CFDP1 regulates the stability of pericentric heterochromatin thereby affecting RAN GTPase activity and mitotic spindle formation.

The densely packed centromeric heterochromatin at minor and major satellites is comprised of H3K9me2/3 histones, the heterochromatin protein HP1α, and histone variants. In the present study, we sought to determine the mechanisms by which condensed heterochromatin at major and minor satellites stabilized by the chromatin factor CFDP1 affects the activity of the small GTPase Ran as a requirement for spindle formation. CFDP1 colocalized with heterochromatin at major and minor satellites and was essential for the structural stability of centromeric heterochromatin. Loss of CENPA, HP1α, and H2A.Z heterochromatin components resulted in decreased binding of the spindle nucleation facilitator RCC1 to minor and major satellite repeats. Decreased RanGTP levels as a result of diminished RCC1 binding interfered with chromatin-mediated microtubule nucleation at the onset of mitotic spindle formation. Rescuing chromatin H2A.Z levels in cells and mice lacking CFDP1 through knock-down of the histone chaperone ANP32E not only partially restored RCC1-dependent RanGTP levels but also alleviated CFDP1-knockout-related craniofacial defects and increased microtubule nucleation in CFDP1/ANP32E co-silenced cells. Together, these studies provide evidence for a direct link between condensed heterochromatin at major and minor satellites and microtubule nucleation through the chromatin protein CFDP1.

MicroRNAs: Harbingers and shapers of periodontal inflammation.

Periodontal disease is an inflammatory reaction of the periodontal tissues to oral pathogens. In the present review we discuss the intricate effects of a regulatory network of gene expression modulators, microRNAs (miRNAs), as they affect periodontal morphology, function and gene expression during periodontal disease. These miRNAs are small RNAs involved in RNA silencing and post-transcriptional regulation and affect all stages of periodontal disease, from the earliest signs of gingivitis to the regulation of periodontal homeostasis and immunity and to the involvement in periodontal tissue destruction. MiRNAs coordinate periodontal disease progression not only directly but also through long non-coding RNAs (lncRNAs), which have been demonstrated to act as endogenous sponges or decoys that regulate the expression and function of miRNAs, and which in turn suppress the targeting of mRNAs involved in the inflammatory response, cell proliferation, migration and differentiation. While the integrity of miRNA function is essential for periodontal health and immunity, miRNA sequence variations (genetic polymorphisms) contribute toward an enhanced risk for periodontal disease progression and severity. Several polymorphisms in miRNA genes have been linked to an increased risk of periodontitis, and among those, miR-146a, miR-196, and miR-499 polymorphisms have been identified as risk factors for periodontal disease. The role of miRNAs in periodontal disease progression is not limited to the host tissues but also extends to the viruses that reside in periodontal lesions, such as herpesviruses (human herpesvirus, HHV). In advanced periodontal lesions, HHV infections result in the release of cytokines from periodontal tissues and impair antibacterial immune mechanisms that promote bacterial overgrowth. In turn, controlling the exacerbation of periodontal disease by minimizing the effect of periodontal HHV in periodontal lesions may provide novel avenues for therapeutic intervention. In summary, this review highlights multiple levels of miRNA-mediated control of periodontal disease progression, (i) through their role in periodontal inflammation and the dysregulation of homeostasis, (ii) as a regulatory target of lncRNAs, (iii) by contributing toward periodontal disease susceptibility through miRNA polymorphism, and (iv) as periodontal microflora modulators via viral miRNAs.

The Glycoprotein/Cytokine Erythropoietin Promotes Rapid Alveolar Ridge Regeneration In Vivo by Promoting New Bone Extracellular Matrix Deposition in Conjunction with Coupled Angiogenesis/Osteogenesis.

The loss of bone following tooth extraction poses a significant clinical problem for maxillofacial esthetics, function, and future implant placement. In the present study, the efficacy of an erythropoietin-impregnated collagen scaffold as an alveolar ridge augmentation material versus a conventional collagen scaffold and a BioOss inorganic bovine bone xenograft was examined. The collagen/Erythropoietin (EPO) scaffold exhibited significantly more rapid and complete osseous regeneration of the alveolar defect when compared to bone xenograft and the collagen membrane alone. The new EPO induced extracellular matrix was rich in Collagen I, Collagen III, Fibronectin (Fn) and E-cadherin, and featured significantly increased levels of the osteogenic transcription factors Runt-related transcription factor 2 (Runx2) and Osterix (Osx). Histomorphometric evaluation revealed a significant two-fold increase in the number of capillaries between the EPO and the BioOss group. Moreover, there was a highly significant 3.5-fold higher level of vascular endothelial growth factor (VEGF) in the collagen/EPO-treated group compared to controls. The significant effect of EPO on VEGF, FN, and RUNX2 upregulation was confirmed in vitro, and VEGF pathway analysis using VEGF inhibitors confirmed that EPO modulated extracellular matrix protein expression through VEGF even in the absence of blood vessels. Together, these data demonstrate the effectiveness of an EPO-impregnated collagen scaffold for bone regeneration as it induces rapid matrix production and osseoinduction adjacent to new capillaries via VEGF.

MicroRNA-21 regulates prostaglandin E2 signaling pathway by targeting 15-hydroxyprostaglandin dehydrogenase in tongue squamous cell carcinoma.

BACKGROUND: Oral tongue squamous cell carcinoma (OTSCC) is one of the most aggressive forms of head and neck/oral cancer (HNOC), and is a complex disease with extensive genetic and epigenetic defects, including microRNA deregulation. Identifying the deregulation of microRNA-mRNA regulatory modules (MRMs) is crucial for understanding the role of microRNA in OTSCC. METHODS: A comprehensive bioinformatics analysis was performed to identify MRMs in HNOC by examining the correlation among differentially expressed microRNA and mRNA profiling datasets and integrating with 12 different sequence-based microRNA target prediction algorithms. Confirmation experiments were performed to further assess the correlation among MRMs using OTSCC patient samples and HNOC cell lines. Functional analyses were performed to validate one of the identified MRMs: miR-21-15-Hydroxyprostaglandin Dehydrogenase (HPGD) regulatory module. RESULTS: Our bioinformatics analysis revealed 53 MRMs that are deregulated in HNOC. Four high confidence MRMs were further defined by confirmation experiments using OTSCC patient samples and HNOC cell lines, including miR-21-HPGD regulatory module. HPGD is a known anti-tumorigenic effecter, and it regulates the tumorigenic actions of Prostaglandin E2 (PGE2) by converts PGE2 to its biologically inactive metabolite. Ectopic transfection of miR-21 reduced the expression of HPGD in OTSCC cell lines, and the direct targeting of the miR-21 to the HPGD mRNA was confirmed using a luciferase reporter gene assay. The PGE2-mediated upregulation of miR-21 was also confirmed which suggested the existence of a positive feed-forward loop that involves miR-21, HPGD and PGE2 in OTSCC cells that contribute to tumorigenesis. CONCLUSIONS: We identified a number of high-confidence MRMs in OTSCC, including miR-21-HPGD regulatory module, which may play an important role in the miR-21-HPGD-PGE2 feed-forward loop that contributes to tumorigenesis.

Lyophilized platelet-rich fibrin (PRF) promotes craniofacial bone regeneration through Runx2.

Freeze-drying is an effective means to control scaffold pore size and preserve its composition. The purpose of the present study was to determine the applicability of lyophilized Platelet-rich fibrin (LPRF) as a scaffold for craniofacial tissue regeneration and to compare its biological effects with commonly used fresh Platelet-rich fibrin (PRF). LPRF caused a 4.8-fold±0.4-fold elevation in Runt-related transcription factor 2 (Runx2) expression in alveolar bone cells, compared to a 3.6-fold±0.2-fold increase when using fresh PRF, and a more than 10-fold rise of alkaline phosphatase levels and mineralization markers. LPRF-induced Runx2 expression only occurred in alveolar bone and not in periodontal or dental follicle cells. LPRF also caused a 1.6-fold increase in osteoblast proliferation (p<0.001) when compared to fresh PRF. When applied in a rat craniofacial defect model for six weeks, LPRF resulted in 97% bony coverage of the defect, compared to 84% for fresh PRF, 64% for fibrin, and 16% without scaffold. Moreover, LPRF thickened the trabecular diameter by 25% when compared to fresh PRF and fibrin, and only LPRF and fresh PRF resulted in the formation of interconnected trabeculae across the defect. Together, these studies support the application of lyophilized PRF as a biomimetic scaffold for craniofacial bone regeneration and mineralized tissue engineering.

The effect of an antioxidant gel compared to chlorhexidine during the soft tissue healing process: An animal study.

BACKGROUND: Prolonged inflammation and oxidative stress can impede healing. To enhance healing efficiency, many solutions have been employed. This is an in vivo study comparing chlorhexidine (CHX) to a commercial antioxidant gel (AO). METHODS: Envelope flaps were created in the lower incisor gingival region of 60 Sprague-Dawley rats, and acellular dermal matrix (ADM) was inserted. Animals were randomly assigned to postsurgical treatment application of AO gel or 0.12% CHX twice daily. A control group received no postsurgical treatment. Data collected (before surgery, 24 h, and 72 h) included surgical images, tissue samples, and weights. Blinded scorers assessed images using a wound healing scale. Real-time polymerase chain reaction (RT-PCR) was used for gene expression of tumor necrosis factor-alpha (TNFα), interleukin-1 (IL-1), myeloperoxidase (MPO), and superoxide dismutase (SOD). RESULTS: The AO group scored higher than the CHX and control groups in clinical evaluation (p < 0.05). At 24 h, TNFα expression was upregulated in the AO group compared to CHX (p = 0.027) and controls (p = 0.018). The AO group had significantly higher expression of antioxidant enzyme (SOD) at 24 h compared to CHX (p = 0.021). All animals lost weight in the first 24 h. Animals treated with AO or CHX regained more weight at 72 h than control animals (p = 0.034 and 0.003, respectively). CONCLUSION: Animals treated with AO healed faster. AO led to earlier upregulation of TNFα and antioxidant enzyme SOD. We hypothesized that AO promoted an earlier inflammatory process while counteracting oxidative stress by increasing antioxidant responses via SOD.

Combinatorial Design of Hydrolytically Degradable, Bone-like Biocomposites Based on PHEMA and Hydroxyapatite.

With advantages such as design flexibility in modifying degradation, surface chemistry, and topography, synthetic bone-graft substitutes are increasingly demanded in orthopedic tissue engineering to meet various requirements in the growing numbers of cases of skeletal impairment worldwide. Using a combinatorial approach, we developed a series of biocompatible, hydrolytically degradable, elastomeric, bone-like biocomposites, comprising 60 wt% poly(2-hydroxyethyl methacrylate-co-methacrylic acid), poly(HEMA-co-MA), and 40 wt% bioceramic hydroxyapatite (HA). Hydrolytic degradation of the biocomposites is rendered by a degradable macromer/crosslinker, dimethacrylated poly(lactide-b-ethylene glycol-b-lactide), which first degrades to break up 3-D hydrogel networks, followed by dissolution of linear pHEMA macromolecules and bioceramic particles. Swelling and degradation were examined at Hank's balanced salt solution at 37 °C in a 12-week period of time. The degradation is strongly modulated by altering the concentration of the co-monomer of methacrylic acid and of the macromer, and chain length/molecular weight of the macromer. 95% weight loss in mass is achieved after degradation for 12 weeks in a composition consisting of HEMA/MA/Macromer = 0/60/40, while 90% weight loss is seen after degradation only for 4 weeks in a composition composed of HEMA/MA/Macromer = 27/13/60 using a longer chain macromer. For compositions without a co-monomer, only about 14% is achieved in weight loss after 12-week degradation. These novel biomaterials offer numerous possibilities as drug delivery carriers and bone grafts particularly for low and medium load-bearing applications.

Epigenetic Repression of RUNX2 and OSX Promoters Controls the Nonmineralized State of the Periodontal Ligament.

The nonmineralized state of the mammalian periodontal ligament is one of the hallmarks of vertebrate evolution as it provides resilient and nontraumatic tooth anchorage for effective predation. Here we sought to determine how the chromatin state of key mineralization gene promoters contributes to the nonmineralized periodontal ligament in the midst of fully mineralized alveolar bone and cementum anchor tissues. In developing mouse periodontal tissues, RUNX2 was localized to alveolar bone-lining cells, while OSX was localized throughout the periodontal ligament's soft tissue. Matching RT-PCR amplification data and western blot comparisons demonstrated that the expression of RUNX2 and OSX bone mineralization transcription factors was at least 2.5-fold elevated in alveolar bone osteoblasts versus periodontal ligament fibroblasts. ChIP enrichment data along the RUNX2 and OSX promoters revealed increased H3K4me3 marks in alveolar bone osteoblasts, while H3K9me3 and H3K27me3 marks were elevated in periodontal ligament fibroblasts. In support of an epigenetic mechanism responsible for the inhibition of mineralization gene expression in periodontal progenitors, histone methylation inhibitors DZNep and Chaetocin reactivated RUNX2 and OSX expression in periodontal progenitors and increased alkaline phosphatase and Alizarin Red, while the in vivo application of DZNep in rat maxillae resulted in aberrant mineralization in the periodontal ligament and a narrowing of the nonmineralized periodontal space. Together, these studies demonstrate that the nonmineralized state of the mammalian periodontal ligament is controlled by an epigenetic regulation of the RUNX2 and OSX key mineralization gene promoters.

Changes in Hox Gene Chromatin Organization during Odontogenic Lineage Specification.

Craniofacial tissues comprise highly evolved organs characterized by a relative lack of expression in the HOX family transcription factors. In the present study, we sought to define the epigenetic events that limit HOX gene expression from undifferentiated neural crest cells to semi-differentiated odontogenic progenitors and to explore the effects of elevated levels of HOX. The ChIP-chip data demonstrated high levels of repressive H3K27me3 marks on the HOX gene promoters in ES and cranial neural crest cells when compared to the H3K4me3 marks, while the K4/K27 ratio was less repressive in the odontogenic progenitors, dental follicle, dental pulp, periodontal ligament fibroblasts, alveolar bone osteoblasts, and cementoblasts. The gene expression of multiple HOX genes, especially those from the HOXA and HOXB clusters, was significantly elevated and many times higher in alveolar bone cells than in the dental follicle cells. In addition, the HOX levels in the skeletal osteoblasts were many times higher in the trunk osteoblasts compared to the alveolar bone osteoblasts, and the repressive mark H3K27me3 promoter occupancy was substantially and significantly elevated in the alveolar bone osteoblasts when compared to the trunk osteoblasts. To explore the effect of elevated HOX levels in craniofacial neural crest cells, HOX expression was induced by transfecting cells with the Cdx4 transcription factor, resulting in a significant decrease in the mineralization markers, RUNX2, OSX, and OCN upon HOX elevation. Promoting HOX gene expression in developing teeth using the small molecule EZH2 inhibitor GSK126 resulted in an increased number of patterning events, supernumerary cusp formation, and increased Hoxa4 and Hoxb6 gene expression when compared to the controls. Together, these studies illustrate the profound effects of epigenetic regulatory events at all stages of the differentiation of craniofacial peripheral tissues from the neural crest, including lineage specification, tissue differentiation, and patterning.

Elongated polyproline motifs facilitate enamel evolution through matrix subunit compaction.

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.

The Hippo Pathway Effectors YAP/TAZ Are Essential for Mineralized Tissue Homeostasis in the Alveolar Bone/Periodontal Complex.

YAP and TAZ are essential transcriptional co-activators and downstream effectors of the Hippo pathway, regulating cell proliferation, organ growth, and tissue homeostasis. To ask how the Hippo pathway affects mineralized tissue homeostasis in a tissue that is highly reliant on a tight homeostatic control of mineralized deposition and resorption, we determined the effects of YAP/TAZ dysregulation on the periodontal tissues alveolar bone, root cementum, and periodontal ligament. Loss of YAP/TAZ was associated with a reduction of mineralized tissue density in cellular cementum and alveolar bone, a downregulation in collagen I, alkaline phosphatase, and RUNX2 gene expression, an increase in the resorption markers TRAP and cathepsin K, and elevated numbers of TRAP-stained osteoclasts. Cyclic strain applied to periodontal ligament cells resulted in YAP nuclear localization, an effect that was abolished after blocking YAP. The rescue of YAP signaling with the heparan sulfate proteoglycan agrin resulted in a return of the nuclear YAP signal. Illustrating the key role of YAP on mineralization gene expression, the YAP inhibition-related downregulation of mineralization-associated genes was reversed by the extracellular matrix YAP activator agrin. Application of the unopposed mouse molar model to transform the periodontal ligament into an unloaded state and facilitate the distal drift of teeth resulted in an overall increase in mineralization-associated gene expression, an effect that was 10-20% diminished in Wnt1Cre/YAP/TAZ mutant mice. The unloaded state of the unopposed molar model in Wnt1Cre/YAP/TAZ mutant mice also caused a significant three-fold increase in osteoclast numbers, a substantial increase in bone/cementum resorption, pronounced periodontal ligament hyalinization, and thickened periodontal fiber bundles. Together, these data demonstrated that YAP/TAZ signaling is essential for the microarchitectural integrity of the periodontium by regulating mineralization gene expression and preventing excessive resorption during bodily movement of the dentoalveolar complex.

Long-acting PFI-2 small molecule release and multilayer scaffold design achieve extensive new formation of complex periodontal tissues with unprecedented fidelity.

The faithful engineering of complex human tissues such as the bone/soft tissue/mineralized tissue interface in periodontal tissues requires innovative molecular cues in conjunction with tailored scaffolds. To address the loss of periodontal bone and connective tissues following periodontal disease, we have generated a polydopamine and collagen coated electrospun PLGA-PCL (PP) scaffold enriched with the small molecule mediator PFI-2 (PP-PFI-pDA-COL-PFI). In vitro 3D studies using PDL progenitors revealed that the PP-PFI-pDA-COL-PFI scaffold substantially enhanced Alizarin Red staining, increased Ca/P ratios 4-fold, and stimulated cell proliferation more than 12-fold compared to PP-controls, suggestive of its potential for mineralized tissue engineering. When applied in our experimental periodontitis model, the PP-PFI-pDA-COL-PFI scaffold resulted in a substantial 34% reduction in alveolar bone defect height, a 25% root-length gain in periodontal attachment, and the formation of highly ordered regenerated acellular cementum twice as thick as in controls. Explaining the mechanism of PFI-2 mineralized tissue regeneration in periodontal tissues, PFI-2 inhibited SETD7-mediated ß-Catenin protein methylation and increased ß-Catenin nuclear localization. Together, dual-level PFI-2 incorporation into a degradable, dopamine/collagen coated PLGA/PCL scaffold backbone resulted in the regeneration of the tripartite periodontal complex with unprecedented fidelity, including periodontal attachment and new formation of mineralized tissues in inflamed periodontal environments.

Structure and function of ameloblastin as an extracellular matrix protein: adhesion, calcium binding, and CD63 interaction in human and mouse.

The functional significance of extracellular matrix proteins in the life of vertebrates is underscored by a high level of sequence variability in tandem with a substantial degree of conservation in terms of cell-cell and cell-matrix adhesion interactions. Many extracellular matrix proteins feature multiple adhesion domains for successful attachment to substrates, such as integrin, CD63, and heparin. Here we have used homology and ab initio modeling algorithms to compare mouse ameloblastin (mAMBN) and human ameloblastin (hABMN) isoforms and to analyze their potential for cell adhesion and interaction with other matrix molecules as well as calcium binding. Sequence comparison between mAMBN and hAMBN revealed a 26-amino-acid deletion in mAMBN, corresponding to a helix-loop-helix frameshift. The human AMBN domain (174Q-201G), homologous to the mAMBN 157E-178I helix-loop-helix region, formed a helix-loop motif with an extended loop, suggesting a higher degree of flexibility of hAMBN compared with mAMBN, as confirmed by molecular dynamics simulation. Heparin-binding domains, CD63-interaction domains, and calcium-binding sites in both hAMBN and mAMBN support the concept of AMBN as an extracellular matrix protein. The high level of conservation between AMBN functional domains related to adhesion and differentiation was remarkable when compared with only 61% amino acid sequence homology.

Ameloblastin regulates cell attachment and proliferation through RhoA and p27.

The matrix adhesion protein ameloblastin (AMBN) is one of the unique components of the mineralizing matrix of bones and teeth. Here we focused on two types of cells expressing AMBN - mouse dental follicle cells (mDF) and mouse periodontal ligament cells (mPDL) - to decipher AMBN function in developing dental, periodontal, and bone tissues. To test AMBN function, cell culture dishes of mDF and mPDL were exposed to either full-length or C-terminal (amino acids 137-407) recombinant Ambn protein. Alternatively, cells were subjected to transient transfection using an Ambn-small hairpin (sh) RNA vector. Our cell culture studies documented that dishes coated with full-length AMBN promoted the attachment of mPDL and mDF cells as early as 1 h after seeding. In order to identify potential intermediaries that might aid the effect of AMBN on adhesion, RhoA expression levels in AMBN-coated and uncoated control dishes were assessed. These studies indicated that AMBN induced RhoA expression 4 h after seeding, especially in mPDL cells. After 4 h of culture, the cell cycle inhibitor p27 was also up-regulated. In addition, exogenous AMBN and its C-terminal fragment reduced the proliferation of mDF and mPDL. Finally, transient transfection of mDF and mPDL cells with the Ambn-shRNA vector resulted in the down-regulation of p27 in mPDL cells. Together, these data indicate that AMBN affects cell adhesion via RhoA and cell cycle progression through p27.

Ameloblastin-rich enamel matrix favors short and randomly oriented apatite crystals.

Molecular evolution studies suggest that amelogenin (AMELX), the principal component of the mammalian enamel matrix, emerged considerably later than ameloblastin (AMBN), and enamelin. Here, we created a transgenic mouse model to ask the question how a conceivable basal enamel lacking AMELX and enriched in the more basal AMBN might compare with recent mouse enamel. To answer this question we overexpressed AMBN using a keratin 14 (K14) promoter and removed AMELX from the genetic background by crossbreeding with amelx(-/-) mice. Enamel coverings of amelx(-/-) mice and of the squamate Iguana iguana were used for comparison. Scanning electron microscopic analysis documented that AMBN transgenic (TG) × amelx(-/-) mouse molars were covered by a 5 µm thin 'enameloid' layer resembling the thin enamel of the Iguana squamate. Transmission electron microscopy revealed that the enamel of developing AMBN TG × amelx(-/-) mouse molars contained short (approximately 70 nm) and randomly oriented crystals, while WT controls, AMBN overexpressors, and AMELX(-/-) mice all featured elongated and parallel oriented crystals measuring between 300 and 600 nm 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.

Polarized, Amelogenin Expressing Ameloblast-Like Cells from Cervical Loop/Dental Pulp Cocultures in Bioreactors.

The growth of long and polarized ameloblast-like cells has long been heralded as a major prerequisite for enamel tissue engineering. In this study, we have designed three-dimensional bioreactor/scaffold microenvironments to propagate and assess the ability of cervical loop derivatives to become long and polarized ameloblast-like cells. Our studies demonstrated that cervical loop/periodontal progenitor coculture in a growth-factor-enriched medium resulted in the formation of ameloblast-like cells expressing high levels of amelogenin and ameloblastin. Coculture of cervical loop cells with dental pulp cells on tailored collagen scaffolds enriched with leucine-rich amelogenin peptide (LRAP) and early enamel matrix resulted in singular, elongated, and polarized ameloblast-like cells that expressed and secreted ameloblastin and amelogenin enamel proteins. Bioreactor microenvironments enriched with enamel matrix and LRAP also proved advantageous for the propagation of HAT-7 cells, resulting in a ∼20-fold higher expression of amelogenin and ameloblastin enamel proteins compared with controls growing on plain scaffolds. Together, studies presented here highlight the benefits of microgravity culture systems combined with ameloblast-specific microenvironments and tailored scaffolds for the growth of ameloblast-like cells.

Propagation of Dental and Respiratory Cells and Organs in Microgravity.

Gravity is one of the key determinants of human cell function, proliferation, cytoskeletal architecture and orientation. Rotary bioreactor systems (RCCSs) mimic the loss of gravity as it occurs in space and instead provide a microgravity environment through continuous rotation of cultured cells or tissues. These RCCSs ensure an un-interrupted supply of nutrients, growth and transcription factors, and oxygen, and address some of the shortcomings of gravitational forces in motionless 2D (two dimensional) cell or organ culture dishes. In the present study we have used RCCSs to co-culture cervical loop cells and dental pulp cells to become ameloblasts, to characterize periodontal progenitor/scaffold interactions, and to determine the effect of inflammation on lung alveoli. The RCCS environments facilitated growth of ameloblast-like cells, promoted periodontal progenitor proliferation in response to scaffold coatings, and allowed for an assessment of the effects of inflammatory changes on cultured lung alveoli. This manuscript summarizes the environmental conditions, materials, and steps along the way and highlights critical aspects and experimental details. In conclusion, RCCSs are innovative tools to master the culture and 3D (three dimensional) growth of cells in vitro and to allow for the study of cellular systems or interactions not amenable to classic 2D culture environments.

An in vivo comparison of wound healing characteristics of two commercial acellular dermal matrices.

OBJECTIVES: Many acellular dermal matrices (ADMs) are available for use in periodontal surgical procedures. However, few studies exist evaluating their in vivo healing properties. The objectives of this study were to compare the wound healing and remodeling of two ADMs used for gingival augmentation procedures in the rat model. MATERIALS AND METHODS: This was a nonrandomized controlled split-mouth study. Envelope flaps were surgically created in the maxillary quadrants of 24 Sprague Dawley rats. Each received either (a) AlloDerm Regenerative Tissue Matrix, or (b) OrACELL. Gingival tissue from one mandibular quadrant served as the untreated control. Six male and six female rats were treated for 7 or 21 days. Biopsies were processed for histologic analysis (H&E, Picro-sirius red, Verhoeff's solution) or RNA analysis (RT-PCR) to analyze the expression of type I collagen (Col1a1), fibronectin (Fn-1) and VEGF-A (Vegf-A). RESULTS: There was a greater density of fibroblasts in OrACELL compared to AlloDerm at both timepoints. There was a greater density of elastin present in AlloDerm compared to OrACELL at 7 days but no differences at 21 days. There were no differences between test groups in the percentage of birefringent collagen or in the expression of Vegf-A or Fn-1. At 7 days, there were significantly more fibroblasts for males in the OrACELL group compared to females. At 21 days, there was a significantly greater expression of Col1a1 for males in the OrACELL group compared to females. CONCLUSIONS: Early wound healing and remodeling of OrACELL appeared to occur more rapidly than AlloDerm and was accelerated in male rats. Whether these results have clinical implications for soft tissue grafting procedures in humans remains to be determined.

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.

Recent advances in periodontal regeneration: A biomaterial perspective.

Periodontal disease (PD) is one of the most common inflammatory oral diseases, affecting approximately 47% of adults aged 30 years or older in the United States. If not treated properly, PD leads to degradation of periodontal tissues, causing tooth movement, and eventually tooth loss. Conventional clinical therapy for PD aims at eliminating infectious sources, and reducing inflammation to arrest disease progression, which cannot achieve the regeneration of lost periodontal tissues. Over the past two decades, various regenerative periodontal therapies, such as guided tissue regeneration (GTR), enamel matrix derivative, bone grafts, growth factor delivery, and the combination of cells and growth factors with matrix-based scaffolds have been developed to target the restoration of lost tooth-supporting tissues, including periodontal ligament, alveolar bone, and cementum. This review discusses recent progresses of periodontal regeneration using tissue-engineering and regenerative medicine approaches. Specifically, we focus on the advances of biomaterials and controlled drug delivery for periodontal regeneration in recent years. Special attention is given to the development of advanced bio-inspired scaffolding biomaterials and temporospatial control of multi-drug delivery for the regeneration of cementum-periodontal ligament-alveolar bone complex. Challenges and future perspectives are presented to provide inspiration for the design and development of innovative biomaterials and delivery system for new regenerative periodontal therapy.