Sustained delivery of bioactive cytokine using a dense collagen gel vehicle collagen gel delivery of bioactive cytokine.
OBJECTIVE
The use of cytokines as localized therapeutic agents is limited by the lack of a satisfactory delivery system. The aim of the current investigation was to determine the release kinetics and bioactivity of a simplified cytokine/collagen gel system designed to achieve extended, local delivery of bioactive cytokines at sites of premature cranial suture fusion (craniosynostosis).


METHODS
Cytokine release was determined by ELISA measurements of Tgf-beta3 collected in media. Cytokine bioactivity was determined by measuring the effect of conditioned media, containing released Tgf-beta3, on mink lung epithelial cell proliferation and osteoblast alkaline phosphatase activity. Osteoblast response was evaluated by measuring proliferation of cells cultured on collagen gel containing Tgf-beta3 using an AlamarBlue assay.


RESULTS
Gels loaded with 100 and 500 ng of Tgf-beta3 produced a sustained release over 14 days with a pattern of initial large release followed by a gradual reduction in the amount released over the time. The reduced release over time was correlated to the amount initially loaded. Mink lung epithelial cell assay results indicated that Tgf-beta3 released from the collagen gel retained its bioactivity following incorporation into the collagen gel and release into the media. This bioactivity was further illustrated by a decreased alkaline phosphatase activity measured in osteoblasts cultured on the gels loaded with Tgf-beta3. Osteoblast proliferation assays demonstrated that the collagen gel has an inherent inhibitory effect on osteoblast cell number.


CONCLUSIONS
This collagen gel/cytokine delivery system can retain and release bioactive cytokine over a prolonged period. These results will allow for better optimization of future in vitro and in vivo studies directed at improving the treatment of craniosynostosis.
Premaraj
Sundaralingam
S
College of Dentistry, New York University, 345 E. 24th Street, 9W, New York, NY 10010, USA.
Mundy
Bethany L
BL
Morgan
David
D
Winnard
Phillip L
PL
Mooney
Mark P
MP
Moursi
Amr M
AM
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002452:Cell Count; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D003094:Collagen; D003398:Craniosynostoses / Q000188:drug therapy*; D017077:Culture Media, Conditioned; D003692:Delayed-Action Preparations / Q000008:administration & dosage / Q000493:pharmacokinetics; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects; D005782:Gels; D008168:Lung / Q000166:cytology / Q000187:drug effects; D008907:Mink; D010006:Osteoblasts / Q000187:drug effects / Q000201:enzymology; D014677:Pharmaceutical Vehicles / Q000008:administration & dosage; D051381:Rats; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000493:pharmacokinetics; D012886:Skull / Q000166:cytology / Q000196:embryology; D013997:Time Factors; D053782:Transforming Growth Factor beta3 / Q000008:administration & dosage* / Q000493:pharmacokinetics
D017077:Culture Media, Conditioned; D003692:Delayed-Action Preparations; D005782:Gels; D014677:Pharmaceutical Vehicles; D011994:Recombinant Proteins; D053782:Transforming Growth Factor beta3; D003094:Collagen; D000469:Alkaline Phosphatase