Suboptimal Level of Bone-Forming Cells in Advanced Cirrhosis are Associated with Hepatic Osteodystrophy

Abstract

Bone loss is common in advanced cirrhosis, although the precise mechanisms underlying bone loss in cirrhosis are unknown. We studied the profile and functionality of bone-forming cells and bone-building proteins in bone marrow (BM) of individuals with cirrhosis (n = 61) and individuals without cirrhosis as normal controls (n = 50). We also performed dual energy X-ray absorptiometry for clinical correlation. BM mesenchymal cells (MSCs) were analyzed for colony-forming units-fibroblasts and their osteogenic (fibronectin-1 [FN1], insulin-like growth factor binding protein 3 [IGFBP3], collagen type 1 alpha 1 chain [COL1A1], runt-related transcription factor 2 [RUNX2], and alkaline phosphatase, liver [ALPL]) and adipogenic ( adiponectin, C1Q, and collagen domain containing [ADIPOQ], peroxisome proliferator-activated receptor gamma [PPARγ], and fatty acid binding protein 4 [FABP4]) potentials. Colony-forming units-fibroblasts were lower in patients with cirrhosis (P = 0.002) than in controls. Cirrhotic BM-MSCs showed >2-fold decrease in osteogenic markers. Compared to controls, patients with cirrhosis showed fewer osteocytes (P = 0.05), osteoblasts, chondroblasts, osteocalcin-positive (osteocalcin+) area, clusters of differentiation (CD)169+ macrophages (P < 0.001, each), and nestin+ MSCs (P = 0.001); this was more apparent in Child-Turcotte-Pugh (CTP) class C than A (P < 0.001). Multivariate logistic regression showed low nestin+ MSCs (P = 0.004) as a predictor of bone loss. Bone-resolving osteoclasts were comparable among CTP groups, but >2-fold decreased anti-osteoclastic and increased pro-osteoclastic factors were noted in patients with CTP C compared to CTP A. Bone-building proteins (osteocalcin [P = 0.008], osteonectin [P < 0.001], and bone morphogenic protein 2 [P = 0.001]) were decreased while anti-bone repair factors (fibroblast growth factor 23 [P = 0.015] and dipeptidyl peptidase 4 [P < 0.001]) were increased in BM and peripheral blood; this was more apparent in advanced cirrhosis. The dual energy X-ray absorptiometry scan T score significantly correlated with the population of osteoblasts, osteocytes, MSCs, and CD169+ macrophages. Conclusion: Osteoprogenitor cells are substantially reduced in patients with cirrhosis and more so in advanced disease. Additionally, increased anti-bone repair proteins enhance the ineffective bone repair and development of osteoporosis in cirrhosis. Hepatology Communications 2018;0:0-0).

Figure 1

Cirrhosis versus control cases. (A) Individual colonies captured at 4× magnification for control and cirrhosis BM cells (left) and bar graph (right) showing average number of CFU‐F colonies (mean ± SE) generated per million BM mononuclear cells in control and cirrhotic BM. (B) Relative mRNA level of indicated osteogenic and adipogenic marker genes in cirrhosis with respect to control BM‐derived MSC after in vitro osteogenic and adipogenic differentiation. (C) Representative images of osterix‐, Runx1‐, and perilipin‐stained sections of control and cirrhosis BM (left); bar graph (right) showing number of osteogenic cells and adipogenic cells (mean ± SE) in cirrhosis and control BM. All immunohistochemistry images ×200; area 1.3 mm². Abbreviations: ADIPOQ, adiponectin, C1Q, and collagen domain containing; ALPL, alkaline phosphatase, liver; CFU‐F, colony‐forming units‐fibroblasts; COL1A1, collagen type I alpha 1 chain; FABP4, fatty acid binding protein 4; FN1, fibronectin‐1; hMSC, human MSC; mRNA, messenger RNA; PPARγ, peroxisome proliferator activated receptor gamma.

Figure 2

Images of BM biopsy in Cirrhosis and Control cases. (A) Panel I. Representative Images of cirrhosis and control BM (left) showing the osteoblast cells and bar graph (right) showing number of osteoblast (mean ± SE) in cirrhosis and control BM (H&E). Panel II. Cirrhosis and control BM (left) showing the osteocytes and bar graph (right) showing number of osteocytes (mean ± SE) in cirrhosis and control BM (H&E). Panel III. Alcian blue‐stained chondroblasts in cirrhosis and control BM biopsy section (left) and bar graph (right) showing number of chondroblasts (mean ± SE) in cirrhosis and control BM. (B) Panel I. Representing osteocalcin+ cells (mean ± SE) in cirrhosis and control BM biopsy section (left) and bar graph (right) showing number of osteocalcin+ cells in cirrhosis and control BM. Panel II. Osteocalcin+ area (mean ± SE) in cirrhosis and control BM biopsy section (left) and bar graph (right) showing percentage osteocalcin+ area in cirrhosis and control BM. Panel III. Showing osteonectin+ cells (mean ± SE) in cirrhosis and control BM biopsy section (left) and bar graph (right) showing osteonectin in cirrhosis and control BM (C) Nestin+ BM MSCs in cirrhosis and control BM biopsy section (left) and bar graph (right) showing number of these cells in cirrhosis and control BM. (D) CD169+ BM macrophage (mean ± SE) in cirrhosis and control BM biopsy section (left) and bar graph (right) showing number of CD169+ cells in cirrhosis and control BM. (All image magnification ×200, area 1.3 mm²). Abbreviations: BM, bone marrow; H&E, hematoxylin and eosin.

Figure 3

Osteoclasts and osteoclast precursors. (A) Representative photomicrographs showing mononucleated osteoclastic progenitor cells (red, with orange arrows) in control and in cirrhosis subgroups CTP A, B, and C (TRAP staining, magnification ×200). (B) Bar graph showing mean ± SE of osteoclastic progenitors in control and cirrhosis subgroups CTP A, B, and C. (C) Multinucleated osteoclasts (red) in control and in cirrhosis subgroups CTP A, B, and C (TRAP staining, magnification ×200). Osteoclast in CTP C shows cytoplasmic podia, indicating the activated osteoclast form (black arrow). Activation is less in CTP B, and there is no activated form in CTP A and control cases. (D) Bar graph showing mean ± SE of osteoclasts in control and in CTP A, B, and C. (E) Relative mRNA level of indicated osteoclast effecter genes in BM tissue of CTP C with respect to CTP A patients with cirrhosis. Abbreviations: CTP, Child‐Turcotte‐Pugh; IGFBP‐1, insulin like growth factor protein‐1; mRNA, messenger RNA; TRAP, tartrate‐resistant acid phosphatase.

Figure 4

Graphs showing BM plasma (left) and peripheral blood plasma (right) of the same patients displaying levels of bone‐building proteins. (A) Osteocalcin, (B) osteonectin, (C) bone morphogenic protein, and (D) anti‐bone building proteins FGF23 and (E) DPP4 in control and patients with cirrhosis with Child‐Turcotte‐Pugh (CTP) A, B, or C { median (interquartile range) }.

Figure 5

Distribution graphs. (A) Panel I. Osteoblasts. Panel II. Osteocytes. Panel III. Chondroblasts. (B) Panel I. Osteocalcin+ cells. Panel II. Percentage osteocalcin+ area. Panel III. Osteonectin+ cells. (C) Nestin+ MSCs. (D) CD169+ BM macrophages (all expressed in mean ± SE) in BM sections of patients with cirrhosis with normal bone density, osteopenia, and osteoporosis; evaluated by T score on bone densitometry scan.

Figure 6

Concept diagram showing the altered status of bone‐forming cells in an advanced stage of cirrhosis.