| Anterior cruciate ligament (ACL), as an important part of the knee, plays an important role in maintaining its stability by cooperating with the PCL and the surrounding soft tissues of the knee. Furthermore, ACL is the primary element in the prevention of possible forward movement of the tibia against femur, and part of fibers of ACL also functions in preventing the excessive retrusion and internal and external rotation of the tibia. As the injury of ACL may cause episodic knee instability, chondral and meniscal injury, and thereafter early osteoarthritis, so the reconstruction of the injured ACL is necessary with various grafts under arthroscope. Although every graft materials have their disadvantages, tissue engineering may compensate for these problems. One of the most frequently studied subjects for ligament tissue engineering is about the selection of seeding cells. Currently, four kinds of cells—SF, MCL, ACL and AT—are proposed to be potential candidates of seeding cells. Some studies in vitro have demonstrated that, SF is the best choice because of its better reproductive activity and secretion ability compared with the others. In our studies, we isolated and cultivated the SF for three passages, then marked and traced SF with two different markers: DiI and BrdU. Finally the SF was combined with fibrin sealant. The composite was wrapped around a frozen ACL in situ in the left knee of rabbit and took the right frozen ACL as control. The middle portion of ACL was harvested, observed and analyzed 12 weeks after the operation. We conclude that SF is a ideal cell source for seeding cells in ACL tissue engineering.Part I: Establishment of the injured model of frozen ACL in situObjective: To provide a reliable ruptured ACL model for the study of intraarticular repairing in vivo. Methods: First, the ACL of rabbit was frozen in situ with liquid nitrogen for 1 min. Second, physiological saline solution was injected into the knee joint cavity under room temperature and followed by 3 min's saline sook. Repeat the procedure mentioned above for 3 times. Result: Eight weeks later, ACLs were harvested from mature New Zealand rabbits and the specimens were examined under microscope with standard hematoxylin-eosin and Sirius red staining respectively. The HE showed that no normal shape nuclei was found in frozen ACL and the Sirius red staining revealed obvious increase of the typeⅢcollagen. Conclusion: The method we use can provide a reliable animal model for the study of the intraarticular repairing of injured ACL. So we concluded that the frozen ACL in situ can eliminate all cells.Part II: Separation culture of SF in vitro and the study of bionomical characteristics of SFObjective: To compare two different methods of SF separation culture and observe the bionomical features of SF in order to lay foundation for the later application of SF as seeding cell in ligament tissue engineering. Methods: The culture of tissue block and enzyme digestion was used for the SF separation respectively. Besides, every passage of SF cells was carefully observed under microscope, and the curves of their growth were measured as well. Results: The time of adherence of cells cultivated with enzyme digestion was shorter than that of cells cultivated with tissue block. The growing speed of the cells obviously slowed down after the fifth passage. Conclusion: Enzyme digestion method is proved to be a better way for cell separation and culture in tissue engineering study, and the first to the fourth passage of SF cells can be used as seeding cells in the study of ACL tissue engineering. Part III: Comparative study of BrdU and CM-DiI in theeffectiveness of marking and tracing SF to repaire ruptured ACLObjective: To find an optimal method between BrdU and CM-DiI in marking and tracing SF in vivo in anterior cruciate ligament (ACL) tissue engineering and to observe whether the SF can survive in the intraarticular environment of knee joint or not, so as to explore the feasibility of using SF as seeding cells in tissue engineering ACL. Methods: The Skin-Fibroblasts were isolated from the skin of a rabbit, then marked with BrdU and CM-DiI respectively, and the fluorescence intensity and morphologic changes of 0, 24, 48, 72 h cells were observed under fluorescence inverted microscope. The proliferation statuses of the cells before and after the mark were measured by using MTT colorimetric method. After that, the compound of the marked SF cells and FS was then implanted into the joint cavity of the frozen ACL and wrapped around the frozen ACL. The middle portions of ACLs were harvested 12 weeks after the operation and were analyzed with HE and immunohistochemistry. Results: The marking effects of DiI and BrdU were proved to be effective in vitro and the act of marking the SF did not influence the growth of the SF cells at all. The early morphological feature of SF cells was characterized with ring-shaped fluorescence. Forty-eight hours later, the fluorescent granules of the ring-shaped fluorescence increased and the fluorescence itself enhanced as well with the nuclei showing no fluorescence stain. Seventy two hours later, everything remained the same with no changes. The experiment in vivo showed that the DiI marker has its defect. Under the fluorescence microscope, the collagen fibre emitted light for being activated by the fluorescence. As a result, the DiI coloration could not be distinguished from the background color of the specimens. HE showed that a lot of fibroblasts gathered around the ACL. The immunohistochemistry also proved that the marking effect of SF by using BrdU is better. Meanwhile, the experiment in vivo also indicated that there were lots of conglomerations of positive staining existing in the FS surrounding the ligament, namely, BrdU is able to trace SF within 12weeks. Conclusions: This study reveals that the way of marking skin fibroblast with BrdU has some superiority over that of marking skin fibroblast with CM-DiI. The SF can survive in intraarticular environment of the knee joint and it is potentially an optimal choice as a kind of seeding cells in tissue engineering ACL.Based on three experiments mentioned previously, the author find a suitable method for separating and cultivating SF cells, demonstrate the bionomical characteristics of SF cells in the intraarticular environment of knee joint, explore a reliable method in marking and tracing SF cells in vivo and choose an ideal cell source for ACL tissue engineering in vivo, which lay foundations for further study in the field of tissue engineering ligament reconstruction.
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