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Hyperbilirubinemia May Activate Histiocytic Osteolysis

Tomoki Nakamura*, Katsuyuki Kusuzaki*, Takao Matsubara*, Haruhiko Satonaka, Ken Shintani*, Touru Wakabayashi*,Akihiko Matsumine*, Atsumasa Uchida*

*Department of Orthopaedic Surgery, Mie University Faculty of Medicine, Tsu, Mie, Japan

Address for Correspondence:

Katsuyuki Kusuzaki MD, Department of Orthopaedic Surgery, 
Mie University Faculty of Medicine,
Edobashi 2-174 Tsu Mie 514-8507, Japan
Tel: +18 59 231 5022
Fax: +81 59 231 5211


We recently encountered a 6-year-old boy with Alagille syndrome, which is characterized by remarkable hyperbilirubinemia caused by cholestasis due to the paucity of interlobular bile ducts in the liver. This patient had a pathological fracture of the femur with local bone atrophy, with malunion and insufficient callus formation. Thus, after liver transplantation, we performed a correction osteotomy. Macroscopically, the femur was stained dark green and histology of the resected bone at the site of the malunion revealed the presence of many histiocytes and osteoclast-like multinucleate giant cells containing bilirubin particles in the cytoplasm. The multinucleate giant cells were found to have caused bone resorption. These findings suggested that bilirubin might activate macrophages to form osteoclast-like multinucleate giant cells, resulting in bone resorption and osteoprosis.

J.Orthopaedics 2007;4(2)e17


Recently, we encountered a rare case of Alagille syndrome, which manifests as severe jaundice due to cholestasis caused by a paucity of interlobular bile ducts in the liver associated with skeletal malformations and cardiovascular dysfunction. The patient, who was a 6-year-old boy, had been known to have marked hyperbilirubinemia since birth. He was found to have a pathological fracture of the shaft of the femur, with malunion due to local bone fragility.  In this context, it has been reported previously that hyperbilirubinemia inhibits osteoblast proliferation and induces osteoprosis6).However, the findings in this case suggested the possibility that hyperbilirubinemia may also activate histiocytes or induce the formation of osteoclast-like multinucleate giant cells. There are no reports in the literature of the histological findings in the bone in patients with marked and persistent hyperbilirubinemia.

Case Report:

The patient was a 6-year-old Indonesian boy. He was taken to the city hospital in Bali at the age of 9 months for persistent jaundice. He had the characteristic face of Alagille syndrome and the liver biopsy findings confirmed the case as one of Alagille syndrome. Although the patient was then initiated on vitamin ADEK supplementation, ursodeoxycholic acid and rifampicin, the liver dysfunction and hyperbilirubinemia had worsened by the time the patient was 5 years old.

He was referred to our hospital for living- donor liver transplantation from his father, after getting private and official financial support from both Japan and Indonesia. Two months prior to his visit to our hospital, he had developed a pathological fracture of the left femoral shaft caused by mild trauma (slipping) and had been given a cast for treatment. However, he could not walk due to the development of an antero-lateral convex deformity caused by delayed union. At the initial visit to our hospital, he was found to be malnourished and exhibited stunted growth, with a height of 80 cm and body weight of 12 kg. He had all of the diagnostic features of Alagille syndrome, including the characteristic face, mild peripheral pulmonary artery stenosis, butterfly vertebrae, posterior embryotoxon, and hyperbilirubinemia. Blood examination revealed anemia (hemoglobin, 9.2g/dl) and liver dysfunction with high serum AST (223U) and ALT (151U), hyperbilirubinemia (12.8mg/dl), high serum total cholesterol (909mg/dl), and high serum ALP (2736U).

A plain x-ray of the left thigh revealed an antero-lateral convex deformity of the left femoral shaft caused by malunion of a fracture with poor callus formation, cortical bone atrophy and osteoprosis, associated with osteolytic lesions (Fig. 1). Osteoprosis of the right femur and spine was also found (Fig. 2). The patient could not move without a wheel- chair. After successful liver transplantation, his parents were anxious for the fracture also to be treated so that the patient could start walking before they returned to Indonesia. Because of satisfactory postoperative recovery of his liver condition, we decided to perform a correction osteotomy and osteosynthesis with internal fixation using a plate and screw. After 6 weeks, partial weight bearing became possible because of complete bone union (Fig. 3), and finally, by two months later, the patient could walk without any support.

Figure 1: X-ray of the left femur with mal-union.There was the antero-lateral convex deformity at the left femur shaft with poor callus formation, cortical bone atrophy and osteoprosis accompanying osteolytic lesions (arrows).

Figure 2: X-ray of the right femur (A) and lumbar spine (B) Both bones are osteoprotic.

 Figure 3: X-ray of the left femur at surgery of correction osteotomyand after 6 weeks. Bone union is completed at 6 weeks after surgery

During the osteotomy, the femoral cortex and callus around the fracture site were macroscopically observed to show dark green staining a very unusual finding, suggesting bilirubin deposition. Coronal sections of the femoral shaft also showed deep green staining of the cortex as well as the callus (Fig. 4).

Histopathologically, numerous osteoclast-like multinucleate giant cells were observed at the surface of the lamellar bone of the cortex and callus, associated with marked histiocytic infiltration of the bone marrow. These cells contained brown pigment in the cytoplasm (Fig. 5). The pigment was identified as bilirubin based on the negative staining with Berlin-blue and positive staining by Hallís method, which is specific for bilirubin (Fig. 6).

Figure 4: Macroscopic finding of the femoral bone at fracture site (A) and cross section of resected bone (B) during surgery of correction osteotomy. The femoral cortex and callus around the fracture site macroscopically shows dark green color. The coronal section of the femur also shows deeply dark green color in cortex as well as callus


Figure 5: Histological findings of resected bone (H.E. A, B: x10 objective lens C: x40 objective lens) There were many multinuclear giant cells like osteoclast at surface of lamellar bone of the cortex and callus and many histiocytes in the bone marrow. These cells contained brown pigments in the cytoplasms (arrows)

Figure 6: Histochemical findings of intracellular pigments after Berlin-blue staining (A) for hemosiderin and Hallís method for bilirubin(B)  The intracellular pigments were not stained with Berlin-blue but stained by Hallís method.

Discussion :

Alagille syndrome is a very rare disease, occurring in 1 in 100,000 births, with an equal gender incidence. An autosomal dominant pattern of inheritance with low penetrance and a great variability of expression have been reported. The Alagille gene has been identified in the 20pl2 region. The syndrome manifests as a multi-system disorder involving the liver, heart, eyes, face, and skeleton. Since it is characterized by cholestasis because of a paucity of interlobular bile ducts 1), 2), patients frequently have hyperbilirubinemia.

It is well known that patients with chronic liver disease have an increased prevalence of osteoporosis because of calcium malabsorption caused by low levels of 25-hydroxy vitamin D3 and hyperbilirubinemia3). However, the underlying mechanism of the relation of osteoprosis to hyperbilirubinemia is still unclear. Guanabens et al reported that patients of primary biliary cirrhosis with osteoporosis had higher serum bilirubin levels than those without osteoporosis4). Ormarsodottir et al also reported that a high serum bilirubin level was associated independently with increased bone loss at the femoral neck in patients with chronic liver disease5). Janes et al demonstrated experimentally that exposure to excessive levels of bilirubin inhibited the proliferation of osteoblasts in cell culture6). However, there are no reports on the relation between the bilirubin level and osteoclast function.

The present case with long-term hyperbilirubinemia showed radiographic evidence of systemic osteoprosis and had a pathological fracture of the femur due to local bone fragility. During surgical correction of the malunited fracture in the femur by osteotomy, we found the femur to be stained dark green, a very unusual finding indeed. This macroscopic color was very similar to that of condensed bile juice. Histological examination revealed the presence of numerous histiocytes in the bone marrow and osteoclast-like multinucleate giant cells lining the resorbed bone surface in the femoral cortex. Both cells contained large amounts of brown pigment in the cytoplasm. Histochemical study revealed that these particles were bilirubin and not hemosiderin or other pigments derived from iron. The multinucleate giant cells closely resembled osteoclasts, however, the number of nuclei was smaller and the cell size was smaller than the corresponding values for ordinary osteoclasts, further they were surrounded by numerous histiocytes. Therefore, we believe that these multinucleate giant cells were histiocytic in origin. We speculate that the histiocytes first phagocytose serum bilirubin in the bone marrow and actively proliferate, with some of them fusing with each other to form multinucleate giant cells, causing bone resorption. This phenomenon suggests that bilirubin may induce osteoprosis by activating histiocytes, a mechanism similar to that of polyethylene- induced particle disease causing loosening of the stem after artificial joint replacement7), 8). The major cause of bone resorption around total joint protheses is the inflammatory response to the wear debris of polyethylene. Particles derived from the wear debris cause macrophage activation and phagocytosis. Gallo et al7) stated that aseptic loosening and osteolysis after hip arthroplasty is caused predominantly by osteoclasts, mediated mainly by an osteoprotegerin ligand(RANKL) and TNF-α. RANKL has been shown to be expressed in activated macrophages, osteoblasts, and lymphocytes7). We believe that bilirubin may also behave similarly to these particles.

This is presumably the first report of the histological findings in the bone in patients with marked and persistent hyperbilirubinemia.

Reference :

  1. Dinesh MD, FRCA, Mohammed MD et al; The Alagilleís syndrome and its anaesthetic considerations: Paediatric Anesthesia Vol 8(1) 79-82, 1998.

  2. G. Maldini, E Torri, A Lucianetti et al; Orthotopic liver transplantation for Alagille syndrome: Transplantation Proceedings Vol 37(2) 1174-1176, 2005.

  3. A. Bagur, C. Mautaken, J. Findor et al; Risk factor for the development of vertebral and total skeleton osteoporosis in patients with primary biliary cirrhosis: Calcif Tissue Int 63 385-390, 1998.

  4. N. Guanabens, A. Pares, I Ros et al; Severity of cholestasis and advanced histological stage but not menopausal status are the major risk factors for osteoporosis in primary biliary cirrhosis: J Hepatol Vol42(4) 573-577, 2005.

  5. S. Ormarsottir, O Ljunggren, H Mallmin et al; Increased rate of bone loss at the femoral neck in patients with chronic liver disease: Europ J Gastroenterol Hepatol Vol 14(1), 2002.

  6. C. Janes, Dickson ER, Bond S; Role of hyperbilirubinemia in the impairment of osteoblast proliferation associated with cholestatic jaundice: J Clin Investigation Inc. Vol(95) 2581-2586, 1995.

  7. Gallo J, Kaminek P, Ticha V et al; Particle disease. A comprehensive theory of periprosthetic osteolysis: a review: Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 146(2):21-28, 2002.

  8. A V Parwani, B Yang, D P Clark et al; Particle disease: cytopathologic findings of an unusual case: Diagn Cytopathol. Vol31(4):259-262, 2004.


This is a peer reviewed paper 

Please cite as :Tomoki Nakamura: Hyperbilirubinemia May Activate Histiocytic Osteolysis

J.Orthopaedics 2007;4(2)e17





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