Insulin-like growth factor I peptide elution profiles from fibrin polymers determined by use of high-performance liquid chromatography.
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.


METHODS
Equine fibrinogen and calcium-activated bovine thrombin were used to form fibrin containing human recombinant IGF-I.


METHODS
Multiple fibrin disks were formed from polymerized fibrinogen and thrombin; 6 disks were loaded with 25 micrograms 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 micrograms 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-I/ml. Retention time for nonprotein-bound IGF-I was 10.3 +/- 0.15 minutes.


CONCLUSIONS
IGF-I (25 micrograms) 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.


CONCLUSIONS
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.
Foley
R L
RL
Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA.
Nixon
A J
AJ
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods / Q000662:veterinary*; D005337:Fibrin / Q000737:chemistry*; D005340:Fibrinogen / Q000032:analysis; D006736:Horses; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000032:analysis*; D011108:Polymers; D011994:Recombinant Proteins / Q000032:analysis; D013917:Thrombin / Q000032:analysis
D011108:Polymers; D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I; D005337:Fibrin; D005340:Fibrinogen; D013917:Thrombin