Enhanced differentiation of dental pulp cells cultured on microtubular polymer scaffolds in vitro.

Dental caries (tooth decay) is the most common chronic disease. Dental tissue engineering is a promising alternative approach to alleviate the shortcomings of the currently available restorative materials. Mimicking the natural extracellular matrix (ECM) could enhance the performance of tissue engineering scaffolds. In this study, we developed microtubular (~20 µm diameter) polymethyl methacrylate (PMMA) scaffolds resembling the tubular (~2.5 µm diameter) structure of dentin, the collagen-based mineralized tissue that forms the major portion of teeth, to study the effect of scaffold architecture on differentiation of mouse dental pulp cells in vitro. Flat (control), plasma-treated solid and microtubular PMMA scaffolds with densities of 240±15, 459±51 and 480±116 tubules/mm2 were first characterized using scanning electron microscopy and contact angle measurements. Dental pulp cells were cultured on the surface of the scaffolds for up to 21 days and examined using various assays. Cell proliferation and mineralization were examined using Alamar Blue and Xylenol Orange (XO) staining assays, respectively. The differentiation of pulp cells into odontoblasts was examined by immunostaining for Nestin and by quantitative PCR analysis for dentin matrix protein 1 (Dmp1), dentin sialophosphoprotein (Dspp) and osteocalcin (Ocn). Our results showed that the highest tubular density scaffolds significantly (p<0.05) enhanced differentiation of pulp cells into odontoblasts as compared to control flat scaffolds, as evidenced by increased expression of Nestin (5.4x). However, mineralization was suppressed on all surfaces, possibly due to low cell density. These results suggest that the microtubular architecture may be a desirable feature of scaffolds developed for clinical applications. LAY SUMMARY: Regenerative engineering of diseased or traumatized tooth structure could avoid the deficiencies of traditional dental restorative (filling) materials. Cells in the dental pulp have the potential to differentiate to dentin-producing odontoblast cells. Furthermore, cell-supporting scaffolds that mimic a natural extracellular matrix (ECM) are known to influence behavior of progenitor cells. Accordingly, we hypothesized that a dentin-like microtubular scaffold would enhance differentiation of dental pulp cells. The hypothesis was proven true and differentiation to odontoblasts increased with increasing density of the microtubules. However, mineralization was suppressed, possibly due to a low density of cells. The results demonstrate the potential benefits of a microtubular scaffold design to promote odontoblast cells for regeneration of dentin.

Lithium-end-capped polylactide thin films influence osteoblast progenitor cell differentiation and mineralization.

End-capping by covalently binding functional groups to the ends of polymer chains offers potential advantages for tissue engineering scaffolds, but the ability of such polymers to influence cell behavior has not been studied. As a demonstration, polylactide (PLA) was end-capped with lithium carboxylate ionic groups (hPLA13kLi) and evaluated. Thin films of the hPLA13kLi and PLA homopolymer were prepared with and without surface texturing. Murine osteoblast progenitor cells from collagen 1α1 transgenic reporter mice were used to assess cell attachment, proliferation, differentiation, and mineralization. Measurement of green fluorescent protein expressed by these cells and xylenol orange staining for mineral allowed quantitative analysis. The hPLA13kLi was biologically active, increasing initial cell attachment and enhancing differentiation, while reducing proliferation and strongly suppressing mineralization, relative to PLA. These effects of bound lithium ions (Li(+) ) had not been previously reported, and were generally consistent with the literature on soluble additions of lithium. The surface texturing generated here did not influence cell behavior. These results demonstrate that end-capping could be a useful approach in scaffold design, where a wide range of biologically active groups could be employed, while likely retaining the desirable characteristics associated with the unaltered homopolymer backbone.

A Site-Specific Integrated Col2.3GFP Reporter Identifies Osteoblasts Within Mineralized Tissue Formed In Vivo by Human Embryonic Stem Cells.

The use of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) for study and treatment of bone diseases or traumatic bone injuries requires efficient protocols to differentiate hESCs/iPSCs into cells with osteogenic potential and the ability to isolate differentiated osteoblasts for analysis. We have used zinc finger nuclease technology to deliver a construct containing the Col2.3 promoter driving GFPemerald to the AAVS1 site (referred to as a "safe harbor" site), in human embryonic stem cells (H9Zn2.3GFP), with the goal of marking the cells that have become differentiated osteoblasts. In teratomas formed using these cells, we identified green fluorescent protein (GFP)-positive cells specifically associated with in vivo bone formation. We also differentiated the cells into a mesenchymal stem cell population with osteogenic potential and implanted them into a mouse calvarial defect model. We observed GFP-positive cells associated with alizarin complexone-labeled newly formed bone surfaces. The cells were alkaline phosphatase-positive, and immunohistochemistry with human specific bone sialoprotein (BSP) antibody indicates that the GFP-positive cells are also associated with the human BSP-containing matrix, demonstrating that the Col2.3GFP construct marks cells in the osteoblast lineage. Single-cell cloning generated a 100% Col2.3GFP-positive cell population, as demonstrated by fluorescence in situ hybridization using a GFP probe. The karyotype was normal, and pluripotency was demonstrated by Tra1-60 immunostaining, pluripotent low density reverse transcription-polymerase chain reaction array and embryoid body formation. These cells will be useful to develop optimal osteogenic differentiation protocols and to isolate osteoblasts from normal and diseased iPSCs for analysis.

Developmental-like bone regeneration by human embryonic stem cell-derived mesenchymal cells.

The in vivo osteogenesis potential of mesenchymal-like cells derived from human embryonic stem cells (hESC-MCs) was evaluated in vivo by implantation on collagen/hydroxyapatite scaffolds into calvarial defects in immunodeficient mice. This study is novel because no osteogenic or chondrogenic differentiation protocols were applied to the cells prior to implantation. After 6 weeks, X-ray, microCT, and histological analysis showed that the hESC-MCs had consistently formed a highly vascularized new bone that bridged the bone defect and seamlessly integrated with host bone. The implanted hESC-MCs differentiated in situ to functional hypertrophic chondrocytes, osteoblasts, and osteocytes forming new bone tissue via an endochondral ossification pathway. Evidence for the direct participation of the human cells in bone morphogenesis was verified by two separate assays: with Alu and by human mitochondrial antigen positive staining in conjunction with co-localized expression of human bone sialoprotein in histologically verified regions of new bone. The large volume of new bone in a calvarial defect and the direct participation of the hESC-MCs far exceeds that of previous studies and that of the control adult hMSCs. This study represents a key step forward for bone tissue engineering because of the large volume, vascularity, and reproducibility of new bone formation and the discovery that it is advantageous to not over-commit these progenitor cells to a particular lineage prior to implantation. The hESC-MCs were able to recapitulate the mesenchymal developmental pathway and were able to repair the bone defect semi-autonomously without preimplantation differentiation to osteo- or chondroprogenitors.

One-step derivation of mesenchymal stem cell (MSC)-like cells from human pluripotent stem cells on a fibrillar collagen coating.

Controlled differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) into cells that resemble adult mesenchymal stem cells (MSCs) is an attractive approach to obtain a readily available source of progenitor cells for tissue engineering. The present study reports a new method to rapidly derive MSC-like cells from hESCs and hiPSCs, in one step, based on culturing the cells on thin, fibrillar, type I collagen coatings that mimic the structure of physiological collagen. Human H9 ESCs and HDFa-YK26 iPSCs were singly dissociated in the presence of ROCK inhibitor Y-27632, plated onto fibrillar collagen coated plates and cultured in alpha minimum essential medium (alpha-MEM) supplemented with 10% fetal bovine serum, 50 uM magnesium L-ascorbic acid phosphate and 100 nM dexamethasone. While fewer cells attached on the collagen surface initially than standard tissue culture plastic, after culturing for 10 days, resilient colonies of homogenous spindle-shaped cells were obtained. Flow cytometric analysis showed that a high percentage of the derived cells expressed typical MSC surface markers including CD73, CD90, CD105, CD146 and CD166 and were negative as expected for hematopoietic markers CD34 and CD45. The MSC-like cells derived from pluripotent cells were successfully differentiated in vitro into three different lineages: osteogenic, chondrogenic, and adipogenic. Both H9 hES and YK26 iPS cells displayed similar morphological changes during the derivation process and yielded MSC-like cells with similar properties. In conclusion, this study demonstrates that bioimimetic, fibrillar, type I collagen coatings applied to cell culture plates can be used to guide a rapid, efficient derivation of MSC-like cells from both human ES and iPS cells.

Wear at the titanium-zirconia implant-abutment interface: a pilot study.

PURPOSE: The purpose of this study was to use a clinical simulation to determine whether wear of the internal surface of a titanium implant was greater following connection and loading of a one-piece zirconia implant abutment or a titanium implant abutment. MATERIALS AND METHODS: Two implants received zirconia abutments and two received titanium abutments. The implants were secured into four fiber-reinforced epoxy resin disks that had been prepared to receive the internal-connection implants. The assemblies were cyclically loaded off-axis for a total of 1,000,000 cycles. At various intervals, the abutments were removed, photographed, examined using scanning electron microscopy (SEM), and returned to the implants for further testing. The area of titanium transfer from the implants to the abutments observed in the SEM images was quantified using image analysis software. RESULTS: The method was able to quantify the area of material transferred to the abutments. There was considerably more wear associated with the zirconia abutments, but the rate of wear slowed after about 250,000 cycles. Parabolic curves were fit to the data. The projected mean ± standard deviation maximum area (wear) values associated with the titanium and zirconia abutments were 15.8 ± 3.3 x 10³ Μm² and 131.8 ± 14.5 x 10³ Μm², respectively, and this difference was statistically significant (P = .0081). CONCLUSIONS: The implants with the zirconia abutments showed a greater initial rate of wear and more total wear than the implants with the titanium abutments following cyclic loading. The amount of titanium transfer seen on the zirconia abutment increased with the number of loading cycles but appeared to be self-limiting. The clinical ramifications of this finding are unknown at this time; however, the potential for component loosening and subsequent fracture and/or the release of particulate titanium debris may be of concern.

Polyphenylene polymers as esthetic orthodontic archwires.

INTRODUCTION: There is continuing interest in an esthetic, effective labial archwire. In this study, we evaluated the potential of new, high-strength polyphenylene polymers to fill this need. METHODS: Polyphenylene (Primospire, Solvay Advanced Polymers, Alpharetta, Ga) polymer was extruded into wires with clinically relevant round and rectangular cross sections. Tensile, flexure, spring-back, stress-relaxation, and formability characteristics were assessed. Arch forms and secondary shapes were formed. RESULTS: Smooth wires with consistent cross-sectional dimensions, high spring-back, and good ductility were produced. Forces delivered were generally similar to typical beta-titanium and nickel-titanium wires of somewhat smaller cross sections. The polyphenylene wire did experience stress relaxation for up to 75 hours. The force magnitudes place polyphenylene wires in the category of an alignment or leveling wire. High formability allowed shape bending similar to that associated with stainless steel wires. CONCLUSIONS: Polyphenylene polymers could serve as esthetic orthodontic archwires; further study is warranted.

Viscoelastic properties of an aesthetic translucent orthodontic wire.

The objective of this study was to evaluate the time-dependent viscoelastic properties of an aesthetic orthodontic archwire. The wire is based on a recently developed translucent polyphenylene thermoplastic, whose rigid molecular structure provides high strength. While the wire has good instantaneous mechanical properties, over time all polymers may relax so it is important to understand the potential impact of the relaxation on orthodontic force systems. Four samples of 0.020 inch round and six samples of 0.021 × 0.025 inch rectangular wire were loaded in tension to a range of initial stresses, and relaxation of the stress was monitored for 7 days. Sixty-three additional samples were maintained in edgewise bracket pairs with vertical displacement for up to 6 weeks. The deformation of these wires was measured immediately after removal from the brackets and for 2 days as the samples recovered. Tensile stress decayed about 10-30 per cent over 24-48 hours depending on the initial stress. The relaxation behaviour was proportional to the initial tensile strain and therefore these data were combined into a single curve using regression. Deformation of the samples placed in the bracket pairs increased with increasing vertical displacement and time, evaluated with analysis of variance, but 19-100 per cent of the deformation was recoverable. The force systems from polyphenylene wires could vary with time and activation, but this behaviour is predictable.

Fabrication and characterization of hydroxyapatite-coated polystyrene disks for use in osteoprogenitor cell culture.

A simple method is reported for fabricating polystyrene disk inserts coated with biomimetic carbonated hydroxyapatite (cHA) to be used for culturing osteoprogenitor cells or other stem cells. Roughened disks cut from tissue-culture polystyrene (TCPS) were coated in simulated body fluid with 5 x normal physiologic ionic concentrations (SBFx5) by a 2-step, 2-day method. The coatings were rigorously characterized by various methods and assessed in cell culture. An adherent, nearly 10 mm thick, relatively uniform layer of single-phase cHA was formed in two days. MC3T3-E1 and mouse calvaria-derived osteoprogenitor cells (pCOBs) were cultured on the cHA for various time points. Despite less initial attachment of both cell types to the cHA, proliferation rates on cHA were similar to that on TCPS. Two-fold greater cell attachment (P < 0.05) of the MC3T3-E1 cells was observed relative to the pCOBs, on both the TCPS and the cHA. Importantly, the coatings were relatively smooth, without the extensive agglomerates observed in other studies and remained adherent and morphologically unchanged after 21 days of culture. This technique can be used to rapidly produce high-quality cHA-coated TCPS disks for cell-culture studies.

A nondestructive method for evaluating in vitro osteoblast differentiation on biomaterials using osteoblast-specific fluorescence.

Transgenic mice with a Col1a1-promoter-driven transgene pOBCol2.3GFP were previously developed to visually identify mature osteoblasts through fluorescent expression. Our goal was to determine if this technology could be used to nondestructively evaluate the in vitro differentiation of osteoprogenitor cells on biomaterials such as biomimetic carbonated hydroxyapatite (cHA). Primary osteoprogenitor cells were harvested from calvaria of neonatal Col2.3GFP transgenic mice and cultured on cHA and a tissue culture polystyrene (TCPS) control. The distribution of intensities and area percentage of green fluorescent protein (GFP)-positive cells were quantified using fluorimetry and image analysis of fluorescent microscopy. At 14 days, an increased area and higher mean intensity of GFP-positive cells was observed on cHA as compared to TCPS, indicating more rapid differentiation on cHA. Notably, there were large continuous regions of GFP-positive osteoblasts on cHA, in contrast to the sparse, nodules of osteoblasts on TCPS, implying that cHA provides an osteogenic cue to cells. Xylenol orange staining was capable of distinguishing osteoblast-initiated mineral from the cHA substrate. With this method the unique pattern of osteoblast differentiation on cHA was clearly observed for the first time. Importantly, the generalized method can be used for rapid, high-throughput, nondestructive screening of biomaterials intended to enhance osteogenic differentiation.

Polypeptide-catalyzed silica for dental applications.

Polypeptides such as polylysine have been shown to catalyze the condensation and direct the structure of silica from precursor solutions under ambient conditions. Several of the reaction parameters have been shown to mediate this activity. Specifically, mechanical perturbation seems to play a role in the formation of hierarchical structures. Most studies have been conducted in solution, but biomedical and particularly dental applications will likely require control of biosilicified coatings, films or particle formation on surfaces. Tetraethylorthosilicate was reacted with polylysine and then spin coated onto a surface. The process parameters catalyst structure, pH, buffer: ethanol ratio and percentage of cocatalyst polyethyleneimine were varied to determine their effects on the formed silica. The chemical nature and morphology of the silica were investigated with FTIR and SEM, respectively and reaction rates were monitored with a colorimetric assay. Our results show that these process parameters had only minor effects on composition, but the catalyst conformation influenced the degree of hydration while the pH, choice of solvent and cocatalyst strongly influenced morphology. We also found that perturbation from spin coating significantly influences the silicification dynamics. The ability to catalyze nano- to micron-sized mineral with different morphologies using polypeptides could have numerous dental applications including, sealing of dentin tubules, in situ reinforcement of resin interfaces or preparation of implant surfaces.

Clinical evaluation of fiber-reinforced fixed bridges.

BACKGROUND: This study evaluated the clinical performance of 39 light and heat polymerized fixed partial bridges made with a substructure of preimpregnated, unidirectional fiber-reinforced composite, or FRC, veneered with a hybrid particu late composite. METHODS: The authors evaluated 22 extracoronal, full-coverage retainer prostheses and 17 intracoronal, partial-coverage retainer prostheses placed over a 37-month period. All substructures initially were fabricated with a low-volume FRC. The authors reevaluated this design after early failures occurred, leading to a substructure with a higher volume of FRC. All prostheses were assessed for surface integrity, anatomical contour, marginal integrity and structural integrity at several intervals. RESULTS: The data show that survival was associated primarily with substructure design volume. When patients with severe parafunctional habits were excluded, the survival rate was 95 percent for prostheses made with a high-volume substructure (survival range, 2.77 to 4.30 years; mean +/- standard deviation survival, 3.75 +/- 0.4 years). Retainer configuration did not have a statistically significant influence on clinical survival. For all surviving prostheses, the authors observed few changes in any clinical parameters from baseline to 48 months. A loss of surface luster was observed in the majority of cases. Repairable surface defects were detected on two prostheses at 24 months. Scanning electron microscopic analyses indicated no exposed fibers on the occlusal surface and minimal wear. CONCLUSIONS: This study shows that a unidirectional, preimpregnated FRC can be used successfully to make bridges of variable retainer designs that last up to four or more years when a high-volume substructure is used. CLINICAL IMPLICATIONS: Short-span polymer prostheses made with particulate composite and unidirectional glass FRC can be used in certain clinical situations in which a metal substructure is not desired.

The design and fabrication of fiber-reinforced implant prostheses.

The use of fiber composite technology in the creation of metal-free implant prostheses may solve many of the problems associated with a metal alloy substructure such as corrosion, toxicity, complexity of fabrication, high cost, and esthetic limitations. Laboratory and clinical research evaluating glass fiber-reinforced composite prostheses used to restore and replace teeth has shown that these materials exhibit excellent mechanical properties and can form a chemical bond to resin-based veneer materials such as those used in the fabrication of certain types of implant prostheses. Two different designs of fiber-reinforced composite implant prostheses have been developed and placed in human subjects. One design (screw-retained, retrievable prosthesis) is used with implant abutments that allow for screw-retained prostheses; the other design is used with abutments that retain prostheses with a luting material. Both designs are described in this article. The prostheses have functioned well in a small group of preliminary subjects, but clinical trials with larger subject populations are needed to more completely evaluate the potential of fiber-reinforced composites in implant prosthodontics.