Preparation And Properties Of Chitosan/β-GP Composite Membranes For Guided Bone Regeneration

Background and purposePeriodontal tissue defects and alveolar bone loss due to age-related physiology changes of the jawbone, surgery, trauma, inflammation and tooth extraction are common clinical problems. How to rebuild the periodontal support tissues and reduce the loss of teeth, to promote long-term success rate and aesthetic effect of dental implants have been a great challenge untile now. The guided bone regeneration (GBR) technique, derived from the guided tissue regeneration (GTR) technique, is regarded as one of the most commonly and effective way for local bone defects surrounding dental implants and teeth. The biological principle of GBR/GTR is to utilize a barrier membrane to maintain space over a bony defect, promoting the ingrowth of osteogenic cells and preventing the migration of undesired cells from the overlying soft tissues. Therefore, the material used for a GBR membrane must have biocompatibility, stability over the required duration the barrier will function, the ability to maintain space, the ability to exclude undesired cell ingrowth, and ease of use. A variety of barrier membranes have been used in alveolar bone augmentation through the GBR technique. However, and have a higher risk of exposure. Clinically, non-resorbable membranes have largely been replaced by bioabsorbable membranes that simplify the surgical procedure and avoid a second surgery. Chitosan (CS) has been used for barrier membranes because of its good film-forming and bioactive properties. However, CS has drawbacks, including poor mechanical strength and low solubility in common dilute acid solutions. CS products therefore usually must be combined with other biomaterials and neutralized in alkali solution to fabricate barrier membranes. These processes are complex and time-consuming, affecting the potential of CS to be incorporated with bioactive factors for promoting tissue regeneration. Interestingly, CS solutions containingβ-GP can remain in solution at low temperature and become a networked solid at physiological pH and body temperature in the form of thermosensitive hydrogel, and the CS/β-GP composite also has good biocompatiblity and biodegradability.Herein, we developed a novel GBR membrane containing CS andβ-GP, fabricated by a sol-gel and dehydrated process around body temperature. This is a simple method of membrane fabrication, and potentially a GBR menbrane which allows the incorporation and sustained release of bioactive molecules, thanks to the relatively neutral and mild solvothermal procedure, that contribute to bone regeneration.MethodsPart I:Preparation and characterization of CS/β-GP thermosensitive hydrogel and composite membranes:CS powder with a 95% deacetylation degree (DD) and a molecular weight (Mw) of about 3.5×106 Da was used in this study.2g CS powder was dissolved in 90mL 0.1M acetic acid solution under stirring and mixing for 2h.0.4g,0.6g, 0.8g, 1.0g, and 1.2g ofβ-GP were dissolved in 1mL deionized water and then sterilized using a 0.22μm filter. CS solutions of 9mL andβ-GP solutions were chilled in an ice bath for 15 mins before being mixed together. Ice-coldβ-GP solutions with different concentrations were then added drop by drop to the ice-cold CS solutions under continuous stirring, and the final formulations were mixed for another 10 mins to obtain a clear and homogeneous liquid solution. Five different combinations of CS andβ-GP were prepared. The final formulations contained about 2% (w/v) of CS and 4-12% (w/v) ofβ-GP. The pH of each solution was tested and gelating time was assessed by the test tube inversion method. Each solution of CS/β-GP (≈10mL) was spread on a plastic dish (?= 40 mm), gelated and dehydrated in an automatic electric thermostat at 37℃to fabricate the membranes. Four kinds of CS/β-GP membranes (M1-M4) were obtained according to their different, respective concentrations ofβ-GP (6-12wt%). Pure CS membrane (M0), prepared as control, was treated with 0.5M NaOH solution for 2h and repeatedly washed with deionized water. Their structural properties, morphology, and tensile strength were investigated by FTIR, XRD, SEM and electronic universal testing machine.PartⅡ:The in vitro experiments:in this part of experiment, the degradation behavior in PBS, swelling behavior in deionized water and the level of mineralization in simulated body fluid (1.5SBF) of the CS/β-GP membranes and pure CS membrane were investigated. For the biological study, two types of CS/β-GP composite membranes (M1,M3) according to different concerntration ofβ-GP were investigated in terms of their in vitro cellular activity using ST2 cells, including cell attachment, proliferation and differentiation, and the results were compared with those obtained using pure CS membrane.PartⅢ:The in vivo experiments:3-month-old female Wistar rats were used for subcutaneous implant and calvarial critical size defects regeneration experiments to assesse the in vivo inflammatory reaction, biodegradation, barrier and guid bone regeneration propertis of the composite membrane.PartⅣ:Preliminary studies on performance improvement of the CS/β-GP composite membrane:Carboxymethyl chitosan calcium (CCC) was synthesized and characterized by FTIR and XRD, and the cellular biocompatibility of the CCC was also studied by MTT assay using ST2 cell. Then, different qualities of the CCC were mixed into CS/β-GP solution and the gelating time of the composite sistems was oberserved. On the other hand, diferent doses of enamel matrix proteins (EMPs) were added into the CS/β-GP solution. And then, gelating times of the CS/β-GP/BMPs composite gel was measured and the in vitro BMSCs (ST2 cell) cellular differentition property of the CS/β-GP/BMPs composite membrane was accessed by ALP activity test. In a word, the purpose of this part of study was to investigate the affactive of the calcium in CS/β-GP thermosensitve hydrogel sistem and bioactive factors in CS/β-GP composite membrane for future GBR application. ResultsPartⅠ:2% (w/v) CS solutions with different concentrations ofβ-GP maintained their liquid state at physiological pH and room temperature, subsequently setting to opaque gelation around 37℃when the quality ofβ-GP is more than 0.4g in lOmL mixed solution. The gelation times and the pH of the mixtures decreased with increasingβ-GP concentration. Statistically, the gelation times of differentβ-GP concentrations were significantly different from one another. After dehydrated in an automatic electric thermostat at 37℃and dried by air, the CS/β-GP composite membaranes were fabricated. SEM photographs showed that the pure CS membrane (M0) had an even, smooth surface, but CS/β-GP composite membranes had a rough and porous structure both at the surface and in sublayers. FTIR analysis indicated that hydrogen bonding and static electric interactions existed between CS andβ-GP. XRD analysis showed that, the introduction of -OH and PO43- groups ofβ-GP disturbed the crystal domain of CS. The concentration ofβ-GP of the composite membrane was proportional to the pore size and thickness but was inversely proportional to the tensile strength of the CS/β-GP membrane. The incorporation ofβ-GP into the CS matrix decreased the tensile strength of the membrane, ranging from 1.13 to 0.78Mpa.PartⅡ:The degradation of the composite membranes in PBS is obviously in the early time, especially in the first day the rate of weight loss of the composite membranes reached 60% to 70%. The degradation rate slowed down after 3 days, and after 14 days the rate of weight loss approximately 80%. The CS/β-GP composite membrane was hydrophilic and absorbent, the rate of swelling behavior of the membranes is versely proportional to the concerntration of theβ-GP. There was no CaP precipitation visible on membrane M0 in 1.5SBF solutions up to 14 days of incubation. However, for the M1 composite membrane, tiny CaP depositions could be seen on the fiber surface. These CaP depositions became more obvious when the immersion period reached 14 days. The CaP deposition process on M3 membrane went much faster. After 14 days of immersion, the surface were fully covered by a layer of nano-textured cauliflower-like CaP coatings. The ST2 cells on the pure chitosan membranes exhibited a spherical or triangular shape but, grew and spread more actively with lamellipodia and ruffling at the leading edge of the cell, and exhibited a flattened, polygonal morphology and intimate adhesion to the rough surface of the composite membranes. Compared with the pure CS membranes, the CS/β-GP composite membranes had much higher bioactivity than the CS membranes, the proliferation and differentiation of the ST2 cells were significantly improved.PartⅢ:After 2 and 4 weeks subcutaneous implantation in vivo, we found that all the implanted membranes had maintained their shapes, and had been surrounded by thin fibrous capsules. The pure CS membrane showed no degradation, and a mild inflammatory reaction at 2 weeks post-implantation. But after 4 weeks implantation, there is a dense accumulation of inflammatory cells with the started degradation from the membrane surface. In contrast, the CS/β-GP composite membrane showed prominent degradation, and some inflammatory cells, including giant cells, at 2 weeks post-implantation. Notably, CS/β-GP composite membrane was degraded to networking structure and decreased inflammatory reaction after 4 weeks of implantation. Compared with the pure chitosan membrane, the calvarial defects which covered by the composite membrane was filled with collagen fibers and some new bone.PartⅣ:CCC was synthesized by CMCS and calcium chloride in the condition of pH 7.0 and room temperature. FTIR and XRD analysis showed that calcium complexed with the COO- and -NH2 of the CMCS and the crystal domain of CMCS was disturbed by the introduce of calcium. When different qualities CCC mixed into CS/β-GP solution the gelation time decreased proportionally. It was indicated that the calcium played a role of physical cross-linking agent in the sistem. When too many CCC (400mg) added into 10mL of the CS/β-GP system, the solution rapid solidified and lost the temperature sensing properties. On the other hand, when a certain amount of EMPs was added and formed CS/β-GP/EMPs composite gel, the gelation time of the CS/β-GP system was no difference. Compared with the CS/β-GP membranes, the CS/β-GP/EMPs composite membranes had much higher bioactivity whose differentiation of the ST2 cells were significantly improved. Conclusion2% (w/v) chitosan (Mw 3.5×106Da, DD 95%) solution containing 6～12%(w/v) of P-GP that formed CS/β-GP sistems with the thermosensitive property. At room temperature, neutral pH conditions the CS/β-GP solutions kept in the liquid and transformed to semi-solid gel at 37℃. And then formed membrane by air drying at 37℃. It is a simple and novel method using the sol-gel phase transition property of the CS/β-GP thermosensitive hydrogel, with the physiologically mild conditions, to prepare GBR barrier membrane. Characteristics and mechanical testing showed that CS/β-GP composite membranes have a rough surface structure, porous internal structure and certain mechanical strength those are required for the application of GBR. In vitro and in vivo experiments indicated that CS/β-GP composite membrane has good degradability, swelling behavior and surfacial mineralization properties in vitro; the CS/β-GP composite membranes had good bioactivity, the proliferation and differentiation of the BMSCs (ST2 cells) were significantly improved. BMSCs adhesion and growth on the surface with normal morphology; the in vivo experiments indicated that the composite membrane with good biocompatibility, biodegradability, barrier property and promote bone healing properties. A certain amount of calcium compounds (CCC) mixed into the CS/β-GP thermosensitive hydrogel system can significantly decreased the gelation time; but, a certain amount of small molecule protein into the CS/β-GP thermosensitive hydrogel system does not affect the gelation time. On the other hand, the CS/β-GP/EMPs composite membrane can promote the ALP activity of BMSCs in vitro expression. Adding calcium or combined growth factors might improve the membrane performance and bone repairing activity. Further bioactive properties of the CS/β-GP membrane in vitro and in vivo are need further experimental investigation.