Steve Wn Ueng

Involvement of Gas7 along the ERK1/2 MAP kinase and SOX9 pathway in chondrogenesis of human marrow-derived mesenchymal stem cells

OBJECTIVE The growth-arrest-specific protein, Gas7, has been shown to be involved in reorganization of the cytoskeleton and for inducing changes in cell shape during cell differentiation. The goals of this study were to investigate the novel role of human Gas7 (hGas7) in chondrogenic differentiation of human mesenchymal stem cells (hMSCs) and to identify the relationship between hGas7, extracellular signal-regulated kinase (ERK1/2) and SOX9 in the chondrogenic pathway. METHODS Bone marrow-derived hMSCs were induced to undergo chondrogenic differentiation with transforming growth factor-beta1 (TGF-beta1) in an aggregate culture system. The expression of hGas7 and SOX9 and phosphorylation of ERK1/2 at multiple time points were investigated. Chondrogenic capacity was evaluated by the size of aggregates, by glycosaminoglycan content, and by type II collagen and proteoglycan deposition after interfering with expression of hGas7, ERK1/2 or SOX9. To delineate the functional role of these genes in chondrogenesis, inhibition of individual genes expression in hMSCs, by antisense oligonucleotides or interference RNA (siRNA), and the effect on chondrogenic differentiation were also investigated. RESULTS Treatment of hMSCs with TGF-beta1 resulted in a transient up-regulation of hGas7b, one of the hGas7 isoforms (day 3-day 5), a transient phosphorylation of ERK1/2 (0.5-4 h) and an up-regulation of SOX9 (2 h to day 14). Transient expression of hGas7b was also detected in hMSCs by reverse transcription-polymerase chain reaction at day 2 and day 3 following TGF-beta1 treatment. Interference with hGas7b production by hGas7b-specific antisense oligonucleotide or inhibition of p-ERK with PD98059, a specific inhibitor of ERK signaling pathway, or interference with SOX9 production by SOX9 siRNA all caused adverse effects of chondrogenic differentiation of hMSCs. Meanwhile, inhibition of p-ERK or SOX9 both blocked the expression of hGas7b. However, the p-ERK and SOX9 pathway was not affected by inhibition of hGas7b. CONCLUSION These results provide evidence that the transient expression of hGas7b, regulated by activation of ERK1/2 and SOX9 pathway, is essential for chondrogenic differentiation of hMSCs.

Elevated intraosseous pressure in the intertrochanteric region is associated with poorer results in osteonecrosis of the femoral head treated by multiple drilling

Multiple drilling is reported to be an effective treatment for osteonecrosis of the head of femur, but its effect on intra-osseous pressure has not been described. We undertook multiple drilling and recorded the intra-osseous pressure in 75 osteonecrotic hips in 60 patients with a mean age of 42 years (19 to 67). At a mean follow-up of 37.1 months (24 to 60), 42 hips (56%) had a clinically successful outcome. The procedure was effective in reducing the mean intra-osseous pressure from 57 mmHg (SD 22) to 16 mmHg (SD 9). Hips with a successful outcome had a mean pressure of 26 mmHg (SD 19). It was less effective in preventing progression of osteonecrosis in hips with considerable involvement and in those with a high intra-osseous pressure in the intertrochanteric region (mean 45 mmHg (SD 25)). This study is not able to answer whether a return of the intra-osseous pressure to normal levels is required for satisfactory healing.

Effects of hyperbaric oxygen on the osteogenic differentiation of mesenchymal stem cells

BackgroundHyperbaric oxygenation was shown to increase bone healing in a rabbit model. However, little is known about the regulatory factors and molecular mechanism involved.We hypothesized that the effect of hyperbaric oxygen (HBO) on bone formation is mediated via increases in the osteogenic differentiation of mesenchymal stem cells (MSCs) which are regulated by Wnt signaling.MethodsThe phenotypic characterization of the MSCs was analyzed by flow cytometric analysis. To investigate the effects of HBO on Wnt signaling and osteogenic differentiation of MSCs, mRNA and protein levels of Wnt3a, beta-catenin, GSK-3beta, Runx 2, as well as alkaline phosphatase activity, calcium deposition, and the intensity of von Kossa staining were analyzed after HBO treatment. To investigate the effects of HBO on Wnt processing and secretion, the expression of Wntless and vacuolar ATPases were quantified after HBO treatment.ResultsCells expressed MSC markers such as CD105, CD146, and STRO-1. The mRNA and protein levels of Wnt3a, β-catenin, and Runx 2 were up-regulated, while GSK-3β was down-regulated after HBO treatment. Western blot analysis showed an increased β-catenin translocation with a subsequent stimulation of the expression of target genes after HBO treatment. The above observation was confirmed by small interfering (si)RNA treatment. HBO significantly increased alkaline phosphatase activity, calcium deposition, and the intensity of von Kossa staining of osteogenically differentiated MSCs. We further showed that HBO treatment increased the expression of Wntless, a retromer trafficking protein, and vacuolar ATPases to stimulate Wnt processing and secretion, and the effect was confirmed by siRNA treatment.ConclusionsHBO treatment increased osteogenic differentiation of MSCs via regulating Wnt processing, secretion, and signaling.

The concentration of antibiotic in fresh-frozen bone graft

We investigated the antibiotic concentration in fresh-frozen femoral head allografts harvested from two groups of living donors. Ten samples were collected from patients with osteoarthritis of the hip and ten from those with a fracture of the neck of the femur scheduled for primary arthroplasty. Cefazolin (1 g) was administered as a pre-operative prophylactic antibiotic. After storage at -80 degrees C for two weeks the pattern of release of cefazolin from morsellised femoral heads was evaluated by an in vitro broth elution assay using high-performance liquid chromatography. The bioactivity of the bone was further determined with an agar disc diffusion and standardised tube dilution bioassay. The results indicated that the fresh-frozen femoral heads contained cefazolin. The morsellised bone released cefazolin for up to four days. The concentration of cefazolin was significantly higher in the heads from patients with osteoarthritis of the hip than in those with a fracture.Also, in bioassays the bone showed inhibitory effects against bacteria.We concluded that allografts of morsellised bone from the femoral head harvested from patients undergoing arthroplasty of the hip contained cefazolin, which had been administered pre-operatively and they exhibited inhibitory effects against bacteria in vitro.

Effects of low-intensity pulsed ultrasound and hyperbaric oxygen on human osteoarthritic chondrocytes

BackgroundAlthough the individual effects of hyperbaric oxygen (HBO) and low-intensity pulsed ultrasound (LIPUS) on osteoarthritic (OA) chondrocytes have been reported, the effects of HBO combined with LIPUS treatment are unknown.MethodsOA chondrocytes were obtained from patients undergoing knee replacement surgery. RNA was isolated for real-time polymerase chain reaction (PCR) analysis of inducible nitric oxide synthase (iNOS), type-II collagen, and aggrecan gene expression. The protein levels of MMP-3 and TIMP-1 were quantified by enzyme-linked immunosorbent assay (ELISA) after LIPUS or HBO treatment. The data are given as mean ± standard deviation (SD) of the results from three independent experiments. A p value less than 0.05 was defined as statistically significant.ResultsOur data suggested that ultrasound and HBO treatment increased cell bioactivity of OA chondrocytes. Real-time PCR analysis showed that HBO treatment increased the mRNA of type-II collagen, aggrecan, and TIMP-1 but suppressed the iNOS expression of OA chondrocytes. LIPUS treatment increased the type-II collagen and iNOS expression of OA chondrocytes. ELISA data showed that HBO or LIPUS treatment increased TIMP-1 production of OA chondrocyte. MMP-3 production was suppressed by HBO treatment. HBO combined with LIPUS treatments resulted in additive effect in TIMP-1 production and compensatory effect in iNOS expression.ConclusionHBO combined with LIPUS treatment-induced increase of the anabolic factor (TIMP-1)/catabolic factor (MMP-3) ratio may provide an additive therapeutic approach to slow the course of OA degeneration.

Shear stresses modulate chondrogenic and osteogenic genes expression in human bone marrow derived stem cells

Background: Mechanical stimuli in the microenvironment may provide cues for differentiation, maintenance of phenotypes, and extracellular matrix homeostasis. This study investigated bone marrow-derived stem cells (BMSCs) response to mechanical stresses in terms of differentiation and the extracellular matrix/matrix metalloproteinase genes expression. Materials & Methods: Human BMSCs were exposed to shear stresses (20 rpm or 50 rpm) for 2, 5, or 10 days with or without osteogenic induction medium. The expression of chondrogenic (type II collagen and aggrecan) and osteogenic (type I collagen, alkaline phosphatase, osteopontin, osteocalcin) phenotypic genes and matrix metalloproteinase genes (aggrecanse, MMP-2, and MMP-9) were analyzed by reverse transcription polymerase chain reaction. In vitro differentiation of BMSCs after shear stress treatment were analyzed by von Kossa staining on monolayer cultures and safarin-O staining on pelleted cells. Results: Shear stress at 20 rpm significantly upregulatedexpression of type II collagen on the 5th day, type I collagen on the 2nd, 5th, and 10th day, osteopontin on the 2nd and 5th day, and osteocalcin on the 5th day as compared to shear stress at 50 rpm. Combination of osteogenic induction medium and shear stress at 20 rpm decreased the mRNA expression levels of chondrogenic genes (type II collagen on all time points and aggrecan on the 5th and 10th day) as compared with shear stress alone. It also decreased the expression of osteogenic genes (type I collagen, alkaline phosphatase, and osteopontin) on the 10th day as compared to osteogenic induction medium. Concomitantly, shear stress upregulated aggrecanase on the 10th day, MMP-2 on the 10th day, and MMP-9 on the 5th and 10th day as compared with osteogenic induction medium. Shear stress increased mineralization of the extracellular matrix as shown by von Kossa staining on the 2nd, 5th, and 10th day. Chondrogenic differentiation by cell pellet analysis found similar amount of proteoglycans but less organized extracellular matrix in shear stress-treated BMSCs as compared to control cells. Discussion: Although shear stress could modulate chondrogenic gene expression as well as osteogenic genes expression in BMSCs, the combination of shear stress inhibited the expression of osteogenic phenotypic genes induced by osteogenic induction medium. Parallel studies revealed that shear stress concomitantly modulate the expression of extracellular matrix genes and the matrix metalloproteinase genes. In vitro differentiation analysis found that shear stress may enhance osteogenesis but interfere with chondrogenesis. Our results may provide data for the design or development of in vitro bioreactor systems to control lineage differentiation of stem cells for tissue engineering. More studies on the mechanotransduction pathways are needed in the future.

Non-tuberculous cold abscess of the psoas muscle : an unusual manifestation of colocutaneous fistula

Abstract We report here a case of colocutaneous fistula drained from the retroperitoneal space mimicking a cold abscess of the psoas muscle. A 60-year-old diabetic woman with a 6-year history of a chronic draining sinus over her right thigh had been treated intermittently with antibiotics. At presentation, she had no systemic toxic signs nor other constitutional symptoms. The patient was inadequately managed by curettage at first under the tentative diagnosis of tuberculous cold abscess. After the correct diagnosis of colocutaneous fistula, right nephrectomy and right hemicolectomy with ileotransverse colostomy were done. The patient was well 5 years later without recurrence. This is an atypical presentation of enterocutaneous fistula in an immunodeficient patient that should be emphasized to facilitate the correct diagnosis and early treatment.