Effects of thrombin on the migration of primary osteogenic cells through fibrin-filled scaffolds.

Description

This thesis is directed towards a bone tissue engineering strategy that would maximize osteogenic cell invasion into the bony defect. Through culturing primary bone marrow cells under osteogenic culture conditions on PLGA 3-D scaffolds and examining the interface with SEM, TEM and EDS, a candidate PLGA scaffold was demonstrated to support direct bone contact. In an attempt to stimulate maximal invasion of host bone tissue in vivo, fibrin, the natural migration matrix of the blood clot, was investigated as a candidate scaffold pore-filling matrix. Scaffolds filled with fibrin, polymerized with either a low thrombin concentration (LTF) or a high thrombin concentration (HTF), were implanted into drill hole defects in the distal femurs of rats. The area of bone formed at 2, 5 and 11 days post implantation was determined histomorphometrically. After 5 days, scaffolds filled with HTF had 2.2 +/- 1.2% of their available area occupied by bone compared to 8.9 +/- 3.0% for empty scaffolds (p = 0.045, n = 3--4), but no statistical difference was found between the empty scaffolds and the scaffolds filled with LTF (p = 0.976, n = 3--4) which had 8.1 +/- 1.4% of their available area occupied by bone. However, after 11 days there was ∼50% less bone formed within the fibrin filled scaffolds compared to the empty scaffolds (p = 0.003, n = 3). Given that the thrombin, used to polymerize the fibrin, can also stimulate cell migration, the role of thrombin in osteogenic cell migration was investigated. It was shown, using a scratch wound assay, that thrombin stimulated a 2.1 +/- 0.7 (p = 0.042, n = 7) fold increase in cell migration and a common motogenic factor, PDGF-BB stimulated a 3.0 +/- 0.4 fold increase (p = 0.004, n = 3). When a modified Boyden chamber assay was combined with a bone nodule assay, primary cells that had migrated in the presence of thrombin formed 50% (p = 0.040, n = 7) more bone nodules compared to the condition without thrombin.This work has shown that the migration of osteogenic cells, through fibrin-filled PLGA scaffolds that support direct bone contact, can be modulated by altering the thrombin concentration present during fibrin polymerization, and that thrombin has the capacity to stimulate the migration of osteoprogenitor cells and increase bone formation by the migrated cells.

Subjects

Links

• URL