| ObjectiveThe main purpose of this study is to develop a new kind of bioabsorbable membrane, which could simplify the film-forming progress, reduce costs and improve the effect of guided bone regeneration by releasing bone promoting factors. Specifically, the purpose is:(1) To compare the physical and chemical properties of the membranes made by different concentration ratio using the sol-gel transition property of chitosan/β-glycerophosphate (CS/β-GP) thermosensitive hydrogel under physiological conditions.(2) To study the protein sustained-release properties of the composite membranes using bovine serum albumin(BSA) as a model protein.(3) To ensure that the main component of the protein extracted by acetic acid is enamel matrix proteins (EMPs) and to explore the guided bone regeneration activity of the composite membranes added EMPs in vivo and in vitro.Method1) Preparation of membranes and alternative of ratio:In the ice bath, three groups of clear and homogeneous solution A (0.6gβ-GP), B (0.8gβ-GP), C (1.0gβ-GP) were prepared by dropwise adding different concentration of β-GP solution, containing0.6g,0.8g and1.0gβ-GP repectively, to the same concentration of CS solution prepared with0.1mol/L acetic acid solution. The PH value and the gel time of the solution was detected in the experiment. The film thickness and tensile strength of the membranes were measured, too.(2) Protein sustained-release experiment of the composite membranes:Three groups of composite membranes which are A (0.6gβ-GP), B (0.8gβ-GP), C (1.0gβ-GP) were prepared added with20mg BSA.1/4of the membranes were immersed in the PBS buffer solution and the protein sustained-release curves were drawn according to the absorbance values obtained at different time point.(3) Study of guided bone regeneration activity of CS/β-GP composite membranes loaded EMPs:(to choose the appropriate ratio of CS/β-GP composite membranes for the guided bone regeneration experiments in vitro and in vivo)1) Extraction and identification of EMPs:EMPs was extracted form porcine tooth germs by acetic and identified by SDS-PAGE electrophoresis.2) In vitro part:CS/β-GP (1.0g) composite membrane loaded EMPs (group A), CS/β-GP (1.0g) composite membrane (group B) and blank control group (group C) were co-cultured with ST2cells. The sustained-release effect of the membranes on ST2cells were detected by comparing the outcome of the MTT assay and alkaline phosphatase (ALP) activity.3) In vivo part:10Wistar rats, male and healthy,were randomly divided into two groups(A and B). Critical size defect (CSD) in a diameter of5mm was created on both sides of the skull of the rats.The left side (the experimental side) was covered by CS/β-GP (1.0g) composite membrane loaded EMPs and the right side (the control side) was smeared with EMPs solution of the same concentration and covered by CS/β-GP (1.0g) composite membrane. The rats of group A and B were killed on the first and the second month seperately.The restoration of the CSD on both sides was compared by gross observation, X-ray and tissue biopsies1month and2months after the operation.Result(1) Preparation of membranes and alternative of ratio:PH value of different CS/β-GP solution was all around7.0, which could increase with the increasing of the concentration of β-GP. All the solution could turn to gel and the gelation time decreased with the increasing of the concentration of β-GP. The tensile strength of the membranes turned from gels through drying method could reach to about1Mpa, which would decrease with the increasing of the concentration of β-GP.(2) Protein sustained-release experiments:Statistically, the difference of the PH value, gelation time of the solution or the depth and tensile strength of the membranes made with or without BSA was not significant (P>0.05). The trends of BSA sustained-release curves of different membranes were similar and the total amount of protein increased with the increasing of the concentration of β-GP.(3)GBR experiment of membranes loaded with EMPs:CS/β-GP (1.0g) was used in this part;1) EMPs:The protein extracted in acetic acid method was white and flocculent through freezing.SDS-PAGE showed that multiple bands were stained, the width and staining extent of the bands were different and the band near20KDa was the most clear one.2) In vitro:Statistically, the difference of the PH value, gelation time of the solution or the depth and tensile strength of the membranes made with or without EMPs was not significant (P>0.05). Morphology and proliferation of ST2co-cultured with membranes were normal. MTT showed that the differences of OD value between A, B and C group were statistically significant and the OD values of group A and B were higher than that of group C, while the value of group A was higher than that of group B(P<0.05). ALP outcome showed the same trend.3) In vivo:Experimental animals were all healthy and were all at the wound healing stage Ⅰ. X-ray showed that one month after the operation, the range of the CSD made in the skull of the rat on the experimental and the control side were both narrowed slightly, while the defect on the experimental side was smaller than that on the control side. It also suggested that two months later, the range of the CSD on both sides were narrowed obviously, while the defect on the experimental side was nearly repaired completely. The data was analyzed by SIDEXIS. The result collected both one and two months after the operation suggested that the density percentage of the new bone in the bone defect area on the experimental side and control side was statistically different (P<0.05). Histological observation predicted that a small amount of new bone proliferative was visible on both side of CSD one month after surgery while the proliferative response of new bone was more notable on the experimental side.Conclusion(1) The phase transition of CS/β-GP thermosensitive hydrogel occurred under physiological conditions whether or not to load protein. The composite membranes acquired in drying method basically meet the need of the physical and chemical properties of barrier membrane. CS/β-GP (1.0gβ-GP) membrane was the better choose for the in vitro and in vivo using.(2)Using BSA as a model protein, different membranes with different concentration showed protein-release ability with slight difference. The total amount of protein sustained-released by CS/β-GP(1.0gβ-GP) membrane was the highest.(3)The dominant component of the EMPs extracted by acetic acid was amelogenin. By comparison, the CS/β-GP (1.0g) composite membrane loaded EMPs performed better guided bone regeneration propeties in vitro and in vivo through protein sustained-release. |
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