Fibro-osseous (FOL) and Degenerative Joint Lesions in Female Outbred NIH Black Swiss Mice

Abstract

A review of spontaneous bone and joint lesions in female aging NIH Black Swiss mice (Cr:NIH BL[S]) revealed a high incidence of fibro-osseous lesions (FOL; 89%) and degenerative joint lesions (90%). FOL was characterized by the replacement of bone marrow by fibrovascular tissue and was first seen at 59 weeks of age, most commonly in the nasal bone, femur, and tibia. FOL in female Black Swiss was often accompanied by reproductive-tract lesions, including ovarian atrophy and uterine cervical dysplasia with hydrometra. Mild degenerative femorotibial joint lesions developed by 59 weeks and progressed to full-thickness articular cartilage ulceration and osteophyte development by 75 weeks; joint inflammation was minimal. Although the underlying etiology of FOL remains unknown, an accurate assessment of FOL and degenerative joint disease as background lesions in this stock is necessary to interpret lesions in genetically engineered mice produced from this outbred line.

The fibro-osseous lesion (FOL) in aging mice is characterized by the replacement of bone marrow with fibrovascular stroma containing prominent fibroblastic cells, osteoblasts, and osteoclasts (Albassam and Courtney, 1996). The lesion has been seen within the tibia, femur, sternum, and vertebrae and is distinguished from fibrous osteodystrophy by the absence of concurrent renal or parathyroid changes. Aging females are predisposed and develop spontaneous age-related FOL that is often accompanied by uterine or ovarian lesions (Albassam et al., 1991; Dodd and Port, 1987). Diethylstilbestrol and misoprostol administration also induces a high incidence of FOL in female mice and a lower rate in males (40% to 100% and 0 to 1%, respectively; Albassam et al., 1991; Highman et al., 1981; McAnulty and Skydsgaard, 2005; Sass and Montali, 1980). FOL may be preneoplastic, reportedly progressing to osteosarcoma in a low number of cases (McAnulty and Skydsgaard, 2005). Although degenerative disease in the femorotibial joint is a commonly cited aging change in certain mouse strains (Schunke et al., 1988; Stanescu et al., 1993; Yamamoto et al., 1999; Yamamoto et al., 2005), previous studies have not reported a high incidence of concurrent FOL and degenerative joint disease.

This study examined the incidence and severity of FOL and degenerative joint disease in aged female NIH Black Swiss mice, a new outbred background strain developed for the production of genetically engineered (GEM) mice.

A cohort of 62 female NIH Black Swiss outbred mice were entered in an aging study that adhered to NIH and National Cancer Institute (NCI) Animal Care and Use Committee guidelines. The mice were developed though a cross of C57BL/6N and N:NIH Swiss outbred mice and received by NCI in 2000. The mice were maintained on standard pelleted dry food (Purina NIH Open 31) and acidified water and housed on corn cob bedding; animals were removed from the study at time of natural death, euthanasia if moribund, or the end of the study at 106 weeks of age (the range of ages at necropsy was 16–106 weeks). Routine tissues were collected at complete necropsy, fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 4 to 6 μM, and stained with hematoxylin and eosin (H&E); bone samples were decalcified with formic acid before processing into paraffin blocks. Tissues examined included all major visceral and thoracic organs, central and peripheral nervous system, ears, nasal cavity, vertebrae, sternebrae, femorotibial joint (transverse and sagittal sections), and any additional grossly observed lesions. H&E sections of femur, tibia, frontal nasal bone, vertebral column, and sternum were scored under light microscopy for the presence and extent of FOL, degenerative joint disease, and any additional bone lesions.

Of the 62 mice on study, 6 were euthanized or died naturally within the first 31 weeks. Although this group of mice did not have FOL in bone sections examined, 4 of the 6 had mild to severe infiltration of lymphoma in at least 1 osseous site, including the frontal nasal bone, vertebrae, sternebrae, femur, and tibia. The remaining 56 mice were on study 59 to 106 weeks; 55 of those mice had FOL in at least 1 site at the time of death, and most mice had lesions in multiple bones (Table 1). The mouse that did not have any FOL in sections examined was euthanized at 83 weeks of age; histological changes in bone and other tissues were otherwise comparable to mice removed from the study between 80 and 90 weeks. The highest incidence of FOL was noted in step sections through the frontal nasal bone, with lesions most commonly in mid- and caudal transverse sections. Sternal FOL was most common in the central portion of the third through fifth sternebrae. Vertebral lesions were most common in the lumbar region and limited to the vertebral bodies. The majority of FOL in sagittal femorotibial and transverse tibial sections was within the metaphysis, distal femoral diaphysis, and proximal tibial diaphysis.

Mild FOL was characterized by limited local replacement of marrow, cancellous, and cortical bone, most often within the inner cortical region of long bones and the central potions of flat and cuboidal bones. Foci were arranged in small clusters connected by ribbons of fibrous stroma (Figure 1A) that occasionally included entrapped hematopoietic tissue; rarely, there were bone spicules and irregular trabeculae lined by prominent osteoblasts. Osteoclasts were present but not numerous, with 1 to 2 prominent osteoclasts per individual focus (Figure 1B). Vascular tissue ranged from capillaries and capillary clusters to large, open vascular channels within the dense, eosinophilic, and poorly organized fibrovascular stroma. Severe FOL involved replacement of the marrow spaces and cortical bones by a solid sheet of fibrovascular tissue. In several older animals, this progressed to complete effacement of sternebrae physeal endplates. Except for focal lymphoma in several of the oldest animals, bone lesions were devoid of nuclear or cellular atypia to suggest a neoplastic or preneoplastic change.

No degenerative femorotibial joint changes were observed grossly in any mice that were removed from the study before 31 weeks. In contrast, each of the 56 older mice had femorotibial degenerative joint changes. Mice older than 59 weeks of age often had firm, raised protuberances visible grossly along the medial and lateral surfaces of femorotibial joints (Figure 1C), resulting in reduced range of motion. Histologically, lesions before 75 weeks were generally mild and included minimal surface roughening and fibrillation of articular cartilage, loss of proteoglycan matrix, and minimal chondrocyte cloning. Lesions after 75 weeks included mild to severe degeneration characterized by focal, full-thickness articular cartilage ulceration within the tibial plateau and milder cartilage degeneration of the proteoglycan matrix and fibrillation of the corresponding femoral condyles (Figure 1D, 1E); the ulceration was often accompanied by severe eburnation and sclerosis of underlying subchondral bone (Figure 1F). The synovial membrane was markedly thickened with minimal numbers of infiltrating lymphocytes and macrophages. Extensive peripheral articular cartilage fibrillation and osteophyte development were prominent.

Additional common nonskeletal lesions in the 62 mice included lymphoma (39%), pheochromocytoma (25%), ovarian atrophy (79%), and cervical dysplasia (57%) with consequential hydrometra (78%); there was no correlation between severity of the reproductive and bone lesions in older animals. Increased splenic weight was seen in cases of lymphoma but had no correlation with severity of FOL replacement of bone marrow. No late-stage renal or parathyroid lesions were noted in any of the 62 mice.

The absence of renal or parathyroid changes that could produce bone lesions supports the characterization of these fibrovascular bone changes as FOL and not fibrous osteodystrophy. The presence of concurrent ovarian and uterine lesions is in accordance with previous experimental studies and lends further evidence to the pathogenesis involving an underlying hormonal imbalance; given the high incidence of FOL in certain strains, specific genetic backgrounds may further enhance a hormonal effect. Previous studies in B6C3F1 mice have noted the development of early fibro-osseous–like lesions by 32 weeks (Albassam et al., 1991); as no mice were removed from study between 31 and 51 weeks, it remains to be determined if FOL develops before 59 weeks in this strain. Potential explanations for the absence of FOL in younger animals include a lack of precursor reproductive-tract lesions and the possibility that initial FOL development was masked by severe lymphoma in younger mice. Since young female NIH Black Swiss mice breed well, it is assumed that ovarian function is normal in the breeding mice and that the ovaries are not atrophied as in aged mice.

The femorotibial degenerative changes produced in this strain are similar to spontaneous human osteoarthritis, which is characterized by a gradual age-related onset and minimal inflammation. Although the slow onset of degenerative joint lesions in NIH Black Swiss mice is more consistent with the development of the human disease than the commonly used rapid-onset surgical models, the length of time needed to produce lesions and variation in lesion severity would likely be limiting for the use of these mice in therapeutic studies. Ultimately, the increased use of NIH Black Swiss in the production of GEM necessitates an accurate assessment of the incidence, distribution, and severity of strain-dependent bone and joint aging changes to prevent attributing background lesions to specific gene effects.