Enhanced Control of In Vivo Bone Formation with Surface Functionalized Alginate Microbeads Incorporating Heparin and Human Bone Morphogenetic Protein-2

Abstract

In this study, we tested the hypothesis that a surface functionalization delivery platform incorporating heparin onto strontium alginate microbeads surfaces would convert this "naive carriers" into "mini-reservoirs" for localized in vivo delivery of recombinant human bone morphogenetic protein-2 (rhBMP-2) that will induce functional bone regeneration. In vitro evaluation confirmed that (1) heparin incorporation could immobilize and prolong rhBMP-2 release for approximately 3 weeks; (2) a significant decrease (p<0.01) in rhBMP-2 burst release is attainable depending on initial protein load; and (3) rhBMP-2 released from surface functionalized microbeads retained bioactivity and stimulated higher alkaline phosphatase activity in cultured C(2)C(12) cells when compared with daily administration of fresh bolus rhBMP-2. Subsequently, surface functionalized microbeads were used for in vivo delivery of rhBMP-2 at local sites of posterolateral spinal fusion surgery in rats. The microbeads were loaded into the pores of medical-grade polyepsilone caprolactone-tricalcium phosphate scaffolds before implantation. Results revealed robust bone formation and a biomechanically solid fusion after 6 weeks. When compared with a control group consisting of an equivalent amount of rhBMP-2 that was directly adsorbed onto bare-surfaced microbeads with no heparin, a 5.3-fold increase in bone volume fraction and a 2.6-fold increase in bending stiffness (flexion/extension) were observed. When compared with collagen sponge carriers of rhBMP-2, a 1.5-fold and a 1.3-fold increase in bone volume fraction and bending stiffness were observed, respectively. More importantly, 3D micro-computed tomography images enabled the visualization of a well-contained newly formed bone at ipsilateral implant sites with surface functionalized rhBMP-2 delivery. This was absent with collagen sponge carriers where newly formed bone tissue was poorly contained and crossed over the posterior midline to contralateral implants. These findings are important because of complications with current rhBMP-2 delivery method, including excessive, uncontrolled bone formation.

Sequestration of rhBMP-2 into Self-Assembled Polyelectrolyte Complexes Promotes Anatomic Localization of New Bone in a Porcine Model of Spinal Reconstructive Surgery

“…This observation is consistent with our previous finding of controlled and contained new bone tissue deposition in rat posterolateral spinal fusion studies with PECs as opposed to collagen carriers. 13 Further evidence of new bone containment in this large-animal ALIF model was obtained via 3D reconstructed m-CT images viewed from the axial projection showing large bone tissue deposition extending anteriorly beyond the implanted scaffold in collagen as opposed to PEC carriers ( b F7 Fig. 7).…”

“…This therefore brings to light the importance of large-animal models in preclinical evaluations as single-layered PECs evaluated previously were able to deliver rhBMP-2 that induced consistent posterolateral spinal fusion in rodents. 13,17 In this study, negatively charged heparin interacted with PLL, a positively charged polyamino acid (via electrostatic, Coulomb, interaction), to form PEC deposits as matrices suitable for rhBMP-2 sequestration and immobilization. This self-assembled polyelectrolyte matrix could be tailored to deliver rhBMP-2 and produce comparable, if not superior, interbody spinal fusion when compared to absorbable collagen sponges.…”

“…Besides, immobilized protein within PEC matrices has been reported to exhibit flexible protein conformation and this could further modulate protein release and support protein bioactivity. 13,17,20 In addition, PEC is a synthetic matrix that offers a key advantage over collagen sponges (commonly derived from animal sources) with respect to elimination of risk of immunogenicity. However, it should be noted that direct FIG.…”

“…We observed that the presence of heparin within PEC substrates was vital for rhBMP-2 bioactivity when used in vivo to facilitate posterolateral spinal fusion in rats. 13 Despite these significant efforts in using layer-by-layer self-assembly of polyelectrolytes to harness the potentials of ECM molecules in tissue engineering, one important challenge remained unresolved; the successful application of this concept in the healing of clinically relevant large-tissue-defect volumes is yet to be achieved as far as we know. 7 Therefore, we hypothesize that PEC deposits could sequester sufficiently bioactive rhBMP-2 to promote bone ingrowth and drive complete healing of a clinically relevant bone defect volume in pigs.…”

Polyelectrolyte Complex Carrier Enhances Therapeutic Efficiency and Safety Profile of Bone Morphogenetic Protein-2 in Porcine Lumbar Interbody Fusion Model

“…[19][20][21] Differing from absorbable collagen sponge (ACS) that delivers BMP-2 by physical adsorption, PEC is tailored to deliver BMP-2 by electrostatic binding and it could prevent elution of the growth factor. In previous studies, glycosaminoglycan molecules such as heparin and heparan sulphate have been explored to deliver labile proteins and cytokines.…”