The poor healing abilities of the anterior cruciate ligament (ACL) in contrast to those of the medial collateral ligament (MCL) are well known. Different intrinsic properties of the constituent cells of these ligaments have been proposed to be one of the factors in the differential repair mechanisms. To examine this hypothesis, we have established primary cell lines of ACL and MCL from the tissue explants of approximately similar dimensions and have studied their behavior in vitro. The outgrowth of cells from ACL explants was slower than from MCL explants, as shown by the size of the surrounding clusters of cells. Both ACL and MCL cultures exhibited typical fibroblastic morphology. No significant differences were observed in either attachment or growth of cells from the attached explants derived from various segments of ACL and MCL. Growth curves of ACL and MCL cultures at both passage numbers 2 and 6 showed a slower rate of proliferation of ACL cells than MCL cells (p less than 0.005). DNA synthesis measured in terms of [3H]thymidine incorporation (CPM/10(3) cells) of both log phase (ACL = 607.5 +/- 5.4 vs. MCL = 1356.4 +/- 11.3) and confluent (ACL = 83.0 +/- 3.6 vs. MCL = 189.8 +/- 5.4) cultures, supports the conclusion that differential proliferation rates of these cells exist in culture. FITC-phalloidin staining (for actin) of later passage cultures (P3-P5) showed a spread-out appearance of ACL cells and an elongated appearance of MCL cells. Relatively more stress fibers were seen within ACL cells. SDS-PAGE and Western blot analysis of cellular proteins revealed higher actin (43 kDa) content in ACL cells than in MCL cells. In vitro wound closure assay was performed by creating a uniform wound of 0.6 mm width in the confluent layer of ACL and MCL cultures. By 48 h postwounding, cell-free zones created in ACL cultures were occupied partially by single cells in a nonconfluent fashion. In contrast, the wounded zone in the MCL cultures was almost completely covered by the cells. Results presented in this report demonstrate a lower proliferation and migration potential of ACL cells in comparison with MCL cells. These differences in intrinsic properties of ACL and MCL cells that were observed in vitro might contribute to the differential healing potentials of these ligaments in vivo.

A complex containing polyoma (py) DNA and protein (py complex) was isolated from polyomainfected mouse-cell cultures. The The small size of the polyoma virus genome (about 3 X 106 daltons) makes it an ideal prototype for studies on the mechanism of viral-induced tumorigenesis. Elucidation of the function of each of the proteins coded by the virus would be a great stride toward an understanding of the molecular basis of cancer (1-3). However, the problem becomes more complex when we consider the fact that polyoma infection stimulates cells to synthesize various enzymes and basic proteins that were repressed prior to infection (4-7). These proteins, as well as those coded by the virus, may play a role in the type of response of the cell to infection; i.e., in determining whether the cell is killed or becomes transformed.In an effort to gain information regarding the mechanism of polyoma replication and development, we have taken the approach of isolating those proteins that remain associated with the viral DNA upon extraction from infected mouse-cell cultures. A modified Hirt procedure (8), employing a neutral detergent (Triton X-100) and low salt concentration, is used to separate a polyoma nucleoprotein complex from cellular DNA. The method is quite similar to our method for the isolation of XDNA-RNA polymerase complex from Escherichia coli (9). MATERIALS AND METHODS Infection of cell culturesLarge-plaque polyoma (py) virus stocks were prepared by infection of secondary mouse embryo (ME) or mouse kidney cells growing in tissue culture (10 ,1 1). Non-purified virus was used for most of the experiments. ME, 3T3 Balb/C, or Ts-a-3T3 cells were used as hosts for polyoma virus. They were grown as monolayers in 100-mm plastic tissue-culture plates in Dulbecco's modification of Eagle's medium containing 10% calf serum, except for the 3T3 Balb/C cells, which were grown with 20% calf serum. Plates were seeded such that confluence was reached within 30 hr after seeding.Cells were infected soon after confluence by adding 0.4 ml of virus [10 plaque-forming units (PFU) per cell] to the cell cultures drained free of medium. After incubation (1 hr) at 37°C for adsorption, the cultures were covered with 5 ml of fresh growth medium that contained 1% horse serum. DNA was labeled with [3H]thymidine (5 .sCi/ml, 12-15 Ci/mmol) at the times designated. from Rohm & Haas], 1 ml/plate, was added at room temperature. The plates were gently swirled for 10 min and NaCl was added to a final concentration of 0.2 M. The salt produced noticeable lysis of the cells. The lysate was poured or scraped with a Teflon "policeman" into a polyallomer centrifuge tube and cleared bycentrifugation in the SW39 rotor (Spinco) at 4°C for 30 min at 20,000 rpm in order to pellet the cellular DNA. The supernatant, containing the py DNA, was decanted and stored in a glass vial at 4°C. It is important not to subject the lysate to shearing forces at any stage in order to prevent the release of cellular DNA into the supernatant.In recent experiments, centr...

No abstract

Cellular migration and proliferation are integral aspects of wound healing. An in vitro assay for cellular migration and proliferation using explants of rabbit anterior cruciate and medial collateral ligaments was developed previously. This study presents the effects of serum-free culture medium supplemented with basic fibroblast growth factor, bovine insulin, transforming growth factor-beta 1, and platelet-derived growth factor-B, added either individually or in combination, on cell outgrowth in explants of rabbit anterior cruciate and medial collateral ligaments. Outgrowth was assessed at 3 and 6 days by counting the number of cells surrounding the tissue explants. For explants of both ligaments, cell outgrowth was dependent on the presence of 10% fetal bovine serum or the combination of growth factors. Little outgrowth occurred in explants of either ligament in the presence of basic fibroblast growth factor, transforming growth factor-beta 1, or bovine insulin. Platelet-derived growth factor-B at concentrations of 20 and 100 ng/ml seemed to increase cell outgrowth from medial collateral ligament explants at 6 days. The outgrowth from the explants of both ligaments was much greater in the presence of all four growth factors than the sum of the outgrowth with the individual factors. This synergistic effect was as much as 10-fold and 20-fold for the anterior cruciate ligament explants at days 3 and 6, respectively, but no more than 3-fold for the medial collateral ligament explants at these times. Medial collateral ligament explants exhibited greater cell outgrowth than anterior cruciate ligament explants in 10% serum and in the presence of the four growth factors.

No abstract