| Polymethylmethacrylate(PMMA),the most common bone cement,has been used as graft substitutes in orthopedic surgeries such as vertebroplasty.However,undesirable minor crack in the bone-cement interface provoked by shrinkage during polymerization and high elastic modulus of conventional PMMA bone cement dramatically increase the risk of vertebral body re-fracture post-surgery.In order to address these problems,various methods of PMMA modification or improvement have been reported.As a superabsorbent polyelectrolyte,polyacrylic acid(PAA)comprises of abundant intramolecular hydrophilic groups.In addition,PAA has been widely used in tissue engineering for owing to its little antigenic reaction in vivo.Our previous studies showed that the PMMA cement had volumetric swelling and lower elastic modulus after blending with PAA.However,this kind of cement was consist of two components.The nonuniformity of water absorption can result in limited volumetric swelling.So the research work became more focus on how to make further optimization on the form of the cement?to improve the volumetric swelling and to reduce the elastic modulus.Acrylic acid(AA)has the same structure with methylmethacrylate(MMA),which is the carbon-carbon double bond in its molecular formula.These two monomermoleculecan be polymerized by opening thecarbon-carbon double bondtoformanewcopolymer;Polystyrene(PSt)has a wide range of applications due to its favorable mechanical performance and adequate chemical inertness that can improve the polymerization and the strength of the polymer.There were studies reported that the homopolymerization of St and methyl methacrylate(MMA)could be successfully obtained from their monomers.In this study,we successfully synthesized hydrophilous expandable bone cements based on PMMA commercial cement,AA,and styrene(St).Four major aspects have been compared between the new cements and PMMA commercial cement: physicochemical properties?mechanical capacity?biological properties? and the injection of cements in the rabbit medialfemoralcondyle cavity defect model to analyze the different bone-cement interfaces.Therefore,the newly synthesized cements were evaluated for bone tissue engineering and OVCF therapeutic applications.Objectives To compare physical properties of two new cements with PMMA commercial cement in polymerization process and investigate the influences of new cements on polymerization;to analysis the variation in mechanical capacity of new cements;to detect the differences in biocompatibility of three different cements with Sprague-Dawly rat-derived osteoblasts and to observe the proliferation and attachment of primary osteoblasts on three different cements;to evaluate the expression of osteogenesis-specific genes on three different cements;to analyze the different bone-cement interfaces and osteogenic capacities by the injection of three different cements in the rabbit medialfemoralcondyle cavity defect model,and to provide the evaluationbasis for newly synthesized expandable bone cement.Methods 1.Physicochemical properties analysis and study on mechanical capacity of newly synthesized expandable bone cements(1)The drainage method was used to determine the change in volume expansion(VE)of the cements.(2)The setting time?setting temperature and doughing time was detected according to standard ISO5833.(3)The compression strength and modulus of the cement were determined by ISO5833.2.Biological characteristics analysis of newly synthesized expandable bone cements(1)The cell proliferation in three different cement extracts was assessed using the Cell Counting Kit-8 assay.(2)Flow cytometry was used to evaluate apoptotic rate of primary osteoblasts in three different cement extracts.(3)The morphology and proliferation of primary osteoblasts on 3-D cement discs were investigated using SEM.(4)Western blotting for protein expression analysis of osteogenesis-specific genes(ALP?Collagen I?Runx2?OC/BGLAP)was conducted on SD rat-derived osteoblasts from both PMMA and new cements films.(5)RT-PCR analysis of osteogenesis-specific genes(ALP?Collagen I?Runx2?OC/BGLAP)was conducted on SD rat-derived osteoblasts from both PMMA and new cements films.3.Cement injection into rabbit femur with cavity defect The New Zealand rabbit medialfemoralcondyle cavity defect model was used to investigate the efficacy of PMMA and new expandable bone cement in filling the defect and promoting repair.27 male New Zealand rabbits(2.0-2.5kg)were divided into three time groups randomized.A radiographic examination consisting of the lateral side of the right femur of each rabbit was carried out after 1 week of cement injection.At post-injection weeks 1,4,and 12,the rabbits were sacrificed,and the right femur with the cement was harvested.The extractedfemoralcondyle was fixed in 4% paraformaldehyde with surrounding soft tissue cleaned.The sections perpendicular to the implants were sliced and stained with Ponceau S to assess the capacity of cements filling in vivo and the formation of the bone-cement interface.Results 1.As compared to the commercial PMMA(expandingrate:-6.5±0.4%),the expandingrate of the new copolymer(PMMA-PAA and PMMA-PAA-PSt)exhibited excellent volumetric swelling of 15.2±0.3% and 87.5±0.5%,respectively;The new copolymer exhibited prolonged doughing time(PMMA-PAA: 9.3±0.2 min;PMMA-PAA-PSt: 11.3±0.3 min),setting time(PMMA-PAA: 49.9±0.9 min;PMMA-PAA-PSt: 55.6±0.8 min),and setting temperature(PMMA-PAA: 46.2±0.5 °C;PMMA-PAA-PSt: 78.9±0.5 °C)than PMMA cement(P<0.05);Compared with PMMA cement(compressive strength: 85.6±5.2MPa,elastic modulus: 1846.0±201.5 MPa),the compressive strength of PMMA-PAA and PMMA-PAA-PSt cements reduced to 58.9±3.1 MPa and 57.4±5.0 Mpa,respectively;the elastic modulus was reduced to 1306.4±118.8 MPa and 1468.2±275.8 Mpa,respectively.The differences between PMMA and new cement were significant(P<0.05);however,there were no differences in the compressive strength or elastic modulus between PMMA-PAA and PMMA-PAA-PSt cements(P>0.05).2.CCK-8 assay showed that the rates of cell proliferation in the PMMA-PAA and PMMA-PAA-PSt cement extracts were increased significantly as compared to the PMMA cement on day 1?4?7(P<0.05);The apoptotic rate of primary osteoblasts in extracts from the PMMA-PAA and PMMA-PAA-PSt cements was significantly lower than that from the PMMA cement(P<0.05).The rate of apoptosis of the primary osteoblasts cultured in PMMA cement extracts was 3.1±0.4% on day 1 and 8.6±1.5% on day 3.In the PMMA-PAA cement extracts,2.4±0.2% of the cells were apoptotic on day 1 and 6.7±0.8% on day 3.In the PMMA-PAA-PSt cement extracts,1.4±0.1% of the cells were apoptotic on day 1 and 3.2±0.3% on day 3.This result was in accordance with those by CCK-8 analysis;SEM revealed that the newly synthesized PMMA-PAA-PSt 3-D cement discs had a specific porous/coralloid morphology.The cells associated with PMMA-PAA and PMMA-PAA-PSt cement discs showed an excellent growth,including elongated,spindle-shaped morphology,and intercellular connections.In contrast,the primary osteoblasts exhibited a poor attachment on PMMA cement discs without spreading;Western blotting analysis showed that on day 3 the protein expressions of Collagen I?Runx2 in PMMA-PAA group were higher than in PMMA group(P<0.05),the expressions of ALP ? Collagen I ? Runx2 in PMMA-PAA-PSt group were higher than in PMMA group(P<0.05);on day 7 the expressions of ALP?Collagen I in PMMA-PAA group were higher than in PMMA group(P<0.05),the expressions of ALP?Collagen I?OC/BGLAP in PMMA-PAA-PSt group were higher than in PMMA group(P<0.05);RT-PCR analysis indicated that on day 3 the gene expression of Runx2 in PMMA-PAA group was higher than in PMMA group(P<0.05),the expressions of ALP?Collagen I?Runx2 in PMMA-PAA-PSt group were higher than in PMMA group(P<0.05);on day 7 the expressions of ALP?Runx2 in PMMA-PAA group were higher than in PMMA group(P<0.05),the expressions of ALP ? Collagen I in PMMA-PAA-PSt group were higher in than in PMMA group(P<0.05),these results suggest that the new cements' films exhibited a tendency to enhance the expression of osteogenesis-specific genes.3.X-ray examinations performed after 1 week cement injection indicated that each cement sufficiently filled the femur cavity defect without fracture or cement leakage.Ponceau S staining assessed the bone-cement contact at 1,4,and 12 weeks and showed that a minor crack was observed at the interface of the PMMA cement at 1 and 4 weeks after implantation.In contrast,direct and cohesive contact with the bone was confirmed at 1,4,and 12 weeks after implantation in the PMMA-PAA and PMMA-PAA-PSt cement groups.On the other hand,as the implantation period increased to 12 weeks,osteogenic-positive staining was observed inside the PMMA-PAA and PMMA-PAA-PSt cement in the proximity to the bone-cement contact.However,no evidence of osteogenic behavior was found in the PMMA cement.Conclusion 1?The newly synthesized PMMA-PAA and PMMA-PAA-PSt cements showed excellent volumetric swelling in vitro with low elastic modulus.2?In comparison to the PMMA commercial cement,PMMA-PAA and PMMA-PAA-PSt cements exhibited a significant low cytotoxicity.Moreover,PMMA-PAA-PSt cement supplied a specific porous/coralloid surface for osteoblasts attachment and proliferation;The newly synthesized PMMA-PAA and PMMA-PAA-PSt cements exhibited a tendency to enhance the expression of osteogenesis-specific genes.3?We also injected the cement into rabbit medialfemoralcondyle cavity defect and revealed direct and cohesive contact with a bone and osteogenic capacity with respect to new cements in contrast to a minor crack in the PMMA cement. |
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