Tenomodulin Research Articles

Electrospun poly(ε-caprolactone)/poly(glycerol sebacate) aligned fibers fabricated with benign solvents for tendon tissue engineering.

The electrospinning technique is a commonly employed approach to fabricate fibers intended for various tissue engineering applications. The aim of this study is to develop a novel strategy for tendon repair through the use of aligned poly(ε-caprolactone) (PCL) and poly(glycerol sebacate) (PGS) fibers fabricated in benign solvents, and further explore the potential application of PGS in tendon tissue engineering (TTE). The fibers were characterized for their morphological and physicochemical properties; amniotic epithelial stem cells (AECs) were used to assess the fibers teno-inductive and immunomodulatory potential due to their ability to teno-differentiate undergoing first a stepwise epithelial to mesenchymal transition, and due to their documented therapeutic role in tendon regeneration. The addition of PGS to PCL improved the spinnability of the polymer solution, as well as the uniformity and directionality of the so-obtained fibers. The mechanical properties were in the range of most TTE applications, specifically in the case of PCL/PGS 4:1 and 2:1 ratios. Compared to PCL alone, the same ratios also allowed a better AECs infiltration and growth over 7 days of culture, and triggered the activation of tendon-related genes (SCX, COL1, TNMD) and the expression of tenomodulin (TNMD) at the protein level. Concerning the immunomodulatory properties, both PCL and PCL/PGS fibers negatively affected the immunomodulatory profile of AECs, up-regulating both anti-inflammatory (IL-10) and pro-inflammatory (IL-12) cytokines over 7 days of culture. Overall, PCL/PGS 2:1 fibers fabricated with benign solvents proved to be the most suitable composition for TTE application based on their topographical cues, mechanical properties, biocompatibility, and teno-inductive properties.

Fibrin Scaffolds Perfused with Transforming Growth Factor-β1 as an In Vitro Model to Study Healthy and Tendinopathic Human Tendon Stem/Progenitor Cells.

A limited understanding of tendon cell biology in healthy and pathological conditions has impeded the development of effective treatments, necessitating in vitro biomimetic models for studying tendon events. We established a dynamic culture using fibrin scaffolds, bioengineered with tendon stem/progenitor cells (hTSPCs) from healthy or diseased human biopsies and perfused with 20 ng/mL of human transforming growth factor-β1 for 21 days. Both cell types showed long-term viability and upregulated Scleraxis (SCX-A) and Tenomodulin (TNMD) gene expressions, indicating tenogenic activity. However, diseased hTSPCs underexpressed collagen type I and III (COL1A1 and COL3A1) genes and exhibited lower SCX-A and TNMD protein levels, but increased type I collagen production, with a type I/type III collagen ratio > 1.5 by day 14, matching healthy cells. Diseased hTSPCs also showed constant high levels of pro-inflammatory cytokines, such as IL-8 and IL-6. This biomimetic environment is a valuable tool for studying tenogenic and inflammatory events in healthy and diseased tendon cells and identifying new therapeutic targets.

Silencing DDX3 Attenuates Interleukin-1β-Induced Intervertebral Disc Degeneration Through Inhibiting Pyroptosis.

Intervertebral disc degeneration (IVDD) is a common disorder associated with chronic inflammation and cell death. In this study, an IVDD rat model was created through Interleukin-1β (IL-1β) injection. The degeneration of intervertebral disc tissues was assessed using magnetic resonance imaging (MRI), followed by hematoxylin and eosin (H&E) and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining. RNA sequencing was performed to identify differentially expressed genes (DEGs) between the IVDD model and control rats. The expression levels of DEGs (DEAD-box polypeptide 3 (DDX3), lysine-specific demethylase 5D (KDM5D), interferon-induced gene-1 (IFIT1), ribosomal protein S10 (RPS10), tenomodulin (TNMD), and pentraxin 3 (PTX3)) were measured by real-time quantitative polymerase chain reaction (RT-qPCR). The regulatory effect of DDX3 on pyroptosis in IL-1β-treated nucleus pulpous (NP) cells was assessed after transfection with siRNA of DDX3. A total of 601 DEGs were identified from the IVDD model rat, and were abundant in extracellular matrix (ECM) organization, ECM-receptor interaction, and inflammatory pathways, including the PI3K-Akt, TNF, and AMPK signaling pathways. DDX3, KDM5D, and IFIT1 levels were notably elevated, whereas RPS10, TNMD, and PTX3 levels were decreased in the IL-1β-induced IVDD rat model. Moreover, silencing DDX3 promoted cell proliferation and abolished IL-1β-induced cell apoptosis and pyroptosis. This study revealed the role of DDX3 in IVDD pyroptosis, providing potential target for IVDD management.

Study on the mechanism of quercetin inducing mesenchymal stem cells to differentiate into fibroblasts through TGF-β1 and IGF-1

BackgroundStem cell differentiation has opened up new avenues for disease treatment, tissue repair, and drug development in the study of regenerative medicine, and has huge application prospects. This study aimed to explore the mechanism of quercetin on the differentiation of mesenchymal stem cells (MSCs) into fibroblasts. MethodsIn this study, cell differentiation experiments and flow cytometry were used to detect the successful isolation of bone marrow MSCs from SD rats. Quercetin at 5, 10, and 20 μM was used as low, medium, and high doses to intervene in MSCs. The cell viability changes of ligament fibroblasts at 24, 48, and 72 hours after quercetin treatment were detected using a CCK-8 cell counting kit. Cell proliferative capacity was determined by flow cytometry. RT-qPCR measured the relative expression levels of TGF-β1, IGF-1, COL-Ⅰ, COL-Ⅲ, FN (fibronectin), and TNMD (Tenomodulin) in different experimental groups. Molecular docking experiments were conducted to explore the binding effect of quercetin on TGF-β1 and IGF-1 proteins. ResultsFlow cytometry verified the successful isolation of MSCs, which had high expression of CD29 and CD73, while lower expression of CD90 and CD45. Experimental results show that low and medium doses of quercetin can enhance cell proliferation, while high doses have no significant effect on cells. Detection of cell proliferation through flow cytometry yielded similar results to CCK-8. Transwell experiments have shown that low and medium doses of quercetin can increase cell migration ability. In addition, RT-qPCR detection showed that quercetin can increase the mRNA expression of TGF-β1 and IGF-1, and promote the expression of COL-Ⅰ, COL-Ⅲ, FN, and TNMD genes in ligament fibroblasts. Molecular docking results showed that quercetin can bind firmly to TGF-β1 and IGF-1. ConclusionOverall, this study revealed the morphological characteristics and identification of MSCs, as well as the regulatory mechanism of quercetin on the behavior of ligament fibroblasts. Quercetin affects the proliferation and gene expression of ligament fibroblasts by regulating the expression of TGF-β1 and IGF-1, which may provide a new perspective for biomedical research on the skeletal system.

NOVEL STEM CELL STRATEGIES FOR TENDON REGENERATIVE MEDICINE

Adipose-derived stem cells (ADSCs) are an effective alternative for Teno-regeneration. Despite their applications in tendon engineering, the mechanisms promoting tendon healing still need to be understood. Since there is scattered information on ovine ADSCs, this research aims to investigate in vitro their teno-differentiation for potential use in preclinical tendon regeneration models.Ovine ADSCs were isolated from the tail region according to FAT-STEM laboratories, expanded until passage six (P6), and characterized in terms of stemness, adhesion and MHC markers by Flow Cytometry (FCM) and immunocytochemistry (ICC). Cell proliferation and senescence were evaluated with MTT and Beta-galactosidase assays, respectively. P1 ADSCs’ teno-differentiation was assessed by culturing them with teno-inductive Conditioned Media (CM) or engineering them on tendon-mimetic PLGA scaffolds. ADSCs teno-differentiation was evaluated by morphological, molecular (qRT-PCR), and biochemical (WesternBlot) approaches.ADSCs exhibited mesenchymal phenotype, positive for stemness (SOX2, NANOG, OCT4), adhesion (CD29, CD44, CD90, CD166) and MHC-I markers, while negative for hematopoietic (CD31, CD45) and MHC-II markers, showing no difference between passages. ICC staining confirmed these results, where ADSCs showed nuclear positivity for SOX2 (≅ 56%) and NANOG (≅ 67%), with high proliferation capacity without senescence until P6. Interestingly, ADSCs cultured with the teno-inductive CM did not express tenomodulin (TNMD) protein or gene. Conversely, ADSCs seeded on scaffolds teno-differentiated, acquiring a spindle shape supported by TNMD protein expression at 48h (p<0.05 vs. ADSCs 48h) with a significant increase at 14 days of culture (p<0.05 vs. ADSCs + fleece 48h).Ovine ADSCs respond differently upon distinct teno-inductive strategies. While the molecules on the CM could not trigger a teno-differentiation in the cells, the scaffold's topological stimulus did, resulting in the best strategy to apply. More insights are requested to better understand ovine ADSCs’ tenogenic commitment before using them in vivo for tendon regeneration.Acknowledgements: This research is part of the P4FIT project ESR5, under the H2020MSCA-ITN-EJD-P4 FIT-Grant Agreement ID:955685.

POLARIZED MACROPHAGE-DERIVED EXTRACELLULAR VESICLES AS POTENTIAL MEDIATORS OF TENDON REPAIR

Macrophages play a critical role in innate immunity by promoting or inhibiting tissue inflammation and repair. Classically, macrophages can differentiate into either pro-inflammatory (M1) or pro-reparative (M2) phenotypes in response to various stimuli. Therefore, this study aimed to address how extracellular vesicles (EVs) derived from polarized macrophages can affect the inflammatory response of tendon cells.For that purpose, human THP-1 cells were stimulated with lipopolysaccharide (LPS), and interleukins -4 and -13 (IL- 4, IL-13), to induce macrophages polarization into M1, M2, and hybrid M1/M2 phenotypes. Subsequently, the EVs were isolated from the culture medium by ultracentrifugation. The impact of these nanovesicles on the inflammation and injury scenarios of human tendon-derived cells (hTDCs), which had previously been stimulated with interleukin- 1 beta (IL-1ß) to mimic an inflammatory scenario, was assessed.We were able to isolate three different nanovesicles populations, showing the typical shape, size and surface markers of EVs. By extensively analyzing the proteomic expression profiles of M1, M2, and M1/M2, distinct proteins that were upregulated in each type of macrophage-derived EVs were identified. Notably, most of the detected pro- inflammatory cytokines and chemokines had higher expression levels in M1-derived EVs and were mostly absent in M2-derived EVs. Hence, by acting as a biological cue, we observed that M2 macrophage-derived EVs increased the expression of the tendon-related marker tenomodulin (TNMD) and tended to reduce the presence of pro-inflammatory markers in hTDCs. Overall, these preliminary results show that EVs derived from polarized macrophages might be a potential tool to modulate the immune system responses becoming a valuable asset in the tendon repair and regeneration fields worthy to be further explored.

Galangin Promotes Tendon Repair Mediated by Tendon-Derived Stem Cells through Activating the TGF-β1/Smad3 Signaling Pathway.

Tendon injury is a prevalent orthopedic disease that currently lacks effective treatment. Galangin (GLN) is a vital flavonoid found abundantly in galangal and is known for its natural activity. This study aimed to investigate the GLN-mediated molecular mechanism of tendon-derived stem cells (TDSCs) in tendon repair. The TDSCs were characterized using alkaline phosphatase staining, alizarin red S staining, oil red O staining, and flow cytometry. The effect of GLN treatment on collagen deposition was evaluated using Sirius red staining and quantitative (q)PCR, while a Western bot was used to assess protein levels and analyze pathways. Results showed that GLN treatment not only increased the collagen deposition but also elevated the mRNA expression and protein levels of multiple tendon markers like collagen type I alpha 1 (COL1A1), decorin (DCN) and tenomodulin (TNMD) in TDSCs. Moreover, GLN was also found to upregulate the protein levels of transforming growth factor β1 (TGF-β1) and p-Smad3 to activate the TGF-β1/Smad3 signaling pathway, while GLN mediated collagen deposition in TDSCs was reversed by LY3200882, a TGF-β receptor inhibitor. The study concluded that GLN-mediated TDSCs enhanced tendon repair by activating the TGF-β1/Smad3 signaling pathway, suggesting a novel therapeutic option in treating tendon repair.

Anoikis resistance of small airway epithelium is involved in the progression of chronic obstructive pulmonary disease.

Anoikis resistance is recognized as a crucial step in the metastasis of cancer cells. Most epithelial tumors are distinguished by the ability of epithelial cells to abscond anoikis when detached from the extracellular matrix. However, no study has investigated the involvement of anoikis in the small airway epithelium (SAE) of chronic obstructive pulmonary disease (COPD). Anoikis-related genes (ANRGs) exhibiting differential expression in COPD were identified using microarray datasets obtained from the Gene Expression Omnibus (GEO) database. Unsupervised clustering was performed to classify COPD patients into anoikis-related subtypes. Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, gene set enrichment analysis (GSEA), and gene set variation analysis (GSVA) were used to annotate the functions between different subtypes. Differential expression analysis and weighted gene co-expression network analysis (WGCNA) were leveraged to identify key molecules. The relative proportion of infiltrating immune cells in the SAE was quantified using the CIBERSORT and ssGSEA computational algorithms, and the correlation between key molecules and immune cell abundance was analyzed. The expression of key molecules in BEAS-2B cells exposed to cigarette smoke extract (CSE) was validated using qRT-PCR. A total of 25 ANRGs exhibited differential expression in the SAE of COPD patients, based on which two subtypes of COPD patients with distinct anoikis patterns were identified. COPD patients with anoikis resistance had more advanced GOLD stages and cigarette consumption. Functional annotations revealed a different immune status between COPD patients with pro-anoikis and anoikis resistance. Tenomodulin (TNMD) and long intergenic non-protein coding RNA 656 (LINC00656) were subsequently identified as key molecules involved in this process, and a close correlation between TNMD and the infiltrating immune cells was observed, such as activated CD4+ memory T cells, M1 macrophages, and activated NK cells. Further enrichment analyses clarified the relationship between TNMD and the inflammatory and apoptotic signaling pathway as the potential mechanism for regulating anoikis. In vitro experiments showed a dramatic upregulation of TNMD and LINC00656 in BEAS-2B cells when exposed to 3% CSE for 48 hours. TNMD contributes to the progression of COPD by inducing anoikis resistance in SAE, which is intimately associated with the immune microenvironment.

Additively manufactured polyethylene terephthalate scaffolds for scapholunate interosseous ligament reconstruction

The regeneration of the ruptured scapholunate interosseous ligament (SLIL) represents a clinical challenge. Here, we propose the use of a Bone-Ligament-Bone (BLB) 3D-printed polyethylene terephthalate (PET) scaffold for achieving mechanical stabilisation of the scaphoid and lunate following SLIL rupture. The BLB scaffold featured two bone compartments bridged by aligned fibres (ligament compartment) mimicking the architecture of the native tissue. The scaffold presented tensile stiffness in the range of 260 ± 38 N/mm and ultimate load of 113 ± 13 N, which would support physiological loading. A finite element analysis (FEA), using inverse finite element analysis (iFEA) for material property identification, showed an adequate fit between simulation and experimental data. The scaffold was then biofunctionalized using two different methods: injected with a Gelatin Methacryloyl solution containing human mesenchymal stem cell spheroids (hMSC) or seeded with tendon-derived stem cells (TDSC) and placed in a bioreactor to undergo cyclic deformation. The first approach demonstrated high cell viability, as cells migrated out of the spheroid and colonised the interstitial space of the scaffold. These cells adopted an elongated morphology suggesting the internal architecture of the scaffold exerted topographical guidance. The second method demonstrated the high resilience of the scaffold to cyclic deformation and the secretion of a fibroblastic related protein was enhanced by the mechanical stimulation. This process promoted the expression of relevant proteins, such as Tenomodulin (TNMD), indicating mechanical stimulation may enhance cell differentiation and be useful prior to surgical implantation. In conclusion, the PET scaffold presented several promising characteristics for the immediate mechanical stabilisation of disassociated scaphoid and lunate and, in the longer-term, the regeneration of the ruptured SLIL.

Micropattern Silk Fibroin Film Facilitates Tendon Repair In Vivo and Promotes Tenogenic Differentiation of Tendon Stem/Progenitor Cells through the α2β1/FAK/PI3K/AKT Signaling Pathway In Vitro.

Tendon injuries are common clinical disorders. Due to the limited regeneration ability of tendons, tissue engineering technology is often used as an adjuvant treatment. This study explored the molecular pathways underlying micropattern SF film-regulated TSPC propensity and their repairing effects to highlight the application value of micropattern SF films. First, we characterized the physical properties of the micropattern SF films and explored their repairing effects on the injured tendons in vivo. Then, we seeded TSPCs on SF films in vitro and determined the micropattern SF film-induced gene expression and activation of signaling pathways in TSPCs through high-throughput RNA sequencing and proteomics assays. The results of in vivo studies suggested that micropattern SF films can promote remodeling of the injured tendon. In addition, immunohistochemistry (IHC) results showed that tendon marker genes were significantly increased in the micropattern SF film repair group. Transcriptomic and proteomic analyses demonstrated that micropattern SF film-induced genes and proteins in TSPCs were mainly enriched in the focal adhesion kinase (FAK)/actin and phosphoinositide 3-kinase (PI3K)/AKT pathways. Western blot analysis showed that the expression of integrins α2β1, tenascin-C (TNC), and tenomodulin (TNMD) and the phosphorylation of AKT were significantly increased in the micropattern SF film group, which could be abrogated by applying PI3K/AKT inhibitors. Micropattern SF films modified by water annealing can promote remodeling of the injured tendon in vivo and regulate the tendon differentiation of TSPCs through the α2β1/FAK/PI3K/AKT signaling pathway in vitro. Therefore, they have great medical value in tendon repair.

N-Acetyl-L-cysteine facilitates tendon repair and promotes the tenogenic differentiation of tendon stem/progenitor cells by enhancing the integrin α5/β1/PI3K/AKT signaling

BackgroundTendon injury is associated with oxidative stress, leading to reactive oxygen species (ROS) production and inflammation. N-acetyl-L-cysteine (NAC) is a potent antioxidant. However, how NAC affects the biological functions of tendon stem/progenitor cells (TSPCs) and tendon repair has not been clarified. MethodThe impacts of NAC on the viability, ROS production, and differentiation of TSPCs were determined with the cell counting kit-8, fluorescence staining, Western blotting, and immunofluorescence. The effect of NAC on gene transcription in TSPCs was analyzed by transcriptomes and bioinformatics and validated by Western blotting. The potential therapeutic effect of NAC on tendon repair was tested in a rat model of Achilles tendon injury.ResultsCompared with the untreated control, treatment with 500 µM NAC greatly promoted the proliferation of TSPCs and significantly mitigated hydrogen peroxide-induced ROS production and cytotoxicity in vitro. NAC treatment significantly increased the relative protein expression of collagen type 1 alpha 1 (COL1A1), tenascin C (TNC), scleraxis (SCX), and tenomodulin (TNMD) in TPSCs. Bioinformatics analyses revealed that NAC modulated transcriptomes, particularly in the integrin-related phosphoinositide 3-kinase (PI3K)/AKT signaling, and Western blotting revealed that NAC enhanced integrin α5β1 expression and PI3K/AKT activation in TSPCs. Finally, NAC treatment mitigated the tendon injury, but enhanced the protein expression of SCX, TNC, TNMD, and COLIA1 in the injured tissue regions of the rats.ConclusionNAC treatment promoted the survival and differentiation of TSPCs to facilitate tendon repair after tendon injury in rats. Thus, NAC may be valuable for the treatment of tendon injury.

Anterior cruciate ligament remnant preservation attenuates apoptosis and enhances the regeneration of hamstring tendon graft

The effects of remnant preservation on the anterior cruciate ligament (ACL) and its relationship with the tendon graft remain unclear. We hypothesized that the co-culture of remnant cells and bone marrow stromal cells (BMSCs) decreases apoptosis and enhances the activity of the hamstring tendons and tenocytes, thus aiding ACL reconstruction. The ACL remnant, bone marrow, and hamstring tendons were surgically harvested from rabbits. The apoptosis rate, cell proliferation, and expression of types I and III collagen, transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF), and tenogenic genes (scleraxis (SCX), tenascin C (TNC), and tenomodulin (TNMD)) of the hamstring tendons were compared between the co-culture medium (ACL remnant cells (ACLRCs) and BMSCs co-culture) and control medium (BMSCs-only culture). We also evaluated the apoptosis, cell proliferation, migration, and gene expression of hamstring tenocytes with exposure to co-culture and control media. Compared to BMSCs-only culture medium, the co-culture medium showed substantially decreased early and late apoptosis rates, attenuation of intrinsic and extrinsic apoptotic pathways, and enhanced proliferation of the hamstring tendons and tenocytes. In addition, the expression of collagen synthesis, TGF-β, VEGF, and tenogenic genes in the hamstring tendons and tenocytes significantly increased in the co-culture medium compared to that in the control medium. In the presence of ACLRCs and BMSCs, the hamstring tendons and tenocytes significantly attenuated apoptosis and enhanced the expression of collagen synthesis, TGF-β, VEGF, and tenogenic genes. This in vitro study suggests that the ACLRCs mixed with BMSCs could aid regeneration of the hamstring tendon graft during ACL reconstruction.Cite this article: Bone Joint Res2023;12(1):9-21.

Therapeutic effects of asperosaponin VI in rabbit tendon disease

AbstarctIntroductionThis study explored the effects and molecular mechanisms of asperosaponin VI in tendon disease.MethodsForty-eight purebred adult male New Zealand white rabbits were randomly divided into the normal group (normal, n = 8); saline group (saline, n = 8) and prostaglandin E2 group (n = 32), which was further divided into four subgroups that were treated with asperosaponin VI doses of 0 mg/kg (model, n = 8), 10 mg/kg (10, n = 8), 20 mg/kg (20, n = 8) and 40 mg/kg (40, n = 8). The expression levels of matrix metallopeptidase 1 (MMP1), metallopeptidase inhibitor 1 (TIMP1), transforming growth factor beta 1 (TGFB1), serpin family E member 1 (SERPINE1), collagen Ⅰ (COL1), collagen Ⅲ (COL3) and tenomodulin (TNMD) in Achilles tendon tissue were determined through Western blot analysis. The histopathological changes in tendon tissue were observed by using Masson staining and haematoxylin–eosin staining.ResultsThe expression levels of MMP1, TIMP1 and COL3 were higher and those of TGFB1, SERPINE1, COL1 and TNMD were lower in the 0 mg/kg group than in the normal group (P < 0.05). Compared with those in the 0 mg/kg group, the levels of MMP1 were lower in the 20 and 40 mg/kg groups. Compared with those in the 0 mg/kg group, the levels of TIMP1 were lower and the levels of TGFB1, COL1 and TNMD were higher in the 10, 20 and 40 mg/kg groups. In addition, compared with those in other groups, the levels of SERPINE1 in the 40 mg/kg group were significantly higher and the levels of COL3 in the 10 and 20 mg/kg groups were significantly lower (P < 0.05). Fibrous tissue arrangements and structures in the 40 mg/kg group were similar to those in the control group.ConclusionThe effects of asperosaponin VI on injured tendons mainly involve eliminating inflammation, restoring balance to extracellular matrix collagen metabolism and inducing tendon cell proliferation. Asperosaponin VI is likely to be an ideal drug for the prevention and treatment of tendon disease.

Associations between the TNMD rs4828038 and ACE2 rs879922 polymorphisms and preeclampsia susceptibility: a case-control study

A case-control study was designed to investigate the association between the angiotensin converting enzyme 2 (ACE2) rs879922, glucose-6-phosphate dehydrogenase (G6PD) rs1050828, and tenomodulin (TNMD) rs4828038 single nucleotide polymorphisms (SNPs), and preeclampsia. A total of 356 Han Chinese pregnant women (170 controls and 186 cases) were recruited into the study. ACE2 rs879922, G6PD rs1050828, and TNMD rs4828038 were tested by the targeted next-generation sequencing technology and the data were analyzed using SPSS version 18. Genotyping of results revealed that patients with the CC/CT genotype in SNP rs4828038 or CC/CG genotype in SNP rs879922 had a significantly decreased susceptibility to late-onset preeclampsia (CC/CT versus TT: OR = 0.543, 95% CI = 0.378 to 0.779, p = .001; CC/CG versus GG: OR = 0.510, 95% CI = 0.038 to 0.860, p = .012). Our study found that the polymorphisms TNMD rs4828038 and ACE2 rs879922 might be associated with late-onset preeclampsia. IMPACT STATEMENT What is already known on this subject? Preeclampsia is associated with multiple SNPs, and ACE2 rs879922, G6PD rs1050828, and TNMD rs4828038 are related to essential hypertension and glucose and lipid metabolism disorders. Essential hypertension, diabetes, and dyslipidemia are risks for preeclampsia. The associations between those three SNPs and preeclampsia have not been reported. What do the results of this study add? The polymorphisms of TNMD rs4828038 and ACE2 rs879922 might be associated with the risk of late-onset preeclampsia. There was no relationship between SNP rs1050828 and preeclampsia. What are the implications of these findings for clinical practice and/or further research? TNMD rs4828038 and ACE2 rs879922 might be target sites for genetic diagnosis and therapy, and the levels of mRNA and protein in pregnant women with preeclampsia should be further tested.

Tenogenic Potential of Equine Fetal Mesenchymal Stem Cells Under The In Vitro Effect of Bone Morphogenetic Protein-12 (BMP-12)

Equine adult bone marrow-derived MSCs (BM-MSCs) may be induced into the tenogenic lineage after exposure with bone morphogenetic protein-12 (BMP-12). Despite fetal BM-MSCs have showed a greater differentiation potential compared to adults, the tenogenic differentiation capacity of equine fetal BM-MSC have not been reported. Thus, the aim of the present study was to evaluate the in vitro tenogenic differentiation potential of equine fetal BM-MSCs under the effect of BMP-12. Equine fetal BM-MSCs were exposed to three concentrations of BMP-12 (25, 50 and 100 ng/mL) during a 21-day culture period. Levels of mRNA of tenogenic markers decorin (DCN), tenomodulin (TNMD), scleraxis (SCX), collagen 1α1 (COL1α1) and protein expression of Col1α1 were evaluated. Plastic adherent cells exhibited specific MSC profile including expression of CD73 and lack of expression of CD34. Gene expression levels of DCN, TNMD, SCX and COL1α1 were increased in equine fetal BM-MSC exposed to three different concentrations of BMP-12 during a 21-day culture period. Equine fetal BM-MSCs displayed specific expression profiles suggesting features of MSCs and multipotent capacity. Furthermore, up-regulation of tenogenic markers DCN, TNMD, COL1α1 and SCX after exposure to different concentrations of BMP-12 suggests that equine fetal BM-MSCs have potential to activate selected genes that control tenogenic differentiation.

Synergistic effects of myogenic cells and fibroblasts on the promotion of engineered tendon regeneration with muscle derived cells

ABSTRACT Aims Tendon development requires the coordinated interaction of muscles and tendons. Muscle-derived cells (MDCs), a mixed cell population containing both myogenic and fibroblastic cell subsets, have been found to be ideal seed cells for tendon regeneration. However, the necessity of these cell types for tendon regeneration has not yet been tested. In this study, we aim to explore the possible synergistic effects of myogenic cells and fibroblasts in engineered tendon regeneration. Methods MDCs were separated into rapidly adhering cell (RAC; fibroblasts) and slowly adhering cell (SAC; myogenic cells) populations. Myogenic- and tenogenic-related molecules were analyzed by immunofluorescent staining, RT-PCR and real-time PCR. The proliferative abilities of MDCs, RACs and SACs were also evaluated. Cell-scaffold constructs were implanted into nude mice, and subsequently evaluated for their histologic, ultrastructure, gene expression, and biomechanical characteristics. Results MDCs have better proliferative activity than RAC and SAC population. RACs could express higher levels of tenogenic-related molecules tenomodulin (TNMD) and scleraxis (SCX) than SACs. Whereas SACs only expressed myogenic-related molecules MyoD. In contrast to the tendons engineered using RACs and SACs, the tendons engineered using MDCs exhibited a relatively more mature and well-organized tissue structure and ultrastructure as well as better mechanical properties. Conclusions Fibroblasts in muscle may be the primary cell population involved in tendon regeneration and that myogenic cells are an important component of the niche and control the fibroblast activity during tendon regeneration. The synergistic effects between fibroblasts and myogenic cells significantly contribute to efficient and effective regeneration of engineered tendons.

Paediatric knee anterolateral capsule does not contain a distinct ligament: analysis of histology, immunohistochemistry and gene expression

ObjectivesThe presence of a discrete ligament within the knee anterolateral capsule (ALC) is controversial. Tendons and ligaments have typical collagens, ultrastructure, transcription factors and proteins. However, these characteristics have not been investigated in paediatric ALC. The purpose of this study was to characterise the paediatric ALC in terms of tissue ultrastructure and cellular expression of ligament markers scleraxis (SCX)—a basic helix-loop-helix transcription factor—and the downstream transmembrane glycoprotein tenomodulin (TNMD), as compared with the paediatric lateral collateral ligament (LCL) and paediatric quadriceps tendon (QT). We hypothesised that, in comparison to the LCL and QT, the ALC would possess poor collagen orientation and reduced SCX and TNMD expression.Methods15 paediatric ALCs (age 6.3±3.3 years), 5 paediatric LCLs (age 3.4±1.3 years) and 5 paediatric QTs (age 2.0±1.2 years) from fresh cadaveric knees were used in this study. Fresh-frozen samples from each region were cryosectioned and then stained with H&E to evaluate collagen alignment and cell morphology. Expression of SCX and TNMD was determined by gene expression analysis and immunohistochemistry.ResultsThe histological sections of the paediatric LCL and QT showed well-organised, dense collagenous tissue fibres with elongated fibroblasts, while the ALC showed more random collagen orientation without clear cellular directionality. The aspect ratio of cells in the ALC was significantly lower than that of the LCL and QT (p<0.0001 and p<0.0001, respectively). The normalised distribution curve of the inclination angles of the nuclei in the ALC was more broadly distributed than that of the LCL or QT, indicating random cell alignment in the ALC. SCX immunostaining was apparent in the paediatric LCL within regions of aligned fibres, while the comparatively disorganised structure of the ALC was negative for SCX. The paediatric LCL also stained positive for TNMD, while the ALC was only sparsely positive for this tendon/ligament cell-surface molecule. Relative gene expression of SCX and TNMD were higher in the LCL and QT than in the ALC.ConclusionIn this study, a distinct ligament could not be discerned in the ALC based on histology, immunohistochemistry and gene expression analysis.Level of evidenceControlled laboratory study.

Comparative Analysis of Tenogenic Gene Expression in Tenocyte-Derived Induced Pluripotent Stem Cells and Bone Marrow-Derived Mesenchymal Stem Cells in Response to Biochemical and Biomechanical Stimuli.

The tendon is highly prone to injury, overuse, or age-related degeneration in both humans and horses. Natural healing of injured tendon is poor, and cell-based therapeutic treatment is still a significant clinical challenge. In this study, we extensively investigated the expression of tenogenic genes in equine bone marrow mesenchymal stem cells (BMSCs) and tenocyte-derived induced pluripotent stem cells (teno-iPSCs) stimulated by growth factors (TGF-β3 and BMP12) combined with ectopic expression of tenogenic transcription factor MKX or cyclic uniaxial mechanical stretch. Western blotting revealed that TGF-β3 and BMP12 increased the expression of transcription factors SCX and MKX in both cells, but the tenocyte marker tenomodulin (TNMD) was detected only in BMSCs and upregulated by either inducer. On the other hand, quantitative real-time PCR showed that TGF-β3 increased the expression of EGR1, COL1A2, FMOD, and TNC in BMSCs and SCX, COL1A2, DCN, FMOD, and TNC in teno-iPSCs. BMP12 treatment elevated SCX, MKX, DCN, FMOD, and TNC in teno-iPSCs. Overexpression of MKX increased SCX, DCN, FMOD, and TNC in BMSCs and EGR1, COL1A2, DCN, FMOD, and TNC in teno-iPSCs; TGF-β3 further enhanced TNC in BMSCs. Moreover, mechanical stretch increased SCX, EGR1, DCN, ELN, and TNC in BMSCs and SCX, MKX, EGR1, COL1A2, DCN, FMOD, and TNC in teno-iPSCs; TGF-β3 tended to further elevate SCX, ELN, and TNC in BMSCs and SCX, MKX, COL1A2, DCN, and TNC in teno-iPSCs, while BMP12 further uptrended the expression of SCX and DCN in BMSCs and DCN in teno-iPSCs. Additionally, the aforementioned tenogenic inducers also affected the expression of signaling regulators SMAD7, ETV4, and SIRT1 in BMSCs and teno-iPSCs. Taken together, our data demonstrate that, in respect to the tenocyte-lineage-specific gene expression, BMSCs and teno-iPSCs respond differently to the tenogenic stimuli, which may affect the outcome of their application in tendon repair or regeneration.

Collagen and chondroitin sulfate functionalized bioinspired fibers for tendon tissue engineering application

Functional tendon tissue engineering depends on harnessing the biochemical and biophysical cues of the native tendon extracellular matrix. In this study, we fabricated highly-aligned poly(L-lactic acid) (PLLA) fibers with surfaces decorated by two of the crucial tendon ECM components, type 1 collagen (COL1) and chondroitin sulfate (CS), through a coaxial stable jet electrospinning approach. Effects of the biomimetic COL1-CS (shell)/PLLA (core) fibers on the tenogenic differentiation of human mesenchymal stem cells (hMSCs) in vitro were investigated. Higher rates of cell spreading and proliferation are observed on the aligned COL1-CS/PLLA fibers compared to that on the plain PLLA fibers. Expression of the tendon-associated genes scleraxis (SCX) and COL1 as well as protein tenomodulin (TNMD) are significantly increased. Introduction of mechanical stimulation gives rise to synergistic effect on tenogenic differentiation of hMSCs. Higher expression of TGF-β2, TGFβR-II, and Smad3 by the cells on the COL1-CS/PLLA fiber substrates are observed, which indicates that COL1-CS/PLLA ultrafine fibers dictate the hMSC tenogenic differentiation through activating the TGF-β signaling pathway. Animal study in rat Achilles tendon repair model corroborated the promoting role of COL1-CS/PLLA in regenerating a tendon-like tissue. Thus, our highly aligned biomimicking fibers may serve as an efficient scaffolding system for functional tendon regeneration.

The importance of plasmin for the healing of the anterior cruciate ligament.

AimsThe anterior cruciate ligament (ACL) is known to have a poor wound healing capacity, whereas other ligaments outside of the knee joint capsule such as the medial collateral ligament (MCL) apparently heal more easily. Plasmin has been identified as a major component in the synovial fluid that varies among patients. The aim of this study was to test whether plasmin, a component of synovial fluid, could be a main factor responsible for the poor wound healing capacity of the ACL.MethodsThe effects of increasing concentrations of plasmin (0, 0.1, 1, 10, and 50 µg/ml) onto the wound closing speed (WCS) of primary ACL-derived ligamentocytes (ACL-LCs) were tested using wound scratch assay and time-lapse phase-contrast microscopy. Additionally, relative expression changes (quantitative PCR (qPCR)) of major LC-relevant genes and catabolic genes were investigated. The positive controls were 10% fetal calf serum (FCS) and platelet-derived growth factor (PDGF).ResultsWCS did not differ significantly among no plasmin versus each of the tested concentrations (six donors). The positive controls with PDGF and with FCS differed significantly from the negative controls. However, we found a trend demonstrating that higher plasmin concentrations up-regulate the expression of matrix metalloproteinase 13 (MMP13), 3 (MMP3), and tenomodulin (TNMD).ConclusionThe clinical relevance of this study is the possibility that it is not solely the plasmin, but also additional factors in the synovial fluid of the knee, that may be responsible for the poor healing capacity of the ACL.Cite this article: Bone Joint Res 2020;9(9):543–553.