Quantitative microcomputed tomography analysis of mineralization within three‐dimensional scaffolds in vitro

Abstract

Synthetic and naturally derived scaffold biomaterials in combination with osteogenic cells or bioactive factors have the potential to serve as bone graft substitutes. Porous poly(l-lactide-co-dl-lactide) (PLDL) scaffolds with mechanical properties comparable to trabecular bone and an oriented, interconnected porosity designed to enhance internal mass transport were recently developed. In this study, PLDL scaffolds were seeded with rat calvarial or rat stromal cells and cultured up to 8 weeks in media containing osteogenic supplements. Cell-seeded human demineralized trabecular bone matrix (DTBM) scaffolds were included for comparison. All constructs were imaged weekly from 4 to 8 weeks using microcomputed tomography (micro-CT) to nondestructively quantify the amount and distribution of mineralized matrix formation. The total mineralized matrix volume increased with time in culture for all construct groups. DTBM constructs contained significantly more mineralized matrix than PLDL constructs. However, an analysis of the acellular DTBM scaffolds exposed to osteogenic media revealed partial remineralization of the demineralized matrix whereas no mineralization was detected in acellular PLDL scaffolds. Differences in mineral distribution were also evident with cell-mediated mineralization found throughout the PLDL constructs but localized to the periphery of the DTBM constructs for both cell types. Expression of bone marker genes indicating osteoblast differentiation was demonstrated in all groups at 8 weeks using a quantitative reverse transcription polymerase chain reaction. Osteocalcin expression was significantly higher for calvarial cell constructs compared to stromal cell constructs, regardless of the type of scaffold. This study demonstrated that micro-CT imaging may be used to nondestructively and quantitatively monitor mineralization within three-dimensional scaffolds in vitro. PLDL scaffolds with an oriented microarchitecture were shown to support cell attachment, differentiation, and cell-mediated mineralization comparable to natural DTBM scaffolds.

Three-Dimensional Characterization of Tissue-Engineered Constructs by Contrast-Enhanced Nanofocus Computed Tomography

“…According to specific study objectives, a customized strategy will be required to extract the necessary information in a robust way, ranging from (1) accurate and detailed (100s of mm scale) identification and quantification of different TE construct components limited to small TE construct samples and using phase-contrast imaging, 25 to (2) using standard desktop micro-or nano-CT as a routine 3D imaging technique for whole TE construct analysis (mm to cm scale) and quantification of mineralization after in vivo implantation. 10,13 The latter approach, without the use of a contrast agent, has also been used for static or bioreactor in vitro cultures, 16,18,19 showing distinct contrast differences between the mineralized matrix and scaffold. Phase-contrast imaging [24][25][26] or micro-CT combined with osmium tetroxide as a contrast agent 28 has shown its potential to assess nonmineralized ECM in an in vitro engineered TE construct.…”

“…In particular, Xray microfocus computed tomography (micro-CT) has been frequently applied as a 3D quantitative imaging technique to assess the scaffold structure, [6][7][8] as well as bone ingrowth after in vivo implantation. 6,[9][10][11][12][13][14] Furthermore, it has been employed for time-lapsed follow-up of mineralization inside scaffolds during in vitro static [15][16][17] or bioreactor cultures. 13,18,19 In most of these studies, polymeric, ceramic, collagen scaffolds, or composites were used, in which, the mineralized extracellular matrix (ECM) could be separated from the scaffold for the purpose of volume calculations and no significant material-dependent artifacts were present.…”

Prediction of spatio-temporal bone formation in scaffold by diffusion equation

“…It involves migration of several cell types [1], proliferation [2], differentiation [3], angiogenesis [4,5], and mineralization [6], as well as the interaction of numerous cytokines and growth factors [7]. Bone formation also depends on the architecture of the scaffold [8], its surface properties [9], the mechanical stimulation [10,11], the implantation site [12], and the boneescaffold interface [13].…”

Investigation of formation processes of porous biocompatible materials based on silicon doped and undoped ?-calcium-phosphate and hydroxyapatite

“…66,67 Biomimetičko taloženje (taloženje biološki sličnog, karbonatnog HA; eng. biomimetic precipitation) na površini implanta je usko povezano sa bioaktivnošdu različitih tipova implantnih materijala (66,68) . Povedana bioaktivnost materijala na kome se dešava biomimetička precipitacija je u vezi sa adsorpcijom i inkorporacijom bioloških moita za koje se veruje da predstavljaju mesta za koja se vežu delije.…”

“…. 68,69 Ograničena bioaktivnost keramike na bazi stehiometrijskog HA leži u velikoj stabilnosti HA u madijumu, što ne pospešuje proces biomimetičkog taloženja uslovljen površinom materijala.…”