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. 2019 Oct 18:10:2443.
doi: 10.3389/fimmu.2019.02443. eCollection 2019.

Compromised Bone Healing in Aged Rats Is Associated With Impaired M2 Macrophage Function

Julia Löffler  1   2 , F Andrea Sass  1   2 , Sebastian Filter  1 , Alexander Rose  1 , Agnes Ellinghaus  1   2 , Georg N Duda  1   2 , Anke Dienelt  1   2
Affiliations

Compromised Bone Healing in Aged Rats Is Associated With Impaired M2 Macrophage Function

Julia Löffler et al. Front Immunol. .

Abstract

Fracture repair is initiated by a multitude of immune cells and induction of an inflammatory cascade. Alterations in the early healing response due to an aged adaptive immune system leads to impaired bone repair, delayed healing or even formation of non-union. However, immuno-senescence is not limited to the adaptive immunity, but is also described for macrophages, main effector cells from the innate immune system. Beside regulation of pro- and anti-inflammatory signaling, macrophages contribute to angiogenesis and granulation tissue maturation. Thus, it seems likely that an altered macrophage function due to aging may affect bone repair at various stages and contribute to age related deficiencies in bone regeneration. To prove this hypothesis, we analyzed the expression of macrophage markers and angiogenic factors in the early bone hematoma derived from young and aged osteotomized Spraque Dawley rats. We detected an overall reduced expression of the monocyte/pan-macrophage markers CD14 and CD68 in aged rats. Furthermore, the analysis revealed an impaired expression of anti-inflammatory M2 macrophage markers in hematoma from aged animals that was connected to a diminished revascularization of the bone callus. To verify that the age related disturbed bone regeneration was due to a compromised macrophage function, CD14+ macrophage precursors were transplanted locally into the osteotomy gap of aged rats. Transplantation rescued bone regeneration partially after 6 weeks, demonstrated by a significantly induced deposition of new bone tissue, reduced fibrosis and significantly improved callus vascularization.

Keywords: CD14+ cells; aging; angiogenesis; bone regeneration; compromised healing; macrophage; monocyte.

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Figures

Figure 1
Figure 1
M2 Macrophage function is impaired in bone hematoma of aged rats. (A) The general monocyte maker CD14 and the general mocrophage maker CD68 show a significant reduced expression 3 and 7 days after osteotomy in hematoma tissue extracted from aged animals compared to young ones. (B) The makers CD80 and CCR7 that are predominantly expressed by M1 macrophages show higher expression levels in fracture callus tissue from aged animals at day 3. (C) Marker genes that are mostly expressed by M2 macrophages (CD163, CD206, Tie-2, Dectin) show a significantly diminished expression in hematoma tissue from aged animals compared to the expression found in fracture hematoma extracted from young animals. n = 4–5, *significant to young d3, *p < 0.05; **p < 0.001; #significant to young d7, #p < 0.05; ###p < 0.001, ANOVA.
Figure 2
Figure 2
Bone callus vascularization is compromised in aged rats. (A) Expression of the angiogenic transcription factor HIF-1α, the angiogenic growth factors PDGFα and VEGF and their respective receptors PDGFRα, PDGFRβ, and VEGFR is significantly reduced in callus tissue from aged rats extracted 3 and 7 days after osteotomy. n = 4–5, *significant to young d3; #significant to young d7, ANOVA on Ranks. (B) Vessel number evaluated in αSMA stained tissue sections. For each condition, the left picture shows the overview of the whole region of interest, the right picture shows the magnification of the region within the square–found in the respective overview mage. Femurs are placed in the same orientation, with the proximal side on the left and distal on the right side. y/d3, y/d7 n = 5, a/d3 n = 3, a/d7 n = 6, *significant to young d3, *p < 0.05, ***p < 0.001; #significant to young d7, #p < 0.05, ###p < 0.001, ANOVA.
Figure 3
Figure 3
Transplantation of CD14+ macrophage precursors partly rescues impaired bone regeneration in aged rats. (A) Exemplary 3D reconstructions from four individual animals receiving either PBMC or CD14+ cells locally at the osteotomy site. Bone healing was induced after CD14+ cell transplantation. (B) Mineral deposition was prone to be increased after CD14+ cell transplantation in the investigated ROI. (C) Formation of new trabeculae was significantly induced after CD14+ cell transplantation as indicated by an increased number and a reduced space between them. Thickness of the single trabeculae were unaffected by cell transplantation. n = 5, *significant to PBMC, p < 0.05, t-test.
Figure 4
Figure 4
Histological assessments verify improved bone healing after CD14+ cell transplantation aged rats. (A) Histomorphometric evaluations of mineralized, fibrotic, and cartilage tissue with the osteotomy gap, as well as distal and proximal to it, were done. The analysis revealed a significant induced deposition of mineralized bone matrix and by trend a diminished formation of fibrotic tissue, when aged animals received CD14+ cells locally at the osteotomy site. Number of αSMA positive vessel increased significantly in the region of interest after CD14+ cell transplantation. PBMCs n = 5, CD14+ n = 7, *significant to PBMC, p < 0.05. (B) Exemplary pictures of Movat-Pentachrom (mineralized bone, yellow/orange; collagen, yellow; cartilage, green/blue; osteoid, dark red; elastic fibers, orange/ref; nuclei, blue-black) and aSMA stained tissue sections. Femurs are placed in the same orientation, with the proximal side on the left and distal on the right side. Bar = 500 μm.
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