The influence of collagen and hyaluronan matrices on the delivery and bioactivity of bone morphogenetic protein-2 and ectopic bone formation
Introduction
Bone morphogenetic protein-2 (BMP-2) is a highly potent osteoinductive agent and a BMP-2 based treatment, INFUSE® Bone Graft, has already been approved by the US Food and Drug Association (FDA) to enhance bone formation in cases of interbody spinal fusion, open tibial fracture and sinus and alveolar ridge bone augmentation [1]. However, in all these applications BMP-2 is required at “supra-physiological” doses to induce osteogenesis [1], [2]. These high doses have contributed to numerous adverse effects, including ectopic bone formation with spinal cord impingement [3], osteoclast activation with transiently elevated bone resorption [4], cyst-like bone void formation and life-threatening cervical swelling. Thus although proven as an effective osteoinducer, these adverse effects, together with the formation of structurally abnormal and mechanically unstable bone tissue, currently limit the overall clinical efficacy of BMP-2.
Supra-physiological doses of exogenous BMP-2 are necessary because of rapid growth factor washout from implants, as well as the short in vivo half-life due to rapid proteolytic degradation. This is compounded by poor release characteristics when bovine collagen sponges are used as a carrier for BMP-2 (INFUSE® Bone Graft) [1], [5]. The collagen biomaterial rapidly releases BMP-2 [6], [7] that in turn initiates an osteogenic fate through recruitment of osteoprogenitor cells [8]. When used to treat bone trauma or disease, this bolus release results in BMP-2 rapidly diffusing away from the implant site before achieving a critical density of newly infiltrating cells [7], so necessitating high doses of BMP-2 to ensure a robust osteogenic affect [1], [9]. In contrast, there is evidence accruing that suggests that a more controlled delivery of BMP-2 can enhance bone regeneration, due to its increased temporal effect on a larger population of osteoprogenitor cells at the fracture site [10], [11], [12], as well as an ancillary role in promoting vasculogenesis [13]. This is corroborated by reports that during normal bone repair, the in situ expression of BMPs (BMP-2, BMP-4, BMP-6 and BMP-7) is up-regulated for longer periods of time, peaking after 21 days [8], [14], [15]. Therefore, the development of improved delivery systems to lower BMP-2 dose requirements through better control of BMP-2 release and bioactivity at the implantation site is an unmet clinical need [7], [16], [17], [18].
Hyaluronic acid (HA) hydrogels are potential candidates for BMP-2 delivery as the structure of hyaluronan is identical in all species, and the subsequent risk of an immune response is small compared to bovine type I collagen materials [19]. Based on its biocompatibility, HA is widely used clinically to treat joint and cartilage diseases, including osteoarthritis [20]. Notably, HA-based hydrogels have been used for delivery of several growth factors, including basic fibroblast growth factor (FGF-2) [21], vascular endothelial growth factor (VEGF) [22] and BMP-2 [23], [24], [25].
We recently reported on two HA-based hydrogel variants for BMP-2 delivery: one containing 0.3% (w/w) thiol-modified heparin relative to thiol-modified HA (Heprasil™) and another containing thiolated HA only (Glycosil™). Except for the presence of covalently immobilized heparin, the chemical composition of both hydrogels was the same. Both hydrogels released bioactive BMP-2 in vitro over 28 days, and were able to induce osteogenesis in an in vivo rat ectopic bone model [26]. In addition, Glycosil, which released BMP-2 with an initial burst followed by a sustained release, promoted significantly more bone formation than Heprasil, which predominantly released BMP-2 in a sustained manner. Thus the greater bone formation with Glycosil appears largely attributable to the higher burst release of BMP-2 that may enhance the recruitment of osteoprogenitor cells into the implant. The lower initial release of BMP-2 from Heprasil might have delayed the recruitment of osteoprogenitor cells. This, together with data from a similar study [12], emphasizes the importance of a burst release of BMP-2 that is followed by a sustained release for efficient bone formation. Despite improvements in BMP-2 carrier systems, there remain significant challenges to their clinical use, especially considering the regulatory issues surrounding growth-factor-based devices. As a result, the bone forming utility of current BMP-2 delivery matrices like collagen need to be more vigorously compared with tunable hydrogel materials to augment the dose-effectiveness of BMP-2. The aim of the present study was to investigate the influence of two matrices having contrasting release profiles on the bioactivity and resultant ectopic bone formation induced by BMP-2. We used a hyaluronon hydrogel that exhibits a low burst followed by a sustained release of BMP-2 and compared it with a collagen matrix approved for BMP-2 delivery.
Section snippets
Materials
All reagents and chemicals were purchased from Sigma (St Louis, MO) unless otherwise stated. Thiol-modified hyaluronan (Glycosil™) and poly(ethylene glycol)-diacrylate were obtained from Glycosan, a division of BioTime Inc. (Alameda, CA). Absorbable collagen sponge (Helistat®) was obtained from Integra LifeSciences Corporation (Plainsboro, NJ). Polycaprolactone (PCL) tubes (4.5 mm inner diameter, 3 mm in height, 1 mm wall thickness) were purchased from Osteopore International (Singapore).
Hydrogel fabrication
The Glycosil hydrogels for this study were prepared with thiolated derivatives of HA. HA is a biocompatible and chemically versatile starting material for preparing clinical-grade hydrogels with a variety of biological and mechanical properties for cell and molecule delivery [29], [30]. These HA hydrogels are synthetic mimics of the extracellular matrix, and have been designed for efficient translation from the laboratory to the clinic [31], [32], [33]. The addition of these thiolated
Discussion
Although the therapeutic effects of BMP-2 are well recognized, an effective means of delivering low-dose BMP-2 is yet to be clinically validated. It is important to understand the ideal release characteristics needed for efficient BMP-2-induced bone formation in order to better develop such carriers. Although collagen-mediated BMP-2 delivery is clinically approved, it is marred by significant adverse effects. In this study, we used a hyaluronan-based hydrogel (Glycosil) that demonstrates a
Conclusions
The results here demonstrate that hyaluronan-based hydrogels and collagen sponge induce bone formation in a non-linear, dose-dependent manner. The study suggests that a release characteristic comprising a sufficient burst followed by a sustained release is more favorable for efficient bone formation than just a high burst and no sustained release. The efficient bone forming ability of Glycosil hydrogels even at lower BMP-2 doses compared to collagen may provide an opportunity to avoid the
Disclosures
G.D.P. declares founders equity in Glycosan and holds stock in BioTime. All other authors confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.
Acknowledgements
The authors would like to acknowledge the funding support of Singapore’s Agency of Science, Technology and Research (A∗STAR) and Institute of Medical Biology (IMB).
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These authors contributed equally to this work.