The molecular mechanism research of cartilage calcification induced by osteoarthritis.

To explore the molecular mechanism of cartilage calcification induced by osteoarthritis (OA) based on distal-less homeobox gene 5 - alkaline phosphatase - integrin-binding sialoprotein - ecto-nucleotide pyrophosphatase 1 (DLX5-ALPL-IBSP-ENPP1) signal axis. Twenty-four rabbits were selected to build models of cartilage calcification induced by OA and randomly divided into 3 groups. The first group was the normal group whose rabbits were injected into 0.9% saline (0.3 mL), and the second group was model group. The third group was model group whose rabbits were injected into DLX5 antibody by caudal vein. Alizarin red calcium staining was used to analyze calcium deposition of cartilage matrix. Immunohistochemical staining was used to analyze the relative expression levels of proteins DLX5 and ENPP1, and western blot was used to analyze the DLX5, ALPL, IBSP, and ENPP1 expression. Calcium salt precipitation was the most serious, and the calcification area increased in the model group. Although calcified nodules appeared in the anti-DLX5 group, they were relatively few. Immunohistochemical staining analysis showed that the protein DLX5 located in the nucleus and the protein ENPP1 located in the extracellular matrix. Western blot analysis showed that the expressions of proteins DLX5, ALPL, IBSP, and ENPP1 were the highest in OA Model group than that of NC group, followed by anti-DLX5 group. The proteins DLX5, ALPL, IBSP, and ENPP1 can promote cartilage calcification induced by OA based on DLX5-ALPL-IBSP-ENPP1 signal axis.

Introduction

Osteoarthritis (OA) is a knee disease that causes articular cartilage fibrosis, chapping, and loss. This is also a common age-related disease characterized by local loss of synovium and articular cartilage, associated with varying degrees of osteophyte formation, subchondral bone alteration, and synovitis [1].

The articular cartilage of knee that covered the surface of the joint is the most important structure to ensure the movement of the joint. The integrity of the articular cartilage is related to the normal movement of the knee [2]. Cartilage of knee joint belongs to hyaline cartilage, which is a kind of special connective tissue, and the thickness is about 1–5 mm and gradually becomes thinner with age increase. Articular cartilage is divided into four layers. The first layer is surface band or tangent band, which is composed of dense collagen fibers arranged along the joint surface. The second transition zone is a thick layer of collagen fibers which are irregularly arranged. The third layer is the deep or radioactive zone and is the main part of articular cartilage. This layer contains the most proteoglycan, the least water, and the most collagen fibers. The fourth layer is called cartilage calcification layer and is rich in hydroxyapatite salt. Collagen fibers are firmly anchored to the underlying bone tissue through the calcified cartilage layer, which separates it from the subchondral bone [3-6].

Calcified cartilage layer plays an important biological role in the articular osteochondral complex tissue. The calcified layer is closely chimed with the hyaluronic layer through the wavy tidal line structure at the upper interface, and the uneven comb tooth structure at the lower interface is closely anchored to the subchondral bone, which not only greatly increases the connection area between the tissue interfaces of each layer but also increases the connection strength between the interfaces. The calcification layer firmly fixes the articular cartilage to the subchondral bone and facilitates the instantaneous stress from the cartilage to conduct and disperse to the subchondral bone. In addition, the dense calcified layer structure prevents the fluid and small molecular organic matter in the medullary cavity from entering the non-calcified layer of cartilage, thus maintaining the stability of the special physiological microenvironment of cartilage and subchondral bone [7,8]. The intact calcified layer plays an important role in maintaining the survival and function of articular hyaline cartilage.

Articular osteochondral defects caused by knee OA are clinically common and often manifest as joint refractory pain and limited joint activity, which seriously affects the quality of life of patients and has become the main cause of limb disability [9]. Cartilage calcification of the knee joint is mainly caused by the stimulation of one or more factors, such as cartilage inflammation, wear, and lack of blood supply. It results in hardening of the local cartilage of the knee joint and increase of calcium content [10]. Protein function analysis reveals that protein DLX5 is involved in osteoblastic differentiation as a transcription factor. Therefore, this research proposes a hypothesis “proteins ALPL and IBSP promoted bone matrix calcification, while protein ENPP1 promotes cartilage calcification.” Meanwhile, further understanding of the role of these proteins is of great significance for inhibiting cartilage calcification-induced knee OA.

Materials and methods

Materials

Twenty-four New Zealand rabbits were purchased from Changchun yisi laboratory animal technology company (Changchun, China). The rabbits were 8–9 weeks of age and were adaptively fed for 2 weeks under SPF level environment, 23 ± 2°C, humidity 50%. The operating protocols for rats were completely obeyed the regulations of the Ethics Committee of Harbin Medical University. Pentobarbital sodium, DLX5 protein, and alizarin calcium staining kit were purchased from sigma (USA); ethylenediam ine tetraacetic acid (EDTA), PBS, FBS, and DMEM were purchased from Gibco (Shanghai, China), DLX5, ALPL, IBSP, and ENPP1 rat anti-rabbit primary antibodies were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Mic-CT and SDS-PAGE electrophoresis were purchased from Thermo Fisher (USA).

Methods

Establishment of OA model

Twenty-four New Zealand rabbits (8–9 weeks of age) were selected to build models of cartilage calcification induced by OA and randomly divided into 3 groups after being fed for 2 weeks [11]. One group was the normal group (n = 8), and the other two groups were established the models (n = 16). The rabbits were narcotized by intravenous injection of 3% pentobarbital sodium (1 mL/kg) and fixed on the operating table in supine position. After routine skin preparation, disinfection, and left knee joint of rabbit slightly flexion, 1 mL syringe was used to insert into the joint cavity below the patella. Then, 4% papain solution (0.2 mL) and 0.03 mol/L L-cysteine (0.1 mL) were injected to build mode groups. The normal group was injected into 0.9% normal saline (0.3 mL) as control by the same method. At the same time, a group of model rabbits were injected with DLX5 protein through caudal vein during the modeling process. The rabbits were killed at the end of the 9th week after initial injection of drug. All animal experiments have passed the review of ethics Committee of Harbin Medical University (No. 20210224).

Calcium deposition analysis of cartilage matrix

Both knee joints and cartilage tissues of experimental rabbits were prepared into paraffin sections, which were 5 μm. The calcium deposition of cartilage matrix was analyzed by alizarin red calcium staining kit. The specific process was that tissue was washed with PBS and fixed with 40 g/L paraformaldehyde at room temperature for 30 min. Then, the samples were washed with PBS after absorbing the fixing solution, dripped with alizarin red staining solution, covered the samples, and stained for 1–5 min. The staining solution was sucked away, washed with PBS twice, and observed under the microscope. After paraffin sections being made, calcium deposition of cartilage matrix was analyzed using VON KOSSA calcium staining kit and the procedures were the same as above [12].

Analysis of proteins DLX5 and ENPP1 expression by immunohistochemical staining

Bilateral knee joint and its cartilage of rabbits were fixed in a 10% neutral formalin solution, and EDTA was used for decalcification [13]. The decalcified specimens were embedded in paraffin and sectioned consecutively with 6 μm. Immunohistochemical staining was used to analyze the pathological features of cartilage tissue of knee joint and the relative expression levels of proteins DLX5 and ENPP1. The procedure of immunohistochemical staining was as follows [14,15]: The sections were sealed with 3% hydrogen peroxide for 10 min, washed with PBS for 3 times, dropped with 100 μL primary antibody in each section, and incubated at 37°C for 30 min. 100 μL second antibody was added and incubated at 37°C for 20 min. The sections were washed with PBS, dyed with DAB color, dehydrated with gradient ethanol, and sealed with transparent xylene.

Western blot analysis of DLX5, ALPL, IBSP, and ENPP1 expression

Total proteins of tissue were extracted from tissue and 20 μg proteins were sampled. 5% concentrated gel and 12% isolated gel were prepared to isolate proteins by SDS-PAGE [16]. Objective and internal proteins were transferred to NC membrane and then closed with 5% skimmed milk powder sealing fluid for 2 h at room temperature. Rat anti-rabbit primary antibody DLX5 (1:500), ALPL (1:500), IBSP (1:500), ENPP1 (1:500), and β-actin (1:1000) were added and incubated at 4°C overnight. TBST (TBS+Tween) was washed 4 times, and then HRP-labeled sheep anti-rat secondary antibody (1:5, 000) was added and incubated at 37°C for 1 h. TBST was washed 4 times. Color was developed with ECL luminescent solution, protein bands were exposed by gel image analysis system, and images were photographed and quantitatively analyzed. The experiment was repeated three times.

Statistical analysis

All experiments were replicated independently at least three times. The data were analyzed using one-way analysis of variance and are presented as the mean ± standard deviation. Statistical significance was defined as P < 0.05.

Results

Calcium deposition analysis of cartilage matrix

Alizarin red staining was performed in different groups, and the red color represented calcium deposition, as shown in Figure 1. Calcium deposition of cartilage matrix analysis of VON KOSSA calcium staining (number of brown cells) in different groups is shown in Figure 2. Calcium salt precipitation was the most serious in the model group, and the calcification area increased significantly compared with the control group. There was a significant difference in OA model. However, calcium salt precipitation stained by alizarin red was reduced in anti-DLX5 group and had a obvious difference. Although calcified nodules were present in anti-DLX5 group, they were relatively few compared with OA model.

Figure 1.

Calcium deposition of cartilage matrix analysis of alizarin red calcium staining in different groups (×50).

Figure 2.

Calcium deposition of cartilage matrix analysis of VON KOSSA calcium staining in different groups (×50).

Analysis of proteins DLX5 and ENPP1 expression by immunohistochemical staining

The protein DLX5 is located in the nucleus, and the protein ENPP1 is located in the extracellular matrix, as shown in Figure 3. There was a significant increase in model histone DLX5 but slightly decreased in the anti-DLX5 group compared with the model group. There was a significant difference between OA model group and anti-DLX5 group. DLX5 as a transcription factor is involved in osteoblast differentiation. DLX5 belongs to the homologous box gene family, and promotes osteoblasts differentiation and mineralized bone matrix by regulating the transcription of osteocalcin, osteopontin, osteopontin, and type I collagen [17-20]. The protein ENPP1 could promote the formation of cartilage calcification, and ENPP1 in model group was significantly increased, but the protein DLX5 in the anti-DLX5 group was slightly decreased compared with the model group.

Figure 3.

Analysis of proteins DLX5 and ENPP1 expression by immunohistochemical staining in different groups.

Western blot analysis of DLX5, ALPL, IBSP, and ENPP1 expression

Western blot analysis showed in Figure 4 that the expressions of proteins DLX5, ALPL, IBSP, and ENPP1 were the highest in OA Model group than that of NC group, followed by anti-DLX5 group. The expressions of proteins DLX5, ALPL, IBSP, and ENPP1 were the lowest in NC group. DLX5 as a transcription factor participates in osteoblastic differentiation. ALPL and IBSP can promote bone matrix calcification, and ENPP1 can promote cartilage calcification. These proteins work together to induce chondro calcification in the knee joint.

Figure 4.

Western blot analysis of expression levels of proteins DLX5, ALPL, IBSP, and ENPP1. **P < 0.01, ***P < 0.001 compared with the OA model.

Discussion

Articular cartilage is the spongy hyaluronic cartilage covering the articular surface and has good elasticity to bear the huge load of joint movement. In addition to compressive resistance, articular cartilage also has mechanical properties such as shock absorption, conduction, and dispersion stress, so as to protect the articular bones and accessory organs from being damaged by sudden huge stimulation [21,22]. This mechanical property of articular cartilage is closely related to its special structure. Mature articular cartilage can be divided into four layers from the deep side of the joint: superficial area, transition area, radiation area, and calcified cartilage area. The first three zones are also known as non-calcified layer of cartilage, while calcified cartilage area is also known as calcified layer [23,24]. The calcified layer of articular cartilage is located between the tidal line and the bonding line, with a thickness from several microns to more than 300 microns. It has a dense structure and a small number of hypertrophic chondrocytes. Chondrocytes in the superficial proliferating area near the articular surface stop proliferating and develop into a spongy hyaluronic layer enveloping the articular surface. The process of endochondral ossification in the deep proliferating area also stops, and the calcium salts deposited in the extracellular matrix are no longer dissolved and absorbed, thus forming the calcified layer structure [25,26].

When the body occurs joint injury, the pathological feature of OA shows that the calcification layer structure is significantly thickened, and the thickest part is more than 900 μm. Calcified cartilaginous layer thickens with vessels growing into the calcified layer of osteochondral or deep cartilage. Non-calcified cartilage and calcified layer occur fibrous change, and calcified layer and deep cartilage occur defect [26]. The longitudinal section of articular cartilage is disorganized, and there are some tidal line fracture, calcification layer thickening, tidal line gap widening, and calcium salt deposit [27]. Calcium salt precipitation was the most serious in the model group, and the calcification area increased significantly compared with the control group. In addition, the expressions of related proteins DLX5, ALPL, and IBSPENPP1 in bone tissue were determined. The results showed that the protein expression in OA Model group was the highest, followed by anti-DLX5 group, and NC group was the lowest. Western blot analysis showed in Figure 4 that the expressions of proteins DLX5, ALPL, IBSP, and ENPP1 were the highest in OA Model group than that of NC group, followed by anti-DLX5 group. These proteins, as transcription factors, participate in osteoblast differentiation, promote calcification of cartilage matrix, and cause cartilage damage. Further understanding of the role of these proteins and the fact that the calcified layer of articular cartilage is an important functional structure in the complex articular osteochondral tissue have profound significance for the treatment of cartilage defects and the restoration of joint function.

Conclusion

The proteins DLX5, ALPL, IBSP, and ENPP1 can promote cartilage calcification-induced OA based on DLX5-ALPL-IBSP-ENPP1 signal axis.