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Comparative Study
. 2013 Aug;471(8):2422-33.
doi: 10.1007/s11999-013-2859-0.

Nano-ceramic composite scaffolds for bioreactor-based bone engineering

Qing Lv  1 , Meng Deng, Bret D Ulery, Lakshmi S Nair, Cato T Laurencin
Affiliations
Comparative Study

Nano-ceramic composite scaffolds for bioreactor-based bone engineering

Qing Lv et al. Clin Orthop Relat Res. 2013 Aug.

Abstract

Background: Composites of biodegradable polymers and bioactive ceramics are candidates for tissue-engineered scaffolds that closely match the properties of bone. We previously developed a porous, three-dimensional poly (D,L-lactide-co-glycolide) (PLAGA)/nanohydroxyapatite (n-HA) scaffold as a potential bone tissue engineering matrix suitable for high-aspect ratio vessel (HARV) bioreactor applications. However, the physical and cellular properties of this scaffold are unknown. The present study aims to evaluate the effect of n-HA in modulating PLAGA scaffold properties and human mesenchymal stem cell (HMSC) responses in a HARV bioreactor.

Questions/purposes: By comparing PLAGA/n-HA and PLAGA scaffolds, we asked whether incorporation of n-HA (1) accelerates scaffold degradation and compromises mechanical integrity; (2) promotes HMSC proliferation and differentiation; and (3) enhances HMSC mineralization when cultured in HARV bioreactors.

Methods: PLAGA/n-HA scaffolds (total number = 48) were loaded into HARV bioreactors for 6 weeks and monitored for mass, molecular weight, mechanical, and morphological changes. HMSCs were seeded on PLAGA/n-HA scaffolds (total number = 38) and cultured in HARV bioreactors for 28 days. Cell migration, proliferation, osteogenic differentiation, and mineralization were characterized at four selected time points. The same amount of PLAGA scaffolds were used as controls.

Results: The incorporation of n-HA did not alter the scaffold degradation pattern. PLAGA/n-HA scaffolds maintained their mechanical integrity throughout the 6 weeks in the dynamic culture environment. HMSCs seeded on PLAGA/n-HA scaffolds showed elevated proliferation, expression of osteogenic phenotypic markers, and mineral deposition as compared with cells seeded on PLAGA scaffolds. HMSCs migrated into the scaffold center with nearly uniform cell and extracellular matrix distribution in the scaffold interior.

Conclusions: The combination of PLAGA/n-HA scaffolds with HMSCs in HARV bioreactors may allow for the generation of engineered bone tissue.

Clinical relevance: In cases of large bone voids (such as bone cancer), tissue-engineered constructs may provide alternatives to traditional bone grafts by culturing patients' own MSCs with PLAGA/n-HA scaffolds in a HARV culture system.

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Figures

Fig. 1
Fig. 1
A flowchart illustrates the experimental design of the study.
Fig. 2A–D
Fig. 2A–D
Three-dimensional (3-D) micro-CT reconstructions of PLAGA/n-HA (A–B) and PLAGA (C–D) scaffolds at the surface (A, C) and cross-sectional interior (B, D). Red dots represent n-HA.
Fig. 3A–F
Fig. 3A–F
PLAGA/n-HA and PLAGA scaffold degradation under dynamic conditions. (A) Average scaffold weight and (B) PLAGA weight-average molecular weight change over time. (C) Scaffold compressive modulus and (D) compressive strength change over time. Scanning electron micrographs of PLAGA/n-HA scaffolds at (E) 3 weeks and (F) 6 weeks.
Fig. 4A–D
Fig. 4A–D
(A) Cell proliferation over time as measured by DNA quantification. (B) Normalized ALP activity of cells seeded on scaffolds over time. SEMs of cells on the surface of PLAGA/n-HA scaffolds at Day 14 (C) and Day 28 (D).
Fig. 5A–E
Fig. 5A–E
Calcium deposition by HMSCs over time. (A) Alizarin red staining of PLAGA/n-HA scaffolds: blank scaffold without cells and scaffolds seeded with cells for 7, 14, 21, and 28 days (left to right). SEM images of the (B) end, (C) side, and (D) cross-sectional interior of Alizarin red-stained cell-seeded PLAGA/n-HA scaffolds at Day 21. (E) Desorbed Alizarin red quantification as measured spectrophotometrically at 550 nm.
Fig. 6A–C
Fig. 6A–C
Immunofluorescent staining of HMSCs on PLAGA/n-HA scaffolds with DAPI (cell nuclei) and TRITC-conjugated phalloidin (polymerized actin) at (A) Day 7 (scaffold surface), (B) Day 21 (scaffold surface), and (C) Day 21 (scaffold center). The scale bars represent 200 μm.
Fig. 7A–C
Fig. 7A–C
After 28 days of dynamic culturing in a HARV bioreactor, HMSC-seeded PLAGA/n-HA scaffolds were stained with (A) H&E, (B) osteocalcin immunostain, or (C) osteopontin immunostain. Each image is representative of three different levels of three independent samples. The scale bars represent 200 μm.
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