Influence of titanium surfaces on attachment of osteoblast-like cells in vitro

Mythili Jayaraman^, , Ulrich Meyer, Martin Bühner, Ulrich Joos and Hans-Peter Wiesmann

Biomineralization Research Unit, Klinik und Poliklinik für Mund- und Kiefer-Gesichtschirurgie der Universität Münster, Waldeyerstraße 30, 48149, Münster, Germany

Received 27 September 2002;  accepted 14 July 2003. ; Available online 10 September 2003.
Biomaterials
Volume 25, Issue 4 , February 2004, Pages 625-631

1. Abstract

Implant surface topography influences osteoblastic proliferation, differentiation and extracellular matrix protein expressions. Studies on preliminary interactions of osteoblast-like cells on implant interface through in vitro systems, can give lucid insights to osseo-integrative efficacies of when in vivo implants. In the present investigation two titanium surfaces of dental implants, a sandblasted and acid-etched surface and an experimental grooved surface were compared through in vitro systems. The titanium implants were seeded with osteoblast-like primary cells and maintained for a period of 1-7 days. Expressions of fibronectin and osteonectin were assessed through immunogold labelling by scanning electron microscopy. The grooved surface, supported better osteoblastic cell adhesion and proliferation than the rough surfaces. Further, osteoblastic cells on the grooved surfaces also displayed a strong labelling for fibronectin at the cytoplasmic extensions coupled with intense osteonectin expression in comparison to the rough surfaced implants. In conclusion, grooved surfaces offered better cell attachment and proliferation than the other rough surfaces studied.

Author Keywords: Osteoblasts; Surface topography; Groove surface; Roughness; Immunogold labelling

2. Article Outline

1. Introduction

2. Materials and methods

2.1. Implants

3. Cell culture

4. Scanning electron microscopic studies

5. Statistical analysis

5.1. Results

5.2. Cell morphology, attachment and proliferation

6. SEM of immuno-gold labelled implants

6.1. Discussion

Acknowledgements

References

3. 1. Introduction

Cell-matrix interaction is dependent on cytoskeletal organization, transmembrane integrin receptor expression and most importantly, the nature of the extracellular matrix (ECM) [1 and 2]. In bone, the ECM is largely composed of ECM proteins, such as collagen [3], fibronectin, laminin [4], vitronectin, osteopontin, osteonectin and other glycoproteins [5 and 6]. The ECM is crucial in mediating cell adhesion to biomaterials, its organization and production modulates the degree of cell attachment to the materials. Success of non-biodegradable implants will first and foremost depend on biocompatibility, followed by the capacity of the surface topography of the implants to evince, desired cell matrix, surface-cell matrix interactions. Although it is well accepted that surface topography of the implants has marked influence in osseointegration, little is known about the effect of surface roughness on cell metabolism or differentiation of osteoblastic cells interacting with the implants. Controlled roughness values of titanium surfaces in dental implants have been associated to increase bone-to-implant contacts [7]. Further, a mineralized osteoblast ECM is necessary for dental implant osseointegration, however, the molecular mechanisms (cytokines, factors up-regulating synthetic activity of osteoblastic cells) associated with osseointegration of osteoblasts to dental implant surfaces are not fully understood. Osseointegration is not only dependent on wound healing process but also on the potential of osteogenic cells to form bone.

The use of endosseous dental implants as transmucosal devices necessitates the successful integration of three different tissues: bone, connective tissue, and epithelium. Previous studies have demonstrated in short-term experiments that sandblasted and acid-etched (SLA) titanium implant had a greater bone-to-implant contact than a titanium plasma-sprayed (TPS) implant in non-oral bone [8 and 9]. Studies done have also shown that surface roughness of titanium implants cannot only have a profound influence on proliferation, differentiation and matrix production of osteoblastic cells but also influence the cytokines and growth factors in the milieu thereby modulating the healing process [10]. The effects of surface roughness on proliferation, matrix synthesis, differentiation, local factor production, cell morphology can also aid in giving valuable insights into implant-cell interface interactions.

In vitro systems offer a model tandem to in vivo applications while studying cell-biomaterial interface interactions. Further, primary osteoblastic cells are better candidates for evaluations of implants as in vitro models to comprehend integrative efficacies of the same when implanted in vivo [11]. In the present study, surface efficacies of two different titanium implants, a SLA surface was compared with a grooved surface.

4. 2. Materials and methods

2.1. Implants

Figs. 1(a) and (b) show the rough SLA surface, Fig. 1(c) and (d) illustrate the experimental grooved surface used during the present study.

(97K)

Fig. 1. (a) and (b) Scanning electron micrographs of rough titanium surface (SLA). (c) and (d) SEM of experimental grooved titanium surface.

5. 3. Cell culture

Initially, osteoblast-like cells were obtained from periost layer of calf metacarpals. Periost, was cut at dimensions of 3-6 mm and placed in culture dishes with the osteogenic layer towards the basal part of culture plate. Osteoprogentior cells migrate from explants [12]. Explants were maintained for a period of 3 weeks in High Growth Enhancement Medium (ICN Biomedicals Gmbh, Eschwege, Germany) supplemented with 10% fetal calf serum, 250 
g/ml Amphotericin B, 10,000 IU/ml penicillin, 10,000 
g/ml streptomycin, 200 m
 l-glutamine, at 37°C and 5% CO₂ in humidified air. Medium was replaced once a week. Cell morphology was monitored through a phase contrast microscope.

Cells were harvested by first washing with Phosphate buffer (PBS), followed by incubation with 6 ml of collagenase (0.4 gm of collagenase in 98.8 mg of Hams F10 in 10 ml of Hepes buffer) for 20 min. Following incubation, cells were washed with PBS twice and incubated with tyrode (300 mg EDTA (Na salt), 200 mg KCl, 8 g NaCl, 50 mg Na₂H₂PO₄·2H₂O, 1000 mg glucose) solution for 10 min. The suspension with cells were centrifuged for 5 min at 500 rpm, counted with a CASY 1 cell counting system and seeded on the implant. In order to study attachment efficacies 60,000 cells were seeded on surface of implants and 20,000 cells were seeded on implants surface to have an insight into the proliferation. For cell attachment and proliferation efficacies, cells/field were counted on the rough/grooved surfaces of the titanium implants, after 4 h and one week, respectively.

6. 4. Scanning electron microscopic studies

After 1st and 7th days, the cells on the implant surface were fixed with 3% paraformaldehyde, followed by immunogold labelling for the localization of fibronectin and osteonectin on the surface of the osteoblasts. Following fixing, the implants were washed in PBS buffer, blocked using a PBS/BSA (1% BSA) solution, incubated with primary antibodies, anti-fibronectin and anti-osteonectin, at a dilution of 1:200, for a period of 45 min at 37°C. After copious washing with buffer the implants were incubated with secondary 20 nm gold conjugated antibody incubation (British Bio-Cell International) for a period of 1 h. Silver enhancement was done for the gold particles using a Silver Enhancement kit (British Bio-Cell International). The samples were then dehydrated through the ascending alcohol series, dried to critical point, carbon coated and viewed through a LEO 1530VP scanning electron microscope (SEM). An acceleration of 20 kV was applied.

7. 5. Statistical analysis

Means and standard deviations (SD) were calculated for descriptive statistical documentation. The unpaired students t-test was applied for analytical statistics. A value p<0.05 was considered significant.

5.1. Results

Cell adhesion is a fundamental process directly involved in cell growth, migration and cell differentiation. It is now clearly established that surface properties of biomaterials play a critical role in the establishment of cell-biomaterial interface. In vitro cyto-compatibility is concerned with the influence of surface topography, roughness, surface energy, adsorbed proteins which in turn influences proliferation, differentiation and matrix synthesis of mesenchymal cells. The purpose of the present study was to evaluate the influence of grooved/rough titanium surface characteristics on cell attachment and possible expression of ECM proteins with reference to osteonectin.

5.2. Cell morphology, attachment and proliferation

Fig. 2(a) and (b), represent the scanning electron micrographs of cells on rough titanium surface of SLA implants at the end of days 1 and 7. Figs. 3(a) and (b) display the cells on grooved titanium surfaces, for the same period. Cell attachment and proliferation was much higher on the experimental grooved surfaces than on rough surfaces, supported in Graph 1.

(68K)

Fig. 2. (a) and (b) SEM of cells on rough titanium surface (SLA).

(63K)

Fig. 3. (a) and (b) SEM of cells on surface on experimental grooved surfaces.

(7K)

Graph 1. Cell attachment and proliferation on the surface of implants.

8. 6. SEM of immuno-gold labelled implants

Figs. 4a and b, display expression for fibronectin in the anti-fibronectin labelled gold coated experimental grooved surfaces, which were not as significant as in the rough surfaced counterparts. Expression of fibronectin appeared on day 1 and sustained till day 7. A closer look at the cytoplasmic extensions of the osteoblastic cells showed rich labelling for fibronectin is shown in Fig. 4c.

(93K)

Fig. 4. (a) and (b) Anti-fibronectin labelled gold coated implants of cells, on grooved titanium surface. (c) Anti-fibronectin labelled gold coated implants, closer look.

On the grooved surfaces, a strong expression for osteonectin was also perceived as shown in Figs. 5a and b, on the anti-osteonectin labelled gold coated grooved titanium surfaces as compared to the rough SLA surfaces, for day 1-7. A closer look of the same is in Fig. 5c. (As the expressions for fibronectin and osteonectin were too insignificant on the rough surface, the data have not been included.)

(102K)

Fig. 5. (a) and (b) Anti-osteonectin labelled gold coated implants of cells, grooved titanium surface. (c) Anti-osteonectin labelled gold coated implants of cells, closer look.

6.1. Discussion

Development of bone-implant interfaces depends directly on the interactions between bone matrix and osteoblasts with the biomaterial. Osteoblastic adhesion then becomes an essential phenomenon for the first bone-biomaterial interaction. Adhesion by itself is essential for embryogenesis, maintenance of tissue integrity, wound healing, immune response, and biomaterial tissue integration. Numerous proteins are involved in adhesion to the ECM proteins (fibronectin, collagen, laminin, vitronectin), cytoskeletal proteins (actin, talin, vinculin), and membrane receptors (integrins). Interactions between these proteins and their specific receptors induce signal transduction which consequently influences cell growth and differentiation.

Earlier in vitro studies of osteoblast-biomaterial interactions were concerned with effect of diverse biomaterials on cell adhesions, proliferation and differentiation without much significance being given to surface characterization [13 and 14]. It is interesting that cells of mesenchymal origin are extremely sensitive to surface properties, roughness and topography. Recently, studies have been made to understand the smooth/grooved surface efficacies on phenotypic expression of osteoblasts [15 and 16], in which grooved surfaces permitted attachment of more cells than a smooth surface. It has also been reported that cell shape and cyto-skeleton alignment was with respect to surface topography of grooved surfaces, which also seem to have a profound influence on osteogenesis. This work investigated on the influence of surface topography of two different titanium surfaces the rough (SLA) surface/grooved surface influence on cell morphology, proliferation and adhesion, in particular on the variation of the expression of specific cell adhesion proteins.

For this study only primary osteoblast-like cells [17], which offer better in vitro models, have been used to comprehend biomaterial interactions when in an in vivo environment. Models drawn through in vitro osteosarcoma cell lines due to varied genesis may show a discrepancy in ECM expressions [18], therefore, may not be the best suited for in vitro evaluation. Our investigations showed that cell attachment and proliferation improved remarkably on grooved surfaces compared to the rough titanium surfaces.

Osteoblastic cells express fibronectin [19 and 20] during early osteogeneis mediating cell attachment and spreading of bone cells in vitro. This is clearly evident at the cytoplasmic extensions of the osteoblastic cells expressing fibronectin, followed by ample cell spreading, which was detected from the first to the 7th day of our study. The rough surfaced titanium implants did not offer a good surface of cell adhesion and fibronectin labelling was undetectable at the end of first day and feebly expressed at the end of 7 days. Osteonectin, another significant bone marker glycoprotein [21], a modulator of mineralising mechanisms, was strongly expressed by the osteoblastic cells on the grooved surfaces. Osteonectin was detected from 1st to 7th day, while on the rough surface a feeble osteonectin expression was seen at the end of 1 day and not much difference was detected at the end of 7 days, as well.

In conclusion, the results of our present investigation is indicative of the fact that grooved titanium experimental surface offered better cell binding affinity with rich ECM protein expression compared to the rough titanium SLA surfaces. In vivo evaluation and studies on osseointegrative efficacies, of these implants are now, in progress.

9. Acknowledgements

We thank Mrs. Grabiniok and Mr. Huda for their useful and informative technical assistance.

10. References

1. C.P. Vary, V. Li, A. Raouf, R. Kitching, I. Kola, C. Franceschi, M. Venanzoni and A. Seth, Involvement of Ets transcription factors and targets in osteoblast differentiation and matrix mineralization. Exp Cell Res 257 1 (2000), pp. 213-222. Abstract | Abstract + References | PDF (262 K)

2. A. Rezania and K.E. Healy, Biomimetic peptide surfaces that regulate adhesion, spreading, cytoskeletal organization, and mineralization of the matrix deposited by osteoblast-like cells. Biotechnol Prog 15 1 (1999), pp. 19-32. Abstract-MEDLINE   | Full Text via CrossRef

3. G.R. Martin, R. Timpl, P.K. Muller and K. Kuhn, The genetically distinct collagens. Trends Biochem Res 251 (1985), pp. 285-287. Abstract

4. T.E. Lallier, R. Yukna and R.L. Moses, Extracellular matrix molecules improve periodontal ligament cell adhesion to anorganic bone matrix. J Dent Res 80 8 (2001), pp. 1748-1752. Abstract-MEDLINE  

5. M.F. Young, J.M. Kerr, K. Ibaraki, A.M. Heegaard and P.G. Robey, Structure, expression, and regulation of the major noncollagenous matrix proteins of bone. Clin Orthop 281 (1992), pp. 275-294. Abstract-MEDLINE | Abstract-EMBASE  

6. R.T. Ingram, B.L. Clarke, L.W. Fisher and L.A. Fitzpatrick, Distribution of noncollagenous proteins in the matrix of adult human bone: evidence of anatomic and functional heterogeneity. J Bone Miner Res 8 9 (1993), pp. 1019-1029. Abstract-MEDLINE | Abstract-EMBASE  

7. J.Y. Martin, Z. Schwartz, T.W. Hummert, D.M. Schraub, J. Simpson, J. Lankford, Jr, D.D. Dean, D.L. Cochran and B.D. Boyan, Effect of titanium surface roughness on proliferation, differentiation, and protein synthesis of human osteoblast-like cells (MG63). J Biomed Mater Res 29 3 (1995), pp. 389-401. Abstract-MEDLINE | Abstract-EMBASE | Abstract-Compendex  

8. D. Buser, R.K. Schenk, S. Steinemann, J.P. Fiorellini, C.H. Fox and H. Stich, Influence of surface characteristics on bone integration of titanium implants. A histomorphometric study in miniature pigs. J Biomed Mater Res 25 7 (1991), pp. 889-902. Abstract-EMBASE | Abstract-MEDLINE  

9. D.L. Cochran, P.V. Nummikoski, F.L. Higginbottom, J.S. Hermann, S.R. Makins and D. Buser, Evaluation of an endosseous titanium implant with a sandblasted and acid-etched surface in the canine mandible: radiographic results. Clin Oral Implants Res 7 3 (1996), pp. 240-252. Abstract-MEDLINE   | Full Text via CrossRef

10. S. Gronthos, K. Stewart, S.E. Graves, S. Hay and P.J. Simmons, Integrin expression and function on human osteoblast-like cells. J Bone Miner Res 12 8 (1997), pp. 1189-1197. Abstract-MEDLINE | Abstract-EMBASE  

11. N. Lumbikanonda and R. Sammons, Bone cell attachment to dental implants of different surface characteristics. Int J Oral Maxillofac Implants 16 5 (2001), pp. 627-636. Abstract-MEDLINE  

12. U. Meyer, M. Terodde, U. Joos and H.P. Wiesmann, Mechanische Stimulation von Osteoblastenin der Zellkultur. Mund Kiefer Gesichts Chir 5 (2001), pp. 166-172. Abstract-MEDLINE   | Full Text via CrossRef

13. R. Hermann, P. Walther and M. Muller, Immunogold-labelling in scanning electron microscopy. Histochem Cell Biol 106 (1996), pp. 31-39. Abstract-Elsevier BIOBASE | Abstract-MEDLINE   | Full Text via CrossRef

14. J.W. Slot and H.J. Geuze, A new method of preparing gold probes for multiple labelling cytochemistry. Eur J Cell Biol 38 (1985), pp. 87-93. Abstract-EMBASE | Abstract-MEDLINE  

15. J. Qu, B. Chehroudi and D.M. Brunette, The use of micromachined surfaces to investigate the cell behavioural factors essential to osseointegration. Oral Dis 2 1 (1996), pp. 102-115. Abstract-MEDLINE  

16. K. Anselme, M. Bigerelle, B. Noel, A. Iost and P. Hardouin, Effect of grooved titanium substratum on human osteoblastic cell growth. J Biomed Mater Res 60 4 (2002), pp. 529-540. Abstract-EMBASE | Abstract-MEDLINE   | Full Text via CrossRef

17. Meyer U, Joos U, Wiesmann HP, Jones DB. Biological and biophysical principles in bone tissue engineering—Part I. Int J Oral Max Surg, in press.

18. T. Kashima, K. Nakamura, J. Kawaguchi, M. Takanashi, T. Ishida, H. Aburatani, A. Kudo, M. Fukayama and A.E. Grigoriadis, Overexpression of cadherins suppresses pulmonary metastasis of osteosarcoma in vivo. Int J Cancer 104 2 (2003), pp. 147-154. Abstract-Elsevier BIOBASE | Abstract-MEDLINE | Abstract-EMBASE   | Full Text via CrossRef

19. R.E. Wiess and A.H. Reddi, Syntheis and localization of fibronectin during collagenous matrix mesenchymal cell interaction and differentiation of cartilage and bone in vivo. Proc Natl Acad Sci USA 108 (1980), pp. 709-721.

20. M.E. Lacouture, J.L. Schaffer and L.B. Klickstein, A comparison of type I collagen, fibronectin, and vitronectin in supporting adhesion of mechanically strained osteoblasts. J Bone Miner Res 17 3 (2002), pp. 481-492. Abstract-MEDLINE | Abstract-EMBASE  

21. H.I. Roach, Why does bone matrix contain Non-collagneous proteins?. The possible roles of osteocalcin, osteonectin, osteopontin and bone sialoprotein in bone mineralization and bone resorption. Cell Biol Int 18 6 (1994), pp. 617-628. Abstract

Corresponding author

---