3D imaging of tissue integration with porous biomaterials

Abstract

Porous biomaterials designed to support cellular infiltration and tissue formation play a critical role in implant fixation and engineered tissue repair. The purpose of this Leading Opinion Paper is to advocate the use of high resolution 3D imaging techniques as a tool to quantify extracellular matrix formation and vascular ingrowth within porous biomaterials and objectively compare different strategies for functional tissue regeneration. An initial over-reliance on qualitative evaluation methods may have contributed to the false perception that developing effective tissue engineering technologies would be relatively straightforward. Moreover, the lack of comparative studies with quantitative metrics in challenging pre-clinical models has made it difficult to determine which of the many available strategies to invest in or use clinically for companies and clinicians, respectively. This paper will specifically illustrate the use of microcomputed tomography (micro-CT) imaging with and without contrast agents to nondestructively quantify the formation of bone, cartilage, and vasculature within porous biomaterials.

Fig. 1

Micro-CT image (A) of a polycaprolactone (PCL) scaffold (5 mm diameter × 9 mm length) created using fused deposition modeling to create a defined porous architecture with 85% porosity. Micro-CT image (B) showing mineralized matrix synthesized within a PCL scaffold by amniotic fluid derived stem cells following 15 weeks of exposure to osteogenic differentiation media under dynamic culture conditions. In this experiment, type I collagen was lyophilized within the PCL scaffold to enhance cell seeding efficiency and six million stem cells were seeded per scaffold. Different volumes of interest may be defined for morphometric analysis to quantify the distribution of mineralized matrix. Whereas static culture conditions create a shell of mineralization isolated to the scaffold periphery, dynamic flow conditions provided by bioreactor systems produce a more homogenously distribution of mineralized matrix production throughout the construct (C).

Fig. 2

Micro-CT image (A) of a 70% L-lactide and 30% DL-lactide co-polymer (PLDL) with oriented porosity created using a fiber-coating and porogen decomposition method [1]. Sustained release of co-delivered recombinant human growth factors (combinations of BMP-2, TGF-β3, and VEGF) is achieved by polymerizing RGD alginate hydrogel within the PLDL scaffold pores prior to implantation into segmental bone defects [2]. Micro-CT images of vascular (B) and bone (C) ingrowth several weeks after implantation. Bone ingrowth can be quantified noninvasively using in vivo micro-CT scanners, while the vascular imaging technique must be done post-mortem.

Fig. 3

Hydrogel constructs containing bovine chondrocytes were equilibrated with contrast agent and scanned using micro-CT every four days until day 16. The contrast agent (Hexabrix) is approved for clinical use in humans and does not affect cell function (unpublished data). Following each scan, therefore, the contrast agent could be desorbed and construct culture continued. The average attenuation within the constructs decreased over time consistent with the accumulation of cartilaginous extracellular matrix.