| [Backgrounds]A variety of materials can be found for the reconstruction of the knee cruciateligament injury, but they all have their own advantages and disadvantages.(PET)Artificial ligament is a kind of graft material which has good applicationprospects for its outstanding capability such as good mechanical property, easy to getand handle and rapid postoperative rehabilitation, etc. The question is that it mayinfluence the long-term treatment effect of the cruciate reconstruction if the PETartificial ligment couldn’t reach the natural healing of the bone-to-tendon structure inthe bone tunnel. According to the report, the short-term effect of LARS artificialligament appears good in the cruciate reconstruction, but its long-term effect seemsunclear. LARS artificial ligament is a kind of inert material which can matter a lot inthe long-term effect of cruciate reconstruction if it can reach a biological healing inthe bone interface. In this study, we use the polypeptide to surface modify PETmaterial, in order to promote the histocompatibility between PET material and bodycells,and then further observe its in vivo biological healing in the bone interface andexplore the feasible biological healing of PET artificial ligaments in the boneinterface.[Objectives]1、Using retrospective analysis to compare the change of bone tunnel before and aftercruciate ligament reconstruction using LARS artificial ligament and find the possiblefactors that cause this change，and then explore the combination of artificial ligamentsand bone interface,thus laying a foundation for further study of the artificial ligamentin tendon bone healing.2、To surface modify PET material using “GRGDSPCpolypeptide” and try to find a simple method of operation which can make it possiblefor GRGDSPC polypeptide and PET material bound with each other covalently.3、Toco-culture polypeptide surface modified PET material and the mesenchymal stemcells in vitro and expore whether the PET material can achieve such biocompatibilitywhen modified with GRGDSPC polypeptide.4、To analyze the in vivo performanceof the “polypeptide surface modified PET material” and observe its biological healingin the bone interface, in order to provide a basis for its future clinical application. [Methods]1.43patients (31males and12females,17-48years old, average age27.5years) whoaccepted anterior cruciate ligament reconstruction using LARS artificial ligamentfrom August2004to April2007were followed up for3years. X-ray and CTexamination were given at1,3,6,12,24and36months respectively after surgery tomeasure the tibial and femoral tunnel width. Lysholm knee score scale was used forknee functional evaluation. We examine the anterior and posterior stability of the kneewith KT-1000.2. H2SO4and KMnO4, and NaOH alkaline hydrolysis methods were applied to treatthe PET material. The treated PET materials and untreated PET materials werecombined with different solubility of GRGDSPC polypeptide solution, using Fouriertransform infrared spectroscopy and X-ray diffraction analysis technique to analyzethe content of polypeptide binding and its covalent binding effect.3. Surface modification of PET material and untreated PET material were co-culturedwith bone marrow mesenchyma stem cells. We use scanning electron microscope toobserve cell adhesion and proliferation. And blood cell plate count method wasapplied to the determination of the adhesion rate of cells. Meanwile, cell alizarin redstaining and alkaline phosphatase were applied to identify the bone marrowmesenchymal stem cells osteogenic differentiation.4. New Zealand rabbits were selected to the establishment of the animal models withanterior cruciate ligament injury. The surface modification of PET material anduntreated PET material were implanted in animals in different time, respectively. HEstaining and scanning electron microscopy was used to observe the interfacecombination of PET material with bone tunnel.[Results]1. Follow-up was36to49months, with an average of39.5months. The results wereevaluated according to the grade method of Peyrache et al（grade0:<2mm, grade1:2-4mm, grade2:4-6mm, grade3:≥6mm）. Bone tunnel enlargemengt wereabserved only in three cases as grade1, which were occurred only in the femoraltunnel near the level of articular surface six months after surgery. The average tunnelexpansion was (2.5±0.3) mm.40cases were evaluated as grade0. The averagetunnel expansion in tibial were (0.8±0.3) mm, and (1.1±0.3) mm in femorl tunnel.There was no statistical significant difference of bone tunnel variation at differentperiod (P>0.05). the measure results of X-ray and CT examination were quite the same. At the last follow-up, the Lysholm score and KT-1000anterior translation valueof the3cases were (94.5±4.6) and (1.5±1.1) mm, respectively, and the40grade0cases were (95.6±4.8) and (1.4±1.5) mm, respectively.2. The method Using NaOH alkaline hydrolysis to treat PET materials is simple andreliable, and efficient. Apply H2SO4and KMnO4to treat PET material can not becombined with GRGDSPC polypeptide covalently. The combined effect ofGRGDSPC polypeptide with the PET material treated by the alkaline hydrolysis isexcellent. FTIR result: the covalent binding efficiency of material and the GRGDSPCpeptide with concentration of7.5mg/ml was much better. And the increase of theconcentration of GRGDSPC peptide did not promote its covalent binding efficiency.XPS result: the nitrogen consistent of7.5mg/ml GRGDSPC polypeptide surfacemodified PET material was2.43%, while the nitrogen consistent of the untreated PETmaterial mixed with GRGDSPC peptide with the concentration of7.5mg/ml,12mg/ml and3mg/ml was1.99%,1.96%, and1.42%, respectively.3. Bone marrow mesenchymal stem cells can achieve successful separation,subculture, cryopreservation, recovery and induced to differentiate into osteoblasts.Early adhesion, proliferation and cell adhesion rate of bone marrow mesenchymalstem cells cultured with PET material were improved with time for each group.48hours after the co-culture, the cells adhesion rate of PET materia with GRGDSPCpolypeptide surface modification was significantly higher.4. After the establishment of the animal models with anterior cruciate ligament injury,PET material with various treating conditions was implanted in vivo. As modelingtime prolonged, the effect of PET group with surface modification of GRGDSPCpolypeptide was excellent. The material and tissue are linked closely together, andcombination of tissue was significantly better. HE staining demonstrated that PETmaterial with surface modification of GRGDSPC polypeptide has a large number offibroblast cell adhesion and proliferation, while untreated PET material is only a smallamount of cell adhesion and growth.[Conclusions]Actually, the expansion of the bone tunnel is not obvious after cruciate reconstructionusing LARS artificial ligament, which indirectly indicate that artificial ligamentdoesn’t experience the “tissue necrosis” and “bone tunnel remodeling” phases asautograft does. The truth about how artificial ligament bind to bone tunnel stillremains unclear, but it is significantly important to further study the factors that promote biological healing of artificial ligaments in bone interface. NaOH Alkalinehydrolysis method is simple, reliable, and efficient and it makes it possible forGRGDSPC polypeptide and PET material bound with each other covalently. Thepolypeptide surface modified PET material can significantly increase the material andcell adhesion and proliferation. In vivo experiments prove that surface modified PETmaterial can bind in the bone interface more tightly. But it still remains longer in vivotest to make clear of the true nature about this binding process and see whether it canachieve the natural bio-binding. Meanwhile, the follow-up study of clinical casesneeds to be continued. The10-year follow-up results of the LARS artificial ligamentreconstruction are especially worth the wait. |
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