Synthesis of a Kunitz-type serine proteinase inhibitory protein that shares homology with bovine pancreatic trypsin inhibitor by ovine intervertebral disc cells in serum-free alginate bead culture.
The objective of this study was to determine whether disc cells could be cultured under serum-free conditions and whether they synthesized bovine pancreatic trypsin inhibitor (BPTI)-like serine proteinase inhibitory proteins (SPIs) previously demonstrated for ovine chondrocytes. Intervertebral discs from 1- to 2-year-old merino wether sheep were dissected into the annulus fibrosus and nucleus pulposus, and the cells isolated by sequential enzymatic digestion. The cells were grown encapsulated in calcium alginate microspheres under serum-free conditions for 10 days. They remained more than 92% viable as assessed using the vital fluorescent dyes chloromethyl fluorescein diacetate and ethidium homodimer-1 to delineate live/dead cells, respectively. Western and affinity blotting identified a 12-16-kDa media SPI and an additional 34-36-kDa BPTI-like species in solubilized bead samples. This study has demonstrated ovine disc cells synthesized BPTI-like SPIs in serum-free alginate bead culture similar to chondrocyte SPIs; however, the 58-kDa precursor SPI form was not detected suggesting differences in the endogenous processing of these SPIs.
Melrose
James
J
Raymond Purves Bone and Joint Research Laboratories, The Institute of Bone and Joint Research, Department of Surgery, The Royal North Shore Hospital, University of Sydney, St. Leonards, New South Wales, Australia. jmelrose@mail.usyd.edu.au
Smith
Susan
S
Ghosh
Peter
P
D000464:Alginates; D000818:Animals; D007611:Aprotinin / Q000378:metabolism*; D015153:Blotting, Western; D016895:Culture Media, Serum-Free; D020723:Glucuronic Acid; D006603:Hexuronic Acids; D007150:Immunohistochemistry; D007403:Intervertebral Disc / Q000166:cytology* / Q000378:metabolism*; D010455:Peptides; D010940:Plant Proteins; D015842:Serine Proteinase Inhibitors / Q000096:biosynthesis*; D012756:Sheep; D014361:Trypsin Inhibitors / Q000096:biosynthesis*
D000464:Alginates; D016895:Culture Media, Serum-Free; D006603:Hexuronic Acids; C053865:Kunitz-type protease inhibitor, plant; D010455:Peptides; D010940:Plant Proteins; D015842:Serine Proteinase Inhibitors; D014361:Trypsin Inhibitors; D020723:Glucuronic Acid; D007611:Aprotinin