| PartⅠThe effects of a hydroxypropylcellulose coated-PET composite on cell compatibility and osteogenic differentiation in vitroAbstractPurpose:To observe the effects of a hydroxypropylcellulose (HPC) polyethylene terephthalate (PET) composite (HPC/PET group) and uncoated PET (PET group) on cell biocompatibility and the osteogenic differentiation of bone marrow stromal stem cells (BMSCs), anterior cruciate ligament (ACL) fibroblasts, and synovial cell line RSC-364 in vitro.Methods:HPC/PET composite and PET from LARS ligament were cut into round 15.5-mm-diameter slices to fit in 24-well culture plates. BMSCs, ACL fibroblasts, and synovial cell suspension (density 2 × 104/ml) were added to the culture plates. The morphology of the BMSCs, ACL fibroblasts, and RSC-364 cells was observed using an inverted microscope after culturing for 7 days, and the cells were counted at different times. The cell proliferation, adhesion, and osteogenic differentiation of BMSCs in the HPC/PET and PET groups were evaluated using a Cell Counting Kit-8 (CCK-8), the integrin-β1 detection method, and the alkaline phosphatase detection method, respectively, and the proliferation and adhesion of fibroblasts and RSC-364 cells were determined using the CCK-8 and integrin-β1 detection.Results:Scanning electron microscopy showed that the PET ligament fiber surface was covered with a thin coating after the PET surface was modified with HPC, whereas the surface of the pure PET fiber was smooth and clean. Fourier transform infrared spectrometry showed in the HPC/PET group, in addition to these characteristic peaks of PET, HPC hydroxyl stretching vibration at 3600-3200 cm-1 and HPC C-H stretching vibration at 3011-2819 cm-1 were observed. The absorbance at 450 nm (A450) of BMSCs in the HPC/PET group was higher than that in the control group after culture for 6,8, and 10 days using the CCK-8 for detection. The A450 of ACL fibroblasts in the HPC/PET group after culture for 6,8, and 10 days was significantly higher than in the control group. The A450 of RSC-364 cells in the HPC/PET group was significantly higher than that in the control after culture for 8 and 10 days. Scanning electron microscopy showed the morphology and adhesion of BMSCs, ACL fibroblasts, and RSC-364 cells cultured for 7 days. No adhesion of these cells was observed on the PET fiber surface in the control group. By contrast, in the HPC/PET group, BMSCs, ACL fibroblasts, and RSC-364 cells showed stretching and adhesion, and a polygonal three-dimensional morphology. Additionally, there were many synapses on these cells. Western blotting showed that the expression of integrin-β1 by BMSCs, ACL fibroblasts, and RSC-364 cells in the HPC/PET group was significantly higher than that in the controls after 7 days of culture. An enzyme-linked immunosorbent assay (ELISA) showed that the alkaline phosphatase (ALP) activity of BMSCs in the HPC/PET group was higher than that in the control group after culture for 5 and 7 days and significantly higher after 10 and 14 days. The expression of VEGF and osteopontin by BMSCs in the HPC/PET group was higher than that in the control group after culture for 7,10, and 14 days. The expression of BMP-2 and BMP-7 by BMSCs in the HPC/PET group was significantly higher than that in the control group after 7 days of culture.Conclusion:The HPC/PET composite showed no toxicity to osteoblasts in vitro and may significantly improve the compatibility and osteogenic differentiation of BMSCs, ACL fibroblasts, and synovial cells.Part IIHydroxypropylcellulose Coating Enhance Polyethylene Terepthalate Artificial Ligament Graft Osteointegration in bone tunnelAbstractPurpose:The purpose of this study was to determine whether hydroxypropylcellulose (HPC) coating of polyethylene terephthalate (PET) artificial ligaments enhances graft osseointegration in the bone tunnel.Methods:Thirty New Zealand white rabbits underwent artificial ligament graft transplantation in the bilateral proximal tibia tunnels. One limb was implanted with an HPC-coated PET graft, and the contralateral limb was implanted with a non-HPC-coated PET graft as a control. The rabbits were then randomly sacrificed at weeks four and eight after surgery for biomechanical testing, histological examination, and histomorphometric and real-time polymerase chain reaction analysis.Results:Regardless of the implantation duration (four or eight weeks), all samples failed when they were pulled out from the bone tunnel. No PET grafts were ruptured. At week eight after surgery, the mean load to failure of the experimental limbs was higher than that of the control limbs. Likewise, at week four after surgery, there was no statistically significant difference in the stiffness values of the experimental and control limbs. At week eight after surgery, the stiffness value of the experimental limbs was higher than that of the control limbs. At week four after surgery, inflammatory cells infiltrated the graft-bone interface in both the control and HPC-coated groups. Thick fibrous scar tissue had formed at the graftto-bone interface at week four after surgery in both the control and HPC-coated groups. At week eight after surgery, some protruding new bone tissue from the host bone to the graft was found in the HPC-coated group, while a thick fibrous tissue band was observed at the interface between the graft and host bone in the control group. Furthermore, no obvious new bone had formed in the control group. Histomorphometric analysis indicated no difference in the graft-bone interface width between the HPC-coated and control groups at week four after surgery. However, the graft-bone interface width in the HPC-coated group was significantly narrower than that in the control group at week eight after surgery. At week four, the mRNA expression levels of BMP-2 and osteopontin in the HPC group were higher than those in the control group. At week eight, the mRNA expression level of BMP-2 in the HPC group was higher than that in the control group. Furthermore, both the mRNA expression levels of BMP-2 and osteopontin decreased with time.Conclusion:The study has shown that HPC coating on the surface of PET artificial ligament grafts may induce artificial ligament graft osseointegration in the bone tunnel.PART ⅢPreparation and biomechanics of PET artificial ligament in beagle dogsAbstractPurpose:To prepare polyethylene terephthalate (PET) artificial ligament for beagle dog anterior cruciate ligament (ACL) reconstruction and to test the biomechanical performance of the artificial ligament.Methods:The structure and organization of artificial LARS ligament were analyzed. Woven fabrics were sewn using existing equipment, and the tensile properties of fabrics sewn under different conditions were examined. PET artificial ligament fabric samples were cleaned using different methods, and cell growth was observed in every group. Semi-finished artificial ligament products were sewn, and then the final PET artificial ligament for use in beagle dogs was made. The mechanical properties of the artificial ligament were measured using tensile strength, static creep (fatigue performance), and porosity tests.Results:When the longitudinal density of the fabric was 17 rings/cm, the breaking strength, theoretical strength, and strength loss rate of the fabric were the highest, while the breaking elongation rate was minimal. In each group, cells grew well, but the cells grew best using cleaning method No.3. The tensile strength of the PET artificial ligament used for the beagle dogs was 2959.53 ± 7.25 N, the maximum elongation was 12.43 ± 0.95 mm, and the maximum elongation rate was 13.8 ± 1.06%. The final elongation rate of this artificial PET ligament was 0.16% after stretching for 24 hours, whereas the final elongation rate of the artificial LARS ligament was<1.5% after stretching for 24 hours. There was no significant difference in the porosity of the PET artificial ligament fabric used for beagle dogs between the cleaning and no-cleaning groups.Conclusion:PET artificial ligament was prepared for beagle dog ACL reconstruction. The biomechanical performance of this artificial ligament was good, and it was suitable for beagle dog experiments.PART ⅣExperimental study of hydroxypropylcellulose coated-PET artificial ligament for beagle dog anterior cruciate ligament reconstructionAbstractPurpose:This study determined whether hydroxypropylcellulose (HPC) coating of polyethylene terephthalate (PET) artificial ligaments enhances graft osseointegration in the bone tunnel.Methods:Twenty-four adult male beagle dogs underwent anterior cruciate ligament (ACL) reconstruction in the left knee; 12 dogs received HPC-coated PET artificial ligament (HPC group), and 12 received untreated PET artificial ligament (PET group). Dogs were sacrificed randomly 4 and 16 weeks after surgery, and the femur-artificial ligament graft-tibia complexes were harvested for biomechanical, histological, and biomolecular tests.Results:Four weeks after surgery, the mean load to failure was higher in the HPC group than in the controls, and the difference was significant at 16 weeks after surgery. There was no significant difference between the HPC and control groups in stiffness at 4 weeks, but the difference was significant at week 16. The mean load to failure and stiffness increased significantly with time in both groups. Masson staining at 4 weeks after the surgery showed many collagen fibers growing into the artificial ligament in the HPC group, whereas only a small amount was observed in the controls. Sixteen weeks after surgery, much new bone tissue extended from the host bone to the artificial ligament in the HPC group, whereas in the control group, the formation of collagen fibers increased, but there was no obvious new bone. Four weeks after surgery, the expression of vascular endothelial growth factor (VEGF) mRNA was higher in the HPC group than in the controls. Four weeks after surgery, there were no significant differences in osteocalcin and RUNX2 mRNA expression between the HPC and control groups, whereas at 16 weeks after surgery, the osteocalcin and RUNX2 mRNA expression were higher in the HPC group. The expression of BMP-7, COL3, and TGF-β1 decreased gradually with time, whereas the expression of osteocalcin, RUNX2, and VEGF increased gradually with time. Four weeks after the surgery, there was a thin layer of synovial tissue covering the free fibers of the PET artificial ligament in the joint cavity of the HPC group, while there was obvious soft tissue covering the free fiber of the PET artificial ligament in the joint cavity in the control group. At week 16 after surgery, there was a thick layer of synovial tissue covering the free fiber of the PET artificial ligament in the joint cavity in the HPC group and a small amount of synovial tissue in the control group. After 4 and 16 weeks, immunohistochemistry showed that the expression of COL1 in the joint cavity was significantly higher in the HPC group than in the controls.Conclusion:The HPC coating on the surface of PET artificial ligament grafts used for ACL reconstruction in beagle dogs may induce PET artificial ligament graft osseointegration in the bone tunnel.PART ⅤThe design and structure of a new artificial suspensory ligamentAbstractTo overcome existing problems with the fixation of LARS artificial ligament, our research group designed several new artificial suspensory ligaments and has obtained the relevant patents for these. A loop of the new artificial suspensory ligament could be connected with an Endobutton. This design was very convenient, shortened the operating time, and improved the biomechanical strength. Most importantly, this design avoids the use of a compression screw, which is conducive to closer contact between the ligament surface and bone tunnel, increasing bone and ligament contact. The new artificial suspensory ligament should promote graft-bone healing. |
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