Examining trabecular morphology and chemical composition of peri-scaffold osseointegrated bone.

BACKGROUND: Porous titanium alloy scaffold fabricated by 3D printing technology could induce osseointegration well to repair bone defect during early postoperative period. However, trabecular histomorphological features and chemical compositions of ingrowth bone in the long term after surgery still lacked in-depth research. METHODS: Fourteen New Zealand rabbits were divided into two groups (7 rabbits in surgery group and 7 rabbits in control group). A 3D-printed porous titanium alloy scaffold was implanted into right femoral condyle of each rabbit in the surgery group. Preload was produced at the surface between bone tissue and scaffold through interference assembly during implantation process. Rabbits in the control group were feed free. All rabbits were sacrificed to extract femoral condyles at week 12 after surgery. All right femoral condyles were performed micro-CT scanning to test bone mineral density (BMD) and trabecular histomorphological parameters, including bone volume fraction (BV/TV), bone surface/volume ratio (BS/BV), bone surface density (BS/TV), structure model index (SMI), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular separation (Tb.Sp), porosity (PO), connectivity density (Conn.Dn), and degree of anisotropy (DA). Scanning electron microscope was used to observe osteogenesis peri-scaffold. Fourier transform infrared spectroscopy (FTIR) scanning was performed to analyze chemical compositions of peri-scaffold trabeculae. All trabecular morphological parameters and BMDs were statistically analyzed between surgery group and control group. RESULTS: The pores of scaffold were filled with ingrowth bone tissues after 12 weeks osseointegration. However, the mean BMD peri-scaffold in surgery group was 800 ± 20 mg/cm3, which was 18.37% lower than that in the control group. There was a significant decrease in BV/TV, Tb.N, and BS/TV, and there was a significant increase in Tb.Sp and PO between the surgery group and control group (p < 0.05). There were no significant differences in Tb.Th, SMI, Conn.Dn, BS/BV, and DA. Although ingrowth of bone tissue was very effective, some fragmented connective tissues were still found instead of bone tissues on the partial beams of scaffolds through SEM images. It was found from FTIR that there was no significant hydroxyapatite peak signal in surgery group. Collagen in the control group mainly existed as cross-link structure, while non-cross-link structure in the surgery group. CONCLUSIONS: Preload could promote the same good osseointegration ability as chemical surface modification method in the early term after surgery, and better osseointegration effect than chemical surface modification method in the mid-long term after surgery. However, histomorphological features of peri-scaffold trabeculae were still in deterioration and low collagen maturity caused by stress shielding. It was suggested from this study that extra physical training should be taken to stimulate the bone remodeling process for recovering to a healthy level.

An efficient method for decellularization of the rat liver.

BACKGROUND/PURPOSE: Using gradient ionic detergent, we optimized the preparation procedure for the decellularized liver biologic scaffold, and analyzed its immunogenicity and biocompatibility. METHODS: EDTA, hypotonic alkaline solution, Triton X-100, and gradient sodium dodecyl sulfate (1%, 0.5%, and 0.1%, respectively) were prepared for continuous perfusion through the hepatic vascular system. The decellularization of the liver tissue was performed with the optimized reagent buffer and washing protocol. In addition, the preservation of the original extracellular matrix was observed. To analyze its biocompatibility, the scaffold was embedded in a heterologous animal and the inflammation features, including the surrounding cell infiltration and changes of the scaffold architecture, were detected. The cell-attachment ability was also validated by the perfusion culture of HepG2 cells with the scaffold. RESULTS: By using gradient ionic detergent, we completed the decellularization process in approximately 5 h, which was shorter than >10 hours in previous experiments (p<0.001). The extracellular matrix was kept relatively intact, with no obvious inflammatory cellular infiltration or structural damage in the grafted tissue. The engraftment efficiencies of HepG2 were 86±5% (n=8). The levels of albumin and urea synthesis were significantly superior to the ones in traditional two-dimensional culture. CONCLUSION: The current new method can be used efficiently for the decellularization of the liver biologic scaffold with satisfying biocomparability for application both in vivo and in vitro.

Cholesterol modulates cell signaling and protein networking by specifically interacting with PDZ domain-containing scaffold proteins.

Cholesterol is known to modulate the physical properties of cell membranes, but its direct involvement in cellular signaling has not been thoroughly investigated. Here we show that cholesterol specifically binds many PDZ domains found in scaffold proteins, including the N-terminal PDZ domain of NHERF1/EBP50. This modular domain has a cholesterol-binding site topologically distinct from its canonical protein-binding site and serves as a dual-specificity domain that bridges the membrane and juxta-membrane signaling complexes. Disruption of the cholesterol-binding activity of NHERF1 largely abrogates its dynamic co-localization with and activation of cystic fibrosis transmembrane conductance regulator, one of its binding partners in the plasma membrane of mammalian cells. At least seven more PDZ domains from other scaffold proteins also bind cholesterol and have cholesterol-binding sites, suggesting that cholesterol modulates cell signaling through direct interactions with these scaffold proteins. This mechanism may provide an alternative explanation for the formation of signaling platforms in cholesterol-rich membrane domains.

Novel functional MAR elements of double minute chromosomes in human ovarian cells capable of enhancing gene expression.

Double minute chromosomes or double minutes (DMs) are cytogenetic hallmarks of extrachromosomal genomic amplification and play a critical role in tumorigenesis. Amplified copies of oncogenes in DMs have been associated with increased growth and survival of cancer cells but DNA sequences in DMs which are mostly non-coding remain to be characterized. Following sequencing and bioinformatics analyses, we have found 5 novel matrix attachment regions (MARs) in a 682 kb DM in the human ovarian cancer cell line, UACC-1598. By electrophoretic mobility shift assay (EMSA), we determined that all 5 MARs interact with the nuclear matrix in vitro. Furthermore, qPCR analysis revealed that these MARs associate with the nuclear matrix in vivo, indicating that they are functional. Transfection of MARs constructs into human embryonic kidney 293T cells showed significant enhancement of gene expression as measured by luciferase assay, suggesting that the identified MARS, particularly MARs 1 to 4, regulate their target genes in vivo and are potentially involved in DM-mediated oncogene activation.

Functional analysis of BnMAR element in transgenic tobacco plants.

Scaffold/matrix attachment regions (S/MARs) are defined as genomic DNA sequences, located at the physical boundaries of chromatin loops. Previous reports suggest that S/MARs elements may increase and stabilize the expression of transgene. In this study, DNA sequence with MAR characteristics has been isolated from B. napus . The BnMARs sequence was used to flank the CaMV35S-GUS-NOS expression cassette within the T-DNA of the plant expression vector pPZP212. These constructs were introduced into tobacco plants, respectively and the GUS reporter gene expression was investigated in stably transformed plants. When the forward BnMARs sequence was inserted into the upstream of CaMV35S promoter, the average GUS activities were much higher than those without BnMARs in transgenic tobacco. The GUS expression of M(+)35S:GUS, M(+)35S:GUSM(+) and M(+)35S:GUSM(-) constructs increased average 1.0-fold, with or without BnMARs located downstream of NOS. The GUS expression would not be affected when reverse BnMARs sequence inserted whether upstream of CaMV35S promoter or downstream of NOS. The GUS expression was affected a little when reverse BnMARs sequence was inserted the downstream of NOS and BnMARs could not act by serving as of promoter. The results showed that the presence of forward BnMARs sequence does have an obvious impact on enhancing downstream gene expression and its effect is unidirectional.

Characterization and heterologous expression of a new matrix attachment region binding protein from the unicellular green alga Dunaliella salina.

Although interactions between the nuclear matrix and special regions of chromosomal DNA called matrix attachment regions (MARs) are implicated in various nuclear functions, the understanding of the regulatory mechanism of MARs is still poor. A few MAR-binding proteins (MARBP) have been isolated from some plants and animals, but not from the unicellular algae. Here, we identify a novel MAR-binding protein, namely DMBP-1, from the halotolerant alga Dunaliella salina. The cDNA of DMBP-1 is 2322-bp long and contains a 1626 bp of an open reading frame encoding a polypeptide of 542 amino acids (59 kDa). The DMBP-1 expressed in Escherichia coli specifically binds A/T-rich MAR DNA. The DMBP-1 fused to green fluorescent protein appears only inside the nuclei of Chinese hamster ovarian cells transfected with the pEGFP-MBP, indicating that the protein is located in the nuclei. The findings mentioned above may contribute to better understanding of the nuclear matrix-MAR interactions.

Patient autoantibodies deplete postsynaptic muscle-specific kinase leading to disassembly of the ACh receptor scaffold and myasthenia gravis in mice.

The postsynaptic muscle-specific kinase (MuSK) coordinates formation of the neuromuscular junction (NMJ) during embryonic development. Here we have studied the effects of MuSK autoantibodies upon the NMJ in adult mice. Daily injections of IgG from four MuSK autoantibody-positive myasthenia gravis patients (MuSK IgG; 45 mg day(1)i.p. for 14 days) caused reductions in postsynaptic ACh receptor (AChR) packing as assessed by fluorescence resonance energy transfer (FRET). IgG from the patients with the highest titres of MuSK autoantibodies caused large (51-73%) reductions in postsynaptic MuSK staining (cf. control mice; P < 0.01) and muscle weakness. Among mice injected for 14 days with control and MuSK patient IgGs, the residual level of MuSK correlated with the degree of impairment of postsynaptic AChR packing. However, the loss of postsynaptic MuSK preceded this impairment of postsynaptic AChR. When added to cultured C2 muscle cells the MuSK autoantibodies caused tyrosine phosphorylation of MuSK and the AChR beta-subunit, and internalization of MuSK from the plasma membrane. The results suggest a pathogenic mechanism in which MuSK autoantibodies rapidly deplete MuSK from the postsynaptic membrane leading to progressive dispersal of postsynaptic AChRs. Moreover, maintenance of postsynaptic AChR packing at the adult NMJ would appear to depend upon physical engagement of MuSK with the AChR scaffold, notwithstanding activation of the MuSK-rapsyn system of AChR clustering.

Modulation of chromatin by MARs and MAR binding oncogenic transcription factor SMAR1.

The orchestration of the events in the cell during the progression of the cell cycle is modulated by various phenomenon which are regulated by structural modules of the cell. The nucleus is a major hub for all these regulatory units which harbour the nuclear matrix, matrix proteins and chromatin. The histone modifications etch a complex code on the chromatin and the matrix proteins in consort with the histone code regulate the gene expression. SMAR1 is a matrix attachment region binding protein that interacts with chromatin modulators like HDAC1, Sin3A and causes chromatin condensation. SMAR1 modulates the chromatin at the Vbeta locus and plays a prominent role in V(D)J recombination. Such indispensable function of SMAR1 by the modulation of chromatin in the context of malignancy and V(D)J recombination emphasizes that MAR binding proteins regulate the complex events of the cell and perturbed expression causes disease conditions.

Scaffold/matrix attachment regions (S/MARs): relevance for disease and therapy.

There is increasing awareness that processes, such as development, aging and cancer, are governed, to a considerable extent, by epigenetic processes, such as DNA and histone modifications. The sites of these modifications in turn reflect their position and role in the nuclear architecture. Since epigenetic changes are easier to reverse than mutations, drugs that remove or add the chemical tags are at the forefront of research for the treatment of cancerous and inflammatory diseases. This review will use selected examples to develop a unified view that might assist the systematic development of novel therapeutic regimens.

Role of gender and anatomical region on induction of osteogenic differentiation of human adipose-derived stem cells.

Adipose-derived stem cells (ASCs) display multilineage plasticity and, under appropriate conditions, can mineralize their extracellular matrix and undergo osteogenesis. The aims of this study are to examine in vitro osteogenic differentiation properties of ASCs to assess the role of gender, fat depot, and optimal duration as variables for differentiation. Human ASCs were isolated from superficial and deep adipose layers of the abdominoplasty specimens obtained from patients undergoing elective surgeries. ASCs were cultured in osteogenic media (OM). After 1, 2, and 4 weeks of differentiation, cultures were assessed for markers of osteogenesis. Alkaline phosphatase (AP), alizarin red (AR) and Masson trichrome (MT) stainings for osteoblastic transformation, matrix mineralization, and collagen production; enzyme-linked immunosorbent assay (ELISA) for Gla-osteocalcin; and Western blot analysis for osteonectin protein expression were performed. Osteogenic differentiation began as early as 1 week. Cells exhibited a vertical growth pattern, lacunae formed in the cultures, matrix volume increased, and mineralization was observed. Differences in AP staining were most evident during the first week. AR activity progressively increased over 4 weeks, and collagen was secreted only by differentiated ASCs. There was no significant difference in the degree of osteogenic differentiation between the ASCs from both depots in the female. In the male, the superficial depot ASCs differentiated faster and more efficiently than those of the deep depot. Male ASCs from both depots differentiated more effectively than female ASCs from both depots. We describe a hierarchy of osteogenic differentiation potential based on gender and anatomic harvest site by layering adipose tissues of the abdominal wall. ASCs derived from male superficial layer were most efficient in achieving osteogenesis. In future clinical applications using stem cells for osseous healing, these gender and depot differences will guide our clinical methods.

Matrix attachment regions as targets for retroviral integration.

BACKGROUND: The randomness of retroviral integration has been debated for many years. Recent evidence indicates that integration site selection is not random, and that it is influenced by both viral and cellular factors. To study the role of DNA structure in site selection, retroviral integration near matrix attachment regions (MARs) was analyzed for three different groups of retroviruses. The objective was to assess whether integration near MARs may be a factor for integration site selection. RESULTS: Results indicated that MLV, SL3-3 MuLV, HIV-1 and HTLV-1 integrate preferentially near MARs, specifically within 2-kilobases (kb). In addition, a preferential position and orientation relative to the adjacent MAR was observed for each virus. Further analysis of SL3-3 MuLV insertions in common integration sites (CISs) demonstrated a higher frequency of integration near MARs and an orientation preference that was not observed for integrations outside CISs. CONCLUSION: These findings contribute to a growing body of evidence indicating that retroviral integration is not random, that MARs influence integration site selection for some retroviruses, and that integration near MARs may have a role in the insertional activation of oncogenes by gammaretroviruses.

S/MAR-binding properties of Sox2 and its involvement in apoptosis of human NT2 neural precursors.

DNA fragmentation in apoptosis, especially in lymphocytic cells, is initiated at scaffold/matrix attachment regions (S/MARs) and is preceded by the degradation of nuclear proteins. The present study was performed to establish whether the same mechanism occurred in human NT2 cells subjected to oxygen and glucose deprivation (OGD). We analyzed the integrity of c-myc S/MAR containing a base-unpairing region (BUR)-like element, which we established to be a binding site of the transcription factor Sox2. An accumulation of DNA breaks in close proximity to this element and a degradation of Sox2 were observed early in the OGD-induced apoptotic response. Identification of Sox2 as a novel c-myc BUR-binding protein was achieved through yeast one-hybrid screening and the Sox2/DNA interaction was confirmed by electrophoretic mobility shift assay and immunoprecipitation with Sox2 antibody. Our data support the notion that early proteolysis of unique BUR-binding proteins might represent a universal mechanism that renders these DNA sites vulnerable to endonucleolysis.

Internal organisation of the nucleus: assembly of compartments by macromolecular crowding and the nuclear matrix model.

Many and possibly all macromolecules in the nucleus are segregated into discrete compartments, but the current model that this is achieved by a fibrillar nuclear matrix which structures the nuclear interior and compartments is not consistent with all experimental observations, as reviewed here. New results are presented which suggest that macromolecular crowding forces play a crucial role in the assembly of at least two compartments, nucleoli and PML bodies, and an in vitro system in which crowding assembles macromolecular complexes into structures which resemble nuclear compartments is described. Crowding forces, which are strong in the nucleus due to the high macromolecule concentration (in the range of 100 mg/ml), vastly increase the association constants of intermolecular interactions and can segregate different macromolecules into discrete phases. The model that they play a role in compartmentalisation of the nucleus is generally consistent with the properties of compartments, including their spherical or quasispherical form and their dynamic and mobile nature.

Breakpoint cluster regions of the AML-1 and ETO genes contain MAR elements and are preferentially associated with the nuclear matrix in proliferating HEL cells.

The spatial organization in interphase nuclei of the breakpoint cluster regions (BCRs) of the AML-1 and ETO genes frequently participating in reciprocal t(8;21) translocations was studied using cytological and biochemical approaches. Both BCRs were found to be localized preferentially, but not exclusively, to the nuclear matrix, as shown by hybridization of specific probes with nuclear halos. This association was not related to transcription, because the transcribed regions of both genes located far from BCRs were located preferentially in loop DNA, as shown by in situ hybridization. The sites of association with the nuclear matrix of the intensely transcribed AML-1 gene were mapped also using the biochemical PCR-based approach. Only the BCR was found to be associated with the nuclear matrix, whereas the other transcribed regions of this gene turned out to be positioned randomly in respect to the nuclear matrix. The data are discussed in the framework of the hypothesis postulating that the nuclear matrix plays an important role in determining the positions of recombination-prone areas.

Lectins induce resistance to proteases and/or mechanical stimulus in all examined cells--including bone marrow mesenchymal stem cells--on various scaffolds.

Transplantation of bone marrow mesenchymal stem cells (MSC), chondrocytes, osteoblasts, or muscle cells promotes regeneration. However, these cells adhere poorly to some scaffolds--depending upon the scaffold material--and are often damaged by proteases or mechanical stimuli at site of transplantation. We found, however, that MSC, chondrocytes, and osteoblasts--along with some other cells--that were exposed to phaseolus vulgaris erythroagglutinin (PHA-E) or concanavalin A (ConA) increased their adhesion capacity on plastic tissue culture dishes and on plates of hydroxyapatite, titanium and poly-DL-lactic-co-glycolic acid (PLGA), and that these cells, moreover, built up resistance to proteases and/or mechanical stimuli. Thus, lectins may have great potential in tissue engineering and cell therapy.