Leptin receptor gene deficiency minimally affects osseointegration in rats

Abstract

Metabolic syndrome represents a cluster of conditions such as obesity, hyperglycaemia, dyslipidaemia, and hypertension that can lead to type 2 diabetes mellitus and/or cardiovascular disease. Here, we investigated the influence of obesity and hyperglycaemia on osseointegration using a novel, leptin receptor-deficient animal model, the Lund MetS rat. Machined titanium implants were installed in the tibias of animals with normal leptin receptor (LepR^+/+) and those harbouring congenic leptin receptor deficiency (LepR^-/-) and were left to heal for 28 days. Extensive evaluation of osseointegration was performed using removal torque measurements, X-ray micro-computed tomography, quantitative backscattered electron imaging, Raman spectroscopy, gene expression analysis, qualitative histology, and histomorphometry. Here, we found comparable osseointegration potential at 28 days following implant placement in LepR^-/- and LepR^+/+ rats. However, the low bone volume within the implant threads, higher bone-to-implant contact, and comparable biomechanical stability of the implants point towards changed bone formation and/or remodelling in LepR^-/- rats. These findings are corroborated by differences in the carbonate-to-phosphate ratio of native bone measured using Raman spectroscopy. Observations of hypermineralised cartilage islands and increased mineralisation heterogeneity in native bone confirm the delayed skeletal development of LepR^-/- rats. Gene expression analyses reveal comparable patterns between LepR^-/- and LepR^+/+ animals, suggesting that peri-implant bone has reached equilibrium in healing and/or remodelling between the animal groups.

Figure 1

Animal model characterisation and assessment of osseointegration. (A) Body weight of LepR^+/+ and LepR^−/− animals on Day 0 and Day 28. (B) Blood glucose levels of LepR^+/+ and LepR^−/− animals on Day 0 and Day 28. (C) Serum sclerostin concentrations on Day 0 and Day 28. (D) Region of interest selected for measurements of bone volume using micro-CT. (E) Bone volume measured in the total volume of interest (BV/TV) using micro-CT. (F) Biomechanical stability of the implants measured by removal torque (RTQ). Inset: average load deformation curves (applied force vs. angular deformation). (G) Example of the histological section made in the sagittal plane of the bone. Scale bar = 500 µm. (H) Bone-implant contact (BIC). (I) Bone area (B.Ar) present within the implant threads. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2

Bone mineral density distribution (BMDD). (A) Left: BMDD curve generated from the histogram data of native bone in LepR^+/+ animals (n = 7). Right: representative qBEI image (with and without 16-level lookup table). Scale bar = 200 µm. (B) BMDD parameters. From left to right: Ca_MEAN, Ca_PEAK, and Ca_WIDTH. (C) Left: BMDD curve generated from the histogram data of native bone in LepR^−/− animals (n = 7). Right: representative qBEI image (with and without 16-level lookup table). Scale bar = 200 µm. **p < 0.01.

Figure 3

Chemical composition of bone. (A) Left: schematic representation of the Raman measurement points (black circles) of bone within the implant threads and native bone. Right: Raman spectra of bone within the implant threads and in the native bone of LepR^−/− (n = 7) and LepR^+/+ (n = 7) animals showing the characteristic peaks for apatite (ν₂PO₄³⁻ at ~420 to 470, ν₁PO₄³⁻ at ~959, and ν₁CO₃²⁻ ~ 1070 cm⁻¹) and collagen (proline at ~855, hydroxyproline at ~876, and amide III at ~1240 to 1270 cm⁻¹). (B) From left to right: mineral crystallinity, carbonate-to-phosphate ratios, and mineral-to-matrix ratios of bone within the implant threads and native bone. *p < 0.05 and **p < 0.01.

Figure 4

Gene expression analysis of bone and implant-adherent cells. Relative expression of genes coding for sclerostin (SOST), receptor activator of nuclear factor-kappa B (RANK), receptor activator of nuclear factor-kappa B ligand (RANKL), osteoprotegerin (OPG), peroxisome proliferator-activated receptor gamma (PPARG), Runt-related transcription factor 2 (RUNX2), bone morphogenetic protein 2 (BMP2), and tumour necrosis factor alpha (TNFa) was analysed for both LepR^+/+ (n = 7) and LepR^−/− animals (n = 7). *p < 0.05, **p < 0.01.

Figure 5

Qualitative evaluation of undecalcified basic fuchsin-stained histological sections of LepR^+/+ (A–D) and LepR^−/− (E–H) animals. (A, E) Representative overview images of LepR^+/+ and LepR^−/− sections showing unicortical placement of the machined Ti implants. Scale bars = 500 µm. (C, G) Large islands of leftover, hypermineralised cartilage (white arrows). Scale bars = 100 µm. (B,F) Recently formed bone is stained intensely blue. The interface between the native and de novo bone is easily discernible (white asterisks). Scale bars = 100 µm. (D,H) Polarised light microscopy of the corresponding threads in (B) and (F), respectively. Woven bone, due to its isotropic character does not transmit light, as observed in threads of both animal types. These areas correspond to areas of intensely stained, recently formed bone. Scale bars = 100 µm.

Figure 6

Newly formed bone within implant threads. Threads of (A) LepR^+/+ and (B) LepR^−/− animals showing the presence of highly disorganised bone matrix as observed under light microscopy (left) and BSE-SEM (right). Scale bars = 50 µm.