Mitochondrial transfer from bone-marrow-derived mesenchymal stromal cells to chondrocytes protects against cartilage degenerative mitochondrial dysfunction in rats chondrocytes.

BACKGROUND: Previous studies have reported that mitochondrial dysfunction participates in the pathological process of osteoarthritis (OA). However, studies that improve mitochondrial function are rare in OA. Mitochondrial transfer from mesenchymal stem cells (MSCs) to OA chondrocytes might be a cell-based therapy for the improvement of mitochondrial function to prevent cartilage degeneration. This study aimed to determine whether MSCs can donate mitochondria and protect the mitochondrial function and therefore reduce cartilage degeneration. METHODS: Bone-marrow-derived mesenchymal stromal cells (BM-MSCs) were harvested from the marrow cavities of femurs and tibia in young rats. OA chondrocytes were gathered from the femoral and tibial plateau in old OA model rats. BM-MSCs and OA chondrocytes were co-cultured and mitochondrial transfer from BM-MSCs to chondrocytes was identified. Chondrocytes with mitochondria transferred from BM-MSCs were selected by fluorescence-activated cell sorting. Mitochondrial function of these cells, including mitochondrial membrane potential (Δψm), the activity of mitochondrial respiratory chain (MRC) enzymes, and adenosine triphosphate (ATP) content were quantified and compared to OA chondrocytes without mitochondrial transfer. Chondrocytes proliferation, apoptosis, and secretion ability were also analyzed between the two groups. RESULTS: Mitochondrial transfer was found from BM-MSCs to OA chondrocytes. Chondrocytes with mitochondrial from MSCs (MSCs + OA group) showed increased mitochondrial membrane potential compared with OA chondrocytes without mitochondria transfer (OA group) (1.79 ±â€Š0.19 vs. 0.71 ±â€Š0.12, t = 10.42, P < 0.0001). The activity of MRC enzymes, including MRC complex I, II, III, and citrate synthase was also improved (P < 0.05). The content of ATP in MSCs + OA group was significantly higher than that in OA group (161.90 ±â€Š13.49 vs. 87.62 ±â€Š11.07 nmol/mg, t = 8.515, P < 0.0001). Meanwhile, we observed decreased cell apoptosis (7.09% ±â€Š0.68% vs.15.89% ±â€Š1.30%, t = 13.39, P < 0.0001) and increased relative secretion of type II collagen (2.01 ±â€Š0.14 vs.1.06 ±â€Š0.11, t = 9.141, P = 0.0008) and proteoglycan protein (2.08 ±â€Š0.20 vs. 0.97 ±â€Š0.12, t = 8.227, P = 0.0012) in MSCs + OA group, contrasted with OA group. CONCLUSIONS: Mitochondrial transfer from BM-MSCs provided protection for OA chondrocytes against mitochondrial dysfunction and degeneration through improving mitochondrial function, cell proliferation, and inhibiting apoptosis in chondrocytes. This finding may offer a new therapeutic direction for OA.

PHF23 negatively regulates the autophagy of chondrocytes in osteoarthritis.

AIM: Osteoarthritis (OA) is the main cause of disability and joint replacement surgery in the elderly. As a crucial cell survival mechanism, autophagy has been reported to decrease in OA. PHF23 is a new autophagy inhibitor which was first reported by us previously. This study aimed to explore the anti-autophagic mechanism of PHF23 to make it a possible therapeutic target of OA. MAIN METHOD: Lentiviral vectors specific to PHF23 were used on chondrocytes (C28/I2) to establish PHF23 overexpressed or knockdown stable cell strains. Interleukin (IL)-1ß (10 ng/mL) and chloroquine (CQ, 25 uM) were used as an inducer of OA and inhibitor of lysosome, respectively. Autophagy was evaluated by autophagosome formation using transmission electron microscopy (TEM) and western blot analysis of P62 and LC3B on different groups of cells. Effects of PHF23 on OA were evaluated by collagen II immunofluorescent staining and western blot analysis of OA-associated proteins MMP13 and ADAMTS5. Effects of PHF23 on AMPK and mTOR/S6K pathways and mitophagy were determined by western blot analysis. KEY FINDINGS: Knockdown of PHF23 enhanced IL-1ß-induced autophagy, while overexpression of PHF23 exerted the opposite effect. Knockdown of PHF23 protected chondrocytes against IL-1ß-induced OA by decreasing the levels of OA-associated proteins and increasing expression of Collagen II. Knockdown of PHF23 also increased mitophagy level and altered the phosphorylation levels of AMPK, mTOR, and S6K. SIGNIFICANCE: PHF23 downregulates autophagy, mitophagy in IL-1ß-induced OA-like chondrocytes and alters the activities of AMPK and mTOR/S6K, which suggests that PHF23 may be a possible therapeutic target for OA.

Rates of periprosthetic infection and surgical revision in Beijing (China) between 2014 and 2016: a retrospective multicenter cross-sectional study.

BACKGROUND: Periprosthetic joint infection (PJI) is a rare but devastating complication after total joint arthroplasty. There is a paucity of data on the incidence and prevalence of periprosthetic infection in mainland China. This study aimed to analyze the rates of surgical revision after arthroplasty due to PJI and the procedures followed in Beijing, China. METHODS: The study involved a retrospective multicenter cross-sectional survey of patients undergoing revisions for periprosthetic infection after hip/knee arthroplasty at nine hospitals in Beijing from 2014 to 2016. Age, gender, body mass index, primary diagnosis, comorbidity, primary surgery, treatment methods, and post-revision complications were analyzed. RESULTS: A total of 38,319 hip/knee arthroplasties and 366 (0.96%) revisions for PJI were identified. Of these, 161 (161/14,110; 1.14%) revisions involved hip arthroplasty, whereas 205 (205/24,209; 0.85%) revisions were due to knee arthroplasty. Procedures for revisions of infected hip included 7 (4.3%) cases of open debridement and prosthesis retention, 32 (19.9%) cases of one-stage exchange, 121 (75.2%) cases of two-stage exchange, and 1 (0.007%) case of hip dissection. As for the infected knee, the procedures included 45 (22.0%) cases of open debridement and prosthesis retention, 13 (6.3%) cases of one-stage exchange, 143 (69.8%) cases of two-stage exchange, and 4 (0.02%) cases of knee fusion. CONCLUSIONS: The study found the rates of revision due to PJI to be low. Nonetheless, the incidence of PJI in mainland China could be higher and calls for more elaborate studies in geographically and socioeconomically diverse health institutions.

Plant homeodomain finger protein 23 inhibits autophagy and promotes apoptosis of chondrocytes in osteoarthritis.

BACKGROUND: Plant homeodomain finger protein 23 (PHF23) is a novel autophagy inhibitor gene that has been few studied with respect to orthopedics. This study was to investigate the expression of PHF23 in articular cartilage and synovial tissue, and analyze the relationship between PHF23 and chondrocyte autophagy in osteoarthritis (OA). METHODS: Immunohistochemical staining and western blot were applied to show the expression of PHF23 in cartilage of different outbridge grades and synovial tissue of patient with OA and healthy control. The normal human chondrocyte pre-treated with rapamycin or 3-methyladenine, treated with interleukin-1ß (IL-1ß). IL-1ß induced expression level of PHF23 and autophagy-related proteins light chain 3B-I (LC3B-I), LC3B-II, and P62, were examined by Western blot. A PHF23 gene knock-down model was constructed with small interfering RNA. Western blot was performed to detect the efficiency of PHF23 and the impact of PHF23 knockout on IL-1ß-induced expression of autophagy-related and apoptotic-related proteins in chondrocyte. RESULTS: The expression of PHF23 was significantly increased in the high-grade cartilage and synovial tissue of patients with OA. The IL-1ß-induced expression of PHF23 was gradually enhanced with time. The level of LC3B-II, P62 changed with time. After knockdown of PHF23, the level of autophagy-related proteins increased and apoptotic-related proteins decreased in IL-1ß-induced OA-like chondrocytes. CONCLUSIONS: The expression of PHF23 increased in human OA cartilage and synovium, and was induced by IL-1ß through inflammatory stress. PHF23 can suppress autophagy of chondrocytes, and accelerate apoptosis.

Intraarticular Injection of Allogenic Mesenchymal Stem Cells has a Protective Role for the Osteoarthritis.

BACKGROUND: Researchers initially proposed the substitution of apoptotic chondrocytes in the superficial cartilage by injecting mesenchymal stem cells (MSCs) intraarticularly. This effect was termed as bio-resurfacing. Little evidence supporting the treatment of osteoarthritis (OA) by the delivery of a MSC suspension exists. The aim of this study was to investigate the effects of injecting allogenic MSCs intraarticularly in a rat OA model and to evaluate the influence of immobility on the effects of this treatment. METHODS: We established a rat knee OA model after 4 and 6 weeks and cultured primary bone marrow MSCs. A MSC suspension was injected into the articular space once per week for 3 weeks. A subgroup of knee joints was immobilized for 3 days after each injection, while the remaining joints were nonimmobilized. We used toluidine blue staining, Mankin scores, and TdT-mediated dUTP-biotin nick end labeling staining to evaluate the therapeutic effect of the injections. Comparisons between the therapy side and the control side of the knee joint were made using paired t-test, and comparisons between the immobilized and nonimmobilized subgroups were made using the unpaired t-test. A P value < 0.05 was considered significant. RESULTS: The three investigative approaches revealed less degeneration on the therapy sides of the knee joints than the control sides in both the 4- and 6-week groups (P < 0.05), regardless of immobilization. No significant differences were observed between the immobilized and nonimmobilized subgroups (P > 0.05). CONCLUSIONS: Therapy involving the intraarticular injection of allogenic MSCs promoted cartilage repair in a rat arthritis model, and 3-day immobility after injection had little effect on this therapy.

[Effect of mitochondrial dysfunction of chondrocyte on cartilage degeneration].

OBJECTIVE: To evaluate the effect of chondrocyte mitochondrial dysfunction on the development of cartilage degeneration. METHODS: In the study, 10 cartilage samples of the knee joint were collected during total knee arthroplasty surgery because of OA from April to October of 2012 in Peking University First Hospital. All the tissues were taken from transmission electron microscope (TEM) observation grouped by Outerbridge classification. Then, TEM observation, quantitative detection of mitochondrial respiratory chain enzyme complex 1,2,2+3,4 and ATPase activity, detection of the mitochondrial membrane potential by JC-1 method were taken with cultured normal and OA chondrocytes. Healthy chondrocytes from 10 normal cartilage samples were divided into 2 groups: the normal control group and rotenone group. The ultrastuctrure alterations of mitochondria, mitochondrial membrane potential, apoptosis rate and collagen II content were compared. RESULTS: With the aggravation of cartilage degeneration, mitochondria swelling, outer membrane rupture, cristae destruction and disappearance were observed in both the tissue and cell TEM examinations. JC-1 staining showed a decreased membrane potential in OA chondrocytes which had a lower red/green fluorescence ratio of 1.50 than that of the normal chondrocytes of 2.58. mitochondrial respiratory chain (MRC) enzyme complex 1,2,2+3,4 and ATPase activity of the OA chondrocytes also represented a decreased tendency compared with the normal chondrocytes although the difference was not significant (P=0.109,0.197,0.098,0.169,0.145). The mitochondria in the Ro group cells showed OA-like changes morphologically by TEM detection. JC-1 staining showed a decreased mitochondrial membrane potential in the Ro group chondrocytes which had a lower red/green fluorescence ratio of 1.78 than that of the normal ones of 2.58. Apoptosis examination represented a higher apoptosis rate of 7.53% in the Ro group chondrocytes than that of the normal ones of 4.38%. Collagen II content of the chondrocytes in the Ro group was (44.63 ± 7.11) µg/L , significantly lower than (72.88 ± 24.3) µg/L in the control group (P=0.044). CONCLUSION: Mitochondrial function is impaired in OA chondrocyte. Mitochondrial function destruction results in an increased chondrocyte apoptosis rate and a decreased collagen II secretion.

[Dermal hypoesthesia after total knee arthroplasty].

OBJECTIVE: To observe the incidence of skin sensory loss after total knee arthroplasty (TKA) and its natural history over time, and to identify the relationship between numbness area and incision length, tourniquet time, age and gender. METHODS: In the study, 132 patients (20 males and 112 females, with an average age of 69.75 years old, 135 cases of TKA) who underwent primary TKA with midline incisions were chosen and grouped chronologically (4 years, 3 years, 2 years, 1 year, 6 months, 1 month) to the investigation time point from Peking University First Hospital. All the operations were done by the same surgeon team with Stryker NRG and Depuy RP (without patellar resurfacing). Numbness incidence, numbness area, scar length, tourniquet time were recorded from the questionnaires sent to the patients and their medical records. RESULTS: 84.44% of the patients received a reduced skin sensory after TKA, 91.22% of which had a smaller numbness area gradually over time. The numbness area was decreased from the 1 month postoperation group to the 4 years postoperation group (P <0.001). The numbness area in 2 years postoperation group and more were significantly smaller than 1 month postoperation group (P=0.042, 0.004, 0.022), however, the skin flap numbness area had little change after 2 years (P>0.05). The hypoesthesia flap was completely lateral to the incision in 88.60% of the patients, and the numbness area covered the lateral skin and part of media skin to the incision in 11.40% of the patients. Numbness size had no relationship with the patients' gender, age, length of scar and tourniquet time (P>0.05). CONCLUSION: Most but not all the patients have a dermal hypoesthesia after total knee arthroplasty. The numbness area will gradually reduce over time. Numbness size is obviously smaller 2 years postoperation and then it will be stable. Gender, age, length of incision, and tourniquet time have no significant relationship with the size of numbness.

Impaired bone healing pattern in mice with ovariectomy-induced osteoporosis: A drill-hole defect model.

OBJECTIVE: To establish a drill-hole defect model in osteoporotic mouse femur by comparing temporal cortical bone healing pattern between OVX-induced osteoporotic bone and sham-operated bone. METHODS: 3-month-old female C57BL/6 mice were randomly divided into an ovariectomy group (OVX) and a sham-operated group (Sham). At 6 weeks post-surgery, 7 mice from each group were sacrificed to examine the distal femur and femoral shaft by both micro-CT and mechanical testing for confirming established osteoporosis induced by OVX. In the remaining mice, a cortical bone defect 0.8mm in diameter was created on the mid-diaphysis of the right femur. The local repair process at days 0, 3, 7, 10, 14 and 21 after creation of the drill-hole was in vivo monitored by high-resolution micro-CT scanning. At each time point, each animal was scanned four times and was removed from the scanner between scans to determine reproducibility. Mice were sacrificed at each time point (n=12 at days 0, 3, 7, 10 and 14; n=20 at day 21). Before sacrifice, sera were collected to examine expression of bone formation marker P1NP (procollagen type I N-terminal propeptide) and bone resorption marker CTX (C-terminal telopeptide of type I collagen). After sacrifice, callus samples were collected and subjected to the following analyses: micro-CT-based angiography; histological examination; immunohistochemical staining to determine estrogen receptor expression; quantitative real-time PCR analysis of collagen type I, collagen type II, collagen type X, osteocalcin, tartrate-resistant acid phosphatase, estrogen receptor alpha (ER alpha) and estrogen receptor beta (ER beta) gene expression; and three-point mechanical testing. RESULTS: At 6 weeks post-surgery, OVX mice had significantly lower bone mass, impaired bone micro architecture and compromised mechanical properties compared to the Sham mice. In vivo micro-CT analysis revealed that the bone volume fraction in the defect region was significantly lower in the OVX group from day 10 to day 21 post-injury as compared to the Sham group, and was significantly lower in the intra-medulla region in the OVX group from day 7 to day 14 as compared to the Sham group, consistent with the histological data. Analysis of bone biochemical markers indicated that circulating P1NP levels normalized by baseline in the OVX mice were significantly lower than in the Sham mice from day 7 to day 10, and that temporal expression of circulating CTX levels normalized by baseline was also lower in the OVX mice as compared to the Sham mice. These results were consistent with quantitative real-time PCR analysis. ER alpha mRNA expression was significantly lower in the OVX mice, whereas ER beta mRNA expression was significantly higher in the OVX mice as compared to the Sham mice at all time points examined, consistent with immunohistochemical staining. The restoration of femoral mechanical property, determined based on ultimate load and energy-to-failure, was significantly lower in the OVX mice than in the Sham mice. In addition, in vivo micro-CT scanning for quantifying new bone formation in the defect site was highly reproducible in this model. CONCLUSION: The bone healing of the drill-hole defect was impaired in mice with OVX-induced osteoporosis. The present study provides a model to investigate the functional role of specific gene in osteoporotic bone healing and may facilitate development of novel therapeutic strategies for promoting osteoporotic bone healing.

Characterization of human primary chondrocytes of osteoarthritic cartilage at varying severity.

BACKGROUND: There is a difficulty in evaluating the in vivo functionality of individual chondrocytes, and there is much heterogeneity among cartilage affected by osteoarthritis (OA). In this study, in vitro cultured chondrocytes harvested from varying stages of degeneration were studied as a projective model to further understand the pathogenesis of osteoarthritis. METHODS: Cartilage of varying degeneration of end-stage OA was harvested, while cell yield and matrix glycosaminoglycan (GAG) content were measured. Cell morphology, proliferation, and gene expression of collagen type I, II, and X, aggrecan, matrix metalloproteinase 13 (MMP-13), and ADAMTS5 of the acquired chondrocytes were measured during subsequent in vitro culture. RESULTS: Both the number of cells and the GAG content increased with increasing severity of OA. Cell spreading area increased and gradually showed spindle-like morphology during in vitro culture. Gene expression of collagen type II, collagen type X as well as GAG decreased with severity of cartilage degeneration, while expression of collagen type I increased. Expression of MMP-13 increased with severity of cartilage degeneration, while expression of ADAMTS-5 remained stable. Expression of collagen type II, X, GAG, and MMP-13 substantially decreased with in vitro culture. Expression of collagen type I increased with in vitro cultures, while expression of ADAMTS 5 remained stable. CONCLUSIONS: Expression of functional genes such as collagen type II and GAG decreased during severe degeneration of OA cartilage and in vitro dedifferentiation. Gene expression of collagen I and MMP-13 increased with severity of cartilage degeneration.

[Acetabular revision using acetabular reinforcement cages with a hook].

OBJECTIVE: To evaluate the effects of cementless revising cup or acetabular reinforcement cages for reconstructing the massive acetabular deficiency. METHODS: From September 2001 to September 2008, 22 loosening acetabular cases (24 hips) were revised using cementless revising cup or acetabular reinforcement cases for reconstructing massive bone defect after particulate bone grafting. There were 2 cases (2 hips) using Lima cementless revising cup, 2 cases (2 hips) using Kerboull ring, and 18 cases (20 hips) using restoration GAP cages. Six cases (6 hips) were male, and 16 cases (18 hips) were female. The mean age was 62 years old (34 - 79 years old). Septic loosening was in 2 cases (2 hips), and aseptic loosening in 20 cases (22 hips). The mean follow-up was 48 months (18 - 84 months). RESULTS: There was no clinical or radiological evidence of loosening for the revising acetabular components at the last follow-up point. The mean Harris hip score was improved significantly from 56 points (44 - 75) before revision to 89 points (78 - 94) at the last follow-up after revision. Excellent and good rate was 95.5% (21/22 cases). The average abduction angle of the three types of acetabular reconstructive cages were 50.1 degrees (39.0 degrees - 66.0 degrees), and almost all cases of the hip rotation center were restored after revision surgery. At the last follow-up, the reinforcement cages were no immigration and breakup, and there was no radiolucent line around the acetabular components. The bone graft integrated well into surrounding acetabular bone. CONCLUSION: The method of revising the massive acetabular bone defect by cementless revising cup and acetabular reinforcement cages restores the normal hip rotation center, supplies the primary stability of the revising component, and protects the bone graft from mechanical overload during its revascularization phase, which is a reliable method for revising the massive acetabular deficiency after total hip arthroplasty.

[Aggrecanases and osteoarthritis].

Osteoarthritis is mainly caused by the degenerative changes of cartilage and cartilage extracellular matrix, while Aggrecanases degradate Proteoglycans which are the major components of cartilage. This review includes three aspects: (1) We have concluded the major enzymes(ADAMTS-4 and ADAMTS-5) which regulate the metabolism of cartilage extracellular matrix. Meanwhile, we have summarized the structure of aggrecanases(ADAMTS-4 and ADAMTS-5) and introduced the function of each regional structure; (2) We have concluded the way cytokines and glycosaminoglycans regulate the metabolism of aggrecanases, and discussed the regulation and control principle of cytokines and glycosaminoglycan; (3) We have summarized the majority of inhibitors to the aggrecanases, introduced the endogenic inhibitors, and put our emphasis on the extrinsic inhibitors (chelating agents, polypeptides and so on). Through deeper research on the enzymes, it will help us further understand the pathogenesis of osteoarthritis, and open up new avenues to clinical treatment.

[Long-term and high-dose bisphosphonate administration causes no bone over-mineralization].

OBJECTIVE: To evaluate the effects of long-term bisphosphonate administration (incadronate) upon the mean degree of secondary bone mineralization. METHODS: Thirty adult beagles were divided randomly into three groups of CNT, YML and YMH based upon their body weights (5 males and 5 females in each group). Animals in CNT were orally given lactose 12 mg x kg(-1) x d(-1) while those in YML and YMH were orally given incadronate disodium at doses of 0.3 mg x kg(-1) x d(-1) and 0.6 mg x kg(-1) x d(-1) respectively. The dosing procedure lasted for three years. Prior to sacrifices, all animals were double-labeled intravenously with oxytetracycline hydrochloride (20 mg x kg(-1)). After necropsy, the left 9th ribs were excised for evaluation. RESULTS: Histomorphometry showed that activation frequency in YML and YMH [(4.93 +/- 0.92)/mm(2) and (2.50 +/- 0.78)/mm(2)] were significantly lower than that in CNT (7.83 +/- 0.96)/mm(2) per year. The mean degree of mineralization in bone in YML and YMH (1.40 +/- 0.12, 1.48 +/- 0.09) were significantly higher than CNT (1.07 +/- 0.06). The increments were 31% and 38% respectively. CONCLUSION: Long-term and high-dose bisphosphonate administration significantly suppresses bone turnover so as to increase the degree of bone mineralization. But there is no resulting bone over-mineralization. (incadronate) administration on the mean degree of secondary mineralization in bone.