Enhanced osteochondral repair by leukocyte-depleted platelet-rich plasma in combination with adipose-derived mesenchymal stromal cells encapsulated in a three-dimensional photocrosslinked injectable hydrogel in a rabbit model.

INTRODUCTION: Intra-articular injection of adipose-derived mesenchymal stromal cells (ASCs) and/or platelet-rich plasma (PRP) have been reported to independently and synergistically improve healing of osteochondral lesions in animal models. However, their independent and combined effects when localized to an osteochondral lesion by encapsulation within a photocrosslinkable methacrylated gelatin hydrogel (GelMA) have not been explored. Herein we investigated a unique combination of allogeneic ASCs and PRP embedded in GelMA as a single-stage treatment for osteochondral regeneration in a rabbit model. METHODS: Thirty mature rabbits were divided into six experimental groups: (1) Sham; (2) Defect; (3) GelMA; (4) GelMA + ASCs; (5) GelMA + PRP; and (6) GelMA + ASCs + PRP.At 12 weeks following surgical repair, osteochondral regeneration was assessed on the basis of gross appearance, biomechanical properties, histological and immunohistochemical characteristics, and subchondral bone volume. RESULTS: In terms of mechanical property reflecting the ability of neotissue to bear stress, PRP only group were significantly lower than the Sham group (p = 0.0098). On the other hand, ASCs only and ASCs combined with PRP groups did not exhibit significantly difference, which suggesting that incorporation of ASCs assists in restoring the ability of the neotissue to bear stresses similarly to native tissue (p = 0.346, p = 0.40, respectively). Safranin O in ASCs combined with PRP group was significantly higher than the Defect and GelMA only groups (p = 0.0009, p = 0.0017, respectively). Additionally, ASCs only and ASCs combined with PRP groups presented especially strong staining for collagen type II. Surprisingly, PRP only and PRP + ASCs groups tended to exhibit higher collagen type I and collagen type X staining compared to ASCs only group, suggesting a potential PRP-mediated hypertrophic effect. CONCLUSION: Regeneration of a focal osteochondral defect in a rabbit model was improved by a single-stage treatment of a photocrosslinked hydrogel containing allogenic ASCs and autologous PRP, with the combination of ASCs and PRP producing superior benefit than either alone. No experimental construct fully restored all properties of the native, healthy osteochondral unit, which may require longer follow-up or further modification of PRP and/or ASCs characteristics.

Talar component subsidence correlates with periprosthetic osteolysis after total ankle arthroplasty.

PURPOSE: This study aimed to investigate the relationship between periprosthetic osteolysis around the talar component and the amount of talar component subsidence after total ankle arthroplasty (TAA). METHODS: This study included forty patients who underwent TAA with a mean follow-up of 67.5 ± 17.0 months. The patients were divided into two groups based on the amount of osteolysis around the talar component, as measured by computed tomography at the latest clinic visit: none to 2 mm (N group, n = 20) and greater than or equal to 2 mm (O group, n = 20). The average amount of talar component subsidence, clinical outcomes, and complications were compared between the two groups. In the O group, the correlation between osteolysis and talar component subsidence was evaluated. RESULTS: The average talar component subsidence was significantly different between the N (0.22 ± 0.94 mm) and O groups (2.12 ± 2.28 mm). Five out of 20 ankles in the O group required revision surgery owing to talar component subsidence. The Japanese Society for Surgery of the Foot scores in the N and O groups were significantly different: 93.5 ± 7.7 and 85.3 ± 15.4, respectively. In the O group, we found that osteolysis tended to develop on the lateral side, and the amount of osteolysis was positively correlated with the talar component subsidence (r = 0.59, P = .007). CONCLUSION: In the O group, a positive correlation between osteolysis and talar component subsidence was found, and five patients required revision surgery.

Medial Pelvic Migration of the Lag Screw after Intramedullary Nailing for Trochanteric Femoral Fracture.

Internal fixation with intramedullary nails has gained popularity for the treatment of trochanteric femoral fractures, which are common injuries in older individuals. The most common complications are lag screws cut-out from the femoral head and femoral fracture at the distal tip of the nail. Herein, we report a rare complication of postoperative medial pelvic migration of the lag screw with no trauma. The patient was subsequently treated by lag screw removal via laparoscopy. This case suggests that optimal fracture reduction, adequate position of the lag screw, and careful attention to set screw insertion are important to prevent complications. Additionally, laparoscopic surgery might be able to remove the lag screw more safely than removal from the femoral side.

Paediatric knee anterolateral capsule does not contain a distinct ligament: analysis of histology, immunohistochemistry and gene expression.

OBJECTIVES: The presence of a discrete ligament within the knee anterolateral capsule (ALC) is controversial. Tendons and ligaments have typical collagens, ultrastructure, transcription factors and proteins. However, these characteristics have not been investigated in paediatric ALC. The purpose of this study was to characterise the paediatric ALC in terms of tissue ultrastructure and cellular expression of ligament markers scleraxis (SCX)-a basic helix-loop-helix transcription factor-and the downstream transmembrane glycoprotein tenomodulin (TNMD), as compared with the paediatric lateral collateral ligament (LCL) and paediatric quadriceps tendon (QT). We hypothesised that, in comparison to the LCL and QT, the ALC would possess poor collagen orientation and reduced SCX and TNMD expression. METHODS: 15 paediatric ALCs (age 6.3±3.3 years), 5 paediatric LCLs (age 3.4±1.3 years) and 5 paediatric QTs (age 2.0±1.2 years) from fresh cadaveric knees were used in this study. Fresh-frozen samples from each region were cryosectioned and then stained with H&E to evaluate collagen alignment and cell morphology. Expression of SCX and TNMD was determined by gene expression analysis and immunohistochemistry. RESULTS: The histological sections of the paediatric LCL and QT showed well-organised, dense collagenous tissue fibres with elongated fibroblasts, while the ALC showed more random collagen orientation without clear cellular directionality. The aspect ratio of cells in the ALC was significantly lower than that of the LCL and QT (p<0.0001 and p<0.0001, respectively). The normalised distribution curve of the inclination angles of the nuclei in the ALC was more broadly distributed than that of the LCL or QT, indicating random cell alignment in the ALC. SCX immunostaining was apparent in the paediatric LCL within regions of aligned fibres, while the comparatively disorganised structure of the ALC was negative for SCX. The paediatric LCL also stained positive for TNMD, while the ALC was only sparsely positive for this tendon/ligament cell-surface molecule. Relative gene expression of SCX and TNMD were higher in the LCL and QT than in the ALC. CONCLUSION: In this study, a distinct ligament could not be discerned in the ALC based on histology, immunohistochemistry and gene expression analysis. LEVEL OF EVIDENCE: Controlled laboratory study.

Development of a large animal rabbit model for chronic periprosthetic joint infection.

AIMS: Periprosthetic joint infections (PJIs) and osteomyelitis are clinical challenges that are difficult to eradicate. Well-characterized large animal models necessary for testing and validating new treatment strategies for these conditions are lacking. The purpose of this study was to develop a rabbit model of chronic PJI in the distal femur. METHODS: Fresh suspensions of Staphylococcus aureus (ATCC 25923) were prepared in phosphate-buffered saline (PBS) (1 × 109 colony-forming units (CFUs)/ml). Periprosthetic osteomyelitis in female New Zealand white rabbits was induced by intraosseous injection of planktonic bacterial suspension into a predrilled bone tunnel prior to implant screw placement, examined at five and 28 days (n = 5/group) after surgery, and compared to a control aseptic screw group. Radiographs were obtained weekly, and blood was collected to measure ESR, CRP, and white blood cell (WBC) counts. Bone samples and implanted screws were harvested on day 28, and processed for histological analysis and viability assay of bacteria, respectively. RESULTS: Intraosseous periprosthetic introduction of planktonic bacteria induced an acute rise in ESR and CRP that subsided by day 14, and resulted in radiologically evident periprosthetic osteolysis by day 28 accompanied by elevated WBC counts and histological evidence of bacteria in the bone tunnels after screw removal. The aseptic screw group induced no increase in ESR, and no lysis developed around the implants. Bacterial viability was confirmed by implant sonication fluid culture. CONCLUSION: Intraosseous periprosthetic introduction of planktonic bacteria reliably induces survivable chronic PJI in rabbits. Cite this article: Bone Joint Res 2021;10(3):156-165.

Gelatin hydrogels with eicosapentaenoic acid can prevent osteoarthritis progression in vivo in a mouse model.

Eicosapentanoic acid (EPA) is an antioxidant and omega-3 polyunsaturated fatty acid that reduces inflammatory cytokine production. Gelatin hydrogel can be used as a carrier of a physiologically active substance that release it gradually for an average of ~3 weeks. Therefore, this study aimed to clarify the effect of EPA-incorporating gelatin hydrogels on osteoarthritis (OA) progression in vivo. Ten-week-old male C57BL/6J mice were randomly divided into six groups (n = 6): Sham, destabilization of the medial meniscus (DMM), Corn: DMM + 2 µL corn oil, EPA injection alone (EPA-I): DMM + 2 µL corn oil + 125 µg/µL EPA, Gel: DMM + gelatin hydrogels, and EPA-G: DMM + 125 µg/µL EPA-incorporating gelatin hydrogels. The mice were euthanized at 8 weeks after DMM or Sham surgery, and subjected to histological evaluation. Matrix-metalloproteinases-3 (MMP-3), MMP-13, interleukin-1ß (IL-1ß), p-IKK α/ß, CD86, and CD163 protein expression in the synovial cartilage was detected by immunohistochemical staining. F4/80 expression was also assessed using the F4/80 score of macrophage. Histological score was significantly lower in EPA-G than in EPA-I. MMP-3-, MMP-13-, IL-1ß-, and p-IKK α/ß-positive cell ratio was significantly lower in EPA-G than in EPA-I. However, CD86- and CD163-positive cell ratio was not significantly different between EPA-I and EPA-G. The average-sum F4/80 score of macrophage in EPA-G was significantly lower than that in EPA-I. EPA-incorporating gelatin hydrogels were shown to prevent OA progression in vivo more effectively than EPA injection alone. Our results suggested that intra-articular administration of controlled-release EPA can be a new therapeutic approach for treating OA.

TGF-ß1 plays a protective role in glucocorticoid-induced dystrophic calcification.

Dystrophic calcification (DC) is the deposition of calcium in degenerated tissue which occurs as a reaction to tissue damage. Sometimes if tissue repair fails, it can progress into heterotopic ossification (HO), a pathological condition of abnormal bone formation. HO happens frequently in severe trauma patients such as in blast injury, central nervous system injury and burn injury, in which excessive endogenous glucocorticoid production has always been found. Glucocorticoids have a big impact on bone and muscle. However, few studies have investigated the impact of glucocorticoids on DC/HO formation in muscle. This study aimed to determine the role of glucocorticoids in DC/HO pathogenesis following muscular injury and the possible underlying mechanism. In this study, we administered a high dose of a synthetic glucocorticoid, dexamethasone (DEX), to animals with muscle injury induced by cardiotoxin (CTX) injection to mimic a glucocorticoid excess state following severe muscle trauma. The findings reported here showed that DEX treatment together with CTX-induced muscle injury led to a significant amount of DC in muscle. This effect was likely related to protein level alterations in the fibrinolytic system and resultant decreased circulating transforming growth factor-beta 1 (TGF-ß1), given that supplementation of recombinant TGF-ß1 markedly rescued this phenomenon. In summary, our results suggest that glucocorticoid excess impairs muscle regeneration and promotes DC/HO, and that TGF-ß1 could be a key factor in modulating this process.

Deletion of p21 expression accelerates cartilage tissue repair via chondrocyte proliferation.

Articular cartilage tissue has a poor healing potential, and when subjected to traumatic damage this tissue undergoes cartilage degeneration and osteoarthritis. The association between the regulation of cell cycle checkpoints and tissue regeneration has been previously investigated, and p21 was initially identified as a potent inhibitor of cell cycle progression. However, the effects of p21 defects on damaged tissue remain controversial. Therefore, the aim of the present study was to evaluate the effects of p21 deficiency on cartilage repair. A mouse model of articular cartilage repair was generated by inducing a patellar groove scratch in 8­week­old p21­knockout (KO) mice and C57Bl/6 wild­type (WT) mice. Mice were sacrificed at 4 and 8 weeks post­surgery. The present study also investigated the effect of p21 deficiency on cartilage differentiation in ATDC5 cells in vitro. Safranin O staining results indicated that cartilage repair initially occurred in p21 KO mice. In addition, immunohistochemical analysis demonstrated that p21 KO upregulated proliferating cell nuclear antigen and increased cell proliferation. However, type II collagen and Sox9 expression levels remained unchanged in p21 KO and WT mice. Moreover, it was identified that p21 downregulation did not affect Sox9 and type II collagen expression levels in vitro. Furthermore, p21 deficiency promoted healing of articular cartilage damage, which was associated with cell proliferation in vivo, and increased chondrocyte proliferation but not differentiation in vitro. Therefore, the present results suggested that p21 does not affect Sox9 or type II collagen expression levels during cartilage differentiation in the repair of cartilage defects.

Point-of-Care Procedure for Enhancement of Meniscal Healing in a Goat Model Utilizing Infrapatellar Fat Pad-Derived Stromal Vascular Fraction Cells Seeded in Photocrosslinkable Hydrogel.

BACKGROUND: Large radial tears of the meniscus involving the avascular region can compromise meniscal function and result in poor healing and subsequent osteochondral degeneration. Augmentation of surgical repairs with adipose-derived stromal vascular fraction (SVF), which contains mesenchymal stromal cells, may improve meniscal healing and preserve function (ie, chondroprotection). PURPOSES: (1) To develop a goat model of a radial meniscal tear with resulting osteoarthritis and (2) to explore the efficacy of a 1-step procedure utilizing infrapatellar fat pad-derived SVF cells seeded in a photocrosslinkable hydrogel to enhance meniscal healing and mitigate osteochondral degeneration. STUDY DESIGN: Controlled laboratory study. METHODS: A full-thickness radial tear spanning 90% of the medial meniscal width was made at the junction of the anterior and middle bodies of the goat stifle joint. Tears received 1 of 3 interventions (n = 4 per group): untreated, repair, or repair augmented with photocrosslinkable methacrylated gelatin hydrogel containing 2.0 × 106 SVF cells/mL and 2.0 µg/mL of transforming growth factor ß3. The contralateral (left) joint served as a healthy control. At 6 months, meniscal healing and joint health were evaluated by magnetic resonance imaging (MRI) and assessed by histological and macroscopic scoring. The Whole-Organ Magnetic Resonance Imaging Score and the presence of a residual tear, as evaluated with T2 MRI sequences, were determined by a single blinded orthopaedic surgeon. RESULTS: When compared with tears left untreated or repaired with suture alone, augmented repairs demonstrated increased tissue formation in the meniscal tear site, as seen on MRI and macroscopically. Likewise, the neotissue of augmented repairs possessed a histological appearance more similar, although still inferior, to healthy meniscus. Osteochondral degeneration in the medial compartment, as evaluated by the Whole-Organ Magnetic Resonance Imaging Score and Inoue (macroscopic) scale, revealed increased degeneration in the untreated and repair groups, which was mitigated in the augmented repair group. Histological evaluation with a modified Mankin score showed a similar trend. In all measures of osteochondral degeneration, the augmented repair group did not differ significantly from the uninjured control. CONCLUSION: A radial tear spanning 90% of the medial meniscal width in a goat stifle joint showed poor healing potential and resulted in osteochondral degeneration by 6 months, even if suture repair was performed. Augmentation of the repair with a photocrosslinkable hydrogel containing transforming growth factor ß3 and SVF cells, isolated intraoperatively by rapid enzymatic digestion, improved meniscal healing and mitigated osteoarthritic changes. CLINICAL RELEVANCE: Repair augmentation with an SVF cell-seeded hydrogel may support successful repair of meniscal tears previously considered irreparable.

Denosumab Treatment Improved Health-Related Quality of Life in Osteoporosis: A Prospective Cohort Study.

Improving patient health-related quality of life (HRQOL) and prevention of bone fracture are important components of the treatment of osteoporosis. Our aim in this study was to evaluate the effect of denosumab treatment in improving HRQOL among patients with osteoporosis. Our analysis was based on 332 patients with osteoporosis, followed for 24 months. All patients received denosumab (60 mg) subcutaneously every 6 months. Bone mineral density (BMD) was assessed at the distal radius, with serum concentration of calcium, phosphate, P1NP, and TRACP5b also measured. HRQOL assessment included pain (visual analogue scale [VAS]) and the EQ-5D questionnaire. A multivariate analysis was performed to identify the possible confounders associated with deterioration in the EQ-5D utility score in response to denosumab treatment. Denosumab treatment yielded a 3.4% increase in BMD at 24 months. Serum levels of TRACP5b and P1NP decreased significantly, from baseline, at 6 months, with no effect on calcium and phosphate levels. Pain VAS and EQ-5D utility score improved significantly, from baseline, at 6 months, with the EQ-5D utility score correlating with the BMD at all time points of measurement over the 24-month period of observation. Knee osteoarthritis and multiple comorbidities were significantly associated with a worse HRQOL in response to denosumab treatment. Denosumab treatment increased BMD, with improvements in BMD correlating with improved HRQOL, supporting a possible benefit of using denosumab for the treatment of osteoporosis. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Dynamic Compressive Loading Improves Cartilage Repair in an In Vitro Model of Microfracture: Comparison of 2 Mechanical Loading Regimens on Simulated Microfracture Based on Fibrin Gel Scaffolds Encapsulating Connective Tissue Progenitor Cells.

BACKGROUND: Microfracture of focal chondral defects often produces fibrocartilage, which inconsistently integrates with the surrounding native tissue and possesses inferior mechanical properties compared with hyaline cartilage. Mechanical loading modulates cartilage during development, but it remains unclear how loads produced in the course of postoperative rehabilitation affect the formation of the new fibrocartilaginous tissue. PURPOSE: To assess the influence of different mechanical loading regimens, including dynamic compressive stress or rotational shear stress, on an in vitro model of microfracture repair based on fibrin gel scaffolds encapsulating connective tissue progenitor cells. STUDY DESIGN: Controlled laboratory study. METHODS: Cylindrical cores were made in bovine hyaline cartilage explants and filled with either (1) cartilage plug returned to original location (positive control), (2) fibrin gel (negative control), or (3) fibrin gel with encapsulated connective tissue progenitor cells (microfracture mimic). Constructs were then subjected to 1 of 3 loading regimens: (1) no loading (ie, unloaded), (2) dynamic compressive loading, or (3) rotational shear loading. On days 0, 7, 14, and 21, the integration strength between the outer chondral ring and the central insert was measured with an electroforce mechanical tester. The central core component, mimicking microfracture neotissue, was also analyzed for gene expression by real-time reverse-transcription polymerase chain reaction, glycosaminoglycan, and double-stranded DNA contents, and tissue morphology was analyzed histologically. RESULTS: Integration strengths between the outer chondral ring and central neotissue of the cartilage plug and fibrin + cells groups significantly increased upon exposure to compressive loading compared with day 0 controls (P = .007). Compressive loading upregulated expression of chondrogenesis-associated genes (SRY-related HGMG box-containing gene 9 [SOX9], collagen type II α1 [COL2A1], and increased ratio of COL2A1 to collagen type I α1 [COL1A1], an indicator of more hyaline phenotype) in the neotissue of the fibrin + cells group compared with the unloaded group at day 21 (SOX9, P = .0032; COL2A1, P < .0001; COL2A1:COL1A1, P = .0308). Fibrin + cells constructs exposed to shear loading expressed higher levels of chondrogenic genes compared with the unloaded condition, but the levels were not as high as those for the compressive loading condition. Furthermore, catabolic markers (MMP3 and ADAMTS 5) were significantly upregulated by shear loading (P = .0234 and P < .0001, respectively) at day 21 compared with day 0. CONCLUSION: Dynamic compressive loading enhanced neotissue chondrogenesis and maturation in a simulated in vitro model of microfracture, with generation of more hyaline-like cartilage and improved integration with the surrounding tissue. CLINICAL RELEVANCE: Controlled loading after microfracture may be beneficial in promoting the formation of more hyaline-like cartilage repair tissue; however, the loading regimens applied in this in vitro model do not yet fully reproduce the complex loading patterns created during clinical rehabilitation. Further optimization of in vitro models of cartilage repair may ultimately inform rehabilitation protocols.

Downregulation of aquaporin 9 decreases catabolic factor expression through nuclear factor­κB signaling in chondrocytes.

Aquaporins (AQPs) are small integral membrane proteins that are essential for water transport across membranes. AQP9, one of the 13 mammalian AQPs (including AQP0 to 12), has been reported to be highly expressed in hydrarthrosis and synovitis patients. Given that several studies have identified signal transduction as an additional function of AQPs, it is hypothesized that AQP9 may modulate inflammatory signal transduction in chondrocytes. Therefore, the present study used a model of interleukin (IL)­1ß­induced inflammation to determine the mechanisms associated with AQP9 functions in chondrocytes. Osteoarthritis (OA) and normal cartilage samples were subjected to immunohistological analysis. In addition, matrix metalloproteinase (MMP)3, MMP13 and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS­5) mRNA and protein analysis was conducted in normal human articular chondrocytes from the knee (NHAC­Kn) stimulated with IL­1ß by reverse transcription­polymerase chain reaction (RT­qPCR) and western blotting, respectively. AQP9 knockdown was also performed by transfection of AQP9­specific small interfering RNA using Lipofectamine. AQP1, 3, 7, 9 and 11 mRNA expression levels were detected in OA human chondrocytes and in IL­1ß­treated normal human chondrocytes. The levels of AQP9, MMP­3, MMP­13 and ADAMTS­5 mRNA were increased by treatment with 10 ng/ml IL­1ß in a time­dependent manner, while knockdown of AQP9 expression significantly decreased the mRNA levels of the MMP3, MMP13 and ADAMTS­5 genes, as well as the phosphorylation of IκB kinase (IKK). Treatment with a specific IKK inhibitor also significantly decreased the expression levels of MMP­3, MMP­13 and ADAMTS­5 in response to IL­1ß stimulation. Furthermore, immunohistochemical analysis demonstrated that AQP9 and inflammatory markers were highly expressed in OA cartilage. In addition, the downregulation of AQP9 in cultured chondrocytes decreased the catabolic gene expression in response to IL­1ß stimulation through nuclear factor­κB signaling. Therefore, AQP9 may be a promising target for the treatment of OA.

Depletion of aquaporin 1 decreased ADAMTS­4 expression in human chondrocytes.

Inflammation serves an important role in the progression of osteoarthritis (OA), and IL­1ß may act as a catabolic factor on cartilage, reducing the synthesis of primary cartilage components type II collagen and aggrecan. Aquaporin 1 (AQP1) is a 28­kDa water channel formed of six transmembrane domains on the cell membrane. AQP1 is highly expressed in the anus, gallbladder and liver, and is moderately expressed in the hippocampus, ependymal cells of the central nervous system and articular cartilage. It was hypothesized that AQP1 may be highly expressed in OA cartilage and that it may increase the expression of catabolic factors during inflammatory OA progression. Therefore, the present study evaluated AQP1 functions in human OA articular chondrocytes. Primary chondrocytes were isolated from human hip and knee cartilage tissues, cultured and transfected with AQP1­specific small interfering RNA with or without subsequent IL­1ß treatment. In vitro explant culture from hip cartilages were also prepared. Reverse transcription­polymerase chain reaction (RT­PCR) was performed to assess the expression of AQP genes in human articular cartilage, AQP1 immunohistochemistry of the cartilages and explant culture, as well as RT­quantitative PCR, western blotting and immunocytochemistry/immunofluorescence of OA chondrocytes to evaluate the expression of AQP1, and catabolic and anabolic factors. RT­PCR results demonstrated that AQP0, 1, 3, 7, 9, and 11 were expressed in OA chondrocytes. Immunohistochemistry revealed that AQP1 was highly expressed in the superficial to middle zones of OA articular cartilages. Additionally, AQP1 mRNA was significantly higher in OA cartilage and IL­1ß treatment significantly increased AQP1 expression in hip explant cartilage. Furthermore, AQP1 downregulation decreased a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS)­4 expression in OA chondrocytes, though it did not affect other associated genes. Immunofluorescence showed that AQP1 and ADAMTS­4 were co­localized. These findings indicated that AQP1 depletion may decrease ADAMTS­4 expression in human OA chondrocytes. Therefore, regulating AQP1 expression may be a strategy to suppress catabolic factors during OA progression.

Cyclin-Dependent Kinase Inhibitor-1-Deficient Mice are Susceptible to Osteoarthritis Associated with Enhanced Inflammation.

Osteoarthritis (OA) is a multifactorial disease, and recent data suggested that cell cycle-related proteins play a role in OA pathology. Cyclin-dependent kinase (CDK) inhibitor 1 (p21) regulates activation of other CDKs, and recently, we reported that p21 deficiency induced susceptibility to OA induced by destabilization of the medial meniscus (DMM) surgery through STAT3-signaling activation. However, the mechanisms associated with why p21 deficiency led to susceptibility to OA by the STAT3 pathway remain unknown. Therefore, we focused on joint inflammation to determine the mechanisms associated with p21 function during in vitro and in vivo OA progression. p21-knockout (p21-/- ) mice were used to develop an in vivo OA model, and C57BL/6 (p21+/+ ) mice with the same background as the p21-/- mice were used as controls. Morphogenic changes were measured using micro-CT, IL-1ß serum levels were detected by ELISA, and histological or immunohistological analyses were performed. Our results indicated that p21-deficient DMM-model mice exhibited significant subchondral bone destruction and cartilage degradation compared with wild-type mice. Immunohistochemistry results revealed p21-/- mice susceptibility to OA changes accompanied by macrophage infiltration and enhanced MMP-3 and MMP-13 expression through IL-1ß-induced NF-κB signaling. p21-/- mice also showed subchondral bone destruction according to micro-CT analysis, and cathepsin K staining revealed increased numbers of osteoclasts. Furthermore, p21-/- mice displayed increased serum IL-1ß levels, and isolated chondrocytes from p21-/- mice indicated elevated MMP-3 and MMP-13 expression with phosphorylation of IκB kinase complex in response to IL-1ß stimulation, whereas treatment with a specific p-IκB kinase inhibitor attenuated MMP-3 and MMP-13 expression. Our results indicated that p21-deficient DMM mice were susceptible to alterations in OA phenotype, including enhanced osteoclast expression, macrophage infiltration, and MMP expression through IL-1ß-induced NF-κB signaling, suggesting that p21 regulation may constitute a possible therapeutic strategy for OA treatment. © 2017 American Society for Bone and Mineral Research.

p21 deficiency is susceptible to osteoarthritis through STAT3 phosphorylation.

INTRODUCTION: Osteoarthritis (OA) is a multifactorial disease, and recent studies have suggested that cell cycle-related proteins play a role in OA pathology. p21 was initially identified as a potent inhibitor of cell cycle progression. However, it has been proposed that p21 is a regulator of transcription factor activity. In this study, we evaluated the role of p21 in response to biomechanical stress. METHODS: Human chondrocytes were treated with p21-specific small interfering RNA (siRNA), and cyclic tensile strain was introduced in the presence or absence of a signal transducer and activator of transcription 3 (STAT3)-specific inhibitor. Further, we developed an in vivo OA model in a p21-knockout background for in vivo experiments. RESULTS: The expression of matrix metalloproteinase (MMP13) mRNA increased in response to cyclic tensile strain following transfection with p21 siRNA, whereas the expression of aggrecan was decreased. Phospho-STAT3 and MMP-13 protein levels increased following downregulation of p21, and this was reversed by treatment with a STAT3 inhibitor. p21-deficient mice were susceptible to OA, and this was associated with increased STAT3 phosphorylation, elevated MMP-13 expression, and elevation of synovial inflammation. The expression of p21 mRNA was decreased and phosphorylation of STAT3 was elevated in human OA chondrocytes. CONCLUSIONS: The lack of p21 has catabolic effects by regulation of aggrecan and MMP-13 expression through STAT3 phosphorylation in the cartilage tissue. p21 may function as a regulator of transcriptional factors other than the inhibitor of cell cycle progression in the cartilage tissue. Thus, the regulation of p21 may be a therapeutic strategy for the treatment of OA.

P21 deficiency delays regeneration of skeletal muscular tissue.

The potential relationship between cell cycle checkpoint control and tissue regeneration has been indicated. Despite considerable research being focused on the relationship between p21 and myogenesis, p21 function in skeletal muscle regeneration remains unclear. To clarify this, muscle injury model was recreated by intramuscular injection of bupivacaine hydrochloride in the soleus of p21 knockout (KO) mice and wild type (WT) mice. The mice were sacrificed at 3, 14, and 28 days post-operation. The results of hematoxylin-eosin staining and immunofluorescence of muscle membrane indicated that muscle regeneration was delayed in p21 KO mice. Cyclin D1 mRNA expression and both Ki-67 and PCNA immunohistochemistry suggested that p21 deficiency increased cell cycle and muscle cell proliferation. F4/80 immunohistochemistry also suggested the increase of immune response in p21 KO mice. On the other hand, both the mRNA expression and western blot analysis of MyoD, myogenin, and Pax7 indicated that muscular differentiation was delayed in p21KO mice. Considering these results, we confirmed that muscle injury causes an increase in cell proliferation. However, muscle differentiation in p21 KO mice was inhibited due to the low expression of muscular synthesis genes, leading to a delay in the muscular regeneration. Thus, we conclude that p21 plays an important role in the in vivo healing process in muscular injury.

Oxidative stress-induced apoptosis and matrix loss of chondrocytes is inhibited by eicosapentaenoic acid.

Eicosapentaenoic acid (EPA) is an antioxidant and n-3 polyunsaturated fatty acid that reduces the production of inflammatory cytokines. We evaluated the role of EPA in chondrocyte apoptosis and degeneration. Normal human chondrocytes were treated with EPA and sodium nitroprusside (SNP). Expression of metalloproteinases (MMPs) was detected by real-time polymerase chain reaction (PCR) and that of apoptosis-related proteins was detected by western blotting. Chondrocyte apoptosis was detected by flow cytometry. C57BL/6J mice were used for the detection of MMP expression by immunohistochemistry and for investigation of chondrocyte apoptosis. EPA inhibited SNP-induced chondrocyte apoptosis, caspase 3 and poly(ADP-ribose) polymerase cleavage, phosphorylation of p38 MAPK and p53, and expression of MMP3 and MMP13. Intra-articular injection of EPA prevented the progression of osteoarthritis (OA) by inhibiting MMP13 expression and chondrocyte apoptosis. EPA treatment can control oxidative stress-induced OA progression, and thus may be a new approach for OA therapy.

Cyclin­dependent kinase inhibitor p21 does not impact embryonic endochondral ossification in mice.

Endochondral ossification at the growth plate is regulated by a number of factors and hormones. The cyclin­dependent kinase inhibitor p21 has been identified as a cell cycle regulator and its expression has been reported to be essential for endochondral ossification in vitro. However, to the best of our knowledge, the function of p21 in endochondral ossification has not been evaluated in vivo. Therefore, the aim of this study was to investigate the function of p21 in embryonic endochondral ossification in vivo. Wild­type (WT) and p21 knockout (KO) pregnant heterozygous mice were sacrificed on embryonic days E13.5, E15.5 and E18.5. Sagittal histological sections of the forearms of the embryos were collected and stained with Safranin O and 5­bromo­2'­deoxyuridine (BrdU). Additionally, the expression levels of cyclin D1, type II collagen, type X collagen, Sox9, and p16 were examined using immunohistochemistry, and the expression levels of p27 were examined using immunofluorescence. Safranin O staining revealed no structural change between the cartilage tissues of the WT and p21KO mice at any time point. Type II collagen was expressed ubiquitously, while type X collagen was only expressed in the hypertrophic zone of the cartilage tissues. No differences in the levels of Sox9 expression were observed between the two groups at any time point. The levels of cyclin D1 expression and BrdU uptake were higher in the E13.5 cartilage tissue compared with those observed in the embryonic cartilage tissue at subsequent time points. Expression of p16 and p27 was ubiquitous throughout the tissue sections. These results indicate that p21 may not be essential for embryonic endochondral ossification in articular cartilage of mice and that other signaling networks may compensate for p21 deletion.

Evaluation of intra-pelvic screw position prior to revision total hip arthroplasty--a report of 2 cases.

Neurovascular injury during total hip revision arthroplasty is rare, but potentially catastrophic. We report two patients requiring revision total hip arthroplasty with intrapelvic screw tips located close to the iliac artery. The screw tips were separated from artery by a retroperitoneal exposure prior to revision surgery. Assessment of screw position by computed tomography (CT) is important prior to revision total hip arthroplasty.