Krüppel-like factor 15 deficiency exacerbates osteoarthritis through reduced expression of peroxisome proliferator-activated receptor gamma signaling in mice.

OBJECTIVE: Krüppel-like zinc finger transcription factors (KLFs) play diverse roles in mammalian cell differentiation and development. In this study, we investigated the function of KLF15 in the progression of osteoarthritis (OA). METHODS: 0Destabilization of the medial meniscus (DMM) surgery was performed in 10-week-old male wild-type control (WT) mice and cartilage-specific KLF15 knockout (KO) mice. Histological analysis, immunohistochemistry, and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling staining were performed. Morphological changes were measured using microcomputed tomography. Six mice from each group were analyzed (total number of mice analyzed: 60). In vitro, immunofluorescence, quantitative reverse transcription-polymerase chain reaction, and western blot analyses were performed. RESULTS: KLF15 KO DMM mice exhibited significant cartilage degradation compared to WT mice. According to the Osteoarthritis Research Society International cartilage OA-histopathology scoring system, the mean sum score in KLF15 KO mice was significantly higher than that in WT mice at 8 weeks after surgery. Immunohistochemistry results revealed KLF15 KO mice exhibited reduced peroxisome proliferator-activated receptor gamma (PPARγ) expression, increased pIKKα/ß, a disintegrin-like and metalloproteinase with thrombospondin motifs (ADAMTS) 5, and Matrix metalloproteinases (MMP13) expression, and reduced Forkhead box O (FOXO1) and Light chain 3B (LC3B) expression. Inhibition of PPARγ phosphorylation accelerated the effects of interleukin (IL) 1ß-treatment in both KLF15 KO and WT chondrocytes, and activation of PPARγ expression canceled the IL1ß-induced catabolic effects. CONCLUSION: Our results indicated that the OA phenotype of KLF15 KO DMM mice was influenced by reduced PPARγ expression, including enhanced pIKKα/ß, ADAMTS5, and MMP13 expression, reduced autophagy, and increased apoptosis. KLF15 regulation may constitute a possible therapeutic strategy for the treating OA.

LIGHT regulated gene expression in rheumatoid synovial fibroblasts.

BACKGROUND: Synovial hyperplasia caused by rheumatoid arthritis (RA), an autoimmune inflammatory disease, leads to the destruction of the articular cartilage and bone. A member of the tumor necrosis factor superfamily, Lymphotoxin-related inducible ligand that competes for glycoprotein D binding to herpes virus entry mediator on T cells (LIGHT) has been shown to correlate with the pathogenesis of RA. METHODS: We used cDNA microarray analysis to compare the expression of genes in rheumatoid fibroblast-like synoviocytes with and without LIGHT stimulation. RESULTS: Significant changes in gene expression (P-values < 0.05 and fold change ≥ 2.0) were associated mainly with biological function categories of glycoprotein, glycosylation site as N-linked, plasma membrane part, integral to plasma membrane, intrinsic to plasma membrane, signal, plasma membrane, signal peptide, alternative splicing, and topological domain as extracellular. CONCLUSIONS: Our results indicate that LIGHT may regulate the expression in RA-FLS of genes which are important in the differentiation of several cell types and in cellular functions.

Paracrine effect of the stromal vascular fraction containing M2 macrophages on human chondrocytes through the Smad2/3 signaling pathway.

The adipose-derived stromal vascular fraction (SVF) is composed of a heterogeneous mix of adipose-derived stem cells (ADSCs), macrophages, pericytes, fibroblasts, blood, and other cells. Previous studies have found that the paracrine effects of SVF cells may be therapeutic, but their role in osteoarthritis treatment remains unclear. This study aimed to investigate the therapeutic effect of SVF cells on chondrocytes. Chondrocytes were seeded on culture plates alone (control) or cocultured with SVF or ADSCs on cell culture inserts. After 48 h of coculture, chondrocyte collagen II, tissue inhibitors of metalloproteinases-3 (TIMP-3), and matrix metalloproteinases-13 (MMP-13) messenger RNA (mRNA) expression levels were evaluated using reverse-transcription polymerase chain reaction, and the transforming growth factor-ß (TGF-ß) levels in the supernatant were measured using ELISA. Immunohistochemical staining and flow cytometry were used to evaluate the macrophages in the SVF. These macrophages were characterized according to phenotype using the F4/80, CD86, and CD163 markers. To determine whether the Smad2/3 signaling pathways were involved, the chondrocytes were pre-treated with a Smad2/3 phosphorylation inhibitor and stimulated with the SVF, and then Smad2/3 phosphorylation levels were analyzed using western blot. The mRNA expression levels of various paracrine factors and chondrocyte pellet size were also assessed. Collagen II and TIMP-3 expression were higher in the SVF group than in the ADSC group and controls, while MMP-13 expression was the highest in the ADSC group and the lowest in the controls. TGF-ß levels in the SVF group were also elevated. Immunohistochemical staining and flow cytometry revealed that the macrophages in the SVF were of the anti-inflammatory phenotype. Western blot analysis showed that the SVF increased Smad2/3 phosphorylation, while Smad2/3 inhibitors decreased phosphorylation. Smad2/3 inhibitors also reduced the expression of various other paracrine factors and decreased chondrocyte pellet size. These findings suggested that the paracrine effect of heterogeneous cells, such as anti-inflammatory macrophages, in the SVF partly supports chondrocyte regeneration through TGF-ß-induced Smad2/3 phosphorylation.

Histopathological changes of synovial tissue in rheumatoid arthritis patients treated with TNF-α inhibitors or IL-6 inhibitors.

OBJECTIVES: To investigate the cell types that undergo apoptosis in TNF-α inhibitor (TNFI)- and IL-6 inhibitor (IL-6I)-treated synovia of RA patients, and to observe and compare histological changes in them. METHODS: Synovial tissue was collected during total knee arthroplasty from 20 RA patients who were divided into three groups based on RA treatment received: conventional synthetic disease-modifying anti-rheumatic drugs (csDMARDs, control group), TNFI, or IL-6I. Tissue samples were subjected to haematoxylin and eosin staining, terminal deoxynucleotidyl transferase fluorescein-deoxyuridine triphosphate nick end labelling (TUNEL), immuno-histochemistry (IHC) and immunofluorescence staining for, respectively, histopathological assessment, apoptosis detection and IHC evaluation and scoring. RESULTS: TUNEL-positive cells were detected surrounding the discoid fibrosis unique to the TNFI group, while those in the IL-6I group were distributed widely, especially surrounding the blood vessels. IHC revealed that in TNFI-treated tissue, CD86- and CD80-positive cells were detected only in the lining and sublining layer, while CD163- and CD206-positive cells were detected more broadly; in the IL-6I-treated tissue, all four were detected widely but their levels were lower than in the control group. Immunofluorescence also revealed macrophages mainly were the apoptotic cells in the lining and sublining layers of TNFI group. TUNEL Expression levels of CD20- and CD3-positive cells were remarkably lower in the IL-6I group, compared with the control and TNFI groups. CONCLUSIONS: TNFIs and IL-6Is target different action sites and synovial cell types, resulting in histopathological features of synovium distinct from one another.

Anterior Cruciate Ligament Deficiency is Not Always a Contraindication for Medial Unicompartmental Knee Arthroplasty: A Retrospective Study in Nondesigner's Japanese Hospital.

BACKGROUND: An intact anterior cruciate ligament (ACL) is thought to be prerequisite for successful unicompartmental knee arthroplasty (UKA), but recent studies reported successful midterm results of UKA in ACL-deficient (ACLD) knees. We hypothesized that ACLD is not always a contraindication for medial UKA when preoperative radiographs showed typical anteromedial knee patterns. METHODS: From April 2012 to March 2016, 401 Oxford mobile-bearing UKAs in 282 patients were retrospectively identified from our database. Patients whose ACL was severely damaged, but preoperative X-rays showed typical anteromedial osteoarthritis patterns, were categorized into the ACLD group. From intraoperative data, those whose ACL was intact were categorized into the ACL functional (ACLF) group. There were 32 and 369 knees in the ACLD and ACLF groups, respectively, and mean follow-up periods were 66.1 and 63.8 months for the ACLD and ACLF groups, respectively. We compared the postoperative clinical outcome and component survivorship, with an endpoint of component revision, between ACLD groups and ACLF groups. RESULTS: In both groups, the Oxford knee score, Knee Society score, Tegner activity score, and knee range of motion in extension were improved after surgery. The UKA component survival rate at five years was 100% in the ACLD group and 98.9% in the ACLF group. There were no significant differences between the groups. CONCLUSION: Mid-term clinical outcomes of Oxford mobile-bearing UKA in ACLD knees were similar to those in ACLF knees. ACL deficiency is not always a contraindication for medial unicompartmental knee arthroplasty in patients with typical anteromedial osteoarthritis radiographs.

The Usability of CAT System for Assessing the Depressive Level of Japanese-A Study on Psychometric Properties and Response Behavior.

BACKGROUND: An innovative measurement system using a computerized adaptive testing technique based on the item response theory (CAT) has been expanding to measure mental health status. However, little is known about details in its measurement properties based on the empirical data. Moreover, the response time (RT) data, which are not available by a paper-and-pencil measurement but available by a computerized measurement, would be worth investigating for exploring the response behavior. PURPOSE: We aimed at constructing the CAT to measure depressive symptomatology in a community population and exploring its measurement properties. Also, we examined the relationships between RTs, individual item responses, and depressive levels. METHOD: For constructing the CAT system, responses of 2061 workers and university students to 24 depression scale plus four negatively revised positive affect items were subjected to a polytomous IRT analysis. The stopping rule was set for standard error of estimation < 0.30 or the maximum 15 items displayed. The CAT and non-adaptive computer-based test (CBT) were administered to 209 undergraduates, and 168 of them administered again after 1 week. RESULTS: On average, the CAT was converged by 10.4 items. The θ values estimated by CAT and CBT were highly correlated (r = 0.94 and 0.95 for the 1st and 2nd measurements) and with the traditional scoring procedures (r's > 0.90). The test-retest reliability was at a satisfactory level (r = 0.86). RTs to some items significantly correlated with the θ estimates. The mean RT varied by the item contents and wording, i.e., the RT to positive affect items required additional 2 s or longer than the other subscale items. CONCLUSION: The CAT would be a reliable and practical measurement tool for various purposes including stress check at workplace.

[Development of the Emotion and Arousal Checklist (EACL)].

We developed the 33-item Emotion and Arousal Checklist (EACL), which consisted of five subscales to assess emotions (Fear, Anger, Sadness, Disgust, and Happiness) and four subscales to assess arousal (Energetic arousal + Energetic arousal -, Tense arousal+, and Tense arousal -). This checklist was developed to assess psychological state, both at a given moment and during the past week. In Study 1, confirmatory factor analyses identified nine subscales, whose internal consistency was indicated by their reliability. In Study 2, the EACL's validity was demonstrated by its correlation with the State-Trait Anxiety Inventory, Multiple Mood Scale, General Arousal Checklist, Japanese UWIST Mood Adjective Checklist, and Profile of Mood States. In Study 3, changes caused by tasks that involved either reading emotion-inducing articles or performing a calculation indicated the validity of the EACL for measuring psychological state at a given moment. Further, the test-retest reliability of the EACL for assessing psychological state during the past week was confirmed. These studies confirmed the reliability and the validity of the EACL.

Influence of Distal Reference Point of the Tibial Mechanical Axis on the Ankle and Hindlimb Alignment Change after Total Knee Arthroplasty.

The alignment philosophy in total knee arthroplasty (TKA) has tended to shift from the gold standard of mechanically aligned technique to personalized alignment, such as the kinematically aligned (KA) technique. However, the influences of different surgical techniques on lower limb alignment relative to the ground are not fully investigated. This study investigated the influence of the ankle and hindlimb alignment change after mechanically aligned TKA and KA-TKA. The varus osteoarthritic patients who underwent TKAs were divided into a mechanically aligned TKA group (group M, n = 50) and a KA-TKA group (group K, n = 50). Radiographic parameters (hip-knee-calcaneus [HKC] angle, hip-knee-ankle [HKA] angle, talar tilt angle [TTA], and tibiocalcaneal angle [TCA]) were investigated using full-length standing radiographs. The deviation angle (ΔTA; angle between the tibial mechanical axis [TMA] and the ground tibial mechanical axis [gTMA]) and the change of ΔTA (cΔTA) were also assessed. These parameters were compared between the two groups, along with the correlation between the preoperative HKA angle and other parameters. ΔTA, TTA, and TCA showed no differences between the groups pre- and postoperatively, and no significant changes were observed postoperatively. The preoperative HKA angle showed a significant negative correlation with cΔTA in both groups (group M: r = -0.33, p = 0.02; group K: r = -0.29, p = 0.04) although no correlation was observed the with preoperative TTA and TCA. Despite no change in ΔTA after surgery, the preoperative varus deformity was associated with a change in the deviation between gTMA and TMA after surgery. A severely varus knee may be inappropriate for ground KA-TKA.

Revealing a Natural Model of Pre-Osteoarthritis of the Hip Through Study of Femoroacetabular Impingement.

Femoroacetabular impingement (FAI) is considered the mechanical cause of hip osteoarthritis (OA). Surgical intervention involves labrum repair and osteochondroplasty to remove the impingement, alleviating symptoms. Nevertheless, some patients progress to hip OA after surgery, indicating that factors other than mechanical abnormality are contributing to hip OA progression. This review article discusses our laboratory's studies on hip FAI and OA, undertaken to identify key molecular players in the progression of hip OA. Transcriptome analysis identified peroxisome proliferator activated receptor gamma (PPARγ) as a crucial molecule in early hip OA. PPARγ, widely expressed in chondrocytes, has a protective role in preventing OA, but its true mechanism remains unknown. We observed a dysregulation of DNA methyltransferase (DNMT) in the progression of hip OA, with high expression of DNMT1 and 3A and downregulation of DNMT3B. Moreover, we established that DNMT3A is the main molecule that is binding to PPARγ promoter CpG area, and hypermethylation of this area occurs during disease progression. This suggests that epigenetic changes are a main mechanism that regulates PPARγ expression. Finally, we developed a novel rabbit model of hip FAI and OA and are currently performing studies to validate our small-animal model to human FAI.

Heterogeneous Cells as well as Adipose-Derived Stromal Cells in Stromal Vascular Fraction Contribute to Enhance Anabolic and Inhibit Catabolic Factors in Osteoarthritis.

The stromal-vascular fraction (SVF), comprising heterogeneous cell populations and adipose-derived stromal cells (ADSCs), has therapeutic potential against osteoarthritis (OA); however, the underlying mechanism remains elusive. This study aimed to investigate the therapeutic effects of heterogeneous cells in rabbit SVF on rabbit chondrocytes. Rabbit SVF and ADSCs were autografted into knees at OA onset. The SVF (1 × 105) and low-dose ADSCs (lADSC; 1 × 104) groups adjusted for their stromal cell content were compared. Animals were euthanized 8 and 12 weeks after OA onset for macroscopic and histological analyses of OA progression and synovitis. Immunohistochemical and real-time polymerase chain reaction assessments were conducted. In vitro, immune-fluorescent double staining was performed for SVF to stain macrophages with F4/80, CD86(M1), and CD163(M2). OA progression was markedly suppressed, and synovitis was reduced in the SVF groups (OARSI histological score 8 W: 6.8 ± 0.75 vs. 3.8 ± 0.75, p = 0.001; 12 W: 8.8 ± 0.4 vs. 5.4 ± 0.49, p = 0.0002). The SVF groups had higher expression of collagen II and SOX9 in cartilage and TGF-ß and IL-10 in the synovium, lower expression of MMP-13, and lower macrophage M1/M2 ratio than the lADSC groups. Immunofluorescent double staining revealed a markedly higher number of M2 than that of M1 macrophages in the SVF. The therapeutic effects of SVF on chondrocytes were superior than those of lADSCs, with enhanced anabolic and inhibited catabolic factors. Heterogeneous cells, mainly M2 macrophages in the SVF, enhanced growth factor secretion and chondrocyte-protective cytokines, thus benefiting chondrocytes and knee joint homeostasis. Overall, the SVF is a safe, relatively simple, and a useful treatment option for OA.

Polyarthritis presented in a patient with untreated chronic hepatitis B infection.

Hepatitis B virus (HBV) infection can cause arthritis, but it is rarely reported. In the current report, we present a case of chronic polyarthritis in a patient with untreated HBV infection. A 63-year-old woman suffering from polyarthritis in her fingers visited our institution. She had experienced exacerbations and remissions of polyarthritis for more than 20 years. She had been diagnosed with rheumatoid arthritis and had been treated with conventional synthetic disease-modifying antirheumatic drugs (csDMARDs) and nonsteroidal anti-inflammatory drugs by her primary care doctor, but the csDMARDs were discontinued at the request of the patient 10 years before the first visit to our hospital. The blood test showed negative for rheumatoid factor and anticyclic citrullinated peptides antibody but positive for hepatitis B surface antigen. Hepatitis B surface antigen and HBV-Deoxyribo Nucleic Acid (DNA) were increased to 312.6 (IU/ml) and 4.6 (log copies/ml), respectively. Based on the results of abdominal computed tomography and echography, she was diagnosed with liver cirrhosis. Treatment for HBV infection was begun with oral tenofovir at 25 mg/day. The polyarthritis in her fingers gradually disappeared and has not relapsed for 6 months after the initiation of treatment for HBV infection. When polyarthritis is diagnosed, the possibility that chronic HBV infection can be one of the causes of polyarthritis should be considered.

The nature of the TRAP-Anti-TRAP complex.

Tryptophan biosynthesis is subject to exquisite control in species of Bacillus and has become one of the best-studied model systems in gene regulation. The protein TRAP (trp RNA-binding attenuation protein) predominantly forms a ring-shaped 11-mer, which binds cognate RNA in the presence of tryptophan to suppress expression of the trp operon. TRAP is itself regulated by the protein Anti-TRAP, which binds to TRAP and prevents RNA binding. To date, the nature of this interaction has proved elusive. Here, we describe mass spectrometry and analytical centrifugation studies of the complex, and 2 crystal structures of the TRAP-Anti-TRAP complex. These crystal structures, both refined to 3.2-A resolution, show that Anti-TRAP binds to TRAP as a trimer, sterically blocking RNA binding. Mass spectrometry shows that 11-mer TRAP may bind up to 5 AT trimers, and an artificial 12-mer TRAP may bind 6. Both forms of TRAP make the same interactions with Anti-TRAP. Crystallization of wild-type TRAP with Anti-TRAP selectively pulls the 12-mer TRAP form out of solution, so the crystal structure of wild-type TRAP-Anti-TRAP complex reflects a minor species from a mixed population.

A genome-wide association study identifying single nucleotide polymorphisms in the PPFIBP2 gene was predictive for interstitial lung disease in rheumatoid arthritis patients.

Objective: Genetic polymorphisms might serve as useful prognostic markers for the timely diagnosis of RA. The purpose of this study was to identify genomic factors predictive of the occurrence of interstitial lung disease (ILD) in RA by performing a genome-wide association study of genetic variants, including single nucleotide polymorphisms (SNPs). Methods: The study population included 306 RA patients. All patients were treated with conventional DMARDs, including 6-16 mg MTX per week. Clinical data and venous blood samples were collected from all patients before administration of DMARDs. A total of 278 347 SNPs were analysed to determine their association with ILD occurrence. Results: Several SNPs were strongly associated with ILD occurrence (P < 10-5). rs6578890, which is located on chromosome 11 in the intronic region of the gene encoding tyrosine phosphatase receptor type F polypeptide-interacting protein-binding protein 2 (PPFIBP2), showed the strongest association with ILD occurrence (odds ratio 4.32, P = 10-7.93). Conclusion: PPFIBP2 could be a useful genetic marker for occurrence of interstitial pneumonia in RA patients and might help to identify the risk of ILD occurrence before RA treatment, thereby improving patient outcomes.

P21 deficiency exhibits delayed endochondral ossification during fracture healing.

INTRODUCTION: Endochondral ossification is a complex biological phenomenon involving a variety of factors and cells. Cyclin-dependent kinase inhibitor 1 (p21) inhibits cell cycle progression and is affected by external stress. We recently reported that embryonic endochondral ossification is unaffected by endogenous p21 deficiency. In this study, we evaluated whether p21 expression affects endochondral ossification during fracture healing. METHODS: Tibial fractures were introduced into p21 knockout (p21-/-) (n = 24) and wild-type C57BL/6 (p21+/+) (n = 24) mice at age 10 weeks. Fracture healing was evaluated using radiological, histological, and immunohistochemical (IHC) analyses. The effect of p21 small interfering RNA (siRNA) on ATDC5 cells was assessed in vitro. RESULTS: The Allen score for fracture healing was lower in p21-/- mice than in p21+/+ mice. In addition, p21-/- mice exhibited larger calluses and lower bone mineral density. IHC analyses showed that p21-/- mice exhibited delayed endochondral ossification via the Ihh-Runx2-Osterix pathway in vivo. Down-regulation of p21 expression in ATDC5 cells delayed endochondral ossification in vitro. CONCLUSIONS: p21 deficiency leads to delayed endochondral ossification by attenuating the Ihh-Runx2-Osterix pathway in vivo, and p21 deficiency in hypertrophic chondrocytes causes delayed differentiation of hypertrophic chondrocytes in vitro. p21 plays a role in endochondral ossification during fracture healing.

Susceptibility of cyclin-dependent kinase inhibitor 1-deficient mice to rheumatoid arthritis arising from interleukin-1ß-induced inflammation.

We recently reported that cyclin-dependent kinase inhibitor 1 (p21) deficiency induces osteoarthritis susceptibility. Here, we determined the mechanism underlying the effect of p21 in synovial and cartilage tissues in RA. The knee joints of p21-knockout (p21-/-) (n = 16) and wild type C57BL/6 (p21+/+) mice (n = 16) served as in vivo models of collagen antibody-induced arthritis (CAIA). Arthritis severity was evaluated by immunological and histological analyses. The response of p21 small-interfering RNA (siRNA)-treated human RA FLSs (n = 5 per group) to interleukin (IL)-1ß stimulation was determined in vitro. Arthritis scores were higher in p21-/- mice than in p21+/+ mice. More severe synovitis, earlier loss of Safranin-O staining, and cartilage destruction were observed in p21-/- mice compared to p21+/+ mice. p21-/- mice expressed higher levels of IL-1ß, TNF-α, F4/80, CD86, p-IKKα/ß, and matrix metalloproteinases (MMPs) in cartilage and synovial tissues via IL-1ß-induced NF-kB signaling. IL-1ß stimulation significantly increased IL-6, IL-8, and MMP expression, and enhanced IKKα/ß and IκBα phosphorylation in human FLSs. p21-deficient CAIA mice are susceptible to RA phenotype alterations, including joint cartilage destruction and severe synovitis. Therefore, p21 may have a regulatory role in inflammatory cytokine production including IL-1ß, IL-6, and TNF-α.

Comparison of plantar pressure distribution during walking and lower limb alignment between modified kinematically and mechanically aligned total knee arthroplasty.

Lower extremity alignment is very important after total knee arthroplasty (TKA). This study aimed to compare the plantar pressure distribution while walking and the overall limb alignment, including the hindfoot, between kinematically (KA) and mechanically aligned (MA) TKA. The plantar pressure distribution was investigated using a pressure plate during walking and one-leg standing among four groups: patients one year after KA-TKA (KA group; n = 25), patients one year after MA-TKA (MA group, n = 25), patients with osteoarthritis (OA) undergoing non-surgical care (OA group, n = 25), and healthy controls (Healthy group; n = 25). Conventional and true mechanical axes (the line from the femoral head to the lowest point of the calcaneus) were evaluated on unipedal standing long-leg radiographs in the KA, MA, and OA groups. Results were compared using analysis of variance. The OA group showed a lateral loading pattern in the mid- and rearfoot, while the MA group showed a medial rearfoot loading pattern during walking. On the contrary, the KA and Healthy groups showed an almost equal pressure distribution between the medial and lateral rearfoot. Moreover, although both mechanical axes in the KA group passed through the knee more medially, a more neutral alignment was achieved in the true mechanical axis compared to that in the MA group. KA-TKA results in more neutral weight-bearing through the true mechanical axis and allows patients to walk while maintaining medial and lateral rearfoot pressure more evenly than MA-TKA.

Cryopreserved human adipose-derived stromal vascular fraction maintains fracture healing potential via angiogenesis and osteogenesis in an immunodeficient rat model.

BACKGROUND: Novel therapeutic strategies for the healing of nonunion, which has serious effects on the quality of life of patients, are needed. We evaluated the therapeutic effect of local transplantation of human stromal vascular fraction (SVF) cells on fracture healing in a rat non-healing fracture model and compared the effects between freshly isolated (F) and cryopreserved (C)-SVFs. METHODS: Non-healing fracture model was induced in the femur of female immunodeficient rats (F344/N Jcl rnu/rnu) with cauterizing periosteum. Immediately after the creation of non-healing fracture, rats received local transplantation of F and C-SVFs suspended in phosphate-buffered saline (PBS) or the same volume of PBS without cells using the same scaffold as a control group. During 8 weeks post-surgery, radiologic, histological, immunohistochemical, and biomechanical analyses were performed to evaluate fracture healing. The comparison of radiological results was performed with a chi-square test, and the multiple comparisons of immunohistochemical, histological, and biomechanical results among groups were made using a one-way analysis of variance. A probability value of 0.05 was considered to denote statistical significance. RESULTS: At week 8, in 60% of animals receiving F-SVF cells and in 50% of animals receiving C-SVF cells, the fracture radiologically healed with bone union whereas nonunion was observed in the control group. The healing potential was also confirmed by histological and biomechanical assessments. One of the mechanisms underlying healing involving intrinsic angiogenesis/osteogenesis was enhanced in F- and C-SVF groups compared with that in the control group. Human cell-derived vasculogenesis/osteogenesis, which was also confirmed in an in vitro differentiation assay, was also enhanced in the F- and C-SVF groups compared with that in the control groups and could be another mechanism for healing. CONCLUSIONS: SVF cells can enhance bone healing and cryopreserved cells have almost equal potential as fresh cells. SVF cells can be used for improving nonunion bone fracture healing as an alternative to other mesenchymal stem cells and the effect of SVF cells can be maintained under cryopreservation.

Comparison of clinical and biomechanical outcomes between the kinematic and mechanical alignment methods in total knee arthroplasty: Protocol for a multicenter randomized controlled trial.

INTRODUCTION: The concept of anatomic restoration has garnered considerable interest in the form of kinematically aligned total knee arthroplasty (KA-TKA). KA-TKAs have been reported to reproduce natural alignment and kinematics. However, few randomized controlled trials (RCTs) have compared the biomechanical outcomes and the long-term clinical outcomes of KA-TKA with those of mechanically aligned TKA (MA-TKA). We aim to investigate the long-term clinical and biomechanical effects of KA-TKA and to determine whether KA-TKA or MA-TKA is more appropriate for primary TKA. METHODS: This trial will compare clinical and biomechanical outcomes of KA-TKA to those of MA-TKA. Two hundred patients will be enrolled in the RCT and randomized into KA-TKA or MA-TKA groups. Both the groups will be evaluated 1 week before the operation, on the day of the operation, 6 months after the operation, and 1, 5, and 10 years after the operation. The primary outcome is the difference between preoperative and 1-year postoperative functional activity scores of the 2011 Knee Society Score (2011 KSS) in both groups as well as the differences between the scores of both groups. The secondary outcomes will include differences in symptom, satisfaction, and expectation scores of the 2011 KSS, intraoperative kinematics evaluation, postoperative clinical outcomes and complications, pre- and postoperative gait analyses and radiograph evaluations between both KA-TKA and MA-TKA.

Prorenin processing enzyme (PPE) produced by Baculovirus-infected Sf-9 insect cells: PPE is the cysteine protease encoded in the acMNPV gene.

In infection cultures of Spodoptera frugiperda (Sf-9) insect cells with a recombinant baculovirus, vhpR, carrying human preprorenin cDNA in the polyhedrin locus of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV), the expressed inactive recombinant human (rh)-prorenin is reported to be proteolytically processed to yield active rh-renin in the very late phase of culture (Takahashi et al., Biosci. Biotechnol. Biochem., 71, 2610-2613 (2007)). To identify the enzyme that catalyzes the processing of rh-prorenin, referred to as prorenin processing enzyme (PPE), we purified potential PPE from virus-infected Sf-9 culture supernatant by the use of an internally quenched fluorescent (IQF) substrate for PPE. The 32-kDa protein band agreed well with PPE activity on the final Mono Q FPLC. By N-terminal amino acid sequence analysis, the protein was revealed to be a cysteine protease encoded by the AcMNPV gene. Enzyme activity was inhibited by cysteine protease inhibitors but not by other protease inhibitors. When the purified rh-prorenin was incubated with the 32-kDa protein, renin activity appeared concomitant with the disappearance of rh-prorenin. The N-terminal amino acid sequence of the activated product was identical to that of the rh-renin that had accumulated in the infection cultures. These results indicate that the 32-kDa cysteine protease derived from the AcMNPV gene is the enzyme PPE of virus-infected Sf-9 cells.

Purification and characterization of aspartic protease derived from Sf9 insect cells.

An aspartic protease that is significantly produced by baculovirus-infected Spodoptera frugiperda Sf9 insect cells was purified to homogeneity from a growth medium. To monitor aspartic protease activity, an internally quenched fluoresce (IQF) substrate specific to cathepsin D was used. The purified aspartic protease showed a single protein band on SDS-PAGE with an apparent molecular mass of 40 kDa. The N-terminal amino acid sequence of the enzyme had a high homology to a Bombyx mori aspartic protease. The enzyme showed greatest affinity for the IQF substrate at pH 3.0 with a K(m) of 0.85 µM. The k(cat) and k(cat)/K(m) values were 13 s(-1) and 15 s(-1) µM(-1) respectively. Pepstatin A proved to be a potent competitive inhibitor with inhibitor constant, K(i), of 25 pM.