A tissue puncture closure device includes a carrier tube and a sealing plug. The carrier tube includes a distal end configured for insertion into the tissue puncture. The sealing plug is positioned in the carrier tube and arranged for ejection from the distal end of the carrier tube into the tissue puncture. The sealing plug includes a first collagen portion having a first cross-linked chemical structure, and a second collagen portion positioned proximal of the first collagen portion in the carrier tube. The second collagen portion has a second cross-linked chemical structure that is different from the first cross-linked chemical structure.