Growing Bone Tissue-Engineered Niches with Graded Osteogenicity: AnIn VitroMethod for Biomimetic Construct Assembly

Abstract

The traditional bone tissue-engineering approach exploits mesenchymal stem cells (MSCs) to be seeded once only on three-dimensional (3D) scaffolds, hence, differentiated for a certain period of time and resulting in a homogeneous osteoblast population at the endpoint. However, after achieving terminal osteodifferentiation, cell viability is usually markedly compromised. On the other hand, naturally occurring osteogenesis results from the coexistence of MSC progenies at distinct differentiative stages in the same microenvironment. This diversification also enables long-term viability of the mature tissue. We report an easy and tunable in vitro method to engineer simple osteogenic cell niches in a biomimetic fashion. The niches were grown via periodic reseeding of undifferentiated MSCs on MSC/scaffold constructs, the latter undergoing osteogenic commitment. Timefractioning of the seeded cell number during differentiation time of the constructs allowed graded osteogenic cell populations to be grown together on the same scaffolds (i.e., not only terminally differentiated osteoblasts). In such cell-dynamic systems, the overall differentiative stage of the constructs could also be tuned by varying the cell density seeded at each inoculation. In this way, we generated two different biomimetic niche models able to host good reservoirs of preosteoblasts and other osteoprogenitors after 21 culture days. At that time, the niche type resulting in 40.8% of immature osteogenic progenies and only 59.2% of mature osteoblasts showed a calcium content comparable to the constructs obtained with the traditional culture method (i.e., 100.03 -29.30 vs. 78.51 -28.50 pg/cell, respectively; p = not significant), the latter colonized only by fully differentiated osteoblasts showing exhausted viability. This assembly method for tissue-engineered constructs enabled a set of important parameters, such as viability, colonization, and osteogenic yield of the MSCs to be balanced on 3D scaffolds, thus achieving biomimetic in vitro models with graded osteogenicity, which are more complex and reliable than those currently used by tissue engineers.

Chemoradiotherapy screening in a novel biomimetic polymer based pancreatic cancer model

“…This approach is hugely benecial in 3D scaffolds as it provides a quantitative indication of the live/dead and apoptotic/ non apoptotic cell distribution within the various scaffold areas for different treatment conditions. 41,86 The latter is particularly important as we have seen that metabolic activity dependent assays like the MTT assay and Alamar Blue assay are affected by various parameters including cytotoxic agents and are not sensitive enough to identify differences in cell population within the scaffolds, especially for very high cell numbers. [87][88][89] In contrast, imaging is much more representative and reproducible.…”

“…2 More recently, 3D in vitro models have emerged and are being developed as low cost, promising alternatives to animals. 7,[29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48] However, most studies focus on the development of the actual 3D model and, to the best of our knowledge, there are very limited 3D studies on PDAC treatment screening (chemotherapy and radiotherapy) with most studies conducted in 3D spheroids (cell aggregates). 14,32,[34][35][36][49][50][51][52] Generally, these studies report a higher resistance of PDAC to treatment in 3D when compared to 2D cultures, a trend which is in closer alignment with in vivo studies.…”

Bioactive and Topographically-Modified Electrospun Membranes for the Creation of New Bone Regeneration Models

“…A cell population with different osteogenic differentiation stages was obtained by culturing undifferentiated cells on the scaffolds with osteogenic medium and by periodically seeding new undifferentiated MSCs (cell shots) into the same scaffold. This method allowed for the development of a stem cell niche with graded osteogenicity characteristics which presented similarities with the bone MSCs populations in in vivo niches [28]. More recently, Muerza-Cascante et al developed melt electrospun polycaprolactone (PCL) calcium-phosphate coated and NaOH-etched scaffolds for bone repair.…”

Fabrication of carbonate apatite honeycomb and its tissue response

“…An interconnected porous structure is attractive for bone substitutes because bone tissue and cells can penetrate the bone substitute . Furthermore, microporous structure should play an important role in cell growth and differentiation, similar to the niche environment or microenvironment produced by the extracellular matrix . Among many porous structures available, the honeycomb (HC) structure is unique due to its fully interconnected and oriented pores having the same pore size, and shows high mechanical strength in the direction of the pores …”

“…[17][18][19][20][21][22][23][24][25] Furthermore, microporous structure should play an important role in cell growth and differentiation, similar to the niche environment or microenvironment produced by the extracellular matrix. [26][27][28][29][30][31][32][33] Among many porous structures available, the honeycomb (HC) structure is unique due to its fully interconnected and oriented pores having the same pore size, [34][35][36][37][38][39] and shows high mechanical strength in the direction of the pores. 40 Therefore, the feasibility to fabricate CO 3 Ap HC was studied herein, based on the HC extrusion method.…”