Introduction: Extensor mechanism reconstruction following massive endoprosthetic replacement for osteosarcomas of the proximal tibia is dependent on functional attachment of the patellar tendon[1]. This is usually accomplished by suturing the tendon into muscle but ideally reattachment back onto the metallic implant surface would be more efficient. Successful re-attachment of the tendon back onto a metallic surface would involve the regeneration of an enthesis that consisting of an interface, of fibrocartilage, mineralised fibrocartilage and bone. HA (Hydroxyapatite) and Silicate-substituted HA (Si-HA) coatings enhance MSC and osteoblast activity, and promote osteogenic differentiation. Tenocytes and tendon stem cells (TSCs) present within the tendon[2] may have a crucial role in the regeneration of the enthesis, and this may be associated with the differentiation and trans-differentiation of these cells. This study hypothesised that HA and Si-HA coatings will promote tendon cell proliferation and TSC osteogenic differentiation in vitro.
Methods: Sandblasted titanium discs were electrochemically coated with HA[3] and Si-HA[4]. Coatings were analysed by light microscopy and scanning electron microscopy. Surface roughness (Ra) of the substrates was measured using 3D optical profilometry. Tendon cells harvested from an ovine tendon were grown on these surfaces. AlamarBlue®, deoxyribonucleic acid and alkaline phosphatase (ALP) assays were performed at 3, 7 and 14 days.
Results: HA and Si-HA coatings were produced using 10 mA/cm2 15min and 27 mA/cm2 60min, respectively. The uncoated roughened surface had the lowest Ra (4891nm, 95% CI 4830-4930) compared with HA (5032nm, 95% CI 4844-5348) and Si-HA (6371nm, 95% CI 5014-6969) substrates. The Ra on Si-HA was significantly higher (p=0.009) than on sand blasted discs. At day 14, AlamarBlue® activity normalised using DNA measurements on roughened and HA surfaces was significantly higher than on Si-HA (p<0.05 in both cases). Osteogenic differentiation did not occur on any of the substrates. ALP activity progressively declined from day 3 to 14.
Figure 1 Comparing alamarBlue® activity normalised per μg of DNA of tendon cells cultured on roughened, HA and SiHA substrates at each time point.
Figure 2 Comparing ALP activity normalised per μg of DNA of tendon cells cultured on roughened, HA and SiHA substrates at each time point.
Discussion: The lack of osteogenic differentiation may be assoacited with immutable tenocytes and the lack of TSCs. While HA and Si-HA coatings are osteogenic for MSCs and osteoblasts, they may not be osteogenic for tendon cells. The coating technique altered the surface chemistry and increased surface roughness. The tendon cells detected small changes in surface roughness and proliferated better on the less rough substrates.
Conclusions: Alterations in surface characteristics when developing tendon-tissue engineered constructs should be considered before seeding cells. Whilst electrochemically deposited HA and Si-HA have potential to be used in musculoskeletal repair the lack of stem cells in the cultures we used may be associated with the failure of the cells to differentiate along the osteogenic lineage.
References:
[1] Oddy, M. J., Pendegrass, C. J., Goodship, A. E., Cannon, S. R., Briggs, T. W. & Blunn, G. W. 2005. Extensor mechanism reconstruction after proximal tibial replacement. J Bone Joint Surg Br, 87, 873-8.
[2] Bi, Y., Ehirchiou, D., Kilts, T. M., Inkson, C. A., Embree, M. C., Sonoyama, W., Li, L., Leet, A. I., Seo, B. M., Zhang, L., Shi, S. & Young, M. F. 2007. Identification of tendon stem/progenitor cells and the role of the extracellular matrix in their niche. Nat Med, 13, 1219-27.
[3] Redepenning, J., Schlessinger, T., Burnham, S., Lippiello, L. & Miyano, J. 1996. Characterization of electrolytically prepared brushite and hydroxyapatite coatings on orthopedic alloys. J Biomed Mater Res, 30, 287-94.
[4] Li, D. H., Lin, J., Lin, D. Y. & Wang, X. X. 2011. Synthesized silicon-substituted hydroxyapatite coating on titanium substrate by electrochemical deposition. J Mater Sci Mater Med, 22, 1205-11.