Elsevier

Biomaterials

Volume 21, Issue 12, June 2000, Pages 1207-1213
Biomaterials

Marrow stromal osteoblast function on a poly(propylene fumarate)/β-tricalcium phosphate biodegradable orthopaedic composite

https://doi.org/10.1016/S0142-9612(99)00254-9Get rights and content

Abstract

The objective of this study was to assess the osteoconductivity of a poly(propylene fumarate)/β-tricalcium phosphate (PPF/β-TCP) composite in vitro. We examined whether primary rat marrow stromal cells would attach, proliferate, and express differentiated osteoblastic function when seeded on PPF/β-TCP substrates. Attachment studies showed that a confluent monolayer of cells had adhered to the substrates within an 8 h time frame for marrow stromal cells seeded at confluent numbers. Proliferation and differentiated function of the cells were then investigated for a period of 4 weeks for an initial seeding density of 42 000 cells/cm2. Rapid proliferation during the first 24 h as determined by 3H-thymidine incorporation was mirrored by an initial rapid increase in total cell number by DNA assay. A lower proliferation rate and a gradual increase in cell number persisted for the remainder of the study, resulting in a final cell number of 128 000 cells/cm2. Differentiated cell function was assessed by measuring alkaline phosphatase (ALP) activity and osteocalcin (OC) production throughout the time course. Both markers of osteoblastic differentiation increased significantly over a 4-week period. By day 28, cells grown on PPF/β-TCP reached a maximal ALP activity of 11 (±1)×10−7 μmol/min/cell, while the OC production reached 40 (±1)×10−6 ng/cell. These data show that a PPF/β-TCP composite exhibits in vitro osteoconductivity similar to or better than that of control tissue culture polystyrene.

Introduction

Current methods to treat skeletal defects focus on biological transplants or synthetic implants. Autograft bone, transferred from one site in the patient's body to another, is limited by the amount of donor bone available. Moreover, its use creates an additional surgical site for the patient, increasing the incidence of further pain and infection. Allograft bone may be a source for disease transfer and is difficult to shape to fill the defect [1]. Synthetic materials, such as poly(methyl methacrylate) bone cement, are permanent implants that may incur complications such as infection or stress shielding leading to bone resorption and requiring revision surgery [2], [3].

With the emergence of the field of tissue engineering, the use of synthetic, biodegradable polymers has gained renowned focus. Biodegradable poly(α-hydroxy esters), such as poly(l-lactic acid), poly(glycolic acid), and poly(dl-lactic-co-glycolic acid) (PLGA), were assayed for osteoblast attachment, proliferation, and migration [4], [5]. Marrow-derived osteoblasts were seen to attach to, migrate upon, and express an osteoblastic phenotype in vitro and in vivo in three-dimensional (3-D) PLGA foams [6], [7], [8]. Although PLGA foams provide an excellent scaffold for 3-D osteoblast culture, they possess mechanical properties inferior to trabecular bone and may not be used at load-bearing sites [9], [10].

In recent studies in our laboratory, we have developed a new injectable, in situ polymerizable, biodegradable orthopaedic material based on poly(propylene fumarate) (PPF) for filling skeletal defects [11]. By combining PPF with a vinyl monomer (N-vinyl pyrrolidone) and an initiator (benzoyl peroxide), an injectable paste can be formulated. This material can be polymerized in situ, filling skeletal defects of any shape or size. Furthermore, the incorporation of solid-phase components β-tricalcium phosphate (β-TCP) and leachable porogen (sodium chloride) can create a porous composite material possessing mechanical properties sufficient for the replacement of human trabecular bone [12]. In addition, PPF/β-TCP scaffolds were shown to maintain the desired mechanical properties over several weeks both in vitro and in vivo [13], [14]. Moreover, by altering this composite formulation, we were able to modulate the handling characteristics of the injectable paste, creating a mixture that hardens within a 5–10 min time span and cures at body temperature. These promising results led us to study PPF/β-TCP further as a potential material for orthopaedic applications.

This paper investigates the osteoconductivity of PPF/β-TCP substrates in vitro and asks whether marrow stromal cells attach, proliferate, and express differentiated osteoblastic function when seeded on PPF/β-TCP substrates.

Section snippets

Materials

Polymer preparation: Propylene glycol, fumaryl chloride, N-vinyl pyrrolidone, potassium carbonate, and benzoyl peroxide were purchased from Acros (Pittsburgh, PA). Fumaryl chloride was purified through distillation at 1 atm with a boiling point range of 161–164°C. Potassium carbonate was ground to a fine powder with mortar and pestle. The remaining chemicals were used as received. Chloroform and petroleum ether for polymer purification were purchased from Fisher (Pittsburgh, PA). Beta-tricalcium

Poly(propylene fumarate)/β-tricalcium phosphate substrate preparation

PPF was synthesized using established techniques described previously [11]. Briefly, a fumaryl chloride (FuCl) solution in chloroform (2 : 1 v/v) was added dropwise to a mixture of propylene glycol (PG), potassium carbonate (K2CO3), and chloroform with 1 : 3 FuCl : PG and 1 : 1.5 FuCl : K2CO3 molar ratios to form a short-chain oligomer. The resulting oligomer solution was centrifuged to remove the K2CO3, and then purified through solution-precipitation in chloroform and petroleum ether. The oligomer was

Results and discussion

The objective of this study was to assess the osteoconductivity of a PPF/β-TCP composite in vitro. We examined whether marrow stromal cells would attach, proliferate, and express differentiated osteoblastic function when seeded on PPF/β-TCP substrates.

To assess cellular attachment, substrates were seeded with a confluent number of cells. Marrow stromal cells attached readily to PPF/β-TCP substrates as compared to TCPS controls (Fig. 1). The cell densities increased significantly (P<0.01) on

Conclusions

Primary rat marrow stromal cells were cultured on a biodegradable PPF/β-TCP composite as a model to assess its osteoconductivity in vitro. The cells were shown to attach to and proliferate on PPF/β-TCP substrates comparable to TCPS controls. The marrow-derived cells exhibited similar or better osteoblastic differentiation on PPF substrates than TCPS controls, as assessed by alkaline phosphatase activity and osteocalcin production over a period of 28 days.

Acknowledgements

This work was completed through funding provided by the National Institutes of Health (R01-AR44381 and R01-DE13031). S.J. Peter acknowledges financial support by the National Institutes of Health Biotechnology Training Grant 5T32GM08362.

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