Phosphorylation of USP27X by GSK3ß maintains the stability and oncogenic functions of CBX2.

Chromobox protein homolog 2 (CBX2) exerts a multifaceted impact on the progression of aggressive cancers. The proteasome-dependent pathway is crucial for modulating CBX2 regulation, while the specific regulatory roles and mechanisms of deubiquitinating enzymes targeting CBX2 remain poorly understood. Mass spectrometry analysis identified ubiquitin-specific peptidase 27X (USP27X) as a deubiquitinating enzyme that targets CBX2. Overexpression of USP27X significantly enhances CBX2 levels by promoting deubiquitination, while deficiency of USP27X leads to CBX2 degradation, thereby inhibiting tumorigenesis. Furthermore, it has been revealed that glycogen synthase kinase 3 beta (GSK3ß) can directly bind to and phosphorylate USP27X, thereby enhancing the interaction between USP27X and CBX2 and leading to further stabilization of the CBX2 protein. Clinically, the co-expression of high levels of USP27X and CBX2 in breast cancer tissues is indicative of a poor prognosis for patients with this disease. These findings collectively underscore the critical regulatory role played by USP27X in modulating CBX2, thereby establishing the GSK3ß-USP27X-CBX2 axis as a pivotal driver of malignant progression in breast cancer.

Association of Combined Exposure to Ambient Air Pollutants, Genetic Risk, and Incident Rheumatoid Arthritis: A Prospective Cohort Study in the UK Biobank.

BACKGROUND: Evidence for a potential link between air pollution and rheumatoid arthritis (RA) is inconsistent, and the modified effect of genetic susceptibility on the relationship between air pollution and RA has not been well studied. OBJECTIVE: Using a general population cohort from the UK Biobank, this study aimed to investigate the associations between various air pollutants and the risk of incident RA and to further estimate the impact of combined exposure to ambient air pollutants on the risk of developing RA under the modification effect of genetic predisposition. METHODS: A total of 342,973 participants with completed genotyping data and who were free of RA at baseline were included in the study. An air pollution score was constructed by summing the concentrations of each pollutant weighted by the regression coefficients with RA from single-pollutant models to assess the combined effect of air pollutants, including particulate matter (PM) with diameters ≤2.5µm (PM2.5), between 2.5 and 10µm (PM2.5-10), and ≤10µm (PM10), as well as nitrogen dioxide (NO2) and nitrogen oxides (NOx). In addition, the polygenic risk score (PRS) of RA was calculated to characterize individual genetic risk. The Cox proportional hazard model was used to estimate hazard ratios (HRs) and 95% confidence intervals (95% CIs) of associations of single air pollutant, air pollution score, or PRS with incident RA. RESULTS: During a median follow-up time of 8.1 y, 2,034 incident events of RA were recorded. The HRs (95% CIs) of incident RA per interquartile range increment in PM2.5, PM2.5-10, PM10, NO2, and NOx were 1.07 (1.01, 1.13), 1.00 (0.96, 1.04), 1.01 (0.96, 1.07), 1.03 (0.98, 1.09), and 1.07 (1.02, 1.12), respectively. We also found a positive exposure-response relationship between air pollution score and RA risk (pTrend=0.000053). The HR (95% CI) of incident RA was 1.14 (1.00, 1.29) in the highest quartile group compared with the lowest quartile group of the air pollution score. Furthermore, the results of the combined effect of air pollution score and PRS on the RA risk showed that the risk of RA incidence in the highest genetic risk and air pollution score group was almost twice that of the lowest genetic risk and air pollution score group [incidence rate (IR) per 100,000 person-years: 98.46 vs. 51.19, and HR= 1.73 (95% CI: 1.39, 2.17) vs. 1 (reference)], although no statistically significant interaction between the air pollution and genetic risk for incident RA was found (pInteraction>0.05). DISCUSSION: The results revealed that long-term combined exposure to ambient air pollutants might increase the risk of RA, particularly in those with high genetic risk. https://doi.org/10.1289/EHP10710.

Association between Meteorological Factors and Outpatient Visits for Herpes Zoster in Hefei, China: A Time-Series Analysis.

This study sought to investigate the relationship between meteorological factors and outpatient visits for herpes zoster. In this time-series analysis, we used data from two major hospitals in Hefei, collected between 2015 and 2019, to evaluate the impact of meteorological factors on the risk of herpes zoster. After controlling for confounders, we adopted a distributed lag nonlinear model to probe the relationship between meteorological factors and outpatient visits for herpes zoster. The analysis was stratified according to age (<40 years, ≥40 years) and sex (male, female). A total of 43,547 cases of herpes zoster were reported, and compared with the median value, a high temperature and high relative humidity had a significant risk effect on the incidence of herpes zoster. The maximum harmful effect of high temperature on herpes zoster occurred on the lag0 (RR: 1.027, 95% CI: 1.002-1.053) and further declined over the following days. The cumulative effect increased with the extension of lag days, and the cumulative RR was the largest on the sixth day of lag (RR1.031, 95% CI: 1.006-1.056) when the relative humidity was 85.7% (77.0% as the reference). The stratified analysis results reveal that females and the elderly (≥40 years) were more susceptible to temperature and relative humidity. This study shows that high-temperatures may lead to herpes zoster, indicating that those infected with varicella zoster virus need to take measures over the course of several days when not exposed to the best appropriate temperature conditions.

Short-term effect of ambient temperature and ambient temperature changes on the risk of warts outpatient visits in Hefei, China: a retrospective time-series study.

Concerns are growing about the adverse health effects of ambient temperature and ambient temperature changes. However, the association between ambient temperature and ambient temperature changes on the risk of warts outpatient visits is poorly understood. Our study used the distributed lag non-linear model (DLNM) aimed to evaluate the association between ambient temperature, ambient temperature changes (including temperature change between neighboring days (TCN) and diurnal temperature range (DTR)), and warts outpatient visits. We also performed subgroup analyses in order to find susceptible populations by gender and age groups. The maximum relative risk (RR) of low ambient temperature (0 °C) for warts outpatient visits was 1.117 (95% CI: 1.041-1.198, lag 04 days), and the maximum RR of high ambient temperature (32 °C) for warts outpatient visits was 1.318 (95% CI: 1.083-1.605, lag 07 days). The large temperature drop (TCN = - 3 °C) decreased the risk of warts visits, with the lowest RR value at the cumulative exposure of lag 7 days (RR = 0.888, 95% CI: 0.822-0.959), and the large temperature rise (TCN = 2 °C) increased the risk of warts visits, with the highest RR value at the cumulative exposure of lag 7 days (RR = 1.080, 95% CI: 1.022-1.142). Overall, both low and high ambient temperatures and large temperature rise can increase the risk of warts visits, while large temperature drop is a protective factor for warts visits. However, we did not find any association between DTR and warts visits. Furthermore, subgroup analyses showed that males and the young (0-17 years old) were more sensitive to low and high ambient temperatures, and the elderly (≥ 65 years old) were more susceptible to TCN. The results may provide valuable evidence for reducing the disease burden of warts in the future.

Implementation of Fault-Tolerant Encoding Circuit Based on Stabilizer Implementation and "Flag" Bits in Steane Code.

Quantum error correction (QEC) is an effective way to overcome quantum noise and de-coherence, meanwhile the fault tolerance of the encoding circuit, syndrome measurement circuit, and logical gate realization circuit must be ensured so as to achieve reliable quantum computing. Steane code is one of the most famous codes, proposed in 1996, however, the classical encoding circuit based on stabilizer implementation is not fault-tolerant. In this paper, we propose a method to design a fault-tolerant encoding circuit for Calderbank-Shor-Steane (CSS) code based on stabilizer implementation and "flag" bits. We use the Steane code as an example to depict in detail the fault-tolerant encoding circuit design process including the logical operation implementation, the stabilizer implementation, and the "flag" qubits design. The simulation results show that assuming only one quantum gate will be wrong with a certain probability p, the classical encoding circuit will have logic errors proportional to p; our proposed circuit is fault-tolerant as with the help of the "flag" bits, all types of errors in the encoding process can be accurately and uniquely determined, the errors can be fixed. If all the gates will be wrong with a certain probability p, which is the actual situation, the proposed encoding circuit will also be wrong with a certain probability, but its error rate has been reduced greatly from p to p2 compared with the original circuit. This encoding circuit design process can be extended to other CSS codes to improve the correctness of the encoding circuit.

Short-term association of NO2 with hospital visits for chronic kidney disease and effect modification by temperature in Hefei, China: A time series study.

BACKGROUND: A large body of evidence has linked air pollution and temperature with chronic kidney disease (CKD) prevalence and hospitalizations. However, most studies have focused on the influence of heat stress on CKD prevalence, and the potential effect modification of temperature on the association between air pollution and CKD has not been well-investigated. In this study, we examined the associations of the whole temperature spectrum and air pollution with CKD-related hospital visits and explored whether temperature modifies the short-term association of air pollution with CKD-related hospital visits. METHODS AND FINDINGS: We collected 40 276 CKD-related hospital visits from the first Affiliated Hospital of Anhui Medical University and Anhui Provincial Hospital in Hefei, China, during 2015-2019. A two-stage time-series design was conducted to investigate the associations of air pollution and daily mean temperature with CKD-related hospital visits. First, we estimated the associations between air pollution and CKD-related hospital visits as well as temperature and CKD-related hospital visits. Second, we analyzed the associations of air pollution with CKD hospital visits at different temperatures. We found that NO2 exposure and low temperature were associated with an increased risk of CKD-related hospital visits. Low temperature enhanced the association between NO2 exposure and CKD-related hospital visits, with an increase of 4.30% (95% CI: 2.47-5.92%) per 10 µg/m3 increment in NO2 at low temperature. Effect modification of the association between NO2 and the risk of CKD-related hospital visits was stronger at low temperature across the whole population. CONCLUSIONS: Our findings indicate that low temperature-related chronic kidney damage should be of immediate public health concern. Impact of NO2 exposure on the risk of CKD-related hospital visits may increase under the low temperature, which suggests the need for NO2 exposure mitigation strategies in the context of climate change and an enhanced understanding of the mechanisms underlying the temperature variance of air pollution effect to help reduce the magnitude of the CKD burden on the healthcare systems.

Influence of chitosan oligosaccharide on the gelling and wound healing properties of injectable hydrogels based on carboxymethyl chitosan/alginate polyelectrolyte complexes.

In situ injectable hydrogels for wound healing based on carboxymethyl chitosan (CMCS) and alginate were developed in this work. The liquid mixture of CMCS and alginate solutions formed a gel by polyelectrolyte complexation after addition of d-glucono-δ-lactone (GDL), which slowly hydrolyzed and donated protons. When chitosan oligosaccharide (COS) was added into the mixture, a two-stage gelling process occurred. The primary gelling process was similar to that of the hydrogel without COS, while the secondary gelling process appeared about 20 min later, and much stronger hydrogels with storage modulus G' about 1 MPa, 104 times higher, were obtained. COS also significantly influenced the microstructure of hydrogels as well as their biological activities. The hydrogels with 0.5% of COS significantly promoted proliferation of human umbilical cord mesenchymal stem cells (HUMSCs). These injectable hydrogels, especially when COS was added, remarkably accelerated the wound healing process in a mouse skin defect model. Microscopic wound analysis showed an increase of the thickness and integrity of epidermal tissue, increased formation of collagen fibers, and enhanced expression of vascular endothelial growth factor as compared to the control group.

Hygroscopicity modulation of hydrogels based on carboxymethyl chitosan/Alginate polyelectrolyte complexes and its application as pH-sensitive delivery system.

Polyelectrolyte complex (PECs) hydrogels with high hygroscopicity modulation ability were successfully prepared based on carboxymethyl chitosan (CMCS)/alginate. The swelling ratio of the hydrogels could be tuned from 1 to 450 simply by changing the weight ratio of CMCS to alginate, and superabsorbent hydrogels were obtained when the weight ratio of CMCS to alginate was below 1/1. Also, the swelling kinetics and water diffusion mechanism in the hydrogels were discussed. In vitro cytotoxicity results indicated that hydrogels had excellent cytocompatibility. The swelling ratio of the hydrogel as well as its BSA releasing profile was notably pH dependent. Compared with the values at pH 1.2, the swelling ratio of hydrogels (CMCS/alginate weight ratio of 1/2) at pH 7.4 was about 34 times higher, and the amount of BSA released at pH 7.4 was also significantly higher. The as-prepared CMCS/alginate PECs hydrogels hold great potential for oral delivery of protein drugs through the intestinal tract.

Protein acetylation as a mechanism for Kandelia candel's adaption to daily flooding.

To explore the adaptation mechanisms of Kandelia candel (L.) Druce in response to daily flooding, a large-scale quantitative lysine acetylome was carried out using immunoaffinity enrichment of Lys-acetylated peptides and liquid chromatography linked to tandem mass spectrometry. A total of 1041 lysine acetylation (LysAc) sites, 1021 Lys-acetylated peptides and 617 Lys-acetylated proteins were identified. Six conserved sequence motifs of the LysAc sites, including a new motif KxxxxK, were detected. Among these proteins, 260 were differentially acetylated in response to flooding, which were preferentially predicted to participate in carbon metabolism and photosynthesis pathways based on KEGG pathway category enrichment analysis. Consistently, the transcriptional level of acetyltransferase and the consumption of acetyl-CoA were up-regulated under flooding conditions. Most of physiological parameters and mRNA expression levels related to carbon metabolism and photosynthesis were found to be insignificantly affected by flooding. Taken together, reversible protein LysAc is likely to be a post-translational mechanism contributing to the mangrove K. candel's adaptation to daily flooding.

Phosphoproteomics unveils stable energy supply as key to flooding tolerance in Kandelia candel.

The mangrove Kandelia candel (L.) Druce experiences daily flooding cycles. To explore the molecular mechanism underlying the physiological adaptation of K. candel to flooding, the potential role of protein phosphorylation in flooding responses was investigated by a large-scale quantitative phosphoproteomic analysis using isobaric tag for relative and absolute quantitation. Total 2141 unique phosphopeptides and 2603 non-redundant phosphorylation sites were identified from 1516 phosphoproteins in K. candel leaves. In addition to known phosphorylation motifs, three new motifs [GSP], [GxxSP] and [RSxS] were discovered. The phosphorylation levels of 96 differentially expressed phosphoproteins, including those involved in pyruvate metabolism and energy production, were identified in response to flooding. The physiological parameters and transcriptional levels relevant to flooding responses including photosynthesis, pyruvate metabolism, and ROS production were investigated and all were found to be robust under flooding conditions. The consistent results of the phosphoproteomic, physiological analyses and transcriptional levels reinforce each other to demonstrate that K. candel adapts to flooding through maintaining sufficient photosynthesis activities, achieving effective anaerobic respiration and increasing pentose phosphate pathway flux. Protein phosphorylation is likely to play a major role in the regulation of these pathways which together contribute to stable energy supply that enhances flooding tolerance in K. candel. BIOLOGICAL SIGNIFICANCE: Flooding stress is one of the major environmental stresses. The woody mangrove Kandelia candel experiences daily flooding cycles in its natural habitat. Protein phosphorylation is a crucial regulatory mechanism in plants' responses to both biotic and abiotic stresses. To analyze phosphorylation levels in critical enzymes involved in key metabolic pathways, we employed phosphoproteomic approach to dissect the adaptive mechanism of K. candel to flooding conditions. To our knowledge, this is the first large-scale quantitative phosphoproteomic analyses of K. candel's flooding responses. Multiplex iTRAQ-based quantitative proteomic and Nano-LC-MS/MS methods were used to construct the phosphorproteome. Our results indicate that K. candel is able to acquire stable energy supply under flooding by maintaining sufficient photosynthesis activities, enhancing effective anaerobic respiration and increasing pentose phosphate pathway (PPP) flux. The protein phosphorylation found in photosynthesis, anaerobic respiration and PPP is likely to play important roles in the flooding tolerance of K. candel.

Loading cisplatin onto 6-mercaptopurine covalently modified MSNS: a nanomedicine strategy to improve the outcome of cisplatin therapy.

In the treatment of cancer patients, cisplatin (CDDP) exhibits serious cardiac and renal toxicities, while classical combinations related to CDDP are unable to solve these problems and may result in worse prognosis. Alternately, this study covalently conjugated 6-mercaptopurine (6MP) onto the surface of mercapto-modified mesoporous silica nanoparticles (MSNS) to form MSNS-6MP and loaded CDDP into the holes on the surface of MSNS-6MP to form MSNS-6MP/CDDP, a tumor-targeting nano-releasing regime for CDDP and 6MP specifically. In the S180 mouse model, the anti-tumor activity and overall survival of MSNS-6MP/CDDP (50 mg·kg-1·day-1, corresponding to 1 mg·kg-1·day-1 of 6MP and 5 mg·kg-1·day-1 of CDDP) were significantly higher than those of CDDP alone (5 mg·kg-1·day-1) or CDDP (5 mg·kg-1·day-1) plus 6MP (1 mg·kg-1·day-1). The assays of serum alanine aminotransferase, aspartate aminotransferase and creatinine, as well as the images of myocardium and kidney histology, support that MSNS-6MP/CDDP is able to completely eliminate liver, kidney and heart toxicities induced by CDDP alone or CDDP plus 6MP.

Simultaneous determination of molecular weights and contents of water-soluble polysaccharides and their fractions from Lycium barbarum collected in China.

Molecular weights and contents of water-soluble polysaccharides and their fractions in fifty batches of fruits of Lycium barbarum (wolfberry) collected from different regions of China, including Qinghai, Ningxia, Inner Mongolia, Xinjiang, and Gansu, were simultaneously determined using high performance size exclusion chromatography (HPSEC) coupled with multi angle laser light scattering (MALLS) and refractive index detector (RID) with the refractive index increment (dn/dc). Results showed that HPSEC chromatograms and molecular weight distributions of polysaccharides in L. barbarum collected from different regions of China were similar. Furthermore, the average contents of each polysaccharide fraction (peaks 1, 2, and 3) in crude polysaccharides of L. barbarum collected from Ningxia were similar with those of Inner Mongolia, Xinjiang, and Gansu, respectively. However, significant difference was found between polysaccharides in L. barbarum collected from Ningxia and Qinghai. Moreover, the average amounts of total polysaccharide fractions (peaks 1, 2, and 3) in the raw material of L. barbarum collected from Ningxia were significantly higher than that of Qinghai. These results may contribute to the rational usage of L. barbarum produced in China, and are beneficial for the improvement of their quality control. Results suggested that HPSEC-MALLS-RID with the dn/dc method could be used as a routine method for the quality evaluation of polysaccharides from natural resources and their products.

A multilevel investigation to discover why Kandelia candel thrives in high salinity.

The halophilic mangrove species Kandelia candel is an excellent model for understanding why halophytes thrive in high salinity. Preliminary transcriptomic analyses revealed that genes involved in diverse functions, such as in phenylpropanoid and amino acid metabolisms, and those in DNA replication and damage repair were highly responsive to salt stress. Proteomic analyses revealed that the proteins involved in light reaction of photosynthesis, amino acid and carbohydrate metabolisms, secondary metabolite biosynthesis and posttranslational modification showed increased levels in response to salt stress. The metabolisms of phenylpropanoids and amino acids under salt stress were systematically examined based on the preliminary omics analyses. The activities of phenylpropanoid biosynthetic enzymes and the contents of phenols, flavonoids, anthocyanins and lignins were significantly increased under salt stress. In the free amino acid pool, glutamate was the most abundant. Together with γ-aminobutyric acid, glutamate levels further increased, while proline levels remained unchanged in response to salt stress. These findings point to the potential importance of phenylpropanoids and free amino acids in salt tolerance of K. candel that have been observed, but not systemically investigated at the levels of gene expression, enzyme activity and metabolite accumulation in glycophytes and non-tree halophytes.

Proteomic approach reveals that starch degradation contributes to anthocyanin accumulation in tuberous root of purple sweet potato.

UNLABELLED: A comparative proteomic approach was carried out to investigate anthocyanin biosynthesis in the tuberous roots of yellow sweet potato (YSP) and purple sweet potato (PSP) cultivars. More than 800 proteins were reproducibly detected through two-dimensional electrophoresis (2-DE), of which 50 proteins with 39 more and 11 less accumulated in PSP were identified through matrix-assisted laser desorption ionization-time of flight/time of flight mass spectrometry (MALDI-TOF/TOF-MS). Most of the analyzed proteins are annotated to be involved in starch metabolism and glycolysis. The more abundant starch phosphorylase (SP) and phosphoglucomutase (PGM) in PSP promoted the synthesis of precursors for anthocyanin synthesis. The results implied that starch degradation provided abundant substrates for anthocyanin biosynthesis in tuberous roots of PSP. 24kDa vacuolar protein (VP24) is related to anthocyanin transport and accumulation in vacuoles. Vacuole-associated annexin protein, VCaB42, is correlated with tonoplast biogenesis. Synergistic action of the two proteins is probably involved in the microautophagy and the intravacuolar trapping of anthocyanins. Interestingly, both VCaB42 and VP24 were more accumulated in PSP, suggesting that anthocyanins generated in the cytosol were transported into and became stored in the vacuoles of PSP. The present study provides new insights into the mechanism of tuberous root-specific anthocyanin accumulation in PSP. BIOLOGICAL SIGNIFICANCE: Sweet potato ranks as the seventh most important crop worldwide. Purple sweet potato, a special sweet potato cultivar, has been extensively investigated because large amounts of anthocyanin accumulate in its tuberous roots. Anthocyanin is well known for its free radical-scavenging activity and beneficial effects on human health. Its biosynthetic pathway has been well characterized in model plants. Although large-scale systematic studies have been performed to identify the proteins present in sweet potato, information on the regulation of anthocyanin synthesis in sweet potato is insufficient. Our proteome study demonstrated that starch degradation may contribute to anthocyanin accumulation in purple sweet potato. To our knowledge, this study is the first to propose that starch degradation may provide precursors of anthocyanin biosynthesis in sweet potato.

Characterization and comparison of polysaccharides from Lycium barbarum in China using saccharide mapping based on PACE and HPTLC.

Water-soluble polysaccharides from 51 batches of fruits of L. barbarum (wolfberry) in China were investigated and compared using saccharide mapping, partial acid hydrolysis, single and composite enzymatic digestion, followed by polysaccharide analysis by using carbohydrate gel electrophoresis (PACE) analysis and high performance thin layer chromatography (HPTLC) analysis, respectively. Results showed that multiple PACE and HPTLC fingerprints of partial acid and enzymatic hydrolysates of polysaccharides from L. barbarum in China were similar, respectively. In addition, results indicated that ß-1,3-glucosidic, α-1,4-galactosiduronic and α-1,5-arabinosidic linkages existed in polysaccharides from L. barbarum collected in China, and the similarity of polysaccharides in L. barbarum collected from different regions of China was pretty high, which are helpful for the improvement of the performance of polysaccharides from L. barbarum in functional/health foods area. Furthermore, polysaccharides from Panax notoginseng, Angelica sinensis, and Astragalus membranaceus var. mongholicus were successfully distinguished from those of L. barbarum based on their PACE fingerprints. These results were beneficial to improve the quality control of polysaccharides from L. barabrum and their products, which suggested that saccharide mapping based on PACE and HPTLC analysis could be a routine approach for quality control of polysaccharides.

A New Modified S-plasty for Skin Defect Closure.

BACKGROUND: Dog-ear, or standing cone deformity, is a common problem during surgical procedures. Multiple methods have been reported to correct the deformity but most create long scars or excessive normal skin loss. METHODS: We designed a simple and convenient procedure to remove small- and medium-sized skin defects. Based on mathematical calculations, the procedure is an easily designed surgical technique. RESULTS: All of our patients healed uneventfully with no significant complications. The procedure not only resulted in a shortened surgical incision and preserved a greater amount of healthy skin, but also left a simple "S-shaped" curvilinear scar with favorable esthetic outcomes. CONCLUSION: This novel modified S-plasty is a simple tool to successfully remove skin defects, with a low deformity risk.

In vitro proliferation and differentiation of adipose-derived stem cells isolated using anti-CD105 magnetic beads.

The present study aimed to investigate the feasibility of isolating adipose-derived stem cells (ADSCs) by selecting cells that express the surface receptor CD105. Surface antigen expression of the unsorted cells was undertaken using FACS analysis. Primary adipose-derived cells were isolated. The second passage cells were incubated with anti-CD105 magnetic beads, and separated using a magnetic separator. Cell growth and colony formation was determined by counting and Giemsa staining, respectively. Cells also underwent histological immunohistochemical, and RT-PCR analyses to determine their chondrogenic, adipogenic and osteogenic potential. Increased cell proliferation and colony formation was observed in CD105-positive (CD105⁺) as compared to the CD105-negative (CD105⁻) cells (P<0.001). Following induction, the expression of type II collagen and the number of calcium deposits and lipid droplets in the CD105⁺ ADCs were markedly higher than in the CD105⁻ ADCs. Furthermore, increased alkaline phosphatase (AKP), leptin and PPARγ2 mRNA expression was detected in the CD105⁺ ADCs (P<0.01). Isolation of CD105⁺ ADSCs by MACS was feasible. Thus, CD105 can be used as a relatively specific marker for the selection of ADSCs. Although the chondrogenic, adipogenic and osteogenic potential of these cells is suggestive of their potential for use in tissue engineering treatments, further in vivo studies are necessary.

Chondrogenesis by co-culture of adipose-derived stromal cells and chondrocytes in vitro.

Adipose-derived stromal cells (ADSCs) could be induced to differentiate into chondrocytes in the presence of cellular factors. In this study, we explored the feasibility of inducing the differentiation of ADSCs into chondrocytes in the presence of chondrocytes. Human ADSCs and porcine auricular chondrocytes were expanded in vitro and then were mixed at the ratio of 7:3. 5.0 × 10(7) mixed cells were seeded onto a polyglycolic acid/polylactic acid scaffold as co-culture group. Chondrocytes and ADSCs with the same cell number were seeded onto the scaffold as positive control group and negative control group. A total of 1.5 × 10(7) chondrocytes were seeded as low-concentration chondrocyte group. After culturing for 8 weeks, gross observation, wet weight, histology, glycosaminoglycan quantification, and collagen II expression were evaluated. Cells in all groups well adhered to the scaffold and could secrete extracellular matrices. In the co-culture group and positive control group, cell-scaffold constructs could maintain the original size and shape during the culture. At the 8th week, cartilage-like tissues were formed, and abundant type II collagen could be detected by immunohistochemistry and reverse transcription-polymerase chain reaction in co-culture and positive control groups. Wet weights and glycosaminoglycan contents of tissues in co-culture group were approximately onefold of those in the negative control group. In the negative control group, constructs shrunk gradually without mature cartilage lacuna formation. In low-concentration chondrocyte group, constructs also shrunk obviously with small amount of cartilage formation. Chondrocytes can provide chondrogenic microenvironment to induce chondrogenic differentiation of ADSCs and thus promote the chondrogenesis of ADSCs in vitro.

In vivo ectopic chondrogenesis of BMSCs directed by mature chondrocytes.

In vivo niche plays an important role in determining the fate of exogenously implanted stem cells. Due to the lack of a proper chondrogenic niche, stable ectopic chondrogenesis of mesenchymal stem cells (MSCs) in subcutaneous environments remains a great challenge. The clinical application of MSC-regenerated cartilage in repairing defects in subcutaneous cartilage such as nasal or auricular cartilage is thus severely limited. The creation of a chondrogenic niche in subcutaneous environments is the key to solving this problem. The current study demonstrates that bone marrow stromal cells (BMSCs) could form cartilage-like tissue in a subcutaneous environment when co-transplanted with articular chondrocytes, indicating that chondrocytes could create a chondrogenic niche to direct chondrogenesis of BMSCs. Then, a series of in vitro co-culture models revealed that it was the secretion of soluble factors by chondrocytes but not cell-cell contact that provided the chondrogenic signals. The subsequent studies further demonstrated that multiple factors currently used for chondroinduction (including TGF-ß1, IGF-1 and BMP-2) were present in the supernatant of chondrocyte-engineered constructs. Furthermore, all of these factors were required for initiating chondrogenic differentiation and fulfilled their roles in a coordinated way. These results suggest that paracrine signaling of soluble chondrogenic factors provided by chondrocytes was an important mechanism in directing the in vivo ectopic chondrogenesis of BMSCs. The multiple co-culture systems established in this study provide new methods for directing committed differentiation of stem cells as well as new in vitro models for studying differentiation mechanism of stem cells determined by a tissue-specific niche.

Potent in vitro chondrogenesis of CD105 enriched human adipose-derived stem cells.

Adipose-derived stem cells (ASCs) are considered as a promising cell source for cartilage regeneration. However, the heterogeneity of this cell source may affect their ability in cartilage formation. It is therefore necessary to establish an efficient method for isolating the cells that have chondrogenic potential. To date, no specific markers have been reported to be able to isolate such a cell population from human adipose tissue. In recent studies, endoglin (CD105) has been known as a relatively specific marker for identifying mesenchymal stem cells, but no studies show it is related to chondrogenic potential of human ASCs. In this study, human cells from adipose tissue were isolated, cultured, and sorted according to CD105 expression. The sorted cells were then subjected to adipogenic, osteogenic, and chondrogenic induction to confirm their multi-potentiality. In adipogenic conditions, CD105- cells showed stronger Oil Red staining and higher expression of adipose-specific genes compared to CD105+ cells. By contrast, CD105+ cells exhibited better osteogenic potential with stronger Alizarin Red staining and higher expression of osteogenic specific genes than CD105- cells. Noticeably, CD105+ cells also exhibited a much stronger chondrogenic potential than CD105- cells, with stronger collagen II staining and higher gene expression of collagen II and aggrecan. Most importantly, CD105+ cells could form a homogeneous cartilage-like tissue when seeded into a biodegradable scaffold and cultured in chondrogenic media for 8 weeks. These results indicate that sorting of ASC subpopulation with CD105 as a marker may allow better in vitro chondrogenesis and thus provide an important implications for cartilage regeneration and reconstruction using autologous cells from adipose tissue.