Clinical value of polymerase chain reaction in the diagnosis of joint tuberculosis by detecting the DNA of Mycobacterium tuberculosis.

OBJECTIVE: To assess the clinical value of polymerase chain reaction (PCR) in the diagnosis and differential diagnosis of joint tuberculosis (TB). METHODS: PCR was used blindly to detect the DNA of Mycobacterium tuberculosis (M.TB) in five specimens of M.TB, 5 of BCG, and 10 of other bacteria. Then, M. TB in 98 samples from patients with joint TB and 100 samples from patients with non-tubercular joint disorders were detected by PCR, acid-fast staining and culture,. The sensitivity, specificity, accuracy, positive predictive value, and negative predictive value of PCR were calculated. The χ2 test was used for statistical analysis of the frequency of various factors. At the same time, some problems with PCR were also systematically analyzed. RESULTS: (1) In the "standard samples", both M. TB and BCG showed positive while other bacteria were negative. (2) In 98 cases from patients with joint TB, 81 were positive by PCR, 6 by acid-fast staining, and 17 by culture. In 100 cases from patients with non-tuberculous joint disorders, 9 were positive by PCR, and none by either acid-fast staining or culture. Sensitivity, specificity, accuracy, positive and negative predictive value of PCR were 82.65% (81/98), 91.00% (91/100), 86.87% (172/198), 90.00% (81/90) and 84.26% (91/108), respectively. (3) The positive rates for PCR, acid-fast staining and culture in detection of M. TB were 82.65% (81/98), 6.12% (6/98), and 17.34% (17/98), respectively. There were statistically significant differences between the three methods (P < 0.001). (4) The process of PCR is automatic, and can be completed within 3 to 6 hours, whereas 4 to 8 weeks are required for the conventional culture of M. TB. CONCLUSION: PCR is a sensitive, specific, rapid, simple and minimally invasive method for detection of M. TB in samples from joint TB, and can play an important role in early and rapid diagnosis and differential diagnosis of joint TB. But it also has some limitations, such as false positivity and false negativity.

[Comparison study on polymerase chain reaction (PCR) and standard culture technique in detecting mycobacterium tuberculosis to diagnose of joint tuberculosis].

OBJECTIVE: To study the role of PCR technique in detection of mycobacterium tuberculosis in the samples from joint tuberculosis, and to evaluate the clinical value of PCR in diagnosis of joint tuberculosis. METHODS: From June 1993 to August 2001, PCR was used to detect DNA of mycobacterium tuberculosis, and the standard culture was applied to detect mycobacterium tuberculosis. Mycobacterium tuberculosis were respectively blindly by the two techniques in the samples obtained from 95 patients with joint tuberculosis (55 males and 40 females, the age ranging from 2 to 75 years, with an average of 34 years). The positive rate of mycobacterium tuberculosis detection was calculated. RESULTS: In the detection of mycobacterium tuberculosis, positive rate was 82% (78/95) in PCR technique, and 16% (15/95) in standard culture technique. There were statistical differences between the two groups (chi2=67, P<0.001). The whole process of PCR amplification was automatic and could be finished within several hours, and the detecting time was considerably shorter. CONCLUSION: PCR technique is a rapid, simple, sensitive and specific method for detection of mycobacterium tuberculosis in the samples of joint tuberculosis, showing more marked advantages than the standard culture technique. It is valuable in the early rapid diagnosis and differential diagnosis of joint tuberculosis.

Tissue engineered cartilage from hTGF beta2 transduced human adipose derived stem cells seeded in PLGA/alginate compound in vitro and in vivo.

Chondrogenic potential of human adipose derived stem cells (hASCs) makes them a possible source of seeding cells for cartilage tissue engineering. We aim to examine the chondrogenic differentiation of human transforming growth factor beta2 (hTGF beta2) transduced hASCs seeded in three-dimensional scaffold in vitro and in vivo. In this study, hASCs were isolated from human subcutaneous adipose tissue and transduced with a replication deficient adenovirus carrying hTGF beta2 (Ad5-hTGF beta2), and then the transduced cells were seeded and cultured in PLGA/alginate compounds. RT-PCR analysis revealed that Ad5-hTGF beta2 transduced hASCs produced aggrecan and collagen type II after 7-day induction in vitro and continued throughout the culture period; this was also demonstrated by the positive staining of Alcian blue and immunohistochemistry for collagen type II. For in vivo study, Ad5-hTGF beta2 transduced hASCs seeded in PLGA/alginate compounds were implanted in subcutaneous pockets of nude mice; after 12 weeks, the implants were harvested and examined by haematoxylin and eosin staining, AB-PAS staining, and immunohistochemical analysis, and the results demonstrated the formation of cartilage tissue. As a control, all these were not observed in the constructs with Ad5-EGFP transduced hASCs. In conclusion, our study demonstrates that adenovirus-mediated hTGF beta2 gene transfer is able to induce the hASCs into chondrogenic lineage both in vitro and in vivo. Ad5-hTGF beta2 transduced hASCs combined with three-dimensional PLGA/alginate compound may be a viable method in treating injuries of cartilage.

Ectopic neocartilage formation from predifferentiated human adipose derived stem cells induced by adenoviral-mediated transfer of hTGF beta2.

Chondrogenic potential of human adipose derived stem cells (hASCs) makes them a possible source of seeding cells for cartilage tissue engineering. In this study, chondrogenic differentiation of hASCs induced by transduction with replication-deficient adenovirus carrying human transforming growth factor beta2 (Ad5-hTGF beta2) was demonstrated by RT-PCR, immunohistochemistry staining, biochemical and western blot analysis. To evaluate if the in vitro differentiated hASCs could keep their chondrocytic phenotype and produce neo-cartilage in vivo, predifferentiated hASCs were seeded in different scaffolds and implanted in subcutaneous pockets on the dorsum of nude mice. After 4 and 12 weeks culture in vivo, specimens were harvested and examined by histological and immunohistochemical analysis, cartilage-like tissue formation was only found in alginate gel and PLGA/alginate compound groups, in PLGA group, fibrous tissues and angiogenesis ingrowth were observed. These findings demonstrated that adenovirus-mediated hTGF beta2 gene transfer could induce hASCs into a chondrogenic lineage in vitro, however, this predifferentiation did not guarantee ectopic cartilage formation in vivo unless appropriate three-dimensional scaffolds were used as the cell carry vehicles.

Neocartilage formation from predifferentiated human adipose derived stem cells in vivo.

AIM: To examine the chondrogenic potential of human adipose derived stem cells (hASC) induced by human transforming growth factor beta2 (hTGF beta2) in vitro, and to investigate if predifferentiated hASC can produce neocartilage in vivo. METHODS: hASC were isolated from subcutaneous adipose tissue and cultured in pellets with the addition of hTGF beta2. Chondrogenic differentiation was assayed by RT-PCR, Western blotting, toluidine blue staining, and immunohistochemistry staining for collagen type II. For the in vivo study, intact induced cell pellets or the released cells embedded in alginate gel with different concentrations were implanted subcutaneously in nude mice. Specimens were harvested at different time points and carried with histological and immunohistochemistry examination to evaluate the cartilage formation. RESULTS: RT-PCR analysis revealed that hASC produced aggrecan and collagen type II after 7 d of induction and continued throughout the culture period. This was also demonstrated by the Western blot analysis, positive staining of toluidine blue, and immunohistochemistry for collagen type II. After reseeding in the monolayer, the cells isolated from the pellets displayed a polygonal morphology compared with the primary spindle shape. hASC were released from the induced cell pellets when embedded in alginate gel (implanted cell concentration=5X10(6) /mL or higher). They produced neocartilage after 12 weeks in vivo culture; however, intact induced cell pellets implanted subcutaneously rapidly lost their differentiated phenotype. CONCLUSION: Chondrogenesis of hASC in vitro can be induced by combining pellet culture and hTGF beta2 treatment. Predifferentiated hASC embedded in alginate gel have the ability of producing neocartilage in vivo.

[A multi-central, randomized, controlled clinical trial of glucosamine hydrochloride/sulfate in the treatment of knee osteoarthritis].

OBJECTIVE: To evaluate the efficacy and safety of glucosamine hydrochloride (GH) in the treatment of patients with knee osteoarthritis (OA) comparing with glucosamine sulfate (GS). METHODS: A multi-central, randomized, parallel-controlled clinical trial of GH vs GS was performed. 142 patients suffering from knee OA were randomized into 2 groups, treated with GH and GS for 480 mg and 500 mg one time respectively. Patients received medicine 3 times daily with total 1440 mg per day for GH group and 1500 mg for GS group, orally for 4 weeks and were assessed for drug efficacy and safety for 2 more weeks. Knee pain at rest, at movement and at pressure, knee swelling, morning stiffness and walking ability were recorded at 1, 2, 3, 4 and 6 week according to Lequesne's criteria. Other features such as therapeutic efficacy, adverse events and laboratory parameters were also recorded. RESULTS: A considerable improvement in OA symptoms and a reduce of total Lequesne's score were observed in both groups after the 4-week treatment. The Lequesne's score decreased to 3.4 +/- 1.9 (P < 0.05) and 3.4 +/- 1.8 (P < 0.05) after 4-week treatment comparing to 0-week in the GH (9.4 +/- 1.8) group and GS (9.5 +/- 1.4) group respectively. There was no significant difference between 2 groups in decreasing the Lequesne's score (P > 0.05). The symptomatic improvement rates in patients were 91.4% and 90.0% at 4-week treatment in the GH and GS group respectively (P > 0.05). There was a remnant therapeutic effect in both groups at 2 weeks after discontinuation of the treatment. And 4.2% (3/71) of patients on GH reported adverse events, vs 7.0% (5/71) adverse events with GS group (P > 0.05), mainly mild stomach discomfort and constipation. CONCLUSION: There were no significant differences in efficacy and safety between GH and GS groups in the treatment of knee OA. Glucosamine hydrochloride is as effective and safe as glucosamine sulfate.

Expression of PDCD5, a novel apoptosis related protein, in human osteoarthritic cartilage.

AIM: To investigate the expression features of PDCD5 (programmed cell death 5 protein) in osteoarthritic and normal human cartilage, and speculate on its potential functions in the pathogenesis of osteoarthritis (OA). METHODS: Articular cartilage specimens were obtained from 30 patients with OA and 16 healthy patients at the time of arthroplasty. Expression of PDCD5 was detected by flow cytometry, immunofluorescence, RT-PCR and immunohistochemical analysis. RESULTS: Enhanced expression and nuclear accumulation of PDCD5 in OA chondrocytes were found. PDCD5-positive chondrocytes were mainly distributed in the superficial and deep zones of OA tissue sections, as opposed to, in the superficial and middle regions of normal healthy tissue sections. CONCLUSION: Since apoptotic chondrocyte death occurs more frequently in OA cartilage than in normal healthy cartilage and PDCD5 is an apoptosis-related protein, the different expression patterns of PDCD5 in OA cartilage from that in normal healthy cartilage indicate that PDCD5 is involved in the pathogenesis of OA.

Effect of hydroxyurea and etoposide on transduction of human bone marrow mesenchymal stem and progenitor cell by adeno-associated virus vectors.

AIM: To study the effect of hydroxyurea and etoposide on transduction of human marrow mesenchymal and progenitor stem cells by adeno-associated virus (AAV). METHODS: Isolated human bone marrow mesenchymal stem and progenitor cells (hMSCs) were cultured in DMEM containing 10% FBS or 5% FBS and dexamethasone 1 micromol/L respectively. After being treated with hydroxyurea and etoposide, hMSCs were transduced by AAV-LUC. After two days luciferase activity (relative light unites per second or RLU/s) were tested, which indirectly reflected the relative transduction efficiency of different groups, and virus DNA was isolated by Hirt extraction for Southern hybridization. RESULTS: Transduction luciferase activity and transduction efficiency in cultures treated with hydroxyurea and etoposide were significantly higher than that in control cultures. Dividing cells had about 20-fold higher transduction efficiency compared with control cells. Transduction efficiency in stationary cells was about 50 times higher than that in control cells. Southern analysis showed that hydroxyurea and etoposide enhanced second-strand DNA synthesis by rAAV. CONCLUSION: Hydroxyurea and etoposide could increase transduction efficiency of hMSCs by AAV vectors, and stationary cells were more sensitive to these drugs than dividing cells.

In vitro chondrogenesis of human bone marrow-derived mesenchymal progenitor cells in monolayer culture: activation by transfection with TGF-beta2.

Bone marrow-derived mesenchymal progenitor cells are capable of chondrogenesis, making them a possible source of cells for cartilage tissue engineering. Because of this, we studied the effect of human transforming growth factor beta2 (TGF-beta2) on mesenchymal progenitor cell chondrogenesis in monolayer culture using gene transfection technology. A recombinant pcDNA3.1(+)/TGF-beta2 construct containing a full-length TGF-beta2 from a human placental cDNA library was created through gene cloning and DNA recombination. The construct was then lipofected into mesenchymal progenitor cells isolated from human bone marrow. RT-PCR, Western blotting, and immunohistochemistry analyses were performed to identify the expression of TGF-beta2 and cartilage-associated genes and proteins. The results showed that TGF-beta2 was expressed throughout the culture period. The transfected progenitor cells expressed and produced collagen type II and aggrecan 48 h after transfection, and the expression and synthesis were upregulated after 4 weeks. In contrast, the tested genes and proteins were not detected in non-transfected cells. This shows that transfection of pcDNA3.1(+)/TGF-beta2 into mesenchymal progenitor cells is able to provide transient and persistent expression of cartilage-specific genes and proteins, and suggests that the differentiation of human marrow-derived mesenchymal progenitor cells into chondrocytes in monolayer culture is feasible and may be induced by TGF-beta2.