Osteoclast-independent bone resorption by fibroblast-like cells

Abstract

To date, mesenchymal cells have only been associated with bone resorption indirectly, and it has been hypothesized that the degradation of bone is associated exclusively with specific functions of osteoclasts. Here we show, in aseptic prosthesis loosening, that aggressive fibroblasts at the bone surface actively contribute to bone resorption and that this is independent of osteoclasts. In two separate models (a severe combined immunodeficient mouse coimplantation model and a dentin pit formation assay), these cells produce signs of bone resorption that are similar to those in early osteoclastic resorption. In an animal model of aseptic prosthesis loosening (i.e. intracranially self-stimulated rats), it is shown that these fibroblasts acquire their ability to degrade bone early on in their differentiation. Upon stimulation, such fibroblasts readily release acidic components that lower the pH of their pericellular milieu. Through the use of specific inhibitors, pericellular acidification is shown to involve the action of vacuolar type ATPases. Although fibroblasts, as mesenchymal derived cells, are thought to be incapable of resorbing bone, the present study provides the first evidence to challenge this widely held belief. It is demonstrated that fibroblast-like cells, under pathological conditions, may not only enhance but also actively contribute to bone resorption. These cells should therefore be considered novel therapeutic targets in the treatment of bone destructive disorders.

Figure 1

Fibroblast-like cells from the synovial-like interface membrane around loose joint arthroplasties produce signs of bone resorption in the severe combined immunodeficient (SCID) mouse model of matrix degradation. (a) and (b) Cultured prosthesis loosening fibroblasts (PLFs) were soaked into a collagen sponge and coimplanted with normal human bone under the renal capsule of SCID mice. After 60 days, histological evaluation showed attachment and invasion of the PLFs into the bone matrix (a, magnification ×630). The presence of PLFs in resorption pits was seen predominantly at sites at which the cells left the collagen sponge (b, magnification ×630). (c) Immunohistochemistry with antivimentin antibodies that recognize human fibroblasts but not mouse cells demonstrated the human origin of the fibroblast-like cells invading the bone. Human, vimentin-positive PLFs (arrowheads) were found abundantly in the SCID mouse sections (c, magnification ×630) and exhibited close contact to the resorption lacunae.

Figure 2

Characterization of cultured human prosthesis loosening fibroblasts (PLFs) by flow cytometry. (a) When compared with isotype control staining, analysis for the macrophage lineage marker CD68 showed no surface expression (<0.1%). (b) Also, no expression of the leucocyte common antigen CD45 was found on the cells (<0.1%). (c) However, fluorescent-activated cell sorter analysis with the fibroblast marker D7-Fib revealed the fibroblast nature of the cells (>99%). (d) In addition, more than 99% of the PLFs stained positive for the fibroblast marker AS02.

Figure 3

Human prosthesis loosening fibroblasts (PLFs) form resorption pits on whale dentin slices in vitro. (a) After 4 weeks of culture on dentin slices, PLFs exhibited morphological signs of bone resorption and produced characteristic signs of resorption pits (closed arrowheads) with remains of PLFs still in situ (open arrowheads). (b) Addition of 10 ng/ml tumour necrosis factor (TNF)-α to the medium enhanced the resorption of dentin, as seen from multiple resorption pits (closed arrowheads). Again, remains of PLFs were seen on the dentin slices (open arrowheads). (c) TNF-α 100 ng/ml also enhanced the size of the resorption pits generated by the PLFs (closed arrowhead). (d) Dentin slices on which no PLFs were cultured showed a clear and smooth surface and no erosions were seen. (e) Freshly differentiated osteoclasts that were used as controls produced characteristic resorption pits after 4 days (closed arrowhead). (f) However, there were considerable differences between osteoclast cultures, with some osteoclasts starting bone resorption only after 2 weeks (closed arrowhead).

Figure 4

Resorption of whale dentin by early differentiation prosthesis loosening fibroblasts (PLFs) from the intracranially self-stimulated (ICSS) Wistar rat model of aseptic prosthesis loosening. (a) Cemented tibial hemiarthroplasties that were implanted into the left knees of ICSS Wistar rats exhibited radiological signs of loosening (arrows) after 12 weeks of stimulated running in a running wheel. (b) Histological evaluation of the interface tissue between the prosthesis and the bone revealed the presence of loose fibrous tissue with fibroblast-like cells, particularly at sites of bone resorption. (c) and (d) Characterization of rat PLFs that were established from this tissue demonstrated the identity of these cells as fibroblasts in that more than 99% of the cells stained positive with specific antibodies against prolyl-4-hydroxylase (d), whereas there was no staining with antibodies against the monocyte lineage marker CD68 (c). (e) and (f) Culturing the rat PLFs on sperm whale dentine slices for 4 weeks produced clear resorption pits (closed arrowheads). Remains of the PLFs were seen in close contact with the resorption pits (open arrowheads).

Figure 5

Release of acidic components by prosthesis loosening fibroblasts (PLFs). (a) and (b) Cytosensor measurement of PLFs stimulated with tumour necrosis factor (TNF)-α (1, 10 and 300 ng/ml) revealed the release of acidic components, with maximal pericellular acidification (rmax/req) of 15% at 10 ng/ml TNF-α (A). Recording of the time curve with 10 ng/ml TNF-α showed a maximal acidification 15 min after the influx of TNF-α was started (b). (c) Incubation of PLFs with the ATPase inhibitors amiloride and bafilomycin A₁ at different concentrations decreased the pericellular acidification by a maximum of 32% with amiloride and 11% with bafilomycin A₁. (d) Inhibition of H ⁺ release by amiloride was recorded after 3 min and remained stable for the total measuring time of 45 min. Pericellular pH returned to the initial values shortly after the perfusion of amiloride was terminated. (e) The specific v-ATPase inhibitor bafilomycin A₁ at concentrations of 10^-6 mol/l showed a clear effect shortly after its addition, and the H⁺ secretion remained low even after discontinuation of bafilomycin A₁ infusion.