| Objective:To construct a new drug pulsatile delivery system that can release of parathyroid hormone(PTH)and to explore the reliability of its promoting effects on the mandibular fracture healing in rabbits.Methods:1.Preparation of PTH local release system and detection its physicochemical properties1.Conduction the monolayer sodium alginate membrane and CMCS/PVA blend membranes and their physicochemical property(1)Carboxymethyl chitosan(CMCs)and polyvinyl alcohol(PVA)were blended according to the volume ratio: 100%CMCS(CMCS)、70%CMCS / 30%PVA(7C3P)、50%CMCS / 50%PVA(5C5P)、30%CMCS / 70%PVA(3C7P).The gels were dripped into 2.5 cm diameter circular mold,and the films were formed after the gels dring naturally.The monolayer films were analyzed morphology,characterization and physicochemical property including the hydrophilicity,the degradation rate and swelling degree in vitro.(2)Preparation PTH local release system and detection its physicochemical property: CMCS/ PVA blend membranes were used as the controlled release layer to regulate the release rate and rhythm;alginate membranes were a drug loading layer carrying PTH active protein.The scanning electron microscope(SEM)were used to observed its section shape.And its degradation rate and p H value were detected in vitro.Then the system was implanted subcutaneously in rats back.The back skin reaction,gross morphological observation and hematoxylin eosin(HE)of the degradation system were evaluated the system degradation rate and biocompatibility within 25 days in vivo.(3)According to above results,the drug release system was constructed by 3C7 P blend membranes,and its release rhythm in vitro was detected.Bovine serum albumin(BSA)was used as a simulated drug and BCA kit was used to evaluate the BSA release rhythm from the release system.PTH release system was constructed.The PTH ELISA kit was used to evaluate the PTH release rhythm from the PTH release system.(4)The PTH release systems were implanted into the mandibular surface of New Zealand white rabbits.The animal blood was collected at the same time every day.The serum PTH concentration was detected by automatic luminescence immunoanalyzer and compared with that of normal rabbits.2.Study on the toxicity and osteogenic differentiation of PTH pulsatile sustained-release system on MC3T3 cells(1)CCK8 was used to detect the effect of the extract of the release system on cells to evaluate the cytotoxicity of the system.(2)MC3T3 cells were co cultured with the sustained-release system.DAPI staining and phalloidin staining were used to detect the biocompatibility and adhesion of the sustained-release system.(3)The effect of the extract of the sustained-release system on the osteogenic differentiation of MC3T3 cells was evaluated by alkaline phosphatase staining and quantification,alizarin red staining and RT-q PCR(Runx2、Opn and Col 1 m RNA).3.Study on the effect of PTH pulsatile sustained-release system on rabbit mandibular fracture healing(1)Experimental grouping.96 New Zealand white rabbits were randomly divided into four groups: 1)Blank control group(group 1): fixed with micro titanium plate and titanium nail;2)Negative control group(group 2): blank local sustainedrelease system(excluding PTH)+ micro titanium plate and titanium nail fixation;3)Positive control group(Group 3): micro titanium plate and titanium nail fixation of fracture + subcutaneous injection of 20 μg PTH every other day;4)Experimental group(Group 4): PTH pulsatile sustained-release system + micro titanium plate and titanium nail fixation.The rabbit mandibular fracture reduction and fixation model was established.The fracture healing was recorded and samples were collected at postoperation 1,2,3 and 4 weeks.(2)The fracture healing situations were assessed by the general observation of the fracture area,x-ray,semi quantitative score and pathological score of the fracture area healing after HE staining.The expression of osteogenesis related factors Runx2,Col Ι,BMP-2 and VEGF were detected by immunohistochemistry and RT-q PCR.Results:1.Preparation PTH local release system and detection its physicochemical property(1)The characterization of CMCS/PVA blend membranes showed that they contained the characteristic bonds and elements of CMCS and PVA by FTIR and energy spectrum.SEM showed that the micro morphology of CMCS film was relatively flat and smooth film,while the CMCS/PVA blend films were granular.The higher the PVA content,the larger the particle size is.The particle size of 3C7 P film is 10 ± 1.21μm.(2)The swelling and degradation of CMCS,7C3 P,5C5P and 3C7 P blend membranes in vitro,the higher the content of CMCS,the faster the swelling and degradation.3C7 P blend membrane decreased about 20% in 24 hours and the swelling degree was about 3 in 3 hours.At the same time,the results showed that the contact angle of CMCS/ PVA blend membrane was smaller than that of pure CMCS membrane or PVA membrane,of which 3C7 P membrane had the smallest contact angle(33.19 ±1.65)°,and the best hydrophilicity.(3)The p H value of the extract of the was alkaline.The p H value of the 3C7 P system was between 7.4 and 7.9 within 10 days,which was closest to the p H value of physiological bone tissue fluid.(4)The degradation rate trend of sustained-release system was consistent with that of monolayer membrane.The degradation of the system constructed by CMCS membrane was the fastest,and the degradation of the system constructed by 3C7 P blend membrane was the slowest.At the same time,multinucleated giant cell phagocytic materials can be seen in the process of degradation in vivo.(5)The biocompatibility of the system in vivo showed that there were no rejection reactions such as abscess,wound dehiscence and pus overflow in the implantation area.According to the above experimental results,we selected the blend membrane with the ratio of 3C7 P to construct the sustained-release system for subsequent experiments.(6)The release results of simulated drug BSA release system and PTH release system showed a pulsatile release rhythm in vitro.At the same time,the amount of PTH released from PTH sustained-release system is about 80% of the whole system within35 days.(7)After the PTH sustained-release system was implanted into rabbit,the serum PTH showed impulse fluctuations,and the average content of serum PTH was higher than that of normal rabbits.This part results show that the sustained-release system presented pulsed drug release rhythm in vitro and in vivo,which was a self-regulated pulsatile release system.2.Study on the toxicity and osteogenic differentiation of PTH pulsatile sustained-release system on MC3T3 cells(1)The results of CCK-8 showed that the extract of sustained-release system was non-toxic to cells.(2)The pulsatile sustained-release system was co cultured with cells,the myofilament protein extended and cells adhered to the surface of the system.(3)The results of alkaline phosphatase staining and quantification,alizarin red staining and RT-q PCR(Runx2、Opn and Col 1 m RNA)were evaluated osteogenic induction.Compared with osteogenic induction solution(negative control group),the speed and effect of osteogenic induction of PTH slow-release system extract were faster and better,and the effect was equivalent to that of osteogenic induction solution added with PTH(positive control group).The results showed that the sustained-release system was conducive to cell adhesion,and its extract was non-toxic to cells.At the same time,the extract of PTH pulsatile sustained-release system could promote the osteogenesis of MC3T3 cells,showing the same effect as that of pure PTH.3.Study on the effect of PTH pulsatile sustained-release system on rabbit mandibular fracture healing(1)The fracture healing of each group was evaluated by grossly,x-ray and histologically.The results showed that the fracture healing speed of PTH local pulsatile sustained-release system group was accelerated,which was equivalent to that of systemic injection of PTH.(2)Osteogenic factors Runx2,Col Ⅰ,BMP-2 and VEGF were detected by immunohistochemistry and RT-q PCR.The PTH pulsatile sustained-release system group and systemic injection PTH group were consistent,which had the effect of promoting osteogenesis.Conclusions:(1)The PTH pulsatile sustained release system constructed by 3C7 P blend membrane have good biocompatibility and can achieve the effect of pulsed release of PTH in vivo and in vitro.(2)The PTH pulsatile sustained release system could be co cultured with MC3T3,has good cell adhesion effect and promotes cell osteogenic differentiation.(3)The PTH pulsatile sustained release system can promote the healing of rabbit mandibular fracture and achieve the same effect as systemic injection of PTH.To sum up,a new PTH pulsatile sustained-release dosage form is constructed in this experiment,which could change the routine subcutaneous injection of PTH.After local application,the dose of PTH can be reduced and the bioavailability of PTH can be improved.At the same time,it has important application basic research value and social benefits for accelerating fracture healing and promoting patients’ early recovery,which can provide theoretical and experimental basis for transformation and application in the field of oral and maxillofacial trauma treatment. |
