Electrospun composite poly(L-lactic acid)/tricalcium phosphate scaffolds induce proliferation and osteogenic differentiation of human adipose-derived stem cells.

Development of tissue-engineered bone constructs has recently focused on the use of electrospun composite scaffolds seeded with stem cells from various source tissues. In this study, we fabricated electrospun composite scaffolds consisting of beta-tricalcium phosphate (TCP) crystals and poly(L-lactic acid) (PLA) at varying loading levels of TCP (0, 5, 10, 20 wt%) and assessed the composite scaffolds' material properties and ability to induce proliferation and osteogenic differentiation of human adipose-derived stem cells (hASCs) in the presence of osteogenic differentiating medium. The electrospun scaffolds all exhibited a nonwoven structure with an interconnected porous network. With the addition of TCP, the fiber diameter increased with each treatment ranging from 503.39 +/- 20.31 nm for 0 wt% TCP to 1267.36 +/- 59.03 nm for 20 wt% TCP. Tensile properties of the composite scaffolds were assessed and the overall tensile strength of the neat scaffold (0 wt% TCP) was 847 +/- 89.43 kPA; the addition of TCP significantly decreased this value to an average of 350.83 +/- 38.57 kPa. As the electrospun composite scaffolds degraded in vitro, TCP was released into the medium with the largest release occurring within the first 6 days. Human ASCs were able to adhere, proliferate and osteogenically differentiate on all scaffold combinations. DNA content increased in a temporal manner for each scaffold over 18 days in culture although for the day 12 timepoint, the 10 wt% TCP scaffold induced the greatest hASC proliferation. Endogenous alkaline phosphatase activity was enhanced on the composite PLA/TCP scaffolds compared to the PLA control particularly by day 18. It was noted that at the highest TCP loading levels of 10 and 20 wt%, there was a dramatic increase in the amount of cell-mediated mineralization compared to the 5 wt% TCP and the neat PLA scaffold. This work suggests that local environment cues provided by the biochemical nature of the scaffold can accelerate the overall osteogenic differentiation of hASCs and encourage rapid ossification.

Mucin gene expression during differentiation of human airway epithelia in vitro. Muc4 and muc5b are strongly induced.

Mucus hypersecretion is characteristic of chronic airway diseases. However, regulatory mechanisms are poorly understood. Human airway epithelial cells grown on permeable supports at the air-liquid interface (ALI) develop a mucociliary morphology resembling that found in vivo. Such cultures provide a model for studying secretory cell lineage, differentiation, and function, and may provide insight regarding events leading to mucus hypersecretion. The mucin gene expression profile of well-differentiated human airway epithelial cells in culture has not yet been established. We compared expression of all the currently described mucin genes in poorly differentiated (conventional cultures on plastic) and well-differentiated (ALI) human nasal and bronchial epithelial cells. Differentiation-dependent upregulation of MUC3, MUC5AC, MUC5B, and MUC6 messenger RNA (mRNA) was demonstrated using reverse transcriptase-polymerase chain reaction (RT-PCR). Northern blot analysis showed a similar increase for MUC4 and demonstrated that induction of MUC4 and MUC5B expression depended on retinoic acid. MUC1, MUC2, MUC7, and MUC8 mRNAs were also detected by RT-PCR, but these genes did not appear to be strongly regulated as a function of differentiation. Mucin gene expression was similar in bronchial and nasal cells. Thus, mucociliary differentiation of human airway epithelia in vitro entails upregulation of several mucin genes.

Suppression of relaxin gene expression by retinoids in squamous differentiated rabbit tracheal epithelial cells.

Northern blot analysis of total RNA from a variety of rabbit tissues indicated that placenta is the primary site of expression of the protein hormone relaxin (previously called SQ10) in rabbits. Relaxin was not detected by this method in other rabbit tissues, including normal trachea and several squamous tissues. However, relaxin is highly induced during squamous cell differentiation in cultured rabbit tracheal epithelial (RbTE) cells. Retinoic acid and retinoids that selectively bind to the nuclear retinoid receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs), and induce RARE- or RXRE-dependent transactivation as well as repression of AP-1-dependent transactivation, were all effective in suppressing relaxin expression. In addition, the retinoid SR11302, which exhibits only anti-AP-1 activity but does not induce RARE- or RXRE-dependent transactivation, was also able to inhibit relaxin expression. These results suggest that the suppression of relaxin expression is related to the anti-AP-1 activity of retinoids. To determine whether the relaxin gene is regulated by retinoids at the level of transcription, a 4.3 kb fragment of the 5' flanking region of the rabbit relaxin gene was cloned and analyzed. This regulatory region included a classic TATA-box as well as consensus sequences for several transcription factors, including CREB, NF-kappaB and AP-1. The ability of the 4.3 kb regulatory region to control the transcription of a luciferase reporter gene was analyzed in transiently transfected, squamous-differentiated RbTE cells. The results demonstrated that this regulatory region caused strong transactivation of the reporter gene. This transactivation was inhibited by retinoic acid, suggesting retinoid control at the transcriptional level. Deletion analysis indicated that multiple regulatory elements are involved in the regulation of relaxin gene expression during squamous differentiation as well as in the suppression by retinoids.

Regulation of transglutaminase type I expression in squamous differentiating rabbit tracheal epithelial cells and human epidermal keratinocytes: effects of retinoic acid and phorbol esters.

In the present study we describe the full length cDNA sequence for rabbit transglutaminase type I as well as the sequence for a 2.9-kilobase (kb) promoter fragment of the gene. Transglutaminase type I mRNA expression was inhibited in squamous differentiating epithelia by retinoic acid (RA) in a dose-dependent (EC50 = 1-2 nM) and transcriptional manner. In human epidermal keratinocytes transglutaminase type I mRNA was induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment, and this induction could be inhibited by bryostatin 1. In contrast, TPA treatment inhibited the expression of c-myc mRNA. Bryostatin 1 but not RA could prevent this decrease in c-myc mRNA expression, indicating that transglutaminase type I mRNA expression was associated with differentiation and not growth arrest. An SP1 element was found within 50 base pairs 5' of the transcription initiation site. A TATA-like element (CATAAAC) was found but was not capable of activating transcription. In addition, putative response elements for C-MYC, Ker1/AP2, 2 AP1 sites, a CK-8-mer, and an AP2 site were present in the 2.9-kb fragment. Transfection of RbTE cells with the 2.9-kb fragment ligated to a promoterless luciferase vector resulted in 2.2-fold more luciferase expression in differentiated vs. undifferentiated cells. Furthermore, luciferase activity was induced 7.4-fold in human epidermal keratinocytes induced to differentiate with TPA. TPA-induced luciferase activity was inhibited by both bryostatin 1 and RA. No known RA response elements were identified in the promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

Cornifin, a cross-linked envelope precursor in keratinocytes that is down-regulated by retinoids.

In this study, we have characterized the cDNA clone SQ37 that was isolated previously from a rabbit squamous cell library. The gene encodes a 14-kDa protein that appears to function as a component of the cross-linked envelope in squamous differentiating cells. The protein, which has been named cornifin, has a high content of proline (31%), glutamine (20%), and cysteine (11%) and contains 13 repeats of an octapeptide (consensus sequence, EPCQPKVP) at its C terminus. SQ37 mRNA and protein are induced during squamous differentiation of rabbit tracheal (RbTE) cells and human epidermal keratinocytes. This induction is repressed by retinoids. Immunohistochemical studies reveal SQ37 immunoreactivity in fragmented cross-linked envelopes from squamous-differentiated RbTE cells and in the suprabasal layers of the epidermis. In situ hybridization analysis showed that the presence of SQ37 mRNA is restricted to the suprabasal layers. Treatment of RbTE cells with a Ca2+ ionophore induces cross-linking of the SQ37 protein into higher molecular weight complexes. This cross-linking reaction appears to be mediated by transglutaminase type I. Our observations suggest that the protein encoded by SQ37 participates in the assembly of the cross-linked envelope.

Expression of a preprorelaxin-like gene during squamous differentiation of rabbit tracheobronchial epithelial cells and its suppression by retinoic acid.

Squamous cell differentiation in tracheobronchial epithelial cells is accompanied by many biochemical and molecular changes. One of the molecular changes in rabbit tracheal epithelial (RbTE) cells is the differential expression of a squamous cell-specific mRNA encoded by the complementary DNA SQ10. In this study, we sequenced SQ10 complementary DNA and showed that this gene encodes a preprorelaxin-like protein. The DNA sequence of the coding region of SQ10 has 68% identity with the human preprorelaxin mRNA, whereas the deduced amino acid sequence exhibits 46% identity with human preprorelaxin. An antiserum (pepIV-Ab) was raised against a synthetic 22-amino acid oligopeptide of the protein encoded by SQ10. Immunoblot analysis of cellular extracts of squamous-differentiated cells showed that this antiserum reacted with proteins of 22 and 20 kilodaltons, possibly constituting prepro- and proforms of this protein. These proteins were undetectable in undifferentiated RbTE cells. In agreement with these observations, PepIV-Ab specifically stained the cytosol of squamous-differentiated RbTE cells but failed to stain undifferentiated cells. PepIV-Ab recognized a 20 and 16 kilodalton polypeptide in medium conditioned by squamous-differentiated RbTE cells, indicating that the prorelaxin-like protein is secreted. The amino acid sequences of three peptides that were obtained after tryptic digestion of the secreted 16 kilodalton protein were identical to sequences encoded by SQ10. Retinoids which have been shown to inhibit squamous differentiation suppressed the induction of SQ10 protein as well as mRNA in a concentration-dependent manner. The concentration at which retinoic acid caused a 50% inhibition of SQ10 mRNA levels was approximately 5 nM.(ABSTRACT TRUNCATED AT 250 WORDS)

Homeobox 1.3 expression: induction by retinoic acid in human bronchial fibroblasts.

Homeobox (Hox) genes code for transcriptional factors and are expressed during many developmental and differentiative processes. In this study, we describe the induction of Hox 1.3 expression by retinoic acid (RA) in human bronchial fibroblasts (HBF) derived from explants of bronchial tissue. Using Northern blot analysis, we show that RA induces Hox 1.3 mRNA 3- to 10-fold over steady-state levels within 2 h after addition of RA to HBF culture medium. The induction was dose dependent, reaching a half-maximal level at approximately 10(-8) M RA. This induction was not seen in human dermal fibroblasts. Immunofluorescent staining of HBF showed a corresponding increase in Hox 1.3 protein levels in the nuclei. The increase in Hox 1.3 transcript levels in HBF was not abolished by cycloheximide treatment, suggesting that synthesis of a protein intermediate is not required for the induction. RA did not significantly alter the rate of degradation of the Hox 1.3 mRNA as determined by actinomycin D treatment, suggesting that the increase in Hox 1.3 mRNA may be due to an increase in the rate of transcription. This study provides further evidence that bronchial fibroblasts are targets for RA. Although downstream target genes for Hox 1.3 have not yet been identified, it is likely that the induction of Hox 1.3 by RA is an early step in a cascade of RA-induced changes in gene expression in bronchial fibroblasts.

Interrelationships of platelet-derived growth factor isoform-induced changes in c-fos expression, intracellular free calcium, and mitogenesis.

Both increases in c-fos proto-oncogene expression and intracellular free calcium ([Ca2+]i) have been implicated as necessary components of the signal transduction pathway by which platelet-derived growth factor (PDGF) stimulates DNA synthesis in cultured BALB/c3T3 fibroblasts. To determine the interrelationship between PDGF-induced increases in c-fos proto-oncogene expression and [Ca2+]i, purified, recombinant BB and AA homodimeric isoforms of PDGF were used to evaluate the dose-response relationships and mechanisms of growth factor-induced changes in these two parameters as well as DNA synthesis. Concentration-dependent increases in [Ca2+]i, c-fos expression, and [3H]thymidine incorporation were observed with both BB and AA PDGF isoforms. BB PDGF was consistently more potent and efficacious than the AA isoform in eliciting a given response. The [Ca2+]i dependency of PDGF-induced increases in c-fos expression and DNA synthesis was determined by pretreatment of cells with agents that inhibit increases in [Ca2+]i: BAPTA, Quin-2, and TMB-8. Under these conditions, PDGF-induced DNA synthesis was blocked, whereas c-fos expression was enhanced. Conversely, in cells made deficient in protein kinase C (PKC) activity by prolonged treatment with phorbol ester, BB and AA PDGF-induced c-fos expression was inhibited by 75-80%, while PDGF-induced increases in [Ca2+]i and DNA synthesis were unaffected or enhanced. Additionally, the PKC-independent component of PDGF-stimulated c-fos expression was found to be independent of increases in [Ca2+]i. These data suggest that 1) both BB and AA PDGF isoforms elicit alterations in [Ca2+]i and c-fos proto-oncogene expression through the same or similar mechanisms in BALB/c3T3 fibroblasts, 2) PDGF-stimulated increases in [Ca2+]i are not required for c-fos expression, and 3) distinct pathways regulate PDGF-induced c-fos expression and mitogenesis, with c-fos expression being substantially PKC-dependent yet [Ca2+]i-independent, while mitogenesis is [Ca2+]i-dependent yet PKC-independent.

Embryonic cellular organization: differential restriction of fates as revealed by cell aggregates and lineage markers.

Cleavage-stage Lytechinus variegatus embryos were dissociated and the cells were aggregated in an experimental system designed to address questions of embryonic organizational capability. Using monoclonal antibodies against stage- and structure-specific antigens, it was determined that germ-layer specific molecules were expressed in the expected locations in aggregates and that the germ layers differentiated in the normal temporal sequence, but the dorsoventral axis of the embryo was not reestablished properly. In other experiments individual rhodamine-labeled blastomeres were incorporated into unlabeled aggregates. Micromeres localized and differentiated normally. Mesomeres, however, which in normal embryos form only ectoderm, were found to change their specified fate and participate in gut formation. The sequence of aggregate organization revealed other properties of the embryo. Ectodermal and endodermal epithelia formed via two temporally distinct epithelialization events. Ectoderm separated from a mass of interior cells at about 12 hrs, and endoderm compacted from the interior cells at about 20 hr. A lathrytic agent that prevents gut formation in normal embryos also prevented gut formation in aggregates; however, it did not affect formation of the ectoderm. Hence, formation of the triploblastic structure in aggregates appears to be dependent upon developmentally regulated, distinct cell adhesion events.