Wijnen, André J. van

The liver is an immunologically active organ with a tolerogenic microenvironment at a quiescent state. The immunoregulatory properties of the liver appear to be retained after transplantation because liver allografts can reduce alloresponses against other organs that are simultaneously transplanted. Mechanisms of this phenomenon remain unknown. Given the known immunomodulatory properties of mesenchymal stromal cells (MSCs), we hypothesized that liver mesenchymal stromal cells (L‐MSCs) are superior immunomodulators and contribute to liver‐mediated tolerance. L‐MSCs, generated from human liver allograft biopsies, were compared with adipose mesenchymal stromal cells (A‐MSCs) and bone marrow mesenchymal stromal cells (BM‐MSCs). Trilineage differentiation of L‐MSCs was confirmed by immunohistochemistry. Comparative phenotypic analyses were done by flow cytometry and transcriptome analyses by RNA sequencing in unaltered cell cultures. The in vitro functional analyses were performed using alloreactive T cell proliferation assays. The transcriptome analysis showed that the L‐MSCs are different than the A‐MSCs and BM‐MSCs, with significant enrichment of genes and gene sets associated with immunoregulation. Compared with the others, L‐MSCs were found to express higher cell surface levels of several select immunomodulatory molecules. L‐MSCs (versus A‐MSCs/BM‐MSCs) inhibited alloreactive T cell proliferation (22.7% versus 56.4%/58.7%, respectively; P < 0.05) and reduced the frequency of interferon ɤ–producing T cells better than other MSCs (52.8% versus 94.4%/155.4%; P < 0.05). The antiproliferative impact of L‐MSCs was not dependent on cell‐to‐cell contact, could be reversed incompletely by blocking programmed death ligand 1, and required a higher concentration of the competitive inhibitor of indoleamine 2,3‐dioxygenase for complete reversal. In conclusion, L‐MSCs appear to be uniquely well‐equipped immunomodulatory cells, and they are more potent than A‐MSCs and BM‐MSCs in that capacity, which suggests that they may contribute to liver‐induced systemic tolerance.

Abstract Although degenerative disc disease (DDD) and related low back pain (LBP) are growing public health problems, the underlying disease mechanisms remain unclear. An increase in the vascular endothelial growth factor (VEGF) levels in DDD has been reported. This study aimed to examine the role of VEGF receptors (VEGFRs) in DDD, using a mouse model of DDD. Progressive DDD was induced by anterior stabbing of lumbar intervertebral discs in wild type (WT) and VEGFR‐1 tyrosine‐kinase deficient mice ( vegfr‐1 TK−/ − ). Pain assessments were performed weekly for 12 weeks. Histological and immunohistochemical assessments were made for discs, dorsal root ganglions, and spinal cord. Both vegfr‐1 TK − / − and WT mice presented with similar pathological changes in discs with an increased expression of inflammatory cytokines and matrix‐degrading enzymes. Despite the similar pathological patterns, vegfr‐1 TK − / − mice showed insensitivity to pain compared with WT mice. This insensitivity to discogenic pain was related to lower levels of pain factors in the discs and peripheral sensory neurons and lower spinal glial activation in the vegfr‐1 TK − / − mice than in the WT mice. Exogenous stimulation of bovine disc cells with VEGF increased inflammatory and cartilage degrading enzyme. Silencing vegfr‐1 by small‐interfering‐RNA decreased VEGF‐induced expression of pain markers, while silencing vegfr‐2 decreased VEGF‐induced expression of inflammatory and metabolic markers without changing pain markers. This suggests the involvement of VEGFR‐1 signaling specifically in pain transmission. Collectively, our results indicate that the VEGF signaling is involved in DDD. Particularly, VEGFR‐1 is critical for discogenic LBP transmission independent of the degree of disc pathology.

AbstractTranscription networks and epigenetic mechanisms including DNA methylation, histone modifications, and noncoding RNAs control lineage commitment of multipotent mesenchymal progenitor cells. Proteins that read, write, and erase histone tail modifications curate and interpret the highly intricate histone code. Epigenetic reader proteins that recognize and bind histone marks provide a crucial link between histone modifications and their downstream biological effects. Here, we investigate the role of bromodomain‐containing (BRD) proteins, which recognize acetylated histones, during osteogenic differentiation. Using RNA‐sequencing (RNA‐seq) analysis, we screened for BRD proteins (n = 40) that are robustly expressed in MC3T3 osteoblasts. We focused functional follow‐up studies on Brd2 and Brd4 which are highly expressed in MC3T3 preosteoblasts and represent “bromodomain and extra terminal domain” (BET) proteins that are sensitive to pharmacological agents (BET inhibitors). We show that small interfering RNA depletion of Brd4 has stronger inhibitory effects on osteoblast differentiation than Brd2 loss as measured by osteoblast‐related gene expression, extracellular matrix deposition, and alkaline phosphatase activity. Similar effects on osteoblast differentiation are seen with the BET inhibitor +JQ1, and this effect is reversible upon its removal indicating that this small molecule has no lasting effects on the differentiation capacity of MC3T3 cells. Mechanistically, we find that Brd4 binds at known Runx2 binding sites in promoters of bone‐related genes. Collectively, these findings suggest that Brd4 is recruited to osteoblast‐specific genes and may cooperate with bone‐related transcription factors to promote osteoblast lineage commitment and maturation.

Chondrosarcomas are chemo- and radiotherapy resistant and frequently harbor mutations in isocitrate dehydrogenase (IDH1 or IDH2), causing increased levels of D-2-hydroxyglutarate (D-2-HG). DNA repair defects and synthetic lethality with poly(ADP-ribose) polymerase (PARP) inhibition occur in IDH mutant glioma and leukemia models. Here we evaluated DNA repair and PARP inhibition, alone or combined with chemo- or radiotherapy, in chondrosarcoma cell lines with or without endogenous IDH mutations. Chondrosarcoma cell lines treated with the PARP inhibitor talazoparib were examined for dose–response relationships, as well as underlying cell death mechanisms and DNA repair functionality. Talazoparib was combined with chemo- or radiotherapy to evaluate potential synergy. Cell lines treated long term with an inhibitor normalizing D-2-HG levels were investigated for synthetic lethality with talazoparib. We report that talazoparib sensitivity was variable and irrespective of IDH mutation status. All cell lines expressed Ataxia Telangiectasia Mutated (ATM), but a subset was impaired in poly(ADP-ribosyl)ation (PARylation) capacity, homologous recombination, and O-6-methylguanine-DNA methyltransferase (MGMT) expression. Talazoparib synergized with temozolomide or radiation, independent of IDH1 mutant inhibition. This study suggests that talazoparib combined with temozolomide or radiation are promising therapeutic strategies for chondrosarcoma, irrespective of IDH mutation status. A subset of chondrosarcomas may be deficient in nonclassical DNA repair pathways, suggesting that PARP inhibitor sensitivity is multifactorial in chondrosarcoma.

Objective. Osteochondral allograft (OCA) transplantation has demonstrated good long-term outcomes in treatment of cartilage defects. Viability, a key factor in clinical success, decreases with peri-implantation storage at 4°C during pathogen testing, matching logistics, and transportation. Modern, physiologic storage conditions may improve viability and enhance outcomes. Design. Osteochondral specimens from total knee arthroplasty patients (6 males, 5 females, age 56.4 ± 2.2 years) were stored in media and incubated at normoxia (21% O 2 ) at 22°C or 37°C, and hypoxia (2% O 2 ) at 37°C. Histology, live-dead staining, and quantitative polymerase chain reaction (qPCR) was performed 24 hours after harvest and following 7 days of incubation. Tissue architecture, cell viability, and gene expression were analyzed. Results. No significant viability or gene expression deterioration of cartilage was observed 1-week postincubation at 37°C, with or without hypoxia. Baseline viable cell density (VCD) was 94.0% ± 2.7% at day 1. At day 7, VCD was 95.1% (37°C) with normoxic storage and 92.2% (37°C) with hypoxic storage ( P ≥ 0.27). Day 7 VCD (22°C) incubation was significantly lower than both the baseline and 37°C storage values (65.6%; P < 0.01). COL1A1, COL1A2, and ACAN qPCR expression was unchanged from baseline ( P < 0.05) for all storage conditions at day 7, while CD163 expression, indicative of inflammatory macrophages and monocytes, was significantly lower in the 37°C groups ( P < 0.01). Conclusion. Physiologic storage at 37°C demonstrates improved chondrocyte viability and metabolism, and maintained collagen expression compared with storage at 22°C. These novel findings guide development of a method to optimize short-term fresh OCA storage, which may lead to improved clinical results.

AbstractNuclear actin plays a critical role in mediating mesenchymal stem cell (MSC) fate commitment. In marrow-derived MSCs, the principal diaphanous-related formin Diaph3 (mDia2) is present in the nucleus and regulates intranuclear actin polymerization, whereas Diaph1 (mDia1) is localized to the cytoplasm and controls cytoplasmic actin polymerization. We here show that mDia2 can be used as a tool to query actin-lamin nucleoskeletal structure. Silencing mDia2 affected the nucleoskeletal lamin scaffold, altering nuclear morphology without affecting cytoplasmic actin cytoskeleton, and promoted MSC differentiation. Attempting to target intranuclear actin polymerization by silencing mDia2 led to a profound loss in lamin B1 nuclear envelope structure and integrity, increased nuclear height, and reduced nuclear stiffness without compensatory changes in other actin nucleation factors. Loss of mDia2 with the associated loss in lamin B1 promoted Runx2 transcription and robust osteogenic differentiation and suppressed adipogenic differentiation. Hence, mDia2 is a potent tool to query intranuclear actin-lamin nucleoskeletal structure, and its presence serves to retain multipotent stromal cells in an undifferentiated state.

Objective This study aims to (1) determine and validate living cartilage allograft transplantation as a novel source for viable osteochondral allograft (OCA) tissues and (2) perform histologic and viability comparisons of living donor cartilage tissues to currently available clinical-grade standard processed grafts. Design Using healthy cartilage from well-preserved contralateral compartments in 27 patients undergoing total knee arthroplasty (TKA) and 10 clinical-grade OCA specimens obtained immediately following operative implantation, standard and living donor OCA quality was evaluated at the time of harvest and following up to 3 weeks of storage on the basis of macroscopic International Cartilage Repair Society grade, histology, and viability. Results Osteochondral samples demonstrated a consistent decrease in viability and histologic quality over the first 3 weeks of storage at 37°C, supporting the utility of an OCA paradigm shift toward early implantation, as was the clinical standard up until recent adoption of transplantation at 14 to 35 days following donor procurement. Samples from the 10 clinical-grade OCAs, implanted at an average of 23 days following graft harvest demonstrated a mean viable cell density of 45.6% at implantation, significantly lower ( P < 0.01) than the 93.6% viability observed in living donor allograft tissues. Conclusions Osteochondral tissue viability and histologic quality progressively decreases with ex vivo storage, even when kept at physiologic temperatures. Currently available clinical OCAs are stored for 2 to 5 weeks prior to implantation and demonstrate inferior viability to that of fresh osteochondral tissues that can be made available through the use of a living donor cartilage program.

ABSTRACTVascularized composite allotransplantation of bone is a possible alternative treatment for large osseous defects but requires life‐long immunosuppression. Surgical induction of autogenous neo‐angiogenic circulation maintains transplant viability without this requirement, providing encouraging results in small animal models [1–3]. A preliminary feasibility study in a swine tibia model demonstrated similar findings [4, 5]. This study in swine tibial allotransplantation tests its applicability in a pre‐clinical large animal model. Previously, we have demonstrated bone vascularized composite allotransplantation (VCA) survival was not the result of induction of tolerance nor an incompetent immune system [1]. Fourteen tibia vascularized bone allotransplants were microsurgically transplanted orthotopically to reconstruct size‐matched tibial defects in Yucatan miniature swine. Two weeks of immunosuppression was used to maintain allotransplant pedicle patency during angiogenesis from a simultaneously implanted autogenous arteriovenous bundle. The implanted arteriovenous bundle was patent in group 1 and ligated in group 2 (a neo‐angiogenesis control). At twenty weeks, we quantified the neo‐angiogenesis and correlated it with transplant viability, bone remodeling, and gene expression. All patent arteriovenous bundles maintained patency throughout the survival period. Micro‐angiographic, osteocyte cell count and bone remodeling parameters were significantly higher than controls due to the formation of a neo‐angiogenic autogenous circulation. Analysis of gene expression found maintained osteoblastic and osteoclastic activity as well as a significant increase in expression of endothelial growth factor‐like 6 (EGFL‐6) in the patent arteriovenous bundle group. Vascularized composite allotransplants of swine tibia maintained viability and actively remodeled over 20 weeks when short‐term immunosuppression is combined with simultaneous autogenous neo‐angiogenesis. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:288‐296, 2020

Aims Options for the treatment of intra-articular ligament injuries are limited, and insufficient ligament reconstruction can cause painful joint instability, loss of function, and progressive development of degenerative arthritis. This study aimed to assess the capability of a biologically enhanced matrix material for ligament reconstruction to withstand tensile forces within the joint and enhance ligament regeneration needed to regain joint function. Materials and Methods A total of 18 New Zealand rabbits underwent bilateral anterior cruciate ligament reconstruction by autograft, FiberTape, or FiberTape-augmented autograft. Primary outcomes were biomechanical assessment (n = 17), microCT (µCT) assessment (n = 12), histological evaluation (n = 12), and quantitative polymerase chain reaction (qPCR) analysis (n = 6). Results At eight weeks, FiberTape alone or FiberTape-augmented autograft demonstrated increased biomechanical stability compared with autograft regarding ultimate load to failure (p = 0.035), elongation (p = 0.006), and energy absorption (p = 0.022). FiberTape-grafted samples also demonstrated increased bone mineral density in the bone tunnel (p = 0.039). Histological evaluation showed integration of all grafts in the bone tunnels by new bone formation, and limited signs of inflammation overall. A lack of prolonged inflammation in all samples was confirmed by quantification of inflammation biomarkers. However, no regeneration of ligament-like tissue was observed along the suture tape materials. Except for one autograft failure, no adverse events were detected. Conclusion Our results indicate that FiberTape increases the biomechanical performance of intra-articular ligament reconstructions in a verified rabbit model at eight weeks. Within this period, FiberTape did not adversely affect bone tunnel healing or invoke a prolonged elevation in inflammation. Cite this article: Bone Joint J 2019;101-B:1238–1247.

ABSTRACTInjuries to flexor tendons can be complicated by fibrotic adhesions, which severely impair the function of the hand. Plasminogen activator inhibitor 1 (PAI‐1/SERPINE1), a master suppressor of fibrinolysis and protease activity, is associated with adhesions. Here, we used next‐generation RNA sequencing (RNA‐Seq) to assess genome‐wide differences in messenger RNA expression due to PAI‐1 deficiency after zone II flexor tendon injury. We used the ingenuity pathway analysis to characterize molecular pathways and biological drivers associated with differentially expressed genes (DEG). Analysis of hundreds of overlapping and DEG in PAI‐1 knockout (KO) and wild‐type mice (C57Bl/6J) during tendon healing revealed common and distinct biological processes. Pathway analysis identified cell proliferation, survival, and senescence, as well as chronic inflammation as potential drivers of fibrotic healing and adhesions in injured tendons. Importantly, we identified the activation of PTEN signaling and the inhibition of FOXO1‐associated biological processes as unique transcriptional signatures of the healing tendon in the PAI‐1/Serpine1 KO mice. Further, transcriptomic differences due to the genetic deletion of PAI‐1 were mechanistically linked to PI3K/Akt/mTOR, PKC, and MAPK signaling cascades. These transcriptional observations provide novel insights into the biological roles of PAI‐1 in tendon healing and could identify therapeutic targets to achieve scar‐free regenerative healing of tendons. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:43–58, 2020