The effect of methotrexate on mouse bone cells in culture.

OBJECTIVE: We have recently shown that administration of long-term, low-dose methotrexate (MTX) causes severe osteopenia in female rats. This osteopenia is characterized both by decreased osteoblast function without a decrease in osteoblast numbers, and by increased bone resorption that is believed to represent a physiologic remodeling response by osteoclasts. The present study investigates the effects of varying doses of MTX on mouse bone cells in culture. METHODS: Cells were obtained by sequential digestion of neonatal mouse calvariae, and cultured with fetal calf serum (10% for osteoblast-like cells and 2% for osteoclast-like cells). After 1 week, MTX was added to each culture in concentrations of 0.6 microM, 0.4 microM, 0.2 microM, 0.1 microM, 1 nM, and 0.5 nM. All experiments were done on 24 wells for each MTX concentration and for the controls. The effect on osteoblastic cells was assessed, at 7 days, by cell counts and by measurement of lysate alkaline phosphatase and supernatant osteocalcin levels, and, at 21 days, by analysis of the calcified matrix production, which was cultured with ascorbic acid and beta-glycerophosphate. For osteoclastic cells, cell count and lysate acid phosphatase levels were determined. RESULTS: Levels of osteoblastic cells and lysate alkaline phosphatase were not changed by any of the concentrations of MTX. Matrix calcification and supernatant osteocalcin levels were diminished by MTX in a dose-responsive manner. Osteoclast-like cell numbers and acid phosphatase levels were not significantly affected by MTX. CONCLUSION: These results suggest that diminished mouse osteoblastic cell function occurs with very low mean concentrations of MTX, in a dose-responsive manner. The mechanism seems to be inability of the cell to synthesize and calcify matrix, possibly through defective osteocalcin production. Thus, low-dose MTX may have an important impact on bone density by slowing osteoblastic matrix production.

Repair of the tarsoligamentous sling in New Zealand white rabbits using polytetrafluoroethylene graft material.

Repair of large full-thickness lower lid defects requires reconstruction of the tarsoligamentous sling. This may necessitate either a tarsus sharing technique or a skin flap with a free cartilage graft. Obtaining tarsus or cartilage in these procedures has the disadvantage of requiring a second surgical site, which may cause further scarring and deformity. Polytetrafluoroethylene (PTFE) is a nonantigenic, inert, highly biocompatible, mechanically strong synthetic material that has been successfully utilized as a vascular and soft tissue patch since the 1970's. PTFE has been used in ophthalmic surgery to wrap orbital implants and as an interpositional graft for the correction of lower lid retraction. We evaluated the usefulness of PTFE in the reconstruction of the tarsoligamentous sling in 24 lids of 12 New Zealand white rabbits. PTFE with internodal spacings of 10 and 30 microns were used initially. Despite a lack of tissue inflammation, the PTFE grafts were uniformly extruded by 2 weeks postoperatively. Four additional lids were reconstructed using PTFE (30 and 60 microns) coated with a biological substrate. This graft material was also extruded. These results suggest that PTFE material may not be satisfactory for reconstruction of the tarsoligamentous sling.

Proliferating cells of human basal cell carcinoma are located on the periphery of tumor nodules.

Study of the growth characteristics of basal cell carcinoma (BCC), a relatively well-organized, slow-growing skin cancer, has been limited because of the lack of methods for propagation of the tumor off the human host. We have used newly developed techniques for transplantation and propagation of BCC on athymic mice to study [3H]thymidine incorporation by nodular BCC. In human BCCs labeled in vitro immediately after removal from the mice and in vivo on the mice, [3H]thymidine during a 4-h pulse was incorporated primarily by cells on the periphery of tumor nodules (labeling indices 6-24%) rather than by the cells more central in tumor nodules (labeling indices 0-2%). Similar results were also seen when samples of tumor freshly removed from patients were labeled in vitro. We conclude that the dividing cells within nodular BCC are primarily the cells at the edges of tumor nodules and that this characteristic is related to the slow, progressive, invasive growth of BCC.

Transplantation of human basal cell carcinomas to athymic mice.

Basal cell carcinomas (BCCs) obtained from 22 subjects undergoing microscopically controlled surgery were transplanted to 40 athymic (nude) mice. With no further immunosuppression of the mice, no tumor growth was noted in the first 14 attempts. When mice were further immunosuppressed with anti-lymphocyte serum (ALS) injections and by splenectomy, successful tumor growth was achieved in 15 of 22 mice by a subcutaneous implantation technique and in 1 of 4 by a superficial grafting technique. Transplanted BCC retained the morphology and basement membrane proteins typical of human BCC. As determined by autoradiography, 3H-thymidine was incorporated primarily in the peripheral palisaded cells of the transplanted tumor. Successful use of the athymic mouse model for study of human BCC requires use of mice further immunosuppressed by splenectomy and ALS, and the use of a subcutaneous implantation technique. With the use of this model, studies of the biology of human BCC may be possible.

Human tumor cell destruction by distilled water. An in vitro evaluation.

A distilled water lavage is sometimes used during tumor surgery in an effort to kill tumor cells spilled into a cavity or wound. To test the efficacy of this technique, a model study utilized nine different human tumor cell lines, subjected in vitro to hypotonic exposure for 1 to 10 minutes. Only the carcinoid, multiple myeloma, leiomyosarcoma cell lines, and normal lymphocytes were destroyed by the treatment. Although breast, ovarian, gastric, bladder, and melanoma cell lines were damaged to varying degrees, viable cells persisted in all cases. These data suggest that hypotonic shock is not an effective method to kill human tumor cells.