Enhanced bone formation by controlled growth factor delivery from chitosan-based biomaterials

https://doi.org/10.1016/S0168-3659(01)00498-9Get rights and content

Abstract

For the purpose of obtaining high bone forming efficacy, development of chitosan was attempted as a tool useful as a scaffolding device. Porous chitosan matrices, chitosan–poly(l-lactide) (PLLA) composite matrices and chitosan coated on PLLA matrices were dealt with in this research. Porous chitosan matrix was fabricated by freeze-drying and cross-linking aqueous chitosan solution. Porous chitosan matrix combined with ceramics and constituents of extracellular matrices were prepared and examined for their bone regenerative potential. Composite porous matrix of chitosan–PLLA was prepared by mixing polylactide with chitosan and freeze-drying. All chitosan based devices demonstrated improved bone forming capacity by increasing mechanical stability and biocompatibility. Release of platelet-derived growth factor-BB (PDGF-BB) from these matrices exerted significant osteoinductive effect in addition to the high osteoconducting capacity of the porous chitosan matrices. The hydrophobic surface of PLLA matrices was modified by chitosan to enhance cell affinity and wettability. The chitosan coated PLLA matrix induced increased osteoblast attachment as compared with intact PLLA surface. Overall results in this study demonstrated the usefulness of chitosan as drug releasing scaffolds and as modification tools for currently used biomaterials to enhance tissue regeneration efficacy. These results may expand the feasibility of combinative strategy of controlled local drug delivery concept and tissue engineered bone formation in reconstructive therapy in the field of periodontics, orthopedics and plastic surgery.

Introduction

Recently, much attention has been given to utilize chitosan in biomedical applications, for example, a wound healing agent, bandage material, skin grafting template, hemostatic agent, hemodialysis membrane and drug delivery vehicle [1], [2], [3], [4], [5], [6]. Chitosan has been applied to conduct the extracellular matrix (ECM) formation in tissue regenerative therapy [7], [8], [9], [10], [11]. The superior tissue compatibility of chitosan may primarily be attributed to its structural similarity to glycosaminoglycan in ECM [8], [9]. Chitosan has been reported to stimulate the activity of growth factors [12], [13], [14]. In vitro studies have clarified the contribution of chitosan in wound healing through its activation of fibrogenic mediators such as growth factors. Increased expression of growth factors enhanced fibroblastic activity and promoted fibrous tissue synthesis.

As bone-forming materials, bone powder [15], [16], ceramics [17], [18] and synthetic polymers [19], [20], [21] have demonstrated effective bone regenerative potency. However, drawbacks of these materials may include disease transmission, inappropriate biodegradation, immune response, low tissue compatibility and poor adaptation. A significant benefit in using chitosan may be that its degradation product is neutral to weak-base sugars. Currently used poly(α-hydroxy acids) generate acidic degradation byproducts at the implanted site which evokes undesirable tissue reaction [22], [23], [24]. The acid by-product may lead to local disturbance due to poor vascularization in the surrounding tissue. Chitosan may be combined with acid-producing biodegradable polymers, so that local toxicity due to the acid byproducts can be alleviated. Chitosan can be utilized in combination with other inorganic ceramics, and bioactive polymers to further enhance tissue regenerative efficacy. Surface coating of common biomaterials using chitosan may enhance both cell adhesion/differentiation and tissue compatibility while maintaining their original physical properties. Recently, an attempt has been made to use chitosan as a gene carrier based on its ionic binding affinity with DNA [25], [26]. For this purpose, functional groups of chitosan may be modified in terms of its charge density and solubility to effectively control formation and dissociation of the chitosan–DNA complex [27].

In this paper, porous chitosan matrices were designed as scaffolds for tissue-engineered bone formation. Additionally, composite matrices of polylactide and chitosan were also prepared to improve mechanical stability while maintaining cell affinity of chitosan. Porous chitosan matrices combined with ceramics and ECM constituent, i.e. chondroitin sulfate were designed and examined for their bone regenerative potential including the release controlling capacity of growth factor, supportive activity of cellular proliferation and differentiation, and bone forming efficacy in case of bone defect. Surface modification of polylactide with chitosan was also attempted. The hydrophobic surface of polylactide matrices may be altered by chitosan coating to enhance wettability and cell affinity.

Chitosan itself is not sufficient to induce rapid bone regeneration at initial status of bone healing. Incorporation of growth factors may be highly beneficial to improve bone forming efficacy. PDGF-BB has demonstrated periodontal tissue regeneration, however, there are limitations in maintaining therapeutic concentrations from injection due to its short halflife in vivo [28], [29], [30], [31], [32], [33]. Carrier systems are required to maintain therapeutic levels of PDGF-BB (1–10 ng/ml) at wound sites for healing periods of up to 4 weeks [28], [29], [30]. Controlled release of growth factors from porous chitosan matrices and PLLA–chitosan composite matrices as scaffolds may be highly effective to enhance bone formation. By releasing growth factors in desirable kinetics, locally implanted chitosan matrices and PLLA–chitosan composite matrices can serve as both drug delivery devices and as physical scaffolding devices, which may shorten the therapeutic period. PDGF-BB release kinetics, osteoblasts proliferation, and in vivo bone formation will be presented in this paper.

Section snippets

Materials

Chitosan (70% deacetylated) was purchased from Showa Chemicals (Osaka, Japan). Chondroitin-4-sulfate (CS) and tetracycline (TC) were obtained from Sigma–Aldrich (St. Louis, MO, USA). Tricalcium phosphate (TCP) and sodium tripolyphosphate (TPP) was purchased from Showa Chemicals. PDGF-BB and 125I-labelled PDGF-BB were purchased from Genzyme (Cambridge, MA, USA) and Amersham (Buckinghamshire, UK), respectively. Collagenase, β-glycerol phosphate, l-ascorbic acid were obtained from Sigma–Aldrich.

Morphology of porous matrices

The porous chitosan matrix showed a three-dimensional porous structure with a pore size of 100–150 μm, and an anastomosing network within its structure (Fig. 1A). Osteoblast proliferation and function was enhanced in three-dimensional culture with spongeous matrices having a pore diameter above 100 μm [37]. Incorporation of CS increased the porosity of the matrix (150–200 μm pore size of both surface and sublayer). Ionic interaction between CS and chitosan led to coprecipitation and,

Conclusion

Porous chitosan matrix and PLLA–chitosan composite porous matrix were developed as bone substitutes and tissue engineering scaffolds. Controlled release of PDGF-BB from chitosan-based scaffolds significantly promoted bone healing and regeneration. This study suggested that chitosan could be utilized as a base material for scaffold devices and as modification tools for currently used biomedical devices in improving tissue regeneration efficacy. Also, these results can expand the feasibility of

Acknowledgements

This work was supported by the grant of the Ministry of Science and Technology, no. 98-N1-02-01-A-13 and IBEC, South Korea.

References (45)

  • Y.K. Park et al.

    Galactosylated chitosan–graft-dextran as hepatocyte-targeting DNA carrier

    J. Controlled Release

    (2000)
  • Y.J. Park et al.

    Controlled release of platelet-derived growth factor-BB from chondroitin sulfate–porous chitosan matrix for guided bone regeneration

    J. Controlled Release

    (2000)
  • D. Tziafas et al.

    Effects of glycosaminoglycans on in vitro mouse dental cells

    Arch. Oral Biol.

    (1988)
  • T. Chandy et al.

    Chitosan — as a biomaterials

    Biomater. Artif. Cell Artif. Org.

    (1990)
  • R.A.A. Muzarelli

    Biochemical significance of exogenous chitins and chitosans in animals and patients

    Carbohydr. Polym.

    (1993)
  • V.R. Patel et al.

    Preparation and characterization of freeze-dried chitosan–poly(ethylene oxide) hydrogels for site-specific antibiotic delivery in the stomach

    Pharm. Res.

    (1996)
  • J.O. Hollinger et al.

    Biodegradable bone repair materials: synthetic polymers and ceramics

    Clin. Orthop.

    (1986)
  • L.G. Cima et al.

    Tissue engineering by cell transplantation using degradable polymer substitutes

    J. Biomech. Eng.

    (1991)
  • C.T. Laurencin et al.

    A highly porous 3-dimensional polyphosphazene polymer matrix for skeletal tissue regeneration

    J. Biomed. Mater. Res.

    (1996)
  • Y. Usami et al.

    Chitin and chitosan induce migration of bovine polymorphonuclear cells

    J. Vet. Med. Sci.

    (1994)
  • B. Peltzman et al.

    Treatment of furcation involvements with fibronectin and intraoral autogenous bone grafts: preliminary observation

    Int. J. Periodont. Restorative Dent.

    (1998)
  • W.W. Sepe et al.

    Clinical evaluation of freeze-dried bone allografts in periodontal osseous defects. Part II

    J. Periodontol.

    (1978)
  • Cited by (0)

    View full text