OBJECTIVE: To investigate a cryophylactic agent (CPA) to protect tissue engineered tendon (TET) in deep low temperature. METHODS: Sixty-four BALB/C inbred nude mice were chosen, which included 4 as blank control group, left sides of 60 as experimental group and their right sides as control group. Transformed human embryonic tendon cells of the 54th passage and artificial materials of carbon fiber (CF) and polyglycolic acid (PGA) were co-cultured in vitro to construct TET. TET was frozen in liquid nitrogen with four kinds of CPA (groups A, B, C, and D) for 2 months. They were thawed quickly and transplanted into hind limbs of nude mice to repair the defects of Achilles tendon, which was 5 mm in length and 65.7% of total Achilles tendon. In control group, no cryopreservation treatment was taken. The morphological, histological, ultrastructure, and immunohistochemistry examinations were made and short tandem repeat loci were detected 2, 4, 6, 8, and 12 weeks later. RESULTS: In the experimental group, the morphological properties of tendon cells resumed gradually and the capability of synthesizing collagen enhanced by degrees. Tendon cells survived and could secret type I collagen and there was less difference between experimental and control groups 12 weeks after transplantation. In group A, vacuole in mitochondrion of tendon cell decreased, tendon cell arranged in order and abundant collagen fibers were found and linked. CONCLUSION: The cryopreservation agent in group A can protect TET in deep low temperature.
OBJECTIVE: To observe the proliferation and differentiation properties of primary human embryonic skeletal myoblasts cultured in vitro. METHODS: The skeletal muscle samples were obtained from 20 to 25-week abortion fetus, the family history of inherited myopathies of parental generation was negative. With a modified method of Blau, the muscle sample was digested with trypsin and collagenase. The isolated cell suspension was a mixture of myoblasts and fibroblasts, the latter was removed by repeated attachment to culture dishes. The morphological, immunohistochemical observation, the proliferation and differentiation of primary myoblasts were studied. RESULTS: The isolated myoblasts were spherical in cell suspension and spindle-like after attached to culture dishes. The myosin specialized immunohistochemical staining was bly positive. A large quantity of skeletal muscle specialized creatine kinase (CK-MM) was synthesized in cultured myoblasts. Additionally, while the cell density of myoblasts increased, the monocyte myoblasts would fused to form multinucleated myotube. All those indicated that the cultured cells were myoblasts. Primary myoblasts proliferated quickly, the doubling time, measured in growth curve, was 4.8 days. CONCLUSION: A large number of myoblasts can be available with digestion and repeated attachment method. The cultured cells can be proved as myoblasts by morphological and immunohistochemical detection. The cultured myoblasts have good ability of proliferation and differentiation.