Stem Cells in Aggregate Form to Enhance Chondrogenesis in Hydrogels

There are a variety of exciting hydrogel technologies being explored for cartilage regenerative medicine. Our overall goal is to explore whether using stem cells in an aggregate form may be advantageous in these applications. 3D stem cell aggregates hold great promise as they may recapitulate the in vivo skeletal tissue condensation, a property that is not typically observed in 2D culture. We considered two different stem cell sources, human umbilical cord Wharton’s jelly cells (hWJCs, currently being used in clinical trials) and rat bone marrow-derived mesenchymal stem cells (rBMSCs). The objective of the current study was to compare the influence of cell phenotype, aggregate size, and aggregate number on chondrogenic differentiation in a generic hydrogel (agarose) platform. Despite being differing cell sources, both rBMSC and hWJC aggregates were consistent in outperforming cell suspension control groups in biosynthesis and chondrogenesis. Higher cell density impacted biosynthesis favorably, and the number of aggregates positively influenced chondrogenesis. Therefore, we recommend that investigators employing hydrogels consider using cells in an aggregate form for enhanced chondrogenic performance

Representative images for immunohistochemistry analysis for collagen I, collagen II, and aggrecan staining for rBMSC groups at weeks 0, 2, and 3.

<p>At week 2, 20M LA had the most intense staining for collagen II and aggrecan. At week 3, 10M LA had the highest staining intensity at collagen II. Scale bar = 200 μm.</p

Explanation of the different experimental groups used in the study and their abbreviations.

<p>Explanation of the different experimental groups used in the study and their abbreviations.</p

DNA content for all the rBMSC and hWJC groups at 0, 2, and 3 weeks expressed as DNA (μg/ scaffold).

<p>(A) DNA content of all the rBMSC LA and LA CS groups. (B) DNA content of all the rBMSC HA and HA CS groups. (C) DNA content of all the hWJC LA and LA CS groups. (D) DNA content of all the hWJC HA and HA CS groups. All aggregate groups had statistically significant increase in DNA over week 0, HA groups had significantly higher values compared to the CS control groups at week 3. Values are reported as mean ± standard deviation, n = 4. (*) represents statistically significant difference from the week 0 value. (#) represents statistically significant difference from the previous time point and (**) represents statistically significant difference from the control at that time point (p < 0.05).</p

GAG content for all the rBMSC and hWJC groups at 0, 2, and 3 weeks expressed as GAG/DNA (A&B).

<p>All rBMSC aggregate groups had statistically significant increase in GAG/DNA over week 0, and select aggregates groups had significantly higher values compared to the control groups at week 3. GAG content for all the hWJC groups at week expressed as GAG/ DNA (C&D) at week 0, 2, and 3. 10 M HA at week 2 exhibited the highest GAG/DNA value and decreased at week 3. Values are reported as mean ± standard deviation, n = 4. (*) represents statistically significant difference from the week 0 value. (#) represents statistically significant difference from the previous time point and (**) represents statistically significant difference from the control at that time point. (p < 0.05).</p

Gene expression of Collagen I, Aggrecan, SOX9, and Runx-2 for hWJC groups at week 0, 2, and 3.

<p>hWJC groups at week 0, 2, and 3. There was a significant difference in SOX9 and Aggrecan gene expression at week 3 by groups 20 M HA and 20M HA CS groups. Values are reported as mean ± standard deviation, n = 4. (*) represents statistically significant difference over the week 0 value. (#) represents statistically significant highest value of the group (**) represents statistically significant difference from the control at that time point. (p < 0.05).</p

Representative images for immunohistochemistry analysis for collagen I, collagen II, and aggrecan staining for hWJC groups at week 0, 2, and 3.

<p>The 10M LA and 20M HA groups at week 0 displayed the highest staining intensity for aggrecan and 20M LA group at week 3 displayed the highest staining intensity for aggrecan. Scale bar = 200 μm.</p

Cell viability at weeks 0 and 3.

<p>(A) Live-dead images of the 10M/mL hWJC aggregates and controls. (B) Live dead images of the 20M/mL hWJC aggregates. Scale bar = 100 μm.</p

Summary of the best performing groups for all the functional assays.

<p>Summary of the best performing groups for all the functional assays.</p

Schematic representation of the hanging down approach and aggregate-encapsulation in agarose hydrogels.

<p>The aggregates/CS is encapsulated in pre-polymer solution and undergoes thermal crosslinking to form the hydrogel.</p