Characterization of Cells for Use in Ligament Tissue Engineering

For potential tissue engineered anterior cruciate ligament (ACL), ACL fibroblasts, medical collateral ligament (MCL) fibroblasts (Lin et al, 1999), mesenchymal stem cells (MSCs) and embryonic stem cells all can serve as cell source theoretically. As ethical debate on embryonic stem cells continues (McLaren, 2001), mesenchymal stem cells (MSCs) are attracting more attention. Not only they have the potential to differentiate into a variety of mesenchymal cell types, including myoblasts and fibroblasts (Pittenger et al, 1999), but also they can easily been obtained by simple aspiration of the anterior superior iliac crest and be amplified in vitro to large amount (Jiang et al, 2002 and Colter et al, 2000). In current study, collagen assay and proliferation of ACL fibroblasts, MCL fibroblasts and MSCs were compared as possible cell sources of tissue engineered ACL. METHODS Harvest and Culture of MSCs, ACL Fibroblasts and MCL Fibroblasts Under general anesthesia, 2-3ml of bone marrow was aspirated from iliac crest of male NZW rabbit (2.2-2.5kg). After centrifugation and wash, cells were cultured in DMEM (Sigma, Ph 7.4) supplemented with 10% FBS (GIBCO, 10270-106), 10,000U/ml penicillin/10,000μl/ml streptomycin and 2mM L-Glutamine (Gibco). Hemopoietic cells were removed by medium changing. Immediately after harvested, ACL and MCL were carefully cut into 1mm×1mm indices and digested with 5ml 0.25% collagenase (Gibco) for 6 hours followed by twice DMEM rinse. Then the systems were incubated at 37o with 5% CO2 until 80% confluence while medium were changed at three days’ interval. Proliferation Assay 200,000 MSCs (Passage 1 or 2) in 5ml of DMEM with same supplement as before were cultured in 25cm flask until 80% confluence. MSCs were trypsined and counted again. Cell doubling times were calculated from formula, TD=t·lg2/ (lgN t-lgN0) in which N0 and Nt meant primary cell number and acquired cell number respectively. Collagen Assay 50,000 cells were loaded in one well of 24-well plate. After 24hrs, medium was changed with 0.8ml of DMEM supplemented with 5% FBS, 10,000U/ml penicillin/10,000μl/ml streptomycin and 2mM LGlutamine (Gibco). Collagen assay were performed according to user manual of Sircol collagen assay kit (Biocolor, UK). Briefly, collected supernatant were centrifuged at 1,500rpm for 4 min to drop extracellular matrix, followed by mixing 100μl supernatant with 1ml of Sircol dye for 30 min and centrifuging at 10,000rpm for 5min to drop formed collagen-dye complex. After decanting suspension, droplets were dissolved in 1ml Sircol alkali reagent and vortexed. 100μl acquired solution were read at 540nm. Immunohistochemistry Cells on chamber slide (Iwaki) were stained with collagen I, II, III and smooth muscle actin antibodies (Sigma), using Ultravision Detectin system (Lab Vision Corporation). RESULTS Proliferation The cell number of ACL fibroblasts and MCL fibroblasts acquired from digestion were not stable. From 3 month old male NZW rabbits, 200,000-500,000 fibroblasts could be acquired after 10-14 days’ culture with current technique. However, after passage, they deteriorated in no time and were hinted by obvious morphological change and decreased proliferation. Starting page #: 1119 2 After 18-20 days’ culture, 1.5-3m MSCs could be acquired with typical fibroblast-like morphology. P1 MSCs kept previous fibroblastlike morphology and doubled in 50 hours. P2 (25 day) and P3 (30days) MSCs stopped to proliferate as whole with obvious morphological changes. MSCs were positively stained with collagen type I, III and asmooth muscle actin which existed in ACL and MCL fibroblasts. Collagen assay P3 MSCs’ collagen excretion decreased slightly when compared with P1 and P2 MSCs, while there is no difference of collagen excretion between P1 and P2 MSCs [Figure 1]. ACL fibroblasts and MCL fibroblasts have obviously lower collagen excretion than P1 MSCs [Figure 2]. Collagen assay of P1, P2 and P3 MSCs