Osteocalcin and osteopontin influence bone morphology and mechanical properties

Abstract

Osteocalcin (OC) and osteopontin (OPN) are major non-collagenous proteins (NCPs) involved in bone matrix organization and deposition. In spite of this, it is currently unknown whether OC and OPN alter bone morphology and consequently affect bone fracture resistance. The goal of this study is to establish the role of OC and OPN in the determination of cortical bone size, shape, and mechanical properties. Our results show that Oc^−/− and Opn^−/− mice were no different from each other or wild type (WT) with respect to bone morphology (P > 0.1). Bones from mice lacking both NCPs (Oc^−/− Opn^−/−) were shorter with thicker cortices and larger cortical areas compared to the WT, Oc^−/−, and Opn^−/− groups (P < 0.05), suggesting a synergistic role for NCPs in the determination of bone morphology. Maximum bending load was significantly different among the groups (P = 0.024), while tissue mineral density (TMD) or measures of stiffness and strength were not different (P > 0.1). We conclude that the removal of both OC and OPN from bone matrix induces morphological adaptation at the structural level to maintain bone strength.

Introduction

An increase in fracture risk is generally attributed to a reduced load-bearing capacity of bones.^1,2 Bone apposition and resorption influence bone quantity and consequently morphology (traits specifying size and shape). It has been shown that slender bones (narrow relative to length) contain a greater amount of interstitial bone tissue compared with robust bones.³ However, a large interstitial bone area will cause the bone to be more susceptible to damage accumulation and to have lower flexibility and reduced material level toughness.⁴ Therefore, functional adaptation, the interaction between bone morphology and bone tissue quality (traits specifying tissue-level material properties) exist to reduce catastrophic failure. Functional adaptation is successful when variation in one trait is compensated by changes in other traits.⁵

The interaction between bone morphology and tissue quality is influenced by biological processes, such as modeling and remodeling. Thus, the mechanical function of bone can be achieved by acquiring a different combination of structural traits. However, identifying traits that are functionally related and determining how they define whole-bone stiffness and strength is complex and challenging. Genetically engineered mouse models offer an opportunity to see the variation of traits in bone when genes encoding for key proteins involved in bone development and homeostasis are removed.⁶

Non-collagenous proteins (NCPs) are integral components of bone extracellular matrix (ECM) and exhibit multifunctional roles. While several NCPs act as structural elements of the ECM,⁷ their contribution to the determination of bone morphology and strength remains largely unknown. Osteocalcin (OC) and osteopontin (OPN) are major NCPs⁸ that play key roles in both the biological and mechanical functions of bone. OC and OPN are produced during bone formation, late in the mineralization process, and they control––directly and/or indirectly–one mass, mineral size, and orientation.^9–11 They are also involved in organizing the ECM, coordinating cell–matrix and mineral–matrix interactions. Both proteins also play structural roles in bone and determine bone’s propensity to fracture.¹² Consequently, these proteins may regulate whole-bone structure and morphology, affecting bone mechanical properties.

The objective of this study is to establish whether OC and OPN play a role in the determination of bone size, shape, and strength. The ability of these proteins to act as structural molecules at the nanoscale (due to their involvement in bone matrix organization) may be translated to the macroscale (affecting bone morphology). This work will improve our current understanding of the contribution of NCPs to bone mechanical properties.

Methods

Specimen preparation

Forty-eight 6-month-old male Oc^−/− Opn^−/−, Oc^−/− Opn^−/−, and C57BL/6J wild-type littermates (WT) mice were used in this study. Animals were fed a normal standard diet and maintained in accordance with Yale animal care guidelines. Fresh frozen forearms were randomly harvested from each animal (n = 12 animals per group), and all soft tissue was removed. The radii were then separated from the respective ulnae and rinsed with 10× phosphate-buffered saline (PBS) and stored in saline-soaked gauze at −20 °C until testing.

Bone geometry

All radii were kept in Eppendorf tubes with saline during scanning. Images were acquired by microcomputed tomography (μCT) at 70 kVp, 114 mA, and 200 ms integration time and at a high-resolution 10.5 μm voxel size (vivaCT 40, Scanco Medical AG, Bassersdorf, Switzerland).¹³ Size and shape of cortical bone were determined by a mid-diaphysis evaluation script, which requires a defined volume of interest (VOI). The VOI for each radius consisted of approximately 40 slices (0.40 mm) of the mid-shaft (beginning at 50% of the bone length, extending 400 μm distally).^13,14 The parameters measured were cortical thickness (Ct.Th, mm), cortical area (Ct.Ar, mm²), marrow area (Ma.Ar, mm²), moment of inertia (I_max and I_min, mm⁴), and tissue mineral density (TMD, mgHA/cm³). TMD was derived from mean gray values of bone within the VOI using hydroxyapatite calibration constants. Gaussian filtration (σ = 0.8, support = 1) was applied to the slices and the segmented cortical bone using a global threshold of 656 mgHA/cm³. Total bone length (Le, mm) was calculated by determining the number of slices of bone per specimen and the thickness of each slice. Slenderness (Tt.Ar/le, mm) defined as the ratio of the total cross-sectional area (cortical and marrow area of VOI) and the total length of each radius, was also calculated.

Mechanical testing

The radii were loaded until fracture in 3-point bending at a displacement-controlled loading rate of 0.001 mm/s (Elf 3200; Enduratec). The distance between the lower supports was 8 mm. The resulting load-displacement curve was used to calculate the structural properties: stiffness (S; N/mm), yield force (F_Y; N), and ultimate force (F_U; N). The yield point was calculated using the 5% secant construction¹⁵ and the ultimate bending strength (σ_U; MPa) of each sample calculated by engineering formulae.¹⁶

Statistical analysis

Bone traits for each group were adjusted for body size using the linear regression method described by Jepsen et al.¹⁷ This takes into account the unique relationship between each trait and body mass. Subsequently, Kruskal-Wallis one-way analysis of variance on ranks was used to determine differences between the groups for the non-normally distributed data followed by Mann-Whitney rank sum tests. Multiple linear regression analysis was used to determine the dependence of mechanical properties on morphological and compositional traits. To identify relationships among morphological traits and whole-bone properties, a correlation matrix was established. Spearman’s ρ correlation coefficients were determined for all combinations of morphological traits and mechanical properties. The analyses were carried out using SPSS 21 (IBM SPSS Statistics) with significance level at P < 0.05.

Results

Kruskal-Wallis analysis revealed that Oc^−/− Opn^−/− bones were more robust (larger total cross-sectional area relative to total length) than those of all three groups (P < 0.01) (Table 1). Additional pairwise comparison test showed that Oc^−/− Opn^−/− mice had a significantly larger cortical area and minimum bending moment of inertia than WTs. There were no differences between Oc^−/− and Opn^−/− mice or between Oc^−/− Opn^−/− and WTs in any other traits (Table 1). The maximum bending load was different between the groups (P = 0.024), with the mutant mice withstanding higher loads than the WTs. A trend of increasing yield load among the mutant mice was also observed (P = 0.055). The multiple-comparisons test confirmed that the yield load was significantly different between WT and Oc^−/− mice (P = 0.013), and between WT and Oc^−/− Opn^−/− mice (P = 0.010). There were no differences in stiffness or ultimate strength among the groups (Fig. 1).

Table 1.

Summary of variables measured from the mid-shaft of each radius after adjustment of body weight.

	Le (mm)	Ct.Th (mm)	Ct.Ar (mm2)	Tt.Ar (mm2)	Ma.Ar (mm2)	Tt.Ar/Le (mm)	Imax (mm4)	Imin (mm4)	TMD (mgHA/ccm)
WT	10.563 ± 1.54	0.155 ± 0.01	0.242 ± 0.02	0.343 ± 0.04	0.101 ±0.02	0.0326 ± 0.003	0.0093 ± 0.002	0.0067 ± 0.001	1195.11 ± 46.38
Oc−/−	10.417 ± 0.23	0.156 ± 0.01	0.251 ± 0.02	0.354 ± 0.02	0.102 ± 0.01	0.0342 ± 0.002b	0.0097 ± 0.001	0.0072 ± 0.001	1194.96 ± 44.80
Opn−/−	10.414 ± 0.25	0.150 ± 0.01	0.243 ± 0.02	0.348 ± 0.03	0.105 ± 0.01	0.0334 ± 0.003c	0.0091 ± 0.003	0.0067 ± 0.001	1181.87 ± 31.48
Oc−/−Opn−/−	10.191 ± 0.31	0.156 ± 0.01	0.258 ± 0.02a	0.371 ± 0.03	0.113 ± 0.02	0.0366 ± 0.003a	0.0104 ± 0.001	0.0077 ± 0.001a	1191.71 ± 45.32

^aDifference between wild type and Oc^−/− Opn^−/− groups.

^bDifference between Oc^−/− Opn^−/− and Oc^−/− groups.

^cDifference between Oc^−/− Opn^−/− and Opn^−/− groups.

Figure 1.

Summary of mechanical properties among the four groups. * indicates significance at P < 0.05.

Correlations were observed between morphological traits and tissue composition (Table 2). Cortical thickness increased linearly with cortical area, slenderness, and TMD (P < 0.01). A negative correlation between length, cortical area, and cortical thickness was only observed for Oc^−/− Opn^−/− mice, indicating that these shorter bones are thicker, with larger cross-sectional size and higher tissue mineral density. Several morphological traits (i.e., cortical area, slenderness, I_min) correlated positively with stiffness, while cortical area and TMD correlated negatively with ultimate strength (σ_U). There were no significant correlations between other structural properties (yield load or ultimate) and bone morphology and TMD.

Table 2.

Combined Spearman ρcorrelation matrix of morphological traits and mechanical properties for all samples.

	Length	Ct.Ar	Ct.Th	TMD	Tt.Ar	Tt.Ar/Le	Ma.Ar	Yield.Load	Max.Load	Stiffness	UltStrength	Imax	Imin
Length	1.000	–0.128	–.0276	–0.252	0.001	–0.171	0.061	–0.018	–0.028	–0.137	–0.019	–0.005	0.061
Ct.Ar		1.000	0.736**	0.389**	0.868**	0.896**	0.551**	0.203	0.160	0.305*	–0.34*	0.639**	0.844**
Ct.Th			1.000	0.610**	0.434**	0.556**	0.075	0.169	0.125	0.186	–0.189	0.268	0.398**
TMD				1.000	0.181	0.297*	0.004	–0.079	–0.180	0.117	–0.367*	0.057	0.140
Tt.Ar					1.000	0.942**	0.867**	0.164	0.139	0.371**	–0.273	0.659**	0.933**
Tt.Ar/Le						1.000	0.749**	0.155	0.157	0.357*	–0.271	0.643**	0.867**
Ma.Ar							1.000	0.168	0.127	0.452**	–0.096	0.498**	0.787**
Yield.Load								1.000	0.864**	0.659**	0.545**	0.026	0.297*
Max.Load									1.000	0.581**	0.701**	0.018	0.258
Stiffness										1.000	0.349*	0.172	0.395**
UltStrength											1.000	–0.260	–0.223
Imax												1.000	0.593**
Imin													1.000

Note:

^*correlation is significant at the 0.05 level;

^**significant at the 0.01 level.

Multiple linear regression analysis was conducted to determine the dependence of bone mechanical properties on bone morphology and tissue quality. There was an overall dependency of tissue mineral density on strength (P = 0.004); however, tissue mineral density, cortical thickness, and slenderness were the only strong predictors of ultimate strength for the WT and Oc^−/− Opn^−/− groups (Table 3). These traits also predicted ultimate load significantly for Oc^−/− Opn^−/− mice. There were no combination of morphological traits and TMD that predicted mechanical properties for the other groups.

Table 3.

Multiple linear regression analysis of ultimate bending strength (Ult. strength) prediction for each genotype.

Equation	R2
Ult. strength WT = 1276.25 − 810.70 Ct.Th − 0.52 TMD − 8239.72 slenderness	0.63
Ult. strength Opn−/− = 846.95 + 7711.25 Ct.Th − 0.757 TMD − 33742.68 Ct.Ar	0.46
Ult. strength Oc−/− = 918.13 − 2363.82 Ct.Th − 0.459 TMD − 13427.07 slenderness	0.40
Ult. strength Oc−/− Opn−/− = 14.89 + 71.51 Ct.Th − 0.015 TMD − 118.54 slenderness	0.72

Discussion

Bone fracture, a major consequence of skeletal fragility, occurs if an applied load exceed its structural strength.²² Whole-bone strength is determined by bone mass, geometry, and tissue material properties.²³ Therefore, both quality and quantity contribute to bone fracture resistance. The goal of this study was to determine whether OC and OPN affect bone morphology and whole-bone strength. This is one of the first studies to compare changes in bone morphology and mechanical properties in the absence of both OC and OPN.

The geometry of bone, such as cortical thickness and external diameters (which incorporates cortical area), plays a major role in determining bone strength.^24–26 In this study, Oc^−/− and Opn^−/− mice adjusted their cortical area and marrow area together such that the absence of either protein resulted in a phenotype that was similar to WT. There were no morphological differences between Oc^−/− and Opn^−/− mice and between Oc^−/−, Opn^−/−, and WT littermates. The loss of both proteins, however, in Oc^−/− Opn^−/− mice resulted in significant changes in morphology, primarily in cortical area and length. In this regard, OC and OPN play synergistic roles in determining bone size and shape. The increase in the cortical area of Oc^−/−Opn^−/− bones may be attributed to the observed increase in outer diameter, since there was no change in the inner diameter compared with the other groups.

An increase in cortical area without an accompanying increase in the inner diameter of bone is usually attributed to inhibition of resorption at the endosteal surface with enhanced bone formation at the periosteal surface.^27,28 Mice lacking OPN have been shown to develop normally, and Opn^−/− bone marrow cells produced a two to four-fold increase in osteoclasts relative to WT cells.²⁹ Ducy et al. demonstrated that Oc^−/− mice had increased bone formation but no impairment of bone resorption through mechanisms that have not yet been fully elucidated.³⁰ Therefore, the net effect on bone formation and resorption due to the activity of these proteins may explain the similarity of the inner diameters among the groups. Oc^−/− mice were previously reported with a significant increase in bone thickness and mineral density compared with WT littermates.³⁰ Here, we observed that the absence of OC in the Oc^−/− (and Oc^−/− Opn^−/−) mice resulted in a similar trend, but no statistical differences were found between the four groups. In the Opn^−/− group, our data is consistent with Thurner et al., who found no differences in bone density and cortical thickness between Opn^−/− and WT mice.³¹

The impact of bone morphology on mechanical properties varied among the groups. Lack of OC in Oc^−/− (and Oc^−/− Opn^−/−) mice showed a significant increase in maximum load. This was also previously observed for Oc^−/− mice by Ducy et al. Duvall et al. showed a significant reduction in maximum load for Opn^−/−, while our results revealed higher loads.³² The discrepancy seen by Duvall et al. could be attributed to the difference in bone size and matrix maturity of the animals (10 weeks vs. 6 months in our study). In our study, the interactions between bone morphological traits in the mutants resulted in similar values for stiffness and ultimate strength such that there was no difference among the four groups.

It is noteworthy that OC and OPN affect bone material properties.^{10–12,35,36} In the absence of one or both proteins, Poundarik et al. demonstrated a loss of bone toughness and a co-localization of these proteins with microdamage accumulation.¹² In this study, bone material properties were not assessed. Here, the goal was to determine the effect of OC and OPN on bone size and strength, which was not previously reported. As such, our investigation provides insight into the roles of OC and OPN at both the material and whole-bone levels. Our results show that, in the absence of both OC and OPN, bone geometry is altered to achieve similar strength as WTs despite poor material quality.

In conclusion, here we show that together osteocalcin and osteopontin play important roles in determining bone size, shape, and strength. Morphological adaptation of mice lacking both OC and OPN can be attributed to three underlying bone traits: cortical thickness, slenderness, and tissue mineral density. These trait combinations are also predictors of whole-bone mechanical properties (stiffness and strength) for Oc^−/− Opn^−/− mice and therefore contribute to functional adaption. While there are inherent differences in microstructure and turnover between mouse and human bones, this approach allows us to identify the contribution of morphological traits on mechanical properties using genetically engineered mouse models.