Elsevier

The Spine Journal

Volume 12, Issue 3, March 2012, Pages 265-272
The Spine Journal

Basic Science
Osteoblasts exhibit a more differentiated phenotype and increased bone morphogenetic protein production on titanium alloy substrates than on poly-ether-ether-ketone

https://doi.org/10.1016/j.spinee.2012.02.002Get rights and content

Abstract

Background context

Multiple biomaterials are clinically available to spine surgeons for performing interbody fusion. Poly-ether-ether-ketone (PEEK) is used frequently for lumbar spine interbody fusion, but alternative materials are also used, including titanium (Ti) alloys. Previously, we showed that osteoblasts exhibit a more differentiated phenotype when grown on machined or grit-blasted titanium aluminum vanadium (Ti6Al4V) alloys with micron-scale roughened surfaces than when grown on smoother Ti6Al4V surfaces or on tissue culture polystyrene (TCPS). We hypothesized that osteoblasts cultured on rough Ti alloy substrates would present a more mature osteoblast phenotype than cells cultured on PEEK, suggesting that textured Ti6Al4V implants may provide a more osteogenic surface for interbody fusion devices.

Purpose

The aim of the present study was to compare osteoblast response to smooth Ti6Al4V (sTiAlV) and roughened Ti6Al4V (rTiAlV) with their response to PEEK with respect to differentiation and production of factors associated with osteogenesis.

Study design

This in vitro study compared the phenotype of human MG63 osteoblast-like cells cultured on PEEK, sTiAlV, or rTiAlV surfaces and their production of bone morphogenetic proteins (BMPs).

Methods

Surface properties of PEEK, sTiAlV, and rTiAlV discs were determined. Human MG63 cells were grown on TCPS and the discs. Confluent cultures were harvested, and cell number, alkaline phosphatase–specific activity, and osteocalcin were measured as indicators of osteoblast maturation. Expression of messenger RNA (mRNA) for BMP2 and BMP4 was measured by real-time polymerase chain reaction. Levels of BMP2, BMP4, and BMP7 proteins were also measured in the conditioned media of the cell cultures.

Results

Although roughness measurements for sTiAlV (Sa=0.09±0.01), PEEK (Sa=0.43±0.07), and rTiAlV (Sa=1.81±0.51) varied, substrates had similar contact angles, indicating comparable wettability. Cell morphology differed depending on the surface. Cells cultured on Ti6Al4V had lower cell number and increased alkaline phosphatase specific activity, osteocalcin, BMP2, BMP4, and BMP7 levels in comparison to PEEK. In particular, roughness significantly increased the mRNA levels of BMP2 and BMP4 and secreted levels of BMP4.

Conclusions

These data demonstrate that rTiAlV substrates increase osteoblast maturation and produce an osteogenic environment that contains BMP2, BMP4, and BMP7. The results show that modifying surface structure is sufficient to create an osteogenic environment without addition of exogenous factors, which may induce better and faster bone during interbody fusion.

Introduction

Currently, spine surgeons have multiple biomaterial choices when performing an interbody fusion. Recently, poly-ether-ether-ketone (PEEK) has gained significant popularity as the biomaterial of choice for interbody fusion, particularly in the lumbar spine because of its radiolucency and reports that it has a modulus similar to that of bone [1], [2], [3]. However, PEEK does not integrate well with the surrounding bone and may form a fibrous connective interface [3], [4], [5]. As a result, micromotion is possible, eventually leading to implant failure [6], [7].

Implant osseointegration, or direct contact between the implant surface and surrounding bone under loading conditions [8], [9], depends on both bone quality and the host environment. Osseointegration is slower in osteopenic bone than in normal bone [10] and has been shown to be 50% slower in osteoporotic animals than in normal animals [11], [12]. Thus, it is important that spinal fusion devices present an osteogenic surface during the fusion process.

Titanium aluminum vanadium (Ti6Al4V) alloys have a well-established history of use as bone graft cages or spacers in lumbar spine fusion procedures. Previous studies have shown that these alloys support good bone to implant contact and are well osseointegrated with the surrounding bone [13], [14], [15]. In vitro experiments comparing the responses of immature osteoblasts to machined and smooth Ti6Al4V (sTiAlV) substrate surfaces indicate that the differentiation of the cells is greater when the surface has a texture with micron-scale roughness [16]. These observations were confirmed using Ti6Al4V that had been grit blasted to create micron-scale roughness [17]. Moreover, when the same surface treatment was applied to Ti6Al4V pedicle screws and tested in vivo in sheep spines, the force required to pull out the screws was doubled compared with screws that had a smooth surface [17].

In the body, osteoblasts mature in osteoclast-conditioned areas of bone that present a micron-scale roughness [18], suggesting that surface texture is an important variable in bone formation. Studies using commercially pure titanium (Ti) substrates have shown that surfaces with micron- and submicron-scale features promote greater osteoblast differentiation, matrix deposition, and production of osteogenic growth factors [19], [20], [21], which regulate the cells via autocrine and paracrine pathways [22], [23], [24], than do cells cultured on smooth surfaces. Similarly, microtextured Ti6Al4V surfaces support increased osteoblastic differentiation compared with sTiAlV surfaces [17]. Moreover, cells on Ti or Ti6Al4V are more differentiated than cells on traditional cell culture plastic [16], [25], [26]. These differences indicate that both surface chemistry and surface microtexture play a role and bring into question whether responses to materials typically used in interbody fusion, Ti6Al4V or PEEK, differ and if so, how.

The purpose of the present study was to compare the osteoblast phenotype of human osteoblast-like MG63 cells to smooth and microtextured Ti6Al4V surfaces with their phenotype on PEEK. MG63 cells are an immature osteoblast cell line used by many laboratories as a model to examine factors that promote osteoblast differentiation [27], [28]. Of particular interest is whether cells grown on these biomaterials contribute to peri-implant bone formation by generating an osteogenic environment through production of osteoinductive factors. To test this, we assessed whether expression of bone morphogenetic proteins (BMPs) and their secretion into the medium were affected by the substrate surface. Because of the high doses used to induce bone formation and side effects derived from the clinical use of BMP2 for spine fusion [29], [30], [31], implant topographies that enhance cell-produced BMPs may enhance the osteogenic microenvironment and improve the stability of the interbody construct through bony on-growth to the interbody device [32].

Section snippets

Disc preparation and characterization

Surgical-grade Ti alloy (Ti6Al4V) and PEEK discs were used in this study (Titan Spine, LLC., Mequon, WI, USA). The discs were 15 mm in diameter and fit snuggly in a well of a 24-well culture plate. Smooth Ti6Al4V discs were machined, tumbled to remove any burs, and passivated through an acid bath, which removes inorganic contaminants on the surface and forms a stable oxide layer that reduces the reactivity of the bulk material with the environment. To create a roughened surface texture

Materials characterization

Scanning electron microscopic images of the surfaces revealed different topographies of the samples (Fig. 1). The PEEK surface exhibited a machined surface finish with no distinct features except for parallel grooves along the entire surface because of processing (Fig. 1A). The high magnification images confirm the lack of smaller features (Fig. 1B). In a similar way, the sTiAlV surface also presented a machined finish with shallower grooves (Fig. 1C) and additional random scratches, evident at

Discussion

Surface properties of implants have been recognized as one of the most important determinants of device success [36]. Scanning electron microscope analyses of the different samples showed that both PEEK and sTiAlV samples were relatively smooth at the micron and submicron levels when compared with rTiAlV samples. These results were confirmed quantitatively by CLM measurements, with rTiAlV samples having a significantly higher average roughness (Sa) than PEEK and sTiAlV samples. Increased

Conclusions

Taken together, this study demonstrates that rTiAlV substrates increase osteoblast maturation and produce an osteogenic environment that contains BMP2, BMP4, and BMP7. The results show that modifying surface structure is sufficient to create an osteogenic environment that could enhance bone formation and implant stability, without addition of exogenous growth factors.

Acknowledgments

This research was supported by National Institutes of Health grant AR052102. Ti6Al4V and PEEK substrates were provided as gifts by Titan Spine, LLC.

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  • Cited by (0)

    FDA device/drug status: Not applicable.

    Author disclosures: RON: Nothing to disclose. RAG: Nothing to disclose. JMS: Stock Ownership: Titan Spine, LLC (4,000 shares, 1%). SLH: Nothing to disclose. DAH: Nothing to disclose. PFU: Royalties: Titan Spine (D); Stock Ownership: Titan Spine (45,000,000 shares, 33%); Trips/Travel: Titan Spine (Nonfinancial); Board of Directors: Titan Spine (Nonfinancial). ZS: Royalties: University of Texas (B); Stock Ownership: SpherIngenics (15%); Research Support (Staff/Materials): MTF ITI (E, Paid directly to institution/employer); Grants: Titan Spine (E, Paid directly to institution/employer). BDB: Stock Ownership: Carticept Medical, Inc. (150,000 shares), SpherIngenics, Inc. (35% ownership), ArthroCare, Inc. (15,000 shares); Private Investments: MedShape Solutions, Inc. (5,000 shares); Consulting: Musculoskeletal Transplant Foundation (Financial and Nonfinancial), Exactech (Financial); Trips/Travel: Titan Spine (A), MTF (A); Board of Directors: Carticept Medical, Inc. (Nonfinancial), SpherIngenics, Inc (Nonfinancial); Scientific Advisory Board: Exactech (A); Research Support (Staff/Materials): MTF (A, Paid directly to institution/employer), Titan Spine (D, Paid directly to institution/employer); Grants: ITI Foundation (F, Paid directly to institution/employer).

    The disclosure key can be found on the Table of Contents and at www.TheSpineJournalOnline.com.

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