Pathological Characters and Molecular Pathogenesis of Diabetic Neuropathic Osteoarthropathy Cartilages Damage

Background: Diabetic neuropathic osteoarthropathy (DNOAP) is a rare and easily missed complication for diabetes that leads to increased morbidity and mortality. DNOAP is characterized by progressive destruction of bone and joint, but its pathogenesis remains elusive. We herein aimed to investigate the pathological features and pathogenesis of the cartilages damage in DNOAP patients. Methods: The articular cartilages of 8 patients with DNOAP and 8 normal controls were included. Masson staining and safranine O/xed green staining (S-O) were used to observe the histopathological characteristics of cartilage, and the ultrastructural changes of chondrocytes were detected by electron microscopy. Chondrocyte were isolated from DNOAP group and control group. The expression of RANKL, OPG, IL-1β, IL-6, TNF-α, Aggrecan protein were evaluated by Western blot. ROS levels were measured using a DCFH-DA probe. The percentage of apoptotic cells was determined by ow cytometry. The chondrocytes were cultured with different glucose concentrations to observe the expression of RANKL and OPG. Results: Compared with the control group, the DNOAP group showed fewer chondrocytes, subchondral bone hyperplasia and structural disorder, and a large number of osteoclasts formed in the subchondral bone area. Moreover, mitochondrial and endoplasmic reticulum swelling were observed in the DNOAP chondrocytes. The chromatin was partially broken and concentrated at the edge of nuclear membrane. The ROS uorescence intensity of chondrocyte in DNOAP group was higher than that in normal control group (28.1 ± 2.3 VS 11.9 ± 0.7, P < 0.05). The expression of RANKL, TNF-α, IL-1β and IL-6 protein in DNOAP group was higher than that in normal control group, while OPG and Aggrecan protein was lower than that in normal control group (both P < 0.05). Flow cytometry showed that the apoptotic rate of chondrocyte in DNOAP group was higher than that in normal control group (P < 0.05). The RANKL/OPG ratio showed signicant upward trend when the concentration of glucose was over than 15mM. Conclusions: DNOAP patients tend to have severe destruction of articular cartilage and collapse of organelle structure including mitochondrion and endoplasm reticulum. Indicators of bone metabolism (RANKL, OPG) and inammatory cytokines (IL-1β, IL-6 and TNF-α) play an important role in promoting the pathogenesis of DNOAP. The glucose concentration higher than 15mM made the RANKL / OPG ratio changed rapidly.


Introduction
Diabetic neuropathic osteoarthropathy (DNOAP) was rst described in1936 by Jordan. It is a serious complication of diabetes, accounting for 0.8-13.0% of all diabetic patients. And, the prevalence of highrisk patients can be as high as 29.0% [1] . As diabetes mellitus has become one of the most common disorders now, there will be an increase in the prevalence of DNOAP. Patients with DNOAP often present with joint subluxations, dislocation, or pathological fractures, which reduce the quality of life and increase the mortality signi cantly [2,3] . Accurate diagnosis and appropriate treatment could avoid the delays in patient condition and operation, improve clinical outcomes and lower the medical costs.
The pathogenesis of DNOAP, however, remains largely unknown. And, new prospects for studying DNOAP are constantly emerging. The investigation of DNOAP pointed out that in ammatory markers and the dynamics of bone metabolism were involved in the pathological process of it [4][5][6] . Among them, the increase of pro-in ammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-1β in serum of DNOAP suffers has been reported multiple times [4,7] . In addition, Jeffcoate et al. proposed that the pathway of receptor-activator of nuclear factor kappaB (RANK), its ligand RANKL, and osteoprotegerin (OPG) played a role in disease progression of Charcot neuroarthropathy rstly in 2004 [8] .
The RANKL/OPG axis is a key mediator which has been used to evaluate osteoclastogenesis and osteolytic processes in numerous diseases such as rheumatoid arthritis, osteoarthritis, and bone tumors.
And the expression of RANKL was increased in diabetes caused by Oxidative stress and in ammation state [9] . Furthermore, La Fontaine et al [10] found that the number of bone trabeculae in patients with DNOAP was signi cantly reduced and the structure was disordered. However, there is no report on the pathological changes of cartilage from DNOAP patients.
In this study, articular cartilage specimens and chondrocytes were used to explore the pathological characteristics and molecular mechanism of DNOAP patients, to open novel avenues for clinical prevention and treatment.

Clinical samples
From March 2017 to June 2018, articular cartilage specimens were collected from 8 clinically con rmed DNOAP patients, and the articular cartilage of matched joints from 8 amputation patients without underlying disease were as control group.
Enrolled controls were patients without diabetes and peripheral neuropathy who underwent amputation due to tra c accident or serious trauma. Exclusion criteria: (1) osteoarthritis, Rheumatoid arthritis, and other degenerative joint disease, (2) open injury or contamination of affected joints. Eight cases of tra c accident or severe injury amputation were recruited as the controls, including 4 males and 4 females, aged 19-65 (57.6 ± 3.7) years old.
The articular cartilages of tibiotalar joint, subtalar joint, and talonavicular joint were taken from two groups. The use of human samples was approved by the Ethical Committee of Honghui Hospital of Xi'an Jiaotong University (approval No.201702003), and informed consent was obtained from each participant.

Pathological examination
The articular cartilage biopsies from donors of DNOAP group and controls were xed in 10% formaldehyde for 24 hours, decalci ed by 15% neutral EDTA-2Na for 15 days, then dehydrated by alcohol gradient of different concentrations and embedded with para n. Cut the para n-embedded specimens into 5μm consecutively. Then, the tissue sections were stained with Masson's Trichrome Stain

Transmission Electron Microscopy
The fresh cartilage specimens of DNOAP group and controls were xed with 2.5% glutaraldehyde at 4 ℃ in a volume of 1 mm 3 overnight. Next day, the cartilage samples were rinsed with ddH 2 O, xed with 1% osmium acid for 1h, stained with 2% uranium acetate for 30min, dehydrated gradiently with 50%, 70%, 90%, 100% ethanol and 100% acetone. After in ltration, embedding and polymerization, the sections were sliced to a thickness of 70nm by ultramicrotome, and then stained with uranium acetate lead citrate. The ultrastructure of organelles in chondrocytes were observed under HITACHIH-7650 transmission electron microscope (Hitachi, Tokyo, Japan).

Chondrocyte maintenance
The cartilage samples were segmented into 3~5mm 3 pieces, and washed into the phosphate buffer solution (PBS) containing penicillin (100U/L) and streptomycin (100mg/L). Then the small pieces were digested with 0.25% trypsin at room temperature for 15-20min, and centrifugated at 1000×g for 5min.
After removing the supernatant, the deposit was washed with PBS for three times. Next, the deposit was digested at 37 ℃ for 8~10h with 0.2% Collagenase Type II (C2-BIOC, Millipore Sigma, USA) in a constant temperature shaker. After ltration by aseptic cell sieves, the cells were washed and collected through centrifugation (1000×g, 10 min). The chondrocytes were cultured with DMEM medium (Invitrogen, USA) supplemented with 10% fetal bovine serum (Thermo Fisher Scienti c, USA) at 37°C in a humidi ed atmosphere containing 5% CO 2 .

Detection of reactive oxygen species (ROS) in chondrocytes
Reactive oxygen species (ROS) production was detected with a Reactive Oxygen Species Assay Kit (Shanghai Beyotime Biotechnology Co., Ltd). Inoculate the chondrocytes into 6-well plate (1×10 6 cells per well) and 3 auxiliary holes were set for each group. When the cell fusion rate reached 70% ~ 80%, the culture medium was removed, and DCFH-DA with the concentration of 10μmol/L was added into each well, and then the plate was placed in dark for 20min at 37℃. Wash the cells with serum-free DMEM for three times. ROS was measured by uorescence microscope at an excitation wavelength of 485nm and an emission of 525nm. The intensity of each group was analyzed by Image Pro Plus image analysis software.

Western blotting
Protein concentration was detected using Pierce BCA Protein Assay Kit (Thermo Fisher Scienti c, USA) in accordance with the manufacturer's instructions. Total proteins were electrophoretically separated on sodium dodecyl sulfate polyacrylamide gels (8-15%) according to the molecular size of the target protein, and were subsequently transferred onto polyvinylidene di uoride membranes. After being blocked with 5% skim milk, the membranes were incubated at 4℃ overnight with the following primary antibodies: anti-RANKL, anti-OPG, anti-IL-1β, anti-IL-6, anti-TNF-α and anti-β-actin (Proteintech, USA).
Then, the membranes were washed thoroughly and incubated with secondary antibodies (1:2000 antimouse/ rabbit, Santa Cruz Biotechnology, USA) at room temperature for 2 hours. The signals were visualized using the enhanced chemiluminescence method (Immobilon Western Chemiluminescent HRP Substrate, Millipore). The samples were analyzed in duplicate, and the experiment was performed three times.

Detection of apoptosis by ow cytometry
The percentage of apoptotic cells was ascertained through Annexin V/FITC-PI apoptosis detection kit (Beijing Sizhengbai Biotechnology Co., Ltd).
Cells were prepared with the concentration of 1×10 6 /ml in 10% bovine serum albumin, according to the manufacturer's instructions. After cultured in the incubator with 5% CO 2 at 37℃ for 24h, the cells were collected in a 10 ml centrifuge tube, centrifuged at 1000×g for 5 min, and washed with precooled PBS twice. Added Annexin V-FITC/ PI and PI (100ug/mL) working solution into cells, and stained for 15min at room temperature in dark. Flow cytometry was used to detect the apoptosis rate of cells, and Cell Quest software was used to obtain and analyze parameters. The experiment was repeated three times.

High glucose induced expression of RANKL and OPG in chondrocytes
Hyperglycemia is a common feature of DNAOP patients, thus, we simulated anomalous level of blood sugar in vitro by treating normal chondrocytes with a wide range of glucose concentrations (5mM, 10mM, 15mM, 20mM, 25 mM, 33mM) for 24 hours. Western blot analyses were used to detect the expression of RANKL and OPG in chondrocytes.

Statistical methods
Statistical analyses were performed with SPSS 19.0 (SPSS Inc., Chicago, IL, USA). Data from cell experiments were analyzed using unpaired Student's t-tests, and images based on the statistical analyses were made in GraphPad Prism 5.0 (GraphPad Software Inc, La Jolla, CA, USA). All hypothetical tests were two-sided, and P-values less than 0.05 were considered statistically signi cant in all tests.

Results
Histopathological characterization of articular cartilage Changes in articular cartilage were explored under the optical microscope. In the articular cartilage from the control group, chondrocytes were in cartilage lacuna, cartilage matrix was stained evenly, and subchondral bone was arranged orderly (Fig. 1A,1B). However, in the tissues of DNOAP patients, S-O staining showed that the continuity of the super cial cartilage was interrupted, the vertical fracture entered the deep layer of cartilage. The chondrocytes around the fracture were rupture and the matrix was light stained. Subchondral bone plates and trabeculae were reduced, and osteoclasts aggregation was observed. The subchondral bone shows the characteristics of reactive bone and the structure is disordered (Fig. 1C). Masson's trichrome staining showed that hyaline cartilage was arranged in a cordlike arrangement, the chondrocytes and osteocytes were decreased. In addition, subchondral bone hyperplasia, structural disorder and cavities were observed (Fig. 1D). The histopathological ndings of all DNAOP patients were consistent.

Ultrastructure of chondrocytes
The integrity of organelles is the primary condition for cellular homeostasis. Next, we observed the ultrastructure of chondrocytes by transmission electron microscope. In the normal chondrocytes, there were abundant rough endoplasmic reticulum and normal condensed chromatin in nucleus ( Fig. 2A,2B). In the cells from DNOAP group, swollen mitochondria were found, some mitochondrial membranes were incomplete, and the arrangement of mitochondrial cristae was disordered. Furthermore, severe expansion of the endoplasmic reticulum and Golgi apparatus were scattered in the cytosol. The nucleus became larger, and the chromatin was partially broken, concentrated, and gathered at the edge of nuclear membrane (Fig. 2C, 2D).

Reactive oxygen species (ROS) increased in chondrocytes of the DNOAP group
Reactive oxygen species (ROS) are highly reactive molecules that provide the normal signals in various cell types. But the accumulation of ROS leads to oxidative damage of biomolecules, which further causes oxidative stress and even cell death. Thus, we detected the level of ROS in chondrocytes by uorescence microscope, and found that the ROS uorescence intensity of cells originated from DNOAP group was obviously higher than the cells from the controls (28.1 ± 2.3 vs. 11.9 ± 0.7, t = 19.059, P < 0.05, Fig. 3)

Apoptosis increased in chondrocytes originated from DNOAP patients
We then compared the cell apoptosis between DNOAP group and the control group. Flow cytometry analysis demonstrated that the proportion of apoptotic chondrocytes was around 2.6 times higher in the DNAOP group than in the control group (3.3 ± 0.2% vs. 1.2 ± 0.1%, P < 0.05, Fig. 4).

The expression of RANKL, OPG, Aggrecan and in ammatory cytokines
Western blot analyses were performed on total protein extracted from articular cartilage specimens of DNOAP patients and control participants. It demonstrated that the tissues of DNOAP group expressed higher levels of RANKL, IL-1β, IL-6 and TNF-α and lower levels of OPG and Aggrecan than that of normal group, respectively (P<0.05, Fig.5). The comparison results of each pair of samples and age-matched controls were consistent. And representative images of each index detected in our study were shown in Fig.5.

High glucose induced the change of RANKL/OPG ratio in chondrocytes
Western blot analyses demonstrated that the expression of RANKL decreased slightly with glucose concentration increase of DMEM medium. However, a sharp decline in OPG was observed, and when the concentration of Glucose was or over than 20mM, the expression of OPG was almost undetectable. Then we found that the ratio of RANKL/OPG remained near 1:1 when the cells were cultured with 5mM glucose, but the ratio showed signi cant upward trend when the concentration of Glucose was over than 15mM. The ratio reached the peak at 20mM of Glucose, and then decreased slightly and almost remained at the same level (Fig.6).

Discussion
Neuroarthropathy, also known as Charcot's joint, refers to the progressive, painless and noninfective destructive disease of bone and joint caused by neuropathy. In 1886, Charcot rst reported the bone and joint lesions of patients with spinal tuberculosis comprehensively. In 1936, Jordan found the relationship between diabetes and Neuroarthropathy, and proposed the concept of DNOAP for the rst time [13] . However, up to now, the pathogenesis of DNOAP have remained unclear. Limited previous studies focused on the changes of synovium and bone trabeculae. In this study, we detected the pathological changes of cartilage rstly.
There were a large number of chondrocytes disintegrated in the cartilage tissue of DNOAP patients, osteoclasts aggregation and subchondral bone remodeling in subchondral bone area. And all of the changes of cartilage above were not found in osteoarthritis or rheumatoid arthritis [14] . Osteoclasts are closely related to bone resorption [15] . Activation and aggregation of osteoclasts will cause bone resorption and destruction, and lead to development of DNOAP. In addition, we observed the ultrastructural features of chondrocytes originated from DNOAP patients with transmission electron microscope, and found mitochondrial swelling and disordered arrangement of mitochondrial cristae, and numerous vacuoles in the endoplasmic reticulum and Golgi. Moreover, the apoptotic rate of chondrocytes in DNOAP group was higher than that in control group. The activity of mitochondrial and endoplasmic reticulum is closely related to the cell viability [16] . Structural damage of mitochondria and endoplasmic reticulum will lead to the changes in cellular metabolism, even the programmed death of chondrocytes, namely apoptosis, and nally induced to the destruction of cartilage tissue structure and matrix.
ROS is the main substance that causes oxidative stress damage in tissues, which plays an important role in bone remodeling by stimulating RANKL and inhibiting OPG expression [17,18] . ROS had a negative effect on osteoblastic differentiation [19] . On the contrary, osteoclast activity was directly stimulated by ROS [20] . Verzijl et al. found that the deposition of advanced glycation end products (AGEs) and ROS were increased in chondrocytes exposed to high-glucose [21] . Consistent with previous study, we found that the production of ROS in DNOAP chondrocytes was more than the healthy controls, accompanied by higher level of RANKL and lower of OPG. These ndings suggested that ROS induced by hyperglycemia may stimulate osteoclast activation by regulating RANKL/OPG pathway in DNOAP patients. RANKL/OPG system, as an important regulatory axis of bone turnover, plays an important role in the activation, aggregation and function of osteoclasts. RANKL belongs to the TNF superfamily and has the ability to promote osteoclast formation. Its main role is to activate the RANK, which regulates osteoclast differentiation, promotes osteoclastogenesis and bone resorption [22] . OPG, the soluble decoy receptor of RANKL, is a cytokine synthesized by activated osteoblasts, commonly known as the "osteoclastogenesis inhibitory factor". OPG antagonizes the RANKL-RANK interactions on the surfaces of osteoclast progenitors and blocks the resultant downstream osteoclastogenic cascade [23] . In a nutshell, osteoclast activity is likely to depend, at least in part, on the relative balance of RANKL and OPG. In general, abnormal RANKL:OPG ratio predicts pathological states, and can lead to an uncontrolled loss of bone mass [24,25] .
Under stable bone metabolism conditions, the secretion and expression of RANKL and OPG are dynamically balanced to maintain the homeostasis of osteogenesis and osteoclast. When the expression of RANKL increased and OPG decreased, osteoclasts would aggregate and activate, leading to bone destruction [26,27] . In the present study, we found that the expression of RANKL in DNOAP group was signi cantly higher than that in normal control group, while an opposite trend was observed for the OPG expression in two groups. These results suggested that the RANKL/OPG pathway may be involved in the pathological changes of cartilage in the patients with DNOAP. Previous studies failed to conclude the relationship between the RANKL/OPG pathway and the progression of DNOAP in peripheral blood [4] . Thus, the change of RANKL and OPG expression in the local cartilage lesion would be a signi cant discovery in the study of DNOAP.
Abnormal in ammatory markers are a common feature in DNOAP patients [28,29] . Generally, proin ammatory cytokines regulate the in ammatory response, and have dynamic interactions with metabolites that could mediate the bone turnover [30] . Kwan Tat et al. reported that the expression of RANKL was increased and OPG decreased in osteoarthritis chondrocytes under the stimulation of IL-1β, TNF-α and PGE2 [31] . In our study, it was found that the expression of IL-1β, IL-6 and TNF-α in DNOAP chondrocytes was signi cantly higher than that in the control group. Previous researches have reached similar conclusions [7,32] . Therefore, we speculated that the effects of these in ammatory indicators on DNOAP may partly depend on the biochemical activation of the RANKL/OPG signaling pathway. Persistent hyperglycemia is a common clinical manifestation in patients with DNOAP [33] . We treated normal chondrocytes with glucose in different concentrations, and found that the expression of RANKL decreased slightly and OPG sharply with glucose concentration increase. Notably, RANKL/OPG ratio showed obvious upward trend when the concentration of Glucose was from 15mM to 20mM. Subsequently, the ratio decreased slightly and almost remained at the same level. Thus, the ndings suggested that 15mM may be a threshold for the imbalance of chondrocyte metabolism, and may also be an important node that triggers the damage of cartilage and even the occurrence of DNOAP.

Conclusion
In this study, we reported the pathological features of cartilage and ultrastructural changes of chondrocytes in DNOAP patients for the rst time. Indicators of bone metabolism (RANKL, OPG) and in ammatory cytokines (IL-1β, IL-6 and TNF-α) play an important role in promoting the pathogenesis of DNOAP. The glucose concentration higher than 15mM made the RANKL / OPG ratio changed rapidly. These results settle a foundation for the further study on the pathogenesis of DNOAP. The broader involvement and clinical relevance of cartilage in the pathogenesis of DNOAP will be the focus of future investigations.

Abbreviations
AGEs: glycation end products, DNOAP, diabetic neuropathic osteoarthropathy, IL-1β: interleukin-1β, IL-6: interleukin-6, OPG: osteoprotegerin, RANK: receptor activator of nuclear factor κβ, RANKL: receptor activator of nuclear factor κβ ligand, ROS: reactive oxygen species, S-O: safranine O/ xed green staining, TNF-α: tumor necrosis factor-α     The protein expression of RANKL, OPG, Aggrecan, IL-1β, IL-6 and TNF-α in cartilage specimens of DNOAP and control group. Representative western blot bands and analysis in samples of DNOAP and control group. β-actin was used as a reference for calculating the relative protein expression. The error bars presented as mean ± Standard Error of Mean (SEM) with analysis of unpaired Student's t-test. *P < 0.05, compared with control group. The expression of RANKL, OPG and RANKL-OPG ratio changed under various concentration of glucose.
Representative western blot bands(A) and analysis (B, C) of RANKL, OPG in chondrocytes under different concentration of glucose. β-actin was used as a reference for calculating the relative protein expression.
Each group was repeated at least three times. The error bars presented as mean ± Standard Error of Mean (SEM) with analysis of unpaired Student's t-test. *P< 0.05, compared with the control group.