A Perfusion Culture System for Assessing Bone Marrow Stromal Cell Differentiation on PLGA Scaffolds for Bone Repair

Abstract

Biomaterials development for bone repair is currently hindered by the lack of physiologically relevant in vitro testing systems. Here we describe the novel use of a bi-directional perfusion bioreactor to support the long term culture of human bone marrow stromal cells (BMSCs) differentiated on polylactic co-glycolic acid (PLGA). Primary human BMSCs were seeded onto porous PLGA scaffolds and cultured in static vs. perfusion culture conditions for 21 days in osteogenic vs. control media. PLGA scaffolds were osteoconductive, supporting a mature osteogenic phenotype as shown by the upregulation of Runx2 and the early osteocyte marker E11. Perfusion culture enhanced the expression of osteogenic genes Osteocalcin and Osteopontin. Extracellular matrix deposition and mineralisation were spatially regulated within PLGA scaffolds in a donor dependant manner. This, together with the observed upregulation of Collagen type X suggested an environment permissive for the study of differentiation pathways associated with both intramembranous and endochondral ossification routes of bone healing. This culture system offers a platform to assess BMSC behavior on candidate biomaterials under physiologically relevant conditions. Use of this system may improve our understanding of the environmental cues orchestrating BMSC differentiation and enable fine tuning of biomaterial design as we develop tissue-engineered strategies for bone regeneration.

Time-lapsed imaging of nanocomposite scaffolds reveals increased bone formation in dynamic compression bioreactors

“…A DNA reduction during culture was also observed for SCPLmade PLGA scaffolds 42 , silk scaffolds 19 , electrospun PLGA with calcium phosphate nanoparticles 12 , and hydrogels cultured under cyclic loading 10 . The DNA reduction observed in this study and literature 10,12,19,42 occurred for scaffolds cultured with (hMSCs) or adipose-derived stem cells under perfusion or static conditions, independent of cell culture medium used. Thus, there seems to be no general trend in DNA change with respect to culture conditions and cell culture medium.…”

“…Thus, there seems to be no general trend in DNA change with respect to culture conditions and cell culture medium. The seeding density used in this study and literature 10,12,19,42 was rather high, with more than one million cells per scaffold. We believe that especially the hydrophobic nature of PLGA scaffolds 43 resulted in weak cell attachment to the scaffold and therefore caused a drop in DNA.…”

“…Cell distribution, time-lapsed micro-CT imaging, and longitudinal monitoring. The DNA amount was quantified after 1 day and at the end of the culture for comparison with previous reports 10,12,19,42 that cultured human cells in bone scaffolds. Both scaffold groups cultured under static and dynamic conditions The spherical hydroxyapatite particles have a smaller specific surface area (SSA) and correspondingly larger equivalent particle size (d BET ) and crystal size (d XRD ) than the needle-like nanoparticles.…”

Recent Advances in Mechanically Loaded Human Mesenchymal Stem Cells for Bone Tissue Engineering

“…Given that oscillatory or pulsatile flow is physiologically generated through daily activities and the circulatory system, some studies have applied oscillatory or pulsatile flow to hMSCs for the development of tissue-engineered bone grafts [ 8 , 45 , 74 , 75 ]. For example, one study applied a long-term, continuous, oscillatory perfusion (0.0123 dyn/cm 2 , 3 weeks) to hMSCs cultivated in a scaffold (200–800 μm) made of cancellous bone powder [ 74 ].…”

Electrodynamic assisted self-assembled fibrous hydrogel microcapsules: a novel 3Din vitroplatform for assessment of nanoparticle toxicity

“…33 Also, the chemical modication and functionalization of alginate hydrogels is costand labor-intensive, and they are not readily remodeled by cells. 34,35 Therefore, current research has been focused to develop 3D hydrogel scaffolds using polymers of both biocompatible and biodegradable origin, such as alginate, 27 chitin, 36 and poly(lactide-co-glycolide) (PLGA) 37 as a potential strategy towards the development of realistic in vivo 3D ECM matrix.…”