Elsevier

Biomaterials

Volume 26, Issue 9, March 2005, Pages 971-977
Biomaterials

Effect of bone extracellular matrix synthesized in vitro on the osteoblastic differentiation of marrow stromal cells

https://doi.org/10.1016/j.biomaterials.2004.04.001Get rights and content

Abstract

Alternative materials for bone grafts are gaining greater importance in dentistry and orthopaedics, as the limitations of conventional methods become more apparent. We are investigating the generation of osteoinductive matrix in vitro by culturing cell/scaffold constructs for tissue engineering applications. The main strategy involves the use of a scaffold composed of titanium (Ti) fibers seeded with progenitor cells. In this study, we investigated the effect of extracellular matrix (ECM) laid down by osteoblastic cells on the differentiation of marrow stromal cells (MSCs) towards osteoblasts. Primary rat MSCs were harvested from bone marrow, cultured in dexamethasone containing medium and seeded directly onto the scaffolds. Constructs were grown in static culture for 12 days and then decellularized by rapid freeze–thaw cycling. Decellularized scaffolds were re-seeded with pre-cultured MSCs at a density of 2.5×105 cells/construct and osteogenicity was determined according to DNA, alkaline phosphatase, calcium and osteopontin analysis. DNA content was higher for cells grown on decellularized scaffolds with a maximum content of about 1.3×106 cells/construct. Calcium was deposited at a greater rate by cells grown on decellularized scaffolds than the constructs with only one seeding on day-16. The Ti/MSC constructs showed negligible calcium content by day-16, compared with 213.2 (±13.6) μg/construct for the Ti/ECM/MSC constructs cultured without any osteogenic supplements after 16 days. These results indicate that bone-like ECM synthesized in vitro can enhance the osteoblastic differentiation of MSCs.

Introduction

Culture of osteoblasts in 3-D is important in developing tissue engineered constructs for regeneration of large bone defects. To date, osteoblasts and osteoprogenitor cells have been cultured on a variety of scaffold materials and their ability to induce bone formation has been examined [1], [2], [3], [4], [5], [6], [7], [8], [9], [10]. While some scaffolds have shown an innate ability to support bone formation during in vitro culture, others have been surface-modified with various molecules to aid their osteoinductive capacities. The most common of these enhancers include molecules present in native bone extracellular matrix (ECM) including hydroxyapatite [4], [6] and collagen type I [11], [12]. Other approaches to enhancing bone formation by cell/scaffold constructs have involved the delivery of certain growth factors that are known to participate in normal osteoblast differentiation including bone morphogenic proteins [3], [12], [13], [14], connective tissue growth factor [15], and transforming growth factors from the TGF-β family [16], [17]. However, an ideal cell/scaffold/signal construct has yet to be developed for use in bone tissue engineering applications.

One approach to development of more osteoinductive materials involves the use of native components of bone ECM to create the scaffold. There has been much focus on the use of bone-derived materials for the 3-D culture of osteoblast type cells, and these can generally be divided into two categories: demineralized bone matrix [3], [18], [19] and ceramics [7], [8], [17], [20], [21], [22]. The first utilizes the organic components of bone ECM while the second utilizes the inorganic components of bone ECM. While both scaffold types have been found to have inherent bone inducing properties, there have been limited studies on the osteoinductive capacity of the combined organic and inorganic bone ECM molecules [2]. We therefore sought to investigate the osteoinductive capacity of a decellularized bone ECM synthesized in vitro on a titanium fiber mesh scaffold. Previously, it has been shown that when rat marrow stromal cells (MSC) are seeded onto titanium fiber mesh and grown in the presence of osteogenic supplements, they will form bone-matrix in vitro [23].

In this study, we investigated the osteoinductive properties of ECM produced by MSCs seeded onto titanium fiber meshes and grown in culture for 12 days. Bone formation on titanium meshes by MSCs has been shown to be successful in the presence of osteogenic supplements, including dexamethasone [2], [3]. We hypothesized that the ECM deposited on the titanium scaffold will induce osteoblastic differentiation of MSCs even in the absence of osteogenic supplements (where osteogenic supplements are defined as β-glycerophosphate and ascorbic acid in the presence of dexamethasone). The aim of this study was to investigate the effect of ECM deposited by osteoblast-like cells on the differentiation of freshly seeded MSCs in static culture. Differentiation of MSCs was determined by analysis of known indicators of the osteoblast phenotype including cell proliferation, production of alkaline phosphatase, secretion of osteopontin, and deposition of a calcified ECM.

Section snippets

Scaffold preparation

The constructs were prepared by die-punching discs from a sheet of titanium (Ti) fiber mesh prepared by Bekaert NV (Zwevegem, Belgium). Scaffolds were 5 mm in diameter and 0.8 mm thick, with fiber size at 20 μm and porosity of 86% according to manufacturer specifications. Scaffolds were sterilized by autoclaving prior to cell seeding.

Marrow stromal cell isolation

MSCs were harvested from the tibiae and femora of 6–8 week old male Wistar rats, weight 150–175 g. Briefly, rats were euthanized using 4% isofluorane in CO2, and the

Results

Fig. 1 illustrates scaffold cellularity. The initial cell number for the Ti/ECM/MSC constructs was higher than expected, however the general trend observed is consistent with the other cultures. With the exception of the Ti/ECM OS group, all groups exhibited an increase in growth, a plateau, then a decrease from day-12 to day-16. The Ti/ECM OS group exhibited a marked decrease in cell number over the entire time period.

Fig. 2 shows the results of ALP activity of the constructs presented on a

Discussion

The aim of this study was to investigate the effect of ECM deposited by osteoblast-like cells on the differentiation of freshly seeded MSCs in static culture. Investigation of the osteoinductive capacity of a decellularized bone ECM yielded results which show that late markers, such as calcium deposition and osteopontin activity, responded positively with increased expression.

Dexamethasone has been shown to direct osteoblastic differentiation of bone MSCs in vitro at both early and late stages

Conclusion

MSCs grown on a decellularized scaffold with a pre-existing ECM exhibited greater cell numbers and calcium deposition, and relative osteopontin levels showed correlation between matrix deposition and detection of the protein. The data indicate that the presence of bone-like ECM deposited on titanium fiber mesh scaffolds can induce osteoblastic differentiation in bone progenitor cells in static culture. The osteoinductive capacity of a decellularized bone ECM was demonstrated by greater calcium

Acknowledgements

This work was supported by a grant from the National Institutes of Health (R01-AR42639) (AGM). Néha Datta also acknowledges support from the Century Scholars Program at Rice University.

References (32)

  • Y. Acil et al.

    Three-dimensional cultivation of human osteoblast-like cells on highly porous natural bone mineral

    J Biomed Mater Res

    (2000)
  • A. Rattner et al.

    Mineralization and alkaline phosphatase activity in collagen lattices populated by human osteoblasts

    Calcif Tissue Int

    (2000)
  • M.A. Attawia et al.

    Proliferation, morphology, and protein expression by osteoblasts cultured on poly(anhydride-co-imides)

    J Biomed Mater Res

    (1999)
  • C. Roehlecke et al.

    Synergistic effect of titanium alloy and collagen type I on cell adhesion, proliferation and differentiation of osteoblast-like cells

    Cells Tissues Organs

    (2001)
  • J.L. Ong et al.

    Osteoblast progenitor cell responses to characterized titanium surfaces in the presence of bone morphogenetic protein-atelopeptide type I collagen in vitro

    J Oral Implantol

    (1999)
  • J. Mahmood et al.

    Geometric effect of matrix upon cell differentiationBMP-induced osteogenesis using a new bioglass with a feasible structure

    J Biochem (Tokyo)

    (2001)
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