Titanium particle-challenged osteoblasts promote osteoclastogenesis and osteolysis in a murine model of periprosthestic osteolysis

Abstract

The current study investigates the interactive behavior of titanium alloy particle-challenged osteoblastic bone marrow stromal cells (BMSCs) and macrophage lineage cells in a murine knee-prosthesis failure model. BMSCs were isolated from male BALB/c mice femurs and induced in osteogenic medium. At 24h after isolation, BMSCs in complete induction medium were challenged with 1, 3 or 5mgml(-1) titanium particles for 7days. Culture media were collected at 2, 4 and 6days and cells were harvested at 7days for alkaline phosphatase (ALP) assay/stains. Cell proliferation in the presence of Ti particles was periodically evaluated by MTT assay. Mice implanted with titanium-pin tibial implants were given an intra-articular injection of 50μl medium containing 5×10(5) Ti particles-challenged bone-marrow-derived osteoblastic cells, followed by a repeat injection at 2weeks post-operation. Control mice with titanium-pin implants received a naïve osteoblastic cell transfusion. After sacrifice at 4weeks, the implanted knee joint of each group was collected for biomechanical pin-pullout testing, histological evaluation and reverse transcriptase polymerase chain reaction analysis of mRNA extracted from the joint tissues. Ti particles significantly stimulated the proliferation of BMSC-derived osteoblastic cells at both high and low particle concentrations (p<0.05), with no marked differences between the particle doses. ALP expression was diminished following Ti particle interactions, especially in the high-dose particle group (p<0.05). In addition, the culture media collected from short-term challenged (48h) osteoblasts significantly increased the numbers of TRAP+ cells when added to mouse peripheral blood monocytes cultures, in comparison with the monocytes cells receiving naïve osteoblasts media (p<0.05). Intra-articular introduction of the osteoblastic cells to the mouse pin-implant failure model resulted in reduced implant interfacial shear strength and thicker peri-implant soft-tissue formation, suggesting that titanium particles-challenged osteoblasts contributed to periprosthetic osteolysis. Comparison of the gene expression profiles among the peri-implant tissue samples following osteoblast injection did not find significant difference in RunX2 or Osterix/Sp7 between the groups. However, MMP-2, IL-1, TNF-α, RANKL, and TRAP gene expressions were elevated in the challenged-osteoblast group (p<0.05). In conclusion, titanium alloy particles were shown to interfere with the growth, maturation, and functions of the bone marrow osteoblast progenitor cells. Particle-challenged osteoblasts appear to express mediators that regulate osteoclastogenesis and peri-prosthetic osteolysis.

Fig. 1

(A) Scanning electron microscopy (SEM) appearance of the particles used in experiments (800x), and (B) the histogram of the particle distribution pattern. Panel (C) shows typical osteoblastic cells when PKH2-labeled bone marrow cells were cultured in osteogenic-induction medium for 7 days (200x magnification). (D) Titanium particles were engulfed into osteoblastic cells (white arrows, 200x).

Fig. 2

(A–D) Immunostain reveals alkaline phosphatase (ALP) expression on bone marrow cells: challenged the cells by different concentrations of titanium particles (0, 0.5, 1, 3 mg/ml) in osteogenic-induction medium (100× magnification); (E) ALP activity assay of cell lysate: The OD values of ALP activity were normalized against the total protein concentration in the sample and expressed as arbitrary unit. The control sample was from cells lysate without particle treatment; (F) MTT assay to periodically estimate the viability and proliferation of BMSCs treated by various concentrated Ti particles. Stimulation indexes were calculated by comparison with baseline proliferation (particle-free controls, *p<0.05, **p<0.01)

Fig. 3

tartrate-resistant acid phosphatase (TRAP) staining of peripheral blood monocytes (PBMCs) following different treatments (200×): (A) cells receiving naïve osteoblasts media; (B) conditioned media from osteoblast culture without titanium particle stimulation was added; (C) cells culturing in osteoblast media with Ti particles stimulation, the insert shows a blowup of multinucleated TRAP+ osteoclasts (400×); (D) summarization of the number of TRAP+ cells in treated PBMCs among groups (*p<0.05, **p<0.01).

Fig. 4

The average of peak pulling force required to pull out the implants at 4 weeks after different treatments (left plot). Plot on the right side summarizes the periprosthetic bone mineral density (BMD) determined using the SCANCO Evaluation program V6.5-1 software to generate isosurfaces of the region of interest (ROI). The insert illustrated a microCT reconstructed 3-D isosurfaces image. (#p<0.05 than@, *p<0.05 than @ and #, **p<0.01 than @ and #)

Fig. 5

(A–C) represent the histological appearances of pin-implanted tibiae at 4 weeks following different treatments (40×): (A) stable pin implantation without particles stimulation; (B) Ti particles treated prosthesis failure control; (C) the injection of Ti particles-challenged OBs significantly result in more severe inflammation with thicker membrane generation. (D) The comparison of periprosthetic membrane thickness in response to different treatments (**p<0.01).

Fig. 6

TRAP positive cell counts in the sections among groups were determined using a computerized image analysis system with ImagePro+ software (*p<0.05, **p<0.01). The inserted image illustrates a TRAP stained peri-implant tissue section with purple stained osteoclastic cells.

Fig. 7

Gene expression profiles of the peri-implant tissue following Ti-particle treatment or the injection of Ti-particles challenged osteoblasts. The data are expressed as comparative gene copies against readings from the stable pin-implantation control (see Material and methods, *p<0.05).