Risk factors for poor progression of addictive internet use across different COVID-19 periods in China.

BACKGROUND AND OBJECTIVES: Addictive behaviors are serious factors for mental health and usually increase during public crises. We identified the vulnerable characteristics for bad prognosis of addictive internet use across different periods of the coronavirus disease 2019 (COVID-19) pandemic. METHODS: Self-reported questionnaires were delivered in three waves through jdh.com during the outbreak (n = 17,960), remission (n = 15,666), and dynamic zero (n = 12,158) periods of COVID-19 pandemic in China. Internet addiction degree was assessed using the Internet Addiction Test. The different progression groups were divided using a latent class growth model among 1679 longitudinal participants. Risk factors for bad progression were identified by two-step logistic regression. RESULTS: A total of 40.16% of participants reported an increase in the addictive degree of internet use compared with prepandemic. Across different COVID-19 periods, the overall trend of addictive internet use was downward among general Chinese study participants (Mslope = -1.56). Childhood traumatic experiences, deterioration of physical health, depression, and anxiety during remission and dynamic periods were the main risk factors for the bad progression of pandemic-induced addictive internet use. DISCUSSION AND CONCLUSIONS: Addictive internet use was remitted following relaxed control policies during the COVID-19 pandemic. Negative childhood experiences and bad mental status during the recovery period were harmful to coping with pandemic-related addictive internet use. SCIENTIFIC SIGNIFICANCE: Our findings profiled the general trend of addictive internet use and the vulnerable characteristics of its bad progression across different periods of the first wave of COVID-19 pandemic in China. Our findings provide valuable insights for preventing the long-term adverse effects of negative public events on Internet addiction.

Atomically Dispersed Co to an End-Adsorbing Molecule for Excellent Biomimetically and Prime Sensitively Detecting O2•- Released from Living Cells.

Single-atom catalysis efficiently exposes the catalytic sites to reactant molecules while rendering opportunity to investigate the catalysis mechanisms at atomic levels for scientific insights. Here, for the first time, atomically dispersed Co atoms are synthesized as biomimetic "enzymes" to monitor superoxide anions (O2•-), delivering ultraordinary high sensitivity (710.03 µA·µM-1·cm-2), low detection limit (1.5 nM), and rapid response time (1.2 s), ranking the best among all the reported either bioenzymatic or biomimetic O2•- biosensors. The sensor is further successfully employed to real-time monitor O2•- released from living cells. Moreover, theoretical calculation and analysis associated with experimental results discover that a mode of end adsorption of the negatively charged O2•- on the Co3+ atom rather than a bridge or/and side adsorption of the two atoms of O2•- on two Co3+ atoms, respectively, plays an important role in the single-atomic catalysis toward O2•- oxidation, which not only facilitates faster electron transfer but also offers better selectivity. This work holds great promise for an inexpensive and sensitive atomic biomimetic O2•- sensor for bioresearch and clinic diagnosis, while revealing that the adsorption mode plays a critical role in single-atom catalysis for a fundamental insight.

Nintedanib inhibits keloid fibroblast functions by blocking the phosphorylation of multiple kinases and enhancing receptor internalization.

Keloid is a benign skin tumor characterized by its cell hyperproliferative activity, invasion into normal skin, uncontrolled growth, overproduction and deposition of extracellular matrices and high recurrence rate after various therapies. Nintedanib is a receptor tyrosine kinase inhibitor targeting VEGF, PDGF, FGF, and TGF-ß receptors with proved efficacy in anti-angiogenesis and in treating various types of cancers. In this study, we investigated the effects of nintedanib on keloid fibroblasts in both in vitro and ex vivo models. Keloid fibroblasts were prepared from 54 keloid scar samples in active stages collected from 49 patients. We found that nintedanib (1-4 µM) dose-dependently suppressed cell proliferation, induced G0/G1 cell cycle arrest, and inhibited migration and invasion of keloid fibroblasts. The drug also significantly inhibited the gene and protein expression of collagen I (COL-1) and III (COL-3), fibronectin (FN), and connective growth factor (CTGF), as well as the gene expression of other pathological factors, such as alpha smooth muscle actin (α-SMA), plasminogen activator inhibitor-1 (PAI-1), FK506-binding protein 10 (FKBP10), and heat shock protein 47 (HSP47) in keloid fibroblasts. Furthermore, nintedanib treatment significantly suppressed the phosphorylation of p38, JNK, ERK, STAT3, and Smad, enhanced endocytosis of various growth factor receptors. Using an ex vivo tissue explant model, we showed that nintedanib significantly suppressed cell proliferation, migration, and collagen production. The drug also significantly disrupted microvessel structure ex vivo. In summary, our results demonstrate that nintedanib is likely to become a potential targeted drug for keloid systemic therapy.

The direct observation of electron backflow in an organic heterojunction formed by two n-type materials.

Many physical processes such as exciton interfacial dissociation, exciton interfacial recombination, and exciton-electron and exciton-hole interactions coexist at the interface of organic solar cells (OSC). In this study, the direction of free charge generation is defined as the direction from the interface to the side where free charges are left. For a p-n type device, the direction of free electron (hole) generation from exciton dissociation at the donor/accepter (D/A) interface is the same as the subsequent transportation direction under the built-in electric field. However, the direction of free electron (hole) generation from exciton-exciton recombination across the D/A interface is opposite to the direction of free charge transportation. Both free charges generated from exciton interfacial dissociation and recombination are contributed to the photocurrent for a p-n type device. In a device with a heterojunction formed by two n-type materials (here it is defined as an n-n type device), the direction of free electron (hole) generation from exciton recombination across the interface is also the same as the subsequent free charge transportation. At the same time, there are also some free electrons (free holes) generated by exciton interfacial dissociation. The direction of free charge generation from exciton dissociation for this n-n type device is also opposite to the direction of free charge transportation. However, only free charges generated from exciton interfacial recombination are contributed to the photocurrent for an n-n type device. But so far there has been no direct experimental evidence to prove the above theories. In this work, an NPB interfacial layer with a high LUMO was introduced in an n-n type OSC to inhibit the backflow of electrons, which are generated from exciton dissociation at the heterojunction formed by two n-type materials, enhancing the device performance accordingly. This work is conducive to interfacial engineering in an OSC to further improve its performance.

Evaluation of Field Portable X-Ray Fluorescence Performance for the Analysis of Ni in Soil.

As a rapid, in-situ analysis method, Field portable X-ray fluorescence spectrometry (FP-XRF) can be widely applied in soil heavy metals analysis field. Whereas, some factors may affect FP-XRF performance and restrict the application. Studies have proved that FP-XRF has poorer performance when the concentration of target element is low, and soil moisture and particle size will affect FP-XRF performance. But few studies have been conducted in depth. This study took an example of Ni, demonstrated the relationship between Ni concentration and FP-XRF performance on accuracy and precision, and gave a critical value. Effects of soil moisture and particle size on accuracy and precision also had been compared. Results show that, FP-XRF performance is related to Ni concentration and the critical value is 400 mg x kg(-1). Relative standard deviation (RSD) and relative uncertainty decrease while the Ni concentration is below 400 mg x kg(-1), hence FP-XRF performance improves with increasing Ni concentration in this range; RSD and relative uncertainty change little while the Ni concentration is above 400 mg x kg(-1), hence FP-XRF performance does not have correlation with Ni concentration any more. For in-situ analysis, the relative uncertainty contributed by soil moisture is 3.77%, and the relative certainty contributed by particle size is 0.56%. Effect of soil moisture is evidently more serious than particle size both on accuracy and precision.

Disentangling the resistant mechanism of Fusarium wilt TR4 interactions with different cultivars and its elicitor application.

Fusarium wilt of banana, especially Tropical Race 4 (TR4) is a major factor restricting banana production. Developing a resistant cultivar and inducing plant defenses by elicitor application are currently two of the best options to control this disease. Isotianil is a monocarboxylic acid amide that has been used as a fungicide to control rice blast and could potentially induce systemic acquired resistance in plants. To determine the control effect of elicitor isotianil on TR4 in different resistant cultivars, a greenhouse pot experiment was conducted and its results showed that isotianil could significantly alleviate the symptoms of TR4, provide enhanced disease control on the cultivars 'Baxi' and 'Yunjiao No.1' with control effect 50.14% and 56.14%, respectively. We compared the infection processes in 'Baxi' (susceptible cultivars) and 'Yunjiao No.1' (resistant cultivars) two cultivars inoculated with pathogen TR4. The results showed that TR4 hyphae could rapidly penetrate the cortex into the root vascular bundle for colonization, and the colonization capacity in 'Baxi' was significantly higher than that in 'Yunjiao No.1'. The accumulation of a large number of starch grains was observed in corms cells, and further analysis showed that the starch content in 'Yunjiao No. 1' as resistant cultivar was significantly higher than that in 'Baxi' as susceptible cultivar, and isotianil application could significantly increase the starch content in 'Baxi'. Besides, a mass of tyloses were observed in the roots and corms and these tyloses increased after application with isotianil. Furthermore, the total starch and tyloses contents and the control effect in the corms of 'Yunjiao No.1' was higher than that in the 'Baxi'. Moreover, the expression levels of key genes for plant resistance induction and starch synthesis were analyzed, and the results suggested that these genes were significantly upregulated at different time points after the application of isotianil. These results suggest that there are significant differences between cultivars in response to TR4 invasion and plant reactions with respect to starch accumulation, tyloses formation and the expression of plant resistance induction and starch synthesis related genes. Results also indicate that isotianil application may contribute to disease control by inducing host plant defense against TR4 infection and could be potentially used together with resistant cultivar as integrated approach to manage this destructive disease. Further research under field conditions should be included in the next phases of study.

A biological product of Bacillus amyloliquefaciens QST713 strain for promoting banana plant growth and modifying rhizosphere soil microbial diversity and community composition.

Introduction: Bananas are not only an important food crop for developing countries but also a major trading fruit for tropical and semitropical regions, maintaining a huge trade volume. Fusarium wilt of banana (FWB) caused by Fusarium oxysporum f. sp. cubense is becoming a serious challenge to the banana industry globally. Biological control has the potential to offer both effective and sustainable measures for this soil-borne disease. Methods: In order to explore the biocontrol effects of the biological agent Bacillus amyloliquefaciens QST713 strain on banana plants, two cultivars, Brazilian and Yunjiao No. 1, with varied resistance to FWB, were used in greenhouse pot experiments. Results: Results showed that the plant height and pseudostem diameter of banana-susceptible cultivar Brazilian increased by 11.68% and 11.94%, respectively, after QST713 application, while the plant height and pseudostem diameter of resistant cultivar Yunjiao No. 1 increased by 14.87% and 12.51%, respectively. The fresh weight of the two cultivars increased by 20.66% and 36.68%, respectively, indicating that this biological agent has potential effects on plant growth. Analysis of the rhizosphere soil microbial communities of two different cultivars of banana plants showed that TR4 infection and B. amyloliquefaciens QST713 strain application significantly affected the bacterial and fungal diversity of Yunjiao No. 1, but not in the cultivar Brazilian. In addition, TR4 infection and QST713 application changed the bacterial community composition of both banana cultivars, and the fungal community composition of Yunjiao No. 1 also changed significantly. Relevance analysis indicated that the relative richness of Bacillus and Pseudomonas in the rhizosphere of both cultivars increased significantly after QST713 application, which had a good positive correlation with plant height, pseudostem girth, aboveground fresh weight, leaf length, and leaf width. Discussion: Therefore, the outcome of this study suggests that the biological agent QST713 strain has potential application in banana production for promoting plant growth and modification of soil microbial communities, particularly in the TR4-infected field.

[In vitro proliferation of rat Leydig cells].

OBJECTIVE: To investigate the feasibility of in vitro proliferation of rat Leydig cells by modifying the cell culture system. METHODS: Leydig cells were isolated from three-week-old rats by a procedure combining collagenase dispersion, stainless steel mesh infiltration and differential adhesion. The isolated cells were cultured in DMEM/F12 and modified media for stem cell proliferation, and the proliferation of the cultured cells was evaluated by cell counting and MTP test. The expression of 3beta-HSD in the cultured cells was detected by immunohistochemistry and flow cytometry, and testosterone productivity in the isolated Leydig cells with or without hCG stimulation was determined at 2 hours and 4 days after cell isolation. RESULTS: The Leydig cells cultured in the modified media proliferated actively, with a doubling time of (2.26 +/- .31) days, as compared with (16.32 +/- 2.14) days for those cultured in the traditional media (P <0.05). The 3beta-HSD positive rate in the cultured cells was (554.3 +/- 7.1)% after 2 hours and (93.6 +/- 4.6)% after 4 days of culture. All the proliferated cells exhibited testosterone productivity, and their testosterone secretion was significantly upregulated by hCG stimulation (P <0.05). CONCLUSION: Leydig cells isolated by differential adhesion proliferate actively in the modified culture media.

Differentiation of human multipotent dermal fibroblasts into islet-like cell clusters.

BACKGROUND: We have previously obtained a clonal population of cells from human foreskin that is able to differentiate into mesodermal, ectodermal and endodermal progenies. It is of great interest to know whether these cells could be further differentiated into functional insulin-producing cells. RESULTS: Sixty-one single-cell-derived dermal fibroblast clones were established from human foreskin by limiting dilution culture. Of these, two clones could be differentiated into neuron-, adipocyte- or hepatocyte-like cells under certain culture conditions. In addition, those two clones were able to differentiate into islet-like clusters under pancreatic induction. Insulin, glucagon and somatostatin were detectable at the mRNA and protein levels after induction. Moreover, the islet-like clusters could release insulin in response to glucose in vitro. CONCLUSIONS: This is the first study to demonstrate that dermal fibroblasts can differentiate into insulin-producing cells without genetic manipulation. This may offer a safer cell source for future stem cell-based therapies.

Degradation or excretion of quantum dots in mouse embryonic stem cells.

BACKGROUND: Quantum dots (QDs) have been considered as a new and efficient probe for labeling cells non-invasively in vitro and in vivo, but fairly little is known about how QDs are eliminated from cells after labeling. The purpose of this study is to investigate the metabolism of QDs in different type of cells. RESULTS: Mouse embryonic stem cells (ESCs) and mouse embryonic fibroblasts (MEFs) were labeled with QD 655. QD-labeling was monitored by fluorescence microscopy and flow cytometry for 72 hours. Both types of cells were labeled efficiently, but a quick loss of QD-labeling in ESCs was observed within 48 hours, which was not prevented by inhibiting cell proliferation. Transmission electron microscope analysis showed a dramatic decrease of QD number in vesicles of ESCs at 24 hours post-labeling, suggesting that QDs might be degraded. In addition, supernatants collected from labeled ESCs in culture were used to label cells again, indicating that some QDs were excreted from cells. CONCLUSION: This is the first study to demonstrate that the metabolism of QDs in different type of cells is different. QDs were quickly degraded or excreted from ESCs after labeling.

[Reconstruction of rabbit corneal stroma with skin fibroblasts].

OBJECTIVE: To explore whether skin fibroblasts could be used as a cell source for reconstruction of the corneal stroma. METHODS: It was an experimental study. Skin fibroblast cells were isolated from newborn rabbits, cultured and expanded in vitro. Cells were labeled with green fluorescence protein (GFP) gene by retro-viral infection. Fibroblasts at passage 3 were seeded on polyglycolic acid (PGA) non-woven fibers to form a cell-scaffold construct. Constructs were then implanted into the adult rabbit corneal stroma layer after being cultured in vitro for 1 week. Engineered stroma were observed continuously and harvested after 8 weeks of transplantation for gross, histological evaluation and Keratocan examination. PGA alone was used as control. RESULTS: The engineered tissue in the cornea became transparent gradually over a period of 8 weeks. Histological analysis showed that engineered stromal lamellar was relatively regular and the orientation of fibers was parallel to the surface of cornea, which is similar to normal cornea. The implanted cells were confirmed by GFP expression under fluorescent microscope, which also express Keratocan. By transmission electron microscopy examination, no significant difference in the diameter of collagen fiber was observed between engineered stroma (33.08 + or - 2.47) nm and normal stroma (t = 1.80, P = 0.0771). CONCLUSION: Skin fibroblast cells could be used as seed cells for reconstruction of the corneal stroma.

High Open-Circuit Voltage of 1.134 V for Inverted Planar Perovskite Solar Cells with Sodium Citrate-Doped PEDOT:PSS as a Hole Transport Layer.

Poly(3,4-ethylenedioxythiophene)/polystyrene sulfonate (PEDOT:PSS) plays an important role in inverted planar perovskite solar cells (IPPSCs) as an efficient hole extraction and transfer layer (HTL). The IPPSCs based on PEDOT:PSS normally display inferior performance with a reduced open-circuit voltage. To address this problem, here sodium citrate-doped PEDOT:PSS is adopted as an effective HTL for improving the performance of IPPSCs. Sodium citrate-doped PEDOT:PSS HTL improves the conversion efficiency of IPPSCs from 15.05% of reference cells to 18.39%. The large increase of the open-circuit voltage ( VOC) from 1.057 to 1.134 V is the main source for this performance enhancement. With the help of characterization analysis of ultraviolet photoelectron spectroscopy, scanning electron microscopy, electrochemical impedance spectroscopy, etc., the higher work function of the doped PEDOT:PSS film and the uniform crystallinity of the perovskite film on it are disclosed as the reasons for the increased VOC and the consequent performance enhancement.

The dependence of in vivo stable ectopic chondrogenesis by human mesenchymal stem cells on chondrogenic differentiation in vitro.

In vivo niche plays an important role in determining the fate of implanted mesenchymal stem cells (MSCs) by directing committed differentiation. An inappropriate in vivo niche can also alter desired ultimate fate of exogenous MSCs even they are in vitro induced to express a specific phenotype before in vivo implantation. Studies have shown that in vitro chondrogenically differentiated MSCs are apt to lose their phenotype and fail to form stable cartilage in subcutaneous environment. We hypothesized that failure of maintaining the phenotype of induced MSCs in subcutaneous environment is due to the insufficient chondrogenic differentiation in vitro and fully differentiated MSCs can retain their chondrocyte-like phenotype and form stable ectopic cartilage. To test this hypothesis, extended in vitro chondrogenic induction and cartilage formation were carried out before implantation. Human bone marrow stem cells (hBMSCs) were seeded onto polylactic acid coated polyglycolic acid scaffolds. The cell-scaffold constructs were chondrogenically induced from 4 to 12 weeks for in vitro chondrogenesis, and then implanted subcutaneously into nude mice for 12 or 24 weeks. The engineered cartilages were evaluated by gross view, glycosaminoglycan content measurement, and histological staining before and after implantation. Histological examination showed typical cartilage structure formation after 8 weeks of induction in vitro. However, part of the constructs became ossified after implantation when in vitro induction lasted 8 weeks or less time. In contrast, those induced for 12 weeks in vitro could retain their cartilage structure after in vivo implantation. These results indicate that a fully differentiated stage achieved by extended chondrogenic induction in vitro is necessary for hBMSCs to form stable ectopic chondrogenesis in vivo.

[Construction of urethra mucosa structure in vitro by tissue engineering].

OBJECTIVE: To construct a structure of urethra mucosa in vitro by tissue engineering. METHODS: Primary porcine urothelial cells (UC) were obtained from the porcine bladder by enzymatic digestion and detected by immunofluorescence and RT-PCR. Bladder acellular matrix grafts (BAMG) were prepared, used as the scaffold and then evaluated by HE staining, Masson's trichrome staining, immunohistology and scanning electron microscopy. After in vitro culture and amplification, the UCs were seeded on the luminal surface the BAMGs. RESULTS: After 1 week of in vitro culture, the UCs formed a multilayer structure on the luminal surface of the BAMGs along the basement membrane. The tissue-engineered urothelium and BAMG complex was well formed and pan cytokeratins were positively expressed in the UCs on the scaffold. CONCLUSION: By tissue engineering, the urethra mucosa structure can be rapidly constructed in vitro, which can be applied to the repair of such urethral defect as hypospadias and urethral stricture.

[Androgen-secreting tissue construction with co-cultured rat testes somatic cells by tissue engineering technique].

OBJECTIVE: To explore the feasibility of constructing androgen-secreting tissue of a certain size and shape using co-cultured somatic cells of rat testis. METHODS: Thirty male Wistar rats were castrated. model and implanted rat model were prepared by resecting bilateral testes. The suspension of mixed testes cells was cultured to obtain various somatic cells of testes and Leydig cells were collected by differential anchorage-dependent method. These two kinds of cells were seeded onto biodegradable scaffolds of polyglycolic acid (PGA) fibers and cultured in vitro. The tissue formation of cell-scaffold constructs was observed by optical microscope and electronic microscope and the level of testosterone in the supernatant was detected regularly. After 7-day culture in vitro, the 2 kinds of cell-scaffold constructs, scaffold with purified Leydig cells or co-cultured testis somatic cells (seed cells), were implanted into the gastrocolic omentum or cavity of tunica vaginalis of the castrated rats. The implants were harvested 4, 6, 9, 12, and 24 weeks later to evaluate the tissue formation of cell-scaffold constructs in vivo. The serum testosterone level of the implanted rats was assayed to evaluate the testosterone secreting function of the regenerative tissue. RESULTS: Both the co-cultured testis somatic cells and Leydig cells had fine compatibility with the PGA fibers and adhered to the scaffolds very well. Testosterone was detected at a certain degree in the supernatant of cell-scaffold constructs, indicating the testosterone secreting function of the constructs. Two months after the implantation both kinds of cell-scaffold constructs formed testosterone secreting tissue in both gastrocolic omentum and cavity of tunica vaginalis of the implanted rats. The regenerative tissues were vascularized very well with a certain size and shape. Six weeks after implantation the serum testosterone level of the Leydig cell group was 0.60 ng/ml +/- 0.04 ng/ml, and that of the co-culture group was 0.84 ng/ml +/- 0.03 ng/ml, both significantly higher than that of the control castrated rats (0.56 ng/ml +/- 0.05 ng/ml, both P < 0.01), and the serum testosterone level of the co-cultured testes somatic cell implantation group was significantly higher than that of the Leydig cell implantation group too (P < 0.01). CONCLUSION: It is completely feasible to construct androgen-secreting tissue in vitro and in vivo using tissue engineering technique. Co-cultured testis somatic cells may serve as the better seed cells for androgen-secreting tissue engineering than purified Leydig cells in terms of the quantity and function of cells.

[Influence of transforming growth factor-beta1 inducing time on chondrogenesis of bone marrow stromal cells (BMSCs): in vitro experiment with porcine BMSCs].

OBJECTIVE: To explore the influence of transforming growth factor (TGF)-beta1 inducing time on the chondrogenesis of bone marrow stromal cells (BMSC), and on the construction of tissue engineering cartilage. METHODS: BMSCs were obtained from the greater trochanters of 3 pigs, cultured, seeded onto the cylindrical scaffolds made of polyglycolic acid at the density of 5.0 x 10(7) cells/ml, and then cultured with chondrogenesis media containing TGF-beta(1) (10 ng/ml), insulin-like growth factor-I (50 microg/L), and dexamethasone (40 microg/L) to be induced by TGF-beta(1) for 2 weeks (Group A), 4 weeks (Group B), 6 weeks (Group C), 8 weeks (Group D), or 10 weeks (Group E) respectively. 10 weeks later the cylindrical scaffolds underwent gross observation and histological examination. Alcin blue method was used to examine the content of proteoglycan (GAG). Immunochemistry and Western blotting were used to examine the type II collagen. The cylindrical scaffolds underwent biomechanical analysis. RESULTS: HE staining showed cartilage lacunae increasing in number from the periphery to center of the cylindrical scaffolds with the extended inducing time, and were arranged more uniformly progressively. Histochemical staining showed GAG accumulation. Immunohistochemistry and Western blotting showed that the content of type II collagen increased gradually, the amount of type II collagen stabilized after 6 weeks' culture, and there was no significant difference in the content of type II collagen between Group C and Group E. Biomechanical analysis showed that the modulus of elasticity and compression strength of the groups induced for more than 6 weeks (Groups C, D, and E) were all higher then those of Groups and B. CONCLUSION: TGF-beta(1) inducing time is correlated with the cartilage engineering characteristics with BMSC as seed cells. Induction for 6 weeks helps construct tissue engineered cartilage with good tissue structure, chemical composition and biomechanical properties.

[Potential of chondrogenesis of bone marrow stromal cells co-cultured with chondrocytes on biodegradable scaffold: in vivo experiment with pigs and mice].

OBJECTIVE: To explore the feasibility of in vivo chondrogenesis of bone marrow stromal cells (BMSCs) co-cultured with chondrocytes on biodegradable scaffold. METHODS: Porcine BMSCs were isolated, expanded and labeled with enhanced green fluorescent protein (EGFP), and then were mixed with articular chondrocytes isolated from porcine knee joint at the ratio of 1:1. The mixed cells were seeded onto polyglycolic acid (PGA) scaffold at the ultimate concentration of 5.0 x 10(7)/ml (co-culture group). Pure chondrocytes and BMSCs of the same ultimate concentration were seeded respectively onto the scaffold as positive control group and negative control group. After two weeks' culture in vitro, they were planted subcutaneously into nude mice respectively. These specimens were collected after in vivo implantation for 8 weeks to undergo microscopy. Laser confocal microscopy was used to observe the distribution of EGFP-labeled cells in the tissue. RT-PCR was used to examine the expression of collagen type II and aggrecan. Immunohistochemistry was used to observe the protein expression of collagen type II. RESULTS: The cell-scaffold constructs of the co-culture group and positive control group, could maintain the original size and shape no matter in vitro or in vivo. After 8 weeks' in vivo implantation, the constructs in both co-culture group and positive control group formed cartilage-like tissue with typical histological structure and extracellular matrix staining similar to those of the normal cartilage. The GAG content and compressive modulus of the co-culture group reached over 80% of those of the positive control group. Confocal microscopy revealed the presence of EGFP-labeled cells in the engineered cartilage lacuna. Histological examination showed that the constructs of the negative control group shrunk gradually after in vivo implantation with no typical cartilage-like tissue formation. CONCLUSION: In vitro co-cultured BMSC-chondrocyte-PGA constructs have the potential to form mature cartilage-like tissue in subcutaneous non-chondrogenesis environment, indicating that chondrocytes still provide enough signals for BMSC chondrogenic differentiation.

[Experimental study of in vitro chondrogenesis by co-culture of bone marrow stromal cells and chondrocytes].

OBJECTIVE: Chondrogenic microenvironments play a very important role in chondrogenesis of bone marrow stromal cells (BMSC). This study explored the feasibility of in vitro chondrogenesis by co-culture of BMSC and chondrocytes so as to confirm the hypothesis that chondrocytes can provide chondrogenic microenvironment to induce chondrogenic differentiation of BMSC and thus promote in vitro chondrogenesis of BMSC. METHODS: Porcine BMSC and auricular chondrocytes were in vitro expanded respectively and then were mixed at a ratio of 8:2 (BMSC:chondrocyte). 200 microl mixed cells(5.0 x 10(7)/ml) were seeded onto a polyglycolic acid/polylactic acid (PGA/PLA) scaffold, 9 mm in diameter and 3 mm in thickness, as co-culture group. Chondrocytes and BMSC with the same cell number were seeded respectively onto the scaffolds as positive control (chondrocyte group) and negative control (BMSC group). 200 microl chondrocytes (1.0 x 10(7)/ml, equal to the chondrocyte number of co-culture group) alone were seeded as low concentration chondrocyte group. There were 6 specimens in each group. All specimens were harvested after in vitro culture for 8 weeks in DMEM plus 10% FBS. Gross observation, average wet weight measurement, glycosaminoglycan (GAG) quantification, histology and immunohistochemistry were used to evaluate the results. RESULTS: Cells in all groups had fine adhesion to the scaffolds and could secrete extracellular matrix. In both co-culture group and positive control group, the cell-scaffold constructs could maintain the original size and shape during in vitro culture and formed homogenous mature cartilage after 8 weeks of in vitro culture. Furthermore, the neo-cartilages in both groups were similar to each other in gross appearance and histological features, and abundant type II collagen was also detected by immunohistochemistry in both groups. The average wet weight and GAG content of co-culture group were both more than 80% of those of positive control group. In negative control group, however, the constructs shrunk gradually during in vitro culture and cartilage-like tissue could only be observed at the edge area of the construct. In low concentration chondrocyte group, the constructs also shrunk gradually during in vitro culture and the average wet weight was below 40% of that of the positive control group although histology showed a small amount cartilage formation. CONCLUSION: Chondrocytes can provide a chondrogenic microenvironment to induce a chondrogenic differentiation of BMSC and thus promote the in vitro chondrogenesis of BMSC.

[Repairing porcine knee joint osteochondral defects at non-weight bearing area by autologous BMSC].

OBJECTIVE: To test the possibility of using bone marrow stromal cells (BMSC) and biodegradable polymers to repair articular osteochondral defects at non-weight bearing area of porcine knee joints. METHODS: Bone marrows were harvested from 18 hybrid pigs. BMSC were cultured and in vitro expanded and induced with dexamethasone (group A) or with dexamethasone and transforming growth factor-beta1 (TGF-beta1) (group B) respectively. Immunohistochemistry and RT-PCR were used to evaluate chondrogenic differentiation of induced cells. Part of BMSC of 2 animals were retrovirally-labeled with green fluorescent protein (GFP). After induction and label, cells were seeded on a construct of polyglycolic acid (PGA) and polylactic acid (PLA) and co-cultured for 1 week before implantation. Total 4 osteochondral defects (8 mm in diameter, 5 mm in depth) in each animal were created at the non-weight bearing areas of knee joints on both sides. The defects were repaired with dexamethasone induced BMSC-PGA/PLA construct in group A, with dexamethasone and TGF-beta1 induced BMSC-PGA/PLA construct in group B, with PGA/PLA construct alone (group C) or left untreated (group D) as controls. Animals were sacrificed at 3 months (n = 6) or 6 months (n = 10) post-repair. Gross observation, histology, glycosaminoglycan (GAG) quantification and biomechanical test were applied to analyze the results. The two animals with GFP-labeled cells were sacrificed at 7 months post-repair to observe with confocal microscope the distribution of GFP-labeled cells in repaired tissue. RESULTS: Stronger expression of type II collagen and aggrecan were observed in BMSCs induced with both dexamethasone and TGF-beta1. At both time points, Gross observation and histology showed that the defects in most of group A were repaired by engineered fibrocartilage and cancellous bone with an irregular surface, minority defects were repaired by engineered hyaline cartilage and cancellous bone. However, in most of group B, the defects were completely repaired by engineered hyaline cartilage and cancellous bone. No repair or only fibrous tissue were observed in groups C and D. Besides, the compressive moduli of repaired cartilage in groups A and B reached 30.37% and 43.82% of normal amount at 3 months and 62.69% and 80.27% at 6 months respectively, which was further supported by the high levels of GAG contents in engineered cartilage of group A (78.03% of normal contents) and group B (no statistical difference from normal contents). More importantly, confocal microscope revealed the presence of GFP-labeled cells in engineered cartilage lacuna and repaired underlying cancellous bone. CONCLUSION: The results demonstrated that implanted BMSC can differentiate into either chondrocytes or osteoblasts at different local environments and repair a complex articular defect with both engineered cartilage and bone. TGF-beta1 and dexamethasone in vitro induction can promote chondrogenic differentiation of BMSC and thus improve the results of repairing articular defects.

[Influence of mechanical stress on chondrogenesis of in vitro cultured porcine bone marrow stem cells: a preliminary study].

OBJECTIVE: To evaluate the influence of mechanical stress on chondrogenesis of in vitro cultured porcine bone marrow stem cells (BMSC). METHODS: Porcine BMSC of passage 2 were seeded onto a cylinder-shaped PGA/PLA scaffold, 8mm in diameter and 3mm in thickness, at a density of 5 x 10(7)/cm(3). After the cell-scaffold constructs were cultured for one week, the primary medium, high-glucose DMEM medium with 10% fetal bovine serum (FBS), was replaced by chondrogenically inductive medium containing TGFbeta(1) (10 ng/ml), IGF-I (50 ng/ml), and dexamethasone (40 ng/ml) in addition to DMEM+10% FBS. The constructs were randomly divided into three groups according to the imposed stress: experimental group A in which a centrifugal stress was imposed at 100 g, 30 min, 2/d; experimental group B in which a rotative stress was imposed at 80 rpm, 8 h/d by a shaker; and control group in which the constructs were statically cultured. The gross view, histology, histochemistry, immunohistochemistry and glycosaminoglycan (GAG) content were evaluated after 4 and 8 weeks respectively. RESULTS: Four weeks later, the constructs in both experimental groups maintained their original sizes and shapes. Histology showed nodular lacuna-like structures, in company with GAG deposition and collagen synthesis. In addition, collagen type II was detected by immunohistochemistry. In the control group, however, the constructs shrunk to a little smaller size than those in the experimental groups, and histological staining showed a little amount of lacuna. Eight weeks later, the constructs in both experimental groups still maintained the original sizes and shapes with good elasticity. HE staining showed massive lacuna-like structures in most areas of the construct and extracellular matrix deposited evenly. Fibrous tissues were only observed in some areas. Safranin-O staining showed massive GAG formation and Masson staining showed much more collagen formation than those in the control group. Immunohistochemical staining of collagen type II showed strong positive expression. In the control group the constructs showed massive fibrous tissues, with a small amount of lacuna-like structures in the peripheral areas. GAG contents in the 2 experimental groups were 5.98 mg/g and 5.62 mg/g respectively, both significantly higher than that in the control group (4.73 mg/g) without a difference between the 2 experimental groups. CONCLUSION: Mechanical stress promotes chondrogenesis and cartilage maturation of BMSC in vitro.