Evaluation of osteoconductive scaffolds in the canine femoral multi-defect model

Abstract

Treatment of large segmental bone defects remains an unsolved clinical challenge, despite a wide array of existing bone graft materials. This project was designed to rapidly assess and compare promising biodegradable osteoconductive scaffolds for use in the systematic development of new bone regeneration methodologies that combine scaffolds, sources of osteogenic cells, and bioactive scaffold modifications. Promising biomaterials and scaffold fabrication methods were identified in laboratories at Rutgers, MIT, Integra Life Sciences, and Mayo Clinic. Scaffolds were fabricated from various materials, including poly(L-lactide-co-glycolide) (PLGA), poly(L-lactide-co-ɛ-caprolactone) (PLCL), tyrosine-derived polycarbonate (TyrPC), and poly(propylene fumarate) (PPF). Highly porous three-dimensional (3D) scaffolds were fabricated by 3D printing, laser stereolithography, or solvent casting followed by porogen leaching. The canine femoral multi-defect model was used to systematically compare scaffold performance and enable selection of the most promising substrate(s) on which to add cell sourcing options and bioactive surface modifications. Mineralized cancellous allograft (MCA) was used to provide a comparative reference to the current clinical standard for osteoconductive scaffolds. Percent bone volume within the defect was assessed 4 weeks after implantation using both MicroCT and limited histomorphometry. Bone formed at the periphery of all scaffolds with varying levels of radial ingrowth. MCA produced a rapid and advanced stage of bone formation and remodeling throughout the defect in 4 weeks, greatly exceeding the performance of all polymer scaffolds. Two scaffold constructs, TyrPC(PL)/TCP and PPF4(SLA)/HA(PLGA) (Dip), proved to be significantly better than alternative PLGA and PLCL scaffolds, justifying further development. MCA remains the current standard for osteoconductive scaffolds.

FIG. 1.

Implanted scaffolds: (A) PLCL/TCP_3DP, (B) TyrPC_PL, (C) PPF2_SLA, and (D) MCA chips (∼3×3 mm). Horizontal scale on lower images is ∼4 mm. (SEM image of the TyrPC_PL scaffold was provided by Joachim Kohn, New Jersey Center for Biomaterials, Rutgers University.) 3DP, three-dimensional printing; MCA, mineralized cancellous allograft; PL, porogen leaching; PLCL, poly(L-lactide-co-ɛ-caprolactone); PPF, poly(propylene fumarate); SLA, stereolithography; TCP, tri-calcium phosphate; TyrPC, tyrosine-derived polycarbonate.

FIG. 2.

(A) Drill guide in position on the lateral surface of the proximal femur. (B) Defect sites before implantation of scaffolds.

FIG. 3.

Micro computed tomography processing technique. (A) The 3D defect volume is defined using a standard 10-mm-diameter×15-mm-long cylinder. Following segmentation, bone volume (BV) data are mapped onto a color 2D contour plot using a scale from 0% to 30% BV. (B) The defect site is divided for analysis into regions for analysis. The pericortical (PC) region and the intramedullary (IM) region are defined based on vertical position from the bottom of the defect. Three regions of depth are defined based on radial distance from the center in millimeters: center (C)=0.25–1.75 mm, middle (M)=1.75–3.25 mm, and outer (O)=3.25–4.75 mm.

FIG. 4.

Percent bone volume (% BV) raw data distribution for the overall defect site is extremely skewed toward low values for all scaffolds except MCA. This was adjusted to enable parametric analysis by applying the logarithm transformation.

FIG. 5.

Two-dimensional contour plots of bone volume percent using color map ranging from 0% (purple) to 30% (red).

FIG. 6.

Percent bone volume (% BV) plotted versus radial position for (A) the entire defect volume, (B) the PC region, and (C) the IM region. BMC, bone marrow clot.

FIG. 7.

Undecalcified histology: Representative images for selected scaffold constructs stained using Goldner's Trichrome. Magnification at 1×, 5×, 10×. The black square box indicates the region of defect where the 5× magnification is presented.

FIG. 8.

Higher magnification of MCA samples stained with Goldner's Trichrome stain.

FIG. 9.

Example of histology slides for TyrPCPL/TCP and PPF2SLA/TCPPLGA spray samples stained with Goldner's Trichrome stain revealing that trabecular bone formation is present and can penetrate deeply into the scaffold constructs in some areas. In the case of the PPF material, no significant resorption is seen and bone formation is most prominent in the pores of the scaffold within the PC region of the defect. In the case of the Tyr-PC scaffold, significant degradation of the scaffold is evident, and trabecular bone formation on the right side of the defect is seen penetrating into the deeper portion of the defect in some areas, while in other areas left side of the defect demonstrate a reactive fibrous tissue response.