Why does intermittent hydrostatic pressure enhance the mineralization process in fetal cartilage?

Abstract

The purpose of this study was to determine which factor is the most likely one to have stimulated the mineralization process in the in vitro experiments of Klein-Nulend et al. (Arth. Rheum., 29, 1002–1009, 1986), in which fetal cartilaginous metatarsals were externally loaded with an intermittent hydrostatic pressure, by compressing the gas phase above the culture medium. Analytical calculations excluded the possibility that the tissue was stimulated by changes in dissolved gas concentration, pH or temperature of the culture medium through compression of the gas phase. The organ culture experiments were also mechanically analyzed using a poroelastic finite element (FE) model of a partly mineralized metatarsal with compressible solid and fluid constituents. The results showed that distortional strains occurred in the region where mineralization proceeded. The value of this strain was, however, very sensitive to the value of the intrinsic compressibility modulus of the solid matrix (K_s). For realistic values of K_s the distortional strain was probably too small (about 2 μstrain) to have stimulated the mineralization. If the distortional strain was not the factor to have enhanced the mineralization process, then the only candidate variable left is the hydrostatic pressure itself. We hypothesize that the pressure may have created the physical environment enhancing the mineralization process. When hydrostatic pressure is applied, the balance of the chemical potential of water across cell membranes may be disturbed, and restored again by diffusion of ions until equilibrium is reached again. The diffusion of ions may have contributed to the mineralization process.

Introduction

The process of endochondral ossification in the fetal skeleton commences in the diaphysis where the primary ossification center is formed by hypertrophied chondrocytes. In a short period of time, a calcium phosphate is deposited in the matrix around these hypertrophied cells. Hence, the chondrocytes hypertrophe before the matrix mineralizes. The processes of hypertrophy and mineralization progress towards the distal ends of the fetal bone, where the epiphyses are formed. Finally, the mineral is resorbed by osteoclasts and replaced by bone tissue. In vivo, the mineralization process in fetal mouse metatarsals starts at 16 days of gestational age. It was observed that this process coincides with the first muscle contractions of the feet (Burger et al., 1991). The deposition of calcium phosphate might, therefore, be stimulated by mechanical loads.

To study the influence of mechanical loads on the mineralization process, Klein-Nulend et al. (1986) performed in vitro culture experiments on 16-day-old fetal mouse metatarsals. The organs were loaded for a period of five days with a cyclic hydrostatic pressure (13 kPa, 0.3 Hz). The pressure was applied through compression of the gas phase (5% CO₂ in air) above the culture medium (Klein-Nulend et al., 1986). The results showed that loaded metatarsals had a mineralized diaphyseal part which was two to three times longer than that of the unloaded controls (Klein-Nulend et al., 1986). Hence, it was concluded that the mineralization process in fetal cartilage is stimulated by mechanical load. However, it is also possible that physical changes of the culture medium affected the results of the experiments.

It is known that the physical conditions of the culture medium may be important for the mineralization process of the fetal metatarsal. For instance, whether the metatarsal mineralizes or not depends, amongst others, on the pH and the temperature of the medium. It has already been shown that chondrocytes in vitro can alter their activity by changes in their biophysical environment, such as osmotic pressure (Urban, 1994; Urban and Hall, 1994), fluid flow (Kim et al., 1994, Kim et al., 1995), hydrostatic pressure (Urban, 1994; Urban and Hall, 1994; Lammi et al., 1994; Parkkinen et al., 1994, Parkkinen et al., 1995), electrical potential gradients (Kim et al., 1995; Frank and Grodzinsky, 1987), and pH (Gray et al., 1988). As the hydrostatic pressure in the in vitro culture experiments was applied through a gas phase, it was suggested that it is possible that the mineralization process was stimulated by changes in dissolved gas concentration or pH, rather than by the pressure itself (Urban, 1994).

Wong and Carter (1990) performed a finite element (FE) stress analysis of the organ culture experiments of Klein-Nulend et al. (1986). They suggested, based on their results, that the mineralization process might have been stimulated by shear stresses at the cartilage/mineralized cartilage interface, as local effects of the external hydrostatic pressure. In this FE analysis, however, they considered the tissue as a linear elastic material. In fact, cartilage consists of a solid phase, mainly collagen and proteoglycans, and a fluid phase of interstitial water. Biphasic tissues like this are known to display strong non-linear, time dependent deformational behavior when loaded (Mow et al., 1980; Spilker et al., 1988). Hence, it is questionable whether the stress patterns determined in a linear-elastic FE analysis are realistic. In addition, not only deformation, but also pressure gradients and interstitial fluid flow play roles that might affect the mineralization process.

The following questions were addressed in this study: (1) Is it likely that the mineralization process in the in vitro organ culture experiments of Klein-Nulend et al. (1986) was stimulated by one of the physical changes in the culture medium, e.g. dissolved gas concentration, pH, or temperature? (2) Is it likely that the mineralization process was stimulated by one of the mechanical factors in the tissue, e.g. internal strain, stress, fluid pressure and fluid flow, as local effects of the external hydrostatic pressure? To answer these questions, we analyzed the physical changes of the culture medium under the experimental loading conditions. In addition, a poroelastic FE analysis was performed to determine the distribution of mechanical variables at the mineralization front. Information about the mineralization process may be important for the prevention and treatment of musculoskeletal developmental deformities.