Plays an Intrinsic Role in Regulating Proliferation of Mesenchymal Cells in the Developing Palate.

Cleft palate is a common congenital abnormality that results from defective secondary palate (SP) formation. The () gene has been linked to abnormalities of craniofacial and kidney development. Our current study examined, for the first time, the specific role of in embryonic mouse SP development.

Analyzing Biological Performance of 3D-Printed, Cell-Impregnated Hybrid Constructs for Cartilage Tissue Engineering.

Three-dimensional (3D) bioprinting of hybrid constructs is a promising biofabrication method for cartilage tissue engineering because a synthetic polymer framework and cell-impregnated hydrogel provide structural and biological features of cartilage, respectively. During bioprinting, impregnated cells may be subjected to high temperatures (caused by the adjacent melted polymer) and process-induced mechanical forces, potentially compromising cell function. This study addresses these biofabrication issues, evaluating the heat distribution of printed polycaprolactone (PCL) strands and the rheological property and structural stability of alginate hydrogels at various temperatures and concentrations.

The Effect of Chondroitin Sulphate and Hyaluronic Acid on Chondrocytes Cultured within a Fibrin-Alginate Hydrogel.

Osteoarthritis is a painful degenerative joint disease that could be better managed if tissue engineers can develop methods to create long-term engineered articular cartilage tissue substitutes. Many of the tissue engineered cartilage constructs currently available lack the chemical stimuli and cell-friendly environment that promote the matrix accumulation and cell proliferation needed for use in joint cartilage repair. The goal of this research was to test the efficacy of using a fibrin-alginate hydrogel containing hyaluronic acid (HA) and/or chondroitin sulphate (CS) supplements for chondrocyte culture.

Strategic design and fabrication of engineered scaffolds for articular cartilage repair.

Damage to articular cartilage can eventually lead to osteoarthritis (OA), a debilitating, degenerative joint disease that affects millions of people around the world. The limited natural healing ability of cartilage and the limitations of currently available therapies make treatment of cartilage defects a challenging clinical issue. Hopes have been raised for the repair of articular cartilage with the help of supportive structures, called scaffolds, created through tissue engineering (TE).

FLRT2 promotes cellular proliferation and inhibits cell adhesion during chondrogenesis.

One of the earliest events during chondrogenesis is the formation of condensations, a necessary pre-requisite for subsequent differentiation of a chondrogenic phenotype. Members of the Fibronectin Lecucine Rich Transmembrane (FLRT) proteins have been shown to be involved in cell sorting and neurite outgrowth. Additionally, FLRT2 is highly expressed at putative sites of chondrogenic differentiation during craniofacial development.

Hoxa2 plays a direct role in murine palate development.

The cleft palate exhibited by Hoxa2 null murine embryos has been described as being secondary to abnormalities of tongue musculature, and Hoxa2 was presumed to not play a direct role in palate development. However, we detected Hoxa2 expression in the developing palate at both the mRNA and protein levels between embryonic day (E) 12.5 and E15.

Regulation of cartilage formation and maturation by mitogen-activated protein kinase signaling.

The majority of bones comprising the adult vertebrate skeleton are generated from hyaline cartilage templates that form during embryonic development. A process known as endochondral ossification is responsible for the conversion of these transient cartilage anlagen into mature, calcified bone. Endochondral ossification is a highly regulated, multistep cell specification program involving the initial differentiation of prechondrogenic mesenchymal cells into hyaline chondrocytes, terminal differentiation of hyaline chondrocytes into hypertrophic chondrocytes, and finally, apoptosis of hypertrophic chondrocytes followed by bone matrix deposition.

Synchrotron FTIR microspectroscopic analysis of the effects of anti-inflammatory therapeutics on wound healing in laminectomized rats.

Peridural scarring, or the excessive formation of scar tissue following spinal surgery, is one of the important contributing factors that result in persistent pain and disability in many individuals who have undergone elective back surgery. Treatment with anti-inflammatory agents following surgery may reduce oxidative stress and scarring, leading to a reduction in post-operative pain. We are using a surgical rat model to test the hypothesis that post-surgical inflammation and oxidative stress following laminectomy can be reduced by systemic administration of L: -2-oxo-thiazolidine-4-carboxylate (OTC) and quercetin.

Fibroblast growth factors 2, 4, and 8 exert both negative and positive effects on limb, frontonasal, and mandibular chondrogenesis via MEK-ERK activation.

Fibroblast growth factors (FGFs) and their receptors play fundamental roles regulating growth, morphogenesis, and cartilage formation in embryonic limbs and facial primordia. However, the intracellular pathways that transduce FGF signals during the differentiation of pluripotent mesenchymal cells into chondrocytes are currently unknown. Our present study demonstrates that FGF8, 4, and 2 treatments exert both inhibitory and stimulatory effects on cartilage differentiation in micromass cultures prepared from mesenchymal cells of the chick embryo wing bud, frontonasal mass, and mandibular arch through activation of the MEK-ERK mitogen-activated protein kinase (MAPK) cascade.

MEK-ERK signaling plays diverse roles in the regulation of facial chondrogenesis.

The extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase pathway, also known as the MEK-ERK cascade, has been shown to regulate cartilage differentiation in embryonic limb mesoderm and several chondrogenic cell lines. In the present study, we employed the micromass culture system to define the roles of MEK-ERK signaling in the chondrogenic differentiation of neural crest-derived ectomesenchyme cells of the embryonic chick facial primordia. In cultures of frontonasal mesenchyme isolated from stage 24/25 embryos, treatment with the MEK inhibitor U0126 increased type II collagen and glycosaminoglycan deposition into cartilage matrix, elevated mRNA levels for three chondrogenic marker genes (col2a1, aggrecan, and sox9), and increased expression of a Sox9-responsive collagen II enhancer-luciferase reporter gene.

The MEK-ERK signaling pathway is a negative regulator of cartilage-specific gene expression in embryonic limb mesenchyme.

The extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase pathway, also known as the MEK-ERK kinase cascade, has recently been implicated in the regulation of embryonic cartilage differentiation. However, its precise role in this complex process remains controversial. To more thoroughly examine the role of the MEK-ERK kinase cascade in chondrogenesis, we analyzed the effects of two structurally different pharmacological inhibitors of MEK, the upstream kinase activator of ERK, on chondrocyte differentiation in micromass cultures of embryonic chick limb mesenchyme cells.

Molecular and cell biology of porcine HSP47 during wound healing: complete cDNA sequence and regulation of gene expression.

Heat shock protein (HSP) 47 is a major stress-inducible protein that is localized to the endoplasmic reticulum of avian and mammalian cells and is thought to act as a molecular chaperone specific for the processing of procollagen. However, limited information is available regarding the regulation of HSP47 during wound healing. Using a polymerase chain reaction strategy, screening of a cDNA library, and RACE-polymerase chain reaction approaches, the sequence of a full-length porcine HSP47 cDNA has been identified.

Molecular and cell biology of skin wound healing in a pig model.

To define the pattern of change at the molecular and cellular levels during the healing of excisional skin wounds in the skeletally immature pig, mRNA levels for relevant molecules were assessed by semiquantitative RT-PCR using porcine specific primer sets and RNA isolated from normal skin and samples at various time post-wounding. Analysis of cellular change was assessed by DNA quantification and histology of tissue sections. The results demonstrated that the changes in the pattern of RNA and DNA content of the scar tissue were consistent with the observed increasing cellularity.

Expression of heat shock protein 47(Hsp47) mRNA levels in rabbit connective tissues during the response to injury and in pregnancy.

Hsp47 (also termed "colligin") is a 47 kDa protein that is localized in the ER and cis-Golgi vesicles of fibrocytes, chondrocytes, and other collagen-secreting cells. Under stress conditions, Hsp47 expression is upregulated as part of the heat shock/stress response that mitigates cell damage from noxious stimuli such as elevated temperature, heavy metals, and oxidative stress. Under non-stress conditions, Hsp47 functions as a collagen-specific molecular chaperone that facilitates intracellular procollagen polypeptide synthesis, and triple helix assembly in connective tissues.

Sox9 expression during chondrogenesis in micromass cultures of embryonic limb mesenchyme.

Sox9 plays a crucial role in chondrogenesis. It encodes an HMG-domain transcription factor that activates an enhancer in the gene for type II collagen (Col2a1), a principal cartilage matrix protein. We have characterized the temporal pattern of Sox9 RNA expression in micromass culture, a widely used in vitro model for the analysis of embryonic cartilage differentiation.

Alcohol promotes in vitro chondrogenesis in embryonic facial mesenchyme.

Ethanol is a well-recognized teratogen in vertebrates that can perturb the development of the facial primordia and various other embryonic structures. However,the mechanisms underlying alcohol's effects on embryogenesis are currently unclear. Recent evidence suggests that the cranial neural crest, which forms the entire facial skeleton, may be a particularly sensitive target of ethanol teratogenicity.

Ethanol exposure stimulates cartilage differentiation by embryonic limb mesenchyme cells.

Studies of neural, hepatic, and other cells have demonstrated that in vitro ethanol exposure can influence a variety of membrane-associated signaling mechanisms. These include processes such as receptor-kinase phosphorylation, adenylate cyclase and protein kinase C activation, and prostaglandin production that have been implicated as critical regulators of chondrocyte differentiation during embryonic limb development. The potential for ethanol to affect signaling mechanisms controlling chondrogenesis in the developing limb, together with its known ability to promote congenital skeletal deformities in vivo, prompted us to examine whether chronic alcohol exposure could influence cartilage differentiation in cultures of prechondrogenic mesenchyme cells isolated from limb buds of stage 23-25 chick embryos.

Cloning and characterization of a cDNA encoding the collagen-binding stress protein hsp47 in zebrafish.

Hsp47 is a major stress-inducible protein that is localized to the endoplasmic reticulum of avian and mammalian cells and is thought to act as a molecular chaperone specific for the processing of procollagen. Although hsp47 is coordinately expressed together with several collagen types, and vertebrate embryos are known to express collagen genes in complex spatial and temporal patterns, limited information is available regarding the function or regulation of hsp47 during early embryonic development. We have initiated an examination of hsp47 in the zebrafish, Danio rerio, which offers a number of features that make it attractive as a model developmental system with which to examine the expression and function of hsp47.

Staurosporine, a protein kinase inhibitor, stimulates cartilage differentiation by embryonic facial mesenchyme.

We have examined the effects of staurosporine, a potent inhibitor of protein kinase C, on cartilage differentiation in cultured mesenchyme of embryonic facial primordia. Mesenchymal cells from the frontonasal, maxillary, and mandibular processes and hyoid arches of stage 24/25 chicken embryos were maintained in high density micromass cell cultures in the presence or absence of 5 nM staurosporine. In cultures of frontonasal and mandibular process mesenchyme, which spontaneously developed numerous chondrogenic cell aggregates, staurosporine treatment enhanced Alcian blue-positive matrix accumulation, increased pericellular sulfated glycosaminoglycan (GAG) deposition by 5.

Type IX collagen gene expression during limb cartilage differentiation.

Changes in the steady-state levels of mRNAs for the alpha 1(IX) and alpha 2(IX) polypeptide chains of cartilage-characteristic type IX collagen were examined during the course of chick limb chondrogenesis in vitro and in vivo. Cytoplasmic type IX collagen mRNAs begin to accumulate at the onset of overt chondrogenesis in high density micromass culture coincident with the crucial condensation phase of the process, in which prechondrogenic mesenchymal cells become closely juxtaposed prior to depositing a cartilage matrix. The initiation of type IX collagen mRNA accumulation at condensation coincides with the initiation of accumulation of cartilage proteoglycan core protein mRNA and with a striking increase in type II collagen mRNA accumulation.

Promotion of embryonic limb cartilage differentiation in vitro by staurosporine, a protein kinase C inhibitor.

Phorbol 12-myristate 13-acetate (PMA), a protein kinase C-activating phorbol ester, is known to inhibit chondrogenic differentiation by embryonic limb mesenchyme cells in vitro. The present study demonstrates that staurosporine, a potent inhibitor of protein kinase C, conversely stimulates cartilage differentiation in cultures of limb mesenchyme cells isolated from whole wing buds of stage 23/24 chick embryos or from the distal subridge region of stage 25 wing buds. In high density micromass cultures, in which limb mesenchyme cells undergo extensive spontaneous cartilage differentiation, exposure to 5-20 nM staurosporine promotes an accelerated accumulation of type II collagen and cartilage proteoglycan mRNA transcripts and a 2- to 3-fold increase in matrix glycosaminoglycan deposition.

Stimulation of limb cartilage differentiation by cyclic AMP is dependent on cell density.

Cyclic AMP (cAMP) has been implicated in the regulation of limb cartilage differentiation. This study represents an attempt to clarify potential mechanisms by which cAMP might regulate chondrogenesis. We have found that the ability of cAMP to stimulate limb cartilage differentiation in vitro is dependent on cell density.

Promotion of embryonic chick limb cartilage differentiation by transforming growth factor-beta.

This study represents a first step in investigating the possible involvement of transforming growth factor-beta (TGF-beta) in the regulation of embryonic chick limb cartilage differentiation. TGF-beta 1 and 2 (1-10 ng/ml) elicit a striking increase in the accumulation of Alcian blue, pH 1-positive cartilage matrix, and a corresponding twofold to threefold increase in the accumulation of 35S-sulfate- or 3H-glucosamine-labeled sulfated glycosaminoglycans (GAG) by high density micromass cultures prepared from the cells of whole stage 23/24 limb buds or the homogeneous population of chondrogenic precursor cells comprising the distal subridge mesenchyme of stage 25 wing buds. Moreover, TGF-beta causes a striking (threefold to sixfold) increase in the steady-state cytoplasmic levels of mRNAs for cartilage-characteristic type II collagen and the core protein of cartilage-specific proteoglycan.

Fibronectin gene expression during limb cartilage differentiation.

A critical event in limb cartilage differentiation is a transient cellular condensation process in which prechondrogenic mesenchymal cells become closely juxtaposed and interact with one another prior to initiating cartilage matrix deposition. Fibronectin (FN) has been suggested to be involved in regulating the onset of condensation and chondrogenesis by actively promoting prechondrogenic aggregate formation during the process. We have performed a systematic quantitative study of the expression of the FN gene during the progression of chondrogenesis in vitro and in vivo.

In situ hybridization analysis of the expression of the type II collagen gene in the developing chicken limb bud.

In situ hybridization with [32P]- or [35S]-labeled double-stranded DNA or single-stranded RNA probes was used to investigate the temporal and spatial distribution of cartilage-characteristic type II collagen mRNA during embryonic chick limb development and cartilage differentiation in vivo. When the type II collagen probes were hybridized to sections through embryonic limb buds at the earliest stages of their development (stages 18-25), an accumulation of silver grains representing type II collagen mRNA first became detectable in the proximal central core of the limb coincident with the prechondrogenic condensation of mesenchymal cells that characterizes the onset of cartilage differentiation. At later stages of development (stage 32; 7 days) intense hybridization signals with the type II collagen probes were localized over the well differentiated cartilage rudiments, whereas few or no silver grains above background were observed over the non-chondrogenic tissues.