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1.  Mussel-inspired human gelatin nanocoating for creating biologically adhesive surfaces 
Recombinant human gelatin was conjugated with dopamine using carbodiimide as a surface modifier. This dopamine-coupled human gelatin (D-rhG) was characterized by 1H-nuclear magnetic resonance, mass spectroscopy, and circular dichroism. D-rhG-coated surface properties were analyzed by physicochemical methods. Additionally, cell attachment and growth on the modified surfaces was assessed using human umbilical endothelial cells. Binding of gelatin onto titanium was significantly enhanced by dopamine conjugation. The thickness of the D-rhG coating depended on the treatment pH; thicker layers were formed at higher pH values, with a maximum thickness of 30 nm. D-rhG enhanced the binding of collagen-binding vascular endothelial growth factor and cell adhesion as compared with gelatin alone, even at the same surface concentration. The D-rhG surface modifier enhanced substrate binding by creating an adhesive nanointerface that increased specific protein binding and cell attachment.
PMCID: PMC4045085  PMID: 24920909
recombinant human gelatin; dopamine; natural catechols; cell adhesion; cell culture; titanium
2.  A novel, visible light-induced, rapidly cross-linkable gelatin scaffold for osteochondral tissue engineering 
Scientific Reports  2014;4:4457.
Osteochondral injuries remain difficult to repair. We developed a novel photo-cross-linkable furfurylamine-conjugated gelatin (gelatin-FA). Gelatin-FA was rapidly cross-linked by visible light with Rose Bengal, a light sensitizer, and was kept gelled for 3 weeks submerged in saline at 37°C. When bone marrow-derived stromal cells (BMSCs) were suspended in gelatin-FA with 0.05% Rose Bengal, approximately 87% of the cells were viable in the hydrogel at 24 h after photo-cross-linking, and the chondrogenic differentiation of BMSCs was maintained for up to 3 weeks. BMP4 fusion protein with a collagen binding domain (CBD) was retained in the hydrogels at higher levels than unmodified BMP4. Gelatin-FA was subsequently employed as a scaffold for BMSCs and CBD-BMP4 in a rabbit osteochondral defect model. In both cases, the defect was repaired with articular cartilage-like tissue and regenerated subchondral bone. This novel, photo-cross-linkable gelatin appears to be a promising scaffold for the treatment of osteochondral injury.
PMCID: PMC3964514  PMID: 24662725
3.  Immobilization of Bone Morphogenetic Protein on DOPA- or Dopamine-Treated Titanium Surfaces to Enhance Osseointegration 
BioMed Research International  2013;2013:265980.
Titanium was treated with 3,4-dihydroxy-L-phenylalanine (DOPA) or dopamine to immobilize bone morphogenetic protein-2 (BMP2), a biomolecule. DOPA and dopamine solutions turned into suspensions, and precipitates were produced at high pH. Both treatments produced a brown surface on titanium that was thicker at high pH than low pH. Dopamine produced a thicker layer than DOPA. The hydrophobicity of the surfaces increased after treatment with dopamine independent of pH. Furthermore, there were more amino groups in the layers formed at pH 8.5 than pH 4.5 in both treatments. Dopamine treatment produced more amino groups in the layer than DOPA. BMP2 was immobilized on the treated surfaces via a coupling reaction using carbodiimide. More BMP2 was immobilized on surfaces treated at pH 8.5 than pH 4.5 in both treatments. The immobilized BMP induced specific signal transduction and alkali phosphatase, a differentiation marker. Thus, the present study demonstrates that titanium treated with DOPA or dopamine can become bioactive via the surface immobilization of BMP2, which induces specific signal transduction.
PMCID: PMC3888698  PMID: 24459666
4.  Novel Microarrays for Simultaneous Serodiagnosis of Multiple Antiviral Antibodies 
PLoS ONE  2013;8(12):e81726.
We developed an automated diagnostic system for the detection of virus-specific immunoglobulin Gs (IgGs) that was based on a microarray platform. We compared efficacies of our automated system with conventional enzyme immunoassays (EIAs). Viruses were immobilized to microarrays using a radical cross-linking reaction that was induced by photo-irradiation. A new photoreactive polymer containing perfluorophenyl azide (PFPA) and poly(ethylene glycol) methacrylate was prepared and coated on plates. Inactivated measles, rubella, mumps, Varicella-Zoster and recombinant Epstein-Barr viruse antigen were added to coated plates, and irradiated with ultraviolet light to facilitate immobilization. Virus-specific IgGs in healthy human sera were assayed using these prepared microarrays and the results obtained compared with those from conventional EIAs. We observed high correlation (0.79–0.96) in the results between the automated microarray technique and EIAs. The microarray-based assay was more rapid, involved less reagents and sample, and was easier to conduct compared with conventional EIA techniques. The automated microarray system was further improved by introducing reagent storage reservoirs inside the chamber, thereby conserving the use of expensive reagents and antibodies. We considered the microarray format to be suitable for rapid and multiple serological diagnoses of viral diseases that could be developed further for clinical applications.
PMCID: PMC3867344  PMID: 24367491
5.  Enhanced in vivo osteogenesis by nanocarrier-fused bone morphogenetic protein-4 
Bone defects and nonunions are major clinical skeletal problems. Growth factors are commonly used to promote bone regeneration; however, the clinical impact is limited because the factors do not last long at a given site. The introduction of tissue engineering aimed to deter the diffusion of these factors is a promising therapeutic strategy. The purpose of the present study was to evaluate the in vivo osteogenic capability of an engineered bone morphogenetic protein-4 (BMP4) fusion protein.
BMP4 was fused with a nanosized carrier, collagen-binding domain (CBD), derived from fibronectin. The stability of the CBD-BMP4 fusion protein was examined in vitro and in vivo. Osteogenic effects of CBD-BMP4 were evaluated by computer tomography after intramedullary injection without a collagen–sponge scaffold. Recombinant BMP-4, CBD, or vehicle were used as controls. Expressions of bone-related genes and growth factors were compared among the groups. Osteogenesis induced by CBD-BMP4, BMP4, and CBD was also assessed in a bone-defect model.
In vitro, CBD-BMP4 was retained in a collagen gel for at least 7 days while BMP4 alone was released within 3 hours. In vivo, CBD-BMP4 remained at the given site for at least 2 weeks, both with or without a collagen–sponge scaffold, while BMP4 disappeared from the site within 3 days after injection. CBD-BMP4 induced better bone formation than BMP4 did alone, CBD alone, and vehicle after the intramedullary injection into the mouse femur. Bone-related genes and growth factors were expressed at higher levels in CBD-BMP4-treated mice than in all other groups, including BMP4-treated mice. Finally, CBD-BMP4 potentiated more bone formation than did controls, including BMP4 alone, when applied to cranial bone defects without a collagen scaffold.
Altogether, nanocarrier-CBD enhanced the retention of BMP4 in the bone, thereby promoting augmented osteogenic responses in the absence of a scaffold. These results suggest that CBD-BMP4 may be clinically useful in facilitating bone formation.
PMCID: PMC3626372  PMID: 23630418
BMP4; bone repair; bone tissue engineering; osteogenesis
6.  Ameloblastin Inhibits Cranial Suture Closure by Modulating Msx2 Expression and Proliferation 
PLoS ONE  2013;8(4):e52800.
Deformities of cranial sutures such as craniosynostosis and enlarged parietal foramina greatly impact human development and quality of life. Here we have examined the role of the extracellular matrix protein ameloblastin (Ambn), a recent addition to the family of non-collagenous extracellular bone matrix proteins, in craniofacial bone development and suture formation. Using RT-PCR, western blot and immunohistochemistry, Ambn was localized in mouse calvarial bone and adjacent condensed mesenchyme. Five-fold Ambn overexpression in a K14-driven transgenic mouse model resulted in delayed posterior frontal suture fusion and incomplete suture closure. Moreover, Ambn overexpressor skulls weighed 13.2% less, their interfrontal bones were 35.3% thinner, and the width between frontal bones plus interfrontal suture was 14.3% wider. Ambn overexpressing mice also featured reduced cell proliferation in suture blastemas and in mesenchymal cells from posterior frontal sutures. There was a more than 2-fold reduction of Msx2 in Ambn overexpressing calvariae and suture mesenchymal cells, and this effect was inversely proportionate to the level of Ambn overexpression in different cell lines. The reduction of Msx2 expression as a result of Ambn overexpression was further enhanced in the presence of the MEK/ERK pathway inhibitor O126. Finally, Ambn overexpression significantly reduced Msx2 down-stream target gene expression levels, including osteogenic transcription factors Runx2 and Osx, the bone matrix proteins Ibsp, ColI, Ocn and Opn, and the cell cycle-related gene CcnD1. Together, these data suggest that Ambn plays a crucial role in the regulation of cranial bone growth and suture closure via Msx 2 suppression and proliferation inhibition.
PMCID: PMC3617155  PMID: 23593111
7.  Long-term remission after multiple bone metastases following cervical cancer: A case report☆ 
•Multiple bone metastases following cervical cancer was completed resolved.•Metastatic lesions were present within a previously irradiated zone of primary external radiation.•Concurrent chemotherapy and bisphosphonate administration is promising.
PMCID: PMC3862314  PMID: 24371687
Bone metastases; Cervical cancer; Bisphosphonate; Chemotherapy
8.  The pH-Sensitive Fusogenic 3-Methyl-Glutarylated Hyperbranched Poly(Glycidol)-Conjugated Liposome Induces Antigen-Specific Cellular and Humoral Immunity 
We examined the ability of a novel liposome, surface modified by 3-methyl-glutarylated hyperbranched poly(glycidol) (MGlu-HPG), to enhance antigen-specific immunity in vitro and in vivo and to function as a vaccine carrier. Murine bone marrow-derived dendritic cells took up ovalbumin (OVA) encapsulated in MGlu-HPG-modified liposomes more effectively than free OVA or OVA encapsulated in unmodified liposomes. Immunization of mice with OVA-containing MGlu-HPG-modified liposomes induced antigen-specific splenocyte proliferation and production of gamma interferon (IFN-γ) more strongly than did immunization with free OVA or OVA encapsulated in unmodified liposomes. The immune responses induced by OVA encapsulated in MGlu-HPG-modified liposomes were significantly suppressed by addition of anti-major histocompatibility complex (MHC) class I and class II monoclonal antibodies, indicating the involvement of antigen presentation via MHC class I and II. Furthermore, delayed-type hypersensitivity responses and OVA-specific antibodies were induced more effectively in mice immunized with OVA encapsulated by MGlu-HPG-modified liposomes than with unencapsulated OVA or OVA encapsulated in unmodified liposomes. These results suggested that MGlu-HPG-modified liposomes effectively induced both cell-mediated and humoral immune responses. Collectively, this study is the first to demonstrate the induction of both cell-mediated and humoral immune responses in vivo by MGlu-HPG-modified liposomes.
PMCID: PMC3428382  PMID: 22815149
9.  Ameloblastin-rich Enamel Matrix Favors Short and Randomly Oriented Apatite Crystals 
European Journal of Oral Sciences  2011;119(Suppl 1):254-260.
Molecular evolution studies suggest that amelogenins (AMEL), the principal components of the mammalian enamel matrix, emerged considerably later than ameloblastin (AMBN), and enamelin. Here we have created a transgenic mouse model to ask the question how a conceivable basal enamel lacking AMEL and enriched in the more basal AMBN might compare to recent mouse enamel. To address this question we have overexpressed AMBN using a K14 promoter and removed AMEL from the genetic background by crossbreeding with AMEL−/− mice. Enamel coverings of AMEL−/− mice and of the squamate Iguana iguana were used for comparison. Scanning electron microscopic analysis documented that AMBN TG × amel−/− mouse molars were covered by a 5μm thin “enameloid” layer resembling the thin enamel of the Iguana squamate. Transmission electron microscopy revealed that the enamel of developing AMBN TG × amel−/−mouse molars contained approximately 70nm short and randomly oriented crystals, while WT controls, AMBN overexpressors, and AMEL−/− mice all featured elongated and parallel oriented crystals measuring between 300nm and 600nm in average length. Together, these studies illustrate that AMBN promotes the growth of a crystalline enamel layer with short and randomly oriented crystals, but lacks the ability to facilitate the formation of long and parallel oriented apatite crystals.
PMCID: PMC3402546  PMID: 22243254
enamel; amelogenin; ameloblastin; evolution; apatite
10.  Genetic PEGylation 
PLoS ONE  2012;7(11):e49235.
Polyethylene glycol (PEG) was genetically incorporated into a polypeptide. Stop-anticodon-containing tRNAs were acylated with PEG-containing amino acids and were then translated into polypeptides corresponding to DNA sequences containing the stop codons. The molecular weights of the PEG used were 170, 500, 700, 1000, and 2000 Da, and the translation was confirmed by mass spectrometry. The PEG incorporation ratio decreased as the molecular weight of PEG increased, and PEG with a molecular weight of 1000 Da was only slightly incorporated. Although improvement is required to increase the efficiency of the process, this study demonstrates the possibility of genetic PEGylation.
PMCID: PMC3493536  PMID: 23145132
11.  Positively charged cholesterol–recombinant human gelatins foster the cellular uptake of proteins and murine immune reactions 
Recombinant human gelatins with defined molecular weights were modified with cholesterol to make them amphiphilic in nature. We investigated the feasibility of these modified human gelatins acting as a carrier of antigenic proteins for inducing cellular immunity. The aim of this study was to synthesize novel and effective compounds for vaccine delivery in vivo.
Two types of cholesterol-modified gelatin micelles, anionic cholesterol-modified gelatin (aCMG) and cationic-cholesterol modified gelatin (cCMG), were synthesized using different cholesterol derivatives such as the cholesterol-isocyanate (Ch-I) for aCMG and amino-modified cholesterol for cCMG. One was anionic and the other cationic, and therefore they differed in terms of their zeta potential. The aCMG and cCMG were characterized for their size, zeta potential, and in their ability to form micelles. Cytotoxicity was also evaluated. The modified human gelatins were then investigated as a carrier of antigenic proteins for inducing cellular immunity both in vitro in DC 2.4 cells, a murine dendritic cell line, as well as in vivo. The mechanism of entry of the polymeric micelles into the cells was also evaluated.
It was found that only cCMG successfully complexed with the model antigenic protein, fluorescein-isothiocyanate ovalbumin (OVA) and efficiently delivered and processed proteins in DC 2.4 cells. It was hypothesized that cCMG enter the cells predominantly by a caveolae-mediated pathway that required tyrosine kinase receptors on the cell surface. Animal testing using mice showed that the cationic cholesterol-modified gelatin complexed with OVA produced significantly high antibody titers against OVA: 2580-fold higher than in mice immunized with free OVA.
Conclusively, cCMG has shown to be very effective in stimulating an immune response due to its high efficiency, stability, and negligible cytotoxicity.
PMCID: PMC3471541  PMID: 23091385
recombinant human gelatin; cholesterol; micelle; protein delivery; caveolae pathway; receptor-mediated endocytosis
12.  Successful Periodontal Ligament Regeneration by Periodontal Progenitor Preseeding on Natural Tooth Root Surfaces 
Stem Cells and Development  2011;20(10):1659-1668.
The regeneration of lost periodontal ligament (PDL) and alveolar bone is the purpose of periodontal tissue engineering. The goal of the present study was to assess the suitability of 3 odontogenic progenitor populations from dental pulp, PDL, and dental follicle for periodontal regeneration when exposed to natural and synthetic apatite surface topographies. We demonstrated that PDL progenitors featured higher levels of periostin and scleraxis expression, increased adipogenic and osteogenic differentiation potential, and pronounced elongated cell shapes on barren root chips when compared with dental pulp and dental follicle cells. When evaluating the effect of surface characteristics on PDL progenitors, natural root surfaces resulted in elongated PDL cell shapes, whereas PDL progenitors on synthetic apatite surfaces were rounded or polygonal. In addition, surface coatings affected PDL progenitor gene expression profiles: collagen I coatings enhanced alkaline phosphatase and osteocalcin expression levels and laminin-1 coatings increased epidermal growth factor (EGF), nestin, cadherin 1, and keratin 8 expression. PDL progenitors seeded on natural tooth root surfaces in organ culture formed new periodontal fibers after 3 weeks of culture. Finally, replantation of PDL progenitor-seeded tooth roots into rat alveolar bone sockets resulted in the complete formation of a new PDL and stable reattachment of teeth over a 6-month period. Together, these findings indicate that periodontal progenitor cell type as well as mineral surface topography and molecular environment play crucial roles in the regeneration of true periodontal anchorage.
PMCID: PMC3210028  PMID: 21250830
13.  Indomethacin Analogues that Enhance Doxorubicin Cytotoxicity in Multidrug Resistant Cells without Cox Inhibitory Activity 
ACS Medicinal Chemistry Letters  2011;2(5):353-357.
Conformationally restricted indomethacin analogues were designed and prepared from the corresponding 2-substituted indoles, which were synthesized by a one-pot isomerization/enamide-ene metathesis as the key reaction. Conformational analysis by calculations, NMR studies, and X-ray crystallography suggested that these analogues were conformationally restricted in the s-cis or the s-trans form due to the 2-substituent as expected. Their biological activities on cyclooxygenase-1 (COX-1) inhibition, cyclooxygenase-2 (COX-2) inhibition, and modulation of MRP-1-mediated multidrug resistance (MDR) are described. Some of these indomethacin analogues enhanced doxorubicin cytotoxicity, although they do not have any COX inhibitory activity, which suggests that the MDR-modulating effect of an NSAID can be unassociated with its COX-inhibitory activity. This may be an entry into the combination chemotherapy of doxorubicin with a MDR modulator.
PMCID: PMC4017980  PMID: 24900317
Cancer; conformation analysis; cytotoxicity; drug design; nitrogen heterocycles
14.  Feeder Cells Support the Culture of Induced Pluripotent Stem Cells Even after Chemical Fixation 
PLoS ONE  2012;7(3):e32707.
Chemically fixed mouse embryonic fibroblasts (MEFs), instead of live feeder cells, were applied to the maintenance of mouse induced pluripotent stem (miPS) cells. Formaldehyde and glutaraldehyde were used for chemical fixation. The chemically fixed MEF feeders maintained the pluripotency of miPS cells, as well as their undifferentiated state. Furthermore, the chemically fixed MEF feeders were reused several times without affecting their functions. These results indicate that chemical fixation can be applied to modify biological feeders chemically, without losing their original functions. Chemically fixed MEF feeders will be applicable to other stem cell cultures as a reusable extracellular matrix candidate that can be preserved on a long-term basis.
PMCID: PMC3292564  PMID: 22396791
15.  NF-Y and USF1 transcription factor binding to CCAAT box and E-box elements activates the CP27 promoter 
Gene  2010;473(2):92-99.
The maintenance and differentiation of embryonic stem cells (ES cells) depends on the regulation of gene expression through the coordinated binding of transcription factors to regulatory promoter elements. One of the genes involved in embryonic development is the chromatin factor CP27. Previously, we have shown that NF-Y interacted with the CP27 proximal promoter CCAAT-box. Here we report that CP27 gene expression in mouse ES cells is controlled by CCAAT and E-box cis-acting regulatory elements and their corresponding transcription factors NF-Y and USF1. Specifically, USF1 interacts with the E-box of the CP27 proximal promoter and NF-Y interacts with the CCAAT box. NF-Y and USF1 also interacted with each other and activated the CP27 promoter in a synergistic fashion. Together, these studies demonstrate that gene expression of the chromatin factor CP27 is regulated through the interaction of the transcription factors NF-Y and USF1 with the CP27 proximal promoter.
PMCID: PMC3039062  PMID: 21078375
Promoter; ES cell; NF-Y; USF1; CP27
16.  Apatite Microtopographies Instruct Signaling Tapestries for Progenitor-Driven New Attachment of Teeth 
Tissue Engineering. Part A  2010;17(3-4):279-290.
Dimension and structure of extracellular matrix surfaces have powerful influences on cell shape, adhesion, and gene expression. Here we show that natural tooth root topographies induce integrin-mediated extracellular matrix signaling cascades in tandem with cell elongation and polarization to generate physiological periodontium-like tissues. In this study we replanted surface topography instructed periodontal progenitors into rat alveolar bone sockets for 8 and 16 weeks, resulting in complete reattachment of tooth roots to the surrounding alveolar bone with a periodontal fiber apparatus closely matching physiological controls along the entire root surface. Displacement studies and biochemical analyses confirmed that progenitor-based engineered periodontal tissues were similar to control teeth and uniquely derived from preimplantation green fluorescent protein (GFP)-labeled progenitors. Together, these studies illustrate the capacity of natural extracellular surface topographies to instruct progenitor cell populations to fully regenerate complex cellular and structural morphologies of tissues once lost to disease. We suggest that our strategy could be used for the replantation of teeth lost due to trauma or as a novel approach for tooth replacement using tooth-shaped replicas.
PMCID: PMC3029000  PMID: 20795795
17.  Design and Synthesis of Binding Growth Factors 
Growth factors play important roles in tissue regeneration. However, because of their instability and diffusible nature, improvements in their performance would be desirable for therapeutic applications. Conferring binding affinities would be one way to improve their applicability. Here we review techniques for conjugating growth factors to polypeptides with particular affinities. Conjugation has been designed at the level of gene fusion and of polypeptide ligation. We summarize and discuss the designs and applications of binding growth factors prepared by such conjugation approaches.
PMCID: PMC3382770  PMID: 22754349
growth factor; immobilization; protein engineering; tissue engineering; collagen; peptide ligation
18.  Extracellular Matrix-Mediated Differentiation of Periodontal Progenitor Cells 
The periodontal ligament (PDL) is a specialized connective tissue that connects the surface of the tooth root with the bony tooth socket. The healthy PDL harbors stem cell niches and extracellular matrix (ECM) microenvironments that facilitate periodontal regeneration. During periodontal disease, the PDL is often compromised or destroyed, reducing the life-span of the tooth. In order to explore new approaches toward the regeneration of diseased periodontal tissues, we have tested the effect of periodontal ECM signals, fibroblast growth factor 2 (FGF2), connective tissue growth factor (CTGF), and the cell adhesion peptide Arg-Gly- Asp (RGD) on the differentiation of two types of periodontal progenitor cells, PDL progenitor cells (PDLPs) and dental follicle progenitor cells (DFCs). Our studies documented that CTGF and FGF2 significantly enhanced the expression of collagens I & III, biglycan and periostin in tissue engineered regenerates after 4 weeks compared to untreated controls. Specifically, CTGF promoted mature PDL-like tissue regeneration as demonstrated by dense periostin localization in collagen fiber bundles. CTGF and FGF2 displayed synergistic effects on collagen III and biglycan gene expression, while effects on mineralization were antagonistic to each other: CTGF promoted while FGF2 inhibited mineralization in PDL cell cultures. Incorporation of RGD peptides in hydrogel matrices significantly enhanced attachment, spreading, survival and mineralization of the encapsulated DFCs, suggesting that RGD additives might promote the use of hydrogels for periodontal mineralized tissue engineering. Together, our studies have documented the effect of three key components of the periodontal ECM on the differentiation of periodontal progenitor populations.
PMCID: PMC2744845  PMID: 19433344
growth factors; extracellular matrix; periodontal regeneration; progenitor cells
19.  Neural Crest Lineage Segregation: A Blueprint for Periodontal Regeneration 
Journal of dental research  2009;88(9):781-791.
During the recent decade, the periodontal attachment apparatus has turned into one of the premier areas of the body for the development of novel tissue engineering strategies. In the present review we are using a developmental biology approach to characterize current concepts in periodontal regeneration and to discuss strategies for future applications in periodontal therapies. In order to decipher the developmental make-up of the periodontal region, we have followed the path of the migratory neural crest as it gives rise to periodontal progenitor tissues, which in turn are subjected to the influence of diverse craniofacial extracellular matrices and peptide growth factors. Based on this developmental perspective, we have conducted a systematic analysis of the factors, progenitor cells, and matrices used in current periodontal tissue engineering approaches. We propose that the developmental history of a tissue is a highly instructive design template for the discovery of novel bioengineering tools and approaches.
PMCID: PMC2749262  PMID: 19767574
20.  The Mosasaur Tooth Attachment Apparatus as Paradigm for the Evolution of the Gnathostome Periodontium 
Evolution & development  2009;11(3):247-259.
Vertebrate teeth are attached to jaws by a variety of mechanisms, including acrodont, pleurodont, and thecodont modes of attachment. Recent studies have suggested that various modes of attachment exist within each sub-category. Especially squamates feature a broad diversity of modes of attachment. Here we have investigated tooth attachment tissues in the late cretaceous mosasaur Clidastes and compared mosasaur tooth attachment with modes of attachment found in other extant reptiles. Using histologic analysis of ultrathin ground sections, four distinct mineralized tissues that anchor mosasaur teeth to the jaw were identified: (i) an acellular cementum layer at the interface between root and cellular cementum, (ii) a massive cone consisting of trabecular cellular cementum, (iii) the mineralized periodontal ligament containing mineralized Sharpey’s fibers, and (iv) the interdental ridges connecting adjacent teeth. The complex, multilayered attachment apparatus in mosasaurs was compared with attachment tissues in extant reptiles, including Iguana and Caiman. Based on our comparative analysis we postulate the presence of a quadruple-layer tissue architecture underlying reptilian tooth attachment, comprised of acellular cementum, cellular cementum, mineralized periodontal ligament, and interdental ridge (alveolar bone). We propose that the mineralization status of the periodontal ligament is a dynamic feature in vertebrate evolution subject to functional adaptation.
PMCID: PMC2704977  PMID: 19469852
21.  Amelogenin Evolution and Tetrapod Enamel Structure 
Frontiers of oral biology  2009;13:74-79.
Amelogenins are the major proteins involved in tooth enamel formation. In the present study we have cloned and sequenced four novel amelogenins from three amphibian species in order to analyze similarities and differences between mammalian and non-mammalian amelogenins. The newly sequenced amphibian amelogenin sequences were from a Red-eyed tree frog (Litoria chloris) and a Mexican axolotl (Ambystoma mexicanum). We identified two amelogenin isoforms in the Eastern Red-backed Salamander (Plethodon cinereus). Sequence comparisons confirmed that non-mammalian amelogenins are overall shorter than their mammalian counterparts, contain less proline and less glutamine, and feature shorter polyproline tripeptide repeat stretches than mammalian amelogenins. We propose that unique sequence parameters of mammalian amelogenins might be a pre-requisite for complex mammalian enamel prism architecture.
PMCID: PMC2763340  PMID: 19828974
22.  Elongated Polyproline Motifs Facilitate Enamel Evolution through Matrix Subunit Compaction 
PLoS Biology  2009;7(12):e1000262.
How does proline-repeat motif length in the proteins of teeth and bones relate to the evolution of vertebrates? Counterintuitively, longer repeat stretches are associated with smaller aggregated subunits within a supramolecular matrix, resulting in enhanced crystal length in mammalian versus amphibian tooth enamel.
Vertebrate body designs rely on hydroxyapatite as the principal mineral component of relatively light-weight, articulated endoskeletons and sophisticated tooth-bearing jaws, facilitating rapid movement and efficient predation. Biological mineralization and skeletal growth are frequently accomplished through proteins containing polyproline repeat elements. Through their well-defined yet mobile and flexible structure polyproline-rich proteins control mineral shape and contribute many other biological functions including Alzheimer's amyloid aggregation and prolamine plant storage. In the present study we have hypothesized that polyproline repeat proteins exert their control over biological events such as mineral growth, plaque aggregation, or viscous adhesion by altering the length of their central repeat domain, resulting in dramatic changes in supramolecular assembly dimensions. In order to test our hypothesis, we have used the vertebrate mineralization protein amelogenin as an exemplar and determined the biological effect of the four-fold increased polyproline tandem repeat length in the amphibian/mammalian transition. To study the effect of polyproline repeat length on matrix assembly, protein structure, and apatite crystal growth, we have measured supramolecular assembly dimensions in various vertebrates using atomic force microscopy, tested the effect of protein assemblies on crystal growth by electron microscopy, generated a transgenic mouse model to examine the effect of an abbreviated polyproline sequence on crystal growth, and determined the structure of polyproline repeat elements using 3D NMR. Our study shows that an increase in PXX/PXQ tandem repeat motif length results (i) in a compaction of protein matrix subunit dimensions, (ii) reduced conformational variability, (iii) an increase in polyproline II helices, and (iv) promotion of apatite crystal length. Together, these findings establish a direct relationship between polyproline tandem repeat fragment assemblies and the evolution and the design of vertebrate mineralized tissue microstructures. Our findings reveal that in the greater context of chordate evolution, the biological control of apatite growth by polyproline-based matrix assemblies provides a molecular basis for the evolution of the vertebrate body plan.
Author Summary
The microstructure of vertebrate bones and teeth is controlled by polyproline-rich protein matrices (such as amelogenin) that serve as a scaffold to control the assembly of biological apatites. In tooth enamel, amphibians have large amelogenin subunits and thin enamel while mammals have smaller amelogenin subunits in tandem with elongated crystals and complex prismatic organization. Using specific peptides and frog amelogenin overexpressed in mice, we confirmed the effect of the length of the elongated polyproline repeat on reduced matrix subunit dimensions and enhanced apatite crystal length. Three-dimensional structures solved by NMR (nuclear magnetic resonance) and surface modeling algorithms indicate that elongated polyproline repeat stretches in amelogenins affect the dimensions of the supramolecular matrix through an increase in polyproline II helices, resulting in a compaction of supramolecular subunit dimensions. We propose that the availability of readily shaped apatites and innovative mechanisms based on amelogenin-repeat motifsthat compartmentalize and shape biological minerals was essential for the rise of early vertebrates, enabling the manufacture of strong teeth and backbones that might have given vertebrates a decisive survival advantage in the competition for food and in the sophistication of locomotion.
PMCID: PMC2787623  PMID: 20027208
23.  Peptide immobilized on gold particles enhances cell growth 
Cytotechnology  2009;58(3):141-144.
A multivalent ligand of thrombopoietin (TPO) was prepared by immobilization of mimetic peptides on gold particles. An effective peptide ligand containing cysteine was designed to enhance the growth of TPO-sensitive cells. The peptide was then immobilized on gold particles by self assembly. The multivalent ligand enhanced the growth of TPO-dependent cells and its activity was more than that of the monovalent ligand.
PMCID: PMC2652554  PMID: 19137449
Thrombopoietin; Multivalency; Gold particle; Bioconjugate; Cell proliferation
24.  A Fusion Protein of Hepatocyte Growth Factor Enhances Reconstruction of Myocardium in a Cardiac Patch Derived from Porcine Urinary Bladder Matrix 
We sought to promote myocardial repair using urinary bladder matrix (UBM) incorporated with a fusion protein which combined hepatocyte growth factor (HGF) and fibronectin collagen-binding-domain (CBD) in a porcine model. CBD acted as an intermediary to promote HGF binding and enhance HGF stability within UBM.
UBM incorporated with CBD-HGF was implanted into the porcine right ventricular wall (F-group) to repair a surgically created defect. Untreated UBM patches (U-group) and Dacron patches (D-group) served as controls (N=5/group). Electromechanical mapping was performed at 60-days after surgery. Linear local shortening (LLS) was used to assess regional contractility, and electrical activity was recorded.
The LLS was significantly improved in the F group compared with controls (F:0.51 ± 1.57%*, U:− 1.06 ± 1.84%, D: −2.72 ± 2.59%; *p<0.05), while they were inferior to the normal myocardium(13.7 ± 4.3%)*. Mean electrical activity was 1.49±0.82mV in the F group, which was statistically greater than control groups (U:0.93 ± 0.71mV; D:0.30 ± 0.22mV)* and less than the normal myocardium (8.24±2.49mV)*. Histological examination showed predominant α-smooth muscle actin positive cells with the F group showing the thickest layer and the D group the thinnest layer, with an endocardial endothelial monolayer. Scattered isolated islands of α-actinin positive cells were observed only in the F group, but not in the controls, suggesting the presence of cardiomyocytes.
The CBD-HGF-UBM patch demonstrated increased contractility and electrical activity compared to UBM alone or Dacron, and facilitated a homogeneous repopulation of host cells. UBM incorporated with CBD-HGF may contribute to constructive myocardial remodeling.
PMCID: PMC2723859  PMID: 19026821
Stem cells and development  2006;15(4):595-608.
As a developmental precursor for diverse periodontal tissues the dental follicle harbors great promise for periodontal tissue regeneration. However, development of optimal therapy awaits the answer to a key question that impinges on many issues in development; do adult progenitor tissues form a homogeneous cell population that differentiates into target tissues when they arrive at the site, or they contain heterogeneous cell populations that are committed to specific fates? In order to address the homogeneity/heterogeneity question we analyzed differentiation pathways and markers in several cloned dental follicle cell lines. Our studies revealed that each of our cloned dental follicle lines featured remarkably unique characteristics indicative of a separate and distinct lineage. One line, DF1 was high in proliferative activity while it did not display any mineralization behavior, suggesting that it might be related to a periodontal ligament-type lineage. DF2 was similar to DF1 but featured remarkably high alkaline phosphatase activity indicative of a highly undifferentiated state. DF3 matched the mineralization characteristics of a same stage alveolar bone line AB1 in terms of gene expression and von Kossa staining indicating that DF3 might be of cementoblastic or alveolar bone osteoblastic lineage. In order to verify the multilineage potential of the dental follicle for purposes of tissue engineering, a series of differentiation induction experiments was conducted. For identification purposes, characteristics of these heterogeneous follicular progenitor cells were compared with follicle components in tissue sections of the postnatal developing periodontium. The presence of heterogeneous cell populations in the dental follicle mirrors individual developmental pathways in the formation of the dental integument. The profound cellular heterogeneity of the dental follicle as an adult progenitor for tissue regeneration also suggests that heterogeneous cellular constituents might play as much of a role in tissue regeneration as the inducible characteristics of individual lineages might do.
PMCID: PMC2738600  PMID: 16978062

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