HOX gene analysis in the osteogenic differentiation of human mesenchymal stem cells

Human bone marrow-derived mesenchymal stem cells (hMSCs) have the capacity to differentiate into osteoblasts during osteogenesis. Several studies attempted to identify osteogenesis-related genes in hMSCs. Although HOX genes are known to play a pivotal role in skeletogenesis, their function in the osteogenesis of hMSCs has not yet been investigated in detail. Our aim was to characterize the expression of 37 HOX genes by multiplex RT-PCR to identify the ones most probably involved in osteogenic differentiation. The results showed that the expression patterns of four HOX genes were altered during this process. In particular, the expression levels of HOXC13 and HOXD13 were dramatically changed. Real-time PCR and Western blot analysis were performed in order to further analyze the expression of HOXC13 and HOXD13. The qRT-PCR results showed that transcription of HOXC13 was up-regulated by up to forty times, whereas that of HOXD13 was down-regulated by approximately five times after osteogenic differentiation. The Western blot results for the HOXC13 and HOXD13 proteins also corresponded well with the real-time PCR result. These findings suggest that HOXC13 and HOXD13 might be involved in the osteogenic differentiation of hMSCs.

Dual knockdown of p65 and p50 subunits of NF-kappaB by siRNA inhibits the induction of inflammatory cytokines and significantly enhance apoptosis in human primary synoviocytes treated with tumor necrosis factor-alpha.

In order to develop an anti-NF-kappaB siRNA as a novel class of anti-inflammatory drug, we have isolated a highly efficient siRNA targeting the p65 (RelA) subunit of NF-kappaB, hereafter named REL1096. To determine whether down-regulation of p65 by REL1096 can block the induction of inflammatory cytokines after treatment with tumor necrosis factor-alpha (TNF-alpha), human primary fibroblast-like synoviocytes were isolated from patients with rheumatoid arthritis. When treated with REL1096, the TNF-mediated induction of downstream target genes such as inflammatory cytokines, chemokines, and anti-apoptosis genes was drastically inhibited. To enhance the inhibitory effect of REL1096, cells were treated with siRNA targeting the p50 subunit of NF-kappaB together with REL1096. In addition to effective downregulation of inflammatory cytokines, knockdown of both p65 and p50 resulted in much more extensive apoptosis when compared to cells treated with either REL1096 or p50-siRNA alone. Thus, our results provide evidence for the potential use of siRNA targeting NF-kappaB as an effective means to treat rheumatoid arthritis. In addition to effective amelioration of synovial inflammation by downregulation of inflammatory cytokines, increased apoptosis by dual knockdown of p65 and p50 may prove advantageous in preventing invasiveness and destructiveness of hyperplastic synoviocytes.

Arthroscopic posterior cruciate ligament augmentation using an autogenous hamstring tendon graft and the posterior-posterior triangulation technique.

We describe a modification of the currently practiced arthroscopic posterior cruciate ligament (PCL) reconstruction techniques. We augmented the injured PCL with quadrupled autogenous hamstring tendon grafts using the arthroscopic posterior-posterior triangulation method and a single-incision transtibial approach. The tibial drill guide was introduced through the anteromedial portal and positioned onto the fossa for the PCL on the tibia under arthroscopic guidance from the posterolateral portal. The femoral tunnel was made 10 mm posterior to the articular cartilage of the medial femoral condyle by use of the anterior cruciate ligament Beath pin (Arthrex, Naples, FL) introduced through the far-inferior anterolateral portal. The tendon graft was positioned in the knee joint by use of the tibial and femoral double-folded silk loops that traversed the bony tunnels. The graft was fixed by use of bioabsorbable Intrafix screw systems (DePuy Mitek, Raynham, MA) at both the ends. The arthroscopic posterior-posterior triangulation method provides adequate exposure of the posterior knee compartment; this allows for convenient instrumentation and safe and accurate placement of the bony tunnels with preservation of the PCL remnants. We believe that retention of the remnant PCL fibers is biologic and contributes to earlier healing and strengthening of the tendon graft.

Synovial chondromatosis of the posterior cruciate ligament managed by a posterior-posterior triangulation technique.

Various causes have been reported for cystic lesions arising from the cruciate ligaments. We present a case of synovial chondromatosis that involved the posterior cruciate ligament and was treated by an arthroscopic posterior-posterior triangulation method. We recommend that synovial chondromatosis be considered in the differential diagnosis of cystic lesions of the cruciate ligaments. Lesions involving the posterior aspect of the posterior cruciate ligament, as in the presented case, can be accessed via the standard anterior arthroscopic portals. However, scattered synovial chondromatosis masses located in the posterolateral compartment are difficult to access via anterior portals. We suggest that the arthroscopic posterior-posterior triangulation technique may be useful for the management of such cases.

Role of tumor necrosis factor-alpha, interleukin-8, and dexamethasone in the focal adhesion kinase expression by human nucleus pulposus cells.

STUDY DESIGN: Human nucleus pulposus cells from intervertebral disc specimens were cultured to study the effects of tumor necrosis factor (TNF)-alpha and interleukin (IL)-8 on the focal adhesion kinase (FAK) expression by these cells. The effect of co-stimulation with dexamethasone on the FAK expression by nucleus pulposus cells was also studied. OBJECTIVES: To evaluate the possible role of activated FAK expressed by the human nucleus pulposus cells and its correlation with inflammatory cytokines (TNF-alpha, IL-8) and dexamethasone. SUMMARY OF BACKGROUND DATA: There have been no reported studies showing the correlation between the activated FAK expression by human nucleus pulposus cells with inflammatory cytokines and dexamethasone. METHODS: The FAK expression in cultured human nucleus pulposus cells was studied, and Western blot and immunofluorescence analysis were performed to assess its relation to TNF-alpha, IL-8, and dexamethasone. RESULTS: Treatments of TNF-alpha and IL-8 up-regulated the activated FAK expression. Dexamethasone attenuated the cytokine-induced FAK expression. The effects of inflammatory cytokines on the FAK expression were found to be concentration dependent, with greater correlation shown by IL-8 than TNF-alpha. CONCLUSION: TNF-alpha and IL-8 stimulation up-regulated the FAK expression of human nucleus pulposus cells, and the coadministration of dexamethasone attenuated it.

Haemorrhagic synovial cyst of the posterior cruciate ligament: a case report.

Cystic lesions arising in relation to the cruciate ligaments of the knee joint may become symptomatic and they can cause restriction of joint movement. We report here on a case of haemorrhagic synovial cyst arising from the posterior cruciate ligament with extension into the posterior compartment of the knee joint. We treated this lesion using the arthroscopic posterior-posterior triangulation technique. The histopathological findings of the synovial cyst were also verified. We would like to suggest that the posterior cruciate ligament synovial cyst should be considered in the differential diagnosis of cystic lesions that arise from the posterior compartment of the knee joint.

Anthropometry of the medial tibial condyle to design the tibial component for unicondylar knee arthroplasty for the Korean population.

Unicondylar knee arthroplasty (UKA) has gained popularity in the recent years for treating medial compartment osteoarthritis of the knee joint in the Asian population. There is little information about the anthropometry of the resected bony surfaces of the knee joint in these population groups for designing the appropriate size-matched UKA components. We studied the anthropometry of the resected medial tibial condyles in 50 male and 50 female Korean cadavers by using three-dimensional computer tomography. We measured the anteroposterior dimension, the mediolateral dimension at defined points and the condylar aspect ratio. These measurements were compared with similar dimensions of the tibial components from five conventionally used UKA designs. Statistical analysis was performed using Student's t-test, Paired t-test and Pearson's correlation coefficient. We found that three of the tibial component designs showed mediolateral overhang for the whole range of measured anteroposterior dimensions of the resected medial tibial condyles, whereas one of the designs (DePuy) showed mediolateral undersizing for the smaller AP dimensions and overhang for the larger AP dimensions of the resected medial tibial condyles. Another design (Smith and Nephew) showed mediolateral undersizing for the whole range of measured anteroposterior dimensions of the resected medial tibial condyle. We found a decrease in the condylar aspect ratio with increasing AP dimension for our cadaver population data. However, the majority of the conventional tibial prosthesis showed either a constant condylar aspect ratio or an increasing aspect ratio (DePuy) with the increasing AP dimension of the resected medial tibial condyle. Our study may provide guidelines for designing appropriate tibial UKA components for a majority of Asian sub-populations and encourage similar studies in other population groups.

Expression of vascular endothelial growth factor and angiogenesis in the diabetic frozen shoulder.

The purpose of this study was to investigate neovascularization and the expression of vascular endothelial growth factor (VEGF) in patients with diabetic frozen shoulders. Eleven patients with diabetic frozen shoulders underwent arthroscopic lysis of adhesions, and we observed the reported findings. VEGF expression was determined by immunohistochemistry and Western blot analysis, and the density of vessels was evaluated based on CD34 immunoreactivity by use of samples of the synovial tissue. For the control group, we took 5 samples of synovium from patients undergoing shoulder arthroscopy. The arthroscopic findings showed hyperemia of the synovial tissue in all cases of diabetic frozen shoulder. This synovium showed stronger immunostaining to VEGF (P = .010) and CD34 (P = .011) than the control synovial tissue. Western blot analysis also showed a stronger VEGF intensity than in the control group. We postulate that VEGF is synthesized and secreted in the synovium of diabetic frozen shoulders and VEGF may have some role in the pathogenesis and neovascularization of frozen shoulders in diabetic patients.

Analysis of neuron-like differentiation of human bone marrow mesenchymal stem cells.

The objective of the study was to evaluate differentiation of human bone marrow mesenchymal stem cells into true or pseudo neurons after treating with chemical induction medium in vitro. The morphological changes were assessed using interference contrast microscopy. Immunocytochemistry and Western blotting were performed using neuronal markers. Further evaluation was conducted with proteomic profiling, DNA microarray analysis and the whole-cell patch clamp test. After three hours of treatment with chemical induction medium, nearly three-fourths of the hMSCs changed to cells with a neuronal phenotype. The results of immunocytochemistry and Western blotting showed a high expression of neuronal markers in these cells at 3 h which decreased at 24 h. The proteomics analysis showed no change of proteins related to neuronal differentiation. DNA microarray showed downregulation of neuron related genes. The patch clamp test was unable to demonstrate any similarity to true neurons. Our findings suggest that neuron-like cells derived from chemical induction of hMSCs are not the genuine neurons as they resemble true neurons phenotypically but are different in genotypic and electrophysiological characteristics.

Identification of BRCA1 and BRCA2 mutations from Korean breast cancer patients using denaturing HPLC.

In order to investigate the presence and function of BRCA mutations in Korean breast cancer patients, mutational analyses using denaturing HPLC (DHPLC) were performed for 1020 breast cancer patients and 167 normal females. We identified 14 deleterious mutations that included 10 frameshift mutations and 4 nonsense mutations. Among these 14 mutations, we found 3 novel BRCA1 and 3 novel BRCA2 mutations. We found that BRCA deleterious mutations were present in 19 of the 1020 patients (1.8%). Three of the 14 deleterious mutations were detected in 7 of these 19 patients. The three mutations were also identified in other studies for BRCA mutations in the Korean population, suggesting that they are possible founder mutations of BRCA for the Korean population. We plan to provide fundamental data for larger population-based studies to establish the frequency and significance of BRCA mutations in Korean breast cancer patients.

KAI1/CD82 suppresses tumor invasion by MMP9 inactivation via TIMP1 up-regulation in the H1299 human lung carcinoma cell line.

We conducted a study on the mechanism of KAI1/CD82-mediated suppression of tumor invasiveness and metastasis, and examined its effect on MMP-9 activity and the TIMP1 levels in H1299 human non-small cell lung carcinoma cells. The H1299 human lung carcinoma cells were transfected with pcDNA3.1-CD82 and stable transfectant clones that had a high KAI1/CD82 expression were obtained. We performed Western blot analysis, cell invasion assay, gelatin zymography, and RT-PCR to assess the KAI1/CD82 expression and tumor invasiveness, the MMP-9 activity, the MMP-9 mRNA and protein levels, and the TIMP1 levels in the H1299/CD82 transfectant cells and compared the results with those of the control groups. The H1299/CD82 transfectants exhibited significant suppression of cell invasion, reduced MMP9 enzyme activity, elevated MMP9 mRNA and MMP-9 protein levels, and elevated TIMP1 levels. It may be postulated that KAI1/CD82 over-expression in the H1299 non-small cell lung carcinoma cells suppresses the tumor invasiveness and metastatic potential by inducing MMP9 inactivation via the up-regulation of TIMP1.

Meniscal hematoma of the knee joint: a case report.

We report here an unusual case of medial meniscal hematoma of the knee joint. A review of the literature has revealed only one case that was previously reported. The authors present the successful decompression of the posterior horn of a medial meniscal hematoma through the direct posterior-posterior triangulation arthroscopic visualization method. We present here the case report.

Posteromedial compartment cement extrusion after unicompartmental knee arthroplasty treated by arthroscopy: a case report.

Unicompartmental knee arthroplasty (UKA) with extrusion of cement into the posterior compartment of the knee is uncommon. Various problems after a UKA procedure, such as aseptic loosening, polyethylene wear and progressive arthritis have been reported. This study will report on a patient with extrusion of cement fragments into the posteromedial compartment of the knee after a UKA procedure. This complication was treated successfully with the direct posterior-posterior triangulation arthroscopic visualization method. In cementing the prosthesis, it is of paramount importance to take caution to completely remove extruded cement remnants in order to prevent this complication during UKA.

Spontaneous and simultaneous rupture of both quadriceps tendons in a patient with chronic renal failure. A case studied by MRI both preoperatively and postoperatively.

Spontaneous bilateral rupture of the quadriceps tendons without a significant history of trauma is an uncommon disease. It is generally associated with chronic metabolic disorders such as chronic renal failure and secondary hyperparathyroidism. Here, we report a case of spontaneous bilateral tendon rupture in a patient on chronic hemodialysis for the past 5 years. We performed a preoperative MRI to confirm the diagnosis; then we repaired the ruptured quadriceps tendons and reviewed the patient both clinically and by performing MRI postoperatively 4 yrs later. The patient attained the full function of the quadriceps tendon.

Transarticular arthroscopic excision of an elbow cyst.

We present the case of a symptomatic anterior elbow cyst supposedly due to repeated (exertional) trauma. Instead of the usual open excision, arthroscopic excision was performed using a Wissinger rod through an additional anterolateral portal for distraction. Histopathology confirmed the lesion to be a ganglion. MRI 12 months postoperatively showed no recurrence.

Two-stage total knee arthroplasty for non-salvageable septic arthritis in diabetes mellitus patients.

Pyogenic knee arthritis in a patient with advanced osteoarthritis is a serious medical problem. We have performed arthroscopic debridement in 136 patients with pyogenic knee arthritis from January 1999 to December 2001. Five of these patients were diabetic, they did not respond to the standard treatment protocol and they continued to have infection. For these patients, we performed open arthrotomy, with implantation of antibiotic cement as a spacer, and staged total knee arthroplasty. The clinical results were evaluated using the Hospital for Special Surgery (HSS) scoring system. At an average follow-up of 38 months (range: 29 to 46), the average pain score was 83 and the functional score was 73 with no patient having recurrence of the infection. This study shows that just as a 2-stage revision is now done for infected total knee arthroplasty, primary uncontrolled infected knees may be treated by a 2-stage arthroplasty as well.

Patellar polyethylene peg fracture: a case report and review of the literature.

The patellar component of a total knee replacement (TKR) is the most frequent source of non-septic complications after total knee arthroplasty. Fracture of patellar pegs in all polyethylene patellar components is a very rare occurrence. We report such a case of a patellar polyethylene fracture in a 72-year-old female patient 10 years after TKR. Due to patellar malalignment and high level of activity, the patellar components failed in this patient. This was treated arthroscopically by removing the components that failed and leaving the patella unresurfaced. We followed up the patient postoperatively and her symptoms were substantially resolved.

Joystick reduction and percutaneous pinning for an acutely anteriorly dislocated coccyx: a case report.

A sacrococcygeal dislocation is a rare occurrence, and the treatment options vary. Initial treatment is nonoperative, consisting of a manual reduction with a gloved finger and local rest. Acute operative treatment of a failed closed reduction is unusual. We report a case of an acute irreducible anteriorly dislocated coccyx successfully treated with a minimally invasive technique: joystick reduction and Steinman pin fixation.

Muscle derived, cell based ex vivo gene therapy for treatment of full thickness articular cartilage defects.

OBJECTIVE: To evaluate the effectiveness of transplanted allogeneic muscle derived cells (MDC) embedded in collagen gels for the treatment of full thickness articular cartilage defects, to compare the results to those from chondrocyte transplantation, and to evaluate the feasibility of MDC based ex vivo gene therapy for cartilage repair. METHODS: Rabbit MDC and chondrocytes were transduced with a retrovirus encoding for the beta-galactosidase gene (LacZ). The cells were embedded in type I collagen gels, and the cell proliferation and transgene expression were investigated in vitro. In vivo, collagen gels containing transduced cells were grafted to the experimental full thickness osteochondral defects. The repaired tissues were evaluated histologically and histochemically, and collagen typing of the tissue was performed. RESULTS: The MDC and chondrocyte cell numbers at 4 weeks of culture were 305 +/- 25% and 199 +/- 25% of the initial cell number, respectively. The initial percentages of LacZ positive cells in the MDC and chondrocyte groups were 95.4 +/- 1.9% and 93.4 +/- 3.4%, and after 4 weeks of culture they were 84.2 +/- 3.9% and 76.9 +/- 4.3%, respectively. In vivo, although grafted cells were found in the defects only up to 4 weeks after transplantation, the repaired tissues in the MDC and chondrocyte groups were similarly better histologically than control groups. Repaired tissues in the MDC group were mainly composed of type II collagen, as in the chondrocyte group. CONCLUSION: Allogeneic MDC could be used for full thickness articular cartilage repair as both a gene delivery vehicle and a cell source for tissue repair.