In vitro healing of avascular meniscal injuries with fresh and frozen plugs treated with TGF-beta1 and IGF-1 in sheep

Abstract

We studied the effect of freezing and inserting meniscal plugs in lesions generated in the avascular area of sheep menisci maintained in vitro, and whether the healing process can be improved by adding growth factors TGF-beta1 and IGF-1. Thirty six menisci obtained from healthy 3 months-old sheep were cultured in 6 well plates and holes were perforated in the avascular area. Meniscal plugs, either fresh or frozen at -20 degrees C for 1 month, were used to fill in the lesions, and then cultured in the presence or absence of TGF-beta1 or IGF-1 for 8 weeks. Samples stained with Massons trichrome were analyzed to evaluate the attachment of the plug and the cell density of the tissue. BrdU immunohistochemistry was performed to identify the proliferation of meniscal cells. Both growth factors improved considerably the cell density of implanted plugs. TGF-beta1 increased significantly the attachment of both fresh and frozen plugs, but it had no effect on meniscal cell proliferation. In contrast, IGF-1 had no effect on the attachment, but did increase significantly the number of proliferating cells in the surface of the host meniscus and the inserted plug. In conclusion, frozen plugs can survive if treated with either TGF-beta1 or IGF-1. The combination of TGF-beta1 and IGF-1 could aid in the repairing of the avascular meniscal injuries, as they are capable of promoting the attachment of tissue, and increasing the proliferation of meniscal cells.

Keywords: IGF1; TGF-β1; avascular injury; meniscus; sheep; tissue engineering.

Figure 1

Analysis of plug adhesion (Masson's Trichrome, ×20) showing a point of tissue union indicated by the asterisk * (M = meniscus, P = plug. Barr: 100 μm).

Figure 2

Analysis of cell density of explants (number of nuclei/mm2 of stained tissue). Samples were divided into vascular, avascular areas and graft (if any).

Figure 3

Quantification of tissue unions between the injured meniscus and the plug for grafted groups.

Figure 4

Immunohistochemistry detection of BrdU. A. Vascular area of an untreated and unfilled explant showing cells in proliferation (×20). B. Frozen plug treated with IGF-1 (×20).

Figure 5

Quantification of positive cells (A) and the distance to the deepest positive cell founded (B).