Effects of Uygur sand therapy on the mechanical properties of femurs in osteoarthritic rabbits

Abstract

Objective:

To investigate the effects of Uygur sand therapy on the mechanical properties of the femur bone of osteoarthritic rabbits.

Methods:

Sixteen rabbits were injected with papain in the right posterior femoral articular cavity on the first, fourth and seventh day to establish the osteoarthritis (OA) rabbit model. Animals were divided into the experimental group and control group (8 rabbits each). The experimental group was treated with sand therapy, and the control group received no sand therapy treatment. Computed tomography (CT) scanning was used to collect the data of the femur before modeling, after modeling and 14 and 28 days after sand treatment. A 3D model of the femur was generated with the MIMIC software the bone layer was divided according to the different gray values and the change of the bone volume was analyzed. The body mesh is divided, and the material properties are given, then the three-point bending simulation is performed in Ansys. Additionally, the three-point bending test was performed on all the rabbits’ femur to obtain the deflection and maximum stress values. And the effects of the sand treatment on the volume and mechanical properties of the bone were analyzed. Finally, the simulation results are compared with the experimental results, and the effects of sand treatment on the volume and mechanical properties of the bone are analyzed.

Results:

(1) there is a tendency in the control group to convert the hard bone into dense bone and soft bone, while in the experimental group, the soft bone is converted into dense bone and hard bone obviously; (2) the morphological parameters of the experimental group are lower than those of the control group, whereas the maximum load, maximum normal stress, maximum shear stress of the experimental group are higher than those of the control group. (3) The mechanical test of three-point bending test was carried out using the three dimensional finite element model of rabbit femur.

Conclusion:

The sand therapy has positive effects on the volume distribution of bone layer and the mechanical properties of the femur of adult osteoarthritic rabbits.

Keywords

Uyghur sand therapy osteoarthritis MIMICS three-point bending test finite element analysis

1. Introduction

Sand treatment in Uyghur medicine is an ancient medical practice in the Xinjiang Turpan area, which can be used for the adjuvant treatment of arthritic diseases. Under the unique climate and geographical conditions, Uyghur sand therapy can effectively relieve various types of arthritis, rheumatism, lumbago and leg pain [1,2]. The mechanism mainly lies in the biophysical and biochemical effects produced by the natural hot sand containing certain minerals [3–5].

Osteoarthritis (OA) is a chronic joint disease characterized by degeneration, destruction of articular cartilage and bone hyperplasia. Its pathogenesis involves the loss of glycoprotein in cartilage matrix, degradation of cartilage on joint surfaces, fibrillation and gradual thinning of the surface cartilage until it disappeared. Under external loads, the loading part of the cartilage will undergo a fracture [6,7]. Accordingly, in order to establish a typical OA model, injection of papain into the rabbit femoral intra-articular bone was adopted to destroy the articular cartilage.

Bone mechanics is an important branch of bio-mechanics, which has been applied to the studies of mechanical properties and biological effects of bone tissue under loads [8]. Bone mechanical properties, such as the maximum normal stress, maximum load and fracture deflection, can reflect the intrinsic properties of bone, and they are not affected by the size of the bone [9]. Therefore, they are important indices for the evaluation of an arthritic femur.

As a matter of fact, researchers have been studying the sand treatment of Uyghur medicine for many years. Through compression and tensile tests, Fu et al. [10] found that the sand therapy helps to improve the mechanical properties of the femur. Later, by studying OA femur bones from rabbits of different age, Hu et al. [11] also found that the sand therapy can improve the mechanical properties of the femur.

A biomechanical study of the femur depends on building finite element model. Because of the irregular structure and uneven distribution of bone materials, Mechanical experiments are sometimes difficult to carry out. At this time, the finite element method is used to simulate the mechanical experiment to be an effective method.

Animal experiment is one necessary means to study OA. The femur of adult rabbits was used as a research object in this study, and was modeled by injecting chemicals into the cavity of the joint. Then this OA model was treated with sand therapy. Finally, with the comprehensive computed tomography (CT) scans and the three-point bending test, the effects of Uygur sand therapy on bone mass and mechanical properties of the osteoarthritic rabbit femur were analyzed.

2. Materials and methods

2.1. Materials

2.1.1. Experimental animals

Seventeen 5 to 6 months old adult New Zealand white male rabbits, weighing 3.8 ± 0.2 Kg, were provided by the Animal Experimental Center of Xinjiang Medical University. The experimental animal production license number is SCXK (Xinxiang) 2003–0002, and the use license number is SCXK (Xinxiang) 2003–0003.

2.1.2. Reagents and instruments

Papain was purchased from Shanghai Sangon biotechnology company; Mimics10.1 (Materialise, Louvain, Belgium);

Ansys15.0 (Canonsburg, PA, USA); Acer A1602; Siemens / Emotion 6 slice spiral CT (minimum slice thickness: 0.3 mm; image pixels: 512 × 512 pixel; Siemens, Munich, Germany); CMT6104 universal testing machine.

2.2. Methods

2.3. Model

Kikuchi et al. proposed that injection of the appropriate amount of collagenase into the joint cavity can cause an inflammatory reaction in the joint in a short period of time [12]. Generally, the establishment of the OA model can be divided into two categories, namely the spontaneous model and the induced model. The materials commonly used in the joint cavity injection to induce arthritis are of three different types, namely the urokinase type plasminogen activator, papaya protease and collagenase enzyme [13]. In this study, we selected the papaya protease to develop the OA model. The experimental subjects had been reared for three months to allow them to reach adulthood and adapt to the rearing environment. The right posterior femoral articular cavities of the 17 rabbits were injected with 3% papain (dosage of 0.3 ml/kg) on the first, fourth and seventh day. Seven days later, a rabbit was randomly selected for scarification, and anatomy images of the knee joint and optical microscope observation of the cartilage of the scarified joint group (right leg, A1, A2) and the normal joint group (left leg, B1, B2) were captured (Fig. 1). Compared with the normal group, the color of the cartilage of the model group was gray–yellow, the surface was ulcerated, the synovium apparently proliferated and the synovial fluid was cloudy. Also, as shown in the histologically stained sections, the tide line and chondrocytes disappeared, cells were significantly lost, and the stroma was mostly unstained y. Averall, the OA model has been established [14].

Fig. 1.

The knee anatomy images and histological stained cartilage tissue sections.

2.3.1. Grouping and sand therapy

The sand therapy was conducted in a man-made sand therapy house in the South Campus of Xinjiang University, and involved heating the sand with 9 infrared lamps to a temperature which was basically consistent with the temperature of the sand field in Turpan between 17:00 to 19:00 in July and August. The OA models were treated with sand therapy twice a day, for 30 minutes each time at 11:30–12:00 and 16:30–17:00 CMT. A comparison of the results of the blood tests before and after modeling revealed that injecting papain on the right posterior femoral articular cavity has no effect on the left leg. Thus, the left hind femur was treated as the normal group. The experimental subjects were divided into three groups: experimental group (8 right legs with sand therapy), control group (8 right legs without sand therapy), and normal group (8 left legs without sand therapy).

2.3.2. CT scanning

Four CT scans were conducted on the hind femur of the 16 rabbits evaluated in this study: before modeling; after modeling, before sand therapy; 14 days after sand therapy; 28 days after sand therapy. Dicom format data were collected for each scan.

2.3.3. Bone stratification

In the scanning of some parts of the body of a certain thickness with a highly collimated X-ray beam, the different density values of the tissue represent different linear attenuation coefficient μ, and typically the relative value of μ is used to represent the CT value.

Bone tissue is composed of trabecular bone and cortical bone. The data collected from of the four CT scans were input into MIMICS, and the data of the rabbit right hind femur was extracted and divided into three layers using the Boolean command in our study: soft bone, compact bone and cortical bone layer (Fig. 2). Then, the volume of each layer was calculated by the bone volume element method. As shown in Fig. 2, in the MIMICS software, the range of the bone CT value was defined in 148-3071 HU, in which the range of soft bone, compact bone and cortical bone were defined in 148-661 HU, 662-1988 HU and 1989-3071 HU, respectively.

Fig. 2.

Distribution of bone stratification.

2.3.4. Three-point bending test and calculation

The three-point bending test was conducted on the CMT6104 universal testing machine (maximum test force: 3 KN; loading speed: 0.5 mm/min), and the experimental materials are bars. The three-point bending test was conducted on the 16 femur specimens (Fig. 3) and the load-deflection curve was obtained.

Fig. 3.

Bone three-point bending test.

The elastic bending deflection and maximum carrying load were extracted from the curve and the cross section of the bone was simplified (Fig. 4). Also, the formulas of the maximum shear stress, maximum bending normal stress and cross-sectional moment of inertia of the bone in the three-point bending test were derived according to the knowledge about the mechanics of the materials, as follows:

Maximum bending normal stress: $\begin{matrix} (1) & σ_{max} = \frac{F_{max} L B}{4 I_{z}} \end{matrix}$

Maximum shear stress: $\begin{matrix} (2) & τ_{max} = \frac{F_{max} (A B^{2} - a b^{2})}{6 I_{z} (a - b)} \end{matrix}$

Cross-sectional moment of inertia: $\begin{matrix} (3) & I_{z} = \frac{π}{64} (A B^{3} - a b^{3}) \end{matrix}$ where, L in formula (1) represents the distance between the two pivot points of the femur in the three-point bending test, $F_{\max}$ represents the maximum applied load.

Fig. 4.

Bone cross-sectional moment of inertia.

2.3.5. Three-point bending test simulation

Import CT scan data into the MIMICS, generate 3D model of the femur, the MIMICS application of the “remesh” command into the grid, the formation of the surface mesh model output to Ansys, using the ANSYS APDL language to write the command stream file to translate the shell element model into the body element model, get the femoral body mesh model, and automatic output LIS, nodes and elements, three format file.

The LIS format file is imported into MIMICS, in order to define material properties, the relationship between the density and the elastic modulus is characterized by using the piecewise function proposed by Kenneth.

Based on the segmented model proposed by Kenneth [12] and other scholars, femur will be divided into four parts. Bone marrow (CT<148HU) is given to a kind of materials; cancellous bone (148HU<CT<661HU) to 10 kinds of materials;compact bone (662HU<CT<1988HU) to 10 kinds of materials; hard bone (CT>1989HU) to a kind of materials.

In the finite element simulation of three-point bending, the span of the fulcrum is 60 mm (the same as three-point bending test setting), left side restrictions as UX, UY, UZ, VELX, VELY, VELZ, right side restrictions as UX, UZ, VELX, VELY, VELZ. Set the vertical concentrated load in the middle part of the span, according to the load of the three-point bending test, the maximum load of the finite element analysis is determined (Fig. 5).

Fig. 5.

Three-point bending test simulation.

2.3.6. The results of statistical processing

The SPSS13.0 statistical software was used for the statistical analysis and a $P < 0.05$ was considered as statistically significant. Measurement of the bone morphology and mechanical data was expressed as $\overline{x} \pm s$ . The comparison between the samples of the two groups was performed using the t test, and the basic mechanical properties of the experimental group and the control group were compared using the single factor analysis of variance test.

3. Results

3.1. Results of bone layer changes

According to the data obtained from the four CT scans, the difference between the volumes of the bone layers of the experimental group and the control group is statistically significant ( $p < 0.05$ ). After sand therapy, as shown in Table 1, the soft bone of the femur from adult rabbits with OA was significantly transformed into compact bone and hard bone. However, a less obvious transformation trend was observed in the ones that did not undergo sand therapy.

Table 1
Volume of each bone layer in the four CT scans ( $\overline{x} \pm s$ $n = 8$ )

Number of CT scan Group Soft bone (mm³) Compact bone (mm³) Cortical bone (mm³) Quantum (mm³)

First EG 2796.156 ± 356.179 3907.451 ± 404.043 722.262 ± 93.204 7354.654 ± 811.948

CG 2422.101 ± 174.911 3430.338 ± 377.347 628.544 ± 173.684 6396.463 ± 549.877

Second EG 3054.456 ± 473.842 3379.448 ± 453.479 560.867 ± 123.989 7008.695 ± 874.444

CG 2653.591 ± 246.602 2952.194 ± 309.663 703.171 ± 142.766 6130.121 ± 517.817

Third EG 2716.668 ± 331.455 3736.092 ± 570.713 764.199 ± 100.476 7221.737 ± 920.062

CG 2381.224 ± 281.975 3256.486 ± 341.684 670.852 ± 202.156 6303.615 ± 565.343

Fourth EG 2433.651 ± 287.572 3953.761 ± 579.937 860.639 ± 139.558 7271.819 ± 970.029

CG 2121.874 ± 256.788 3434.981 ± 543.799 760.374 ± 160.184 6332.872 ± 748.329

Number of CT scan	Group	Soft bone (mm³)	Compact bone (mm³)	Cortical bone (mm³)	Quantum (mm³)
First	EG	2796.156 ± 356.179	3907.451 ± 404.043	722.262 ± 93.204	7354.654 ± 811.948
CG	2422.101 ± 174.911	3430.338 ± 377.347	628.544 ± 173.684	6396.463 ± 549.877
Second	EG	3054.456 ± 473.842	3379.448 ± 453.479	560.867 ± 123.989	7008.695 ± 874.444
CG	2653.591 ± 246.602	2952.194 ± 309.663	703.171 ± 142.766	6130.121 ± 517.817
Third	EG	2716.668 ± 331.455	3736.092 ± 570.713	764.199 ± 100.476	7221.737 ± 920.062
CG	2381.224 ± 281.975	3256.486 ± 341.684	670.852 ± 202.156	6303.615 ± 565.343
Fourth	EG	2433.651 ± 287.572	3953.761 ± 579.937	860.639 ± 139.558	7271.819 ± 970.029
CG	2121.874 ± 256.788	3434.981 ± 543.799	760.374 ± 160.184	6332.872 ± 748.329

(Annotation: EG represents experimental group, CG represents control group, identical below)

3.2. Results of the three-point bending test

The morphological parameters of the femur section were extracted in MIMICS (Table 2), and all the data are statistically significant ( $p < 0.05$ ). The statistical analysis of the data read from the three-point bending test machine and that obtained with the formulas above, indicate that the cross-sectional moment of inertia and deflection are not statistically significant ( $p > 0.05$ ). Nevertheless, the maximum bending normal stress, maximum shear stress and maximum load are statistically significant ( $p < 0.05$ ). In addition, as shown in Table 2, the morphological parameters of the femur section in the experimental group were lower than those in the control group, indicating that the sand therapy suppressed the femoral marrow cavity expansion caused by OA. On the other hand, as shown in Table 3, the maximum load, maximum bending normal stress and maximum shear stress of the cross section of the femurs in the experimental group are higher than those in the control group, which suggests that sand therapy improves the OA-affected mechanical properties of the femur. The increase of the maximum bending normal stress can increase the strength of the femur. The maximum shear stress increases the femur’s deformation but does not cause fracture of the femur. Therefore, the mechanical properties of the femur are improved.

Table 2
Measurement of the morphology of the EG and CG femurs cross-section ( $\overline{x} \pm s$ $n = 8$ )

Group Fracture bone inter section major axis A (mm) Fracture bone inter section short axis B (mm) Fracture bone outer section major axis a (mm) Fracture bone outer section short axis b (mm) Cross sectional area (mm²)

EG 10.51 ± 0.81 8.38 ± 0.51 5.06 ± 0.34 3.46 ± 0.29 55.49 ± 9.48

CG 10.92 ± 0.71 8.69 ± 0.49 5.61 ± 0.48 3.96 ± 0.41 57.13 ± 7.87

Group	Fracture bone inter section major axis A (mm)	Fracture bone inter section short axis B (mm)	Fracture bone outer section major axis a (mm)	Fracture bone outer section short axis b (mm)	Cross sectional area (mm²)
EG	10.51 ± 0.81	8.38 ± 0.51	5.06 ± 0.34	3.46 ± 0.29	55.49 ± 9.48
CG	10.92 ± 0.71	8.69 ± 0.49	5.61 ± 0.48	3.96 ± 0.41	57.13 ± 7.87

Table 3

Basic mechanical properties of the EG and CG of femurs ( $\overline{x} \pm s$ $n = 8$ )

Group	Maximum load (N)	Deflection (mm)	Maximum shear stress (MPa)	Maximum bending normal stress (MPa)	Cross-sectional moment of inertia
EG	266.09 ± 33.64	1.03 ± 0.21	123.67 ± 20.31	65.03 ± 7.25	297.99 ± 50.03
CG	257.72 ± 50.52	1.19 ± 0.35	109.69 ± 26.31	58.01 ± 4.99	339.25 ± 82.04

3.3. The result of three-point bending simulation

According to the displacement images from the ANSYS analysis (Fig. 6) shows that the middle part of the femoral shaft, the region of the maximum displacement is consistent with the applied load, and femoral fracture location in test. At the same time, according to the equivalent stress distribution map (Fig. 7) from analysis shows that the stress is mainly concentrated in the femur at both ends of the restricted area and the load.

Fig. 6.

Displacement image.

Fig. 7.

Equivalent stress distribution.

4. Discussion

4.1. Effect of the sand therapy on the bone layer of osteoarthritic femur

In this study, the right-hind leg of the rabbit was injected with 3% of papain to damage the articular cartilage in order to develop a typical OA model. In the CT scanning, the dense bone volume of the control group decreased by 5.65%, while the hard bone volume decreased by 12.93%, indicating that OA inhibited the formation of hard bone and compact bone and led to the bone layer volume changes. On the other hand, for the experimental group, the CT scan data collected after 14 days sand therapy showed that the volume of soft bone declined by 11.07%, while dense bone and hard bone increased by 9.56% and 26.70%, respectively. Likewise, after 28 days of sand therapy, the volume of soft bone declined by 10.42%, while dense bone and hard bone increased by 5.49% and 11.16%, respectively. Accordingly, sand therapy exhibited repairing effects on the reduction of the volume of hard bone and bone mass caused by OA. The biophysical and biochemical effects of the minerals in the sand treatment can effectively activate bone remodeling, promote bone mineralization and reconstruction, and transform soft bone into compact bone and hard bone. In addition, the data analysis revealed that the sand treatment effect is more evident at 0∼14 days rather than at 14∼28 days.

For adult rabbits, the bone volume of each bone layer was stable. Through the preliminary determination, sand therapy can effectively promote soft bone calcification, transforming soft bone into cancellous bone and hard bone, which are harder. Additionally, it can also alleviate the pathological effects of OA on each bone layer volume.

4.2. The influence of sand therapy on mechanical properties of OA femur

In this study, we show that sand therapy can improve the basic mechanical properties of osteoarthritic rabbit femur. In the control group, the load balance of the bone was broken in the bone affected by OA. These would change the fibrous structure of the bone, the trabecular bone will become thinner and fractured, thus leading to a decrease of the creep mechanical properties index. Also, the cortical bone will be progressively thinning, and the bone marrow cavity will be constantly expanding [15,16]. In the three-point bending test, the maximum bending normal stress and the maximum bending shear stress of the experimental group were 12.12% and 12.75% higher than those of the control group, respectively. The average value of the maximum load of the femoral fracture in the experimental group was 3.55% higher than that of the control group. These results indicate that sand therapy improves the stress balance of the bone, and reduces the damage caused by OA, as well as the absorption and formation of compact bone and hard bone.

In the experimental group, the average value of the cross-sectional area of the bone fracture was 2.95% lower than that of the control group, which is consistent with the Wolff law [17]. Sand therapy can activate osteoblasts, promote the energy metabolism pathways in osteoblasts, improve the lubrication of articular cartilage, accelerate the reabsorption and deposition of bone materials, improve the bone tissue volume density and bone microstructural changes, so as to improve not only the bone strength [18], but also the bearing capacity of the non-load bearing area.

4.3. The significance of finite element analysis

Effect of sand treatment in Uyghur medicine on the mechanical properties of the femur was studied by three dimensional finite element modeling method based on CT value. The effect of sand therapy on the mechanical properties of bone can be further verified. This provides a reference method and theoretical basis for the study of bone biomechanics.

5. Conclusion

The sand therapy has positive effects on the volume distribution of the bone layer and mechanical properties of femurs of adult osteoarthritic rabbits, which can provide theoretical basis for the research of knee OA.

Ethical questions

Laboratory animals underwent all operations under anesthesia, and we made every effort to minimize the pain, pain and death of animals.

Footnotes

Acknowledgement

The authors gratefully acknowledge the support of National Natural Science Foundation of China (No. 81160542) and the State Key Laboratory for Manufacturing Systems Engineering (Xi’an Jiaotong University) for this study.

Conflict of interest

The authors have no conflict of interest to report.

References

Rong

and Mahemuty

, Theoretical discussion of uygur sand therapy on rheumatism [J], Journal of Xinjiang Medical University 31(10) (2008), 1460–1461.

Chunhui

Hua

and Upur

, Clinical trial on treatment of knee osteoarthritis by the instrument of heat conduction sand treatment in uyghur medicine [J], Journal of Xinxiang Medical College 29(5) (2012), 637–638.

Rexati

and Chunyao

, Trace Elements and Biological Heat Transfer of Uygur Sand Therapy [M], Xinjiang People’s Health Publishing House, Urumqi, 2003, pp. 16–17.

Saidula

Kadir

et al., Uygur sand therapy with other regional sand therapy sand characteristics comparative study [J], Journal of Medicine and Pharmacy of Chinese Minorities 19(8) (2013), 39–40.

Saidula

and Kadir

, Characteristics and study progress of sand therapy [R], in: First International Symposium on Natural Medicine, 2011.

Qiong

, The Role of Osteopontin in the Pathogenesis of Rheumatoid Arthritis [D], Anhui Medical University, 2008.

Huawei

Liu

et al., Study on the pathology shape structure of calcified cartiage zone in osteoarthritis knee joint [J], Orthopedic Journal of China 12(4) (2009), 30–39.

Xiaogang

Daping

et al., Application and research progress of bone biomechanical [J], Chinese Journal of Osteoporosis 18(9) (2012), 850–853.

Baohua

, The Influence of Vertical Jump on Bone Mineral Density and Biomechanical Indicators and RUNX2MRNA Expressing to Suspended Rat Femur [D], East China Normal University, 2010.

10.

Rong Chang

Mahemuti

et al., Experimental research of effect of uygur sand therapy on femur mechanical properties of rabbits [J], Journal of Xinjiang University 27(4) (2010), 401–404.

11.

Xiaoxin

Maitirouzi

et al., Change of volume and mechanical properties of femoral bone in rabbits at different ages with knee osteoarthritis under sand-therapy [J], Journal of Biomedical Engineering 32(5) (2015), 1038–1043.

12.

Kikuchi

Satuta

and Yamaguchi

, Intra-articular injection of collagenase induces experimental osteoarthritis in mature rabbits [J], Osteoarthritis Cartilage/OARS 6(3) (1998), 177–186. doi:10.1053/joca.1998.0110.

13.

Xintai

, Modern Medical Laboratory Animal Science [M], People’s Military Medical Press, Beijing, 2000.

14.

Jianxiang

Jingyuan

and Shuhua

, Establishment of rat experimental osteoarthritis model and pathological features [J], Acta Medicinae Universitatis Science of Technologiae Huazhong 38(1) (2009), 98–102.

15.

Qiong

Tie

and Liao

, Effect of aspirin on cancellous bone in cyclophosphamide-treatedrats [J], Chinese Medical Journal 32(2) (2011), 221–225.

16.

Keshawarz

N.M.

and Recker

R.R.

, Expansion of the medullary cavity at the expanse of cortex in postmenopausal osteoarthritis [M], Metab Bone Dis Relat Res 5(5) (1984), 223–228. doi:10.1016/0221-8747(84)90063-8.

17.

Woff

, The Law of Bone Remodeling [M], Springer-Verlag, Berlin, 1986, pp. 1–126.

18.

Chunyuan

Mahemuti

and Rong

, Effect of sand therapy on femur mechanical properties of arthritis in rabbits and finite element analysis [J], Journal of Central South University (Medical Sciences) 34(1) (2009), 8–12.