In Vitro Studies On The Seeding Cells For Anterior Cruciate Ligament Tissue Engineering

Anterior cruciate ligament (ACL) is an important structure for the knee stability. Poor healing capacity of the ACL after injury has led to necessity of reconstruction with various grafts under arthroscope in most of cases. However, those graft materials own varied disadvantages. Tissue engineering may potentially provide solution for these problems. Seeding cells, scaffold and tissue construction are three essential elements of tissue engineering. Selecting seeding cells is nowadays one of difficult subjects for ligament tissue engineering. In these studies, we have observed histology and cytology of normal ACL in rabbit model first. Then comparison of four different body cells (anterior cruciate ligament cell, skin fibroblast, Achilles tendon cell and medial collateral ligament cell) has been done by cell growth, proliferation and collagen secretion of type I and type III. Lastly, in vitro induction and differentiation from bone marrow stromal stem cells into fibroblasts was undertaken in attempt to provide another cell source for seeding cells in ACL tissue engineering.Partâ… : Histological and cytological observation on anterior cruciate ligamentObjective : To observe the characteristics of anterior cruciate ligament(ACL)at proximal, middle and distal region in rabbit by histological study, to identify cell isolation culture and observe biological features of ACL cells in vitro. Methods: Twelve ACLs were harvested from mature New Zealand rabbits. The specimens were examined under microscope with standard hematoxylin-eosin, toluidine blue and Sirius red staining respectively. Differences at proximal, middle and distal region were addressed. ACL cells were isolated and cultured by tissue pieces and enzyme digestion. Cells in each passage were observed under phase-contrast light microscope, growth curve were descripted, and type I collagen was detected by immunohistochemistry staining. Results: Spindle-shape fibroblasts with short-rod shape nuclei were found at the middle region and aligned along with the bundles of collagenous fibers. Toluidine blue staining revealed negative. In contrast, cells at the proximal or distal region showed round shape with ovoid or round nuclei and Toluidine blue staining was positive. Sirius red staining was positive at proximal, middle and distal region. Cells, which were isolated by enzyme digestion, adhered to wall faster, but proliferation slowed down and declined in expression of collagen type I after 4th passage obviously. Conclusions: our study suggests that the ACL has different histological and cytological characteristics at proximal, middle and distal region, this distinct cellular distribution implies that construction of ACL like a natural one is impossible by any techniques. Enzyme digestion is efficient method to isolate seeding cells from tissue and P1-P4 ACL cells can be used as seeding cells in ACL tissue engineering.Partâ…¡: Preliminary study on selection of seeding cells for anterior cruciate ligament tissue engineeringObjective: The aim of this study was to determine the optimal cell source for anterior cruciate ligament (ACL) tissue engineering. Methods: Fibroblasts were isolated and cultured from anterior cruciate ligament, medial collateral ligament (MCL), Achilles tendon (AT) as well as skin and compared by the following parameters in vitro: proliferation rate, collagen excretion and expression of collagen type I and III. Results: Four kinds of cells grew well in DMEM supplemented with 10% calf serum and demonstrated similar cell morphology. Skin fibroblasts (SK) owned the highest rate in proliferation and followed by AT, MCL and ACL cells. ACL, MCL and Skin fibroblasts were positively stained with antibodies for type I and III collagen. AT fibroblasts were well stained with antibodies for type I collagen while negatively stained with antibodies for type III collagen. The staining densities of collagen type I and III of SK were the highest. Conclusions: This study shows that skin fibroblast has some superiority over ACL, MCL and AT fibroblast regarding to proliferation and expression of collagen type I and III, and also skin fibroblasts are easily to be harvested. Therefore, skin fibroblasts are recommended to be potentially a better cell source for ACL tissue engineering than ACL, MCL or AT fibroblast.Partâ…¢: Epidermal growth factor inducing fibroblast differentiation from rabbit bone marrow stromal cells in vitroObjective: To investigate the effect of epidermal growth factor on proliferation and differentiation of rabbit bone marrow stromal stem cells (BMSCs) into fibroblasts in vitro for ideal seeding cells of ligament tissue engineering. Methods: After inducing and culturing with 2ng/ml, 20ng/ml EGF and a control group without EGF, BMSCs were determined by the utilization of morphological observation, MTT assay and immunohistochemistry. Results: On day of 7th or 14th, the group with EGF developed a more homogeneous fibroblast-like, spindle-shaped morphology with long cell processes, and reached higher cell densities. Groups with EGF had higher rate in proliferation. On day of 10th, EGF groups and control group were positively stained with antibodies for type I and III collagen, but the staining densities type I and III collagen were higer in treatment groups, there was no difference between two EGF treatment groups. Both the EGF group and control group were negatively stained with antibodies for typeâ…¡collagen. Conclusions: EGF has the capacity to stimulate cell proliferation and expression of extracellular matrix proteins for the tissue engineering of tendons and ligaments. But there is no difference between 2ng/ml to 20ng/ml doses.According to our three parts of experiment, we have found a suitable method of isolation and culture of ACL cells, demonstrated biological features of ACL cells in vitro, selected an ideal cell source of ACL tissue engineering, detected the effect of different dose epidermal growth factor on proliferation and differentiation of rabbit bone marrow stromal stem cells (BMSCs) into fibroblasts and provided some basic references for tissue engineering ligament construction.