Wijnen, André J. van

Objective Cartilage formation is stimulated in mixtures of chondrocytes and human adipose–derived mesenchymal stromal cells (MSCs) both in vitro and in vivo. During coculture, human MSCs perish. The goal of this study is to elucidate the mechanism by which adipose tissue–derived MSC cell death occurs in the presence of chondrocytes. Methods Human primary chondrocytes were cocultured with human MSCs derived from 3 donors. The cells were cultured in monoculture or coculture (20% chondrocytes and 80% MSCs) in pellets (200,000 cells/pellet) for 7 days in chondrocyte proliferation media in hypoxia (2% O 2 ). RNA sequencing was performed to assess for differences in gene expression between monocultures or coculture. Immune fluorescence assays were performed to determine the presence of caspase-3, LC3B, and P62. Results RNA sequencing revealed significant upregulation of >90 genes in the 3 cocultures when compared with monocultures. STRING analysis showed interconnections between >50 of these genes. Remarkably, 75% of these genes play a role in cell death pathways such as apoptosis and autophagy. Immunofluorescence shows a clear upregulation of the autophagic machinery with no substantial activation of the apoptotic pathway. Conclusion In cocultures of human MSCs with primary chondrocytes, autophagy is involved in the disappearance of MSCs. We propose that this sacrificial cell death may contribute to the trophic effects of MSCs on cartilage formation.

AbstractThe catalytic subunit of DNA‐dependent protein kinase (DNA‐PKcs) is a pleiotropic enzyme involved in DNA repair, cell cycle control, and transcription regulation. A potential role for DNA‐PKcs in the regulation of osteoblastogenesis remains to be established. We show that pharmacological inhibition of DNA‐PKcs kinase activity or gene silencing of Prkdc (encoding DNA‐PKcs) in murine osteoblastic MC3T3‐E1 cells and human adipose‐derived mesenchymal stromal cells markedly enhanced osteogenesis and the expression of osteoblast differentiation marker genes. Inhibition of DNA‐PKcs inhibited cell cycle progression and increased osteogenesis by significantly enhancing the bone morphogenetic protein 2 response in osteoblasts and other mesenchymal cell types. Importantly, in vivo pharmacological inhibition of the kinase enhanced bone biomechanical properties. Bones from osteoblast‐specific conditional Prkdc‐knockout mice exhibited a similar phenotype of increased stiffness. In conclusion, DNA‐PKcs negatively regulates osteoblast differentiation, and therefore DNA‐PKcs inhibitors may have therapeutic potential for bone regeneration and metabolic bone diseases.

Significance Statement The arteriovenous fistula (AVF) is the preferred vascular access for hemodialysis, but several studies have shown that the primary patency rate at 1 year is approximately 60%. Percutaneous transluminal angioplasty (PTA) is the first line of treatment for AVF stenosis. However, the rate of restenosis after angioplasty is high and the primary patency rate after PTA treatment in AVFs at 6, 12, and 24 months is 61%, 42%, and 35%, respectively. Currently, no durable therapies prolong patency after angioplasty. This paper describes the use of adipose-derived mesenchymal stem cells to reduce stenosis after PTA treatment in a murine AVF model and provides rationale for potential clinical use. Background Percutaneous transluminal angioplasty (PTA) is the first line of treatment for stenosis in the arteriovenous fistula (AVF) created to provide access for hemodialysis, but resenosis still occurs. Transplants of adipose-derived mesenchymal stem cells (AMSCs) labeled with green fluorescent protein (GFP) to the adventitia could reduce pro-inflammatory gene expression, possibly restoring patency in a murine model of PTA for venous stenosis. Methods Partial nephrectomy of male C57BL/6J mice induced CKD. Placement of the AVF was 28 days later and, 14 days after that, PTA of the stenotic outflow vein was performed with delivery of either vehicle control or AMSCs (5×10 5 ) to the adventitia of the vein. Mice were euthanized 3 days later and gene expression for interleukin-1 beta (IL-1 β ) and tumor necrosis factor-alpha TNF- α ) analyzed, and histopathologic analysis performed on day 14 and 28. GFP (+) AMSCs were tracked after transplantation for up to 28 days and Doppler ultrasound performed weekly after AVF creation. Results Gene and protein expression of IL-1 β and TNF- α, fibrosis, proliferation, apoptosis and smooth muscle actin decreased, and the proportions of macrophage types (M2/M1) shifted in a manner consistent with less inflammation in AMSC-transplanted vessels compared to controls. After PTA, AMSC-treated vessels had significantly higher wall shear stress, average peak, and mean velocity, with increased lumen vessel area and decreased neointima/media area ratio compared to the control group. At 28 days after delivery, GFP (+) AMSC were present in the adventitia of the outflow vein. Conclusions AMSC-treated vessels had improved vascular remodeling with decreased proinflammatory gene expression, inflammation, and fibrotic staining compared to untreated vessels.

AbstractScapholunate interosseous ligament tears are a common wrist injury in young and active patients that can lead to suboptimal outcomes after repair. This research aims to assess a multiphasic scaffold using 3D‐printing for reconstruction of the dorsal scapholunate interosseous ligament. The scaffold was surgically implanted in vivo in the position of the native rabbit medial collateral ligament. Two branches of treatment were implemented in the study. In the first group, the rabbits (n = 8) had the knee joint fixed in flexion for 4 weeks using 1.4 mm K‐wires prior to sample harvesting. The second group (n = 8) had the rabbit knee joint immobilized for 4 weeks prior to K‐wire removal and mobilization for an additional 4 weeks prior to sample harvesting. Overall, samples were harvested at 4 weeks post‐surgery (immobilized group) and eight weeks post‐surgery (mobilized group). Mechanical tensile testing (n = 5/group) and histology (n = 3/group) of the constructs were conducted. Tissue integration and maturation were observed resulting in increased mechanical strength of the operated joint at 8 weeks (P < .05). Bone and ligament tissues were regenerated in their respective compartments with structural and mechanical properties approaching those reported for the human dorsal SLIL ligament. Clinical Significance: This proof of concept study has demonstrated that the synthetic multiphasic scaffold was capable of regenerating both bone and ligament while also withstanding the physiological load once implanted in the rabbit knee. The artificial scaffold may provide an alternative to current techniques for reconstruction of scapholunate instability or other ligament injuries in the hand and wrist.

Aims Arthrofibrosis is a relatively common complication after joint injuries and surgery, particularly in the knee. The present study used a previously described and validated rabbit model to assess the biomechanical, histopathological, and molecular effects of the mast cell stabilizer ketotifen on surgically induced knee joint contractures in female rabbits. Methods A group of 12 skeletally mature rabbits were randomly divided into two groups. One group received subcutaneous (SQ) saline, and a second group received SQ ketotifen injections. Biomechanical data were collected at eight, ten, 16, and 24 weeks. At the time of necropsy, posterior capsule tissue was collected for histopathological and gene expression analyses (messenger RNA (mRNA) and protein). Results At the 24-week timepoint, there was a statistically significant increase in passive extension among rabbits treated with ketotifen compared to those treated with saline (p = 0.03). However, no difference in capsular stiffness was detected. Histopathological data failed to demonstrate a decrease in the density of fibrous tissue or a decrease in α-smooth muscle actin (α-SMA) staining with ketotifen treatment. In contrast, tryptase and α-SMA protein expression in the ketotifen group were decreased when compared to saline controls (p = 0.007 and p = 0.01, respectively). Furthermore, there was a significant decrease in α-SMA (ACTA2) gene expression in the ketotifen group compared to the control group (p < 0.001). Conclusion Collectively, these data suggest that ketotifen mitigates the severity of contracture formation in a rabbit model of arthrofibrosis. Cite this article: Bone Joint Res 2020;9(6):302–310.

Abstract Objective Chondrocyte apoptosis plays a vital role in osteoarthritis (OA) progression. Angelica sinensis polysaccharide (ASP), a traditional Chinese medicine, possesses anti-inflammatory and anti-apoptotic properties in chondrocytes. This study aimed to determine the protective role of ASP on sodium nitroprusside (SNP)-induced chondrocyte apoptosis, and explore the underlying mechanism. Method Human primary chondrocytes isolated from articular cartilage of OA patients were treated with SNP alone or in combination with different dose of ASP. Cell viability and apoptosis were assessed, and apoptosis-related proteins including Bcl-2 and Bax were detected. Autophagy levels were evaluated by light chain 3 (LC3)-II immunofluorescence staining, mRFP-GFP-LC3 fluorescence localisation and western blot (LC3II, p62, Beclin-1, Atg5). Meanwhile,activation of ERK 1/2 pathway was determined by western blot. The autophagy inhibitors 3-Methyladenine (3-MA), chloroquine (CQ) and a specific inhibitor of ERK1/2, SCH772984, were used respectively to confirm the autophagic effect of ASP. Results The results showed that SNP-induced chondrocyte apoptosis was significantly rescued by ASP, whereas ASP alone promoted chondrocytes proliferation. The anti-apoptotic effect of ASP was related to the enhanced autophagy and depended on the activation of ERK1/2 pathway.Conclusion ASP markedly rescued SNP-induced chondrocyte apoptosis by activating ERK1/2-dependent autophagy in chondrocytes, and it made ASP a potential therapeutic supplementation for OA treatment.

Skeletal development is controlled by epigenetic mechanisms that regulate chromatin compaction and suppress access to gene regulatory sequences. Post‐translational modifications in the N‐termini of histone 3 (H3), including tri‐methylated H3 lysine 9 (H3K9Me3) or lysine 27 (H3K27Me3) represent repressive chromatin marks that are recognized by a family of heterochromatin‐associated Chromobox (Cbx) proteins (i.e., Cbx1 to Cbx8). We show using RNA‐seq analysis that each of these Cbx isoforms is actively expressed in bone and osteoblasts with variable expression of distinct members (between 1 and 100 reads per million). The most prominently expressed Cbx member is Cbx3, which encodes a member of the heterochromatin protein 1 (HP1) family that interacts with H3K9me3. Cbx3 is known to be important for normal development while promoting neurogenesis, as well as renal and smooth muscle development. Therefore, we postulated that CBx3 may perform a similar role in osteoblastogenesis. We tested this hypothesis by both transient and stable knock‐down of Cbx3 by respectively siRNA or shRNA. RNA interference in MC3T3 osteoblasts effectively decreases Cbx3 mRNA levels by 2 to 5 fold, while increasing expression of bone‐related markers by RT‐qPCR (e.g., Alpl/alkaline phosphatase, Ibsp/bone sialoprotein, Bglap/osteocalcin, and Col1a1/Collagen Type I α1 chain). In addition, Cbx3 depletion increases mineral deposition as measured by Alizarin Red staining. Thus, Cbx3 normally appears to suppress osteoblastic differentiation, unlike its essential function in the neuronal differentiation. Because Cbx3 recognizes H3K9me3, we examined whether Cbx3 controls the levels of this histone mark. Loss of Cbx3 did not affect total H3K9me3 levels unless Cbx1 and Cbx5 were simultaneously depleted by siRNA. We propose that Cbx3 may suppress osteoblastogenesis by controlling select loci with H3K9me3 marks.

AbstractThe dense formation of abnormal scar tissue after total knee arthroplasty results in arthrofibrosis, an unfortunate sequela of inflammation. The purpose of this study was to use a validated rabbit model to assess the effects on surgically‐induced knee joint contractures of two combined pharmacological interventions: celecoxib (CXB) loaded on an implanted collagen membrane, and subcutaneously (SQ) injected ketotifen. Thirty rabbits were randomly divided into five groups. The first group received no intervention after the index surgery. The remaining four groups underwent intra‐articular implantation of collagen membranes loaded with or without CXB at the time of the index surgery; two of which were also treated with SQ ketotifen. Biomechanical joint contracture data were collected at 8, 10, 16, and 24 weeks. At the time of necropsy (24 weeks), posterior capsule tissue was collected for messenger RNA and histopathologic analyses. At 24 weeks, there was a statistically significant increase in passive extension among rabbits in all groups treated with CXB and/or ketotifen compared to those in the contracture control group. There was a statistically significant decrease in COL3A1, COL6A1, and ACTA2 gene expression in the treatment groups compared to the contracture control group (P < .001). Histopathologic data also demonstrated a trend towards decreased fibrous tissue density in the CXB membrane group compared to the vehicle membrane group. The present data suggest that intra‐articular placement of a treated collagen membrane blunts the severity of contracture development in a rabbit model of arthrofibrosis, and that ketotifen and CXB may independently contribute to the prevention of arthrofibrosis. Statement of clinical significance: Current literature has demonstrated that arthrofibrosis may affect up to 5% of primary total knee arthroplasty patients. For that reason, novel pharmacologic prophylaxis and treatment modalities are critical to mitigating reoperations and revisions while improving the quality of life for patients with this debilitating condition.

Abstract Osteolineage cell‐derived extracellular vesicles (EVs) play a regulatory role in hematopoiesis and have been shown to promote the ex vivo expansion of human hematopoietic stem and progenitor cells (HSPCs). Here, we demonstrate that EVs from different human osteolineage sources do not have the same HSPC expansion promoting potential. Comparison of stimulatory and non‐stimulatory osteolineage EVs by next‐generation sequencing and mass spectrometry analyses revealed distinct microRNA and protein signatures identifying EV‐derived candidate regulators of ex vivo HSPC expansion. Accordingly, the treatment of umbilical cord blood‐derived CD34 + HSPCs with stimulatory EVs‐altered HSPC transcriptome, including genes with known roles in cell proliferation. An integrative bioinformatics approach, which connects the HSPC gene expression data with the candidate cargo in stimulatory EVs, delineated the potentially targeted biological functions and pathways during hematopoietic cell expansion and development. In conclusion, our study gives novel insights into the complex biological role of EVs in osteolineage cell‐HSPC crosstalk and promotes the utility of EVs and their cargo as therapeutic agents in regenerative medicine.

ABSTRACT During bone marrow stromal cell (BMSC) differentiation, both Wnt signaling and the development of a rigid cytoskeleton promote commitment to the osteoblastic over adipogenic lineage. β-catenin plays a critical role in the Wnt signaling pathway to facilitate downstream effects on gene expression. We show that β-catenin was additive with cytoskeletal signals to prevent adipogenesis, and β-catenin knockdown promoted adipogenesis even when the actin cytoskeleton was depolymerized. β-catenin also prevented osteoblast commitment in a cytoskeletal-independent manner, with β-catenin knockdown enhancing lineage commitment. Chromatin immunoprecipitation (ChIP)-sequencing demonstrated binding of β-catenin to the promoter of enhancer of zeste homolog 2 (EZH2), a key component of the polycomb repressive complex 2 (PRC2) complex that catalyzes histone methylation. Knockdown of β-catenin reduced EZH2 protein levels and decreased methylated histone 3 (H3K27me3) at osteogenic loci. Further, when EZH2 was inhibited, β-catenin's anti-differentiation effects were lost. These results indicate that regulating EZH2 activity is key to β-catenin's effects on BMSCs to preserve multipotentiality. © 2020 American Society for Bone and Mineral Research.