Elsevier

Bone

Volume 42, Issue 3, March 2008, Pages 554-564
Bone

Human fetal bone cells associated with ceramic reinforced PLA scaffolds for tissue engineering

https://doi.org/10.1016/j.bone.2007.10.018Get rights and content

Abstract

Fetal bone cells were shown to have an interesting potential for therapeutic use in bone tissue engineering due to their rapid growth rate and their ability to differentiate into mature osteoblasts in vitro. We describe hereafter their capability to promote bone repair in vivo when combined with porous scaffolds based on poly(l-lactic acid) (PLA) obtained by supercritical gas foaming and reinforced with 5 wt.% β-tricalcium phosphate (TCP).

Bone regeneration was assessed by radiography and histology after implantation of PLA/TCP scaffolds alone, seeded with primary fetal bone cells, or coated with demineralized bone matrix. Craniotomy critical size defects and drill defects in the femoral condyle in rats were employed. In the cranial defects, polymer degradation and cortical bone regeneration were studied up to 12 months postoperatively. Complete bone ingrowth was observed after implantation of PLA/TCP constructs seeded with human fetal bone cells. Further tests were conducted in the trabecular neighborhood of femoral condyles, where scaffolds seeded with fetal bone cells also promoted bone repair.

We present here a promising approach for bone tissue engineering using human primary fetal bone cells in combination with porous PLA/TCP structures. Fetal bone cells could be selected regarding osteogenic and immune-related properties, along with their rapid growth, ease of cell banking and associated safety.

Introduction

Fetal bone cells were shown to proliferate, differentiate and finally to mineralize their extra cellular matrix in vitro[1]. For these reasons, we decided to test their association with porous scaffolds for bone repair in vivo.

Ceramic reinforced poly(l-lactic acid) (PLA) structures obtained by melt-extrusion followed by supercritical gas foaming offer suitable conditions for primary human osteoblasts to achieve a full differentiation process in vitro[2]. Moreover, open frames are designed with defined parameters to improve the osteoconductivity, such as porosity, pore size, and connectivity, as well as enhanced mechanical properties [3]. Furthermore, this technique allows producing biocompatible samples without the use of potentially toxic organic solvents [4], [5]. These structures were recently tested in vivo for host tissue induced reactions as well as for their osteoconductive properties [6].

Critical size defect models (CSD) are often used to study orthopaedic materials [7]. A CSD is defined to be the smallest in situ bone wound that would not heal spontaneously by bone formation during the lifespan of the animal [8]. Thus, the CSD created on the bony vault of the cranium (calvaria) represents a severe test for bone graft substitutes. CSDs in the calvaria have been established for different mammalian species [9], [10], [11], [12], [13], [14]. Modelling on parietal bone has been applied on rats from different strains and at various ages [10], [15], [16], [17], [18]. Three months old Sprague–Dawley rats did not spontaneously fill 8 mm diameter lacunae 18 weeks post-surgery [6].

Several animal models are described to test implants in cancellous bone [19], [20], [21] and the femoral condyle is often used for this purpose [22], [23]. Due to its vascularization and the presence of precursor cells, this location is of particular interest to assess in parallel biodegradation and bone repair processes [24].

For cell-based approaches in bone tissue engineering, bone marrow preparations [25], mesenchymal stem cells (MSCs) [26], [27], and osteoprogenitor cells [28] have been used. Recently, fetal bone cells were characterized in vitro to investigate their potential use for tissue engineering [1]. They were shown to be able to proliferate and differentiate into mature osteoblasts in vitro when seeded on PLA/TCP scaffolds [2].

In the present study, we describe the combination of fetal bone cells with PLA/5 wt.% β-tricalcium phosphate (PLA/TCP) implants for cortical and trabecular bone repair. Fetal bone cells were used in their proliferating phase without induction of differentiation to evaluate their osteogenic potential in vivo. In rat cranial defects, cortical bone regeneration was assessed with PLA/TCP implants with and without fetal bone cells as well as with β-tricalcium phosphate matrices (designated as β-TCP Mathys hereafter), corresponding to the composition and structure of chronOS™ scaffolds already used in clinical applications (Mathys, Bettlach, Switzerland). In parallel, both scaffolds were coated with demineralized bone matrix (DBM) to improve their osteoinductivity and to compare them for cortical bone regeneration with scaffolds seeded with cells. To further evaluate the osteoconduction of PLA/TCP implants and the osteogenesis induced by fetal bone cells, PLA/ceramic structures with or without cells were implanted in the metaphyseal trabecular network of rat femoral condyles.

We demonstrate here for the first time, the high potential of human fetal bone cells associated with PLA/ceramic composite structures processed by supercritical gas foaming for their use in cortical and trabecular bone repair.

Section snippets

Cell sources

Human fetal bone cells were obtained from our cell bank comprising eight donors at the end of April 2007. Biopsies were obtained in accordance with the Ethics Committee of the University Hospital in Lausanne (Ethical Protocol 51/01). In this study, bone cells from a fetus of 15 weeks gestational age were used following voluntary interruption of pregnancy. Primary osteoblast cultures were established by rinsing the tissue first with PBS (containing penicillin and streptomycin for washing only).

Scaffold generation and quality control

Porosity and pore size were verified to be in the expected range of 75–90% and 200–500 μm, respectively, for the PLA/TCP scaffold (Fig. 1A) using micro-CT and scanning electron microscopy. The following values were obtained: porosity: 83.4 ± 2.5%; pore diameter: 390 ± 180 μm; modulus: 121.0 ± 12.1 MPa (expected: value: 150 MPa). Furthermore, the β-TCP Mathys scaffolds (Fig. 1C) were examined showing a porosity of 75.4 ± 0.3%, a pore diameter of 410 ± 30 μm and a modulus of 2.70 ± 0.44 GPa.

Coating of the

Discussion

The aim of this study was to test the association of fetal bone cells with ceramic reinforced PLA scaffolds for tissue engineering. As animal models, the CSD craniotomy and the femoral condyle approaches in rats were chosen to follow cortical and trabecular bone repair processes. Key findings were the observation of complete bone bridging 12 months after implantation in skulls of PLA porous structures seeded with human fetal bone cells, and a strong induction of trabecular bone ingrowth in

Summary

The in vivo behaviour of PLA/ceramic foams in combination with human fetal cells was assessed in terms of scaffold resorption as well as cortical and trabecular bone repair. The degradation rate was coupled to the rate of tissue regeneration, leading to a structural integrity of the constructs. The presence of human fetal bone cells did improve the overall healing process when compared to the scaffold material alone. A complete reconstruction of the cranial vault was observed within the porous

Acknowledgments

We thank PD Dr. Brigitte von Rechenberg, Katalin Zlinsky, Käthi Kämpf and Jens Langhoff from the Musculoskeletal Research Unit, Equine Clinic, University of Zürich, Switzerland, for scientific discussions, Veronique Garea from the Laboratory for Regenerative Medicine and Pharmacobiology (EPFL-LMRP), Ecole Polytechnique Fédérale de Lausanne, Switzerland, for assistance during surgery, Sandra Jaccoud from the Laboratory of Biomechanical Orthopedics (EPFL-HOSR), Ecole Polytechnique Fédérale de

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