Elsevier

Biomaterials

Volume 29, Issue 31, November 2008, Pages 4195-4204
Biomaterials

Sequential growth factor delivery from complexed microspheres for bone tissue engineering

https://doi.org/10.1016/j.biomaterials.2008.07.017Get rights and content

Abstract

Aim of the study was to design a 3D tissue-engineering scaffold capable of sequentially delivering two bone morphogenetic proteins (BMP). The novel delivery system consisted of microspheres of polyelectrolyte complexes of poly(4-vinyl pyridine) (P4VN) and alginic acid loaded with the growth factors BMP-2 and BMP-7 which themselves were loaded into the scaffolds constructed of PLGA. Microspheres carrying the growth factors were prepared using polyelectrolyte solutions with different concentrations (4–10%) to control the growth factor release rate. Release kinetics was studied using albumin as the model drug and the populations that release their contents very early and very late in the release study were selected to carry BMP-2 and BMP-7, respectively. Foam porosity changed when the microspheres were loaded. Bone marrow derived stem cells (BMSC) from rats were seeded into these foams. Alkaline phosphatase (ALP) activities were found to be lowest and cell proliferation was highest at all time points with foams carrying both the microsphere populations, regardless of BMP presence. With the present doses used neither BMP-2 nor BMP-7 delivery had any direct effect on proliferation, however, they enhanced osteogenic differentiation. Co-administration of BMP enhanced osteogenic differentiation to a higher degree than with their single administration.

Introduction

Bone defects are caused by a variety of routes including trauma, congenital deformity or pathological deformation. The currently used approaches for the treatment of these defects include use of bone substitutes and grafts [1], [2]. However, certain disadvantages such as donor site morbidity, limited donor tissue availability for autografts and risk of donor pathogen transmission, rejection due immunogenic response have led the scientists to focus on the emerging interdisciplinary field of biomaterials and tissue engineering as an alternative treatment approach. Developing implantable cellular constructs to enable and/or accelerate repair and regeneration of bone tissue at the defect site is a major goal of bone tissue engineering.

Use of mesenchymal stem cells (MSC) in bone tissue engineering is preferred because of reduced immunoreactivity [3], [4], [5], [6], resistance to low oxygen conditions [7], rapid proliferation and, especially, high differentiation potential under the influence of certain bioactive agents [8], [9]. BMP are regulatory molecules involved in skeletal tissue formation during embryogenesis, growth, adulthood, and healing [10] and are known to be very important regulators of proliferation and osteogenic differentiation of the MSC [11] and it was demonstrated that their withdrawal during differentiation cascade can result in the loss of the osteogenic differentiation potential [12]. BMP-2 and BMP-7 are two critical biosignalling molecules with various roles in natural bone regeneration cascade. The proliferative roles of BMP-2 [13], [14], and BMP-7 [15], [16] on bone marrow derived MSC are controversial. However, they have been demonstrated to enhance bone regeneration in various in vitro [15], [16], in vivo [17], [18] and clinical studies [19]. In a specific study BMP-2 when added to the proliferation medium increased the osteogenic activity of MSC [20].

Controlled delivery is the process of delivering certain molecules at a determined rate achieving their prolonged availability in addition to providing protection for the bioactive agent which might otherwise be rapidly metabolized. Since tissue formation and repair is a complex cascade of events in which a number of growth factors are involved, controlled delivery of combinations of growth factors from scaffolds appears to be a logical strategy in mimicking nature in applications such as tissue engineering. This has been employed by various researchers in a number of potential tissue engineering applications such as in the case of simultaneous delivery of insulin-like growth factor-1 (IGF-1) and transforming growth factor-h1 (TGF-h1) for repair of injured cartilage tissue employing the water soluble polymer hydrogels, oligo(poly(ethylene glycol) fumarate) encapsulating gelatin microparticles [21]. Another is the case of sequential delivery of BMP-2 and TGF-β from glutaraldehyde crosslinked gelatin layers for the purpose of tissue engineering [22]. The two major challenges in growth factor delivery from tissue engineering scaffolds are the selection of proper growth factor cocktails and fine control of the relationship between concentration and timing. The significance of enhanced osteogenic effect with multiple growth factors has been well established by a comprehensive study which demonstrated that certain bone morphogenetic proteins with very low or no osteogenic activity exhibited strong osteogenic activity when co-expressed in stem cells [23]. In various studies enhanced bone formation upon co-administration of growth factors was reported. For instance, enhanced bone formation was observed with application of TGF-β1 and IGF-1 simultaneously [24]. Similarly, TGF-β3 and BMP-2 loaded alginate scaffolds had significantly enhanced bone formation in mice in comparison to single growth factor loaded scaffolds [25]. However, as mentioned earlier, selection of the right combinations of growth factors is crucial and they might not always yield positive results; there are cases where decreased bone formation was reported when BMP-2 and bFGF were co-administered from collagen sponges [26]. It has been suggested that multiple growth factor use can also be cost effective by yielding better results with reduced amounts of growth factor utilization when compared to much higher doses of single growth factors required for comparable results [25], [27]. Various strategies are being employed in the design of 3D scaffolds capable of delivering multiple growth factors in a controlled manner and preferentially with different release kinetics because it could be more advantageous in comparison to their simultaneous delivery. This concept was demonstrated with sequential delivery of BMP-2 and IGF-1, respectively, from bilayered gelatin coatings which led to accelerated and enhanced osteogenic differentiation of BMSC in comparison to their simultaneous delivery as well as sequential delivery in the reverse order [22]. Although direct loading into the scaffold structure is a widely used approach in multiple growth factor delivery [26], [28], it does not provide a means to control the relative release rates of the growth factors for a sequential delivery. Alternative strategies to improve this situation can, therefore, be employed such as in the case of bilayered coatings with different release rates from the top and bottom layers [22] or growth factor release from oligo(poly(ethylene glycol) fumarate) hydrogels with a faster release of TGF-β1 directly from gel phase and a slower release of IGF-1 from gelatin microparticles embedded in the gel thus enabling the control over relative timing and concentration of the growth factors in the healing tissue [29]. Delivery from scaffold embedded micro or nanoparticles could overcome the disadvantages of direct delivery from scaffolds which have poor control over release rates due to the open pore structure and exposure of the growth factors to the medium leading to a loss of growth factor bioactivity. It has previously been demonstrated that release kinetics from microsphere-loaded scaffolds could be adjusted by using microspheres prepared under different conditions and attaining different release behaviors [30]. In the same study it was suggested that delivery of multiple growth factors with different release kinetics could be achieved by preparing scaffolds which encapsulate mixed microsphere populations and thus promising precise control over relative release kinetics. The delivery system in the present study was based on such an approach which to the best of our knowledge has not been previously employed for sequential delivery of two different growth factors. Among the emerging approaches is the manufacture of scaffolds from fused microparticles leading to different release behaviors. A recent study has reported fusion of PLGA microsphere populations displaying different release kinetics into 3D scaffolds which can sequentially deliver IGF-1 and TGF-β1 for cartilage tissue engineering [31].

The purpose of this study was to sequentially deliver BMP-2 and BMP-7 via a controlled release system and to investigate in vitro whether it is possible to improve osteogenic activity of MSC. The BMP were entrapped in microspheres of polyelectrolyte complexes of alginic acid and poly(4-vinyl pyridine) (P4VN) in order to achieve the encapsulation under very mild conditions since alginic acid can be easily crosslinked in an aqueous Ca+2 solution thus avoiding the use of organic solvents and any other chemical treatments that may harm and reduce bioactivity of the growth factors. The kinetics of release from the microspheres was studied by microBradford using a model protein, bovine serum albumin (BSA) to represent the growth factors. Growth factor loaded microspheres were then introduced into scaffolds of poly(lactic acid-co-glycolic acid) (PLGA), a FDA approved and widely used scaffold material in bone tissue engineering studies. The microsphere loaded foams were then seeded with bone marrow stem cells. The proliferative and differentiative effects of BMP-2 and BMP-7 on BMSC were studied by Alamar Blue and Alkaline Phosphatase assays, respectively.

Section snippets

Microsphere preparation

Microspheres were prepared by complexation of polyelectrolytes and crosslinking with CaCl2 according to a modified version of an earlier study [32]. P4VN (MW 150,000–200,000, Polysciences, USA) was dissolved in 1:1 dioxane/water (4, 6, 8, 10%, w/v). Alginic acid (Sigma–Aldrich Co., USA) and BSA (Fluka Biochemica, Switzerland) as the model protein for BMP were dissolved in distilled water (4, 6, 8, 10%, w/v). P4VN and Alginate–BSA solutions of the same concentrations were mixed in 3:1 volume

In situ BSA release

An initial burst effect was followed by a much lower BSA release for all types of microspheres. The microspheres prepared from 4% and 6% polymer solutions released all of their content in 10 and 15 days, respectively, (Fig. 1). The 8% and 10% microspheres, on the other hand released only 59% and 41% of their content, respectively, in 7 days and no further release was detected in the following 9 days. This plateau could be due to high levels of retention of BSA in the denser matrices prepared

Conclusion

There was a difference in the pore size distribution profiles of the constructs loaded with single and double microsphere populations. Cell proliferation was highest and total ALP activities were lowest at all time points for foams containing both types of microspheres regardless of BMP presence or absence. When BMP-2 and/or BMP-7 were included in the constructs, proliferation decreased and differentiation increased in comparison to their corresponding BMP-free controls. When the contribution

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