Early osteoimmunomodulatory effects of magnesium-calcium-zinc alloys

Abstract

Today, substantial attention is given to biomaterial strategies for bone regeneration, and among them, there is a growing interest in using immunomodulatory biomaterials. The ability of a biomaterial to induce neo vascularization and macrophage polarization is a major factor in defining its success. Magnesium (Mg)-based degradable alloys have attracted significant attention for bone regeneration owing to their biodegradability and potential for avoiding secondary removal surgeries. However, there is insufficient evidence in the literature regarding the early inflammatory responses to these alloys in vivo. In this study, we investigated the early body responses to Mg-0.45wt%Zn-0.45wt%Ca pin-shaped alloy (known as ZX00 alloy) in rat femora 2, 5, and 10 days after implantation. We used 3D micro computed tomography (µCT), histological, immunohistochemical, histomorphometrical, and small angle X-ray scattering (SAXS) analyses to study new bone formation, early macrophage polarization, neo vascularization, and bone quality at the implant bone interface. The expression of macrophage type 2 biological markers increased significantly after 10 days of Mg alloy implantation, indicating its potential in stimulating macrophage polarization. Our biomineralization results using µCT as well as histological stained sections did not indicate any statistically significant differences between different time points for both groups. The activity of alkaline phosphatase (ALP) and Runt-related transcription factor 2 (Runx 2) biological markers decreased significantly for Mg group, indicating less osteoblast activity. Generally, our results supported the potential of ZX00 alloy to enhance the expression of macrophage polarization in vivo; however, we could not observe any statistically significant changes regarding biomineralization.

Keywords: Magnesium; biomineralization; calcium; macrophage polarization; zinc.

Figure 1.

Immunohistochemical analysis of macrophage polarization over time. (a) Representative images of antibody staining against CD68, 80, and 206 macrophage markers (red color) 10 days after implantation in the Mg-based alloy and sham groups, scale bar = 200 µm. CD68, 80 were used as the markers of macrophage type 1, while CD80 and CD206 were the markers for macrophage type 2. Quantitative histomorphometrical data of macrophage polarization using the percentage of antibodies against CD68 (b), CD80 (c), and CD206 (d) markers over time. The percentage of CD80 decreased significantly after 10 days in both groups (p < 0.05). However, the percentage of CD206 marker significantly increased only in Mg group after 10 days, compared with day 2 and 5 (p < 0.05). After 5 and 10 days, macrophage phenotype changed in both groups from predominantly macrophages type 1–2 with significant changes for Mg group (b–d). We observed a significantly higher number of CD206 positive macrophages (type 2 macrophages) for Mg group compared with sham over time. Values represent the mean ± standard deviation. Significant differences were presented as *p < 0.05.

Figure 2.

Immunohistochemical evaluation of early blood vessel formation and neo-vascularization of Mg-based alloy and sham groups over time. (a) Representative images of α-SMA antibody staining (red color) for early blood vessel formation 2, 5, and 10 days after implantation in both groups. Quantitative histomorphometrical data of neo-vascularization using Factor VIII antibody (b), and blood vessel formation using alpha smooth muscle Actin (α-SMA) antibody percentage over time, scale bar = 200 µm (c). An insignificant decrease in the percentage of positive blood vessels was observed for both factor VIII and α-SMA positive blood vessels over time in sham group. The percentage of factor VIII positive blood vessels decreased significantly for Mg group at day 5 compared to day 2 (p < 0.05). (d) Representative bright field and fluorescent images of α-SMA antibody staining used for evaluating the blood vessel regularity over time using a 3-point scale system, scale bar = 200 µm. The round shape vessels were categorized as regular type 1 vessels, black arrow (d, e), small to moderate oval shape ones as regular type 2, blue arrow, (d, f) and big vessels in oval or other undefined shapes as irregular type 3 vessels, green arrow (e, g). Values represent the mean ± standard deviation. Significant differences were presented as *p < 0.05.

Figure 3.

Analyzing the bone mineralization in Mg-based alloy and sham groups over time. (a) Representative images of 3D µCT analysis of bone mineralization in Mg group over time, in the transverse and coronal planes of µCT (left and right panels, respectively), scale bar = 500 µm. (b) Representative images of 2D evaluation of mineralized (MB, black color) versus non-mineralized bone matrix percentage (NMB, pink to red color) in both groups over time, scale bar = 200 µm. The brown color represents bone marrow (BM). Quantitative data of bone volume to tissue volume percentage (BV/TV) (c) and bone surface to bone volume (BS/BV) (d) using 3D µCT analysis. The BV/TV and BS/BV did not change significantly over time for both groups. Quantitative histomorphometrical data of mineralization (e) and new bone formation (f) using Von Kossa/Van Gieson staining. Changes in mineralization and non-mineralization using Von Kossa/Van Gieson staining were also insignificant in both groups. However, changes in the non-mineralization had more fluctuations in both groups, by decreasing and increasing after 5 and 10 days, subsequently. Values represent the mean ± standard deviation.

Figure 4.

Analyzing the bone mineralization and cartilage formation in Mg-based alloy and sham groups over time. (a) Representative images of Movat Pentachrome histology staining. Mineralized (MB) and non-mineralized bone (NMB) as well as cartilage formation (c) were evaluated in both groups over time, scale bar = 200 µm. Descriptive analysis of tissue homogeneity (b) and integrity (c) as well as defect closure (d) in both groups using a 3-point scale system (good, fair, poor for 1–3, respectively). Quantitative histomorphometrical data of mineralization (e), non-mineralization (f) and cartilage distribution percentage (g) using Movat Pentachrome histology staining. There was no significant differences between groups regarding their tissue homogeneity, integrity, and defect closure. Although the percentage of mineralized and non-mineralized bone tissue did not significantly change over time for both groups, cartilage formation significantly decreased after 5 days for sham group (p < 0.05). Values represent the mean ± standard deviation. Significant differences were presented as *p < 0.05.

Figure 5.

Enzyme histochemical and immunohistochemical analyses of bone metabolism activities in Mg-based alloy and sham groups over time. (a) Representative images of osteoblast activity (purple color) using alkaline phosphatase (ALP) enzyme histochemistry in Mg-based alloy and sham groups 10 days after implantation. (b, c) Quantitative data of the Mg-based alloy retention and osteoblast activity over time based on ALP stained sections, respectively. (b) Mg had a significant degradation after 5 and 10 days compared to day 2 with p values of p < 0.05 and p < 0.01, subsequently. (c) The ALP activity decreased significantly after 5 and 10 days for sham group (p < 0.05) indicating less osteoblast activity; however, changes were not significant for Mg group. (d) Representative images of immunohistochemistry staining against SRY-Box Transcription Factor 9 (Sox 9) and runt-related transcription factor 2 (Runx 2) biological markers, scale bar = 200 µm. (e, f) Quantitative histomorphometrical data of Sox 9 and Runx 2 biological markers percentage. (e) An insignificant decrease in the percentage of Sox 9 was observed in both groups over time. (f) Although the percentage of Runx 2 decreased significantly after 5 days (p < 0.01) in Mg group, it started to increase after 10 days. Values represent the mean ± standard deviation. Significant and highly significant differences were presented as *p < 0.05 and **p < 0.01, respectively.

Figure 6.

Studying collagen fiber properties in the Mg-based alloy and sham groups over time. (a) Representative images of Sirius red staining 2, 5, and 10 days after implantation in both groups, scale bar = 200 µm. Quantitative histomorphometrical data of collagen fiber length (b), width (c), angle (d), and straightness (e) using Sirius red staining. Although the collagen fiber length, width, and straightness remained almost unchanged over time for the sham group, we observed increasing the fiber length as well as decreasing the fiber width, angle, and straightness toward 90° orientation. A significant decrease in the collagen straightness for Mg group was observed after 5 days (p < 0.05). Values represent the mean ± standard deviation. Significant differences were presented as *p < 0.05.

Figure 7.

Studying the collagen/hydroxyapatite (HAp) orientation and the size of hydroxyapatite plates in the sham group over time using small angle X-ray scattering (SAXS) analysis. Samples are scanned in a region of approximately 5 mm by 5 mm and each pixel corresponds to a probed region of 60 µm in radius. The shown information are a result of evaluating the scattering data and, therefore, test specific features at other length scales. Representative images of descriptively analyzing the scattered intensity (a, d, g) as well as the HAp orientation in degree (b, e, h) and size of the platelets in nm (c, f, i). No residuals of the defect were detected after day 10 for sham group (g, h). The HAp size was homogenous along the bone and around 2 nm for all time points (c, f, i). The color code in the HAp orientation analysis (b, e, h) shows the orientation degree, which corresponds to the inset, for example, red is along the y-axis of the image.

Figure 8.

Studying the collagen/hydroxyapatite (HAp) orientation and the size of hydroxyapatite platelets in the Mg-based alloy group over time using small angle X-ray scattering (SAXS) analysis. Samples are scanned in a region of approximately 5 mm by 5 mm and each pixel corresponds to a probed region of 60 µm in radius. The shown information are a result of evaluating the scattering data and, therefore, test specific features at other length scales. Representative images of descriptively analyzing the scattered intensity (a, d, g) as well as the HAp orientation in degree (b, e, h) and size of the platelets in nm (c, f, i). Only a minor degree of HAp orientation was observed at the Mg interface after 5 days (d–f). However, the HAp platelets size increased at the interface. The bone orientation at day 5 was in the horizontal direction; however bone was always orientated vertically at other time points. The color code in the HAp orientation analysis (a, d, g) shows the orientation degree, which corresponds to the inset, for example, red is along the y-axis of the image.