Partially Demineralized Macroporous Allograft as a Tissue Engineering Scaffold

Objectives: There is potential for processing a partially demineralized macroporous (PDM) allograft to demonstrate appropriate mechanical properties as a stem-cell carrying scaffold for cranioplasty applications. The objectives of this project are to: 1) investigate the effects of degree of demineralization and porosity on the mechanical properties of allograft, 2) model the deformation of the allograft through finite-element analysis (FEA), and 3) evaluate the biocompatibility and mineralization behaviors of mesenchymal stem cells cultured on PDM allografts.
Methods: Bone disks of 2 mm thickness and 8 mm diameter harvested from pig tibia were used. Macropores of 600 μm diameter at 30% to 60% were created in the bone disk, following demineralization in 1N HCl for 0.5 to 3.5 hours. The stiffness of the bone disk was measured on a material testing machine at 1 mm/min loading rate. The deformation behavior of the disks was modeled using FEA software LS-DYNA (LSTC, Livermore, CA). Cytocompatibility of these bone disks were investigated using canine mesenchymal stem cells (MSCs) and XTT and ALP assay kit.
Results: Within each demineralization time, the stiffness of the allograft decreases linearly with the level of porosity, with non-demineralized bone disk decreasing from 600 N/mm to 100 N/mm as the porosity increases from 30% to 60%. Within each porosity level, the stiffness decreases exponentially with the demineralization time. The FEA results show the von Mises stress distribution with the characteristic stress concentration in the contact area between the pushing rod and the bone allograft specimen. The axial effect through holes with respect to stress concentration is less prominent. In the in vitro assay, the cells demonstrated increased XTT and ALP activities when cultured on the PDM allograft as compared to culturing on tissue culture plates.
Conclusions: We have demonstrated that we can process PDM allograft to demonstrate stiffness appropriate for clinical handling. Furthermore, we have shown that PDM allograft scaffolds support cell proliferation and osteogenic differentiation of canine MSCs. The in vivo performance of these PDM allograft scaffolds will be evaluated in the future.