The effects of amphiregulin induced MMP-13 production in human osteoarthritis synovial fibroblast.

Osteoarthritis (OA) belongs to a group of degenerative diseases. Synovial inflammation, cartilage abrasion, and subchondral sclerosis are characteristics of OA. Researchers do not fully understand the exact etiology of OA. However, matrix metalloproteinases (MMPs), which are responsible for cartilage matrix degradation, play a pivotal role in the progression of OA. Amphiregulin (AREG) binds to the EGF receptor (EGFR) and activates downstream proteins. AREG is involved in a variety of pathological processes, such as the development of tumors, inflammatory diseases, and rheumatoid arthritis. However, the relationship between AREG and MMP-13 in OA synovial fibroblasts (SFs) remains unclear. We investigated the signaling pathway involved in AREG-induced MMP-13 production in SFs. AREG caused MMP-13 production in a concentration- and time-dependent manner. The results of using pharmacological inhibitors and EGFR siRNA to block EGFR revealed that the EGFR receptor was involved in the AREG-mediated upregulation of MMP-13. AREG-mediated MMP-13 production was attenuated by PI3K and Akt inhibitors. The stimulation of cells by using AREG activated p65 phosphorylation and p65 translocation from the cytosol to the nucleus. Our results provide evidence that AREG acts through the EGFR and activates PI3K, Akt, and finally NF-kappaB on the MMP-13 promoter, thus contributing to cartilage destruction during osteoarthritis.

Hypoxia enhances chondrogenesis and prevents terminal differentiation through PI3K/Akt/FoxO dependent anti-apoptotic effect.

Hypoxia, a common environmental condition, influences cell signals and functions. Here, we compared the effects of hypoxia (1% oxygen) and normoxia (air) on chondrogenic differentiation of human mesenchymal stem cells (MSCs). For in vitro chondrogenic differentiation, MSCs were concentrated to form pellets and subjected to conditions appropriate for chondrogenic differentiation under normoxia and hypoxia, followed by the analysis for the expression of genes and proteins of chondrogenesis and endochondral ossification. MSCs induced for differentiation under hypoxia increased in chondrogenesis, but decreased in endochondral ossification compared to those under normoxia. MSCs induced for differentiation were more resistant to apoptosis under hypoxia compared to those under normoxia. The hypoxia-dependent protection of MSCs from chondrogenesis-induced apoptosis correlated with an increase in the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/FoxO pathway. These results suggest that the PI3K/Akt/FoxO survival pathway activated by hypoxia in MSCs enhances chondrogenesis and plays an important role in preventing endochondral ossification.

Injectable laminin-functionalized hydrogel for nucleus pulposus regeneration.

Cell delivery to the pathological intervertebral disc (IVD) has significant therapeutic potential for enhancing IVD regeneration. The development of injectable biomaterials that retain delivered cells, promote cell survival, and maintain or promote an NP cell phenotype in vivo remains a significant challenge. Previous studies have demonstrated NP cell - laminin interactions in the nucleus pulposus (NP) region of the IVD that promote cell attachment and biosynthesis. These findings suggest that incorporating laminin ligands into carriers for cell delivery may be beneficial for promoting NP cell survival and phenotype. Here, an injectable, laminin-111 functionalized poly(ethylene glycol) (PEG-LM111) hydrogel was developed as a biomaterial carrier for cell delivery to the IVD. We evaluated the mechanical properties of the PEG-LM111 hydrogel, and its ability to retain delivered cells in the IVD space. Gelation occurred in approximately 20 min without an initiator, with dynamic shear moduli in the range of 0.9-1.4 kPa. Primary NP cell retention in cultured IVD explants was significantly higher over 14 days when cells were delivered within a PEG-LM111 carrier, as compared to cells in liquid suspension. Together, these results suggest this injectable laminin-functionalized biomaterial may be an easy to use carrier for delivering cells to the IVD.

Isolation of mesenchymal stem cells from human ligamentum flavum: implicating etiology of ligamentum flavum hypertrophy.

STUDY DESIGN: To demonstrate the existence of mesenchymal stem cells (MSCs) in ligamentum flavum (LF) and their pathogenic role in LF hypertrophy. OBJECTIVE: To isolate and characterize LF-derived MSCs and their response to transforming growth factor-beta 1 (TGF-ß1) and trichostatin A (TSA), a histone deacetylase inhibitor (HDACi). SUMMARY OF BACKGROUND DATA: LF is a connective tissue, of which hypertrophic changes induce spinal stenosis. The pathogenic role of TGF-ß1 in spinal stenosis has been implicated. TSA has been shown to suppress TGF-ß1-induced alpha-smooth muscle actin (α-SMA), type I and III collagen synthesis in a variety of cells. MSCs have been isolated from a variety of adult tissues, except LF. Whether MSCs exist in LF and their response to TGF-ß1 and TSA is not clear. METHODS: The MSCs from LF were isolated and cultured. Their phenotypic character, linage differentiation potential, and response to TGF-ß1 and TSA were analyzed. RESULTS: LF-derived MSCs have the similar profile of surface markers as bone marrow MSCs. They were demonstrated to have the potential to be differentiated into osteoblasts, adipocytes, and chondrocytes. Administration of TGF-ß1 stimulated cell proliferation, enhanced the gene expression of type I and III collagen, and increased the gene expression and protein level of α-SMA. TSA blocked the fibrogenic effects of TGF-ß1. CONCLUSION: The current results demonstrated the isolation of MSCs from LF. The cellular response to TGF-ß1 implied that these cells might play an important role in the pathogenesis of LF hypertrophy. TSA, which blocks the effects of TGF-ß1, may be a potent therapeutic choice for inhibiting LF hypertrophy.

Trichostatin A inhibits TGF-ß1 induced in vitro chondrogenesis of hMSCs through Sp1 suppression.

Trichostatin A (TSA) is a histone deacetylase inhibitor (HDACi) known to modulate differentiation of many cells. However, its effect on chondrogenesis remains elusive. This study was aimed to investigate the effects of TSA on in vitro transforming growth factor-ß1 (TGF-ß1)-induced chondrogenesis of human mesenchymal stem cells (hMSCs). The pellet cultures of hMSCs in a chondrogenic medium were exposed to TGF-ß1 and TSA. Quantitative reverse transcription/polymerase chain reaction (PCR) analysis, Alcian blue staining, and immunohistochemistry staining were used to confirm and compare the differences in chondrogenesis by analyzing the mRNA of chondrogenic genes (Sox9, Aggrecan, and Col2A1), synthesis of chondrogenic proteins and type II collagen, respectively. TGF-ß1 signaling and its downstream targets were determined by western blot analysis. TGF-ß1 led to significant increases in chondrogenic gene expression and the synthesis of chondrogenic proteins. However, TSA significantly decreased chondrogenic gene expression and the synthesis of chondrogenic proteins in a dose-dependent manner. TGF-ß1 increased phosphorylation of Smad 2/3 and Sp1 expression around half an hour after induction. The increase of Sp1, but not Smad 2/3 activation was almost completely blocked by the addition of TSA. The chondrogenic effect of TGF-ß1 was also suppressed by the Sp1-binding inhibitor mithramycin A. Finally, overexpression of Sp1 abolished TSA-mediated inhibition of TGF-ß1-induced chondrogenesis. Our study showed that TSA inhibited chondrogenesis through inhibition of TGF-ß1-induced Sp1 expression. Furthermore, Sp1 could be a useful tool in future studies looking into biological mechanisms by which chondrogenesis of hMSCs can be augmented, especially in the area of clinical application.

Treatment of congenital scoliosis with single-level hemivertebrae.

INTRODUCTION: The natural history of congenital scoliosis with hemivertebrae is unpredictable and the management is also controversial. MATERIALS AND METHODS: Between 1986 and 2004, 22 patients (eight male and fourteen female, mean 19.3 years old) with single-level hemivertebrae related congenital scoliosis underwent non-operative or operative treatment at our institution with an average follow-up period of 8.8 years. RESULTS: Only a 5 degrees curve progression was noted in upper thoracic hemivertebrae after followed up 6 years. By one stage combined anterior hemivertebrae excision, posterior instrumentation, and arthrodesis, up to 61% curve correction can be achieved. Posterior instrumentation, correction and arthrodesis showed a 25% correction. The result of pain relief is promising in skeletal-matured patients. CONCLUSIONS: Surgical instrumentation, correction and arthrodesis showed good results. The optimal treatment of choice may differed from one to the other.

Diaphyseal locking hip arthroplasty for treatment of failed fixation of intertrochanteric hip fractures.

Between 1997 and 2004, 18 patients (8 men and 10 women, with a mean age of 73.2 years) with failed treatment of intertrochanteric hip fractures underwent hip arthroplasty as salvage procedures at our institution. Cementless, 5/8-porous coated, 6-in. primary diaphyseal locking femoral stems were used. Prospective follow-up ranging from 2 to 5 years (mean, 37.1 months) showed improvement of hip function without prosthesis loosening. Complications included 1 case of postoperative infection, 2 cases of dislocation, and 2 cases of stem subsidence. The clinical results were satisfactory. The 5/8-porous coated, 6-in. cementless femoral stems could be used in the salvage procedures for failed fixation of intertrochanteric hip fractures.