Influences of osteoarthritis and osteoporosis on the electrical properties of human bones as in vivo electrets produced due to Wolff’s law

Abstract

We characterized the electrical properties of living bone obtained from patients who had undergone total hip arthroplasty (THA) or hemiarthroplasty by means of analysis of the electrically polarized and nonpolarized bone specimens, and we discussed the role of an organic and inorganic matrix of human bone in bone piezoelectricity.

We used human femoral neck bone that was harvested during THA for advanced osteoarthritis of the hip joint (OA group) and hemiarthroplasty for femoral neck fracture (FNF group). The specimens were scanned to evaluate the cancellous bone structures using micro-computed tomography, and we quantified the carbonic acid by attenuated total reflection (ATR) spectra to estimate carbonate apatite. The stored electrical charge in the electrically polarized and nonpolarized bone specimens were calculated using thermally stimulated depolarized current (TSDC) measurements.

Each TSDC curve in the groups had peaks at 100°C, 300°C and 500°C, which may be attributed to collagen, carbonate apatite and hydroxyapatite, respectively. It is suggested that organic substances are more effectively electrically polarized than apatite minerals by the polarization at room temperature and that the stored charge in living bone may be affected not only by total bone mass but also by bone quality, including 3-dimensional structure and structural component.

Keywords

Electrical property human bone electrical polarization osteoarthritis osteoporosis proximal femur

1. Introduction

When mechanical stress is impressed upon bone, an electrical potential is induced; the area of bone under compression develops negative potential, whereas that under tension develops positive potential. This phenomenon is generated by collagen piezoelectricity [1,2], and the electrical potential generated in bone by collagen displacement has been well documented [3]. This fact suggests that a polarized region may exist in human bone; that is, bone is an electret, and mineral crystals play a cooperative role with collagen in bone piezoelectricity. Yamashita et al. first reported that bone-like crystals were grown at an accelerating rate in a simulated body fluid on the negatively charged surface of electrically polarized hydroxyapatite (HA) ceramics [4]. We further reported that proton transport polarization induces a large surface charge on HA ceramics [5]. We confirmed via implantation tests into animals that osteobonding is enhanced by negative surface charges of electrically polarized HA [6] and additionally that bone ingrowth is favored by the cooperative interaction between the osteoconductivity of HA ceramics with interconnecting pores and large charges stored on their pore surfaces with electrical polarization [7]. Therefore, it is expected that an ideal alternate material of autogenous bone with the 3-dimensional structure of human bone, on which osteogenic cells are facilitated to attach, can be developed by the polarization of allogenic bone. We previously developed a technique to electrically polarize living bone at room temperature, and reported that the composite structure of organic and inorganic constituents collaboratively played an important role in the electrical properties of human bone based on empirical data of the electrically polarized bone [8].

Due to increased life span, certain age-specific joint and spine disorders in both genders, osteoarthritis and osteoporosis, have become major social problems. However, they are quite different conditions. Osteoarthritis involves the whole joint and causes subsequent changes to the subchondral surface involving bone remodeling. Radiographic changes include narrowing of the joint space, osteophytes, subchondral cysts and evidence of overloading in the form of bony sclerosis. Therefore, osteoarthritis exhibits increase in bone mineral density (BMD) but reduced bone mineral content and increased osteoid content, as well as alterations in subchondral bone microstructure. Meanwhile, osteoporosis is a systemic skeletal disorder, in which an increase in the rate of remodeling is observed, together with incomplete filling of individual bone remodeling units by osteoblasts. As a result, a decrease in BMD with alterations in bone microstructure and a reduction in the bone mineral component occur [9]. When severe coxalgia disturbs the activity of daily living, total hip arthroplasty (THA) is indicated for advanced osteoarthritis of the hip joint. Additionally, one of the most frequent fragile fractures, femoral neck fracture (FNF), is treated by hemiarthroplasty. Femoral heads removed by each surgery are usually discarded after the operation. In this study, we characterized the electrical properties of these bone specimens before and after electrical polarization and discussed the role of an organic matrix, including collagen, and an inorganic matrix, including apatite minerals, of human bone in bone piezoelectricity.

2. Materials and methods

2.1. Preparation of bone specimens

We used human femoral neck bone that was removed by THA for advanced osteoarthritis of hip joint surgeries (Fig. 1(A), (B)) and hemiarthroplasty for FNF (Fig. 1(C), (D)) from September 2012 to August 2015 at Kawakita General Hospital (OA and FNF group, respectively). The research protocol was approved by the ethics committees of the institutions with which the authors are affiliated and was conducted according to the Declaration of Helsinki. Informed written consent was obtained from each candidate. Thirty-three patients in the OA group (mean age $71.6 \pm 11.4$ years) and thirty-seven patients in the FNF group (mean age $81.9 \pm 9.9$ years) participated in this study. After temporary storage at −20°C, the specimens were sliced into 1-mm thick pieces using a diamond saw (IsoMet^® Low Speed Saw, Buehler, Illinois, USA), washed with distilled water, dried at 40°C and used for the experiment (Fig. 2).

Fig. 1.

Radiographs of THA for advanced osteoarthritis of the hip joint and hemiarthroplasty for FNF. (A) Osteoarthritis of the hip joint. The radiograph shows subluxation of the femoral head with polycystic lesions and a large spur known as the tear drop (arrow), which causes stenosis and sclerosis of the hip joint. (B) After THA surgery. (C) Femoral neck fracture which is indicated with an arrow. (D) After hemiarthroplasty surgery.

Fig. 2.

Preparation of the femoral neck human bones that are removed by THA or hemiarthroplasty surgery. (A) The harvested femoral neck bone. (B) The bone specimen used for the experiment, which is sliced into 1-mm thick pieces, washed with distilled water and dried at 40°C. (C) The structure of human femoral neck bone that is constructed by micro-CT scan.

2.2. Micro-computed tomography scanning

The specimens were scanned to evaluate the cancellous bone structures using micro-computed tomography (CT) (smx100, Shimazu, Tokyo, Japan), and analyzed using computer analysis software (3D-BON, LATOC, Tokyo, Japan). Trabecular bone was examined using the parallel plate model. The primary parameters that were evaluated included the bone mineral content (BMC), bone volume (BV) and tissue volume (TV). The secondary parameters that were calculated included the BMD, BV/TV and BMC/TV values.

2.3. Quantification of the carbonic acid to estimate carbonate apatite in bone specimens

We attempted to quantify the carbonic acid by attenuated total reflection (ATR) spectra to estimate carbonate apatite, which was expected to be characteristic of human bone. ATR spectra of the bone specimens were recorded in the range of 400 to 4000/cm using an ATR spectrophotometer (Thermo Scientific, Nicolet is50, MA, USA). The peak intensity of the carbonate, which is characteristic of type B carbonate apatite [10], and of the phosphate ion, which correlates with the amount of phosphate [11], were recorded at 871/cm and 559/cm, respectively (Fig. 3). The peak intensities of the carbonate and phosphate ions were analyzed using the instrument software (Thermo Scientific, OMNIC, MA, USA). The ratio of the two peak intensities was calculated to estimate the ratio of carbonate ions to phosphate ions (CO₃/PO₄ ratio) in the bone specimens.

Fig. 3.

Representative ATR spectrum of the bone specimen. The peak intensity of the carbonate and phosphate ions is recorded at 871/cm and 559/cm, respectively.

2.4. Polarization treatment of human bone to evaluate the faculty as an electret

The bone specimens, which bordered the sliced bones, were divided into two groups; one was treated for polarization and the other was untreated (polarized and nonpolarized group, respectively). For the polarization treatment bone specimens were clamped with a pair of platinum electrodes and electrically polarized in direct current (DC) electric fields of 5 kV/cm in air at 25°C.

2.5. Measurement of thermally stimulated depolarization current

The bone specimens in both the polarized and nonpolarized groups were clamped tightly with a pair of platinum electrodes and were covered with alumina plates to form a bilayer. The thermally stimulated depolarized current (TSDC) measurements were performed using an electric current measuring unit (6514 system electrometer, Keithley, Ohio, USA). The sample temperature was increased at a rate of 5.0°C/min from 25°C to 600°C, and the depolarization current was measured. The stored charges in the bone specimens around each peak and total stored charges were calculated using the following formula: $\begin{matrix} Q = \frac{1}{β} \int J (t) d t, \end{matrix}$ where Q is the stored charge (nC/cm²), β is the temperature ramp rate (K/s²), and $J (t)$ is the electric current density (nA/cm²).

2.6. Statistical analysis

The age, secondary parameters calculated using the trabecular bone analysis, CO₃/PO₄ ratio and the stored charge before and after electrical polarization in the FNF and OA groups were compared using a Mann–Whitney test. Correlative analyses of these values for the bone specimens in the groups were performed using Spearman’s rank correlation coefficient test. Furthermore, the best-fit model was computed using a simple regression analysis, which compared the parameters with a significant difference. A p value of <0.05 was considered to be statistically significant.

3. Results

Baseline characteristics of patients in the OA and FNF groups, including gender, the average age and calculated parameters of bone structure of specimens are summarized in Table 1. The average age was significantly younger in the OA group than in the FNF group ( $p < 0.01$ ).

Table 1
Gender, the average age and calculated parameters of bone structure of specimens in the OA and FNF groups

Type of bone Gender Age (years old) BMD (mg/cm³) BV/TV (%) BMC/TV (mg/cm³)

Male Female

OA 3 30 71.6 ± 11.4^* 586.7 ± 92.3 23.9 ± 9.1 144.3 ± 65.0

FNF 6 31 81.9 ± 9.9^* 559.0 ± 132.1 24.4 ± 13.4 146.4 ± 114.7

Type of bone	Gender	Age (years old)	BMD (mg/cm³)	BV/TV (%)	BMC/TV (mg/cm³)
OA	3	30	71.6 ± 11.4^*	586.7 ± 92.3	23.9 ± 9.1	144.3 ± 65.0
FNF	6	31	81.9 ± 9.9^*	559.0 ± 132.1	24.4 ± 13.4	146.4 ± 114.7

Average ± SD.

$p < 0.01$ .

Each TSDC curve of bone specimens in the OA and FNF groups had peaks at 100°C, 300°C and 500°C before and after electrical polarization (Fig. 4(A), (B)). The CO₃/PO₄ ratios and the stored charge in the bone specimens before and after polarization in the groups are summarized in Table 2. The stored charge around 100°C, 300°C and 500°C peak as well as the total stored charge in the OA group were significantly larger than those in the FNF group before polarization ( $p < 0.01$ , $p < 0.01$ , $p < 0.05$ and $p < 0.05$ , respectively). After polarization, the stored charge around the 100°C and 500°C peaks, and the total stored charge in the OA group were significantly larger than those in the FNF group (each $p < 0.05$ ).

There was a significant correlation between BMD and the CO₃/PO₄ ratio as well as the stored charge around the 500°C peak and the total stored charge in the nonpolarized bone specimens (each $p < 0.01$ ). The equations for these parameters are shown, respectively, as shown below: $\begin{matrix} Y = - 0.00052 X + 0.89295 (R^{2} = 0.11009), \end{matrix}$ where Y is the CO₃/PO₄ ratio and X is BMD (mg/cm³) (Fig. 5(A)). $\begin{matrix} Y = 0.02418 X + 0.02823 (R^{2} = 0.14245), \end{matrix}$ where Y is the stored bone charge around the 500°C peak (μC/cm²) and X is BMD (mg/cm³) (Fig. 5(B)). $\begin{matrix} Y = 0.02872 X + 2.35549 (R^{2} = 0.13828), \end{matrix}$ where Y is the total stored bone charge (μC/cm²) and X is BMD (mg/cm³) (Fig. 5(C)).

However, there was no correlation between the examined parameters in the polarized bone specimens in the groups.

Fig. 4.

Representative TSDC curve pattern before and after electrical polarization treatment. (A) OA group. A broad peak is found at 300°C before polarization; however, large peaks are shown clearly at 100°C and 300°C after polarization. (B) FNF group. Although a peak cannot be found at 100°C before polarization, a clear peak is shown at 100°C after polarization.

Table 2

The CO₃/PO₄ ratios and the stored charge in the bone specimens before and after polarization in the OA and FNF groups

	Type of bone	CO₃/PO₄	Q (100°C) (μC/cm²)	Q (300°C) (μC/cm²)	Q (500°C) (μC/cm²)	Total Q (μC/cm²)
Before polarization	OA	$0.58 \pm 0.18$	1.9 ± 2.2^**	3.6 ± 2.2^**	16.3 ± 9.4^*	21.8 ± 11.3^*
Before polarization	FNF	$0.62 \pm 0.26$	0.9 ± 1.7^**	2.6 ± 3.4^**	11.3 ± 8.4^*	16.2 ± 10.6^*
After polarization	OA	–	45.1 ± 67.6^*	10.5 ± 10.4	48.1 ± 60.9^*	103.8 ± 116.4^*
After polarization	FNF	–	23.8 ± 28.6^*	9.5 ± 11.8	27.6 ± 30.6^*	60.9 ± 53.8^*

Average ± SD.

$p < 0.05$ .

^**

$p < 0.01$ .

Fig. 5.

Correlation between BMD, CO₃/PO₄ ratio and the stored bone charge in the nonpolarized bone specimens. (A) There is a negative correlation between BMD and the CO₃/PO₄ ratio. (B, C) There is a positive correlation between BMD and the stored charge around the 500°C peak and between BMD and the total stored charge.

4. Discussion

Since the discovery of bone piezoelectricity [12], there has been a concern that the mechanical stress-induced electric charges in bone may influence bone remodeling through Wolff’s law [13]. The previous report that rabbit and bovine bone TSDC curves have two significant peaks at 100°C and 500°C [14], has successfully demonstrated that bone is electrically charged, an electret. The report stated that the peak at 100°C may be attributed to collagen fibrils and the peak at 500°C to apatite minerals. Meanwhile, we have found that the TSDC curve of human bone has 3 peaks at 100°C, 300°C and 500°C. Considering that human bone consists of an organic matrix, including collagen, and an inorganic matrix, including apatite minerals, the peaks at 300°C and 500°C are expected to be attributed to 2 different types of apatite minerals. Because the minerals in calcined bone specimens are converted to hydroxyapatite with a loss of organic content and carbonate ions during the calcification, the peak at 500°C is expected to be attributed to hydroxyapatite. Additionally, calcium phosphate and calcium carbonate are the primary mineral components of human bone. Therefore, it is predicted that the peak at 300°C is derived from carbonate apatite. Furthermore, there is a possibility that carbonate apatite is involved in forming the peaks at both 300°C and 500°C. There was a negative correlation between the CO₃/PO₄ ratio and BMD, however, there was no correlation between the CO₃/PO₄ peak ratio and the stored charges at approximately 300°C in this study. More than one peak or a broad peak was found at 300°C in both polarized and nonpolarized bone specimens. These phenomena may result from an inhomogeneous distribution of carbonate ions in human bone: (1) individual differences in contained amounts of carbonic acid, (2) carbonate apatite formation by carbonation of apatite minerals due to cauterization of protein substance during TSDC and (3) decarboxylation of carbonate apatite between 100°C and 400°C in the process of TSDC. Thus, the peak at 300°C of human bone may become deformed compared with that of refined carbonate apatite.

No peak or a very small peak at 100°C was observed in the nonpolarized or natural bone specimens. This phenomenon may derive from the fact that the peak of organic substances, including collagen, is recorded under 100°C and is very small compared with the large peak of inorganic substances, including hydroxyapatite, at 500°C. On the other hand, the peaks at 100°C increased ten- to twentyfold after polarization when compared with those before polarization. In contrast, the peaks at 300°C and 500°C increased only three- to fourfold after polarization. Furthermore, there was a positive correlation between the stored bone charge and the BMD before polarization; however, no significant correlation was found between these parameters after polarization. These results may indicate that organic substances are more effectively electrically polarized than apatite minerals by the polarization treatment at room temperature. Charge carriers tend to align in the same direction in organic substances, although, they move in various directions in apatite minerals. Resolution of carriers for electrical polarization of human bone and the roles they play in each substance in the polarization process at low temperature is an issue that we plan to address in the future.

Considering that stored charge is significantly larger and the BMD value tended to be larger in bone specimens obtained in cases of THA than those of hemiarthroplasty, the stored charge in living bone may be affected by total bone mass as well as bone quality, including 3-dimensional structure and structural components. Autogenous bone grafts are the clinical gold standard to fill bone defects. However, when allogenic bone, that is obtained from cases of osteoarthritic joint surgery and cryopreserved aseptically, is electrically polarized and grafted into bone defects, enhancement of new bone formation and facilitation of bone healing comparable to autograft may be expected. We would like to verify this hypothesis by an implantation test into animals in the near future.

Conflict of interest

The authors have no conflict of interest to report. No benefit of any kind will be received either directly or indirectly by the authors.

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