(627e) Injectable, Biodegradable Polyurethane Scaffolds with Local Lovastatin Delivery for Enhanced Bone Regeneration

While autologous bone may be the ideal graft material for use in reconstructive orthopaedic surgery, its harvesting often causes donor site morbidity and has limited availability. Thus, synthetic or biological material substitutes have been increasingly studied for bone tissue repair. We have developed injectable biodegradable polyurethane (PUR) scaffolds with high porosity (>90%) that support cell migration and proliferation, and degrade to non-cytotoxic products. Lovastatin (LV) has been shown to promote osteogenesis by upregulating BMP2, and local delivery of LV could optimize its efficacy in vivo. In this study, we have fabricated PUR scaffolds to simultaneously provide a template for cellular infiltration and new bone formation, as well as a vehicle for local LV delivery to further effective bone regeneration.

Injectable PUR scaffolds were synthesized by syringe-mixing of two phases: a prepolymer of lysine triisocyanate (LTI) and 200-MW PEG, and a hardener consisting of a polyester triol, water, catalyst, pore opener. This reactive liquid mixture expanded by gas foaming to form porous scaffolds. Lovastatin was incorporated into the hardener phase before foaming at 20 μg, 200 μg, and 800 μg per gram of foam. In vitro release of LV in PBS at 37 ºC was measured daily from 1 to 30 days. The released LV was quantified by HPLC at 237 nm. We also examined in vitro biocompatibility of PUR scaffolds incorporated with LV: MC3T3-E1 cell attachment at 4 hours after seeding and cell viability at 5 days (5 x 104 cells/implant in 24-well dish) by MTT assay. In vitro biocompatibility of PUR was examined using preosteoblastic MC3T3-E1 cells. The cells were statically seeded onto thin foam discs (5×104 cells/implant) in 24-well plate, and cultured for 5 days under normal cell culture conditions. Cell viability was assessed qualitatively by fluorescent images using Live/Dead Viability/Cytotoxity assay kit. In vivo behavior of the scaffolds (without LV) was preliminarily evaluated for biocompatibility, osteoconductivity, and tissue regeneration. The materials were then either implanted (pre-made) or injected to cure in situ in 3-mm femoral plug defects in male Sprague-Dawley rats. The scaffolds were assessed at 2 and 4 weeks for biocompatibility, biodegradation, cellular infiltration, and bone regeneration. Mechanical properties were also ascertained by dynamic mechanical analysis (DMA). The stimulatory activity of LV released from PUR scaffolds (r-LV) in BMP2 expression was evaluated. MC3T3-E1 cells were cultured with r-LV (0, 0.2, 1 μM) and LV (positive control: 1 μM) for 24 and 48 hours. Total RNA was isolated using TriZOL reagent, and mRNA was transcribed to cDNA using Superscript II. To measure the expression of BMP2 expression, Taqman® gene expression assays were used to perform qRTPCR, using 18S as an endogenous control.

MC3T3-E1 cells substantially permeated and adhered to the scaffold interstices, and viable cells were well visible at 5 days after seeding in fluorescent microscope images. In the LV release assay, 10-20% of the incorporated LV eluted by 30 days, with a nearly linear release profile and nearly constant daily elution. The low elution rate may be due to the highly hydrophobic nature of lovastatin. Cell attachment and cell viability in PUR-LV scaffolds was comparable to control PUR scaffolds without LV. BMP2 expression in MC3T3-E1 cells treated with r-LV was enhanced in a dose-dependent manner, although its effect was lower than LV. The rat femoral plug implants showed good biocompatibility and osteoconductivity. After 2 and 4 weeks, microCT images showed new bone formation within the foams, which are not independently radio-opaque. Hematoxilin & eosin staining of decalicified histological sections also showed evidence of scaffold biodegradation coincident with cellular infiltration and new bone formation. Surprisingly, the presence of lovastatin did not significantly increase rate or amount of new bone formation with the defects, although the high-LV group demonstrated significantly higher density of the newly formed bone. A greater effect from LV may have been seen at a longer time point. Although previous studies showed that LV is effective for enhanced bone formation (both in vitro and in vivo, in a calvarial model) in non-wound cases where osteoblasts were most likely already present, this study may suggest that lovastatin may not have the chemoattractive effect of BMP-2.

These biodegradable PUR scaffolds show promise as possible therapies for bone regeneration. Their elastomeric and resilient properties may improve adhesion and integration at the bone-material interface. With >90% porosity, cells readily infiltrated the materials and produced new tissue, and in vivo studies demonstrate the osteoconductivity and biocompatibility of the scaffolds, as well as preliminary biodegradation. In vitro LV release experiments reveal a linear, controlled elution profile from the scaffolds. LV released from PUR enhances BMP2 expression in vitro and does not negatively affect cell attachment and viability. This scaffold therefore provides a structural support for bone ingrowth, and also a matrix from which biologicals can be released locally to enhance bone formation. However, further investigation is required to develop these materials in combination with lovastatin as a therapeutic platform.