| Objective:As oral implant technology getting more and more mature,dental implants become the first choice for patients of dentition defect or edentulous jaw.However,lack of bone mass at implant sites caused by Inflammation,trauma,anatomical factors,congenital factors,disuse atrophy,et al.prevents those patients from choosing dental implants to a large degree.Guided bone regeneration refers to a bone graft technology for repairing local bone defect at implant sites,and barrier membrane of guided bone regeneration is one of the key factors to the success of implant surgery.Recent years,tissue-derived extracellular matrix materials gradually become a research hot spot of tissue engineering and regenerative medicine.The most obvious advantage of extracellular matrix materials lies in that they maximally retain the natural 3D structure and bioactive components in the extracellular matrix,while maximally removal those immune inflammatory cell components,which is more beneficial for tissue repair and regeneration.The present study selected sheep periosteum tissue as the tissue source of guided bone regeneration membrane,1.Explore and optimize the decellularization protocol for sheep periosteum tissue,and evaluate the decellularization effectiveness and conservation of extracellular matrix structure and components.2.Evaluate the effect of acellular sheep periosteum on cell adhesion,proliferation and osteogenesis differentiation through in vitro experiments by cultivating MC3T3-E1 cells together with the acellular sheep periosteum material.3.Investigate the potential general and local immune inflammatory response induced by acellular xenogeneic sheep periosteum through the in vivo experiment of rat subcutaneous implantation test.4.Further evaluate the effect of acellular sheep periosteum material on guided bone regeneration through establishing a rabbit model of cranial defect.Methods: 1.After a combined application of physical agent(multigelation),chemical agent(Triton X-100?SDS),enzymatic agent(DNase and RNase)to remove the cell components from sheep periosteum tissues,HE and DAPI staining of histological sections were conducted to conform cell residual degree in the tissues,DNA components were extracted from the sheep periosteum to quantitative test their contents,the size of the extracted DNA fragments was detected by agarose gel electrophoresis.Residual amounts of ?-Gal epitopes in acellular periosteum were quantified by enzyme-linked immunoabsorbent assay using a sheep ?-Gal ELISA kit in comparison with the amount in fresh periosteum.Extract liquor of acellular periosteum was prepared and tested the residual concentration of SDS.Masson and safranin O staining of were performed to evaluate the retention situations of collagen and glycosaminoglycan,and Hydroxyproline Assay Kit and DMMB Colorimetry Kit were used to quantify the collagen and glycosaminoglycan content in the acellular periosteum material.The surface microarchitecture of fresh periosteum and acellular periosteum were identified by scanning electron microscopy(SEM).Swelling behavior was tested to indicate the difference of water absorption ability between fresh periosteum and acellular periosteum.Tensile mechanical property test was utilized to evaluate those mechanical performance index of acellular periosteum.2.MC3T3-E1 cells were cultivated in extract medium of acellular sheep periosteum with different concentrations,and CCK-8 test was performed to evaluate the influence of acellular sheep periosteum on cell proliferation.Then,MC3T3-E1 cells were seeded directly onto acellular sheep periosteum,the process of cell adhesion was observed by SEM and fluorescence microscope;effect of acellular sheep periosteum on cell early differentiation was evaluated by alkaline phosphatase activity;influence of acellular sheep periosteum on the expression level of Col-I ? Runx2 ? OCN in the osteoblasts was detected by quantitative real-time PCR technique.Fresh periosteum,acellular periosteum and Bio-Gide were randomly implanted subcutaneously into the rat backs,sacrifice was conducted at 3,7,14,28 and 56 days after surgery.Blood was sampled from rat eyes and serum was harvested to detect the concentrations of,IL-2,IFN-? and IL-4 by ELISA test.Local tissues at the implant sites were collected,paraffin sections were made and dyed with HE and immunohistochemical staining to evaluate the degree of local immune inflammatory response in different groups.3.A rabbit model of cranial defect was established,four round full-thickness defects with a critical diameter of 8 mm were drilled in each rabbit calvarium,four groups were set as below:(1)Control group with nothing filled inside.Mucosal layer and geometric skin were sutured layer by layer;(2)Only acellular periosteum was covered(P group);(3)Only autogenous bone granules of one bone block were placed(B group);(4).Autogenous bone granules of one bone block were implanted into the defect and acellular periosteum was covered upon the implanted bone(B+P group).After 4,8,12 weeks,X-ray was performed to identify if there was any infection in different groups,micro-computed tomography was used to evaluate the bone formation and the percentage of bone volume in bone defect,mean bone trabecula number and spacing were quantificationally measured.Undecalcified sections were made and toluidine blue staining was performed to evaluate the effect of acellular periosteum on guided bone regeneration.Results: 1.No macroscopic cell nucleus debris was observed both in the pictures of HE and DAPI staining,the DNA content of fresh periosteum was 580.37±33.92ng/mg,while the DNA content of acellular periosteum was only 32.52±5.31ng/mg,no visible bands of DNA were observed after agarose gel electrophoresis in acellular periosteum compared with the whole genome bands in FP.The quantitative analysis result of ?-Gal epitopes showed that the ?-Gal content of FP was 35.80±5.07ng/mg,while the ?-Gal content of AP was only 7.08±1.64 ng/mg,no significant statistical difference was observed between the two groups(p<0.05).Residual SDS amount of AP was 0.0025%,which was far less than absolute safe dose of 0.005%.The result of Masson and safranin O staining showed that,the structure,distribution and integrity of collagen and glycosaminoglycan,which are major components of extracellular matrix,presented no significant difference between AP and FP,the collagen contents of AP and FP(627.02±56.76?g/mg vs.609.91±38.15?g/mg)and the glycosaminoglycan contents of AP and FP(1.801±0.058?g/mg vs.1.748±0.038?g/mg)also had no significant difference.Compared to AP,the SEM picture of FP presented to be more loose and porous,but the structure of each collagen fiber itself still remained intact.The water absorption value of FP was 2.11±0.43mg/mg dry weight,while the water absorption value of AP was 3.41±0.34 mg/mg dry weight,no significant difference was found between two groups(p<0.05).The elastic modulus of AP(E=38.22 ± 5.71 MPa)was significantly lower than the value of FP(E = 45.89 ± 3.27 MPa)(p < 0.05),but here was no significant difference in the values of yield stress(40.79 ± 1.22 MPa and 39.11 ± 0.90 MPa)between FP and AP(p > 0.05).2.SEM and fluorescence microscope results showed that MC3T3-E1 cells stretched well on AP,they gradually turned from sphere to fusiform and polygon,and linked with each other to form a new cell layer on the whole AP.The result of CCK-8 test showed that at day 5 and 7,the group of 100% extract exhibited better cell viability than the group of 50% and then the group of 25%,the OD values of the 100% group and the 50% group were significantly bigger than the values of the control group(p < 0.05)?The ALP activity of cells seeded on AP was detected higher than each value in the control group,the superiority was quite obvious especially at day 3,7 and 10(p < 0.05).The result of quantitative real-time PCR test showed that AP could promote cell osteogenesis differentiation by increasing the expression of COL-I?Runx2?OCN to different degree.In the rat model of back subcutaneous implantation,FP caused rises of IL-2 and IFN-? in serum,which were significantly higher than the AP group and the Bio-Gide group at day 7,14 and 28(p < 0.05),while the IL-4 values of the three groups existed no significant difference between any two of then(p > 0.05).The HE and immunohistochemical staining showed that a big quantity of inflammatory cells was infiltrating around the implanted FP with a large area of hemangiectasis and hyperemia,which showed earlier and longer than AP and Bio-Gide,and there was no significant difference between AP and Bio-Gide.Quantification of immunohistochemical staining results showed that significant differences could be found at each time point between AP and the other two groups.3.The X-ray image indicated no infection was found in the bone defect at any time point.The Micro-CT results showed that time-dependent bone growth can be observed in each group,and at each time point,a larger quantity of new bone was found in the P group than that in the control group,and more bone formation with more compact bone was found in the B+P group than that in the B group.At 8 weeks and 12 weeks,there appeared differences in the percentages of bone volume in the bone defects between the P group and the control group,and between the B+P group and the B group.The values of mean bone trabecula number at 12 weeks was significantly larger than the control group(p < 0.05),and conversely,the mean bone trabecula spacing in the P group was significantly lower than the control group(p < 0.05).The result of toluidine blue staining showed that the invasion images of fibrous connective tissue with different degrees were observed in the control group and the B group,while in the P group and the B+P group,favourable osteogenesis trend that the leading bone edge stretched forward exactly along the bone surface of AP,the top edge was smooth and intact,and the bone remolding speeds were better than other two groups.Conclusions: 1.The modified decellularization protocol could effectively remove the cell components in the AP materials with good retention the extracellular matrix,the water absorption property got better due to the pore within AP became bigger,but the mechanical properties were not significantly influenced.2.AP presented good biocompatibility,it could not only promote MC3T3-E1 cell adhesion,proliferation and osteogenesis differentiation,but also would not induce severe immune inflammatory response with xenogeneic implantation.3.AP could protect the growth of bone tissues in the bone defects from the invasion of those fibrous connective tissues,promote the growth of bone tissues,and play an active role in guided bone regeneration. |
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