Lucy Di Silvio

Optimizing HAPEX™ Topography Influences Osteoblast Response

HAPEX (hydroxyapatite-reinforced polyethylene composite) is a second-generation orthopedic biomaterial designed as a bone analog material, which has found clinical success. The use of topography in cell engineering has been shown to affect cell attachment and subsequent response. Thus, by combining bioactivity and enhancing osteoblast response to the implant surface, improved tissue repair and implant life span may be achieved. In this study a primary human osteoblast-like cell model has been used to study the influence of surface topography and chemistry produced by three different production methods. Scanning electron microscopy, fluorescence microscopy, and confocal scanning laser microscopy have been used to study cell adhesion; tritiated thymidine uptake has been used to observe cell proliferation; and the reverse transcriptase-polymerase chain reaction and biochemical methods have been used to study phenotypic expression. Transmission electron microscopy has also been used to look at more long-term morphology. The results show that topography significantly influences cell response, and may be a means of enhancing bone apposition on HAPEX.

Pre-warming of dental composites

OBJECTIVES Cavity lining with flowable composites have been proposed to improve initial marginal adaptation and minimize shrinkage stresses. The purpose of this study was to evaluate if prewarming of composites would influence the flow and enhance marginal adaptation thus the effect of pre-warming different types of composites on their properties are reported. METHODS Six different composites were used in this study including a flowable and a polyacid modified composite. Uncured composites were pressed between two glass plates with a known load and the film thickness was measured to determine flow. Polymerization shrinkage was measured by means of a one-dimension contacting transducer. Flexural strength was determined using a three-point bend test. Microleakage was determined in human lower third molars on both enamel and dentin restoration interfaces. Cytocompatibility was analyzed with an Alamar Blue redox cell proliferation assay. The flow properties, linear shrinkage, flexural strength, microleakage and cytocompatibility were evaluated at 22 °C and 60 °C. RESULTS The results indicated that the film thickness for each of the materials was significantly lower at 60 °C and the linear shrinkage was greater as a result of the higher degree of polymerization. The flexural strength of Spectrum TPH and Wave were found to be statistically significantly higher with pre-warming, however the other composites did not exhibit any differences. Microleakage studies showed that pre-warming had no significant bearing on the results and alamarBlue(®) results showed that the pre-heating did not have an effect on the cytotoxicity however the levels of cytotoxicity varied between the composites that can be attributed to the composition. SIGNIFICANCE Pre-warming of the composites studied enhanced flow as observed by measuring film thickness and did not significantly affect other properties.

A novel jet-based nano-hydroxyapatite patterning technique for osteoblast guidance

Surface topography is well known to play a crucial role in influencing cellular responses to an implant material and is therefore important in bone tissue regeneration. A novel jet-based patterning technique, template-assisted electrohydrodynamic atomization spraying, was recently devised to control precisely the surface structure as well as its dimensions. In the present study, a detailed investigation of this patterning process was carried out. A range of nano-hydroxyapatite (nHA) line-shaped patterns <20 µm in width were successfully deposited on a commercially pure Ti surface by controlling the flow of an nHA suspension in an electric field. In vitro studies showed that the nHA patterns generated are capable of regulating the human osteoblast cell attachment and orientation.

In vitro response of human osteoblasts to multi-step sol-gel derived bioactive glass nanoparticles for bone tissue engineering.

A multi-step sol-gel process was employed to synthesize bioactive glass (BG) nanoparticles. Transmission electron microscopy (TEM) revealed that the BG nanoparticles were spherical and ranged from 30 to 60 nm in diameter. In vitro reactivity of the BG nanoparticles was tested in phosphate buffer saline (PBS), Tris-buffer (TRIS), simulated body fluid (SBF), and Dulbeccos modified Eagles medium (DMEM), in comparison with similar sized hydroxyapatite (HA) and silicon substituted HA (SiHA) nanoparticles. Bioactivity of the BG nanoparticles was confirmed through Fourier transform infrared spectroscopy (FTIR) analysis. It was found that bone-like apatite was formed after immersion in SBF at 7 days. Solutions containing BG nanoparticles were slightly more alkaline than HA and SiHA, suggesting that a more rapid apatite formation on BG was related to solution-mediated dissolution. Primary human osteoblast (HOB) cell model was used to evaluate biological responses to BG nanoparticles. Lactate dehydrogenase (LDH) cytotoxicity assay showed that HOB cells were not adversely affected by the BG nanoparticles throughout the 7day test period. Interestingly, MTS assay results showed an enhancement in cell proliferation in the presence of BG when compared to HA and SiHA nanoparticles. Particularly, statistically significant (p<0.05) alkaline phosphatase (ALP) activity of HOB cells was found on the culture containing BG nanoparticles, suggesting that the cell differentiation might be promoted by BG. Real-time quantitative PCR analysis (qPCR) further confirmed this finding, as a significantly higher level of RUNX2 gene expression was recorded on the cells cultured in the presence of BG nanoparticles when compared to those with HA and SiHA.

Tissue engineering technology and its possible applications in oral and maxillofacial surgery

Tissue engineering is a rapidly advancing discipline that combines the attributes of biochemical and biomaterial engineering with cell transplantation to create bio-artificial tissues and organs. For the oral and maxillofacial surgeon, the reconstruction of maxillofacial defects in hard and soft tissues is an ongoing challenge. While autologous grafts and vascularised free flaps are the current gold standard, they are not without complications at both the donor and reconstructed sites. Tissue engineering, which aims to create tissue-matched, prefabricated, prevascularised bony or soft tissue composite grafts, or both, therefore has the potential to revolutionise practice in maxillofacial surgery. We review the technology of tissue engineering and its current and future applications within the specialty, and discuss contemporary obstacles yet to be overcome.

Assessment of novel chemical strategies for covalent attachment of adhesive peptides to rough titanium surfaces: XPS analysis and biological evaluation

Bioactive molecules have been proposed to promote beneficial interactions at bone-implant interfaces for enhancing integration. The main objective of this study was to develop novel methods to functionalize oxidized titanium surfaces by the covalent immobilization of bioactive peptides, through selective reaction involving single functional groups. In the first protocol, an aminoalkylsilane was covalently linked to the Ti oxide layer, followed by covalent binding of glutaric anhydride to the free NH(2) groups. The carboxylic group of glutaric anhydride was used to condense the free N-terminal group of the side-chain protected peptide sequence. Finally, the surface was treated with trifluoroacetic acid to deprotect side-chain groups. In the second protocol, the peptide was directly anchored to the Ti oxide surface via UV activation of an arylazide peptide analogue. X-ray photoelectron spectroscopy analyses confirmed that modifications induced onto surface composition were in agreement with the reactions performed. The peptide density of each biomimetic surface was determined on the basis of radiolabeling and XPS derived reaction yields. The in vitro cellular response of the biomimetic surfaces was evaluated using a primary human osteoblast cell model. Cell adhesion, proliferation, differentiation, and mineralization were examined at initial-, short-, and long-time periods. In was shown that the biomimetic surface obtained through photoprobe-marked analogue that combines an easily-performed modification provides a favorable surface for an enhanced cellular response.

Nanohydroxyapatite shape and its potential role in bone formation: an analytical study

Bone cells (osteoblasts) produce a collagen-rich matrix called osteoid, which is mineralized extracellularly by nanosized calcium phosphate (CaP). Synthetically produced CaP nanoparticles (NPs) have great potential for clinical application. However few studies have compared the effect of CaP NPs with different properties, such as shape and aspect ratio, on the survival and behaviour of active bone-producing cells, such as primary human osteoblasts (HOBs). This study aimed to investigate the biocompatibility and ultrastructural effects of two differently shaped hydroxyapatite [Ca10(PO4)6(OH)2] nanoparticles (HA NPs), round- (aspect ratio 2.12, AR2) and rice-shaped (aspect ratio 3.79, AR4). The ultrastructural response and initial extracellular matrix (ECM) formation of HOBs to HA NPs were observed, as well as matrix vesicle release. A transmission electron microscopy (TEM)-based X-ray microanalytical technique was used to measure cytoplasmic ion levels, including calcium (Ca), phosphorus (P), sodium (Na) and potassium (K). K/Na ratios were used as a measure of cell viability. Following HA NP stimulation, all measured cytoplasmic ion levels increased. AR2 NPs had a greater osteogenic effect on osteoblasts compared with AR4 NPs, including alkaline phosphatase activity and matrix vesicle release. However, they produced only a moderate increase in intracellular Ca and P levels compared with AR4. This suggests that particular Ca and P concentrations may be required for, or indicative of, optimal osteoblast activity. Cell viability, as measured by Na and K microanalysis, was best maintained in AR2. Initial formation of osteoblast ECM was altered in the presence of either HA NP, and immuno-TEM identified fibronectin and matrilin-3 as two ECM proteins affected. Matrilin-3 is here described for the first time as being expressed by cultured osteoblasts. In summary, this novel and in-depth study has demonstrated that HA NP shape can influence a range of different parameters related to osteoblast viability and activity.

P16/p53 expression and telomerase activity in immortalized human dental pulp cells

Introduction:Residing within human dental pulp are cells of an ectomesenchymal origin which have the potential to differentiate into odontoblast-like cells. These cells have a limited growth potential owing to the effects of cell senescence.This study examines the effects of immortalizing odontoblast-like cells on cell proliferation and mineralization by comparing transformed dental pulp stem cells (tDPSCs) and non-transformed dental pulp stem cells(nDPSCs). Methods: Using a retroviral vector, exogenous human telomerase reverse transcriptase (hTERT) was expressed in tDPSCs. Both cell groups were cultured and their telomerase activities is determined using a telomerase quantification assay. Also examined were the expression of genes involved in proliferation and mineralization such as human alkaline phosphatase (ALP), B-actin, collagen 1 (col-1), core binding factor (cbfa-1), dentin matrix protein (DMP-1),dentin sialophosphoprotein (DSPP), GAPDH, hTERT,osteocalcin (OCN), osteopontin (OPN) as well as oncoproteins involved in senescence (p16,p21 and p53) using RT-PCR. DNA and alkaline phosphatase activity was assayed in both cell groups. Results: With the exogenous expression of hTERT, tDPSCs maintained a continued expression of odontogenic markers for cell proliferation and mineralization (ALP,COL-1,DMP-1,DSPP,OCN amd OPN)as did nDPScs. Oncoprotein expression was ssen in both groups except for a noted absence of p16 in the tDPSCs.nDPSCs also showed lower levels of total ALP and DNA activity in comparison to tDPSCs when assayed as well as low telomerase activity readings. Conclusions: These results indicate maintainance of odontoblast-like differentiation characteristics after retroviral transformation with hTERT and suggest a possible link with a reduced p16 expression.

In vitro osteoinductive potential of porous monetite for bone tissue engineering

Tissue engineering–based bone grafts are emerging as a viable alternative treatment modality to repair and regenerate tissues damaged as a result of disease or injury. The choice of the biomaterial component is a critical determinant of the success of the graft or scaffold; essentially, it must induce and allow native tissue integration, and most importantly mimic the hierarchical structure of the native bone. Calcium phosphate bioceramics are widely used in orthopaedics and dentistry applications due to their similarity to bone mineral and their ability to induce a favourable biological response. One such material is monetite, which is biocompatible, osteoconductive and has the ability to be resorbed under physiological conditions. The osteoinductive properties of monetite in vivo are known; however, little is known of the direct effect on osteoinduction of human mesenchymal stem cells in vitro. In this study, we evaluated the potential of monetite to induce and sustain human mesenchymal stem cells towards osteogenic differentiation. Human mesenchymal stem cells were seeded on the monetite scaffold in the absence of differentiating factors for up to 28 days. The gene expression profile of bone-specific markers in cells on monetite scaffold was compared to the control material hydroxyapatite. At day 14, we observed a marked increase in alkaline phosphatase, osteocalcin and osteonectin expressions. This study provides evidence of a suitable material that has potential properties to be used as a tissue engineering scaffold.

Gene Expression Responses to Mechanical Stimulation of Mesenchymal Stem Cells Seeded on Calcium Phosphate Cement

INTRODUCTION The aim of the study reported here was to investigate the molecular responses of human mesenchymal stem cells (MSC) to loading with a model that attempts to closely mimic the physiological mechanical loading of bone, using monetite calcium phosphate (CaP) scaffolds to mimic the biomechanical properties of bone and a bioreactor to induce appropriate load and strain. METHODS Human MSCs were seeded onto CaP scaffolds and subjected to a pulsating compressive force of 5.5±4.5 N at a frequency of 0.1 Hz. Early molecular responses to mechanical loading were assessed by microarray and quantitative reverse transcription-polymerase chain reaction and activation of signal transduction cascades was evaluated by western blotting analysis. RESULTS The maximum mechanical strain on cell/scaffolds was calculated at around 0.4%. After 2 h of loading, a total of 100 genes were differentially expressed. The largest cluster of genes activated with 2 h stimulation was the regulator of transcription, and it included FOSB. There were also changes in genes involved in cell cycle and regulation of protein kinase cascades. When cells were rested for 6 h after mechanical stimulation, gene expression returned to normal. Further resting for a total of 22 h induced upregulation of 63 totally distinct genes that were mainly involved in cell surface receptor signal transduction and regulation of metabolic and cell division processes. In addition, the osteogenic transcription factor RUNX-2 was upregulated. Twenty-four hours of persistent loading also markedly induced osterix expression. Mechanical loading resulted in upregulation of Erk1/2 phosphorylation and the gene expression study identified a number of possible genes (SPRY2, RIPK1, SPRED2, SERTAD1, TRIB1, and RAPGEF2) that may regulate this process. CONCLUSION The results suggest that mechanical loading activates a small number of immediate-early response genes that are mainly associated with transcriptional regulation, which subsequently results in activation of a wider group of genes including those associated with osteoblast proliferation and differentiation. The results provide a valuable insight into molecular events and signal transduction pathways involved in the regulation of MSC osteogenic differentiation in response to a physiological level of mechanical stimulation.