Isolation and phenotyping of pro- versus anti- inflammatory equine macrophages for chronic tendinopathy research Marguerite Meeremans1 2, Sarah Bairiot1, Sandra Van Vlierberghe2, Mario Van Poucke3, Luc Peelman3, Kristel Demeyere4, Evelyne Meyer4, Bert Devriendt5, Catharina De Schauwer1 1Veterinary Stem Cell Research Unit, Department of Translational Physiology, Infectiology and Public Health, Faculty of Veterinary Medicine, Ghent, Belgium; 2Polymer Chemistry and Biomaterials Group, Centre of Macromolecular Chemistry, Faculty of Sciences, Ghent, Belgium; 3Laboratory of Animal Genetics, Department of Veterinary and Biosciences, Faculty of Veterinary Medicine, Ghent, Belgium; 4Laboratory of Biochemistry, Department of Veterinary and Biosciences, Faculty of Veterinary Medicine, Ghent, Belgium; 5Laboratory of Immunology, Department of Translational Physiology, Infectiology and Public Health, Faculty of Veterinary Medicine, Ghent, Belgium Introduction Inflammation and immune cells play important roles in tissue homeostasis and regeneration. In chronic degenerative tendino- pathies, the balance between pro- and anti-inflammatory macro- phages is a crucial parameter determining tendon repair outcomes. When disturbed, fibrosis occurs impairing tendon structure and elasticity. In both human and equine athletes ten- don injuries are a major cause of poor performance and impaired quality of life. In contrast to human, however, a robust protocol to isolate equine pro- and anti-inflammatory macrophages is lacking. Additionally, the interaction between macrophages and tendon cells was explored to improve our understanding of the role of macrophages in tendon repair and regeneration. Methods Equine peripheral blood-derived monocytes (PBMCs) were isolated using gradient density separation (Lymphoprep� 1.077 g/mL) and magnetic-activated cell sorting based on CD172a expression. After 4 days of culture, cells were cultured another 4 days in medium sup- plemented with either IFN-c/LPS or IL-4 to induce pro- and anti- inflammatory macrophage polarization, respectively. Polarization was evaluated by assessing mRNA expression (pro: TNFA, CXCL8, IL6, CD80, CD86, and IL1B; anti: CD206, ARG2, VEGFA, IL10, and TGFB1). Furthermore, CD86 and CD206 protein expression was evaluated with flow cytometry, while VEGFa protein expres- sion was assessed by immunofluorescent staining, complemented with a NO and an arginase activity assay. Finally, a Luminex multi- plex cytokine assay was performed to measure the secreted cyto- kines (TNFa, CXCL8, IL-6, IL-1b, IL-10 and TGFb1) within the culture medium upon macrophage polarization (= conditioned medium, CM). As proof of concept, CM was supplemented to equine tendon cells after which their proliferation (MTT assay) was evaluated. Results On average 2.6x106 monocytes/mL were isolated from venous blood samples with a purity of 87–5% post-sorting. PBMCs treated with IFN-c/LPS showed an enlarged cell shape with a round cell body and numerous cytoplasmic extensions in contrast to IL-4 treated PBMCs, that demonstrated both spindle-shaped and multi- nucleated giant cells. Increased TNFA, CXCL8, CD80 and IL1B mRNA expression was observed after treatment with IFN-c/LPS, while IL-4 treatment increased CD206, VEGFA and TGFB1 tran- script levels. Flow cytometry and immunofluorescence con- firmed this polarization. No differences in NO production or arginase activity were observed. Cytokines upregulated in the CM of pro-inflammatory macrophages included TNFa, CXCL8 and IL-1b, while TGFb1 was increased in anti-inflammatory macrophages. Next, varying concentrations of the CM were applied to equine tendon cells for 24 or 72 hours and compared to control media, containing the same supplements but not exposed to macrophages. Applying 50% CM (both pro- and anti- inflammatory) for 72 hours increased cell proliferation com- pared to control media. Conclusions An efficient isolation protocol to generate equine macrophages is described, followed by the in-depth characterization of their induced pro- versus anti-inflammatory phenotypes. As the horse is the most relevant preclinical orthopaedic animal model, this research holds both a strong species-specific as well as transla- tional value. Co-culturing these cells with tendon cells will pro- vide novel insights into chronic degenerative pathologies and facilitate the development of regenerative therapies in both horse and man. Topic: Submit to SYMPOSIUM Subtopic: Veterinary TERM – results from preclinical science, clinical studies and patient treatments to facilitate the “One health” concept Type: Oral presentation

Researchers have been exploring alternative methods for bone tissue engineering, as current management of critical bone defects may be a significant challenge for both patient and surgeon with conventional surgical treatments associated with several potential complications and drawbacks. Recent studies have shown mesenchymal stem cell sheets may enhance bone regeneration in different animal models. We investigated the efficacy of implanted scaffold-free bone marrow-derived mesenchymal stem cell (BMSC) sheets on bone regeneration of a critical bone defect in a weight-bearing rat model. BMSCs were isolated from the femora of male Sprague-Dawley rats 5-6 weeks of age and cell sheets were produced on temperature-responsive culture dishes. Nine male Sprague-Dawley rats 6-8 weeks of age were utilized. A bilateral femoral critical bone defect was created with a bridge plate serving as internal fixation. One side was randomly selected and BMSC sheets were implanted into the bone defect (BMSC group), with the contralateral side receiving no treatment (control). Rats were anesthetized and radiographs were performed at 2-week intervals. At the 8-week time point, rats were euthanized, femurs harvested, and microcomputed tomography and histological analysis was performed. We found a statistically significant increase in new bone formation and bone volume fraction compared with the control. Histomorphometry analysis revealed a larger percent of newly formed bone and a higher total histological score. Our results suggest that scaffold-free BMSC sheets may be used in the management of large weight-bearing bone defects to complement a different surgical technique or as a standalone approach followed by internal fixation. However, further research is still needed.

The Hippo signaling pathway inhibits cell growth, and its components and functions are highly conserved in mammals. LATS1 is a core component of the Hippo signaling pathway associated with lymphatic invasion, astrogliosis, apoptosis, and autophagy. Nevertheless, the role of Hippo/LATS1 in osteogenesis remains unclear. In this study, we used ribonucleic acid (RNA) lentiviruses to inhibit the expression of Lats1 in bone marrow-derived stem cells (BMSCs) and distraction osteogenic regions in rats. Increased osteogenic, proliferative, and migratory abilities of BMSCs were observed in Lats1-inhibited BMSCs, while these phenotypes were partially reversed by YAP1 inhibition. In vivo, we found that the LATS1/YAP1 axis promoted osteogenesis during distraction osteogenesis (DO). β-catenin was positively correlated with YAP1 expression in vivo and in vitro. When YAP1 was strongly positive in the nucleus, β-catenin expression was upregulated; when YAP1 expression was inhibited by verteporfin, β-catenin was not expressed in the nucleus. These findings suggest that the LATS1/YAP1 signaling axis promotes DO by activating the Wnt/β-catenin signaling pathway. This study provides insights into the molecular mechanism of osteogenesis and a potential therapeutic strategy for bone regeneration in DO by associating with LATS1/YAP1-β-catenin.

From a literary perspective, the concept of tissue engineering and regenerative medicine dates back several thousand years. However, from a scientific aspect, the current state of the field owns its initial origin to the discovery of cell culture methods and the ability to maintain cells outside the body in the early 1900s, to later discoveries surrounding stem cells. The science of biomaterials evolved more recently, from the use of degradable natural biomaterials in the 1970's to artificial biomaterials in the 1980s, and bioprinting hydrogels this century. Tissue engineering, originally involving the combination of cells and biomaterials, owes its roots to the early attempts in the 1960s to create artificial skin grafts as temporary wound covers for burn patients. Much has transpired since, with an increasing number of technologies reaching patients. Academia, industry, government agencies, societies, and nonprofit organizations have all played a role in advancing the field to where it is today. This overview, presented at the Rice Short Course on Advances in Tissue Engineering, highlights some of the historical aspects, as well as past and future challenges and opportunities. At the current pace of discovery, the field is poised to continue its exponential growth.

For functional reconstruction of fibrocartilage, it is necessary to reproduce the essential mechanical property exhibited by natural fibrocartilage. The distinctive mechanical property of fibrocartilage is originated from the specific histological features of fibrocartilage composed of highly aligned type I collagen (Col I) and an abundant cartilaginous matrix. While the application of tensile stimulation induces highly aligned Col I, our study reveals that it also exerts an antichondrogenic effect on scaffold-free tissues constructed with meniscal chondrocytes (MCs) and induces downregulation of Sox-9 expression and attenuated glycosaminoglycan production. Modulation of mechanotransduction by blocking nuclear translocation of Yes-associated protein (YAP) ameliorated the antichondrogenic effect in the presence of tensile stimulation. Since MCs subjected to mechanical doses either by surface stiffness or tensile stimulation showed reversibility of YAP status even after a long-term exposure to mechanotransduction, fibrocartilage tissue was constructed by sequentially inducing tissue alignment by tensile stimulation followed by inducing cartilaginous matrix production in a tension-released state. The minimal tensile dose to constitute durable tissue alignment was screened by investigating the alignment of cytoskeleton and Col I after culturing the scaffold-free tissue constructs with various tensile doses (10% static tension for 1, 3, 7, and 10 days) followed by maintaining in a released state for 5 days. Fluorescence-conjugated phalloidin binding and immunofluorescence of Col I indicated that the duration of static tension for more than 7 days resulted in durable tissue alignment for at least 5 days in the tension-released state. The tissues subjected to tensile stimulation for 7 days followed by 14 days in a released state in chondrogenic media resulted in abundant cartilaginous matrix as well as uniaxial anisotropic alignment. Our results show that the optimized tensile dose can facilitate the successful reconstruction of fibrocartilage by modulating the characteristics of matrix production by MCs.

Skeletal muscle is highly adaptive to mechanical stress due to its resident stem cells and the pronounced level of myotube plasticity. Herein, we study the adaptation to mechanical stress and its underlying molecular mechanisms in a tissue-engineered skeletal muscle model. We subjected differentiated 3D skeletal muscle-like constructs to cyclic tensile stress using a custom-made bioreactor system, which resulted in immediate activation of stress-related signal transducers (Erk1/2, p38). Cell cycle re-entry, increased proliferation, and onset of myogenesis indicated subsequent myoblast activation. Furthermore, elevated focal adhesion kinase and β-catenin activity in mechanically stressed constructs suggested increased cell adhesion and migration. After 3 days of mechanical stress, gene expression of the fusogenic markers MyoMaker and MyoMixer, myotube diameter, myonuclear accretion, as well as S6 activation, were significantly increased. Our results highlight that we established a promising tool to study sustained adaptation to mechanical stress in healthy, hypertrophic, or regenerating skeletal muscle. Impact statement Sustained adaption to mechanical stress presents a key feature for skeletal muscle functionality and growth. Knowledge of these processes, however, is mostly based on in vivo or 2D cell culture models, both of which entail significant shortcomings. Herein, we generated highly hypertrophic tissue-engineered skeletal muscle-like constructs that are comparable to the results of successful in vivo models of adaption to mechanical stimuli, achieving an outcome that only few in vitro approaches have reached. Second, we aimed at studying the underlying molecular mechanisms, which is of interest since there is little knowledge of the intracellular events during hypertrophy upon mechanical stimulation.

Bioprinting, a technology that allows depositing living cells and biomaterials together into a complex tissue architecture with desired pattern, becomes a revolutionary technology for fabrication of engineered constructs. Previously, we have demonstrated that EphrinB2-modified dental pulp stem cells (DPSCs) are expected to be promising seed cells with enhanced osteogenic differentiation capability for alveolar bone regeneration. In this study, we aimed to bioprint EphrinB2-overexpressing DPSCs with low-concentrated Gelatin methacrylate (GelMA) hydrogels into three-dimensional (3D) constructs. The printability of GelMA (5% w/v) and the structural fidelity of bioprinted constructs were examined. Then, viability, proliferation, morphology, and osteogenic differentiation of DPSCs in bioprinted constructs were measured. Finally, the effect of EphrinB2 overexpression on osteogenic differentiation of DPSCs in bioprinted constructs was evaluated. Our results demonstrated that GelMA (5% w/v) in a physical gel form was successfully bioprinted into constructs with various shapes and patterns using optimized printing parameters. Embedded DPSCs showed round-like morphology, and had a high viability (91.93% ± 8.38%) and obvious proliferation (∼1.9-fold increase) 1 day after printing. They also showed excellent osteogenic potential in bioprinted constructs. In bioprinted 3D constructs, EphrinB2-overexpressing DPSCs expressed upregulated osteogenic markers, including ALP, BMP2, RUNX2, and SP7, and generated more mineralized nodules, as compared with Vector-DPSCs. Taken together, this study indicated that fabrication of bioprinted EphrinB2-DPSCs-laden constructs with enhanced osteogenic potential was possible, and 3D bioprinting strategy combined with EphrinB2 gene modification was a promising way to create bioengineered constructs for alveolar bone regeneration.

Abdominal aortic aneurysms (AAAs) represent a multifactorial, proteolytic disorder involving disintegration of the matrix structure within the AAA wall. Intrinsic deficiency of adult vascular cells to regenerate and repair the wall elastic matrix, which contributes to vessel stretch and recoil, is a major clinical challenge to therapeutic reversal of AAA growth. In this study, we investigate the involvement of epidermal growth factor receptor-mitogen activated protein kinase (EGFR-MAPK) pathway in the activation of aneurysmal smooth muscle cells (SMCs) by neutrophil elastase, and how EGFR can be targeted for elastic matrix regeneration. We have demonstrated that neutrophil elastase activates EGFR and downregulates expression level of key elastin homeostasis genes (elastin, crosslinking enzyme-lysyl oxidase, and fibulin4) between a dose range of 1-10 μg/mL (p < 0.05). It also incites downstream proteolytic outcomes by upregulating p-extracellular signal-regulated kinase (ERK)1/2 (p < 0.0001) and matrix metalloprotease 2 (MMP2) at a protein level, which is significantly downregulated upon EGFR-specific inhibition by tyrosine kinase inhibitor AG1478 (p-ERK1/2 and MMP2 [p < 0.05]). Moreover, we have shown that EGFR inhibition suppresses collagen amounts in aneurysmal SMCs (p < 0.05) and promotes robust formation of elastic fibers by enhancing its deposition in the extracellular space. Hence, the EGFR-MAPK pathway in aneurysmal cells can be targeted to provide therapeutic effects toward stimulating vascular matrix regeneration. Impact statement Proteolytic disorders such as aortal expansions, called abdominal aortic aneurysms (AAAs), are characterized by naturally irreversible enzymatic breakdown and loss of elastic fibers, a problem that has not yet been surmounted by existing tissue engineering approaches. In this work, we show, for the first time, how epidermal growth factor receptor (EGFR) inhibition provides downstream benefits in elastic fiber assembly and deposition in aneurysmal smooth muscle cell cultures. This work can open future possibilities for development of EGFR-targeted drug-based therapies not only for vessel wall repair in AAAs but also other proteolytically compromised elastic tissues.

The chronic overexpression of matrix metalloproteases leading to consequent degradation and loss of the elastic matrix with the reduction in tissue elasticity is central to the pathophysiology of proteolytic disorders, such as abdominal aortic aneurysms (AAAs), which are localized rupture-prone aortic expansions. Effecting tissue repair to alleviate this condition is contingent on restoring elastic matrix homeostasis in the aortic wall. This is naturally irreversible due to the poor elastogenicity of adult and diseased vascular cells, and the impaired ability to assemble mature elastic fibers, more so in the context of phenotypic changes to medial smooth muscle cells (SMCs) owing to the loss of nitric oxide (NO) signaling in the AAA wall tissue. In this study, we report the benefits of the exposure of primary human aneurysmal SMCs (aHASMCs) to NO donor drug, sodium nitroprusside (SNP), in improving extracellular matrix homeostasis, particularly aspects of elastic fiber assembly, and inhibition of proteolytic degradation. SNP treatment (100 nM) upregulated elastic matrix regeneration at both gene (p < 0.05) and protein levels (p < 0.01) without affecting cell proliferation, improved gene, and protein expression of crosslinking enzyme, lysyl oxidase (p < 0.05), inhibited the expression of MMP2 (matrix metalloprotease 2) significantly (p < 0.05) and promoted contractile SMC phenotypes in aHASMC culture. In addition, SNP also attenuated the expression of mitogen-activated protein kinases, a significant player in AAA formation and progression. Our results indicate the promise of SNP for therapeutic augmentation of elastic matrix regeneration, with prospects for wall repair in AAAs. Impact Statement Chronic and naturally irreversible enzymatic degradation and loss of elastic fibers are centric to proteolytic disorders such as abdominal aortic aneurysms (AAAs). This is linked to poor elastogenicity of adult and diseased vascular cells, compromising their ability to assemble mature elastic fibers. Toward addressing this, we demonstrate the phenotype-modulatory properties of a nitric oxide donor drug, sodium nitroprusside on aneurysmal smooth muscle cells, and its dose-specific proelastogenic and antiproteolytic properties for restoring elastic matrix homeostasis. Combined with the development of vehicles for site-localized, controlled drug delivery, this can potentially lead to a new nonsurgical approach for AAA wall repair in the future.

Severe uterine injury is a major cause of endometrial scar formation and female infertility. At present, the methods for accelerating injured uterine healing are still lacking. Genetic engineering modification of mesenchymal stem cells (MSCs) has been shown great promise in preclinical studies on regeneration. Here, we constructed a type of umbilical cord MSCs (UC-MSCs) with overexpressed basic fibroblast growth factor (UCMSC-bFGF) and investigated the effects of the UCMSC-bFGF/scaffold on functional regeneration of the full-thickness defect uterus of the rat model. At days 7, 14, and 30 after treatments, the rats were killed and the injured uterus was observed. The structural and functional change of uterine was assessed by hematoxylin and eosin staining, immunohistochemical staining, and fertility experiment. The UCMSC-bFGF/scaffold group exhibited anti-inflammatory effect, and the number of CD45+ cell in the UCMSC-bFGF/scaffold group was significantly less than that in UC-MSCs/scaffold group and scaffold group, but higher than sham-operated group at day 7 postmending. At day 14, the UCMSC-bFGF/scaffold group exhibited dramatically proangiogenesis efficacy compared with UC-MSCs/scaffold group and scaffold group. At day 30, the endometrial thickness, structure of myometrium, and blood vessels in the UCMSC-bFGF/scaffold were better than those of the UC-MSCs/scaffold group and scaffold group, even close to sham-operated group. Implantation rate at injury region postoperation 30 days in the UCMSC-bFGF/scaffold group (8/16) was significantly higher than that in UC-MSCs/scaffold group (1/16) and scaffold group (0/16). Taken together, the UCMSC-bFGF/scaffold system suppressed local inflammation, promoted angiogenesis, and accelerated regeneration of the defected uterine wall, and thereby greatly shortened the healing time of the injured uterus. Impact statement In this study, we used umbilical cord mesenchymal stem cells (UC-MSCs) with stably overexpressed basic fibroblast growth factor (UCMSC-bFGF) to repair the full-thickness defect uterine wall of the rat model and found that the UCMSC-bFGF/scaffold system suppressed early acute inflammation after uterus injury, promoted angiogenesis, and accelerated regeneration of the injured uterine wall.