Elsevier

Acta Biomaterialia

Volume 71, 15 April 2018, Pages 184-199
Acta Biomaterialia

Full length article
Localization and promotion of recombinant human bone morphogenetic protein-2 bioactivity on extracellular matrix mimetic chondroitin sulfate-functionalized calcium phosphate cement scaffolds

https://doi.org/10.1016/j.actbio.2018.01.004Get rights and content

Abstract

Localization of recombinant human bone morphogenetic protein-2 (rhBMP-2) with continuous and effective osteogenic stimulation is still a great challenge in the field of bone regeneration. To achieve this aim, rhBMP-2 was tethered on chondroitin sulfate (CS)-functionalized calcium phosphate cement (CPC) scaffolds through specific noncovalent interactions. CS, one of the core glycosaminoglycans, was covalently conjugated onto CPC scaffolds with the assistance of polydopamine (PDA) and further immobilized rhBMP-2 in a biomimetic form. The CPC-PDA-CS scaffolds not only controlled the release kinetics and presentation state of rhBMP-2 but also effectively increased the expression levels of bone morphogenetic protein receptors (BMPRs) and enhanced the recognitions of the remaining rhBMP-2 to BMPRs. Strikingly, the rhBMP-2-loaded CPC-PDA-CS significantly promoted the cellular surface translocation of BMPRs (especially BMPR-IA). In vivo studies demonstrated that, compared with the rhBMP-2 upon CPC and CPC-PDA, the rhBMP-2 upon CPC-PDA-CS exhibited sustained release and induced high quality and more ectopic bone formation. Collectively, these results suggest that rhBMP-2 can be localized within CS-functionalized CPC scaffolds and exert continuous, long-term, and effective osteogenic stimulation. Thus, this work could provide new avenues in mimicking bone extracellular matrix microenvironment and localizing growth factor activity for enhanced bone regeneration.

Statement of Significance

A bioinspired chondroitin sulfate (CS)-functionalized calcium phosphate cement (CPC) platform was developed to tether recombinant human bone morphogenetic protein-2 (rhBMP-2), which could exhibit continuous, long-term, and effective osteogenic stimulation in bone tissue engineering. Compared with rhBMP-2-loaded CPC, the rhBMP-2-loaded CPC-polydopamine-CS scaffolds induced higher expression of bone morphogenetic protein receptors (BMPRs), greater cellular surface translocation of bone morphogenetic protein receptor-IA, higher binding affinity of BMPRs/rhBMP-2, and thus higher activation of the drosophila gene mothers against decapentaplegic protein-1/5/8 (Smad1/5/8) and extracellular-regulated protein kinases-1/2 (ERK1/2) signaling. This work can provide new guidelines for the design of BMP-2-based bioactive materials for bone regeneration.

Introduction

The increasing demand for bone tissue repair remains a significant clinical challenge worldwide. In the realm of bone tissue regeneration, one of the major focuses is sustainable delivery of growth factors with great bioactivity. The bone morphogenetic protein-2 (BMP-2) belongs to the transforming growth factor-β (TGF-β) superfamily; BMP-2 is a strong osteoinductive growth factor and has become an essential component in the fabrication of highly active bone regeneration scaffolds [1], [2], [3]. Unfortunately, even in the US Food and Drug Administration-approved absorbable collagen sponge carrier, owing to low in vivo bioactivity, recombinant human BMP-2 (rhBMP-2) must be delivered at a notably high dose, which leads to the risk of adverse events such as osteoarthritis, sclerosteosis, and inflammatory response [2], [4], [5]. Therefore, it is crucially important to explore, understand, and promote the bioactivity of rhBMP-2 upon orthopedic materials.

During the past decades, physical adsorption and chemical immobilization are commonly used delivery approaches of growth factors [6], [7], [8]. The physical adsorption of rhBMP-2 frequently led to a burst release of the loaded proteins, thus unable to localize the loaded growth factors within the implant region [4], [9]. In the chemical immobilization approaches, although the burst release of loaded growth factors could be eliminated, the chemical linkage can directly decrease the bioactivity of growth factors (e.g., by disabling protein active sites) [3], [10]. Recently, loading growth factors in a biomimetic way is a promising and useful approach to deliver proteins and drugs in tissue engineering. For example, Crouzier et al. showed that cross-linked poly(l-lysine)/hyaluronan layer-by-layer film can serve as a biomimetic reservoir for rhBMP-2 delivery [6]. Lee et al. demonstrated that, by mimicking features of the extracellular environment, fibronectin-mimetic peptide-amphiphile nanofibers can amplify the regenerative capacity of growth factors [11]. Thus, loading rhBMP-2 in a biomimetic form might provide a more favorable platform for delivering rhBMP-2 and realize sustained and continuous osteogenic stimulation to defective sites. As a matter of fact, a few previous investigations have reported delivery of rhBMP-2 through silk fibroin [2], gelatin hydrogel [7], carbon nanotubes [12], poly(lactic-co-glycolic acid)/calcium phosphate cement (CPC) microparticles [13], and heparan sulfate [14]. Our group also has successfully tethered rhBMP-2 upon CPC scaffolds through alendronate/heparin [15]. These approaches reduced the burst release of rhBMP-2 and prolonged half-life of rhBMP-2 to some extent, but the osteoactivity of rhBMP-2 was barely improved (still not satisfying clinical needs). Therefore, it is of urgent need to develop new and more desirable platforms for highly efficient and active immobilization of rhBMP-2.

In recent years, chondroitin sulfate (CS) has gained much more interest owing to its roles in osteoblast maturation, osteoarthritis therapy, and its specific noncovalent interactions with growth factors [16], [17], [18]. For instance, Altgärde et al. validated that, even when CS is covalently immobilized on lipid bilayers, it could exhibit specific noncovalent interactions with extracellular matrix proteins [19]. It was reported that noncovalent interactions between CS and midkine were involved in midkine-induced migration of macrophages [20]. A polyethylene glycol-functionalized CS hydrogel could produce robust adhesive strength with cartilage tissue and minimal inflammatory response in a rat subcutaneous model [21]. These studies imply the possibility of using CS for protein, drug, and gene delivery systems in tissue engineering [22]. More recently, it has been reported that biomaterials that were specifically engineered to mediate growth factor bioactivity might, in turn, regulate cellular response during tissue regeneration [5], [10], [23]. Thus, more researchers endeavor to develop materials that actively control and localize bioactivity of proteins or growth factors through specific noncovalent interactions [24], [25], [26]. Motivated by these studies, it is interesting and meaningful to engineer CS-functionalized scaffolds that can actively mediate immobilization and bioactivity of rhBMP-2 for bone tissue regeneration.

On the basis of this rationale, in the present study, we endeavored to localize and promote rhBMP-2 bioactivity through CS-functionalized CPC scaffolds. Dopamine, similar to the composition of adhesive proteins in mussels, was introduced to self-polymerize on CPC scaffolds [27], [28]. CS was covalently grafted onto the resulting surfaces by adipic acid dihydrazide (ADH), which exhibited superior stability compared to the immobilization produced through physical interactions. The osteogenic bioactivity of rhBMP-2 was examined by alkaline phosphatase (ALP) activity assay, western blot assay, and real-time polymerase chain reaction (RT-PCR) assay. The osteogenic performance of rhBMP-2-loaded scaffolds was validated by using a mouse thigh muscle pouch model. In addition, the underlying mechanism was elucidated by exploring the expression and distribution of bone morphogenetic protein receptors (BMPRs) in bone mesenchymal stem cells (BMSCs) and the recognition of rhBMP-2 to BMPRs.

Section snippets

Materials

CS (extracted from bovine cartilage, molecular weight 10–50 kDa) and p-nitrophenyl phosphate were purchased from Sangon Biotech., Shanghai, China. Escherichia coli-derived rhBMP-2 (carrier-free, >95% purity) was obtained from Shanghai Rebone Biomaterials Co. Ltd., Shanghai, China. Dopamine, N-hydroxysuccinimide (NHS), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), adipic acid dihydrazide (ADH), fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC),

Characterization of CS-ADH

Fig. 1 shows that ADH was covalently conjugated to CS molecules through an amide reaction. It can be found that the side groups of CS were tailored with the chain ends of ADH (Fig. 1A). FTIR spectra (Fig. 1B) revealed that the intensity of ν(Cdouble bondO) at 1633 cm–1, β(Osingle bondH) at 1416 cm–1, ν(Csingle bondO) at 1237 cm–1, and γ(Osingle bondH) at 1064 cm–1 was obviously weakened in the CS-ADH sample, indicating that the amide reaction consumed carboxylic acid groups. In 1H NMR spectra (Fig. 1C), the CS-ADH sample exhibited

Discussion

Among the growth factors involved in bone healing, rhBMP-2 is one of the most potent bone inductors because it participates in different phases of repair [1], [5], [36]. Nevertheless, in vivo administration of rhBMP-2 often suffers from the rapid clearance of growth factors (known as short half-life), which is ascribed to the poor retention of rhBMP-2 by the most current carriers [9], [37], [38]. Apart from short half-life, another serious issue of rhBMP-2 application is that the released

Conclusion

Inspired by the natural extracellular matrix, we covalently tethered CS on CPC scaffolds, endeavoring to actively localize and mediate rhBMP-2 bioactivity through specific noncovalent interactions. The engineered CPC-PDA-CS/rhBMP-2 scaffolds revealed distinguished osteogenic ability in vitro and in vivo, which was ascribed to the localization of rhBMP-2 and the promoted expression and recognition of BMPRs. This study illustrates the mechanism of localizing rhBMP-2 bioactivity at the

Acknowledgements

The authors wish to express their gratitude for the financial support received from the National Natural Science Foundation of China for Innovative Research Groups (No. 51621002) and the National Natural Science Foundation of China (No. 31330028 and No. 31470924). This study was also supported by the 111 project (B14018).

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