Bone tissue engineering evaluation based on rat calvaria stromal cells cultured on modified PLGA scaffolds

doi:10.1016/j.biomaterials.2005.07.002

Biomaterials

Volume 27, Issue 6, February 2006, Pages 896-904

https://doi.org/10.1016/j.biomaterials.2005.07.002 Get rights and content

Abstract

Using natural materials to coat the scaffolds used for tissue-engineered bone-repair techniques is expected to increase osteoblast adhesion to the scaffold and to express normal physiological function. To test this hypothesis, we therefore modified poly(dl-lactic-co-glycolic acid) (PLGA) substrate by coating it with natural biomaterial solutions of collagen, chitosan, or N-succinyl-chitosan, and then used these three combinations as scaffolds to evaluate their effects on osteoblast attachment, proliferation, and differentiation. The results demonstrated that the pore size of scaffolds ranging from 125–500 μm did not affect the osteoblast phenotype; however, the surface modification of the scaffolds coated with these natural biomaterials did. Collagen increased cell attachment and proliferation, but chitosan and N-succinyl-chitosan decreased them. Chitosan and N-succinyl-chitosan increased differentiation, but collagen decreased it. These results provide us a new strategy for modifying microenvironments to increase osteoblast adhesion, proliferation, and differentiation on PLGA scaffolds, a strategy that might be useful for tissue regeneration.

Introduction

Traditional bone-defect therapies (autograft, allograft, and synthetic materials) have limitations [1], [2]. There are many clinical reasons to develop advanced bone substitutes, for example, more biocompatibility and more-compatible mechanics [3], [4], [5], [6]. Bone tissue engineering is a promising method for repairing bone defects. Recently, culturing donor cells into poly(lactic acid) (PLA), poly(glycolic acid) (PGA), or poly-dl-lactic-co-glycolic acid (PLGA) scaffolds has resulted in the development of in vivo substitutes for bone and cartilage [7], [8], [9], [10], [11], [12], [13]. Although a PLGA scaffold seems to be suitable for tissue engineering, its mechanical strength, small pore size, and hydrophobic surface properties have limited its usage. The surface treatment of a scaffold, therefore, is an important method for achieving good surface characteristics for bone-cell attachment and proliferation.

In a previous study [14], we prepared PLGA scaffolds with a mixture of ammonium bicarbonate and sodium-chloride salt particulate, used as a porogen additive. The polymer scaffolds fabricated using this method demonstrated a highly open, interconnected macroporous structure and better mechanical properties than those made using the standard solvent casting/particle leaching (SC/PL) technique. The good mechanical properties and open pore structure of the PLGA scaffold suggested a promising advantage for bone tissue engineering. However, the hydrophobic surface of PLGA was not adequate for cell attachment and growth. In addition, the relationship between cellular behavior and the pore size of scaffolds prepared using this method needs further study.

Coating a PLGA scaffold with natural material may change its surface properties and improve its physiological function. An extracellular matrix of natural bone tissue is composed of a collagen type I network that deposits calcium phosphate. Such a matrix directly or indirectly influences the regenerated bone cell phenotype and activity. Type I collagen implants have been proved suitable for healing bony defects, which suggests a positive potential for bone tissue engineering in bone regeneration [15], [16]. Chitosan, a copolymer of glucosamine and N-acetylglucosamine, is the deacetylated product of chitin, a ubiquitous biopolymer found in the exoskeleton of insects and marine invertebrates. Chitosan carries positive charges that attract bFGF or VEGF, which help wound healing [17]. Little is known about the utility of chitosan in propagating osteoblasts, however [18], [19], [20], [21]. Negatively charged N-succinyl-chitosan was prepared by acylating amino groups of chitosan with succinic anhydride [22]. The effect of N-succinyl-chitosan on osteoblast growth has not been studied before.

In this study, three different natural biomaterials, collagen, chitosan, and N-succinyl-chitosan, were used to coat PLGA scaffolds with two different pore sizes (125–180 and 300–500 μm). Both uncoated and coated PLGA scaffolds were examined for the effect of the coatings on their water absorption ability and degradation rate, and on the attachment, proliferation, and differentiation of osteoblasts.

Section snippets

Scaffold preparation

PLGA (75:25) was purchased from Aldrich Chemical Company Inc. (Milwaukee, WI). Preparation details were previously described [14]. Briefly, a viscous polymer solution (0.1 g/mL) was prepared by dissolving PLGA polymer in chloroform. Sieved NH₄HCO₃/NaCl (1:1) particulates with two different particle sizes (125–180 and 300–500 μm) were added to the PLGA solution and mixed thoroughly using a glass rod. The weight ratio of salt particulates to polymer was 9:1. The paste mixture of polymer, salts, and

Surface properties of scaffolds

SEM showed that the pore size of the scaffolds was comparable to that of the salt size. Scaffolds with pore sizes of 300–500 and 125–180 μm had a well-interconnected open-pore structure (Fig. 1(a, b) and (c, d), respectively). The scaffold with pore size 300–500 μm was referred as a large-pore scaffold and the one with pore size 125–180 μm was referred as a small-pore scaffold.

The untreated PLGA surface was very smooth (Fig. 1(e)), but the surfaces of the modified scaffolds were rough and had

Discussion

Using SEM, we found that PLGA scaffolds coated with natural biomaterials changed some of their surface characteristics, for example, microstructure, hydrophilic property, and degradation rate. Chitosan and collagen treatment significantly improved the surface hydrophilic property of PLGA scaffolds. The degradation rate was slightly increased in collagen- and N-succinyl-chitosan-treated scaffolds, and was decreased in chitosan-treated scaffolds. It has been reported that water molecules can act

Conclusion

The in vitro degradation of surface-modified PGLA scaffolds demonstrated that chitosan prevented the rapid degradation of PLGA. The pore size of scaffolds did not affect osteoblast behavior, but the surface modification of the scaffold with collagen did modulate osteoblast attachment and proliferation, and modification with chitosan did affect its differentiation. Tissue engineering requires not only the recruitment of appropriate progenitor cells, but also induction of progenitor-cell

Acknowledgements

This work was supported in part by Grant NSC 89-2314-B-006-147 from the National Science Council of Taiwan. We thank Bill Franke for help in manuscript preparation.

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Bone tissue engineering evaluation based on rat calvaria stromal cells cultured on modified PLGA scaffolds

Abstract

Introduction

Section snippets

Scaffold preparation

Surface properties of scaffolds

Discussion

Conclusion

Acknowledgements

Biomaterials

Biochem Biophys Res Commun

J Control Release

Carbohydrate Polym

Life Sci

Biomaterials

Biomaterials

Biomaterials

Donor site pain from the ilium. A complication of lumbar spine fusion

J Bone Joint Surg Br

Morbidity at bone graft donor sites

J Orthop Trauma

Autogenous cortical bone grafts in the reconstruction of segmental skeletal defects

J Bone Joint Surg Am

Bone and cartilage transplantation in orthopaedic surgery. A review

J Bone Joint Surg Am

Bridging large defects in bone by demineralized bone matrix in the form of a powder. A radiographic, histological, and radioisotope-uptake study in rats

J Bone Joint Surg Am

The role of a composite, demineralized bone matrix and bone marrow in the treatment of osseous defects

Orthopedics

Synthetic polymers seeded with chondrocytes provide a template for new cartilage formation

Plast Reconstr Surg

Tissue-engineered growth of bone and cartilage

Transplant Proc