Effects of protein dose and delivery system on BMP-mediated bone regeneration
Introduction
Large bone defects associated with high-energy trauma, fracture nonunion, and bone tumor resection present a difficult challenge to orthopaedic surgeons accentuated by the limited effectiveness of current treatment options. The gold standard of care, the autograft, in which bone graft particles are surgically transplanted from the patient’s iliac crest, is limited by the available volume of graft material and significant donor site morbidity [1], [2]. Allografts are therefore often used to bridge the defects; however, these frequently fail to revascularize and remodel, resulting in graft fracture or tissue necrosis, requiring debridement and retreatment [3], [4], [5].
Biomaterials-mediated delivery of biologic agents including growth factors, stem cells, and genes has been used to stimulate regeneration of the structure and function of various tissues and has specifically emerged as a promising alternative to bone grafting techniques [6]. Delivery of recombinant proteins is a particularly attractive therapeutic strategy to promote endogenous repair mechanisms and tissue regeneration [6]. For a given protein, the delivery system may affect regenerative response by modulating protein stability and release kinetics. Langer and Folkman first demonstrated in 1976 the possibility of sustaining protein release via encapsulation in biocompatible polymers [7]. Since then, investigators have explored numerous materials and encapsulation and tethering techniques for tissue regenerative applications. For example, Griffith and colleagues have tethered growth factors to biomaterial substrates to regulate spatiotemporal presentation to mesenchymal stem cells and hepatocytes [8], [9], [10], and Phelps et al. covalently linked PEG hydrogels with vascular endothelial growth factor (VEGF) and cell adhesive peptides to enhance vascular network formation in vivo [11]. Others, such as Stayton and Mooney, have focused on modifying the degradation properties of various hydrogels to modulate growth factor delivery [12], [13]. These inexhaustive examples illustrate the variety and power of the biomaterial delivery approach; however, degradation properties, release kinetics, and other material properties must be designed and tailored for each application [14], [15].
Delivery of recombinant human osteoinductive growth factors is one of the most successful and clinically-applicable bone tissue engineering strategies to date [14]. The principle of bone induction dates back to Marshal Urist’s seminal discovery in 1965 of the potential of devitalized, decalcified allografts to induce heterotopic bone formation [16]. Subsequently, Urist, Reddi, and others extracted and identified the active biological agents, the bone morphogenetic proteins (BMPs), which belong to the transforming growth factor-β (TGF-β) supergene family [17], [18], [19], [20], [21], [22]. Identification of the genetic sequence of BMP-2 by Wozney and colleagues enabled production of highly purified BMPs through recombinant gene technology, which has facilitated its use as a clinical therapy [21], [23], [24], [25]. To date, two of the BMPs have been approved by the FDA for use in humans: BMP-2 and BMP-7, also known as human osteogenic protein-1 (hOP-1) [26].
Portending the tissue engineering paradigm in the early 1980’s, Reddi and colleagues first isolated and combined these soluble osteoinductive factors with insoluble substrata to induce bone formation [17], [19]. This approach has seen continued success and aims to stimulate the endogenous regenerative potential of the host by recapitulating the molecular cascades that lead to bone formation during development [27], [28]. However, as animal model and clinical data accumulate, the importance of the biomaterial carrier has become increasingly evident [26], [29], and while hOP-1 and rhBMP-2 have been successfully used in spinal fusion and open tibial fractures [30], [31], [32], [33], [34], significant limitations to current delivery systems remain [29]. In current clinical practice, rhBMP-2 is delivered by implanting an absorbable collagen sponge soaked in water-solubilized protein [29]. However, complications associated with rapid protein degradation and diffusion (such as soft tissue inflammation and ectopic bone formation) [35], [36], [37], the cost of the high doses required for efficacy [38], [39], [40], [41], [42], and concerns over a correlation between extremely high doses of rhBMP-2 and cancer incidence [43] suggest that spatiotemporal delivery strategies may improve the efficacy, efficiency, and safety of recombinant growth factor delivery.
Of particular importance for growth factor delivery vehicles is the release profile of the protein from the scaffold, which must maintain a sufficient concentration to induce the desired response for a long enough time to promote recruitment of endogenous progenitor cells [14]. Development and assessment of such delivery vehicles requires systematic evaluation of protein dose-response relationships as well as comparison to the current clinical standard for both protein release and function. Such studies will facilitate comparison between different carrier systems, animal models, and associated protein doses.
The goal of this study was therefore to characterize and evaluate the dose-response of rhBMP-2 in a recently described protein delivery system designed to provide controlled spatial and temporal protein delivery [44], to compare this system with the clinically-used collagen sponge, and to explain the differences in response by quantifying the in vivo protein release profile of each. We hypothesized that bone regeneration responds in a dose-dependent manner to recombinant rhBMP-2 delivery in the nanofiber mesh/alginate delivery system and that this delivery system enhances bone regeneration over the currently-used collagen sponge delivery method due to sustained protein release, thereby reducing the necessary effective dose.
Section snippets
Surgical procedure
Bilateral, critically-sized (8 mm) segmental defects were surgically created in femora of 13 week-old SASCO Sprague Dawley rats, as previously described [44], [45], [46]. Limbs were stabilized by custom radiolucent fixation plates that allowed in vivo monitoring with X-ray and microcomputed tomography (microCT). The experimental design featured 8 groups (Table 1, n = 9–10 per group). In 6 groups, the dose-response of bone regeneration to rhBMP-2, when delivered in an alginate hydrogel, was
Results: dose-dependency
First, the dose-response of rhBMP-2 in the nanofiber mesh/alginate delivery system was evaluated over 12 weeks in critically-sized rat femoral bone defects.
Results: Delivery system comparison
Next, we compared the nanofiber mesh/alginate delivery system at a non-bridging dose (0.1 μg) and a bridging dose (1.0 μg) with the clinically-used collagen sponge delivery system at the same doses. Finally, we compared the in vivo protein release kinetics of the two delivery methods using fluorophore-tagged rhBMP-2.
Discussion
Delivery of recombinant proteins carries great promise for the field of regenerative medicine; however, optimal doses and delivery vehicles have not yet been determined. This study presents the dose-response relationships for rhBMP-2 delivered in a controlled-release hydrogel in comparison to the currently-used collagen sponge carrier, and revealed a reduction in the necessary effective dose for the spatiotemporal delivery system.
Conclusions
These data demonstrate the dose-response and temporal release of rhBMP-2 in a spatiotemporal protein delivery system, in comparison to the clinical standard collagen sponge. This work demonstrates an improvement in bone formation over current rhBMP-2 delivery methods, and highlights the importance of quantification of release kinetics and scaffold degradation properties for evaluating novel recombinant protein carriers.
Acknowledgements
This work was supported by grants from the NIH (R01 AR051336), AFIRM, and DoD. The authors would like to thank the following individuals: Dr. David Mooney, for providing the RGD-alginate and Dr. Laura O’Farrell, for assistance with animal studies. We gratefully acknowledge Angela Lin, Dr. Tamim Diab, Dr. Mela Johnson, Christopher Dosier, Jessica O’Neal, Jason Wang, Tanushree Thote, Alice Li, and Ashley Allen for their assistance in surgeries.
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