Biomaterials
Volume 24, Issue 25 , November 2003, Pages 4585-4589

Osteoblast response to phospholipid modified titanium surface

Arpan Satsangi^{a, b}, Neera Satsangi^a, Renee' Glover^a, Rajiv K. Satsangi^{, , b} and Joo L. Ong<sup>a

a</sup> Department of Restorative Dentistry, Division of Biomaterials, University of Texas Health Science Center, San Antonio, TX, 78229, USA
^b Rann Research Corporation, 7410 John Smith Drive Suites 214 and 215, San Antonio, TX, 78229, USA

Available online 26 June 2003.

1. Abstract

The objective of this study was to evaluate the effect of different phospholipid coatings on osteoblast responses in vitro. Commercially available phospholipids [phosphatidylcholine (PC), phosphatidyl-serine (PS) and phosphatidylinositol (PI)] were converted to their Ca-PL-PO₄ and were coated on commercially pure titanium (Ti) grade 2 disks. Using uncoated Ti surfaces as controls, cell responses to phospholipid-coated surfaces were evaluated using the American Type Culture Collection (Manassas, VA, USA) CRL-1486 human embryonic palatal mesenchyme cells (HEPM), an osteoblast precursor cell line, over a 14-day period. Total protein synthesis and alkaline phosphatase specific activity at 0, 7, and 14 days were measured. It was observed that Ti surfaces coated with PS exhibited enhanced protein synthesis and alkaline phosphatase specific activity compared to other phospholipids and uncoated surfaces. These results indicate the possible usefulness of PS-coated Ti surfaces for inducing enhanced bone formation and are very encouraging for bone and dental implantology.

Author Keywords: Surface modified titanium; Phospholipids; Calcium-phospholipid-phosphate complex; Osteoblast differentiation; Alkaline phosphatase specific activity; Mineralization

2. Article Outline

1. Introduction

2. Materials and methods

2.1. Materials

2.1.1. Synthesis of calcium-phospholipid-phosphate complex (Ca-PL-PO₄) for Ti surface modification

2.2. Cell culture

2.2.1. Determination of protein production

2.2.2. Alkaline phosphatase specific activity

2.3. Mineralization on Ca-PL-PO₄ modified Ti surface in simulated body fluid and physico-chemical properties of the mineralized surface

3. Results and discussion

4. Conclusion

Acknowledgements

References

3. 1. Introduction

Titanium (Ti) is the implant material of choice for use in dental and orthopedic applications. The stable oxide that formed readily on Ti surfaces was reported to attribute to its excellent biocompatibility [1]. However, it was also reported that bone response to implant surfaces was dependent on the chemical and physical properties of Ti surfaces, thereby affecting implant success [2]. As such, attention has been focused on the surface preparation of the Ti implant.

Several techniques such as plasma spraying, laser deposition, ion beam dynamic mixing, ion beam deposition, magnetic sputtering, hot isostatic pressing, electrophoretic deposition, sol-gel, ion implantation, NaOH treatment, and electrochemical methods have been employed to deposit hydroxyapatite (HA) or calcium phosphate coatings on Ti surfaces [3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18]. Among the different processes described, plasma spraying of HA and Ti has been the most common method for modifying implant surfaces. However, numerous problems with the plasma-sprayed coatings have also been cited, including variation in bond strength between the coatings and the metallic substrates, non-uniformity in coating density as a result of the process, poor adhesion between the coatings and metallic substrates, and microcracks on the coating surface [19, 20, 21 and 22]. However, these problems do not reflect shortcomings inherent in the rationale for HA coating, but rather in the plasma spray and other technologies currently used to apply the coatings.

Recently, an alternative implant surface modification using phospholipids coatings have been suggested. It has been reported that various kind of calcium deposition processes involve the use of phospholipids [23, 24, 25, 26 and 27]. It has also been reported that a complex between calcium-inorganic phosphate and the phospholipid was essential for inducing the deposition of calcium phosphate [28]. Despite numerous chemical and structural characterizations, cellular responses to these phospholipids-coated implants anodized surfaces have yet to be evaluated. As such, the effect of different phospholipid coatings on in vitro osteoblast responses was evaluated in this study.

4. 2. Materials and methods

2.1. Materials

Phospholipids [phosphatidylcholine (PC), phosphatidylserine (PS, and phosphatidylinositol (PI); Fig. 1] were purchased from Sigma Chemical Company, St. Louis, MO. Commercial pure titanium (Ti) grade 2 disks were obtained from Metal Samples, Munford, AL. Plastic cell culture 24 and 96 well cluster plates were obtained from Costar, Corning, New York. Human Embryonic Palatal Mesenchyme cell suspension (HEPM; the osteoblast precursor cell line; Catalog #CRL-1486) was purchased from American type Culture Collection, Manassas, VA. Vitamin D3 was from Biomol Research Lab Inc., Plymouth Meeting, PA. Triton, NaOH, Magnesium chloride, 2-A2-M1-PP (2-amino-2-methyl-1-propanol), PNPP (p-nitrophenyl phosphate substrate), and PNP (p-nitrophenol) were bought from Sigma Chemical Company (St. Louis, MO). All labwares were prewashed and, wherever needed, were sterilized by UV light for 48 h.

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Fig. 1. Common natural phospholipids.

2.1.1. Synthesis of calcium-phospholipid-phosphate complex (Ca-PL-PO₄) for Ti surface modification

The aforesaid phospholipids were converted to their respective calcium-phospholipid-phosphate complex by the method of Boskey and Posner [28]. Briefly, 2.5 mg of phospholipid was taken in 5 ml of a solution containing 1 m
CaCl₂ and 1 m
(NH₄)₂HPO₄ in 0.05 
Tris buffer (pH 7.45) and the mixture was sonicated in ice for 1 min. The dispersion was agitated at 37°C for 24 h. The solution was phased by the addition of mixture of chloroform and methanol (2:1, V/V, 15 ml) followed by centrifugation at 2000 rpm for 15 min at 5°C. The lower phase, thus obtained, was washed three times with 10⁻⁵ 
HCl to remove the unchanged phospholipid. The Ca-PL-PO₄, recovered in the lower phase was finally used for coating on to the Ti surface to create the in vitro model of the surface modified biomaterial. It was then left under UV light (253.7 nm) for 48 h. The solvent was fully evaporated by then. The modified surface was rinsed with deionized water. Cleaned Ti surface prior to phospholipids coatings was used as the control. All coated and uncoated surfaces were sterilized under UV light (253.7 nm) for 48 h prior to mineralization and cell culture study.

2.2. Cell culture

One milliliter of HEPM cell suspension (osteoblast progenitor cells) at 1.5×104 cells/cm² was seeded in each well containing surface coated Ti disks, in a 24 well cluster plate. In one group, uncoated Ti disks were used (control). Alpha modified Eagle's medium containing 7% fetal bovine serum, 1% antibiotic-antimycotic solution, 50 mg/ml ascorbic acid, and 4 m
b-glycerophosphate was used. The cultures were maintained in a humidified 95% air, 5% CO₂ atm at 37°C. The medium was changed the day after seeding to remove nonadherent cells. Triplicates were conducted for each experimental point. The enzymatic assay on culture was performed on 0, 7, and 14 days.

2.2.1. Determination of protein production

Triplicate samples from each group were analyzed for total protein synthesis. Protein synthesis was performed using the Pierce BCA protein assay (Pierce, IL). The cell layer suspension (30 
l) was added to 200 
l of working reagent (sodium carbonate, sodium bicarbonate, BCA detection reagent, sodium tartrate in 0.1 
NaOH, and 4% copper sulfate). The samples were incubated for 30 min at 37°C and read using a microplate reader at 600 nm. The absorbance for the cell layer suspension was correlated to a standard protein curve and differences in protein synthesis were statistically compared using the ANOVA test (p<0.05), with differences assessed using the Student Newman-Keuls post hoc test.

2.2.2. Alkaline phosphatase specific activity

Triplicate samples from each treatment were used for measuring the ALP activity. The cell layer suspension (50 
l) was added to 50 
l of working reagent (1.5 
2-amino-2 methyl-1-propanol, 20 m
p-nitrophenol phosphate, and 1 m
magnesium chloride). The samples were then incubated for 3 h at 37°C. After 3 h incubation, the reaction was stopped with the addition of 100 
l of 1 
NaOH and read using a Titertek Multiscan Plus MK II microplate reader (MTX Labsystems, Inc., VA, USA) at 410 nm. The absorbance for the cell layer suspension was correlated to a standard ALP activity curve prepared using p-nitrophenol stock standard. Alkaline phosphatase specific activity was calculated using the previously determined protein values. Differences in alkaline phosphatase specific activity were statistically compared using the ANOVA test (p<0.05), with differences assessed using the Student Newman-Keuls post hoc test.

2.3. Mineralization on Ca-PL-PO₄ modified Ti surface in simulated body fluid and physico-chemical properties of the mineralized surface

All coated and uncoated Ti disks were sterilized under UV light (253.7 nm) for 48 h prior to immersion in the SBF consisting of 93.9 m
NaCl, 1.24 m
K₂HPO₄, 0.66 m
KH₂PO₄, 0.94 m
MgCl₂, 1.48 m
CaCl₂, 18 m
KHCO₃ (pH 7.4 at 37°C). Twelve coated and 12 uncoates disks were kept immersed in this solution under sterilized conditions for 14 days to observe the mineral deposition. After that period the disks were rinsed with deionized water and followed by the mixture of chloroform and methanol (1:1) to remove the solubilizable salts as well as the phospholipids. The residual surface was studied with scanning electron microscopy. The mineral deposited on the surface were solubilized in 100 
l of 1 
aqueous hydrochloric acid and mixed together in one test tube for phospholipids-coated disks and in another tube for uncoated disks. The presence of calcium and phosphate ions was checked by the previously described method [22].

5. 3. Results and discussion

Matrix proteins in bone have been shown to play a crucial role in the calcification and architectural construction of these hard tissues [29]. As evident from Fig. 2, only phosphatidylserine was able to enhance total protein production on coated surface on day 7 and day 14.

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Fig. 2. Protein production by osteoblast cells on different Ca-PS-PO₄ coated on Ti surface. The uncoated Ti surface was used as control.

The alkaline phosphatase specific activity is widely recognized as a biochemical marker for the osteoblast phenotype, and may be considered an important factor in bone mineralization. As shown in Fig. 3, the alkaline phosphatase specific activity observed in this study was significantly greater on PS-coated surfaces after 14 days incubation compared to the other phospholipids and control surfaces, indicating expression of the osteoblast phenotype by this osteoblast precursor cell line [30].

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Fig. 3. Specific alkaline phosphatase activity of osteoblast cells on different Ca-PS-PO₄ coated Ti surfaces. The uncoated Ti surface was used as control.

As evident from Fig. 4, an impressive process of calcium phosphate deposition on Ca-PS-PO₄-coated Ti surface after 14 days was observed by the scanning electron microscopic examination (Figs. 4B and C), whereas the uncoated surface clearly did not induce any crystal deposition ( Fig. 4A). After proper rinsing of the surface with limited amount of water and appropriate organic solvent to remove unwanted salts and phospholipids, the residual surface was studied with scanning electron microscopy and active mineralization was suggested. These were then solubilized in 100 
l of 1 
aqueous hydrochloric acid and their chemical analyses [22] revealed that it contained both calcium and phosphate ions.

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Fig. 4. SEM of experimental Ti-disks treated with simulated body fluid for 14 days: (A) Ti-without PS-control (×100); (B) Ti-with PS (×100); (C) Ti-with PS (×1500).

6. 4. Conclusion

The purpose of this research was to investigate if Ti surfaces modified with the calcium phosphate complex of different natural phospholipids induce a substantial enhancement in osteoblast differentiation and growth from their progenitor cells in culture, as compared to non-coated surface. Based on the data and results obtained in this study, it can be concluded that the variation in polar head group of the phospholipid part of Ca-PL-PO₄, imparts a pronounced effect on osteoblast differentiation and growth. Ti surfaces coated with Ca-PS-PO₄ exhibited enhanced alkaline phosphatase specific activity compared to uncoated surface. This indicates that Ca-PS-PO₄-coated Ti surface induced better osteoblast differentiation from its progenitor cells than the uncoated Ti control. The Ti surface coated with Ca-PS-PO₄ registered a 150% increment in the specific alkaline phosphatase activity compared to control uncoated Ti surface after day 14. Further, the mineralization of coated surface when immersed in simulated body fluid and the presence of calcium and phosphate ions in the mineralized surface, leads to postulations that the Ti implants coated with the same materials will be able to form hydroxyapatite nodules and will be able to help in osseointegration of implant. These results indicate the possible usefulness of Ca-PS-PO₄-coated Ti surfaces for inducing enhanced bone formation and are very encouraging for bone and dental implantology.

7. Acknowledgements

The authors are very thankful to National Institutes of Health, National Institute for Dental and Cranofacial Research for financial supported to carry out this research (NIH/NIDCR grant# 1 R43 DE13996-01A1).

8. References

1. W.R. Lacefield, Hydroxyapatite coating. Ann NY Acad Sci 523 (1988), pp. 72-80. Abstract-MEDLINE  

2. L.L. Hench, Biocamics. J Am Ceram Soc 81 (1998), pp. 1705-1728. Abstract-Compendex  

3. P. Ducheyne, W.V. Raemdonck, J.C. Heughebaert and M. Heughebaert, Structural analysis of hydroxyapatite coating on titanium. Biomaterials 7 (1986), pp. 97-103. Abstract

4. K. de Groot, R. Geesink, C.P.A.T. Klein and P. Selekion, Plasma sprayed coating of hydroxyaptite. J Biomed Mater Res 21 (1987), pp. 1375-1381. Abstract-MEDLINE | Abstract-Compendex  

5. R.K. Singh, F. Qian, V. Ngaushnam, R. Damodaran and B.M. Moudgil, Excimer laser deposition of hydroxyapatite thin films. Biomaterials 15 (1994), pp. 522-528. Abstract

6. C.M. Cotell, D.B. Chrisey, K.S. Graowski and J.A. Spreue, Pulsed laser deposition of hydroxyapatite thin films on Ti-6A1-4V. J Appl Biomater 8 (1992), pp. 87-92.

7. Y. Ohtsuka, M. Matsuura, M. Chida N Yoshinari, T. Sumii and T. Derand, Formation of hydroxyapatite coating on pure titanium substrates by ion beam dynamic mixing. Surface Coat Technol 65 (1994), pp. 224-230. Abstract-Compendex  

8. J.L. Ong, L.C. Lucas, W.R. Lacefield and E.D. Rigney, Structure, solubility and bond strength of thin calcium phosphate coatings produced by ion beam sputter deposition. Biomaterials 13 (1992), pp. 249-254. Abstract

9. J.G.C. Wolke, K. van Dijk, H.G. Schaeken, K. de Groot and J.A. Jansen, Study of the surface characteristics of magnetron-sputter calcium phosphate coatings. J Biomed Mater Res 28 (1994), pp. 1477-1484. Abstract-MEDLINE | Abstract-Compendex | Abstract-EMBASE  

10. H. Hero, H. Wie, R.B. Jorgensen and I.E. Ruyter, Hydroxyapatite coatings on Ti produced by hot isostatic pressing. J Biomed Mater Res 28 (1994), pp. 343-348. Abstract-EMBASE | Abstract-Compendex  

11. I. Zhitomirsky and L. Gal-Or, Electrophortic deposition of hydroxyapatite. J Mater Sci Mater Med 8 (1997), pp. 213-219. Abstract-Compendex   | Full Text via CrossRef

12. W. Weng and J.L. Baptista, Sol-gel drived porous hydroxyapatite coatings. J Mater Sci Mater Med 9 (1998), pp. 159-163. Abstract-Compendex | Abstract-EMBASE   | Full Text via CrossRef

13. E. Wieser, I. Tsyganov, W. Matz, H. Reuther, S. Oswald, T. Pham and E. Richter, Modification of titanium by ion implantation of calcium and or phosphorus. Surf Coating Technol 111 (1999), pp. 103-109. SummaryPlus | Full Text + Links | PDF (387 K)

14. T. Kokubo, F. Miyaji, H.M. Kim and T. Nakamura, Spontaneous apatite formation on chemically treated titanium metals. J Am Ceram Soc 79 (1996), pp. 1127-1129. Abstract-Compendex | Abstract-INSPEC  

15. J. Redepending and J.P. McIsaac, Electrocrystallization of rushite coatings on prosthetic alloys. Chem Mater 2 (1990), pp. 625-627.

16. M. Shirkhanzadeh, Bioactive calcium phosphate coatings prepared by electrodeposition. J Mater Sci Lett 10 (1991), pp. 1415-1417. Abstract-INSPEC  

17. P. Royer and C. Rey, Calcium phosphate coatings for orthopaedic prosthesis. Surf Coat Technol 45 (1991), pp. 171-177. Abstract

18. S. Ban and S. Maruno, Deposition of calcium phosphate on titanium by electrochemical process in simulated body fluid. Jpn J Appl Phys 32 10B (1993), pp. L1577-L1580. Abstract-INSPEC  

19. M.J. Filiaggi, N.A. Coombs and R.M. Pilliar, Characterization of the interface in plasma-sprayed HA coating/Ti-6A1-4V implant system. J Biomed Mater Res 25 (1991), pp. 1211-1229. Abstract-MEDLINE | Abstract-EMBASE | Abstract-Compendex  

20. V. Palka, E. Postrkova and H.K. Koerten, Some characteristics of hydroxylapatite powders after plasma spraying. Biomaterials 19 (1998), pp. 1763-1772. Abstract | PDF (1301 K)

21. L.G. Ellies, D.G. Nelson and J.D. Featherstone, Crystallographic changes in calcium phosphates during plasma-spraying. Biomaterials 13 (1992), pp. 313-316. Abstract

22. S.A. Bender, J.D. Bumgardner, M.D. Roach, K. Bessho and J.L. Ong, Effect of protein on the dissolution of HA coatings. Biomaterials 21 (2000), pp. 299-305. SummaryPlus | Full Text + Links | PDF (322 K)

23. A.L. Boskey, Models for matrix vesicle calcification. Inorg Perspect Biomed 2 (1980), pp. 51-92.

24. Wuthier RE. In: Anghilery LJ, Tuffet-Anghilery AM, editors. The role of phospholipid calcium phosphate complexes in biological mineralization. The role of calcium in biological system, vol. I. Boca Raton, FL: CRC Press; 1982. p. 41-63.

25. B.D. Boyan-Sayler and A.L. Boskey, Relationship between proteolipids and calcium phospholipid phosphate complexes in bacterionema matrucotti calcification. Calcif Tissue Internat 30 (1980), pp. 167-174.

26. N. Vyavahare, D. Hirsch, E. Lerner, J.Z. Baskin, F.J. Schoen, R. Bianco, H.S. Kruth, R. Zand and R.J. Levy, Prevention of bioprosthetic Heart valve calcification by ethanol preincubation. Circulation 95 (1997), pp. 479-488. Abstract-MEDLINE | Abstract-EMBASE  

27. L.L. Demer, Lipid hypothesis of cardiovascular calcification. Circulation 95 (1997), pp. 297-298. Abstract-MEDLINE | Abstract-EMBASE  

28. A.L. Boskey and A.S. Posner, Optimal condition for Ca-acidic phospholipid-PO₄ formation. Calcif Tissue Internat 34 (1982), pp. S1-S7. Abstract-MEDLINE  

29. M.J. Glimcher, Mechanism of calcification: role of collagen fibrils and collagen-phospho-protein complexes in vitro and in vivo. Anat Rec 224 (1989), pp. 139-153. Abstract-MEDLINE | Abstract-EMBASE  

30. R.S. Thies, M. Bauduy, B.A. Ashton, L. Kurtzberg, J.M. Wozney and V. Rosen, Recombinant human bone morphogenetic protein-2 induces osteoblastic differentiation in W-20-17 stromal cells. Endocrinology 130 (1992), pp. 1318-1324. Abstract-MEDLINE | Abstract-EMBASE   | Full Text via CrossRef

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