Mocetinostat activates Krüppel-like factor 4 and protects against tissue destruction and inflammation in osteoarthritis.

Osteoarthritis (OA) is the most common joint disorder, and disease-modifying OA drugs (DMOADs) represent a major need in OA management. Krüppel-like factor 4 (KLF4) is a central transcription factor upregulating regenerative and protective functions in joint tissues. This study was aimed to identify small molecules activating KLF4 expression and to determine functions and mechanisms of the hit compounds. High-throughput screening (HTS) with 11,948 clinical-stage compounds was performed using a reporter cell line detecting endogenous KLF4 activation. Eighteen compounds were identified through the HTS and confirmed in a secondary screen. After testing in SW1353 chondrosarcoma cells and human chondrocytes, mocetinostat - a class I selective histone deacetylase (HDAC) inhibitor - had the best profile of biological activities. Mocetinostat upregulated cartilage signature genes in human chondrocytes, meniscal cells, and BM-derived mesenchymal stem cells, and it downregulated hypertrophic, inflammatory, and catabolic genes in those cells and synoviocytes. I.p. administration of mocetinostat into mice reduced severity of OA-associated changes and improved pain behaviors. Global gene expression and proteomics analyses revealed that regenerative and protective effects of mocetinostat were dependent on peroxisome proliferator-activated receptor γ coactivator 1-α. These findings show therapeutic and protective activities of mocetinostat against OA, qualifying it as a candidate to be used as a DMOAD.

Targeting FoxO transcription factors with HDAC inhibitors for the treatment of osteoarthritis.

OBJECTIVES: Osteoarthritis (OA) features ageing-related defects in cellular homeostasis mechanisms in articular cartilage. These defects are associated with suppression of forkhead box O (FoxO) transcription factors. FoxO1 or FoxO3 deficient mice show early onset OA while FoxO1 protects against oxidative stress in chondrocytes and promotes expression of autophagy genes and the essential joint lubricant proteoglycan 4 (PRG4). The objective of this study was to identify small molecules that can increase FoxO1 expression. METHODS: We constructed a reporter cell line with FoxO1 promoter sequences and performed high-throughput screening (HTS) of the Repurposing, Focused Rescue and Accelerated Medchem (ReFRAME) library . Hits from the HTS were validated and function was assessed in human chondrocytes, meniscus cells and synoviocytes and following administration to mice. The most promising hit, the histone deacetylase inhibitor (HDACI) panobinostat was tested in a murine OA model. RESULTS: Among the top hits were HDACI and testing in human chondrocytes, meniscus cells and synoviocytes showed that panobinostat was the most promising compound as it increased the expression of autophagy genes and PRG4 while suppressing the basal and IL-1ß induced expression of inflammatory mediators and extracellular matrix degrading enzymes. Intraperitoneal administration of panobinostat also suppressed the expression of mediators of OA pathogenesis induced by intra-articular injection of IL-1ß. In a murine OA model, panobinostat reduced the severity of histological changes in cartilage, synovium and subchondral bone and improved pain behaviours. CONCLUSION: Panobinostat has a clinically relevant activity profile and is a candidate for OA symptom and structure modification.

Runx2 and Runx3 differentially regulate articular chondrocytes during surgically induced osteoarthritis development.

The Runt-related transcription factor (Runx) family plays various roles in the homeostasis of cartilage. Here, we examined the role of Runx2 and Runx3 for osteoarthritis development in vivo and in vitro. Runx3-knockout mice exhibited accelerated osteoarthritis following surgical induction, accompanied by decreased expression of lubricin and aggrecan. Meanwhile, Runx2 conditional knockout mice showed biphasic phenotypes: heterozygous knockout inhibited osteoarthritis and decreased matrix metallopeptidase 13 (Mmp13) expression, while homozygous knockout of Runx2 accelerated osteoarthritis and reduced type II collagen (Col2a1) expression. Comprehensive transcriptional analyses revealed lubricin and aggrecan as transcriptional target genes of Runx3, and indicated that Runx2 sustained Col2a1 expression through an intron 6 enhancer when Sox9 was decreased. Intra-articular administration of Runx3 adenovirus ameliorated development of surgically induced osteoarthritis. Runx3 protects adult articular cartilage through extracellular matrix protein production under normal conditions, while Runx2 exerts both catabolic and anabolic effects under the inflammatory condition.

Krüppel-like factor-4 and Krüppel-like factor-2 are important regulators of joint tissue cells and protect against tissue destruction and inflammation in osteoarthritis.

OBJECTIVES: Analysing expression patterns of Krüppel-like factor (KLF) transcription factors in normal and osteoarthritis (OA) human cartilage, and determining functions and mechanisms of KLF4 and KLF2 in joint homoeostasis and OA pathogenesis. METHODS: Experimental approaches included human joint tissues cells, transgenic mice and mouse OA model with viral KLF4 gene delivery to demonstrate therapeutic benefit in structure and pain improvement. Mechanistic studies applied global gene expression analysis and chromatin immunoprecipitation sequencing (ChIP-seq). RESULTS: Several KLF genes were significantly decreased in OA cartilage. Among them, KLF4 and KLF2 were strong inducers of cartilage collagen genes and Proteoglycan-4. Cartilage-specific deletion of Klf2 in mature mice aggravated severity of experimental OA. Transduction of human chondrocytes with Adenovirus (Ad) expressing KLF4 or KLF2 enhanced expression of major cartilage extracellular matrix (ECM) genes and SRY-box transcription factor-9, and suppressed mediators of inflammation and ECM-degrading enzymes. Ad-KLF4 and Ad-KLF2 enhanced similar protective functions in meniscus cells and synoviocytes, and promoted chondrocytic differentiation of human mesenchymal stem cells. Viral KLF4 delivery into mouse knees reduced severity of OA-associated changes in cartilage, meniscus and synovium, and improved pain behaviours. ChIP-seq analysis suggested that KLF4 directly bound cartilage signature genes. Ras-related protein-1 signalling was the most enriched pathway in KLF4-transduced cells, and its signalling axis was involved in upregulating cartilage ECM genes by KLF4 and KLF2. CONCLUSIONS: KLF4 and KLF2 may be central transcription factors that increase protective and regenerative functions in joint tissue cells, suggesting that KLF gene transfer or molecules upregulating KLFs are therapeutic candidates for OA.

Chronological Changes in Anterior Knee Stability after Anatomical Anterior Cruciate Ligament Reconstruction Using Bone-Patellar Tendon-Bone and Hamstring Grafts.

The clinical outcomes of anterior cruciate ligament (ACL) reconstruction are typically evaluated at specific time points only. This study aimed to characterize the chronological changes in anterior knee stability after anatomical ACL reconstruction and to compare the anterior knee stability achieved with bone-patellar tendon-bone (BPTB) and hamstring tendon (HT) grafts. A total of 59 patients underwent anatomical rectangular tunnel ACL reconstruction using the BPTB graft and 23 patients underwent anatomical double-bundle ACL reconstruction using the HT graft. Anterior knee stability was quantitatively assessed using the KneeLax 3 arthrometer at 6 months, 1 year, and 2 years after surgery using side-to-side differences. The values for anterior knee stability using the BPTB graft were 0.3 mm after 6 months, 0.2 mm after 1 year, and 0.2 mm after 2 years, and no significant differences were observed during the postoperative study period. Meanwhile, the values for anterior knee stability using the HT graft were -0.3 mm after 6 months, 0.5 mm after 1 year, and 1.2 mm after 2 years, and anterior knee stability decreased chronologically from 6 months up to 2 years. Regarding anterior stability, the HT graft showed significant laxity compared with the BPTB graft only after 2 years. No chronological changes in anterior stability were observed from 6 months up to 2 years after ACL reconstruction using the BPTB graft, whereas anterior laxity developed during the same period after ACL reconstruction using the HT graft. This is a Level IV, therapeutic case series study.

Type of bone graft and primary diagnosis were associated with nosocomial surgical site infection after high tibial osteotomy: analysis of a national database.

PURPOSE: Although several small-scale studies have reported risk factors for surgical site infection (SSI) after high tibial osteotomy (HTO), no study has collectively analysed risk factors in a large cohort. The present study aimed to clarify the risk factors for SSI after HTO using a national database. METHODS: Data of inpatients who underwent HTO from 2010 to 2017 were obtained from the Diagnosis Procedure Combination database in Japan. Outcome measures were the incidence of SSI and deep SSI after HTO. Associations between SSI and patient data were examined with multivariable logistic regression analysis. RESULTS: Among 12,853 patients who underwent HTO, 195 developed SSI (1.52%) and 50 developed deep SSI (0.39%). Univariate analysis showed that male sex, smoking, and longer anaesthesia duration were associated with higher incidences of SSI, whereas a primary diagnosis of osteonecrosis and use of natural bone grafts were associated with lower incidences. In multivariable analysis, SSI was positively associated with male sex, anaesthesia duration longer than 210 min (vs. 150-210 min), and use of artificial bone graft (vs. natural bone graft). SSI was negatively associated with age ≤ 49 years (vs. 50-59 years) and a primary diagnosis of osteonecrosis (vs. osteoarthritis). CONCLUSION: The present study revealed novel risk factors for SSI after HTO that previous studies have failed to find, including use of artificial bone graft and longer anaesthesia duration; primary diagnosis of osteonecrosis and younger age were novel protective factors. These findings will help surgeons assess risks of SSI after HTO in individual patients. LEVEL OF EVIDENCE: III.

Unicompartmental knee arthroplasty is associated with lower proportions of surgical site infection compared with total knee arthroplasty: A retrospective nationwide database study.

BACKGROUND: This study aimed to clarify the association between types of knee arthroplasty (KA) (total knee arthroplasty (TKA) or unicompatmental knee arthroplasty (UKA)) and surgical site infection (SSI) with adjustment for various factors, using a Japanese national database. METHODS: Data on 181,608 patients who underwent unilateral primary KA for osteoarthritis from 2010 to 2017 were obtained from the Japanese Diagnosis Procedure Combination database. SSI was identified based on International Classification of Diseases 10th Revision codes. Deep SSI (i.e. periprosthetic joint infection (PJI)) was identified as SSI treated with surgical procedures. Multivariable logistic regression analyses for SSI and PJI were performed, in which dependent variables included types of KA, patient backgrounds (sex, age, body mass index (BMI), smoking status, comorbidities), and seasonality. RESULTS: Eight percent of analyzed patients underwent UKA, while 92% underwent TKA. The proportions of SSI and PJI after UKA were 0.9% and 0.3%, respectively, both of which were lower than those after TKA (1.9% and 0.6%) (P < 0.001). Multivariable analyses showed lower proportions of SSI for UKA (adjusted odds ratio, 0.47; 95% confidence interval, 0.37-0.60; P < 0.001) and PJI (adjusted odds ratio, 0.47; 95% confidence interval, 0.34-0.65; P < 0.001) than TKA. Other factors associated with both SSI and PJI included male sex, BMI >30 kg/m2, renal dysfunction and summer season. CONCLUSION: UKA was associated with lower proportions of SSI and PJI than TKA. Surgeons should carefully consider the indication of UKA before performing TKA, especially in patients with knee unicompartmental osteoarthritis who are at a high risk for SSI or PJI.

Length of the Tendon within the Tibial Tunnel Affects Tibial Tunnel Widening following Anatomic Anterior Cruciate Ligament Reconstruction Using a Bone-Patellar Tendon-Bone Graft.

The purpose of this study was to retrospectively investigate the effects of variations in the length of the tendon within the tibial tunnel on tunnel widening (TW) following anatomical anterior cruciate ligament (ACL) reconstruction using a bone-patellar tendon-bone (BTB) graft. In total, 68 patients who underwent isolated ACL reconstructions using BTB grafts were included in this study. The patients were divided into two groups according to the length of the tendon within the tibial tunnel: group S (n = 30; tendon length, < 10 mm) and group L (n = 38; tendon length, ≥ 10 mm). Tunnel aperture area was measured using three-dimensional computed tomography (3D CT) at 1 week and 1 year postoperatively, and tibial TW (%) was calculated. The correlation coefficient between the length of the tendon within the tibial tunnel and tibial TW was also calculated. Clinical assessment was performed 1 year postoperatively, corresponding to the period of CT assessment, which involved the evaluation of the Lysholm's score, measurement of anterior knee stability using a KneeLax3 arthrometer, and the pivot-shift test. A weak positive correlation was observed between the length of the tendon within the tibial tunnel and tibial TW (r = 0.270, p = 0.026). Mean tibial tunnel aperture area increased by 19.3 ± 17.4% and 35.8 ± 25.4% in the groups S and L, respectively. TW in the group L was significantly greater than that in the group S (p = 0.004). No significant difference was observed between the two groups in any clinical outcomes. In conclusion, a longer tendinous portion within the tibial tunnel resulted in a greater tibial TW following anatomical ACL reconstructions using a BTB graft.

Simple and Robust Differentiation of Human Pluripotent Stem Cells toward Chondrocytes by Two Small-Molecule Compounds.

A simple induction protocol to differentiate chondrocytes from pluripotent stem cells (PSCs) using small-molecule compounds is beneficial for cartilage regenerative medicine and mechanistic studies of chondrogenesis. Here, we demonstrate that chondrocytes are robustly induced from human PSCs by simple combination of two compounds, CHIR99021, a glycogen synthase kinase 3 inhibitor, and TTNPB, a retinoic acid receptor (RAR) agonist, under serum- and feeder-free conditions within 5-9 days. An excellent differentiation efficiency and potential to form hyaline cartilaginous tissues in vivo were demonstrated. Comprehensive gene expression and open chromatin analyses at each protocol stage revealed step-by-step differentiation toward chondrocytes. Genome-wide analysis of RAR and ß-catenin association with DNA showed that retinoic acid and Wnt/ß-catenin signaling collaboratively regulated the key marker genes at each differentiation stage. This method provides a promising cell source for regenerative medicine and, as an in vitro model, may facilitate elucidation of the molecular mechanisms underlying chondrocyte differentiation.

Meniscal bearing dislocation while rolling over in sleep following Oxford medial unicompartmental knee arthroplasty.

Meniscal bearing dislocation while rolling over in sleep has never been reported in Oxford unicompartmental knee arthroplasty (UKA). This study reports two cases of meniscal bearing dislocation into the intercondylar ridge while rolling over in sleep. In the case of one patient, closed reduction of the bearing was performed, and the use of a knee brace was effective in preventing re-dislocation. In the second patient, closed reduction was possible; however, bearing dislocation was repeated. Therefore, revision surgery was performed by replacing the tibial component and using a thicker bearing. The common features in dislocation during rolling over while sleeping in both cases were dislocation into the intercondylar ridge, the combination of small femur and AA-size tibia components, and osteonecrosis. As determined by intraoperative testing, valgus position of the knee while rolling over in sleep could induce bearing dislocation into the intercondylar ridge. LEVEL OF EVIDENCE: Retrospective case series, Level IV.

Atopic dermatitis is a novel demographic risk factor for surgical site infection after anterior cruciate ligament reconstruction.

PURPOSE: Although various risk factors for surgical site infection after anterior cruciate ligament reconstruction (ACLR) have been reported, the number of studies with large sample sizes on this topic is limited. The aim of the present study was to clarify the risk factors for early surgical site infection after ACLR in a large cohort using a national database in Japan. METHODS: The data of patients who underwent ACLR from 2010 to 2015 were obtained from the Diagnosis Procedure Combination database, which covers approximately half of all hospital admissions in Japan. The outcome measures were the prevalences of surgical site infection and deep surgical site infection after ACLR during hospitalization. The association between the occurrence of surgical site infection and patients' demographic data, including sex, age, body mass index (BMI), smoking status, preoperative steroid use, and comorbidities such as diabetes, hepatic dysfunction, renal dysfunction, and atopic dermatitis, were examined using a multivariable logistic regression model. RESULTS: Among 30,536 patients who underwent ACLR, 288 patients with surgical site infection (0.94%) and 86 with deep surgical site infection (0.28%) were identified. The univariate analysis showed that higher prevalences of surgical site infection and deep surgical site infection were associated with male sex, a higher BMI, atopic dermatitis, and preoperative steroid use. Patients with diabetes or hepatic dysfunction had a significantly higher prevalence of surgical site infection. The multivariable analysis showed that surgical site infection was significantly associated with male sex vs. female sex; odds ratio (OR), 2.90; 95% confidence interval (CI), 2.17-3.89, age of ≤ 19 vs. 20-29 years; OR, 1.56; 95% CI 1.13-2.15, BMI of ≥ 30.0 vs. 18.5-22.9 kg/m2; OR, 1.72; 95% CI 1.16-2.54, diabetes (OR, 2.70; 95% CI 1.28-5.71), atopic dermatitis (OR, 7.19; 95% CI 2.94-17.57), and preoperative steroid use (OR, 6.18; 95% CI 2.32-16.52). CONCLUSION: Atopic dermatitis, preoperative steroid use, young age (≤ 19 years), obesity (BMI of ≥ 30.0 kg/m2), male sex, and diabetes were independent demographic risk factors for surgical site infection after ACLR. The present study will be useful when surgeons evaluate the risk of SSI after ACLR in terms of demographic aspects. LEVEL OF EVIDENCE: III.

Annual trends in arthroscopic meniscus surgery: Analysis of a national database in Japan.

BACKGROUND: The importance of meniscus preservation is widely recognized. There have been a few studies describing trends in meniscectomy and meniscus repair in the United States; however, they presented differing results. We reported annual trends in meniscus surgery, using a national database in Japan. METHODS: We interrogated the Diagnosis Procedure Combination database, which represents approximately half of all hospital admissions in Japan. We included the patients who underwent meniscectomy and meniscus repair between July 2007 and March 2015. The diagnosis, age and sex of each patient were recorded. RESULTS: We identified 83,105 patients: 69,310 underwent meniscectomy; 13,416 underwent meniscus repair and 379 underwent both in a single admission. The proportion of patients undergoing meniscus repair rose from 7.0% in 2007 to 25.9% in 2014 (p < 0.001), while the proportion undergoing meniscectomy fell from 92.8% in 2007 to 73.3% in 2014 (p < 0.001). Among patients under 30 years old, the proportions undergoing meniscus repair or meniscectomy in 2014 were 50.3% versus 48.3%, respectively. A bimodal age distribution was observed for meniscectomy, with peaks at 10-19 years of age and 60-69 years of age, whereas most patients undergoing meniscus repair were 10-19 years of age. CONCLUSIONS: We found characteristic trends where the popularity of meniscus repair increased rapidly at the expense of meniscectomy in Japan during the study period. In the last survey year, the proportion of meniscus repair exceeded that of meniscectomy in those younger than 30 years. Meniscectomy was undertaken most often in adolescents and early old age, while meniscus repair was undertaken most often in adolescents.

Surgical timing of anterior cruciate ligament reconstruction to prevent associated meniscal and cartilage lesions.

BACKGROUND: The purpose of this study was to analyze the association between the prevalence of meniscal and chondral lesions and the timing of surgery in patients undergoing primary anterior cruciate ligament (ACL) reconstruction to determine a safe time for surgery. METHODS: This retrospective study involved 226 patients (91 females and 135 males; median age, 29 years) undergoing primary ACL reconstruction. Time interval from ACL injury to surgery (median, 4 months; range, 1-420 months) and concomitant meniscal and cartilage lesions in ACL reconstruction were reviewed. Receiver operating characteristic (ROC) curve analysis was used to determine the precise threshold interval to surgery to prevent meniscal or cartilage lesions. The risk of lesion occurrence after each cutoff period was determined using odds ratio (OR). RESULTS: The incidences of medial meniscus (MM), lateral meniscus (LM), and cartilage lesions were 43.8%, 32.7%, and 27.4%, respectively. ROC analysis revealed that patients who waited for more than 6, 4, and 5 months for ACL reconstruction had a significantly greater risk of associated MM, LM, and chondral lesions, respectively. Patients who underwent ACL reconstruction ≥7 months after injury had OR of 4.1 (p < 0.001) for the presence of MM lesion as compared with those who underwent reconstruction within 6 months. Similarly, patients who underwent ACL reconstruction ≥5 months after injury had OR of 1.9 (p = 0.023) for the presence of LM lesion as compared with those who underwent reconstruction within 4 months, and patients who underwent ACL reconstruction ≥6 months after injury had OR of 2.9 (p < 0.001) for chondral lesion as compared with those who underwent reconstruction within 6 months. CONCLUSION: ACL reconstruction should be performed within approximately 6 months after the injury to prevent associated meniscal or chondral lesions.

Annual trends in knee arthroplasty and tibial osteotomy: Analysis of a national database in Japan.

BACKGROUND: Various nationwide studies have reported differing annual trends in utilization of knee arthroplasty and tibial osteotomy. Using the Diagnosis Procedure Combination database in Japan, the present series examined annual trends and demographics in total knee arthroplasty (TKA), unicompartmental knee arthroplasty (UKA) and tibial osteotomy. METHODS: All patients were identified who underwent TKA, UKA or tibial osteotomy for osteoarthritis, osteonecrosis or rheumatoid arthritis of the knee between July 2007 and March 2015. RESULTS: A total of 170,433 cases of TKA, 13,209 cases of UKA and 8760 cases of tibial osteotomy were identified. The proportion of patients undergoing UKA rose from 4.0% in 2007 to 8.1% in 2014 (P<0.001), and that of tibial osteotomy from 2.6% in 2007 to 5.5% in 2014 (P<0.001); the proportion undergoing TKA fell from 93.4% in 2007 to 86.3% in 2014 (P<0.001). Between 2007 and 2014 the proportions of patients with osteonecrosis who underwent UKA and tibial osteotomy increased from 34.7% and 11.6% to 38.6% and 16.2%, respectively (P=0.001 for UKA and P=0.004 for tibial osteotomy). The proportions of patients with osteonecrosis undergoing UKA or tibial osteotomy were significantly greater than those with other diagnoses (P<0.001 for both). CONCLUSIONS: The popularity of UKA and tibial osteotomy in Japan increased during the period 2007-2014 at the expense of TKA. The proportions of UKA and tibial osteotomy in patients with osteonecrosis also increased, and were larger than those in patients with other causative diseases.

Different regulation of limb development by p63 transcript variants.

The apical ectodermal ridge (AER), located at the distal end of each limb bud, is a key signaling center which controls outgrowth and patterning of the proximal-distal axis of the limb through secretion of various molecules. Fibroblast growth factors (FGFs), particularly Fgf8 and Fgf4, are representative molecules produced by AER cells, and essential to maintain the AER and cell proliferation in the underlying mesenchyme, meanwhile Jag2-Notch pathway negatively regulates the AER and limb development. p63, a transcription factor of the p53 family, is expressed in the AER and indispensable for limb formation. However, the underlying mechanisms and specific roles of p63 variants are unknown. Here, we quantified the expression of p63 variants in mouse limbs from embryonic day (E) 10.5 to E12.5, and found that ΔNp63γ was strongly expressed in limbs at all stages, while TAp63γ expression was rapidly increased in the later stages. Fluorescence-activated cell sorting analysis of limb bud cells from reporter mouse embryos at E11.5 revealed that all variants were abundantly expressed in AER cells, and their expression was very low in mesenchymal cells. We then generated AER-specific p63 knockout mice by mating mice with a null and a flox allele of p63, and Msx2-Cre mice (Msx2-Cre;p63Δ/fl). Msx2-Cre;p63Δ/fl neonates showed limb malformation that was more obvious in distal elements. Expression of various AER-related genes was decreased in Msx2-Cre;p63Δ/fl limb buds and embryoid bodies formed by p63-knockdown induced pluripotent stem cells. Promoter analyses and chromatin immunoprecipitation assays demonstrated Fgf8 and Fgf4 as transcriptional targets of ΔNp63γ, and Jag2 as that of TAp63γ. Furthermore, TAp63γ overexpression exacerbated the phenotype of Msx2-Cre;p63Δ/fl mice. These data indicate that ΔNp63 and TAp63 control limb development through transcriptional regulation of different target molecules with different roles in the AER. Our findings contribute to further understanding of the molecular network of limb development.

Regulation of Chondrocyte Survival in Mouse Articular Cartilage by p63.

OBJECTIVE: Transcription factor p63, of the p53 family, regulates cell proliferation, survival, and apoptosis in various cells and tissues. This study was undertaken to examine the expression and roles of p63 transcript variants in the mouse growth plate and articular chondrocytes. METHODS: For in vivo analyses, we generated Cre-mediated TAp63α-transgenic and TAp63γ-transgenic mice. To induce tissue-specific overexpression or deletion in chondrocytes, chondroprogenitor cells, or early limb bud mesenchymal cells, we used Col2a1-Cre, Sox9-Cre, and Prx1-Cre mice, respectively. We analyzed osteoarthritis (OA) development with aging or surgically induced instability in Prx1-Cre;p63fl/fl (P-conditional knockout) mice. RESULTS: Among major variants, TAp63α and TAp63γ are highly expressed in mouse primary costal and articular chondrocytes. The p63 protein was predominantly localized in the hypertrophic zone of the embryonic limb cartilage, and in the middle zone of articular cartilage. No obvious change was observed in skeletal growth of TAp63α-transgenic mice, Sox9-Cre;p63fl/fl , or P-conditional knockout mice, while that of TAp63γ-transgenic mice was impaired due to ectopic apoptosis and the resulting decreased number of chondrocytes. Expression of proapoptotic genes including bax, noxa, puma, and fas was increased in TAp63γ-transgenic mouse chondrocytes, and their transcription was probably sustained by p53 in p63-conditional knockout mouse chondrocytes because both proteins were coexpressed in the growth plate. In contrast, p53 was expressed in the superficial zone of articular cartilage, differently from p63. Notably, P-conditional knockout mice showed significant resistance to OA development, with suppression of chondrocyte apoptosis in the aging and surgical models. CONCLUSION: We demonstrated regulation of chondrocyte survival in articular cartilage by p63.

Ultrasound biomicroscopic findings of the iridocorneal angle in live healthy and glaucomatous dogs.

By using ultrasound biomicroscopy (UBM), the cross-sectional structures of the entire iridocorneal angle (ICA) which are unable to assess with gonioscopic examination were evaluated objectively and quantitatively in live healthy and glaucomatous dogs. The ICAs of normotensive eyes in healthy dogs with normal open angle (NOR), a predisposition to primary closed angle glaucoma (PCAG) (PREDIS) and suffering from unilateral PCAG (UNI), as well as the ICAs of hypertensive eyes with acute and chronic PCAG (ACG and CRG), were assessed. The opening of the ciliary cleft in PREDIS was smaller than that in NOR. In UNI, the opening and area of the ciliary cleft were significantly decreased compared with those of NOR and PREDIS. ACG had widespread structural abnormalities including marked decrease in the ciliary cleft and scleral venous plexus, and a thinner sclera than those in normotensive eyes, whereas the ICA collapsed in CRG with the thinnest sclera. Medical therapy-responsive glaucomatous cases had wider ciliary cleft and scleral venous plexus than unresponsive ones. These findings suggest that the ciliary cleft and scleral venous plexus of the ICA are key structures contributing to not only the pathophysiology of canine glaucoma but also the responsiveness to medical therapy in glaucomatous eyes, and cross-sectional entire structures of the ICA should be evaluated quantitatively with UBM when diagnosing and managing canine glaucoma.

Hyaline cartilage formation and tumorigenesis of implanted tissues derived from human induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) are a promising cell source for cartilage regenerative medicine. Meanwhile, the risk of tumorigenesis should be considered in the clinical application of human iPSCs (hiPSCs). Here, we report in vitro chondrogenic differentiation of hiPSCs and maturation of the differentiated hiPSCs through transplantation into mouse knee joints. Three hiPSC clones showed efficient chondrogenic differentiation using an established protocol for human embryonic stem cells. The differentiated hiPSCs formed hyaline cartilage tissues at 8 weeks after transplantation into the articular cartilage of NOD/SCID mouse knee joints. Although tumors were not observed during the 8 weeks after transplantation, an immature teratoma had developed in one mouse at 16 weeks. In conclusion, hiPSCs are a potent cell source for regeneration of hyaline articular cartilage. However, the risk of tumorigenesis should be managed for clinical application in the future.

Transcription factor Hes1 modulates osteoarthritis development in cooperation with calcium/calmodulin-dependent protein kinase 2.

Notch signaling modulates skeletal formation and pathogenesis of osteoarthritis (OA) through induction of catabolic factors. Here we examined roles of Hes1, a transcription factor and important target of Notch signaling, in these processes. SRY-box containing gene 9 (Sox9)-Cre mice were mated with Hes1(fl/fl) mice to generate tissue-specific deletion of Hes1 from chondroprogenitor cells; this deletion caused no obvious abnormality in the perinatal period. Notably, OA development was suppressed when Hes1 was deleted from articular cartilage after skeletal growth in type II collagen (Col2a1)-Cre(ERT);Hes1(fl/fl) mice. In cultured chondrocytes, Hes1 induced metallopeptidase with thrombospondin type 1 motif, 5 (Adamts5) and matrix metalloproteinase-13 (Mmp13), which are catabolic enzymes that break down cartilage matrix. ChIP-seq and luciferase assays identified Hes1-responsive regions in intronic sites of both genes; the region in the ADAMTS5 gene contained a typical consensus sequence for Hes1 binding, whereas that in the MMP13 gene did not. Additionally, microarray analysis, together with the ChIP-seq, revealed novel Hes1 target genes, including Il6 and Il1rl1, coding a receptor for IL-33. We further identified calcium/calmodulin-dependent protein kinase 2δ (CaMK2δ) as a cofactor of Hes1; CaMK2δ was activated during OA development, formed a protein complex with Hes1, and switched it from a transcriptional repressor to a transcriptional activator to induce cartilage catabolic factors. Therefore, Hes1 cooperated with CaMK2δ to modulate OA pathogenesis through induction of catabolic factors, including Adamts5, Mmp13, Il6, and Il1rl1. Our findings have contributed to further understanding of the molecular pathophysiology of OA, and may provide the basis for development of novel treatments for joint disorders.