Osteocytes Influence on Bone Matrix Integrity Affects Biomechanical Competence at Bone-Implant Interface of Bioactive-Coated Titanium Implants in Rat Tibiae

Abstract

Osseointegration is a prerequisite for the long-term success of implants. Titanium implants are preferred for their biocompatibility and mechanical properties. Nonetheless, the need for early and immediate loading requires enhancing these properties by adding bioactive coatings. In this preclinical study, extracellular matrix properties and cellular balance at the implant/bone interface was examined. Polyelectrolyte multilayers of chitosan and gelatin or with chitosan and Hyaluronic acid fabricated on titanium alloy using a layer-by-layer self-assembly process were compared with native titanium alloy. The study aimed to histologically evaluate bone parameters that correlate to the biomechanical anchorage enhancement resulted from bioactive coatings of titanium implants in a rat animal model. Superior collagen fiber arrangements and an increased number of active osteocytes reflected a significant improvement of bone matrix quality at the bone interface of the chitosan/gelatin-coated titan implants over chitosan/hyaluronic acid-coated and native implants. Furthermore, the numbers and localization of osteoblasts and osteoclasts in the reparative and remodeling phases suggested a better cellular balance in the chitosan/Gel-coated group over the other two groups. Investigating the micro-mechanical properties of bone tissue at the interface can elucidate detailed discrepancies between different promising bioactive coatings of titanium alloys to maximize their benefit in future medical applications.

Keywords: bioactive coating; gelatin; hyaluronic acid; osseointegration; osteocytes; titanium implants.

Figure 1

Higher amount of mineralized bone matrix seen within bone-implant interface after 8 W. Changes in matrix mineralization were evaluated using histological analysis. (A) Modified Masson Goldner stain helped in the quantification of mineralized and non-mineralized bone matrix area. (B) Chi/Gel group showed significantly higher mineralized bone matrix after 8 W compared with 3 W. Although not as apparent as the Chi/Gel group, Ti and Chi/HA groups also showed a higher mineralized bone matrix area after 8 W, (C) Chi/Gel showed a significantly lower portion of non-mineralized bone matrix area after 8 W compared with 3 W. Chi/HA group also showed lower non-mineralized bone area after 8 W (Ti: n = 5 (3 W), n = 3 (8 W); Chi/Gel: n = 5/time point; Chi/HA: n = 4 (3 W), n = 5 (8 W); *: p ≤ 0.05).

Figure 2

Quantitative evaluation of osteoblasts and osteoclasts within bone-implant interface showed higher osteoblasts/osteoclasts count in Ti and Chi/Gel groups after 8 W. Bone remodeling is governed by balanced osteoblast and osteoclast activity. Therefore, osteoblasts and osteoclasts were quantitatively evaluated for all groups. Higher osteoblasts and osteoclasts count were seen in Ti and Chi/Gel groups after 8 W compared with 3 W. Whereas, a lower number of osteoblasts and osteoclasts were seen in the Chi/HA group after 8 W compared with 3 W (Ti: n = 5 (3 W), n = 3 (8 W); Chi/Gel: n = 5/time point; Chi/HA: n = 4 (3 W), n = 5 (8 W).

Figure 3

Quantitative evaluation of osteocytes within bone-implant interface showed higher spindle-shaped and lower empty lacunae in all groups after 8 W. Silver nitrate stain was used to visualize osteocytes within bone-implant interface. Morphologically, osteocytes are categorized as (A1) spindle-shaped, (A2) intermediate, and (A3) empty lacunae. (B) Total osteocytes were lower in Chi/Gel and Chi/HA groups after 8 W compared with 3 W. (C) Higher spindle-shaped osteocytes were seen in all groups after 8 W. Spindle-shaped osteocytes in the Chi/Gel group were higher compared with Ti and Chi/HA groups after 8 W. (Ti: n = 5 (3 W), n = 3 (8 W); Gel: n = 5/time point; HA: n = 4 (3 W), n = 5 (8 W); *: p ≤ 0.05, scale bar: 10 µm).

Figure 4

Collage fibers properties at the bone implant interface indicate bone matrix quality. (A) and (B) Sirius red-stained images were used to visualize collagen type around the implant. (C) collagen fibers properties were investigated on monochromic pictures using CT-FIRE software. (D) Number of collagen fibers was less at 8 W without significant difference to 3 W. (E) Fiber alignment was highest in the Chi/Gel group at 3 W, with progression of tissue maturation no significant change in fiber alignment was seen in any group. (F) Orientation angle of fibers was around or below 50°, at all time-points in all groups. (Ti: n = 5 (3 W), n = 3 (8 W); Chi/Gel: n = 5/time point; Chi/HA: n = 4 (3 W), n = 5 (8 W), *: p ≤ 0.05).

Figure 5

Enhanced collagen fiber properties led to enhanced micro-mechanical quality in Chi/Gel at 3 W and 8 W. CT-FIRE was used to quantify the collagen fibers from Sirius red-stained images. Collagen fibers orient mostly at smaller angles, <55°. (A) The fibers were mostly between 3 and 5 µm in length, the Chi/Gel group showed longer fibers at the early time point and shorter at the later time point, the Chi/Ha showed an inverse pattern. (B) Fiber width reflects crosslinking and intramolecular spacing, all groups showed comparable width within the range of healthy tissue. (C) Fiber straightness indicates anisotropic nature of the matrix, at 8 W the Chi/Gel and Chi/HA show the highest values. (Ti: n = 5 (3 W), n = 3 (8 W); Gel: n = 5/time point; HA: n = 4 (3 W), n = 5 (8 W), *: p ≤ 0.05).