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.</p

Elongated Polyproline Motifs Facilitate Enamel Evolution through Matrix Subunit Compaction

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

Periodontal Bioengineering: A Discourse in Surface Topographies, Progenitor Cells and Molecular Profiles.

Periodontal Bioengineering: A Discourse in Surface Topographies, Progenitor Cells and Molecular Profiles

Successful Periodontal Ligament Regeneration by Periodontal Progenitor Preseeding on Natural Tooth Root Surfaces

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

Apatite Microtopographies Instruct Signaling Tapestries for Progenitor-Driven New Attachment of Teeth

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

Successful Periodontal Ligament Regeneration by Periodontal Progenitor Preseeding on Natural Tooth Root Surfaces

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

Differentiation of Neural-Crest-Derived Intermediate Pluripotent Progenitors into Committed Periodontal Populations Involves Unique Molecular Signature Changes, Cohort Shifts, and Epigenetic Modifications

Intermediate progenitor populations play a crucial role in the regional specification and differentiation of the cranial neural crest. On the basis of global gene expression profiles, gene cohort expression levels, and epigenetic modifications, we have defined key factors involved in the differentiation of dental follicle (DF) intermediate progenitors into periodontal lineages, including alveolar bone (AB) osteoblasts, cementoblasts, and periodontal ligament (PDL) cells. When comparing differentially expressed genes, PDL cells most closely resembled DF progenitors, followed by AB osteoblasts and cementoblasts as the most distant population. According to gene ontology analyses, extracellular matrix-adhesion proteins were substantially increased in PDL cells, osteogenesis factors were elevated in AB osteoblasts, and gene expression levels were lower in cementoblasts, especially in the cytokine group. Unique signature proteins included interleukin 6, paired-like homeodomain transcription factor 2, thrombospondin 2, and glial cell line-derived neurotrophic factor for DF progenitors; asporin and prostaglandin-H2 D-isomerase for AB osteoblasts; and keratin 18, Netrin 4, Jagged 1, and Dickkopf1 for cementoblasts, as verified by western blot analysis. Secreted frizzled-related protein 1 was preferentially expressed in PDL cells, whereas matrix Gla-protein, bone sialoprotein, and insulin-like growth factor binding protein 5 were higher in AB osteoblasts than in cementoblasts. On an epigenetic level, DF progenitors featured high levels of the euchromatin marker H3K4me3, whereas PDL cells, AB osteoblasts, and cementoblasts contained high levels of the transcriptional repressor H3K9me3. Together, our data indicate that in addition to changes in signature gene expression, unique shifts in gene cohort expression levels, epigenetic modifications, and changes in cell morphology contribute to the individuation of tissue populations from a common neural-crest-derived ancestor

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 &lt; 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

Platelet-Rich Fibrin Promotes Periodontal Regeneration and Enhances Alveolar Bone Augmentation

In the present study we have determined the suitability of platelet-rich fibrin (PRF) as a complex scaffold for periodontal tissue regeneration. Replacing PRF with its major component fibrin increased mineralization in alveolar bone progenitors when compared to periodontal progenitors, suggesting that fibrin played a substantial role in PRF-induced osteogenic lineage differentiation. Moreover, there was a 3.6-fold increase in the early osteoblast transcription factor RUNX2 and a 3.1-fold reduction of the mineralization inhibitor MGP as a result of PRF application in alveolar bone progenitors, a trend not observed in periodontal progenitors. Subcutaneous implantation studies revealed that PRF readily integrated with surrounding tissues and was partially replaced with collagen fibers 2 weeks after implantation. Finally, clinical pilot studies in human patients documented an approximately 5 mm elevation of alveolar bone height in tandem with oral mucosal wound healing. Together, these studies suggest that PRF enhances osteogenic lineage differentiation of alveolar bone progenitors more than of periodontal progenitors by augmenting osteoblast differentiation, RUNX2 expression, and mineralized nodule formation via its principal component fibrin. They also document that PRF functions as a complex regenerative scaffold promoting both tissue-specific alveolar bone augmentation and surrounding periodontal soft tissue regeneration via progenitor-specific mechanisms