SUMMARY
Objective
To develop a high-performance liquid chromatography (HPLC) assay for insulin-like growth factor I (IGF-I) and to use it to quantify elution of IGF-I from polymerized fibrin in an in vitro system.
Sample Population
Equine fibrinogen and calcium-activated bovine thrombin were used to form fibrin containing human recombinant IGF-I.
Procedure
Multiple fibrin disks were formed from polymerized fibrinogen and thrombin; 6 disks were loaded with 25 μg of recombinant human IGF-I at the time of polymerization, and 4 remained as unladen controls. The resultant clots were incubated at 37 C and 90% humidity for 22 days. Phosphate-buffered saline solution in each well was replaced daily, and IGF-I content was assayed by HPLC. Solid-phase separation was used to assay free IGF-I peptide peaks. A scan was done to determine optimal wavelength for IGF-I absorbance. Commercially pure IGF-I was used to construct a standard curve, and the IGF-I content of medium removed from all 10 wells each day was assayed.
Results
Pure unbound IGF-I eluted from the fibrin polymers for 22 days; initial rapid daily release of 1.6 to 1.7 μg of IGF/ml of medium changed after day 3 to commence an asymptotic decrease to 110 ng of IGF/ml by day 22. The fibrin disks had dissolved by day 22, and the experiment was terminated. Control disks did not have detectable IGF-I content at any time. Limit of the HPLC assay was 25 ng of IGF-l/ml. Retention time for nonprotein-bound IGF-I was 10.3 ± 0.15 minutes.
Conclusion
IGF-I (25 μg) can be added to polymerized fibrin and eluted as free ligand over a 22-day period of culture at 37 C. Release of IGF-I was initially independent of fibrin dissolution, but later appeared to follow a pattern consistent with fibrin degradation.
Clinical Relevance
IGF-I can be incorporated as a depot form in polymerized fibrin and is released over time in sufficient concentration to effectively stimulate articular chondrocyte metabolic activity. (Am J Vet Res 1997;58:1431–1435)
