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

Microsphere-Based Osteochondral Scaffolds Carrying Opposing Gradients Of Decellularized Cartilage And Demineralized Bone Matrix

This document is the Accepted Manuscript version of a Published Work that appeared in final form in ACS Biomaterials Science & Engineering, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://doi.org/10.1021/acsbiomaterials.6b00071.Extracellular matrix (ECM) “raw materials” such as demineralized bone matrix (DBM) and cartilage matrix have emerged as leading scaffolding materials for osteochondral regeneration owing to their capacity to facilitate progenitor/resident cell recruitment, infiltration, and differentiation without adding growth factors. Scaffolds comprising synthetic polymers are sturdy yet generally lack cues for guiding cell differentiation. We hypothesized that opposing gradients of decellularized cartilage (DCC) and DBM in polymeric microsphere-based scaffolds would provide superior regeneration compared to polymer-only scaffolds in vivo. Poly(D,L-lactic-co-glycolic acid) (PLGA) microsphere-based scaffolds were fabricated, either with opposing gradients of DCC and DBM encapsulated (GRADIENT) or without DCC and DBM (BLANK control), and implanted into rabbit osteochondral defects in medial femoral condyles. After 12 weeks, gross morphological evaluation showed that the repair tissue in about 30% of the implants was either slightly or significantly depressed, hinting toward rapid polymer degradation in scaffolds from both of the groups. Additionally, no differences were observed in gross morphology of the repair tissue between the BLANK and GRADIENT groups. Mechanical testing revealed no significant differences in model parameter values between the two groups. Histological observations demonstrated that the repair tissue in both of the groups was fibrous in nature with the cells demonstrating notable proliferation and matrix deposition activity. No adverse inflammatory response was observed in any of the implants from the two groups. Overall, the results emphasize the need to improve the technology in terms of altering the DBM and DCC concentrations, and tailoring the polymer degradation to these concentrations.R01 AR056347Kansas Bioscience Authority Rising Star Awar

Cell activation-based screening of natively paired human T cell receptor repertoires

Adoptive immune therapies based on the transfer of antigen-specific T cells have been used successfully to treat various cancers and viral infections, but improved techniques are needed to identify optimally protective human T cell receptors (TCRs). Here we present a high-throughput approach to the identification of natively paired human TCRα and TCRβ (TCRα:β) genes encoding heterodimeric TCRs that recognize specific peptide antigens bound to major histocompatibility complex molecules (pMHCs). We first captured and cloned TCRα:β genes from individual cells, ensuring fidelity using a suppression PCR. We then screened TCRα:β libraries expressed in an immortalized cell line using peptide-pulsed antigen-presenting cells and sequenced activated clones to identify the cognate TCRs. Our results validated an experimental pipeline that allows large-scale repertoire datasets to be annotated with functional specificity information, facilitating the discovery of therapeutically relevant TCRs

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

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

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

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

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

<p>All rBMSC aggregate groups had statistically significant increase in collagen over week 0. At week 3, 20 M HA also displayed the highest collagen/DNA. Collagen content for all the hWJC groups expressed as collagen/DNA (C&D) at week 0, 2, and 3. We noticed that the 20 M HA at week 0 had the highest collagen/DNA value. 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 particular group and (**) represents statistically significant difference from the control at that time point. (p < 0.05).</p