Stromal-Cell-Derived Factor-1 Antibody Decreased Cancellous Osseointegration Strength in a Murine Tibial Implant Model

Abstract

Background: Active recruitment of osteogenic cells by secreted signaling factors, such as stromal-cell-derived factor 1 (SDF-1), has recently been proposed as a novel strategy to enhance osseointegration. However, the intrinsic importance of the SDF-1/C-X-C chemokine receptor type 4 (CXCR4) axis in promoting osseointegration is unknown. To study the role of SDF-1/CXCR4 in osseointegration, we blocked the SDF-1/CXCR4 pathway in a murine tibial implant model through repeated administrations of an antibody against SDF-1. Methods: Using our previously described murine tibial implant model (N = 24), mice were randomized into an anti-SDF-1 group and a control group (N = 12/group). Intraperitoneal injections of CXCL12/SDF-1 monoclonal antibody (84 µg/mouse) or mouse IgG1 isotype were administered on days 2, 4, 7, 10, 13, 16, 19, 22, and 25 post-surgery. Mice were euthanized 4 weeks post-surgery. Peri-implant bone mass and architecture were determined through microcomputed tomography (µ-CT). Bone implant strength was detected through implant pull-out testing. Results: Inhibition of the SDF-1/CXCR4 pathway significantly reduced host bone–implant interface strength but did not significantly change the cancellous architecture surrounding the implant. Conclusion: SDF-1/CXCR4 is an important pathway to achieve maximum implant osseointegration. However, inhibition of the pathway did not completely eliminate osseointegration.

1. Introduction

The longevity of cementless joint arthroplasties relies on initial stability and rapid osseointegration, the structural and functional connection between the bone and the implant [1]. The host response after insertion and mechanical fixation of cementless implants begins with hematoma formation, where platelets at the implant interface are activated to release cytokines and growth factors [2]. Activated platelets also form a fibrin matrix, which acts as a scaffold for the migration and differentiation of osteogenic cells [3,4]. Subsequently, osteogenic cells (namely, osteoblasts and mesenchymal cells) migrate and attach to the implant’s surface. These cells deposit bone-related proteins and created an approximately 55 µm thick matrix that is rich in calcium, phosphorous, bone sialoprotein, and osteopontin [4,5]. The deposited calcium-rich matrix on the implant’s surface forms woven bone, which fills the gap at the implant–bone interface and provides implant fixation. The final stage of osseointegration involves the remodeling of woven bone to highly mineralized lamellar bone over the course of several months [6,7].

Osseointegration is affected by multiple factors, which range from intrinsic properties of the implants, such as surface topography, chemical composition, and geometric design [8], to comorbidities of the patients, such as osteoporosis, rheumatoid arthritis, nutritional deficiency, smoking, and renal insufficiency [9,10,11,12]. Osseointegration can be enhanced through the use of bone grafts [13], osteogenic biological coatings [14,15], biophysical stimulation [16], and pharmacological agents, such as simvastatin and bisphosphonates [17]. On the other hand, implant micromotion [18,19], narrow implant porosity [20], radiation therapy [21,22], and use of cyclosporine A, methotrexate, cis-platinum [23,24], warfarin, low-molecular-weight heparin [25], and non-steroidal anti-inflammatory drugs [26,27] have been shown to inhibit osseointegration.

Several growth and differentiation factors have been shown to enhance osseointegration, including bone morphogenic proteins BMP-2 and BMP-7 [28], osteogenic protein-1 (OP-1) [29], platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), and transforming growth factor-beta 1 (TGFβ-1) [12]. A recently proposed strategy to enhance osseointegration consisted of actively recruiting osteogenic cells through the creation of a chemotactic gradient using released signaling molecular factors [30]. One of the most well-studied recruitment factors is the cytokine stromal-derived growth factor-1 (SDF-1α), also known as CXC chemokine ligand-12 (CXCL-12) [30]. SDF-1α-bound CXCR4 is crucial in directing the migration of hematopoietic cells during embryogenesis [31], recruiting endothelial progenitor cells for angiogenesis in adulthood [32], influencing tumor metastasis [33], and acting as a chemotactic factor for mesenchymal stem cells (MSCs) [34,35]. SDF-1α has been shown to enhance bone tissue regeneration by increasing the local recruitment of stem cells through activation of their CXCR4 cells both in vitro and in vivo [36,37].

While the findings above confirm the benefit of local delivery of SDF-1α for bone formation [38], it is unclear if a lack of SDF-1α inhibits osseointegration. The purpose of our study is to evaluate the effects of SDF-1α inhibition on tibial cancellous osseointegration in a novel murine model. We inhibited the activity of SDF-1α systemically through administration of anti-SDF-1α to test the hypothesis that SDF-1α inhibition would result in reduced bone implant interface strength and inferior peri-implant bone architecture.

2. Materials and Methods

2.1. Materials and Reagents

Both a monoclonal anti-human/mouse SDF-1/CXCL12 neutralizing antibody (catalog number: 779014) and an isotype-matched control (11711) were purchased from R&D systems (Minneapolis, MN, USA). We utilized a three-dimensionally printed tibial implant composed of the same Ti-6Al-4V alloy used in human prosthetic joint components. The implant has a smooth tibial plateau (2.0 × 1.5 mm oval with a mean surface roughness of 8.4 μm) and a 2.0 mm long intramedullary stem textured with titanium spheres that are 40 μm in diameter. Prior to implantation, the implant’s surface was passivated with 25% nitric acid for 24 h, in accordance with ASTM F86 (Standard Practice for the Surface Preparation and Marking of metallic Surgical Implants), neutralized with phosphate-buffered saline (PBS), and then autoclaved.

2.2. Murine Model

All animal experiments followed protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the Hospital Special Surgery (approval code 2018-0001; approved: 26 May 2018). Twenty-four 16-week-old C57BL/6 female mice underwent right proximal tibial implantation through a previously described surgical technique (Figure 1) [39]. Following surgery, the mice were randomized into the anti-SDF-1 group (N = 12) receiving SDF-1 monoclonal antibody and the control group (N = 12) receiving mouse IgG1 isotype. The antibodies (84 µg/mouse, i.p.) were administered on days 2, 4, 7, 10, 13, 16, 19, 22, and 25 post-surgery. Mice were provided with buprenorphine (0.05 mg/kg, s.q., bid) for 48 h postoperatively and not restricted in activity. The right tibiae were collected immediately after euthanasia 4 weeks after implantation.

2.3. Microcomputed Tomography (MicroCT)

Right tibia of all mice (N = 24) were retrieved en bloc four weeks after implantation. MicroCT (µCT 35; SCANCO Medical, Bassersdorf, Zurich, Switzerland) scans were performed on all samples to analyze cancellous bone architecture. Scan settings were 6 µm voxel size, 55 kVp, 145 mA, and 0.36° rotation step with a 180° angular range, as previously described [39]. Two volumes of interest (VOI), peri-implant and distal to implant, were examined (Figure 1b) [39]. The peri-implant VOI consisted of the cancellous bone along the distal 500 µm of the stem. The distal to the implant VOI consisted of the 500 µm cancellous bone distal to the stem tip. Both VOI were located within the metaphysis of the tibia to avoid including the growth plate. A 60 µm thick volume around the surface of the implant was excluded to minimize the effect of beam-hardening artifacts caused by the metallic implant. Bone volume fraction, trabecular thickness, trabecular number, and tissue mineral density were calculated using the software provided by the manufacturer of the MicroCT.

2.4. Biomechanical Testing

The strength of the bone–implant interface was measured through pull-out testing of the implant from each tibia. After the MicroCT scan, specimens (n = 12/group) were wrapped in 0.9% saline-solution-soaked gauze and frozen at −20 °C. Prior to testing, each tibia was thawed to room temperature and potted in polymethylmethacrylate. The bone at the proximal end of the tibia was dissected with a number-11 scalpel to allow for clamping of the implant under the tibial plateau. A pull-out loading at a displacement of 0.03 mm/s (EnduraTEC ELF 3200 system, Eden Prairie, MN, USA) was applied along the axis of the implant stem until complete failure. Maximum pull-out load, load to failure, and stiffness were calculated from the load displacement data.

2.5. Statistical Analysis

Data are reported as mean ± standard deviation. Statistical analysis was performed using a Student’s t-test. Significance was assigned to p < 0.05.

3. Results

3.1. SDF-1 Inhibition Decreased Bone–Implant Interface Strength

Four weeks of anti-SDF-1 treatment decreased the maximum load of pull-out testing by 17.9% (p = 0.027) from 24.2 ± 5.0 N in controls to 19.9 ± 4.1 N (Figure 2a). Furthermore, the anti-SDF-1 decreased the load to failure by 33.6% (p < 0.030) from 15.5 ± 8.3 N in controls to 10.3 ± 4.9 N (Figure 2b). No significant difference was observed in stiffness between the anti-SDF-1 treatment group and the controls.

3.2. SDF-1 Inhibition Did Not Change the Mass or Architecture of the Peri-Implant Cancellous Bone

Four weeks of anti-SDF-1 treatment did not significantly change the trabecular bone fraction, trabecular thickness, trabecular number, or tissue mineral density measured through MicroCT (

Table 1. Effects of SDF-1 antibody administration on peri-implant bone architecture.

	Control	SDF-1 Ab	t-Test (p-Value)
BV/TV	0.178 ± 0.034	0.195 ± 0.029	0.234
Trabecular Number	6.07 ± 1.57	6.27 ± 1.22	0.745
Trabecular Thickness	0.050 ± 0.006	0.049 ± 0.006	0.710
Tissue Mineral Density	1096.27 ± 50.71	1093.18 ± 59.45	0.894

).

MicroCT was used to measure both the distal-to-implant and peri-implant trabecular bone fraction (BV/TV), trabecular thickness, trabecular number, and tissue mineral density. The measurements demonstrated similar BV/TV, trabecular thickness, trabecular number, and tissue mineral density between the anti-SDF-1 group and the control group.

4. Discussion

Failure of osseointegration results in the formation of fibrous tissue at the bone–implant interface, causing low interface strength and subsequent loosening of the implant [17]. Aseptic loosening of the implant results in pain and instability [40]. Enhancing osseointegration by actively recruiting mesenchymal stem cells has been proposed recently to secure initial mechanical stability and to increase long-term implant survival [30].

In this study, we investigated the effect of inhibiting the SDF-1/CXCR4 pathway of osseointegration on the bone–implant interface. Our results demonstrate that inhibition of SDF-1 significantly reduces the bone–implant interface strength. Upon exposure to invasive procedures, such as implantation of a metal prosthesis, SDF-1α is locally released within the bone marrow [41] or in the liver [42]. The released SDF-1α then enhances bone tissue regeneration by increasing local recruitment of stem cells through activation of CXCR4 receptors in stem cells [36]. Binding of SDF-1α to CXCR4 receptors in stem cells causes selectin-mediated cells to roll on the endothelial walls, followed by a firm stop via activation of cell integrins, and extravascular migration into the injured local tissues [43]. In addition to recruitment of stem cells through the SDF-1/CXCR4 pathway, SDF-1α has also been shown to increase the efficacy of BMP-2 in generating new bone [35] and induce the formation of blood vessels through its ability to induce the secretion of VEGF in endothelial cells [44,45].

The results of this study also further demonstrate the crucial role of SDF-1/CXCR4 in enhancing implant osseointegration. Previous studies of two groups showed that local delivery of SDF-1 enhances stem cells’ recruitment and osseous tissue regeneration [46,47]. In addition, local delivery of SDF-1α in a murine model has been shown to increase bone formation [37] and improve bone–implant osseointegration [38].

Interestingly, this difference in the bone–implant interface did not coincide with the peri-implant cancellous bone mass or architecture as measured through MicroCT. This is consistent with the study by Karlsson, et al. [38], which did not find significant enhancement of osseointegration with local delivery of SDF-1 alone. There are several possible reasons for this observation. First, the BMP-regulated local stem cells may compensate for the lack of recruitment of SDF-1-regulated blood-circulating mesenchymal stem cells. Kumagai, et al. 2008 demonstrated that local stem cells contribute to 94% of total bone fracture healing, while the SDF-1-regulated blood-circulating mesenchymal stem cells only contribute to 6% of total bone fracture healing [48]. Additionally, in this experiment, tissue directly adherent to the implant (within the 60 µm region) was excluded to avoid the effect of a beam hardening artifact caused by the presence of the metal implant. This very local interface tissue could be a major contributor to the strength of the bone–implant interface; changes in its composition would not be detectable using our assay.

This study has potential limitations. The use of antibodies instead of knockout mice may have resulted in incomplete inhibition of the SDF-1/CXCR4 pathway. The statistically significant reduction in the bone–implant interface shear strength, without significant changes in peri-implant bone mass or architecture as measured through MicroCT, could be attributed to this incomplete inhibition of the SDF-1/CXCR4 pathway.

5. Conclusions

In conclusion, we demonstrated that inhibition of the SDF-1/CXCR4 pathway in vivo through administration of anti-SDF-1 reduces the strength of the bone–implant interface. Interestingly, the reduction in interface strength is not accompanied by macroscopic cancellous architectural change, presumably due to the ability of other lineages of progenitor cells to partially compensate for the inhibition of the SDF-1/CXCR4 pathway. Further research is needed to evaluate the role of multiple lineages of skeletal progenitor cells in peri-implant bone formation.