Fibroblasts from the inner granulation tissue of the pseudocapsule in hips at revision arthroplasty induce osteoclast differentiation, as do stromal cells

Abstract

Background: It has previously been shown that many osteoclast precursors are included in the granulation tissue within the pseudocapsule obtained at revision arthroplasty from hips with osteolysis. In vitro culture of only cells isolated from the granulation tissue has been previously shown to generate many mature osteoclasts.

Objective: To investigate the presence or otherwise of supporting cells, similar to stromal cells, which differentiate osteoclasts within the granulation tissue.

Methods: Cells isolated from the granulation tissue were cultured alone, and after four weeks fibroblast-like cells (granulation fibroblasts) remained. Rat non-adherent bone marrow cells (NA-BMCs) were co-cultured with the granulation fibroblasts with or without 1alpha,25(OH)2D3 (10(-8) M) or heat treated ROS 17/2.8 cell conditioned medium (ht ROSCM), or both. Multinucleated cells (MNCs), which formed, were assessed by biochemical and functional characterisation of osteoclasts. Receptor activator of NFkappaB ligand (RANKL) was investigated by immunohistochemistry.

Results: Co-culture of NA-BMCs and granulation fibroblasts caused the formation of tartrate resistant acid phosphatase (TRAP) positive MNCs, which had the calcitonin receptor (CTR), the Kat-1 antigen, which is specific to the surface of rat osteoclasts, and the ability to form pits in the presence of both 1alpha,25(OH)2D3 and ht ROSCM or in the presence of just ht ROSCM. RANKL was detected in fibroblast-like cells in the granulation tissue.

Conclusion: These data suggest that granulation fibroblasts support osteoclast differentiation, as do osteoblasts/stromal cells, and may play a part in aseptic loosening.

Figure 1

Preparation of granulation fibroblasts. Cells isolated from the granulation tissue staining for TRAP (A) after culture for one week and (B) after culture for four weeks. Bar = 10 µm. TRAP positive MNCs (arrow) appeared in the culture of the isolated cells from the granulation tissue after culture for one week (A). After culture for four weeks, neither TRAP positive mononuclear cells nor MNCs were seen, only the granulation fibroblasts remaining in the culture (B).

Figure 2

Expression of CTR mRNA in granulation fibroblasts. Expression of CTR mRNA by RT-PCR (50 cycle). Lane 1, giant cell tumour tissue (positive control); lane 2, granulation tissue; lane 3–6, granulation fibroblast culture (lane 3, with no supplementation; lane 4, with 1α,25 (OH)₂D₃; lane 5, with ht ROSCM; lane 6, with 1α,25 (OH)₂D₃ and ht ROSCM). Giant cell tumour tissue and granulation tissue showed two signals (346 bp and 412 bp) of CTR, but the culture of granulation fibroblasts alone did not show any signal under any of the conditions.

Figure 3

Stromal cell-like activity of granulation fibroblasts. Three different groups of cells were cultured; NA-BMCs alone (A–D), NA-BMCs and skin fibroblasts (E–H), NA-BMCs and granulation fibroblasts (I–L). This figure shows cultured cells stained by TRAP on day 2 (A, E, I), day 4 (B, F, J), day 7 (C, G, K), or day 14 (D, H, L). Bar = 10 µm. In the culture of NA-BMCs alone and NA-BMCs and skin fibroblasts, TRAP positive mononuclear cells (small arrows) survived until day 4 (A, B, F). Only in the culture of NA-BMCs and granulation fibroblasts, could we observe TRAP positive MNCs (large arrows) (J).

Figure 4

Effect of 1α,25(OH)₂D₃ supplementation on the number of TRAP positive MNCs. In the co-culture of NA-BMCs and granulation fibroblasts in the presence of 1α,25(OH)₂D₃ and ht ROSCM or in the presence of just ht ROSCM, TRAP positive MNCs appeared at two days and the number of TRAP positive MNCs increased until four days, reaching a maximum, before decreasing with time until the end of the co-culture. There was no significant difference in the number of TRAP positive MNCs (in the presence of just ht ROSCM compared with the presence of 1α,25(OH)₂D₃ and ht ROSCM).

Figure 5

Presence of CTR on the TRAP positive MNCs formed. The cells cultured with 1α,25(OH)₂D₃ and ht ROSCM for four days were incubated with [¹²⁵I]sCT for one hour, either without (A) or with (B) an excess amount of unlabelled sCT, followed by slight staining for TRAP. Bar = 10 µm. Grains were seen over TRAP positive MNCs (black arrow) and TRAP positive mononuclear cells (A). No grains were detected over TRAP positive MNCs or TRAP positive mononuclear cells in the same culture system when the cultured cells were incubated with an excess amount of unlabelled sCT (B).

Figure 6

Immunostaining by mAb Kat-1. The cultured cells (A) and human osteoclast-like cells from the giant cell tumour (B) which had been cultured for four days were stained with mAb Kat-1 followed by detection, using an ABC-AP kit. Bar = 10 µm. The MNCs formed were stained with mAb Kat-1 (A). Human osteoclast-like cells were unreactive with mAb Kat-1 (B).

Figure 7

Pit formation by SEM. The cultured cells (A) and NA-BMCs alone (B) were cultured on a dentine slice for seven days. Pits were formed by the cultured cells (A). In the culture of NA-BMCs, pits were not seen (B).

Figure 8

Localisation of RANKL in the granulation tissues. The granulation tissues inside the pseudocapsule were immunostained by anti-RANKL antibody (A) and control (B). Bar = 10 µm. RANKL was localised at vascular cells (arrow) and fibroblast like cells (arrow heads).