Cell Distribution In Scaffolds Research Articles

Optical projection tomography can be used to investigate spatial distribution of chondrocytes in three-dimensional biomaterial scaffolds for cartilage tissue engineering.

Biomaterial scaffolds have been used in autologous chondrocyte implantation to facilitate the repair of large lesions and to advance the formation of articular cartilage [Exp. Biol. Med. (Maywood) 237(1) (2012), 10-17]. Biomaterial scaffolds are usually three-dimensional (3-D) porous structures consisting of biodegradable materials to support articular cartilage formation. Adequate porosity of the scaffold is necessary for uniform cell distribution and cell attachment, and the density of the cells in the scaffold should be appropriate for cartilage formation [Cartilage 3(2) (2012), 108-117]. There have been only a restricted number of studies on the spatial distribution of cells in scaffolds, and on the role of this to cartilage formation [J. Biotechnol. 129 (2007), 516-531; Biotechnol. Progr. 14 (1998), 193-202; Biotechnol. Bioeng. 84 (2003), 205-214]. This may be due to the limited availability of appropriate visualization methods. Acquiring 3-D images throughout the scaffold by histology or confocal methods are not applicable to all types of scaffolds, and moreover, they are time consuming, laborious and thus not very feasible for a large scale analysis. To make the visualization of the spatial distribution of the cells easier in biomaterial scaffolds we have applied optical projection tomography (OPT). OPT microscope produces high-resolution 3-D images of both fluorescent and non-fluorescent specimens [Science 296(5567) (2002), 541-545]. Here we demonstrate that the OPT method can be used for the evaluation and visualization of the cell seeding method, spatial distribution and density of cells in biomaterial scaffolds and thus establish the OPT as a valid tool for analysis of cell distribution in cartilage tissue engineering samples.

Mathematical model and numerical simulation of the cell growth in scaffolds

A scaffold is a three-dimensional matrix that provides a structural base to fill tissue lesion and provides cells with a suitable environment for proliferation and differentiation. Cell-seeded scaffolds can be implanted immediately or be cultured in vitro for a period of time before implantation. To obtain uniform cell growth throughout the entire volume of the scaffolds, an optimal strategy on cell seeding into scaffolds is important. We propose an efficient and accurate numerical scheme for a mathematical model to predict the growth and distribution of cells in scaffolds. The proposed numerical algorithm is a hybrid method which uses both finite difference approximations and analytic closed-form solutions. The effects of each parameter in the mathematical model are numerically investigated. Moreover, we propose an optimization algorithm which finds the best set of model parameters that minimize a discrete l(2) error between numerical and experimental data. Using the mathematical model and its efficient and accurate numerical simulations, we could interpret experimental results and identify dominating mechanisms.

Analysis of Collagen and Glucose Modulated Cell Growth within Tissue Engineered Scaffolds

The strategy of tissue engineering includes seeding cells onto porous scaffolds. The cellular construct is cultured in vitro for a period of time before transplantation for the patient. Because of the intrinsic complexity of biological systems, it is valuable to have models of simulation that can assess the culture conditions and optimize experiments. This work presents a mathematical model to account for the effects of glucose and type II collagen on chondrocyte growth under static culture conditions. Dependence of cell growth on collagen was assumed as a biphasic function of collagen quantity, whereby the cell growth rate increases and then decreases with increasing collagen content. Results from simulation were compared with experimental data in literature. The model was then applied to investigate the effects of cell seeding area, demonstrating the spatiotemporal evolution of cell distribution in scaffolds. Results show that the conventional uniform seeding method may not be a good way of gaining uniform and large cell number densities at the final stage of cultivation. A seeding mode that has cells reside initially in the middle area of scaffold was shown to be able to not only reduce the diffusion limitation of nutrients but also weaken the inhibiting impact of aggregated collagen on cell growth. Therefore the middle seeding mode may result in better cell amounts and uniformities for developing tissue engineered constructs.

Adipose tissue-derived stem cell expansion on collagen/chitosan scaffolds in bioreactor

Recent reports indicate that adipose tissue is a novel source of multipotent stem cells, which can be used in cell therapy and tissue engineering. However, the static amplification of adipose tissue-derived stem cells(ADSCs) is a time-consuming procedure and prone to contamination, and it's hard to meet the need of clinical applications. To obtain the stem cells in a great number of quantity and with stemness well reserved, we plated ADSCs in collagen/chitosan scaffolds and compared the proliferation of ADSCs in three dimensional (3D) static environment and 3D dynamic environment. hADSCs were isolated and cultivated in T-flask, in collagen/chitosan scaffolds statically or in the scaffolds placed in spinner flasks for 21 days. During the culture period, the growth dynamic parameters and viability of ADSCs were examined by CCK-8 kit every other day; metabolic rates of glucose and lactic acid were analyzed every day. After 21 days, the cells were evaluated with scanning electron microscope (SEM) for histological examination of the aggregates. The surface markers (CD13, CD29, CD34, CD44, CD45, CD105, CD166 and HLA-DR), the specific transcription factors (Oct-4, Sox-2, Rex-1) and the multi-differentiation potential (adipogenic, osteogenic and chondrogenic) were also assayed to identify the stemness of proliferated cells. The results showed that in spinner flask, the cells in scaffolds could be expanded by more than ten times, and they presented better morphology and vitality and stronger differentiation ability than cells cultivated in scaffolds statically. And the expansion of the both cases for cells in scaffolds was better than that in T-flask. All cells maintained stem cell characteristics after proliferation. From the results, we found that dynamic spinner flask cultivation of hADSC/scaffold constructs resulted in increased proliferation, differentiation and distribution of cells in scaffolds. Therefore, spinner flask cultivation is an easy-to-use inexpensive system for expanding hADSCs in 3D scaffolds.

Mesenchymal stem cell ingrowth and differentiation on coralline hydroxyapatite scaffolds

Culture of osteogenic cells on a porous scaffold could offer a new solution to bone grafting using autologous human mesenchymal stem cells (hMSC) from the patient. We compared coralline hydroxyapatite scaffolds with pore sizes of 200 and 500 μm for expansion and differentiation of hMSCs. We cultivated the hMSC statically or in spinner flasks for 1, 7, 14 and 21 days and found that the 200-μm pore scaffolds exhibited a faster rate of osteogenic differentiation than did the 500-μm pore scaffolds as shown by an alkaline phosphatase activity assay and real-time reverse transcriptase polymerase chain reaction for 10 osteogenic markers. The 500-μm scaffolds had increased proliferation rates and accommodated a higher number of cells (shown by DNA content, scanning electron microscopy and fluorescence microscopy). Thus the porosity of a 3D microporous biomaterial may be used to steer hMSC in a particular direction. We found that dynamic spinner flask cultivation of hMSC/scaffold constructs resulted in increased proliferation, differentiation and distribution of cells in scaffolds. Therefore, spinner flask cultivation is an easy-to-use inexpensive system for cultivating hMSCs on small to intermediate size 3D scaffolds.

Fabrication of tubular tissue constructs by centrifugal casting of cells suspended in an in situ crosslinkable hyaluronan‐gelatin hydrogel

Achieving the optimal cell density and desired cell distribution in scaffolds is a major goal of cell seeding technologies in tissue engineering. In order to reach this goal, a novel centrifugal casting technology was developed using in situ crosslinkable hyaluronan-based (HA) synthetic extracellular matrix (sECM). Living cells were suspended in a viscous solution of thiol-modified HA and thiol-modified gelatin, a polyethyleneglycol diacrylate crosslinker was added, and a hydrogel was formed during rotation. The tubular tissue constructs consisting of a densely packed cell layer were fabricated with the rotation device operating at 2000 rpm for 10 min. The majority of cells suspended in the HA mixture before rotation were located inside the layer after centrifugal casting. Cells survived the effect of the centrifugal forces experienced under the rotational regime employed. The volume cell density (65.6%) approached the maximal possible volume density based on theoretical sphere packing models. Thus, centrifugal casting allows the fabrication of tubular constructs with the desired redistribution, composition and thickness of cell layers that makes the maximum efficient use of available cells. Centrifugal casting in this sECM would enable rapid fabrication of tissue-engineered vascular grafts, as well as other tubular and planar tissue-engineered constructs.

Fabrication of tubular tissue constructs by centrifugal casting of cells suspended in an in situ crosslinkable hyaluronan-gelatin hydrogel

Achieving the optimal cell density and desired cell distribution in scaffolds is a major goal of cell seeding technologies in tissue engineering. In order to reach this goal, a novel centrifugal casting technology was developed using in situ crosslinkable hyaluronan-based (HA) synthetic extracellular matrix (sECM). Living cells were suspended in a viscous solution of thiol-modified HA and thiol-modified gelatin, a polyethyleneglycol diacrylate crosslinker was added, and a hydrogel was formed during rotation. The tubular tissue constructs consisting of a densely packed cell layer were fabricated with the rotation device operating at 2000 rpm for 10 min. The majority of cells suspended in the HA mixture before rotation were located inside the layer after centrifugal casting. Cells survived the effect of the centrifugal forces experienced under the rotational regime employed. The volume cell density (65.6%) approached the maximal possible volume density based on theoretical sphere packing models. Thus, centrifugal casting allows the fabrication of tubular constructs with the desired redistribution, composition and thickness of cell layers that makes the maximum efficient use of available cells. Centrifugal casting in this sECM would enable rapid fabrication of tissue-engineered vascular grafts, as well as other tubular and planar tissue-engineered constructs.