Citizen science meets eDNA: A new boom in research exploring urban wetland biodiversity.

•eDNA citizen science provides a comprehensive picture of the biodiversity.•Non-native species reduced the local fish diversity in urban wetlands.•Expanding water area can improve wetland biodiversity.

Pold4 is dispensable for mouse development, DNA replication and DNA repair.

The DNA polymerase delta (Pol δ), a heterotetramer of four subunits (Pol δ4), plays a pivotal role in DNA replication, as well as in DNA damage repair. Pold4, as the smallest subunit of Pol δ, is degraded in response to DNA damage or when entering into S-phase. This leads to the conversion of Pol δ4 to the trimeric complex Pol δ3. However, the contribution of Pold4 has not been fully elucidated in mammals. Cdm1, the Pold4 ortholog in Schizosaccharomyces pombe, is dispensable for cell growth and DNA damage repair, and there are no Pold4 orthologs in Saccharomyces cerevisiae. We previously generated a knockout mouse model of Pold3 and revealed its essential role in genome stability. Unexpectedly, we here found that Pold4 knockout mice are viable and fertile. In addition, Pold4 knockout mice do not exhibit any pathologic changes in the lung and spleen, tissues with the most abundant expression of Pold4. Moreover, Pold4 knockout mouse tail tip fibroblasts (TTF) exhibited normal cell growth, cell cycle, DNA replication, DNA damage and DNA repair capacity. These results suggested that Pol δ3 but not Pol δ4 may be responsible for these processes in normal cells. Interestingly, 19-month-old wild-type (WT) mice had tumors in the liver, while Pold4 knockout mice did not, and Pold4 knockout mice showed increased longevity. In further, this provided evidence suggested that Pold4 could be a potential novel target for lung carcinoma because its depletion does not affect normal cells but does affect cancer cells.

A bivalent vaccine containing D614G and BA.1 spike trimer proteins or a BA.1 spike trimer protein booster shows broad neutralizing immunity.

The newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant, sublineages BA.1 and BA.2, recently became the dominant variants of concern (VOCs) with significantly higher transmissibility than any other variant appeared and markedly greater resistance to neutralization antibodies and original ancestral WA1 spike-matched vaccine. Therefore, it is urgent to develop vaccines against VOCs like Omicron. Unlike the new booming messenger RNA (mRNA) vaccine, protein vaccines have been used for decades to protect people from various kinds of viral infections and have advantages with their inexpensive production protocols and their relative stability in comparison to the mRNA vaccine. Here, we show that sera from BA.1 spike protein vaccinated mice mainly elicited neutralizing antibodies against BA.1 itself. However, a booster with BA.1 spike protein or a bivalent vaccine composed of D614G and BA.1 spike protein-induced not only potent neutralizing antibody response against D614G and BA.1 pseudovirus, but also against BA.2, other four SARS-CoV-2 VOCs (Alpha, Beta, Gamma, and Delta) and SARS-CoV-2-related coronaviruses (pangolin CoV GD-1 and bat CoV RsSHC014). The two recombinant spike protein vaccines method described here lay a foundation for future vaccine development for broad protection against pan-sarbecovirus.

Monitoring Rotator Cuff Muscle Fatty Infiltration Progression by Magnetic Resonance Imaging T1 Mapping: Correlation With Direct Evaluation Findings in Rats.

BACKGROUND: Monitoring the fatty infiltration (FI) process in rotator cuff muscles is of value in establishing a treatment plan and predicting the postoperative prognosis. Quantitative T1 mapping shows promise for evaluating muscle degeneration, while its validity in monitoring rotator cuff muscle FI progression needs further investigation. PURPOSE: To determine the validity of T1 mapping in monitoring FI progression of rotator cuff muscles. STUDY DESIGN: Controlled laboratory study. METHODS: Sprague-Dawley rats (N = 108) underwent left supraspinatus (SS) and infraspinatus (IS) tenotomy only (TT), suprascapular nerve transection only (NT), or SS and IS tenotomy plus suprascapular nerve transection (TT+NT). Sham surgery on the right shoulder served as the control. The magnetic resonance imaging examination included T1 mapping performed at 12, 16, and 20 weeks postoperation. SS and IS muscles were harvested to quantitatively evaluate FI via direct evaluation (triglyceride quantification assay and histological analysis) at the same predetermined intervals. The correlation of the imaging data with direct evaluation of rotator cuff muscles was analyzed. RESULTS: T1 values were significantly lower in left SS and IS muscles at 12, 16, and 20 weeks postoperation as compared with those on the right side. T1 values of the left SS and IS muscles were continuously decreased in all groups. The TT+NT group had a greater decrease in T1 value than did the TT and NT groups. Triglyceride quantification assay and histological analysis demonstrated significant and progressive FI of the left SS and IS muscles in the 3 groups. The most serious FI changes were observed in the TT+NT group. T1 values were also well correlated with triglyceride contents and area fractions of fat. CONCLUSION: T1 mapping can be an effective imaging modality for sensitive and quantitative monitoring of FI progression in rotator cuff muscles. CLINICAL RELEVANCE: The findings of this study provide a tool for researchers to noninvasively and quantitatively monitor the process of muscle degeneration, contributing to the evaluation of surgical indication and postoperative prognosis.

Patient-specific hemodynamic analysis of IVCS-induced DVT.

The aim of this study is to perform patient-specific hemodynamic simulations of patients with iliac vein compression syndrome (IVCS) and evaluate the deep venous thrombosis (DVT) potential, with clinical observations as reference. 15 patient-specific IVCS models were reconstructed from computed tomography venography (CTV) data, and divided into three groups, i.e. two groups with thrombosis: Group A (complete obstruction) and Group B (incomplete obstruction), and a third group without DVT, Group C. Hemodynamic simulations were conducted with patient-specific inlet flow rates. The blood residue was predicted using the blood stasis model. Time histories of old blood volume fraction (OBVF) was obtained, in addition to conventional hemodynamic parameters such as wall shear stress (WSS). The mean area-averaged WSS of the stenosis region for Group A and Group B were 3.68 Pa and 1.78 Pa, respectively. For the telecentric end region, the WSS were 0.76 Pa and 0.58 Pa, respectively. For Group C, the WSS at these two regions were 4.61 Pa and 1.57 Pa, respectively. The OBVF was 74.0% at the stenosis region and 76.2% at the telecentric end region for Group A, much higher than 4.8% and 43.1% of Group B. For Group C, the OBVF at the two regions were close to 0. This corresponded well with clinical observations. The potential of DVT can be predicted through patient-specific hemodynamic simulations in combination of blood stasis model. The findings of this study are of great significance for the preoperative evaluation and treatment prognosis of IVCS patients with DVT.

FGF14-AS2 accelerates tumorigenesis in glioma by forming a feedback loop with miR-320a/E2F1 axis.

Glioma is the most common primary tumour in the central nervous system in adults, and at present, there is no effective treatment to cure this malignancy. Long noncoding RNAs (lncRNAs) are closely related to tumour progression and have attracted increasing attention in tumour research. However, the role of lncRNA FGF14-AS2 in glioma tumorigenesis has not been determined. In the present study, we found that FGF14-AS2 expression was significantly elevated in glioma tissues and was associated with poor survival in glioma patients. Silencing FGF14-AS2 inhibited the proliferation, migration and invasion ability of glioma cells. In vivo assay showed that silencing FGF14-AS2 led to inhibition of tumour growth. In addition, FGF14-AS2 was observed to promote glioma progression via the miR-320a/E2F1 axis. Moreover, E2F1 could bind to the promoter region of FGF14-AS2, thereby enhancing FGF14-AS2 expression. In conclusion, FGF14-AS2 could accelerate tumorigenesis of glioma by forming a feedback loop with the miR-320a/E2F1 axis which suggested that FGF14-AS2 could serve as a therapeutic target for glioma.

Impact of Platelet Glycoprotein Ia/IIa C807T Gene Polymorphisms on Coronary Artery Aneurysms of KD Patients.

BACKGROUND: Kawasaki disease (KD) is a systemic vasculitis of unknown etiology in children. Coronary artery abnormalities are the most common complications of KD. Recent evidence showed that genetic polymorphisms may lead to susceptibility to KD. Genetic variants in platelet glycoprotein have been reported to be associated with coronary artery disease. The aim of the present study is to investigate the correlation between the role of platelet glycoprotein and coronary artery aneurysms in KD patients. METHODS: We did a case-control study that enrolled 818 KD patients and 1401 healthy children with the same age and sex from January 2013 to December 2016. Analysis of single-nucleotide polymorphism (rs1126643) of the platelet glycoprotein Ia/IIa C807T was performed by multiplex polymerase chain reactions in this study. RESULTS: A significant difference in the genotype distribution between KD cases and controls was observed for the glycoprotein Ia/IIa C807T (rs1126643) polymorphism (p=0.026). Compared with the healthy children, the rs1126643T allele carriers had odds ratio (OR) of 0.63 for developing KD (TT vs. CC: adjusted OR = 0.62, 95% confidence interval (CI) = 0.43-0.88,p=0.0078; TT vs. CT/CC: adjusted OR = 0.63, 95% CI = 0.44-0.889,p=0.0093). Furthermore, we also found that children less than 60 months of age and female patients with rs1126643 T allele carriers had an adjusted OR of 0.66 (95% CI = 0.46-0.95) for noncoronary artery aneurysm patients (p=0.0242). Single-nucleotide polymorphism rs1126643 TT seems to represent a protective factor against KD in coronary artery aneurysm formation in multivariate analysis. CONCLUSIONS: The platelet glycoprotein Ia/IIa T allele carriers may have a protective effect on the risk of coronary artery aneurysms of KD patients, especially in females and children aged less than 60 months. These results may provide evidence for platelet glycoprotein Ia/IIa gene polymorphisms in the pathogenesis of KD patients.

Conditioned medium of human bone marrow-derived stem cells promotes tendon-bone healing of the rotator cuff in a rat model.

Rotator cuff repair is a common surgery in sports medicine. During the surgery, torn tendon was re-fixed onto the bony surface. The majority of patients gain good results. However, re-tear occurs in some patients. The reason under this phenomenon is that the normal tendon-bone enthesis cannot be reconstructed. In order to strengthen the tendon-bone healing and promote enthesis regeneration, numerous manners are tested, among which stem cell related therapies are preferred. Stem cells, due to the ability of multi-lineage differentiation, are widely used in regenerative medicine. However, safety and ethics concerns limit its clinical use. Recent studies found that it is the secretome of stem cells that is biologically effective. On ground of this, we, in the current study, collected the conditioned medium of human bone marrow-derived stem cells (hBMSC-CM) and tested whether this acellular method could promote tendon-bone healing in a rat model of rotator cuff repair. By using histological, radiological, and biomechanical methods, we found that hBMSC-CM promoted tendon-bone healing of the rat rotator cuff. Then, we noticed that hBMSC-CM exerted an impact on macrophage polarization both in vivo and in vitro by inhibiting M1 phenotype and promoting M2 phenotype. Further, we proved that the benefit of hBMSC-CM on tendon-bone healing was related to its regulation on macrophage. Finally, we proved that, hBMSC-CM influenced macrophage polarization, which was, at least partially, related to Smad2/3 signaling pathway. Based on the experiments above, we confirmed the benefit of hBMSC-CM on tendon-bone healing, which relied on its immune-regulative property. Considering the accessibility and safety of acellular hBMSC-CM, we believe it is a promising candidate clinically for tendon-bone healing.

Integrin α2 gene polymorphism is a risk factor of coronary artery lesions in Chinese children with Kawasaki disease.

BACKGROUND: Kawasaki disease (KD) is a systemic vasculitis, and the formation of coronary artery lesions(CAL) is its most common sequela. Both genetic and environmental factors are considered to be important factors of in KD. Integrin α2 (ITGA2) is a transmembrane receptor that is associated with susceptibility to several diseases, but its relevance to KD with CAL is unclear. METHODS: We genotyped ITGA2 rs1126643 in 785 KD patients with the CAL and no-CAL(NCAL) (300 patients with CAL, and 485 age- and sex-matched patients with NCAL). OR (95% CI) and adjusted OR (95% CI) were used to evaluate the intensity of the association. RESULTS: We found a significantly increased risk of KD with CAL associated with ITGA2 rs1126643 genotypes (CT vs CC: adjusted OR = 1.57, 95% CI = 1.16-2.12, P = 0.0032; CT/TT vs CC: adjusted OR = 1.49, 95% CI = 1.12-2.00, P = 0.0068; T vs C: adjusted OR = 1.66, 95% CI = 1.16-2.51, P = 0.0165). Moreover, we found that carriers of the CT/TT genotype had a significant risk of KD with coronary artery lesion susceptibility for children ≤60 months of age, and the CT/TT genotype was significantly associated with an increased risk of SCAL formation and MCAL formation when compared with the CC genotype. CONCLUSION: ITGA2 rs1126643 was associated with increased susceptibility and severity of CAL in KD.

The rs1051931 G>A Polymorphism in the PLA2G7 Gene Confers Resistance to Immunoglobulin Therapy in Kawasaki Disease in a Southern Chinese Population.

Background: Kawasaki disease (KD) is a common cardiovascular disease in infants and young children, with fever, rash, and conjunctivitis as the main clinical manifestations, which can lead to the occurrence of coronary aneurysms. Intravenous immunoglobulin (IVIG) is the preferred treatment for KD patients, but 10-20% of patients are resistant to IVIG. Lipoprotein-associated phospholipase A 2 (Lp-PLA2) is a potential therapeutic target for coronary atherosclerotic heart disease, and the polymorphism of Phospholipase A2 Group VII (PLA2G7) is closely related to the activity of Lp-PLA2, of which rs1051931 is the strongest. Therefore, the rs1051931 polymorphism may be a predictor of IVIG resistance in KD patients. Methods: A total of 760 KD cases, including 148 IVIG-resistant patients and 612 IVIG-responsive patients, were genotyped for rs1051931 in PLA2G7, we compared the effects of rs1051931 on IVIG treatment in KD patients by odds ratios (OR) and 95% confidence interval (CI). Results: The homozygous mutation AA may be a protective factor for IVIG resistance in KD patients (adjusted OR = 3.47, 95% CI = 1.14-10.57, P = 0.0284) and is more evident in patients with KD aged <60 months (adjusted OR = 3.68, 95% CI = 1.10-12.28, P = 0.0399). Conclusions: The PLA2G7 rs1051931 G>A polymorphism may be suitable as a biomarker for the diagnosis or prognosis of IVIG resistance in KD in a southern Chinese population.

Levels of Soluble CD30 and CD26 and Their Clinical Significance in Patients with Primary Immune Thrombocytopenia.

BACKGROUND: sCD30 and sCD26 are correlated with autoimmune diseases. However, little research has been done on the relationship between them and primary immune thrombocytopenia (ITP). METHODS: This study enrolled 47 patients diagnosed with ITP in the Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences (Tianjin, China), from January 2015 to August 2015. The peripheral blood of all subjects was collected. The mRNA expression of CD30 was quantified by RT-PCR, and concentrations of sCD30 and sCD26 were measured by ELISA. Patient characteristics, CD30 mRNA levels, and sCD30 and sCD26 concentrations were analyzed. RESULTS: The concentration of sCD30 was higher in active ITP patients (median, 35.82 ng/mL) than in remission ITP patients (median, 23.12 ng/mL; P = 0.021) and healthy controls (median, 25.11 ng/mL; P = 0.002). Plasma sCD26 levels decreased in remission ITP patients compared with that in healthy controls (median, 599.4 ng/mL vs. 964.23 ng/mL; P = 0.004). Ratios of sCD26/sCD30 in active ITP patients decreased compared with those in controls (P = 0.005). Increased sCD30 was positively correlated with hemorrhage (r = 0.493, P = 0.017) in ITP patients while little relationship was identified between sCD26 and ITP. CONCLUSION: Since sCD30 levels and sCD26/sCD30 ratios may contribute to the activity of the disease, they may be used to assess ITP disease activity.

Association between P2RY12 Gene Polymorphisms and IVIG Resistance in Kawasaki Patients.

Children with Kawasaki disease (KD) resistant to intravenous immunoglobulin (IVIG) have a higher incidence of coronary artery lesions (CAL). Despite the association between Purinergic receptor P2Y12 (P2RY12) polymorphism, KD genetic susceptibility, and CAL complications being proved, few studies have assessed the relationship between P2RY12 polymorphisms and IVIG resistance in patients with KD. We recruited 148 KD patients with IVIG resistance and 611 with IVIG sensitivity and selected five P2RY12 polymorphisms: rs9859538, rs1491974, rs7637803, rs6809699, and rs2046934. A significant difference in the genotype distributions between patients was only observed for the rs6809699 A > C polymorphism (AC vs. AA: adjusted odds ratio (OR) = 0.48, 95% confidence interval (CI) = 0.27-0.84, P=0.011; AC/CC vs. AA: adjusted OR = 0.47, 95% CI = 0.27-0.83, P=0.0084). After adjusting for age and gender, the carriers of the rs6809699 C allele had OR of 0.44 to 0.49 for IVIG sensitivity (AC vs. AA: adjusted OR = 0.48, 95% confidence interval (CI) = 0.27-0.84, P=0.011; AC/CC vs. AA: adjusted OR = 0.47, 95% CI = 0.27-0.83, P=0.0084) compared to the carriers of a rs6809699 AA genotype, suggesting the protective effect of this SNP against IVIG resistance. Moreover, individuals with all five protective polymorphisms experienced a significantly decreased IVIG resistance compared to that of individuals with up to three protective polymorphisms (adjusted OR = 0.27, 95% CI = 0.13-0.57, P=0.0006). Our results suggest that the P2RY12 rs6809699 polymorphism could be used as a biomarker to predict IVIG resistance in KD patients.

Association between the miRNA-149 rs2292832 T>C polymorphism and Kawasaki disease susceptibility in a southern Chinese population.

BACKGROUND: Kawasaki disease (KD), which is characterized by vasculitis, is prone to occur in patients under 5 years of age, has an ambiguous etiology, and displays coronary artery lesions as the chief complication. Previous studies have linked miRNA-149 to cancers, and rs2292832 T>C is related to allergic diseases and inflammatory bowel disease, which both show immune system disorders and coronary artery disease. Therefore, we performed a study concentrating on the association between the miRNA-149 rs2292832 T>C polymorphism and KD susceptibility. METHODS: The subjects enrolled were 532 children with KD and 623 controls. We used TaqMan real-time PCR to obtain the genotypes of the rs2292832 T>C polymorphism. RESULTS: Ultimately, no significant association was found between the miRNA-149 rs2292832 T>C polymorphism and KD susceptibility, even in stratification analysis. CONCLUSION: Our results indicated that in southern Chinese patients, the miRNA-149 rs2292832 T>C polymorphism did not affect KD susceptibility, which needs to be further confirmed.

BMP9 promotes osteogenic differentiation of SMSCs by activating the JNK/Smad2/3 signaling pathway.

Synovial mesenchymal stem cells (SMSCs) with high proliferation and multi differentiation ability, and low immunogenicity have attracted research attention for their potential application in tissue engineering. Once their ability of osteogenesis is strengthened, it will be of practical value to apply the SMSCs in the field of bone regeneration. The current study aimed to investigate the osteogenic characteristics of SMSCs induced by bone morphogenetic protein 9 (BMP9) both in vitro and in vivo and to elucidate the mechanism underlying these characteristics. Specifically, different BMPs were assessed to determine the protein that would be the most favorable for stimulating osteogenic differentiation of SMSCs following their separation. The BMP9-enhanced osteogenesis of SMSCs was fully investigated in vitro and in vivo, and the c-Jun N-terminal kinase (JNK)/Smad2/3 signaling pathway stimulated by BMP9 was further explored. Our data suggested that BMP9 could significantly promote gene and protein expression of runt-related transcription factor 2, alkaline phosphatase, osteopontin, and osteocalcin, and SP600125, a JNK-specific inhibitor, could effectively decrease this tendency. Similar results were also confirmed in rats with cranial defects. In conclusion, our study indicated that BMP9 promotes bone formation both in vitro and in vivo possibly by activating the JNK/Smad2/3 signaling pathway.

Effects of hypoxia­inducible factor­1α on the proliferation and apoptosis of human synovial mesenchymal stem cells.

Hypoxia is a constant feature of the synovial microenvironment. How synovial mesenchymal stem cells (SMSCs) proliferate and differentiate in a hypoxic environment over a long period of time has aroused the interest of researchers. The aim of the present study was to explore the effects of hypoxia­inducible factor­1α (HIF­1α) on the proliferation and apoptosis of human SMSCs. SMSCs were harvested and cultured under different concentration of oxygen, normoxia (21% O2), hypoxia (5% O2) and severe hypoxia (0.5% O2) to determine its effect on the expression of HIF­1α. Then, the cells were collected and cell proliferation and apoptosis were detected at severe hypoxia (0.5% O2) and hypoxia (5% O2) conditions following HIF­1α siRNA transfection. There were no significant changes in cellular proliferation or apoptosis when cultured in normoxia (21% O2), hypoxia (5% O2) or severe hypoxia (0.5% O2). However, the mRNA and protein expression of HIF­1α were markedly upregulated in the hypoxic conditions. Further experiments suggested that the proliferation of SMSCs was obviously suppressed and apoptosis was markedly increased under severe hypoxic (0.5%) and hypoxic (5% O2) conditions following HIF­1α siRNA transfection. In conclusion, HIF­1α effectively improved the tolerance of SMSCs to hypoxia, which may promote cellular proliferation and prevent the apoptosis of SMSCs under hypoxic conditions.

Co-culture with synovial tissue in patients with rheumatoid arthritis suppress cell proliferation by regulating MAPK pathway in osteoblasts.

There is growing evidence that synovial tissue affects osteoblasts although the mechanisms behind the aberrant bone metabolism in rheumatoid arthritis (RA) are unclear. The aim of this study is to preliminarily establish a co-culture system of rheumatoid arthritis-derived synovial tissue (RAS) and osteoblasts in vitro and to investigate the potential mechanism of RAS on osteoblasts. A consistent volume of approximately 85 mm3 of RAS was cultured isolated and co-cultured with Hfob1.19 cells for up to 21 days. Equal volume of normal synovial tissue (NS) was co-cultured as a control group. Cell proliferation, cell cycle and bone markers were valued and the mechanisms underlying MAPK pathway have been fully delineated. Our findings suggested that co-cultures with RAS exhibited decreased proliferation of Hfob1.19 cells. Moreover, gene and protein expressions of GLUT3 in cells were suppressed, and the cell cycle was also down-regulated. The expressions of related proteins of MAPKs (JNK and p38) signaling pathway were found to be inhibited. Rescue experiments demonstrated that co-cultures with RAS could decrease the growth and cell cycle of Hfob1.19 cells, which were reversed by p-JNK and p-p38 over expression. In conclusion, this study suggested that synovial tissue in patients with RA may negatively regulate osteoblasts proliferation by declining MAPK pathway.

Biosynthesis of Silver nanoparticles using Bauhinia acuminate flower extract and their effect to promote osteogenesis of MSCs and improve meniscus injury healing.

The latent utilization of biomaterials that are osteo-conducive in the advancement of healing bone fracture has fascinated extensive consideration. This work includes the synthesis of silver nanoparticles (AgNPs) with the help of a Bauhinia acuminate plant flower extract through an ecofriendly synthetic process without any use of harmful reductants. In the fabrication of AgNPs, Bauhinia acuminate plant flower extract bio constituents acts as both stabilizing and reducing agent. The studies of Fourier transform infrared (FTIR) and X-ray diffraction (XRD) techniques confirmed the formation of AgNPS. TEM images revealed that AgNPs are uniform with average particle size of 17 nm. Further, this work explored if silver nanoparticles (AgNPs) might endorse the osteogenesis and proliferation of mesenchymal stem cells (MSCs) and advance the curing of bone fractures. We also exhibited that the prepared AgNPs could promote the in -vitro osteogenic differentiation and proliferation of MSCs'. Also, the prepared AgNps could stimulate the proliferation of mMSCs at specific concentrations of 6-20 µM. Further, cell viability studies showed that AgNPs exhibited no reduction in mouse mesenchymal stem cell viability at <4 µM. Further, these results indicated the induction effects of AgNPs on osteogenic differentiation and proliferation on MSCs, as well as the advancement of meniscus injury healing.

Stem Cell-Conditioned Medium Promotes Graft Remodeling of Midsubstance and Intratunnel Incorporation After Anterior Cruciate Ligament Reconstruction in a Rat Model.

BACKGROUND: Stem cell-conditioned medium (CM) has been increasingly used in regenerative medicine. However, its effect on graft-host integration after anterior cruciate ligament (ACL) reconstruction (ACLR) remains unclear. PURPOSE: To examine the effect of human bone marrow stem cell (hBMSC)-CM on graft-bone integration and graft midsubstance ligamentization in a rat model of ACLR. STUDY DESIGN: Controlled laboratory study. METHODS: CM was obtained from the supernatant of commercially available hBMSCs in serum-free Dulbecco's modified Eagle medium (DMEM). In a rat model of an ACL injury, isometric ACLR was performed. Three groups were established: CM injection group (CM; n = 40), control injection group (CI; n = 40) with serum-free DMEM injections, and no injection group (NI; n = 40). An intra-articular injection was performed weekly. Micro-computed tomography was conducted at 2, 4, and 8 weeks postoperatively. Histological and biomechanical analyses were conducted at 4 and 8 weeks postoperatively. The NIH3T3 fibroblast was utilized as a model in vitro to examine the effect of CM using the cell counting kit-8 (CCK-8) assay and immunofluorescence staining of Ki-67, α-smooth muscle actin (α-SMA), and collagen 1 (Col 1). RESULTS: At 4 and 8 weeks, the femoral and tibial bone tunnel areas as well as the interface between the graft and host bone were smaller, while the bone volume/total volume ratio was higher, in the CM group. Sharpey-like fibers formed at 8 weeks in the CM group. At 4 and 8 weeks, more Col 1 was noticed in the CM group than in the NI group (both P < .001) or CI group (both P < .001). Immunohistochemically, the α-SMA-positive area was up-regulated at the graft-bone interface at 4 weeks (P < .001) and declined at 8 weeks (P < .001) in the CM group compared with the other 2 groups. At the midsubstance, α-SMA expression decreased from 4 to 8 weeks in all groups and was significantly lower in the CM group than in the NI group (P < .01) or CI group (P < .05) at 8 weeks. The CCK-8 assay showed that CM increased NIH3T3 viability (P < .001) and the level of Ki-67 (P < .05), α-SMA (P < .001), and Col 1 (P < .001) in CM-educated NIH3T3 cells. CONCLUSION: hBMSC-CM accelerates graft-bone incorporation and midsubstance ligamentization and enhances the proliferation, differentiation, and collagen synthesis of fibroblasts. CLINICAL RELEVANCE: Graft-host integration is essential after ACLR. The current study identified a novel agent, that is, hBMSC-CM, as a candidate for promoting integration.

Conditioned medium of mesenchymal stem cells delays osteoarthritis progression in a rat model by protecting subchondral bone, maintaining matrix homeostasis, and enhancing autophagy.

Evidence accumulated that mesenchymal stem cell (MSC) therapy ameliorated osteoarthritis (OA) via paracrine effect, whereas conditioned medium (CM) of MSCs contains all the secretomes. In vitro studies have proved its therapeutic effect in OA, but few in vivo evidences were unveiled. This study investigated the effect of MSCs-CM in an animal model of OA. OA was induced by anterior cruciate ligament transaction and destabilization of the medial meniscus in 12 rats bilaterally. The CM group (N = 6) was administered with intraarticular injection of MSCs-CM weekly, whereas the phosphate-buffered saline (PBS) group (N = 6) was injected with PBS. Six rats served as normal control and received sham operation with weekly PBS injection. Rats were sacrificed 8 weeks postoperatively. Gross and histological morphology were analysed. Microcomputed tomography was applied to assess the subchondral bone. Components of extracellular matrix (ECM) including type II collagen (Col II) and aggrecan, and ECM homeostasis-related enzymes (metalloproteinase-13 [MMP-13] and tissue inhibitor of metalloproteinase-1 [TIMP-1]), as well as autophagy markers (Beclin-1 and microtubule-associated protein light chain 3) were evaluated immunohistochemically. Chondrocyte apoptosis was measured by terminal deoxynucleotidyl transferase dUTP nick-end labelling staining. Gene expression of Col II, aggrecan, MMP-13, and TIMP-1 was confirmed by real-time polymerase chain reaction. Morphological outcomes demonstrated remarkable articular-protective effect of MSCs-CM. Well-maintained subchondral bone structure, significantly more abundant cartilage matrix, notably decreased ratio of MMP-13 to TIMP-1, and inhibited chondrocyte apoptosis with enhanced autophagy were observed in the CM group compared with the PBS group. In conclusion, MSCs-CM demonstrated satisfactory effect in alleviating OA in rats via protecting the microarchitecture of subchondral bone, balancing the ratio of MMP-13 to TIMP-1 in cartilage, and enhancing autophagy, which might provide a new remedy against OA.

FAK mediates BMP9-induced osteogenic differentiation via Wnt and MAPK signaling pathway in synovial mesenchymal stem cells.

Objective: Focal adhesion kinase (FAK) has critical functions in proliferation and differentiation of many cell types, however, the role of FAK on BMP9-induced osteogenic differentiation in SMSCs has not been characted. The purpose of current study is to explore the mechanism of FAK on the BMP9-induced osteogenesis of SMSCs in vitro and in vivo. Methods: The optimal dose of BMP9 was determined by incubation in different BMP9 concentrations, then cells were transfected with siRNA-induced FAK knockdown in BMP9-induced osteogenesis. Cell proliferation, migration, the osteogenic capacity, and the underlying mechanism were further detected in vitro. Imaging and pathological examination were conducted to observe the bone formation in vivo. Results: Our findings suggested that BMP9 could obviously promote FAK phosphorylation in osteogenic conditions. In contrast, FAK knockdown significantly decreased the cell proliferation, migration, the osteogenic capacity of SMSCs. To be specific, FAK knockdown could markedly inhibit the Wnt and MAPK signal pathway of SMSCs induced by BMP9. Besides, FAK knockdown could also effectively inhibit BMP-9-induced bone formation in vivo. Conclusion: FAK plays a pivotal role in promoting BMP9-induced osteogenesis of SMSCs, which is probably via activating Wnt and MAPK pathway.