Trans-differentiation via Epigenetics: A New Paradigm in the Bone Regeneration

In regenerative medicine, growing cells or tissues in the laboratory is necessary when damaged cells can not heal by themselves. Acquisition of the required cells from the patient's own cells or tissues is an ideal option without additive side effects. In this context, cell reprogramming methods, including the use of induced pluripotent stem cells (iPSCs) and trans-differentiation, have been widely studied in regenerative research. Both approaches have advantages and disadvantages, and the possibility of de-differentiation because of the epigenetic memory of iPSCs has strengthened the need for controlling the epigenetic background for successful cell reprogramming. Therefore, interest in epigenetics has increased in the field of regenerative medicine. Herein, we outline in detail the cell trans-differentiation method using epigenetic modification for bone regeneration in comparison to the use of iPSCs.

Erratum: Institutions, Correspondence, Figures & Legends Correction. Hyperglycemia increases the expression levels of sclerostin in a reactive oxygen species- and tumor necrosis factor-alpha-dependent manner

Some parts of published paper were misprinted.

Hyperglycemia increases the expression levels of sclerostin in a reactive oxygen species- and tumor necrosis factor-alpha-dependent manner

PURPOSE: Sclerostin, an inhibitor of Wnt/beta-catenin signaling, exerts negative effects on bone formation and contributes to periodontitis-induced alveolar bone loss. Recent studies have demonstrated that serum sclerostin levels are increased in diabetic patients and that sclerostin expression in alveolar bone is enhanced in a diabetic periodontitis model. However, the molecular mechanism of how sclerostin expression is enhanced in diabetic patients remains elusive. Therefore, in this study, the effect of hyperglycemia on the expression of sclerostin in osteoblast lineage cells was examined. METHODS: C2C12 and MLO-Y4 cells were used in this study. In order to examine the effect of hyperglycemia, the glucose concentration in the culture medium was adjusted to a range of levels between 40 and 100 mM. Gene expression levels were examined by quantitative reverse transcription-polymerase chain reaction and Western blot assays. Top-Flash reporter was used to examine the transcriptional activity of the beta-catenin/lymphoid enhanced factor/T-cell factor complex. Tumor necrosis factor-alpha (TNFalpha) protein levels were examined with the enzyme-linked immunosorbent assay. The effect of reactive oxygen species on sclerostin expression was examined by treating cells with 1 mM H2O2 or 20 mM N-acetylcysteine. RESULTS: The high glucose treatment increased the mRNA and protein levels of sclerostin. High glucose suppressed Wnt3a-induced Top-Flash reporter activity and the expression levels of osteoblast marker genes. High glucose increased reactive oxygen species production and TNFalpha expression levels. Treatment of cells with H2O2 also enhanced the expression levels of TNFalpha and sclerostin. In addition, N-acetylcysteine treatment or knockdown of TNFalpha attenuated high glucose-induced sclerostin expression. CONCLUSIONS: These results suggest that hyperglycemia increases sclerostin expression via the enhanced production of reactive oxygen species and TNFalpha.

A Novel Osteogenic Activity of Suberoylanilide Hydroxamic Acid is Synergized by BMP-2

BACKGROUND: Many histone deacetylase (HDAC) inhibitors are well recognized as potential anti-cancer drugs. Inhibition of HDACs induces temporal transcription or epigenetic control, thus regulating many different biological responses. Here, we investigated the osteogenic effect of the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA; vorinostat). METHODS: The effects of SAHA on osteoblast differentiation were examined in the 6XOSE-Luc reporter assay for determination of runt-related transcription factor 2 (Runx2) activity and alkaline phosphatase (ALP) activity and in an immunoprecipitation assay to determine the Runx2 acetylation state. The osteogenic activity of SAHA in vivo was studied in and receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoporotic mouse model. RESULTS: SAHA increased the transcriptional activity of Runx2 in a dose-dependent manner in the 6XOSE-Luc reporter assay. SAHA by itself was unable to induce ALP activity; however, SAHA enhanced ALP activity induced by bone morphogenetic protein-2 (BMP-2). The degree of acetylation of Runx2 was increased with SAHA treatment, which suggests that the increase in Runx2 transcriptional activity might be dependent on stabilization by acetylation. Also, SAHA successfully reversed soluble RANKL-induced osteoporotic bone loss. CONCLUSIONS: Our study shows an intriguing osteogenic potential of SAHA in a BMP-2-dependent manner and suggests that SAHA could be used at lower doses along with BMP-2 to treat osteoporosis.

Msx2 mediates the inhibitory action of TNF-alpha on osteoblast differentiation

TNF-alpha, a proinflammatory cytokine, inhibits osteoblast differentiation under diverse inflammatory conditions; however, the underlying mechanisms in terms of the TNF-alpha signaling pathway remain unclear. In this study, we examined the role of Msx2 in TNF-alpha-mediated inhibition of alkaline phosphatase (ALP) expression and the signaling pathways involved. TNF-alpha down-regulated ALP expression induced by bone morphogenetic protein 2 (BMP2) in C2C12 and Runx2-/- calvarial cells. Over-expression of Msx2 suppressed BMP2-induced ALP expression. Furthermore, TNF-alpha induced Msx2 expression, and the knockdown of Msx2 by small interfering RNAs rescued ALP expression, which was inhibited by TNF-alpha. TNF-alpha activated the NF-kappaB and the JNK pathways. Inhibition of NF-kappaB or JNK activation reduced the inhibitory effect of TNF-alpha on ALP expression, whereas TNF-alpha-induced Msx2 expression was only suppressed by the inhibition of the NF-kappaB pathway. Taken together, these results indicate that Msx2 mediates the inhibitory action of TNF-alpha on BMP2-regulated osteoblast differentiation and that the TNF-alpha-activated NF-kappaB pathway is responsible for Msx2 induction.

Osteoblast differentiation of human bone marrow stromal cells (hBMSC) according to age for bone tissue engineering

Tissue engineered bone (TEB) can replace an autogenous bone graft requiring an secondary operation site as well as avoid complications like inflammation or infection from xenogenic or synthetic bone graft. Adult mesenchymal stem cells (MSC) for TEB are considered to have various ranges of differentiation capacity or multipotency by the donor site and age. This study examined the effect of age on proliferation capacity, differentiation capacity and bone morphogenetic protein-2 (BMP-2) responsiveness of human bone marrow stromal cells (hBMSC) according to the age. In addition, to evaluate the effect on enhancement for osteoblast differentiation, the hBMSC were treated with Trichostatin A (TSA) and 5-Azacitidine (5-AZC) which was HDAC inhibitors and methyltransferase inhibitors respectively affecting chromatin remodeling temporarily and reversibly. The young and old group of hBMSC obtained from the iliac crest from total 9 healthy patients, showed similar proliferation capacity. Cell surface markers such as CD34, CD45, CD90 and CD105 showed uniform expression regardless of age. However, the young group showed more prominent transdifferentiation capacity with adipogenic differentiation. The osteoblast differentiation capacity or BMP responsiveness was low and similar between young and old group. TSA and 5-AZC showed potential for enhancing the BMP effect on osteoblast differentiation by increasing the expression level of osteogenic master gene, such as DLX5, ALP. More study will be needed to determine the positive effect of the reversible function of HDAC inhibitors or methyltransferase inhibitors on enhancing the low osteoblast differentiation capacity of hBMSC.

Trichostatin A-mediated upregulation of p21

Histone deacetylase inhibitors (HDIs), a new class of anti-cancer agents, have been reported to suppress formation of osteoclast precursors and their fusion into multinucleated cells. However, little is known about the effect of HDIs on mature osteoclasts, which may have significance for their therapeutic use. Here, we demonstrate a novel action of HDIs on osteoclast apoptosis. Primary multinucleated mature osteoclasts were prepared from mouse bone marrow cells. Treatment of osteoclasts with the HDI trichostatin A (TSA) caused apoptosis, as confirmed by annexin V staining and caspase activation. TSA caused the upregulation of p21WAF1 in osteoclasts. To understand the role of p21(WAF1) upregulation in TSA-treated osteoclasts, shRNA against p21(WAF1)-containing lentivirus was introduced into osteoclasts. The suppression of p21(WAF1) decreased TSA-directed osteoclast apoptosis. Collectively, our results provide evidence that TSA causes osteoclast apoptosis, which involves, in part, TSA-induced upregulation of p21(WAF1), and strongly supports HDIs as potential therapeutic agents for excessive bone resorption.

Clinical Analysis Of Metastatic Spinal Cord Compression / 대한내과학회지

BACKGROUND: Spinal cord compression is a common neurologic complication of advanced cancer and it is a medical emergency because delay in treatment often results in irreversible neurologic dysfunction. We analyzed the clinical characteristics, treatment outcome, favorable factors which affect survival and the result of radiation therapy and surgery. METHODS: The records of 50 patients with epidural cord compression during the period from January 1994 to December 2003 were reviewed retrospectively. RESULTS: The most common cause of metastatic spinal cord compression was lung cancer (28%), followed by hepatoma (22%), and colorectal cancer (14%). 44 patients received radiation therapy and 2 patients were treated with surgery. The median survival was 2.9 months irrespective of treatment. The factors which affect survival were initial performance status, radiotherapy and ambulatory function (p<0.05). At diagnosis, 31 (62%) of 50 patients presented with paralytic status. Radiation therapy affected preservation of ambulatory function significantly (p<0.05). Among the patients treated with radiotherapy, 14 of 18 patients who were ambulatory and 5 of 26 paralytic patients before treatment remained ambulatory or became ambulatory (p<0.01). Surgery permitted patients to remain ambulatory, but only 2 patients were received surgery. CONCLUSION: We confirmed that radiation therapy is effective palliative treatment for patients with epidural cord compression. Initial performance status and ambulatory function were identified as important prognostic factors. Prompt diagnosis and treatment were necessary, if not, neurologic function was not regained in the majority of patients. Indivisualized and more aggressive therapy including surgery should be considered.

Role of Transcription Factors in Bone and Vascular Mineralization / 대한내분비학회지

No Abstract available.

Clinical Results of Postoperative Chemoradiation on Advanced Rectal Cancers: Tumor Response, Toxicities, and Morbidity

PURPOSE: Adjuvant chemotherapy and radiotherapy have been considered effective treatments in advanced rectal cancers. Recently, several studies have reported that preoperative chemoradiation (CRT) may have advantages over postoperative CRT, particularly in reducing local recurrence and preserving the anal sphincter. We studied the short-term efficacy of preoperative CRT for locally advanced rectal cancers. METHODS: Between Jun. 2000 and Aug. 2003, 23 patients were treated with preoperative CRT, followed by surgery (pre-CRT) and 31 patients were treated with chemoradiation postoperatively (post-CRT). We compared these two groups for the incidence and degree of side effects from CRT, postoperative complications, type of surgery, including anal sphincter preservation, and short-term recurrence. RESULTS: The average age and male-to-female ratio of the pre- and the post-CRT groups were 58+/-11, years and 13:10, and 61+/-14 and 14:17, respectively. T downstagings were observed in 17 of 23 (74%) pre-CRT patients. On the RTOG-EORTC scale, the patients who showed hematological, intestinal and dermal side effects in the pre-CRT group and in the post-CRT group were 5, 5, 2 and 5, 2, 4, respectively and the difference was not statistically significant (P=0.41). Anal sphincter preserving surgical procedures were performed 91.3% (21/23) and 83.9% (26/31) of the patients in the pre- and the post-CRT groups, respectively. But this difference was not statistically significant (P=0.4). Postoperative complications in the pre-CRT group were anastomosis site leakages (n=3) and rectovaginal fistula (n=1). In the post-CRT group, complications were two anastomosis site leakages. Four of the 31 post-CRT group patients had recurrences such as locoregional area (n=2), liver (n=1), and lung (n=1) while no patient was observed in pre- CRT group. CONCLUSIONS: Although pre-CRT group showed higher incidence of complications than post-CRT group, these were managed easily and safely. Pre-CRT seems to be an effective modality for treating advanced rectal cancers particularly for preserving anal sphincter. Long-term follow- up data are needed to clarify the effect of pre-CRT.

A Case of Pure Red Cell Aplasia Associated with B-cell Chronic Lymphocytic Leukemia / 대한혈액학회지

Pure red cell aplasia (PRCA) is a rare disorder, which is characterized by severe anemia associated with reticulocytopenia and absence of erythroid precursor cells in the bone marrow. Recently we experienced a patient with B-cell chronic lymphocytic leukemia associated with PRCA, which was successfully treated with prednisone. A 50-year-old man was admitted because of lymphocytosis. The leukocyte count was 26,800/nL with 82% abnormal mature lymphocytes. Neither anemia nor thrombocytopenia was present. Many abnormal lymphocytes and erythroblasts were seen in the bone marrow. The surface markers of these cells were positive for CD5, CD19, CD20, CD22, and surface immunoglobulin. The patient was diagnosed of B-CLL in Binet-Rai stage A (II), and was treated with chlorambucil. Six months later, severe anemia (hemoglobin 5.9g/dL) with reticulocytopenia developed. In the bone marrow at that time, neutrophils and megakaryocytes besides leukemic cells were preserved, but marked decrease of erythroblasts, a pattern of PRCA was observed. After one-month's observation, we prescribed prednisone. After four weeks of such therapy the patient's hemoglobin reached 10.9g/dL, and he needed no further transfusion.

Brain metastasis: Clinical characteristics and prognosis / 대한내과학회지

BACKGROUND: Brain metastasis is a common complication in cancer patients. We evaluated the clinical characteristics, treatment outcome and prognostic factors for patients with metastatic brain tumor. METHODS: The records of 97 patients with metastatic brain tumor during the period from January 1991 to November 1997 were reviewed retrospectively. RESULTS: The most common primary tumor is lung cancer (61 cases, 63%) followed by metastatic cancer unknown primary site (15 cases, 16%), gastrointestinal cancer (13 cases, 13%), breast cancer (6 cases, 6%) and renal cancer (2 cases, 2%). There were 44 patients with a single brain metastasis and 53 patients with multiple brain metastases. The median survival was 3.0 months and one-year survival rate was 8% irrespective of treatment. Favorable prognostic factors which affect survival were ambulatory status (p<0.01) and functional neurologic class 1, 2 (p<0.01). Median survival was 3.7 months for patients with steroid therapy and 1.1 months with no therapy (p<0.01). Median survival was 4.8 months for patients with steroid therapy plus whole brain radiotherapy (WBRT) and 2.2 months with steroid therapy alone (p<0.01). Additional chemotherapy did not appear to affect the survival. The patients treated with surgery had median survival time of 8.8 months compared with 2.5 months for patients treated with steroid therapy plus WBRT (p<0.05). CONCLUSION: In present study, we confirmed that whole brain irradiation and corticosteroid administration are effective palliative treatment for patients with metastatic brain tumor. Initial performance status and neurological function were identified as important prognostic factors. Although confounded by the limitations of retrospective study, more aggressive treatments including surgery and chemotherapy could be regarded to have a significant role to achieve better treatment outcome in some selected cases.

Spatio-temporal expression patterns of Runx2 isoforms in early skeletogenesis

Skeletogenesis occurs through either intramembranous or endochondral ossification. In addition, some parts of the skeletal components maintain their cartilaginous characteristics throughout life without mineralization. Runx2 is known to be a pivotal transcription factor for all skeletogenic processes. In this study, we examined the expression patterns of two major isoforms of Runx2 in early skeletogenesis. During intramembranous bone formation, Runx2-type I (Runx2-I) was widely expressed in osteoprogenitor cells and active osteoblasts, while Runx2-type II (Runx2-II) expression was stringently restricted to cells lining mineralized bones. Cells in permanent cartilage expressed collagen type II (Col-II) but never expressed Runx2 or Col-X. These permanent cartilages were well circumscribed by Runx2-I positive cells, in which Runx2-II was negative. In endochondral bone formation, Runx2 expression temporarily disappeared in Col-II-positive proliferating chondrocytes, but a secondary surge of Runx2-I expression occurred in the prehypertrophic zone before the mineralization of cartilage. Collectively, both Runx2 isoforms showed very similar expression patterns in active bone forming areas; however, Runx2-I has an exclusive role in the early commitment stage of intramembranous or endochondral bone forming processes or in cells surrounding permanent cartilage.

Role of BMP-2 in Osteoblast Differentiation / 대한내분비학회지

No abstract available.

Localization of bone matrix gene mrna in regenerating bone tissue during the guided bone regeneration

The investigation of microvasculature changes in osseous regeneration by guided tissue regeneration procedure

Dynamics of gingival crevicular fluid volume and enzyme activities after application of orthodontic force / 대한치과교정학회지

The aim of this investigation was to study the effect of orthodontic force on the flow of gingival crevicular fluid and activities of arylsulfatase and brta-glucuronidase in crevicular fluid. The material consisted of 12 persons between the ages of 13 years and 22 years and all were categorized Class I, 4-4 extraction cases Crevicular fluids were sampled from distal crevis of each canine before treatment (phase 1), after bracketing (phase 2), after application of force (phase 3) and after run out of orthodontic force (phase 4). Crevicular fluid flow did not show any significant changes during the period of treatment. The activities of arylsulfatase increased significantly after setting of orthodontic appliance without application of force, but did not show any significant difference after application of force. The activities of beta-glucuronidase increased significantly after application of orthodontic force and decreased with force deminished. These indicated that beta-glucuronidase was good indicator of bone remodelling resulted from initial orthodontic force.