BV), bone volume/tissue volume (BV/TV), removal torque value and bone-implant contact (BIC) than Ti/HAp, Ti/Hep/BMP-2 or pristine Ti in vivo. Histological evaluations showed that many desirable features of bone remodelling existed at the interface between Ti/HAp/Hep/BMP-2 and the host bone. Consequently, Ti/HAp/Hep/BMP-2 may have potential for clinical use as dental or orthopaedic implants.

Poly(L-lactic acid)/hydroxyapatite nanocylinders as nanofibrous structure for bone tissue engineering scaffolds.

Poly (L-lactic acid) (PLLA), a biodegradable and biocompatible polyester, has been used as material of for tissue engineered scaffold and regenerative medicine. In this study, hydroxyapatite (HAp) and PLLA composite was electrospun. PLLA fibers were treated with an amino groups containing base in order to fabricate amino groups modified polymeric microcylinders. Simvastatin was loaded into the PLLA/HAp microcylinders. Our study focuses on the incorporation HAp and Simvastatin into the fibrous and cylindrical structure, its effects on the loading and release of simvastatin, and biological responses to osteoblast cells. The aspect ratio of microcylinders was tunable by varying the aminolysis treatment time and density of the base. The effects of the PLLA/HAp composites on osteoblast cell proliferation were evaluated. The PLLA/HAp may interact with osteoblast and obtain desired effects that were stimulating osteoblast function and restraining the osteoclast function for bone tissue regeneration. The potential of the developed composite microcylinders could be a promising way to fabricate the microfibrous structural scaffold that accelerates cell proliferation and functions for bone tissue engineering.

Burn-wound healing effect of gelatin/polyurethane nanofiber scaffold containing silver-sulfadiazine.

Despite the fact that advances of burn treatment have led to reduction in the morbidity caused by burns, burn infection is still a serious problem. In this study, we designed blended synthetic and natural polymers nanofiber scaffolds using polyurethane (PU) and gelatin, which were prepared by an electrospinning method. Silver-sulfadiazine (SSD) was co-mixed to the blended polymer solution for being incorporated into the nanofibers after the electrospinning, followed by examination of burn-wound healing effect. The nanofiber scaffolds containing SSD should not only serve as a substrate for skin regeneration, but may also deliver suitable drugs, within a controlled manner during healing. The SSD release was able to prevent the growth of a wide array of bacteria and accelerate the wound healing by preventing infection. Therefore it could accelerate the burn-wound closure rate. We confirmed that PU/gelatin nanofiber scaffolds containing SSD lead to enhanced regeneration of burn-wounds.

ZrO2 surface chemically coated with hyaluronic acid hydrogel loading GDF-5 for osteogenesis in dentistry.

The objective of this study was to modify zirconium dioxide (ZrO(2)) with photo-cured hyaluronic acid hydrogel (pcHAgel), and to subsequently evaluate the bone regeneration potential of the modified ZrO(2). In the present study, HA grafted onto a ZrO(2) substrate was investigated for its biocompatibility and other properties. We describe the positive influences of ZrO(2) surface-modified with pcHAgel (Zr-3) containing two different loads of growth and differentiation factor-5 (GDF-5) to aid new bone formation as compared to the same amount of BMP-2 (Zr-4-7). We characterized the Zr-3 for their surface morphology and chemical properties. Atomic force microscopy (AFM), scanning electron microscope (SEM), and X-ray photoelectron spectroscopy (XPS) showed that the pcHAgel was successfully grafted onto the ZrO(2) surface. The sustained release of GDF-5 and BMP-2 were observed to occur in the Zr-4-7. In vitro cell tests showed a higher level of MG63 cell proliferation and differentiation on Zr-4-7 than on Zr-3. The Zr-3 is a good biomaterial to deliver osteogenic differentiation factors such as BMP-2 and GDF-5, and GDF-5 can be useful as an effective alternative to aid new bone formation as compared to BMP-2.

Surface and chemical properties of surface-modified UHMWPE powder and mechanical and thermal properties of its impregnated PMMA bone cement, IV: effect of MMA/accelerator on the surface modification of UHMWPE powder.

In our previous study, we manufactured a reinforced poly(methylmethacrylate) (PMMA) bone cement with 3 wt% of the surface-modified ultra high molecular weight polyethylene (UHMWPE) powder to improve its poor mechanical and thermal properties resulting from unreacted methylmethacrylate (MMA), the generation of bubble and shrinkage, and high curing temperature. In the present study, the effect of ratios of MMA and N,N'-dimethyl-p-toluidine (DMPT) solutions in redox polymerization system was investigated for the surface modification of UHMWPE powder. We characterized physical and chemical properties of surface-modified UHMWPE powder and reinforced bone cements by a scanning electron microscope, ultimate tensile strength (UTS) and curing temperature (Tmax). It was found that UTSs (41.3-51.3 MPa) of the reinforced PMMA bone cements were similar to those (44.5 MPa) of conventional PMMA bone cement (control), as well as significantly higher (P < 0.05) than those (33.8 MPa) of 3 wt% unmodified UHMWPE powder-impregnated bone cement. In particular, the UTS of redox polymerization system using MMA/DMPT solution was better than that of radical system using MMA/xylene solution. Also, Tmax of the reinforced PMMA bone cements decreased from 103 to 72-84 degrees C. From these results, we confirmed that the surface-modified UHMWPE powder can be used as reinforcing agent to improve the mechanical and thermal properties of conventional PMMA bone cement.

Surface and chemical properties of surface-modified UHMWPE powder and mechanical and thermal properties of it impregnated PMMA bone cement, III: effect of various ratios of initiator/inhibitor on the surface modification of UHMWPE powder.

From our previous study, 3 wt% of ultra-high-molecular-weight polyethylene (UHMWPE) powder surface-modified by various ratios of methyl methacrylate (MMA) and poly(methyl methacrylate) (PMMA) solution was impregnated to improve the poor mechanical and thermal properties of conventional PMMA bone cement. In this study, various amounts of benzoyl peroxide (BPO) and hydroquinone were used for the adhesion reinforcement of UHMWPE powder with PMMA polymerized from MMA monomer (polyMMA) by the mixture of BPO and hydroquinone and ultimately to strengthen the poor mechanical and thermal properties of conventional PMMA bone cement. The tensile strengths of 3 wt% of UHMWPE powders surface-precoated with polyMMA prepared by various amounts of BPO- and hydroquinone-impregnated composite PMMA bone cements were similar to that of conventional PMMA bone cement. In particular, 3 wt% of UHMWPE powder surface precoated with polyMMA prepared with 0.75 wt% of BPO and 300 ppm of hydroquinone impregnated composite PMMA bone cement revealed the maximum tensile strength. However, no obvious significant difference was revealed, although the curing temperatures of the composite PMMA bone cements decreased from 103 degrees C to 91-97 degrees C. From these results, it was determined that the mixture of BPO and hydroquinone plays an important role in improving the poor mechanical properties of conventional PMMA bone cement. However, the thermal properties of the composite PMMA bone cements were not remarkably improved. The mechanical, chemical and thermal properties were individually confirmed using a scanning electron microscope (SEM), universal transverse mercator (UTM), Fourier transform infrared-attenuated total reflectance (FT-IR-ATR) and digital thermometer, respectively.