Reconstruction of bone fenestration on mandiblar by the guided bone regeneration methods with beta-TCP/PLGC membranes

Guided Bone Regeneration (GBR) is a method for bone tissue regeneration. In this method, membranes are used to cover bone defects and to block the invasion of the surrounding soft tissues. It would provide sufficient time for the osteogenic cells from bone marrow to proliferate and form new bony tissues. In spite of the potential usefulness of this method, no appropriate materials for the GBR membrane have been developed. Here we design the ideal mechanical properties of the GBR membranes and created novel materials, which is the composite of beta-tricalcium phosphate (beta-TCP) and block copolymer of L-lactide, glycolide, and epsilon-caplolactone (PLGC). In the animal experiments with the use of the GBR membranes for large bone defects, we observed significant enhancement in the bone regeneration after 12 weeks implantation and proved the effectiveness of the materials.     

Carbon nanohorns accelerate bone regeneration in rat calvarial bone defect

A recent study showed that carbon nanohorns (CNHs) have biocompatibility and possible medical uses such as in drug delivery systems. It was reported that some kinds of carbon nanomaterials such as carbon nanotubes were useful for bone formation. However, the effect of CNHs on bone tissue has not been clarified. The purpose of this study was to evaluate the effect of CNHs on bone regeneration and their possible application for guided bone regeneration (GBR). CNHs dispersed in ethanol were fixed on a porous polytetrafluoroethylene membrane by vacuum filtration. Cranial defects were created in rats and covered by a membrane with/without CNHs. At two weeks, bone formation under the membrane with CNHs had progressed more than under that without CNHs and numerous macrophages were observed attached to CNHs. At eight weeks, there was no significant difference in the amount of newly formed bone between the groups and the appearance of macrophages was decreased compared with that at two weeks. Newly formed bone attached to some CNHs directly. These results suggest that macrophages induced by CNHs are related to bone regeneration. In conclusion, the present study indicates that CNHs are compatible with bone tissue and effective as a material for GBR.     

Alginate film prepared on polyethylene nonwoven sheet and its function for ellagic acid release in response to sodium ions

Calcium alginate film containing ellagic acid as a functional material was formed on polyethylene nonwoven sheet by the reaction of sodium alginate with calcium chloride in the presence of ellagic acid. The film had the intelligent function of release of ellagic acid triggered by sodium ion. This response resulted from conversion of the water-insoluble calcium alginate to water-soluble sodium alginate. The optimal conditions for the intelligent calcium alginate film prepared on the polyethylene surface were 0.5% CaCl₂ solution and 0.05 or 0.1% sodium alginate solution.     

Plasmid DNA-loaded asymmetrically porous membrane for guided bone regeneration

Although bone defects can be restored spontaneously,bone reconstruction with sufficient strength and volume continues to be a challenge in clinical practices.In recent years,the use of a variety of biomaterials with bioactivity has been attempted to compensate for this limitation.Herein,we fabricated a pDNA(encoding for BMP-2)-loaded asymmetrically porous polycaprolactone(PCL)/Pluronic F127 membrane as a bioactive guided bone regeneration(GBR)membrane,using a modified immersion-precipitation method.It was observed that the GBR membrane allows continuous release of pDNA for more than20 weeks.The pDNA was sufficiently transfected into human bone marrow stem cells(h BMSCs)without significant cytotoxicity and the gene-transfected cells showed prolonged synthesis of BMP-2.From in vitro osteogenic differentiation and in vivo animal studies,the effective induction of osteogenic differentiation of h BMSCs and enhanced bone regeneration by the pDNA-loaded asymmetrically porous PCL/Pluronic F127 membrane was observed,suggesting that the pDNA-loaded membrane as a bioactive GBR membrane can be an alternative therapeutic technique for effective bone regeneration.     

Preparation of electrospun siloxane-poly(lactic acid)-vaterite hybrid fibrous membranes for guided bone regeneration

A nonwoven fabric scaffold for guided bone regeneration (GBR) consisting of siloxane-poly(lactic acid) (PLA)-vaterite hybrid material (SiPVH) was prepared by an electrospinning method. To improve the cellular compatibility of the fabric, the fibers were completely coated with hydroxyapatite (HA) by soaking in simulated body fluid. The HA-coated SiPVH nonwoven fabric contained large-sized spaces and showed the ability of releasing soluble silica and calcium species, which have previously been reported to stimulate osteogenic cells at the genetic level. A new type of GBR membrane was prepared by bonding SiPVH fabric with PLA nonwoven fabric with small-sized pores for preventing the intrusion of soft tissue. The resultant bi-layered membrane was expected to be effective not only for having an open structure for bone formation and a barrier to soft tissue, but also for enhancing bone growth by the release of ionic species.     

Physicochemical and biological characteristics of BMP-2/IGF-1-loaded three-dimensional coaxial electrospun fibrous membranes for bone defect repair

Coaxial electrospun fibrous membranes show favorable mechanical properties for use in guided bone regeneration (GBR). We used coaxial electrospinning technology to fabricate three-dimensional nanofiber membranes loaded with BMP-2 and IGF-1, and assessed the physicochemical and biological properties of these novel membranes in vitro. We fabricated four experimental groups of BMP-2/IGF-1/BSA-loaded membranes with different flow ratios (shell/core). Membrane characteristics were assessed by scanning and transmission electron microscopy, and laser confocal microscopy. Physicochemical and drug release properties were evaluated based on contact angle, mechanical property testing, X-ray diffraction analysis, and ELISA. The membranes were seeded with bone marrow-derived mesenchymal stem cells (BMMSCs) to estimate their biological properties based on cell viability and alkaline phosphatase (ALP) activity. The four membrane groups presented uniform diameters and core-shell structures. Acceleration of the shell solution flow rate increased the contact angle and mechanical properties of the fibrous membrane, while dual-factor addition did not impact fiber structure. Each drug-loaded membrane showed a gradually increasing release curve, with varying degrees of burst and sustained release. Compared to the other groups, the membranes with a core-shell flow ratio of 1:10 showed better drug-loading capacity and sustained release performance, higher biological properties and good barrier function. Optimal parameters were chosen based on the physical and chemical characteristics and biological properties of the membrane. Our results imply that the BMP-2/IGF-1/BSA-loaded coaxial electrospun fibrous membrane with optimum parameters is a suitable barrier membrane for GBR, and releases multiple factors promoting osteoconduction and osteoinduction.     

Biomimetic coating of an apatite layer on poly(<Emphasis Type="SmallCaps">l</Emphasis>-lactic acid); improvement of adhesive strength of the coating

A biodegradable polymer coated with a bonelike apatite layer on its surface would be useful as a scaffold for bone tissue regeneration. In this study, poly(l-lactic acid) (PLLA) was treated with oxygen plasma to produce oxygen-containing functional groups on its surface. The plasma-treated specimen was then alternately dipped in aqueous CaCl₂ and K₂HPO₄·3H₂O solutions three times, to deposit apatite precursors onto the surface. The surface-modified specimen then successfully formed a dense and uniform bonelike surface apatite layer after immersion for 24 h in a simulated body fluid with ion concentrations approximately equal to those of human blood plasma. The adhesive strength between the apatite layer and the specimen surface increased as the power density of the oxygen plasma used increased. The maximum adhesive strength of the apatite layer to the specimen was significantly higher than that to the commercially available artificial bone, HAPEX^TM. The resultant bonelike apatite–PLLA composite would be useful as a scaffold for bone tissue regeneration.     

Human amniotic membrane for guided bone regeneration of calvarial defects in mice

Due to its biological properties, human amniotic membrane (hAM) is widely studied in the field of tissue engineering and regenerative medicine. hAM is already very attractive for wound healing and it may be helpful as a support for bone regeneration. However, few studies assessed its potential for guided bone regeneration (GBR). The purpose of the present study was to assess the potential of the hAM as a membrane for GBR. In vitro, cell viability in fresh and cryopreserved hAM was assessed. In vivo, we evaluated the impact of fresh versus cryopreserved hAM, using both the epithelial or the mesenchymal layer facing the defect, on bone regeneration in a critical calvarial bone defect in mice. Then, the efficacy of cryopreserved hAM associated with a bone substitute was compared to a collagen membrane currently used for GBR. In vitro, no statistical difference was observed between the conditions concerning cell viability. Without graft material, cryopreserved hAM induced more bone formation when the mesenchymal layer covered the defect compared to the defect left empty. When associated with a bone substitute, such improved bone repair was not observed. These preliminary results suggest that cryopreserved hAM has a limited potential for GBR.

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Analysis of bone formation and membrane resorption in guided bone regeneration using deproteinized bovine bone mineral versus calcium sulfate

Guided Bone Regeneration (GBR) is a technique based on the use of a physical barrier that isolates the region of bone regeneration from adjacent tissues. The objective of this study was to compare GBR, adopting a critical-size defect model in rat calvaria and using collagen membrane separately combined with two filling materials, each having different resorption rates. A circular defect 8?mm in diameter was made in the calvaria of Wistar rats. The defects were then filled with calcium sulfate (CaS group) or deproteinized bovine bone mineral (DBBM group) and covered by resorbable collagen membrane. The animals were killed 15, 30, 45 and 60 days after the surgical procedure. Samples were collected, fixed in 4% paraformaldehyde and processed for paraffin embedding. The resultant sections were stained with H&E for histological and histomorphometric study. For the histomorphometric study, the area of membrane was quantified along with the amount of bone formed in the region of the membrane. Calcium sulfate was reabsorbed more rapidly compared to DBBM. The CaS group had the highest percentages of remaining membrane at 15, 30, 45 and 60 days, compared to the DBBM group. The DBBM group had the highest amount of new bone at 45 and 60 days compared to the CaS group. Based on these results, it was concluded that the type of filling material may influence both the resorption of collagen membrane and amount of bone formed.

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Preparation and characterization of nano-hydroxyapatite/polyamide 66 composite GBR membrane with asymmetric porous structure

In this study, a nano-hydroxyapatite/polyamide 66 (nHA/PA66) composite with good biocompatibility and high bioactivity is employed to develop novel asymmetric structure porous membranes for guided bone regeneration (GBR). FT-IR and XRD analyses suggest that chemical bonds are formed between nHA and PA66 both in composite powders and membranes. The fabricated membranes show gradient porous structure. SEM analysis reveal that pores less than 10 μm and pores with a size ranging from 30 μm to 200 μm distribute in the micropore layer and the spongy structure layer, respectively. The surface energy determination also reveals that the fabricated membranes have asymmetric surface properties on the two sides of the membrane. The incorporation of nHA in PA66 matrix improves the properties of the membrane. The elongation at break and the tensile strength of nHA/PA66-40 suggest that the composite membrane has good strength and toughness. The rough porous structure surface with high surface energy of nHA/PA66 composite membrane may be beneficial to promote cells immobility and differentiation into a mature phenotype producing mineralized matrix. The biocompatibility, bioactivity, osteoconductivity, asymmetric porous structure, mechanical properties and hydrophilicity of the composite membrane can meet the requirement of GBR technique.     

Sorption, diffusion, and pervaporation characteristics of dimethylformamide/water mixtures using sodium alginate/polyvinyl pyrrolidone blend membranes

Separation of aqueous dimethylformamide (DMF) solutions in the concentration range of 0-100 wt% were studied using sodium alginate (NaAlg)/polyvinyl pyrrolidone (PVP) blend membranes. The NaAlg was blended in different ratios with PVP. Prepared membranes were crosslinked with CaCl₂ for testing in pervaporation (PV) separation of DMF/water mixtures. Effects of feed composition (0-100 wt%), operating temperature (30-50 °C), and membrane thickness were investigated. Best results were obtained at the conditions of 75/25 NaAlg/PVP blend ratio (w/w), 40 °C temperature, 20 wt% DMF concentration, and 70 μm membrane thickness. Blending of PVP with NaAlg increased permeation flux whereas it decreased the separation factor. NaAlg/PVP membranes gave separation factors of 5.5-27 for permeation flux of 0.96-1.81 kg/m²h depending on the operating temperature and the feed mixture composition. Arrhenius plot of permeation flux data versus reciprocal of temperature exhibited linear trends. Permeation activation energy of DMF and water in the PV was calculated as 6.76 and 1.88 kcal/mol, respectively, using an Arrhenius type relationship. Sorption-diffusion properties of the NaAlg/PVP membranes were also investigated at the operating temperature and the feed composition.     

纳米羟基磷灰石/聚酰胺复合材料屏障膜的体外生物相容性

用相转移法制备了用于引导骨再生的纳米磷灰石/聚酰胺66(n-HA/PA66)和载银纳米羟基磷灰石/二氧化钛/聚酰胺66(Ag-HA-TiO₂/PA66)复合生物材料屏障膜, 并通过与骨髓基质干细胞(BMSC)共培养评价了其体外生物相容性。扫描电子显微镜(SEM)观察显示,制备的复合材料膜为不对称多孔膜,一面是孔径小于10μm的微孔层,另一面是孔径在30～200μm的大孔结构。四唑盐比色(MTT)和流式细胞术(FCM)试验结果表明：n-HA/PA66膜具有良好的细胞亲和力,有利于BMSC的黏附、生长和减少凋亡;Ag-HA-TiO₂/PA66 膜也具有良好的生物相容性,但加速了BMSC的凋亡。2种膜的结构和生物相容性能够满足引导骨组织再生膜材料的要求。     

Properties and in vitro biological evaluation of nano-hydroxyapatite/chitosan membranes for bone guided regeneration

Nano-hydroxyapatite(n-HA)/chitosan(CS) composite membranes were prepared by solvent casting and evaporation methods for the function of guided bone regeneration (GBR). The effect of n-HA content and solvent evaporation temperature on the properties of the composite membranes was studied. The n-HA/CS membranes were analyzed by scanning electron microscopy, Fourier transformed infrared spectroscopy, X-ray diffraction, swelling measurement, mechanical test, cell culture and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenylte-2H-tetrazolium bromide) assay. The results show that the surface roughness and micropores of the composite membranes increase with the rise of n-HA content, suitable for adhesion, crawl and growth of cells. The hydroxyapatite holds nano size and distributes uniformly in the composite membranes. Chemical bond interaction exists between Ca ions and –OH groups of n-HA and –NH₂ or –OH groups of CS. The n-HA content and solvent evaporation temperature have obvious influence on the swelling ratio, tensile strength and elongation rate of the composite membranes. Cell culture and MTT assays show that n-HA and its content can affect the proliferation of cells. The n-HA/CS composite membranes have no negative effect on the cell morphology, viability and proliferation and possess good biocompatibility. This study makes the n-HA/CS composite membrane be a prospective biodegradable GBR membrane for future applications.     

Poly(Vinylidene Fluoride-Trifluorethylene)/barium titanate membrane promotes de novo bone formation and may modulate gene expression in osteoporotic rat model

Osteoporosis is a chronic disease that impairs proper bone remodeling. Guided bone regeneration is a surgical technique that improves bone defect in a particular region through new bone formation, using barrier materials (e.g. membranes) to protect the space adjacent to the bone defect. The polytetrafluorethylene membrane is widely used in guided bone regeneration, however, new membranes are being investigated. The purpose of this study was to evaluate the effect of P(VDFTrFE)/BT [poly(vinylidene fluoride-trifluoroethylene)/barium titanate] membrane on in vivo bone formation. Twenty-three Wistar rats were submitted to bilateral ovariectomy. Five animals were subjected to sham surgery. After 150 days, bone defects were created and filled with P(VDF-TrFE)/BT membrane or PTFE membrane (except for the sham and OVX groups). After 4 weeks, the animals were euthanized and calvaria samples were subjected to histomorphometric and computed microtomography analysis (microCT), besides real time polymerase chain reaction (real time PCR) to evaluate gene expression. The histomorphometric analysis showed that the animals that received the P(VDF-TrFE)/BT membrane presented morphometric parameters similar or even better compared to the animals that received the PTFE membrane. The comparison between groups showed that gene expression of RUNX2, BSP, OPN, OSX and RANKL were lower on P(VDF-TrFE)/BT membrane; the gene expression of ALP, OC, RANK and CTSK were similar and the gene expression of OPG, CALCR and MMP9 were higher when compared to PTFE. The results showed that the P(VDF-TrFE)/BT membrane favors bone formation, and therefore, may be considered a promising biomaterial to support bone repair in a situation of osteoporosis.     

Preparation of alginate–CaCl2 microspheres as resveratrol carriers

Resveratrol-loaded calcium alginate microspheres for prolonged drug release were prepared by ionic gelation of alginate with calcium chloride (CaCl₂). Further, resveratrol-loaded calcium alginate microspheres were developed using two concentrations of alginate (0.5 and 1 % w/v) and CaCl₂ (0.5 and 1 M) and an encapsulator equipped with a 300-μm nozzle. The mean particle size of the microspheres was between 175.52 and 244.03 μm, and an encapsulation efficiency (EE) of over 95 % was observed. FTIR spectroscopy indicated a polyelectrolyte interaction between alginate and CaCl₂; alginate microsphere thermograms were analyzed by differential scanning calorimetry. X-ray diffraction shows the crystalline change of microspheres by cross linking. The release profiles and EE increased depending on the CaCl₂ concentration, and a slow initial burst release was observed on freeze-dried microspheres. These results indicate that resveratrol-loaded calcium alginate microspheres can be used as a potential resveratrol delivery system in the food industry.     

In vivo behaviour of a biodegradable poly(trimethylene carbonate) barrier membrane: a histological study in rats

The aim of the present study was to evaluate the response of surrounding tissues to newly developed poly(trimethylene carbonate) (PTMC) membranes. Furthermore, the tissue formation beneath and the space maintaining properties of the PTMC membrane were evaluated. Results were compared with a collagen membrane (Geistlich BioGide), which served as control. Single-sided standardized 5.0 mm circular bicortical defects were created in the mandibular angle of rats. Defects were covered with either the PTMC membrane or a collagen membrane. After 2, 4 and 12 weeks rats were sacrificed and histology was performed. The PTMC membranes induced a mild tissue reaction corresponding to a normal foreign body reaction. The PTMC membranes showed minimal cellular capsule formation and showed signs of a surface erosion process. Bone tissue formed beneath the PTMC membranes comparable to that beneath the collagen membranes. The space maintaining properties of the PTMC membranes were superior to those of the collagen membrane. Newly developed PTMC membranes can be used with success as barrier membranes in critical size rat mandibular defects.     

Poly(ε-caprolactone)-based membranes with tunable physicochemical,bioactive and osteoinductive properties

In contrast to currently used materials, membranes for the treatment of bone defects should actively promote regeneration of bone tissue beyond their physical barrier function. What is more, both material properties and biological features of membranes should be easily adaptable to meet the needs of particular therapeutic applications. Therefore, the role of preparation methods (non-solvent-induced phase separation and thermal-induced phase separation) of poly(ε-caprolactone)-based membranes and their modification with gel-derived bioactive glass (BG) particles of two different sizes (<45 and <3 μm) in modulating material morphology, polymer matrix crystallinity, surface wettability, kinetics of in vitro bioactivity and also osteoblast response was investigated. Both surfaces of membranes were characterised in terms of their properties. Our results indicated a possibility to modulate microstructure (pore size ranging from submicron to hundreds of micrometres), wettability (from hydrophobic to fully wettable surface) and polymer crystallinity (from 19 to 60%) in a wide range by the use of various preparation methods and different BG particle sizes. Obtained composite membranes showed excellent in vitro hydroxyapatite forming ability after incubation in simulated body fluid. Here we demonstrated that bioactive layer formation on the surface of membranes occurred through ACP–OCP–CDHA–HCA transformation, that mimic in vivo bone biomineralization process. Composite membranes supported human osteoblast proliferation, stimulated cell differentiation and matrix mineralization. We proved that kinetics of bioactivity process and also osteoinductive properties of membranes can be easily modulated with the use of proposed variables. This brings new opportunities to obtain multifunctional membranes for bone regeneration with tunable physicochemical and biological properties.     

Evaluation of alginate-chitosan semi IPNs as cartilage scaffolds

In this study, alginate and alginate:chitosan semi interpenetrating polymer network (IPN) scaffolds were prepared by freeze-drying process. Alginate scaffolds were crosslinked with different concentrations of CaCl₂, i.e. 0.5, 1 or 3% (w/v), in 96% (v/v) ethanol solutions for two different periods, i.e. 4 and 24 h, after freeze-drying. Scanning electron microscope (SEM)/ Energy Dispersive Analysis by X-ray (EDAX) analysis and swelling studies indicated that crosslinking of scaffolds with 3% (w/v) CaCl₂ for 24 h was effectively created suitable alginate scaffolds in terms of optimum porosity and mechanical stability. This is why, alginate:chitosan semi IPN scaffolds were prepared at the crosslinking condition mentioned above in 70:30, 60:40 and 50:50% (v/v) alginate:chitosan ratios. Besides the attachment and proliferation abilities of ATDC5 murine chondrogenic cells on alginate, 70:30% (v/v) alginate:chitosan and 50:50% (v/v) alginate:chitosan scaffolds, their cellular responses were assessed for chondrogenic potential. These structural and cellular outcomes demonstrate potential utility of chitosan semi IPNs in alginate scaffolds. Comparative results found in relation to alginate scaffolds, support the necessity for alginate:chitosan scaffolds for improved cartilage tissue engineering.     

Thermoreversible behavior of κ-carrageenan and its apatite-forming ability in simulated body fluid

Osteoconductive materials with self-setting ability have received much attention because their properties allow developing injectable materials for bone defects. Thermosensitive hydrogel with ability of bone-like apatite formation in a body environment is a candidate of injectable bone fillers with osteoconductivity because the apatite formation on materials is an essential to show osteoconduction. The present study focused on the development of a thermosensitive hydrogel through modifications of the sulphonic groups of the polysaccharide, κ-carrageenan, with potassium chloride (KCl) and calcium chloride (CaCl₂). We found that the gelation temperature of κ-carrageenan solutions increased with increasing amounts of K⁺ ions. Apatite formation was observed on the gel after exposure to simulated body fluid for 0.5 day when the gel was prepared with a molar ratio of Ca²⁺/sulfonic groups = 1.5. These results indicate that a thermosensitive κ-carrageenan hydrogel with apatite-forming ability was obtained through the incorporation of K⁺ and Ca²⁺ ions into the solution.     

Guided bone regeneration using osteopatite® granules and polytetrafluoroethylene membranes

Granules of a modified hydroxyapatite, Osteopatite®, were implanted in the right posterior tibiae of adult rabbits. We studied the extent of bone regeneration in bone holes. In the right tibiae, that were filled with granules of this biomaterial covered with a polytetrafluoro-ethylene (PTFE) membrane using, as a control, uncovered granules. In the left tibia, an empty hole was covered with PTFE membrane and a second hole was left empty to be used as a control. A histomorphometric study was carried out using light microscopy, four and eight weeks after the surgery. The covered granules presented a higher percentage of bone contact than the uncovered ones, and it was also possible to observe a better bone tissue organization, mainly produced by the immobilization action of the PTFE membrane. Empty bone defects covered with PTFE membranes, two months after implantation, presented large areas of Haversian bone and direct bone contact to the PTFE membrane.