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1.
J Mater Sci Mater Med ; 29(11): 166, 2018 Nov 03.
Article in English | MEDLINE | ID: mdl-30392028

ABSTRACT

Design of cell-free scaffolds for endogenous cell recruitment requires an intimate knowledge of precise relationships between structure and biological function. Here, we use morphological analysis by Micro-CT to identify the key structural features necessary for periodontal ligament fibroblast recruitment into collagen scaffolds. By the combined use of time-lapse imaging and end-point invasion analysis, we distinguish the influences of pore size, pore wall alignment, and pore transport pathways (percolation diameter) on the individual cell migration and bulk invasion characteristics of these fibroblasts. Whereas maximising percolation diameter increased individual cell speed, elongation and directionality, and produced the most rapid bulk cell invasion, a pore size of 100 µm was found to be necessary to ensure an even distribution of cells across the scaffold cross-section. These results demonstrate that control of percolation diameter and pore size may be used respectively to tune the efficiency and uniformity of invasion through macroporous scaffolds. Crucially, however, these observations were subject to the condition of pore wall alignment, with low alignment in the direction of travel producing relatively low cell speeds and limited invasion in all cases. Pore wall alignment should therefore be carefully optimised in the design of scaffolds for cell recruitment, such as that required for periodontal ligament regeneration, as a key determining factor for cell movement.


Subject(s)
Collagen/chemistry , Fibroblasts/physiology , Periodontal Ligament/cytology , Tissue Scaffolds , Cell Movement/physiology , Humans , Tissue Engineering
2.
Dev Dyn ; 245(9): 947-62, 2016 09.
Article in English | MEDLINE | ID: mdl-27264541

ABSTRACT

BACKGROUND: Lineage tracing has shown that most of the facial skeleton is derived from cranial neural crest cells. However, the local signals that influence postmigratory, neural crest-derived mesenchyme also play a major role in patterning the skeleton. Here, we study the role of BMP signaling in regulating the fate of chondro-osteoprogenitor cells in the face. RESULTS: A single Noggin-soaked bead inserted into stage 15 chicken embryos induced an ectopic cartilage resembling the interorbital septum within the palate and other midline structures. In contrast, the same treatment in stage 20 embryos caused a loss of bones. The molecular basis for the stage-specific response to Noggin lay in the simultaneous up-regulation of SOX9 and downregulation of RUNX2 in the maxillary mesenchyme, increased cell adhesiveness as shown by N-cadherin induction around the beads and increased RA pathway gene expression. None of these changes were observed in stage 20 embryos. CONCLUSIONS: These experiments demonstrate how slight changes in expression of growth factors such as BMPs could lead to gain or loss of cartilage in the upper jaw during vertebrate evolution. In addition, BMPs have at least two roles: one in patterning the skull and another in regulating the skeletogenic fates of neural crest-derived mesenchyme. Developmental Dynamics 245:947-962, 2016. © 2016 Wiley Periodicals, Inc.


Subject(s)
Bone Morphogenetic Proteins/metabolism , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/metabolism , Mesoderm/cytology , Mesoderm/physiology , Stem Cells/cytology , Stem Cells/physiology , Animals , Bone Morphogenetic Proteins/genetics , Carrier Proteins/pharmacology , Chick Embryo , Face/embryology , Gene Expression Regulation, Developmental/genetics , Gene Expression Regulation, Developmental/physiology , Mesoderm/metabolism , SOX9 Transcription Factor/genetics , SOX9 Transcription Factor/metabolism , Signal Transduction/drug effects , Stem Cells/metabolism
3.
Tissue Eng Part C Methods ; 22(5): 409-17, 2016 05.
Article in English | MEDLINE | ID: mdl-26888449

ABSTRACT

Interconnecting pathways through porous tissue engineering scaffolds play a vital role in determining nutrient supply, cell invasion, and tissue ingrowth. However, the global use of the term "interconnectivity" often fails to describe the transport characteristics of these pathways, giving no clear indication of their potential to support tissue synthesis. This article uses new experimental data to provide a critical analysis of reported methods for the description of scaffold transport pathways, ranging from qualitative image analysis to thorough structural parameterization using X-ray Micro-Computed Tomography. In the collagen scaffolds tested in this study, it was found that the proportion of pore space perceived to be accessible dramatically changed depending on the chosen method of analysis. Measurements of % interconnectivity as defined in this manner varied as a function of direction and connection size, and also showed a dependence on measurement length scale. As an alternative, a method for transport pathway parameterization was investigated, using percolation theory to calculate the diameter of the largest sphere that can travel to infinite distance through a scaffold in a specified direction. As proof of principle, this approach was used to investigate the invasion behavior of primary fibroblasts in response to independent changes in pore wall alignment and pore space accessibility, parameterized using the percolation diameter. The result was that both properties played a distinct role in determining fibroblast invasion efficiency. This example therefore demonstrates the potential of the percolation diameter as a method of transport pathway parameterization, to provide key structural criteria for application-based scaffold design.


Subject(s)
Biocompatible Materials/chemistry , Cell Movement/physiology , Collagen/chemistry , Tissue Engineering/methods , Tissue Scaffolds/chemistry , X-Ray Microtomography/methods , Achilles Tendon/cytology , Achilles Tendon/metabolism , Animals , Biological Transport , Cattle , Cells, Cultured , Fibroblasts/cytology , Fibroblasts/metabolism , Image Processing, Computer-Assisted
4.
Adv Healthc Mater ; 4(9): 1317-21, 2015 Jun 24.
Article in English | MEDLINE | ID: mdl-25881025

ABSTRACT

The relationship between biological scaffold interconnectivity and cell migration is an important but poorly understood factor in tissue regeneration. Here a scale-independent technique for characterization of collagen scaffold interconnectivity is presented, using a combination of X-ray microcomputed tomography and percolation theory. Confocal microscopy of connective tissue cells reveals this technique as highly relevant for determining the extent of cell invasion.


Subject(s)
Cell Movement , Collagen/chemistry , Fibroblasts/metabolism , Models, Biological , Tissue Scaffolds/chemistry , Cells, Cultured , Fibroblasts/cytology , Humans , X-Ray Microtomography
5.
Bone ; 57(2): 405-12, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24076022

ABSTRACT

The osteogenic and osseointegrative potential of a small molecule was examined to assess its usefulness in regenerative procedures. Purmorphamine was used to stimulate bone growth and repair in an in vitro cell-based assay and an in vivo chick embryo CAM-assay with and without the presence of an implant. Purmorphamine adhered to precipitated hydroxyapatite coating, could activate the sonic hedgehog pathway and thereby stimulated osteodifferentiation. Porous calcium phosphate beads were used to deliver this small molecule in vivo and showed that purmorphamine increased the trabecular bone to bone area significantly. The assay showed purmorphamine failed to induce any significant difference in osseointegration on titanium coated PTFE implants. This suggests that, while a small molecule can enhance osteogenesis and might be useful in regenerative procedures, it failed to enhance the osseointegration of a Ti coated implant, suggesting that this sort of stimulation might be useful for enhancing bone regeneration where bone loss due to disease exists, but not for enhancing early stability of an implant.


Subject(s)
Bone Regeneration/drug effects , Implants, Experimental , Morpholines/pharmacology , Osseointegration/drug effects , Purines/pharmacology , Titanium/pharmacology , Alkaline Phosphatase/metabolism , Animals , Animals, Newborn , Bone Marrow/drug effects , Bone Marrow/physiology , Calcium Phosphates/pharmacology , Chick Embryo , Coated Materials, Biocompatible/pharmacology , Femur/drug effects , Femur/physiology , Mice , Osteogenesis/drug effects , Spectrum Analysis, Raman
6.
Acta Biomater ; 7(6): 2672-7, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21354477

ABSTRACT

The aim of this study was to quantify and provide evidence as to how addition of tricalcium phosphate (ß-TCP) and the Hedgehog agonist purmorphamine to a degradable bone adhesive affects cell attachment/proliferation and Hedgehog pathway activation. Fourier transform infrared spectroscopy demonstrated that high levels (75 wt.%) of ß-TCP addition reduced the photocure rate of the chosen poly(propylene glycol-co-lactide) dimethacrylate (PPLM) bone adhesive, but this problem was overcome by increased light exposure. In phosphate-buffered saline the total surface mass loss of set 15 mm diameter PPLM films was ∼3.2 mg in 12 weeks, irrespective of thickness (200 or 400 µm) or ß-TCP level (50 or 75 wt.%). With 400 µm samples there was additional bulk material loss. Proliferation of pre-osteoblast cells (MC3T3-E1) on the set adhesive surfaces was enhanced by decreased sample thickness or filler content increase. Degradation evidence suggested that both effects were due to reduced acidic polymeric degradation products. Activation of the Hedgehog pathway was quantified by measuring Gli expression in Light II reporter cells. The 0.01 and 0.1 wt.% purmorphamine in composite discs (400 µm, 75 wt.% ß-TCP) enhanced Gli expression of attached cells 2- and 5-fold, respectively, without influencing their number. Pre-storage of the composite samples in culture medium had no detrimental effect on this response. Furthermore, sample storage medium gave no enhanced Gli expression in cells on tissue culture plastic. This suggests drug release levels were very low. Purmorphamine and ß-TCP incorporation in PPLM adhesives might, therefore, provide prolonged enhancement of in vivo bone repair without systemic drug side-effects.


Subject(s)
Adhesives/pharmacology , Calcium Phosphates/pharmacology , Cell Adhesion/drug effects , Morpholines/pharmacology , Purines/pharmacology , 3T3 Cells , Animals , Cell Division/drug effects , Mice
7.
Acta Biomater ; 6(3): 845-55, 2010 Mar.
Article in English | MEDLINE | ID: mdl-19800424

ABSTRACT

A poly(propylene glycol-co-lactide) dimethacrylate adhesive with monocalcium phosphate monohydrate (MCPM)/beta-tricalcium phosphate (beta-TCP) fillers in various levels has been investigated. Water sorption by the photo-polymerized materials catalyzed varying filler conversion to dicalcium phosphate (DCP). Polymer modulus was found to be enhanced upon raising total calcium phosphate content. With greater DCP levels, faster release of phosphate and calcium ions and improved buffering of polymer degradation products were observed. This could reduce the likelihood of pH-catalyzed bulk degradation and localized acid production and thereby may prevent adverse biological responses. Bone-like MG-63 cells were found to attach, spread and have normal morphology on both the polymer and composite surfaces. Moreover, composites implanted into chick embryo femurs became closely apposed to the host tissue and did not appear to induce adverse immunological reaction. The above results suggest that the new composite materials hold promise as clinical effective bone adhesives.


Subject(s)
Bone Cements/chemistry , Bone Cements/therapeutic use , Calcium Phosphates/chemistry , Calcium Phosphates/therapeutic use , Femoral Fractures/therapy , Methacrylates/chemistry , Methacrylates/therapeutic use , Osteoblasts/physiology , Animals , Cell Adhesion , Cell Line , Chick Embryo , Compressive Strength , Femoral Fractures/pathology , Hardness , Humans , Materials Testing , Osteoblasts/cytology , Osteogenesis , Treatment Outcome
8.
Biorheology ; 43(3,4): 355-70, 2006.
Article in English | MEDLINE | ID: mdl-16912408

ABSTRACT

The development of the skull is characterised by its dependence upon epigenetic influences. One of the most important of these is secondary chondrogenesis, which occurs following ossification within certain membrane bone periostea, as a result of biomechanical articulation. We have studied the genesis, character and function of the secondary chondrocytes of the quadratojugal of the chick between embryonic days 11 and 14. Analysis of gene expression revealed that secondary chondrocytes formed coincident with Sox9 upregulation from a precursor population expressing Cbfa1/Runx2: a reversal of the normal sequence. Such secondary chondrocytes rapidly acquired a phenotype that is a compound of prehypertrophic and hypertrophic chondrocytes, exited from the cell cycle and upregulated Ihh. Pulse and pulse/chase experiments with BrdU confirmed the germinal region as the highly proliferative source of the secondary chondrocytes, which formed by division of chondrocyte-committed precursors. By blocking Hh signalling in explant cultures we show that the enhanced proliferation of the germinal region surrounding the secondary chondrocytes derives from this Ihh source. Additionally, in vitro studies on membrane bone periosteal cells (nongerminal region) demonstrated that these cells can also respond to Ihh, and do so both by enhanced proliferation and precocious osteogenesis. Despite the pro-osteogenic effects of Ihh on periosteal cell differentiation, mechanical articulation of the quadratojugal/quadrate joint in explant culture revealed a negative role for articulation in the regulation of osteocalcin by germinal region descendants. Thus, the mechanical stimulus that is the spur to secondary chondrocyte formation appears able to override the osteogenic influence of Ihh on the periosteum, but does not interfere with the cell cycle-promoting component of Hh signalling.


Subject(s)
Chondrogenesis/physiology , Animals , Cell Differentiation/physiology , Cell Proliferation , Chick Embryo , Chondrocytes/metabolism , Chondrocytes/physiology , Core Binding Factor Alpha 1 Subunit/metabolism , Epigenesis, Genetic , Gene Expression Regulation, Developmental , Hedgehog Proteins , High Mobility Group Proteins/metabolism , Mechanotransduction, Cellular/physiology , SOX9 Transcription Factor , Skull/cytology , Skull/embryology , Stress, Mechanical , Tissue Culture Techniques , Trans-Activators/metabolism , Transcription Factors/metabolism
9.
Genes Dev ; 20(10): 1365-77, 2006 May 15.
Article in English | MEDLINE | ID: mdl-16702409

ABSTRACT

Talpid3 is a classical chicken mutant with abnormal limb patterning and malformations in other regions of the embryo known to depend on Hedgehog signaling. We combined the ease of manipulating chicken embryos with emerging knowledge of the chicken genome to reveal directly the basis of defective Hedgehog signal transduction in talpid3 embryos and to identify the talpid3 gene. We show in several regions of the embryo that the talpid3 phenotype is completely ligand independent and demonstrate for the first time that talpid3 is absolutely required for the function of both Gli repressor and activator in the intracellular Hedgehog pathway. We map the talpid3 locus to chromosome 5 and find a frameshift mutation in a KIAA0586 ortholog (ENSGALG00000012025), a gene not previously attributed with any known function. We show a direct causal link between KIAA0586 and the mutant phenotype by rescue experiments. KIAA0586 encodes a novel protein, apparently specific to vertebrates, that localizes to the cytoplasm. We show that Gli3 processing is abnormal in talpid3 mutant cells but that Gli3 can still translocate to the nucleus. These results suggest that the talpid3 protein operates in the cytoplasm to regulate the activity of both Gli repressor and activator proteins.


Subject(s)
Avian Proteins/genetics , Chick Embryo/abnormalities , Chickens/genetics , Polydactyly/genetics , Trans-Activators/metabolism , Amino Acid Sequence , Animals , Avian Proteins/analysis , Avian Proteins/metabolism , Chick Embryo/metabolism , Cytoplasm/chemistry , Cytoplasm/metabolism , Gene Expression , Gene Expression Regulation, Developmental/genetics , Hedgehog Proteins , Kruppel-Like Transcription Factors/metabolism , Molecular Sequence Data , Neural Tube Defects/embryology , Neural Tube Defects/genetics , Physical Chromosome Mapping , Protein Transport , Signal Transduction , Somites/cytology
10.
Biol Cell ; 98(4): 203-14, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16545076

ABSTRACT

Skeletal muscle is one of the few adult tissues that possesses the capacity for regeneration (restoration of lost functional tissue) as opposed to repair. This capacity is due to the presence of 'muscle stem cells' known as satellite cells. Detailed investigation of these cells over the past 50 years has revealed that both these and other cells within the skeletal muscle complex are capable of regenerating both muscle and other cell types as well. Here, we review this information, and suggest that skeletal muscle is an exciting reservoir of cells for regenerating skeletal muscle itself, as well as other cell types.


Subject(s)
Muscle, Skeletal/physiology , Regeneration , Satellite Cells, Skeletal Muscle/physiology , Stem Cell Transplantation , Stem Cells/physiology , Tissue Engineering/methods , Adipose Tissue/physiology , Animals , Bone and Bones/physiology , Cartilage/physiology , Cell Differentiation , Hematopoietic Stem Cells/physiology , Humans , Models, Biological , Muscle Development , Muscle, Skeletal/cytology , Muscle, Smooth/physiology , Myocardium/pathology , Nervous System
11.
Differentiation ; 72(7): 348-62, 2004 Sep.
Article in English | MEDLINE | ID: mdl-15554946

ABSTRACT

The talpid(3) chicken mutant has a pleiotropic phenotype including polydactyly and craniofacial abnormalities. Limb polydactyly in talpid(3) suggests a gain of Hedgehog (Hh) signaling, whereas, paradoxically, absence of midline facial structures suggests a loss of Hh function. Here we analyze the status of Shh signaling in the talpid(3) mutant head. We show that Shh expression domains are lost from the talpid(3) head--in hindbrain, midbrain, zona limitans intrathalamica, and stomodeal ectoderm--and that direct targets of Hedgehog signaling, Ptc1, Ptc2, and Gli1, are also absent even in areas associated with primary Shh expression. These data suggest that the talpid(3) mutation leads to defective activation of the Shh pathway and, furthermore, that tissue-to-tissue transduction of Shh expression in the developing head depends on Hh pathway activation. Failure to activate the Shh pathway can also explain absence of floor plate and Hnf-3beta and Netrin-1 expression in midbrain and hindbrain and absence of Fgf-8 expression in commissural plate. Other aspects of gene expression in the talpid(3) head, however, suggest misspecification, such as maintenance of floor plate-like gene expression in telencephalon. In branchial arches and lower jaw, where Shh is expressed, changes in expression of genes involved in patterning and mesodermal specification suggest both gain and loss of Hedgehog function. Thus, analysis of gene expression in talpid(3) head shows that, as in talpid(3) limb, expression of some genes is lost, while others are ectopically expressed. Unlike the limb, many head regions depend on Hh induction of a secondary domain of Shh expression, and failure of this induction in talpid(3), together with the inability to activate the Shh pathway, explain the loss-of-function head phenotype. This gene expression analysis in the talpid(3) head also confirms and extends knowledge of the importance of Shh signaling and the balance between activation and repression of Shh targets in many aspects of craniofacial morphogenesis.


Subject(s)
Chick Embryo/growth & development , Chickens/genetics , Craniofacial Abnormalities/etiology , Mutation , Trans-Activators/metabolism , Animals , Body Patterning/genetics , Brain/metabolism , Brain Chemistry , Chickens/metabolism , Craniofacial Abnormalities/genetics , Craniofacial Abnormalities/metabolism , Gene Expression Regulation, Developmental , Head/embryology , Hedgehog Proteins , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Morphogenesis/genetics , RNA, Messenger/analysis , RNA, Messenger/metabolism , Signal Transduction/genetics , Trans-Activators/genetics , Transcription Factors/genetics , Transcription Factors/metabolism
12.
Development ; 130(19): 4729-39, 2003 Oct.
Article in English | MEDLINE | ID: mdl-12925598

ABSTRACT

The development of the skull is characterised by its dependence upon epigenetic influences. One of the most important of these is secondary chondrogenesis, which occurs following ossification within certain membrane bone periostea, as a result of biomechanical articulation. We have studied the genesis, character and function of the secondary chondrocytes of the quadratojugal of the chick between embryonic days 11 and 14. Analysis of gene expression revealed that secondary chondrocytes formed coincident with Sox9 upregulation from a precursor population expressing Cbfa1/Runx2: a reversal of the normal sequence. Such secondary chondrocytes rapidly acquired a phenotype that is a compound of prehypertrophic and hypertrophic chondrocytes, exited from the cell cycle and upregulated Ihh. Pulse and pulse/chase experiments with BrdU confirmed the germinal region as the highly proliferative source of the secondary chondrocytes, which formed by division of chondrocyte-committed precursors. By blocking Hh signalling in explant cultures we show that the enhanced proliferation of the germinal region surrounding the secondary chondrocytes derives from this Ihh source. Additionally, in vitro studies on membrane bone periosteal cells (non-germinal region) demonstrated that these cells can also respond to Ihh, and do so both by enhanced proliferation and precocious osteogenesis. Despite the pro-osteogenic effects of Ihh on periosteal cell differentiation, mechanical articulation of the quadratojugal/quadrate joint in explant culture revealed a negative role for articulation in the regulation of osteocalcin by germinal region descendants. Thus, the mechanical stimulus that is the spur to secondary chondrocyte formation appears able to override the osteogenic influence of Ihh on the periosteum, but does not interfere with the cell cycle-promoting component of Hh signalling.


Subject(s)
Cell Division/physiology , Chondrocytes/metabolism , Chondrogenesis/physiology , Periosteum/cytology , Trans-Activators/metabolism , Animals , Cell Cycle/physiology , Cell Differentiation/physiology , Cell Lineage , Chick Embryo , Chondrocytes/cytology , Culture Techniques , Embryonic Induction/physiology , Gene Expression Regulation, Developmental , Hedgehog Proteins , Joints/embryology , Periosteum/metabolism , Signal Transduction/physiology
13.
Development ; 130(15): 3503-14, 2003 Aug.
Article in English | MEDLINE | ID: mdl-12810597

ABSTRACT

The limb musculature arises by delamination of premyogenic cells from the lateral dermomyotome. Initially the cells express Pax3 but, upon entering the limb bud, they switch on the expression of MyoD and Myf5 and undergo terminal differentiation into slow or fast fibres, which have distinct contractile properties that determine how a muscle will function. In the chick, the premyogenic cells express the Wnt antagonist Sfrp2, which is downregulated as the cells differentiate, suggesting that Wnts might regulate myogenic differentiation. Here, we have investigated the role of Wnt signalling during myogenic differentiation in the developing chick wing bud by gain- and loss-of-function studies in vitro and in vivo. We show that Wnt signalling changes the number of fast and/or slow fibres. For example, in vivo, Wnt11 decreases and increases the number of slow and fast fibres, respectively, whereas overexpression of Wnt5a or a dominant-negative Wnt11 protein have the opposite effect. The latter shows that endogenous Wnt11 signalling determines the number of fast and slow myocytes. The distinct effects of Wnt5a and Wnt11 are consistent with their different expression patterns, which correlate with the ultimate distribution of slow and fast fibres in the wing. Overexpression of activated calmodulin kinase II mimics the effect of Wnt5a, suggesting that it uses this pathway. Finally, we show that overexpression of the Wnt antagonist Sfrp2 and DeltaLef1 reduces the number of myocytes. In Sfrp2-infected limbs, the number of Pax3 expressing cells was increased, suggesting that Sfrp2 blocks myogenic differentiation. Therefore, Wnt signalling modulates both the number of terminally differentiated myogenic cells and the intricate slow/fast patterning of the limb musculature.


Subject(s)
Cell Differentiation/physiology , Muscle Development/physiology , Proto-Oncogene Proteins/physiology , Wings, Animal/embryology , Zebrafish Proteins , Animals , Chick Embryo , Embryonic Induction/physiology , Multigene Family , Proto-Oncogene Proteins/biosynthesis , Proto-Oncogene Proteins/genetics , Wnt Proteins
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