Effect of Cell Density on Osteoblastic Differentiation and Matrix Degradation of Biomimetic Dense Collagen Scaffolds

Abstract

Plastic compression of hyperhydrated collagen gels produces tissue-like scaffolds of enhanced biomechanical properties. By increasing collagen density, these scaffolds could be developed into highly Biomimetic cell-seeded templates. When utilizing three-dimensional (3-D) scaffold systems for tissue repair, and indeed when investigating the cytocompatibility of two-dimensional (2-D) surfaces, the cell seeding density is often overlooked. In this study, we investigated this potentially critical parameter using MG-63 cells seeded in the dense collagen scaffolds. This is conducted within the overall scope of developing these scaffolds for bone repair. Cell proliferation, osteoblastic differentiation, and matrix remodelling capacity in relation to various seeding densities, ranging from 10(5) to 10(8) cells/ml compressed collagen, were evaluated in vitro. This was performed using the AlamarBlue assay, quantitative polymerase chain reaction (qPCR), and tensile mechanical analysis respectively. Variations in cell seeding density significantly influenced cell proliferation where lower initial seeding density resulted in higher proliferation rates as a function of time in culture. Gene transcription levels for alkaline phosphatase (ALPL), runt-related transcription factor 2 (RUNX2), and osteonectin (SPARC) were also found to be dependent on the cell density. While ALPL transcription was down-regulated with culturing time for all seeding densities, there was an increase in RUNX2 and SPARC transcription, particularly for scaffolds with cell densities in the range 10(6)-10(7) cells/ml collagen. Furthermore, this range of seeding density affected cell capacity in conducting collagenous matrix degradation as established by analyzing matrix metalloproteinase 1 (MMP1) transcription and scaffold mechanical properties. This study has shown that the seeded cell population in the three-dimensional dense collagen scaffolds clearly affected the degree of osteoblastic cell proliferation, differentiation, and some aspects of matrix remodelling activity. The seeding density played a major role in influencing the corresponding cell differentiation and cell-matrix interaction.

Rheological characterization of the nucleus pulposus and dense collagen scaffolds intended for functional replacement

“…21 Our technique of plastic compression differs from earlier studies, because we use cell culture inserts with 3 mm pores to reduce the liquid content instead of nylon and stainless steel meshes. [7][8][9][10]21 Furthermore, the dimensions of our scaffolds are much larger than reported earlier and the compression times therefore longer. The latter reduces the attractivity to enrich scaffolds with cells, which should not easily survive in such large constructs.…”

“…Plastic compression of collagen solutions leads to significant densification and viscoelastic properties that closely approach those of skeletal tissue and has already been investigated for its potential in cartilage and bone engineering. [7][8][9][10] In the current study, we characterize the viscoelastic properties of the NP by rheology, 11,12 and screen dense collagen type I scaffolds to determine which collagen density matches to these properties. Our overall scope is to develop a collagen scaffold that could be used in in situ therapy in patients with a herniated NP.…”

Osteocyte differentiation and the formation of an interconnected cellular network in vitro

“…We previously reported that MC3T3-E1 cells cultured at low density (0.1 × 10 4 cells/ cm 2 ) had higher ALP activity compared to those at high density (1 × 10 4 cells/cm 2 ), albeit that higher amounts of mineralisation were reported for the low seeding density (Mullen et al, 2013). Human marrow-derived MSC cells and MG-63 osteosarcoma cells encapsulated at low densities (2 × 10 6 /3 × 10 6 cells/mL) within 3D collagen/ alginate gels had higher ALP activity than cells seeded at higher densities (1 × 10 8 /15 × 10 6 cells/mL) (Bitar et al, 2008;Maia et al, 2014). Here we showed that a low density (0.4 × 10 4 cells/cm 2 ~ 0.25 × 10 6 cells/mL) lead to higher ALP production after 3 d, compared to cells seeded at a high density (1.6 × 10 4 cells/cm 2 ~ 2 × 10 6 cells/mL).…”

“…In vivo, osteoblasts and osteocytes primarily exist within a complex three dimensional (3D) environment (Boukhechba and Balaguer, 2009), and it is known that 3D environment has a significant effect on cell morphology and geometry, as shown in NIH 3T3 fibroblast (Legant et al, 2010), cardiac cells (Soares et al, 2012) and MC3T3-E1 osteoblasts (Murshid et al, 2007). The process of osteocyte dendrite formation within a 3D environment is highly dynamic, as the embedding cells repeatedly extend and retract their dendrites.…”

Cell cytoskeletal changes effected by static compressive stress lead to changes in the contractile properties of tissue regenerative collagen membranes

“…Static compressive stress of cell-seeded hyperhydrated type I collagen gels can lead to a change in the microenvironment and structure to produce tissue-like scaffolds with enhanced biomechanical properties (Bitar et al, 2007;Bitar et al, 2008;Brown et al, 2005;Mudera et al, 2007). In addition to possible changes in cell survival and differentiation, a shift in microfilament and dendrite distribution could lead to a favourable change in the relationship between cell contractility and subsequent contraction of the collagen gel.…”