Bone quality and biomechanical function: A lesson from human ossicles

Highlights

• Bone quality from ossicles of human middle ear was rarely investigated.

• Morphology, microarchitecture, and mechanical and intrinsic properties were assessed in incuses in inflammatory (cholesteatoma) and healthy conditions.

• Noninflammatory incuses were composed of compact, rarely remodeled, more calcified tissue and were harder than femoral cortical bone.

• In inflammatory incuses, microarchitecture was degraded and organic tissue was abundant in bone cavities.

• Bone quality of incuses, well adapted to their unique function of transmission of vibration, is impaired in inflammatory conditions (cholesteatoma).

Abstract

In humans, the middle ear contains a chain of three ossicles with a major highly specific mechanical property (transmission of vibrations) and modeling that stops rapidly after birth. Their bone quality has been rarely studied either in noninflammatory ossicles or in those from ears with chronic inflammation. Our primary goal was to assess bone microarchitecture, morphology and variables reflecting bone quality from incuses, in comparison with those from human femoral cortical bone as controls. Secondly, the impact of chronic inflammation on quality of ossicles was documented. The study was performed on 15 noninflammatory incuses from 15 patients (35 ± 32 years, range: 2–91). Comparisons were performed with 13 inflammatory incuses from 13 patients (55 ± 20 years, range: 1–79) with chronic inflammation of the middle ear, essentially cholesteatoma. Microarchitecture and bone mineral density (BMD) were assessed by microcomputed tomography. Microhardness was measured by microindentation. Mineral and organic characteristics were investigated by Fourier transform infrared microspectroscopy. Noninflammatory incuses were composed of a compact, well mineralized bone without bone marrow and with sparse vessels. Remodeling activity was rarely observed. Woven or lamellar textures and numerous osteocytes were observed. In inflammatory incuses, architecture was degraded, organic tissue was abundant and bone cavities contained fibrocellular tissue and adipocytes. BMD of noninflammatory incuses was significantly higher than BMD from both control bones (4 embedded cortical femoral bone samples; age: 72 ± 15 years, range: 50–85) and inflammatory incuses. Noninflammatory incuses were less hard than both control bone (8 cortical femoral bone samples; age: 49 ± 18 years, range: 24–74) and inflammatory incuses. All incuses were more mineralized and less mature than controls. In conclusion, bone quality of incuses (dense, well mineralized, hard) is well adapted to their function of sound transmission. In inflammatory condition, incuses were degraded, thus explaining the decline of hearing. Moreover, microhardness was found higher than in noninflammatory incuses. Compared to bone with remodeling, the mineralization index in incuses does not explain variation of microhardness. Interestingly, a linear multiple regression model indicated that a combination of two variables, i.e., crystallinity index (crystal size/perfection) and carbonation (incorporation of carbonate ions in apatite) explains 26% of the increase in microhardness variability. Because the low remodeling level of ossicles could not prevent the reversibility of their degradation which impacts audition quality, an early management of ear inflammation in chronic otitis is recommended.

Introduction

In humans, the middle ear contains three ossicles (malleus, incus and stapes). They are special tiny bones whose major function is sound transmission. The vibrations concerned have a frequency range from 20 to 20,000 Hz (mostly 250 to 8000 Hz, voice is 1000 to 4000 Hz) and an amplitude of 10^− 9 m. Furthermore, a unique particularity of ossicles is that their final size is obtained early during life of human beings [1]. It is a bone tissue rapidly modeled after birth (tissue volume is not correlated with age) then poorly remodeled. Features such as the presence of a dense meshwork of collagenous fibers organized in an interlacing fashion (woven texture), a smooth fibrous appearance and limited vascular channels and osteocytes have previously been described in stapes crura and ossicles [2], [3].

Ossicles, like other bones, are complex and structured materials. Usually, bone mechanical behavior results from an interaction between the properties of each level of its structural hierarchy, i.e., macroscopic morphology, biochemical composition (collagenous and noncollagenous proteins), organization of tissues, and mineralization quality and quantity [4]. The degree of mineralization of bone and the characteristics of the mineral deposited are major determinants of bone strength in the elastic domain, whereas organic matrix and crosslinks are more implicated in plastic behavior [5], [6]. Bone remodeling activity acts as a regulator of the degree of mineralization and of the distribution of mineral at the tissue level, directly impacting bone mechanical properties. An augmentation of the remodeling induces a decrease of the lifespan of BSUs (Bone Structural Units, i.e., osteons in cortical bone or cancellous packets), increasing the time available for the secondary mineralization. Remodeling also defines the average collagen maturity and mineral crystallinity. A comprehension of bone quality in terms of bone density, mineralization, crystallinity, collagen and mineral maturities, is thus needed for incuses, to characterize the consequences of changes at various levels on whole bone comportment and function. Primary and secondary osteons, as well as bone packets, are observed in incus from both young and old patients, regardless of the presence or absence of inflammation.

Cholesteatoma is a benign tumor of the temporal bone resulting in abnormal presence of proliferating squamous epithelium from the tympanic membrane in the middle ear. The pathogenesis of acquired cholesteatoma is in a vast majority of cases, caused by migration of this epithelium from lateral (external auditory canal) to medial (middle ear spaces) of the auditory apparatus. It was shown that during inflammatory processes such as chronic otitis media with or without cholesteatoma, deterioration of the ossicles could seriously attenuate sound conduction [7], [8]. However, only few data exist concerning the bone quality of ossicles. Morphological studies carried out on ear stapes [2] and ossicles [3] in humans have shown that these bones display peculiar features when viewed both macroscopically and microscopically. Their external shape and inner architecture cannot be classified neither as long bones nor as flat or even short bones, and they are exclusively made up of compact bone. As regards their texture, they are made up mainly of woven-fibered bone tissue and, to a lesser extent, of parallel-fibered and lamellar bone tissue. In all instances, it is important to emphasize that incuses are bone laid down during bone growth and modeling, and that these bones are thus only partly remodeled. Remodeling is restricted to certain regions, namely at the head of the stapes, the body of the incus and the head of the malleus.

To evaluate the relationship between the quality of the ossicles and their function, our primary goal was to assess bone microarchitecture, morphology and various variables reflecting intrinsic bone quality in both noninflammatory and inflammatory incuses, and to compare data with those obtained from human femoral cortical bone samples. Secondly, the impact of chronic inflammation on the quality of ossicles was documented. To do this, different variables reflecting bone quality were investigated such as bone and tissue mineral densities, histological features (calcified and osteoid tissues, texture of tissue, bone cells, bone marrow), mineralization, mineral quality at the tissue and crystal levels, quality of organic matrix and the microhardness of ossicles.