BACKGROUND:Periprosthetic joint infection is the most common cause of early failure after total knee replacement.The current methods of preventing periprosthetic joint infection by improving the surface of the prosthesis have limitations to varying degrees.OBJECTIVE:To construct a coating material that can stably improve the surface of the implant,prevent the initial floating bacterial infection of periprosthetic infection,and regulate the bone transfer function around the implant.METHODS:(1)Material preparation:YGF polypeptide(which promotes bone formation),LL-37 polypeptide(with antibacterial properties)and YGF+LL-37 composite peptide were prepared by Fmoc solid phase peptide synthesis technology.The titanium-based materials were immersed in the three polypeptide solutions for 2 hours to obtain YGF coating,LL-37 coating and composite peptide coating coated titanium sheets.(2)In vitro experiment:Uncoated titanium sheets and coated titanium sheets were co-cultured with Escherichia coli(or Staphylococcus aureus)and the colonies were counted by plate method.MC3T3 cells were inoculated on the surface of uncoated titanium sheet and coated titanium sheet,respectively.Alizarin red staining was used to observe the calcium salt deposition on the surface of the material.Western blot assay was used to detect the protein expression of RUNX2,osteocalcin,osteopontin,and bone morphogenetic protein 2.(3)Animal experiment:24 SD rats were randomly divided into three groups:the blank group(n=8)was implanted with uncoated titanium nails in the femoral medullary canal;the control group(n=8)was implanted with uncoated titanium nails in the femoral medullary canal+intra-articular injection of Staphylococcus aureus suspension;the experimental group(n=8)was implanted with composite peptide coated titanium nails in the femoral medullary canal+intra-articular injection of Staphylococcus aureus suspension.After 5 weeks of implantation,micro-CT examination,hematoxylin-eosin staining and immunohistochemical staining of femur specimens were performed.RESULTS AND CONCLUSION:(1)In vitro experiment:Compared with uncoated titanium sheet and YGF coated titanium sheet,LL-37 coated and composite peptide coated titanium sheet could significantly inhibit the growth and reproduction of Escherichia coli and Staphylococcus aureus.Compared with uncoated titanium sheets and LL-37-coated titanium sheets,YGF-coated and composite peptide-coated titanium sheets could promote calcium salt deposition in osteoblasts and increase the protein expression of RUNX2,osteocalcin,osteopontin and bone morphogenetic protein 2.(2)Animal experiment:Micro-CT test showed that the control group had less bone mass than the blank group and the experimental group.Hematoxylin-eosin staining showed that there was a large amount of fibrous tissue around the nail channel in the control group,only a small amount of tissue fibrosis around the nail channel in the blank group,and only a small amount of tissue fibrosis around the nail channel in the experimental group.Immunohistochemical staining showed that the protein expression of interleukin 1β and tumor necrosis factor α in the control group was higher than that in the blank group and the experimental group,and the expression of osteocalcin,RUNX2 and osteopontin in the experimental group was higher than that in the blank group and the control group.(3)The results show that the titanium-based material coated with YGF+LL-37 composite peptide coating has good antibacterial ability and can promote bone transfer around the implant.

BACKGROUND:Periprosthetic joint infection is the most common cause of early failure after total knee replacement.The current methods of preventing periprosthetic joint infection by improving the surface of the prosthesis have limitations to varying degrees.OBJECTIVE:To construct a coating material that can stably improve the surface of the implant,prevent the initial floating bacterial infection of periprosthetic infection,and regulate the bone transfer function around the implant.METHODS:(1)Material preparation:YGF polypeptide(which promotes bone formation),LL-37 polypeptide(with antibacterial properties)and YGF+LL-37 composite peptide were prepared by Fmoc solid phase peptide synthesis technology.The titanium-based materials were immersed in the three polypeptide solutions for 2 hours to obtain YGF coating,LL-37 coating and composite peptide coating coated titanium sheets.(2)In vitro experiment:Uncoated titanium sheets and coated titanium sheets were co-cultured with Escherichia coli(or Staphylococcus aureus)and the colonies were counted by plate method.MC3T3 cells were inoculated on the surface of uncoated titanium sheet and coated titanium sheet,respectively.Alizarin red staining was used to observe the calcium salt deposition on the surface of the material.Western blot assay was used to detect the protein expression of RUNX2,osteocalcin,osteopontin,and bone morphogenetic protein 2.(3)Animal experiment:24 SD rats were randomly divided into three groups:the blank group(n=8)was implanted with uncoated titanium nails in the femoral medullary canal;the control group(n=8)was implanted with uncoated titanium nails in the femoral medullary canal+intra-articular injection of Staphylococcus aureus suspension;the experimental group(n=8)was implanted with composite peptide coated titanium nails in the femoral medullary canal+intra-articular injection of Staphylococcus aureus suspension.After 5 weeks of implantation,micro-CT examination,hematoxylin-eosin staining and immunohistochemical staining of femur specimens were performed.RESULTS AND CONCLUSION:(1)In vitro experiment:Compared with uncoated titanium sheet and YGF coated titanium sheet,LL-37 coated and composite peptide coated titanium sheet could significantly inhibit the growth and reproduction of Escherichia coli and Staphylococcus aureus.Compared with uncoated titanium sheets and LL-37-coated titanium sheets,YGF-coated and composite peptide-coated titanium sheets could promote calcium salt deposition in osteoblasts and increase the protein expression of RUNX2,osteocalcin,osteopontin and bone morphogenetic protein 2.(2)Animal experiment:Micro-CT test showed that the control group had less bone mass than the blank group and the experimental group.Hematoxylin-eosin staining showed that there was a large amount of fibrous tissue around the nail channel in the control group,only a small amount of tissue fibrosis around the nail channel in the blank group,and only a small amount of tissue fibrosis around the nail channel in the experimental group.Immunohistochemical staining showed that the protein expression of interleukin 1β and tumor necrosis factor α in the control group was higher than that in the blank group and the experimental group,and the expression of osteocalcin,RUNX2 and osteopontin in the experimental group was higher than that in the blank group and the control group.(3)The results show that the titanium-based material coated with YGF+LL-37 composite peptide coating has good antibacterial ability and can promote bone transfer around the implant.

Objective:To evaluate the effects of low-frequency ultrasound on antibiotic susceptibility and biofilm formation of methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli).Methods:After MRSA and E. coli were treated with low-frequency ultrasound with different parameters, they were divided into a group with different ultrasound durations and a group with different ultrasound powers. With the power parameter set at 100%, the former was divided into 5 subgroups: control, 1.0 min, 2.5 min, 5.0 min, and 10.0 min subgroups. The bacteria were sonicated for 0, 1.0, 2.5, 5.0, 10.0 min, respectively. The group with different ultrasound powers was also divided into 5 subgroups: control, 25% power, 50% power, 75% power, and 100% power subgroups. The bacteria were treated with ultrasonic powers of 0, 25%, 50%, 75%, and 100% for 5.0 min. The MRSA and E. coli corresponded to antibiotic susceptibility testing using vancomycin and meropenem. The number of bacteria surviving was assessed by colony counts. Confocal microscopy was used to observe the changes in biofilms co-cultured with 1/2 minimum inhibitory concentration (MIC) antibiotics after sonication.Results:The E. coli enhanced its susceptibility to meropenem after 5.0 min of high-power sonication while the susceptibility of MRSA to vancomycin was unaffected. The number of E. coli decreased significantly with increasing ultrasound time and power: the numbers of E. coli in the 1.0 min, 2.5 min, 5.0 min, and 10.0 min subgroups [(51.00±18.73), (30.00±9.17), (5.33±4.04), and (0.23±0.03)×10 4 CFU/mL] were significantly smaller than that in the control subgroup [(120.00±7.81)×10 4 CFU/mL], and the numbers of E. coli in the 25%, 50%, 75%, and 100% subgroups [(25.00±3.00), (8.00±2.65), (5.00±2.00), and (5.33±4.04)×10 4 CFU/mL] were significantly smaller than that in the control subgroup [(120.00±7.81)×10 4 CFU/mL] ( P<0.05). However, the number of MRSA was not significantly affected. After treatment with ultrasound combined with 1/2 MIC meropenem, the ratio of live/dead biofilm areas of E. coli decreased significantly with increasing ultrasound time and power: the proportions of E. coli in the 1.0 min, 2.5 min, 5.0 min and 10.0 min subgroups (66.10%±1.78%, 50.84%±7.99%, 60.98%±2.23%, and 29.20%±16.49%) were significantly smaller than those in the control subgroup (93.73%±0.44%), and the proportions of E. coli in the 25%, 50%, 75%, and 100% subgroups (75.23%±2.21%, 65.10%±1.25%, 57.34%±11.21%, and 60.98%±2.23%) were significantly smaller than that in the control subgroup (93.73%±0.44%) ( P<0.05). However, the MRSA live/dead biofilm area ratio was not significantly affected by the treatment with ultrasound combined with 1/2 MIC vancomycin. Conclusions:Low frequency ultrasound can effectively inhibit the growth of E. coli and significantly enhance its sensitivity to antibiotics, and its combination with antibiotics can inhibit the formation of bacterial biofilm. However, low frequency ultrasound or its combination with antibiotics has no significant effect on MRSA.

Objective:To evaluate the effects of low-frequency ultrasound on antibiotic susceptibility and biofilm formation of methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli).Methods:After MRSA and E. coli were treated with low-frequency ultrasound with different parameters, they were divided into a group with different ultrasound durations and a group with different ultrasound powers. With the power parameter set at 100%, the former was divided into 5 subgroups: control, 1.0 min, 2.5 min, 5.0 min, and 10.0 min subgroups. The bacteria were sonicated for 0, 1.0, 2.5, 5.0, 10.0 min, respectively. The group with different ultrasound powers was also divided into 5 subgroups: control, 25% power, 50% power, 75% power, and 100% power subgroups. The bacteria were treated with ultrasonic powers of 0, 25%, 50%, 75%, and 100% for 5.0 min. The MRSA and E. coli corresponded to antibiotic susceptibility testing using vancomycin and meropenem. The number of bacteria surviving was assessed by colony counts. Confocal microscopy was used to observe the changes in biofilms co-cultured with 1/2 minimum inhibitory concentration (MIC) antibiotics after sonication.Results:The E. coli enhanced its susceptibility to meropenem after 5.0 min of high-power sonication while the susceptibility of MRSA to vancomycin was unaffected. The number of E. coli decreased significantly with increasing ultrasound time and power: the numbers of E. coli in the 1.0 min, 2.5 min, 5.0 min, and 10.0 min subgroups [(51.00±18.73), (30.00±9.17), (5.33±4.04), and (0.23±0.03)×10 4 CFU/mL] were significantly smaller than that in the control subgroup [(120.00±7.81)×10 4 CFU/mL], and the numbers of E. coli in the 25%, 50%, 75%, and 100% subgroups [(25.00±3.00), (8.00±2.65), (5.00±2.00), and (5.33±4.04)×10 4 CFU/mL] were significantly smaller than that in the control subgroup [(120.00±7.81)×10 4 CFU/mL] ( P<0.05). However, the number of MRSA was not significantly affected. After treatment with ultrasound combined with 1/2 MIC meropenem, the ratio of live/dead biofilm areas of E. coli decreased significantly with increasing ultrasound time and power: the proportions of E. coli in the 1.0 min, 2.5 min, 5.0 min and 10.0 min subgroups (66.10%±1.78%, 50.84%±7.99%, 60.98%±2.23%, and 29.20%±16.49%) were significantly smaller than those in the control subgroup (93.73%±0.44%), and the proportions of E. coli in the 25%, 50%, 75%, and 100% subgroups (75.23%±2.21%, 65.10%±1.25%, 57.34%±11.21%, and 60.98%±2.23%) were significantly smaller than that in the control subgroup (93.73%±0.44%) ( P<0.05). However, the MRSA live/dead biofilm area ratio was not significantly affected by the treatment with ultrasound combined with 1/2 MIC vancomycin. Conclusions:Low frequency ultrasound can effectively inhibit the growth of E. coli and significantly enhance its sensitivity to antibiotics, and its combination with antibiotics can inhibit the formation of bacterial biofilm. However, low frequency ultrasound or its combination with antibiotics has no significant effect on MRSA.