Cartilage regeneration using transforming growth factor-beta 3-loaded injectable crosslinked hyaluronic acid hydrogel.

Cartilage defects can be difficult to heal, potentially leading to complications such as osteoarthritis. Recently, a tissue engineering approach that uses scaffolds and growth factors has been proposed to regenerate new cartilage tissues. Herein, we investigated the application of hyaluronic acid (HA) gel loaded with transforming growth factor-beta 3 (TGF-ß3) for enhanced cartilage regeneration. We assessed the clinical conditions required to efficiently enhance the ability of the modified HA gel to repair defective cartilage. Based on our findings, the prepared HA gel exhibited good physicochemical and mechanical properties and was non-toxic and non-inflammatory. Moreover, HA gel-loaded TGF-ß3 (HAT) had improved biocompatibility and promoted the synthesis of cartilage-specific matrix and collagen, further improving its ability to repair defects. The application of HAT resulted in an initial burst release of HA, which degraded slowly in vivo. Finally, HAT combined with microfracture-inducing bone marrow stem cells could significantly improve the cartilage microenvironment and regeneration of cartilage defects. Our results indicate that HA is a suitable material for developing growth factor carriers, whereas HAT is a promising candidate for cartilage regeneration. Furthermore, this differentiated strategy provides a rapid and effective clinical approach for next-generation cartilage regeneration.

Preparation of Foam Dressings Based on Gelatin, Hyaluronic Acid, and Carboxymethyl Chitosan Containing Fibroblast Growth Factor-7 for Dermal Regeneration.

Wound recovery close to the function of the native skin is the goal of wound healing. In this study, we prepared foam dressings (FDs; 2-GHC-FD-1-9, 5-GHC-FD-1-9, and 10-GHC-FD-1-9) composed of various concentrations of gelatin, hyaluronic acid, and carboxymethyl chitosan, which are chemically interconnected through amide bond formation, for evaluating wound healing. Tensile and cell proliferation tests showed that 2-GHC-FD-1-9 are suitable for wound dressing. For further evaluation, three types of FDs, 2-GHC-FD-1, 2-GHC-FD-4, and 2-GHC-FD-8 were chosen. The results of animal intradermal reactivity, water vapor transmission rate, and absorption rate of the three FDs indicated that 2-GHC-FD-8 is the most appropriate scaffold for wound healing. For wound healing acceleration, various concentrations of fibroblast growth factor-7 (FGF-7) was soaked in 2-GHC-FD-8 (2-GHC-FD-8/F1-6) and evaluated by using scanning electron microscopy, cell proliferation, release behavior, and in vivo animal tests. The FDs showed interconnected porous structures, increased cell proliferation until 8.0 × 10-11 M, controlled release with initial burst within 1 h, and sustained release for 48 h. The results of the animal test showed an appropriate concentration of FGF-7 for wound healing. In addition, 2-GHC-FD-8 is a suitable scaffold for wound healing. Therefore, we suggest that 2-GHC-FD-8/F3 is a useful wound dressing for accelerating wound healing.

Frameshift Mutations and Loss of Expression of CLCA4 Gene are Frequent in Colorectal Cancers With Microsatellite Instability.

Chloride channel calcium-activated (CLCA) genes encode regulators for chloride transport across the cell membrane. As for cancer development, some CLCA genes are considered putative tumor suppressor genes. The aim of this study was to explore whether CLCA4 gene would have mutations in its nucleotide repeats in colorectal cancer (CRC). In a public database, we found that CLCA4 gene had mononucleotide repeats in the coding sequences that might be mutational targets in the cancers with microsatellite instability. For this, the current study studied 146 CRCs for mutation and expression analyses by single-strand conformation polymorphism analysis, DNA sequencing, and immunohistochemistry. Overall, we found CLCA4 frameshift mutations in 12/101 (11.8%) CRCs with high-microsatellite instability (MSI-H), but none in microsatellite stable CRCs (0/45) (P<0.01). In addition, we analyzed intratumoral heterogeneity of the CLCA4 frameshift mutations and found that 1 CRC harbored regional intratumoral heterogeneity of the CLCA4 frameshift mutation. Loss of CLCA4 protein expression was identified in 50% of CRCs. Also, cancers with MSI-H harboring CLCA4 frameshift mutations showed lower CLCA4 immunostaining than those with the wild-type. Our data indicate that the CLCA4 gene harbors alterations both in somatic mutation and expression, suggesting their roles in tumorigenesis of CRC with MSI-H.

Intratumoral Heterogeneity of Somatic Mutations for NRIP1, DOK1, ULK1, ULK2, DLGAP3, PARD3 and PRKCI in Colon Cancers.

Both NRIP1 and DOK1 genes are considered candidate tumor suppressor genes (TSGs). Also, cell polarity-related genes PARD3, PRKCI and DLGAP3, and autophagy-related genes ULK1 and ULK2 genes are considered to play crucial roles in tumorigenesis. The aim of our study was to find whether these genes were mutated in colorectal cancer (CRC). In a genome database, we observed that each of these genes harbored mononucleotide repeats in the coding sequences, which could be mutated in cancers with high microsatellite instability (MSI-H). For this, we studied 124 CRCs for the frameshift mutations of these genes and their intratumoral heterogeneity (ITH). NRIP1, DOK1, PARD3, PRKCI, DLGAP3, ULK1 and ULK2 harbored 18 (22.8%), 2 (2.5%), 2 (2.5%), 2 (2.5%), 5 (6.3%), 2 (2.5%) and 2 (2.5%) of 79 CRCs with MSI-H, respectively. However, we found no such mutations in microsatellite stable (MSS) cancers in the nucleotide repeats. We also studied ITH for the frameshift mutations in 16 cases of CRCs and detected that the frameshift mutations of NRIP1, DOK1, PARD3, PRKCI, DLGAP3, ULK1 and ULK2 showed regional ITH in 5 (31.3%), 2 (12.5%), 0 (0%), 0 (0%), 1 (6.3%), 1 (6.3%) and 3 (18.8%) cases, respectively. Our data exhibit that candidate cancer-related genes NRIP1, DOK1, PARD3, PRKCI, DLGAP3, ULK1 and ULK2 harbor mutational ITH as well as the frameshift mutations in CRC with MSI-H. Also, the results suggest that frameshift mutations of these genes might play a role in tumorigenesis through their inactivation in CRC.

Intratumoral Heterogeneity of Frameshift Mutations of GLI1 Encoding a Hedgehog Signaling Protein in Colorectal Cancers.

GLI1 is a transcription factor for hedgehog signaling that plays a crucial role in signaling pathways for controlling cell proliferation, alterations of which are known to contribute to tumorigenesis. Aim of this study was to explore whether GLI1 gene is mutated in gastric (GC) and colorectal cancers (CRC). In a public database, we found that GLI1 had a G7 mononucleotide repeat in the coding sequences that could be a mutation target in the cancers with microsatellite instability (MSI). In this study, we analyzed frameshift mutation of GLI1 in 79 GCs and 129 CRCs (high MSI (MSI-H) or microsatellite stable (MSS)) by single-strand conformation polymorphism analysis and DNA sequencing. We found 10 frameshift mutations in the repeat, nine for CRCs and one for GC. All of the mutations were detected in cancers with MSI-H and there was a statistical difference in the frameshift mutation frequencies between the cancers with MSI-H (10/113) and MSS (0/90). We also analyzed intratumoral heterogeneity (ITH) of the frameshift mutation in 16 CRCs and found that the mutations exhibited regional ITH in three of the CRCs (18.8%). Our data indicate GLI1 harbored not only frameshift mutation but also its mutational ITH, which together could be a feature of GC and CRC with MSI-H.

Mutational and expressional alterations of ZMPSTE24, DNA damage response-related gene, in gastric and colorectal cancers.

Loss of ZMPSTE24 is related to progeroid phenotypes in human. Cells in zmpste24-deficient mice show delayed DNA damage response, increased aneuploidy and increased genomic instability, which are considered features of cancer cells. The aim of this study was to address whether ZMPSTE24 gene was mutated in colorectal cancers (CRCs) and gastric (GCs), and its expression was altered. ZMPSTE24 possesses a T9 mononucleotide repeat in an exon, which could be mutated in cancers with defects in mismatch repair that can result in microsatellite instability (MSI). For this, the current study studied 124 CRCs and 79 GCs for mutation and expression analyses. For mutations in the T9, CRCs (16.4%) and GCs (8.8%) with high MSI (MSI-H), but not microsatellite stable/low MSI (MSS/MSI-L), exhibited frameshift mutations. Also, the ZMPSTE24 mutations showed intratumoral heterogeneity (ITH) in 4 of 16 CRC cases. Downregulation of ZMPSTE24 protein expression was found in 16.9% of CRCs and 8.9% of GCs by immunohistochemistry. Our study found frameshift mutation and its ITH in ZMPSTE24 gene as well as downregulation of ZMPSTE24 expression. Based on these observations, the present study suggests that inhibition of ZMPSTE24 by both mutational and expressional pathways might together play a role in tumorigenesis of CRC and GC harboring MSI-H phenotype.

Frameshift mutations of a tumor suppressor gene ZNF292 in gastric and colorectal cancers with high microsatellite instability.

A transcription factor-encoding gene ZNF292 is considered a candidate tumor suppressor gene (TSG). Its mutations have been identified in cancers from liver, colon, and bone marrow. However, ZNF292 inactivating mutations that might suppress the TSG functions have not been reported in gastric (GC) and colorectal cancers (CRC) with microsatellite instability (MSI). In a public database, we found that ZNF292 gene had mononucleotide repeats in the coding sequences that might be mutation targets in the cancers with MSI. In this study, we analyzed 79 GCs and 124 CRCs including high MSI (MSI-H) and microsatellite stable/low MSI (MSS/MSI-L) cases for the detection of somatic mutations in the repeats. Overall, we identified frameshift mutations of ZNF292 in 3 (8.8%) GCs and 11 (13.9%) CRCs with MSI-H (14/113), but not in MSS/MSI-L cancers (0/90) (p < 0.001). Also, we studied intratumoral heterogeneity (ITH) of the ZNF292 frameshift mutations in 16 CRCs and found that two (12.5%) had regional ITH of the mutations. Our data show that ZNF292 gene harbors not only frameshift mutations but also mutational ITH, which together may be features of GC and CRC with MSI-H. Based on this, the ZNF292 frameshift mutations may possibly contribute to tumorigenesis by altering its TSG functions in GC and CRC.

Frameshift mutation of WISP3 gene and its regional heterogeneity in gastric and colorectal cancers.

WISP3 is involved in many cancer-related processes including epithelial-mesenchymal transition, cell death, invasion, and metastasis and is considered a tumor suppressor. The aim of our study was to find whether WISP3 gene was mutated and expressionally altered in gastric (GC) and colorectal cancers (CRCs). WISP3 gene possesses a mononucleotide repeat in the coding sequence that could be mutated in cancers with high microsatellite instability (MSI-H). We analyzed 79 GCs and 156 CRCs, and found that GCs (8.8%) and CRCs (10.5%) with MSI-H, but not those with microsatellite stable/low MSI, harbored a frameshift mutation. We also analyzed intratumoral heterogeneity (ITH) of the frameshift mutation in 16 CRCs and found that the WISP3 mutation exhibited regional ITH in 25% of the CRCs. In immunohistochemistry, loss of WISP3 expression was identified in 24% of GCs and 21% of CRCs. The loss of expression was more common in those with WISP3 mutation than with wild-type WISP3 and those with MSI-H than with microsatellite stable/low MSI. Our data indicate that WISP3 harbored not only frameshift mutation but also mutational ITH and loss of expression, which together might play a role in tumorigenesis of GC and CRC with MSI-H by inhibiting tumor suppressor functions of WISP3. Our data also suggest that mutation analysis in multiregions is needed for a proper evaluation of mutation status in GC and CRC with MSI-H.

Frequent frameshift mutations in 2 mononucleotide repeats of RNF43 gene and its regional heterogeneity in gastric and colorectal cancers.

RNF43, an E3 ligase, inhibits Wnt signaling by removing Wnt receptors and behaves as a candidate tumor suppressor. Recent studies identified that RNF43 gene was frequently mutated in gastric (GC), colorectal (CRC), and endometrial cancers with high microsatellite instability (MSI-H). The aim of this study is to explore whether RNF43 gene is mutated in GC and CRC in Korean patients and whether the mutations show regional intratumoral heterogeneity (ITH). We analyzed 2 exonic repeats (C6 and G7) of RNF43 in 78 GCs and 130 CRCs by single-strand conformation polymorphism and DNA sequencing analyses. Also, we analyzed regional ITH of RNF43 mutation in 16 CRCs. We found RNF43 frameshift mutation in MSI-H (50/118), the incidence of which was significantly higher than that in microsatellite stable/low microsatellite instability (1/90). GCs showed a significantly higher incidence of the mutation than CRCs (66.7% of GC and 32.9% of CRC with MSI-H). Also, we found that all of the 7 CRCs with the mutations harbored mutational ITH. By immunohistochemistry, we observed that loss of RNF43 expression was significantly more common in those with RNF43 frameshift mutation than those with wild-type RNF43. Our data indicate that RNF43 gene harbored not only exceedingly high mutations but also mutational ITH, which together might play a role in tumorigenesis of GC and CRC. We suggest that regional analysis is required for a more comprehensive evaluation of the mutation status in these tumors.

A Korean kindred with autosomal dominant nocturnal frontal lobe epilepsy and mental retardation.

BACKGROUND: A Korean family had distinctive clinical and neuroimaging features and carried the same genetic mutation that was found in a previously described Japanese kindred with autosomal dominant nocturnal frontal lobe epilepsy. OBJECTIVE: To describe the first Korean family with autosomal dominant nocturnal frontal lobe epilepsy. METHODS: Members of a large family, including 9 affected individuals from 3 generations, underwent a comprehensive genetic, clinical, electroencephalographic, neuropsychological, and neuroimaging evaluation. Affected members were tested for possible mutations in transmembrane regions 1 through 3 of the neuronal nicotinic acetylcholine receptor alpha4 subunit (CHRNA4) by direct sequencing and subsequent restriction analysis. RESULTS: Seizures began in childhood, presenting as nocturnal episodes of staring, confusion, shouting, perioral movements, unintelligible speech, and hand waving. Some patients had ictal or interictal epileptiform activity in the temporal and/or frontocentral areas. Neurological examination and brain magnetic resonance imaging results showed no abnormalities, except that all patients available for testing had mild to moderate mental retardation. Fluorodeoxyglucose F 18 with positron emission tomography showed mild decreased glucose uptake in the superior and middle frontal regions, more so on the left than on the right. Patient response to carbamazepine was poor. All affected members were heterozygous for the CHRNA4 Ser252Leu mutation. CONCLUSIONS: Disorders associated with mutations in the transmembrane region 2 of CHRNA4 are genetically and phenotypically heterogeneous. Distinctive features of this kindred include (1) mental retardation in all affected members available for testing, (2) abnormal brain findings on fluorodeoxyglucose F 18 with positron emission tomography, (3) poor response to carbamazepine, and (4) full penetrance.