Substrate Stiffness And the Timing of Cell Differentiation Regulate Bone Tissue
Formation Within Gelatin Scaffolds
Myles Mc Garrigle, BEng, Matthew Haugh, PhD, Muriel Voisin, PhD, Laoise McNamara, PhD.
National University of Ireland Galway, Galway, Ireland.
Disclosures:
M. Mc Garrigle: None. M. Haugh: None. M. Voisin: None. L. McNamara: None.
Introduction: Cell-seeded tissue engineered (TE) scaffolds have a limited capacity to regenerate bone in vivo, due to
complications associated with diffusion of nutrients and waste, cell death in scaffold cores [1] and failure to integrate with the
host tissue [2]. A particular problem is that cell differentiation at the scaffold periphery produces a matrix that acts as a barrier
to remodelling and osseointegration [3]. Cell distribution throughout a TE scaffold can influence the consistency of tissue
formation [4], whereby scaffolds with a dispersed cell population have consistent tissue formation throughout the construct
whereas partial cellular infiltration can lead to localising tissue formation, particularly at the periphery of the scaffold [5].
Scaffold stiffness plays an important role in regulating cell distribution [6], as higher stiffness retain the overall structure and
allow cells to migrate further throughout, whereas softer scaffolds are subject to collapse under cell-mediated contraction.
Furthermore the timing of delivering differentiation factors is important, as the cells will no longer proliferate or migrate after
differentiation occurs. Therefore a tissue regeneration approach that strives to optimise (a) the scaffold stiffness and (b) delivers
differentiation cues at the most appropriate time might enhance cell distribution and ensure uniform tissue formation within
cell seeded scaffolds. However, to date the optimum cellular distribution and mechanical properties required for bone
regeneration within TE scaffolds have not been defined. The objectives of this research are to investigate whether an approach
that 1) optimizes the scaffold stiffness and 2) delays the addition of differentiation cues to facilitate differentiation after the
scaffold has been fully populated, can enhance cell distribution and mineralisation of TE scaffolds.
Methods: Gelatin glycosaminoglycan (GG) scaffolds (Fig. 1) were produced via a freeze-drying process. Crosslinking was carried
out with varying concentrations of 1-Ethyl-3-3-dimethylaminopropyl carbodiimide (EDAC) to produce scaffolds of different
mechanical stiffness. Unconfined compressive testing was used to determine the mechanical properties of the crosslinked GG
scaffolds. Samples were hydrated and held in a bath of phosphate buffered saline (PBS) during testing. Three different
mechanical stiffness (0.5, 0.8, 1.4 kPa) from the EDAC group were used to examine the effects of mechanical properties on
mineralisation of scaffolds. Scaffolds were seeded with 2x106 MC3T3 cells and then were either (a) allowed to differentiate
immediately, through addition of osteogenic growth factors (ascorbic acid, β-glycerolphosphate and dexamethasone) to cell
culture media, or (b) cell differentiation was delayed for 7 days, during which time cells proliferated in expansion media (10%
foetal bovine serum, 2% penicillin/streptomycin and 1% L-glutamine). Both groups were cultured from Day 7 in osteogenic
media up to 28 days. Biochemical and histological analyses were performed to determine cell number, distribution and
mineralisation at specific time points (0, 7, 21 and 28 days). Cell distribution throughout the constructs was quantified by
staining with Propidium iodide (PI). Images were captured using an upright fluorescent microscopy. ImageJ software [7] was
used to generate stitched images of the sectioned construct allowing for particle analysis to be performed, thus quantitatively
assessing cell distribution throughout the entire scaffold cross section. Fixed sections are stained with 2% Alizarin red stain to
determine calcium distribution throughout the scaffold. Images were captured through a brightfield microscope.
Results: Mechanical testing showed a significantly higher compressive modulus in scaffolds crosslinked with EDAC compared to
untreated GG scaffolds (p 0.05) in the 0.8 and 1.4 kPa groups (> 4.5x106) compared to the 0.5 kPa group (> 3 x106) (Fig. 1 (A)). PI
staining for cell distribution showed an increase in cell infiltration and distribution throughout the scaffold from day 0 to 7 in the
delayed and immediate osteogenic media (Fig 2). Cell distribution in the 0.5 kPa delayed osteogenic group showed enhanced cell
infiltration and proliferation throughout the scaffold while the 0.5 kPa immediate osteogenic group showed uneven cell
distribution and infiltration from the scaffold periphery (Fig. 2). Results for mineral content showed no significant difference in
the calcium content in all the groups at day 28 (Fig. 1 (B)). However Alizarin red staining for mineral distribution in the delayed
osteogenic groups showed positive staining throughout the constructs compared to the immediate osteogenic group, in which
mineral localised to the interior of the constructs (Fig 2).
Discussion: In this study we observe that GG scaffolds of high stiffness (0.8 and 1.4 kPa) were more effective in inducing cell
proliferation compared to low stiffness scaffolds (0.5 kPa). We propose that scaffolds with the higher stiffness retain their pore
structure, thereby allowing cells to migrate and facilitate diffusion of waste products and nutrients, whereas softer scaffolds are
subject to collapse and thereby prevent cell migration and nutrient supply. Results for mineral content showed similar amounts
of calcium in all stiffness groups. However in the lowest stiffness group (0.5 kPa) a significantly lower cell number was observed
compared to the higher stiffness scaffolds. These results are believed to have occurred due to increased cell contraction within
the lower stiffness which positively increase osteoblast differentiation and decrease cell proliferation [4]. Results for mineral
distribution showed signs of enhanced mineral distribution in the delayed osteogenic constructs compared to higher amounts of
mineral localised to the construct interiors in the immediate osteogenic group. Other TE studies have shown that high amounts
of mineralisation can be achieved in TE scaffolds, however these results were achieved at the expense of losing their pore
structure and contracting in size [3-4]. This approach looks to optimise the scaffold stiffness and deliver differentiation cues at
the most appropriate time to enhance cell distribution and ensure uniform tissue formation within cell seeded scaffolds.
Ongoing work is further quantifying distribution and mineralisation of the Immunofluorescent samples to investigate whether
these properties are enhanced by a delayed differentiation approach.
Significance: The results of these experiments provide an enhanced understanding of optimal scaffold stiffness and timing of cell
differentiation for bone regeneration.
Acknowledgments: This project was supported by the European Research Council Grant 258992 (BONEMECHBIO).
References: [1] L. S. Gardel, journal of biomedical materials research. Part B, Applied biomaterials, 2013, pp.1-10. [2] S Kadiyala,
Tissue engineering, 3(2), 1997. [3] F. Lyons, Biomaterials, 35(1), 2010, pp.9232-43. [4] M. B. Keogh, Acta biomaterialia, 6(11),
2010, pp.4305-13. [5] M. B. Keogh, Biotechnology and bioengineering, 108(5), 2011, pp.1203-10. [6] M. G Haugh, Tissue
Engineering: Part A, 17, 2010, pp.0590. [7] S. Preibisch, Bioinformatics, 25(11), 2009, pp. 1463-
1465.
ORS 2014 Annual Meeting
Poster No: 0650