Mitochondrial Transplantation Ameliorates the Development and Progression of Osteoarthritis

Abstract

Osteoarthritis (OA) is a common degenerative joint disease characterized by breakdown of joint cartilage. Mitochondrial dysfunction of the chondrocyte is a risk factor for OA progression. We examined the therapeutic potential of mitochondrial transplantation for OA. Mitochondria were injected into the knee joint of monosodium iodoacetate-induced OA rats. Chondrocytes from OA rats or patients with OA were cultured to examine mitochondrial function in cellular pathophysiology. Pain, cartilage destruction, and bone loss were improved in mitochondrial transplanted-OA rats. The transcript levels of IL-1β, TNF-α, matrix metallopeptidase 13, and MCP-1 in cartilage were markedly decreased by mitochondrial transplantation. Mitochondrial function, as indicated by membrane potential and oxygen consumption rate, in chondrocytes from OA rats was improved by mitochondrial transplantation. Likewise, the mitochondrial function of chondrocytes from OA patients was improved by coculture with mitochondria. Furthermore, inflammatory cell death was significantly decreased by coculture with mitochondria. Mitochondrial transplantation ameliorated OA progression, which is caused by mitochondrial dysfunction. These results suggest the therapeutic potential of mitochondrial transplantation for OA.

Keywords: Autophagy; Mitochondrial dysfunction; Mitochondrial transplantation; Necroptosis; Osteoarthritis.

Figure 1. Attenuation of OA by mitochondrial transplantation. (A) Bar graph shows the amount of isolated mitochondria from 1x10⁶ of L6 cells. (B) Representative immunoblots show the expression of Ki-67, STIM1, tubulin, and COX4 in the nuclear, ER, cytosolic, and mitochondrial fractions, respectively. (C) Isolated mitochondira stained with NAO. Representative FACS plot shows percentage of NAO positive mitochondria. (D) Bar graph shows average ATP content of isolated mitochondria. MIA-induced OA rats were observed and evaluated for 29 days (n=5 animals per group). The mitochondrial injection was performed intra-articularly twice weekly. Kinetics of PWL, PWT (E), and weight-bearing (F). (G) Bar graph shows the average amount of CTX-II in serum of each group. (H) Representative images of Safranin O-stained cartilage. Scale bar = 100 μm. (I) Average total Mankin score (left) and OARIS score (right). (J) Representative micro CT images of bone. (K) Percentage of bone surface (left) and BV: TV ratio (right). Data are means ± SEM.

NAO, nonyl acridine orange. ^*p<0.05, ^**p<0.01.

Figure 2. Improvement of mitochondrial function in chondrocytes of OA rats by mitochondrial transplantation. Chondrocytes were isolated from normal, MIA-induced OA rat, and mitochondrial transplanted-MIA-induced OA rats. (A) Representative transmission electron microscopy images show mitochodria in chondrocytes of each group. Red arrow, mitochondria. Scale bar, 2 μm (upper panels) and 0.5 μm (lower panels). (B) Bar graph shows averaged number of mitochondria (left) and mitochondrial diameter (right) in each group. (C) Mitochondrial membrane potential was measured by JC-1 staining. Data are means ± SEM.

^*p<0.05, ^**p<0.01.

Figure 3. Improvement of mitochondrial function in human OA chondrocytes by mitochondrial transplantation. MTDR-stained mitochondria were transferred into human chondrocytes with MTG-labeled mitochondria. (A) Representative FACS plot shows percentages of MTDR-positive cells. (B) Representative images show MTG (greed), MTDR (red), and DAPI (blue) positive cells. Scale bar = 20 μm. (C) mtOXPHOS Complex genes were determined by RT-qPCR in OA chondrocytes incubated in the presence or absence of IL-1β (20 ng/mL) and mitochondria (5 μg) for 24 h. Average transcript levels of complex I, III, IV, and V. (D) Mitochondrial membrane potential analyzed by JC-1 staining. (E) Transmission electron micrographs of chondrocytes from OA patients in the presence or absence of IL-1β (20 ng/mL) and mitochondria (5 μg) for 24 h. Arrowheads, mitochondria. Scale: 2 μm (upper panels), 0.5 μm (lower panels). (F) The average number of mitochondria (left) and mitochondrial diameter (right). Data are means ± SEM.

^*p<0.05, ^**p<0.01, ^***p<0.001.

Figure 4. Regulation of autophagy activity by mitochondrial transfer. Human OA chondrocytes were cultured with mitochondria (5 μg) in the presence or absence of IL-1β (20 ng/mL) for 24 h. (A) Mitochondrial ROS was measured using MitoSOX dye and flow cytometry. Bar graph shows averaged amount of ROS in each condition. (B) Representative immunoblots show LC3, p62, and LAMP1 in each condition. Average LC-3I and LC-3II (left), p62 and GAPDH (middle), and LAMP1 and GAPDH (right) ratio. (C) Confocal micrographs show colocalization of p62 (FITC) and LC3 (APC), and LAMP1 (PE) and LC3 (APC) for autophagosome and autophagolysosome, respectively. Scale bar = 20 μm. (D) Percentages of cells with autophagosomes and autophagolysosomes. (E) Autophagy was analyzed by TEM. Red arrows, autophagosomes; yellow arrows, autophagolysosomes. Scale bar = 2 μm (upper panels), 0.5 μm (lower panels). (F) Percentages of cells with autophagosomes and autophagolysosomes. Data are means ± SEM.

^*p<0.05, ^**p<0.01.

Figure 5. Expression of cell death and cartilage regulatory markers following mitochondria administration. Human OA chondrocytes were cultured in the presence or absence of IL-1β (20 ng/mL) or TNF-α (50 ng/ml) and mitochondria (5 μg) for 24 h. (A) mRNA levels of catabolic factors were analyzed by qPCR. (B) Necrosis analysis by annexinV/PI staining. (C) Expression of phospho- or total of RIPK1, RIPK3, MLKL in OA chondrocytes was analyzed by western blotting. (D) mRNA levels of chondrogenesis markers in OA chondrocytes. Data are means ± SEM.

^*p<0.05, ^***p<0.001.

Figure 6. Mitochondrial transplantation from OA patient muscle tissue in MIA-induced rat joint. MIA-induced OA rats were evaluated for 21 days (n=3 animals per group). Intra-articular mitochondria injection was performed twice weekly. Kinetics of PWL, PWT (A), and weight-bearing (B). (C) Representative images show Safranin O-stained cartilage. Bar graphs show averaged total Mankin score (left) and OARIS score (right). (D) Representative micro-CT images show bone of each group. Bar graphs show percentage of bone surface (left) and BV: TV ratio (right). (E) IL-1β, TNF-α, MCP-1, MMP-13, and pMLKL expression determined immunohistochemically in the synovium of MIA-induced OA rats. Data are means ± SEM.

^*p<0.05, ^**p<0.01.