Original study - ZZI 03/2009

In vitro studies to screen for implant surface properties*

H.-L. Graf1, J. Hofmann1, U. Tröger1, J. Schreckenbach2, H. Hilbig3

In a short-term (ten days in vitro) culture of cells derived from human maxillae the expression of the non-collagenous bone matrix proteins osteonectin, osteocalcin and bone sialoprotein at the surface of four clinically-employed and three experimentally-designed implants was estimated quantitatively. The surfaces “titanium-calcium-magnesium ANOF” and “titanium-calcium-magnesium-fluoride ANOF” induced high expression levels of bone-derived cell markers. Theses results suggest further investigation magnesium – and fluorid modifications including.

Both the chemical composition of the implant and the microstructure of the implant surface influence protein expression.

The experimental design is suitable for preliminary and quick screening to select experimentally designed implant surfaces for further testing.

Keywords: Bone Sialoprotein, Osteonectin, Osteocalcin, Screening, Magnesium Fluoride, Selenium

Introduction

The insertion of endosteal implants as a fixation element for dentures is currently the greatest development in prosthetic dentistry.

All of the usual implant systems are based on the endosteal insertion of rotationally symmetrical prefabricated components, the surfaces of which vary considerably with regard to microstructure, nanostructure and chemical composition. These differences are regarded as the crucial modulator for the rate of bone-implant contact, and the duration of clinical treatment depends directly on this. For obvious reasons, this should be shortened, which is why surface development is one of the main subjects of research in implantology. The present study is intended to make an indirect contribution by testing an in vitro method of screening known surfaces and applying it to experimental surfaces that are not in clinical use.

In vitro studies of osteocompatibility are usually performed using human or animal osteoblast precursors or osteoblasts. The expression of collagenous bone matrix proteins, such as collagen type I [1, 5], and of non-collagenous bone matrix proteins such as osteocalcin [18, 20], osteonectin [13, 14] or bone sialoprotein (BSP) [5, 13, 14] is regarded as a measure of osteoblast differentiation. Mature osteoblasts secrete a matrix, which contains nucleation sites on which nanocrystalline calcium phosphate can be deposited.

Osteonectin is believed to modulate cell-cell and cell-matrix interactions and is therefore important for the organization of this extracellular matrix. This and the high affinity for calcium and collagen make measurement of osteonectin interesting.

Osteocalcin is a calcium-binding protein and is regarded as a marker of differentiated osteoblasts. An increase in osteocalcin secretion is interpreted as further differentiation. Greater roughness of sand-blasted and etched surfaces correlates with increased osteocalcin production [2, 3].

Determination of the amount of osteocalcin expressed appears helpful for drawing conclusions about the stage of osteoblast differentiation and investigating the described association with implant surfaces having different degrees of roughness.

BSP was found in the collagen-free matrix produced by the osteoblasts, which is the first layer to be deposited on implant surfaces [6]. The ability of BSP to bind in vitro to hydroxyapatite nuclei and be expressed during the early mineralization phase of the bone tissue indicates its involvement in initiating tissue mineralization [6, 12].

These proteins were therefore used to characterize human bone cell cultures on different test specimens.

 

Material and methods

Tissue harvesting and cell culture

After obtaining informed consent from the male patient and the approval of the ethics committee of Leipzig University, during an oral surgical procedure at the Clinic and Polyclinic for Oro-maxillary and Facial Plastic Surgery Clinic of Leipzig University, bone tissue was removed, transferred to sterile, isotonic phosphate-buffered saline solution (PBS) with penicillin and streptomycin, cut up and rinsed with sterile PBS to remove residual blood. To accelerate the growth of bone cells from the harvested tissue, the bone fragments were incubated with 0.25 % collagenase in PBS in an incubator at 37°C, 5 % CO2 and 95 % humidity. After a reaction time of 30 minutes, the collagenase was aspirated, discarded and replaced by fresh solution for two more hours. The preparation was then centrifuged for ten minutes at 300 g. The supernatant was pipetted, and the resulting pellet resuspended with 1 ml of nutrient medium, then transferred to a cell culture flask (25 cm²) and 2 ml of nutrient medium were added.

The cell culture was left in the incubator for seven days. After that, the nutrient medium was exchanged every third day and cell growth checked microscopically. After confluent growth was obtained on the surface of the culture after six weeks at the earliest, the first passage of the cells could be performed. This meant that the nutrient medium had to be aspirated and the cells rinsed carefully with sterile PBS. The cells were separated from the floor of the culture vessel enzymatically by incubation for two minutes with 0.5 % trypsin in PBS. To interrupt the trypsinization, 5 ml of nutrient medium were added after the incubation period. The resulting cell suspension was pipetted, transferred to a centrifuge tube and centrifuged for ten minutes at 300 g. The supernatant was then aspirated and the cell pellet carefully resuspended with 5 ml of fresh nutrient medium. Each 2.5 ml of this cell suspension was pipetted into a 75 cm² cell culture flask, 14 ml of nutrient medium were added and left in the incubator with a change of medium every three days until there was confluent growth.

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