Search for: All records

Recreating articular cartilage tri-layered patterning for an engineered in vitro cell construct holds promise for advancing cartilage repair efforts. Our approach involves the development of a mul-tichambered perfusion tissue bioreactor that regulates fluid shear stress levels similar to the gradated hydrodynamic environment in articular cartilage. COMSOL modeling reveals our ta-pered cell chamber design will produce three different shear levels, high in the 22 – 41 mPa range, medium in the 4.5 – 8.4 mPa range, and low in the 2.2 – 3.8 mPa range and distributed across the surface of our mesenchymal stromal cell (MSC) encapsulated construct. In a 14-day bioreactor culture, we assess how fluid shear magnitude and cell vertical location within a 3D construct influence cell chondrogenesis. Notably, Sox9 expression for MSCs cultivated in our reactor shows spatially patterned gene upregulations coding for key chondrogenic marker pro-teins. Beginning with the high shear stress region, lubricin and type II collagen gene increases of 410 and 370-fold indicate cell movement towards a superficial zone architype which is further supported by histological and immunohistochemical stains illustrating the formation of a dense proteoglycan matrix enriched with lubricin, versican, and collagen types I and II molecules. For the medium shear stress region high aggrecan and type II collagen gene expressions of 2.3 and 400-fold, respectively, along with high proteoglycan analyses show movement toward a superfi-cial/mid-zone cartilage architype. For low shear stress regions higher collagen types II and X gene upregulations of 550 and 8,300-fold, the latter being 2x of that for the high shear regime, indicate cell movement with deep zone characteristics. Collectively, biochemical analysis, histol-ogy, and gene expression data demonstrated that our fluid shear bioreactor induced a stratified structure within tissue engineered constructs, demonstrating the feasibility of using this ap-proach to recapitulate the structure of native articular cartilage. 

Osteoarthritis, a chronic disease, remains an issue for adults that causes cartilage degradation within a joint . According to the Centers forDisease Control and Prevention (2023), over 32.5 million adults in the US are affected by osteoarthritis (OA). In this study we seek tounderstand the connection between tissue engineering and genetics to regenerate human articular cartilage (hAC). We purpose to validatea protocol for RNA isolation and characterize the transcriptome of hAC in a tri-layer fashion via bulk RNA sequencing (bulk-RNA-seq).Additionally, we aim to analyze the transcriptome of normal articular cartilage in comparison to the hAC chemical composition and physicalproperties. We are relating these properties to the tri-layers of hAC through histological staining with Safranin O—Fast green and imagingwith differential interference contrast (DIC) microscopy. We are relating these properties to superfic ial, middle, and deep zone with acryotome procedure, RNA extracted, and qualified by Bioanalyzer. Next, we generate bulk RNA sequencing of hAC layer-by-layer andcompare results to early passaging of Mesenchymal Stromal Cells (MSC) and tissues intended for Matrix-Induced Autologous ChondrocyteImplantation (MACI). We will use differential gene expression (DE) analysis by DESeq2 R package software for bulk-RNA-seq. The resultwill be interpreted in terms of differentiation from MSCs to gene expression patterns of tri-layer hAC. We will report on development andvalidation of protocols for isolating cells and their subsequent characterization with application in regenerating the tri-layered hACtranscriptome stimulatory bioreactors used in our laboratory and corresponding properties of the extracellular matrix (ECM) 

Introduction: Directing mesenchymal stem cell (MSC) chondrogenesis by bioreactor cultivation provides fundamental insight towards engineering healthy, robust articular cartilage (AC). The mechanical environment is represented by compression, fluid shear stress, hydrostatic pressure, and tension which collectively contribute to the distinct spatial organization of AC. Mimicking this cell niche is necessary for dictating cell growth, fate, and role. Researchers have shown that different mechanical stimulus types improve MSC chondrogenic commitment demonstrated by increases in key chondrogenic gene and protein markers. However, challenges remain in manufacturing spatially, anisotropic AC consisting of defined regions such as native tissue. Our strategy towards furthering this effort involves exposing MSC-laden alginate scaffolds in a multi-chambered, perfusion bioreactor with controlled fluid shear stress magnitudes to better mimic the native AC microenvironment leading to defined regions throughout the scaffold marked by varied cellular phenotypes. Validations made from assessing biochemical content, mRNA expression, western blot analysis, and cell viability will provide meaningful insight towards regulating MSC chondrogenesis. Methods: MSCs grown up to passage 4 were expanded to confluency in a T-175 flask then released from the surface using trypsin. Cells were stored in -80 ℃ freezer until experimentation. Our bioreactor system was sterilized by UV radiation for 4 hours then perfused with 70% ethanol overnight. Cell-laden scaffolds were prepared by first dissolving 1.5% alginate into deionized water. The polymeric solution was sterilely filtered and stored until usage. Cryopreserved MSCs were thawed and suspended in α-MEM medium containing essential supplements. Cells were counted and resuspended in alginate at a density of 106 cells/mL. The mixture was transferred to our multi-chambered bioreactor where they were allowed to crosslink in CaCl2 solution for 45 min. Separate scaffolds (N = 3) were molded within an identical reactor system and removed to serve as a control to compare effects of fluid shear stress on MSC differentiation. All, structures were washed with PBS then supplied with DMEM/F-12 medium containing 10% FBS, 1% penicillin/streptomycin , 1% L-glutamine, 100 nM dexamethasone, 50 µg/mL L-ascorbic acid, and10 ng/mL TGF-β3. The flowrate for the bioreactor was adjusted to 20 mL/min which provided desired fluid shear ranges of 2-87 mPa to stimulate the cells . Cell cultures were grown for 7 days, and medium changed every 3 days. Sectioned samples were analyzed for biochemical content, mRNA expression, and western blot to understand the impact of fluid shear stress magnitudes on MSC differentiation. Results: Directed fluid shear stress across a cell-laden alginate scaffold contained within an individual chamber in our bioreactor indicates varied cellular behavior within the superficial and deep regions of the construct marked by spatially secreted biochemical content as well as mRNA expression. This observation is supported by superficial MSCs stimulated by high and medium mechanical stimulation which indicates a 1.3 and 1.2-fold increase in total collagen production, respectively, when directly compared to cells deep in the construct. A similar effect is supported by total GAG secretion where high and medium shear stress across the fluid hydrogel interface yielded 1.2 and 1.3-fold upregulation of protein secretion, respectively, when observed under similar conditions. Perfused MSCs show upregulation to 3 and 20-fold for Sox9 and aggrecan, respectively, compared to a static culture. Shear ranges distributed throughout our cell-laden alginate scaffold correlates to differential chondrogenic commitment shown by variance of Sox9 expression when assessed by location and depth. Additional information on COL10A1 expression demonstrates mechanical stimulation that reduces hypertrophic cell differentiation contrary to a static culture. Discussion: In this investigation we emphasize that cells respond differently to mechanical stimulation when located in either the superficial or deep region of an alginate scaffold. This observation is supported by enhanced matrix production of chondrogenic protein for cells near the perfused fluid and hydrogel interface compared to deeper areas when stimulated by high and medium fluid shear loading regimes. Most importantly, maintenance of a healthy fluid shear gradient in our TBR provides evidence of promoting MSC chondrogenesis by spatially upregulating anabolic cartilage-like markers in addition to diminishing the onset of cell hypertrophy. Our efforts in monitoring mRNA expression of our samples reveals enhancement of chondrogenic cell differentiation for a perfused sample marked by increases in Sox9 and aggrecan genes; whereas a static sample stimulated only by TGF-β3 leads to undesirable expression of COL10A1. Key takeaways from our study support the contributions from previous researchers in recreating the native AC mechanical environment to encourage MSC differentiation. The development of our TBR system for controlled delivery of fluid shear stresses to MSCs furthers efforts in spatially guiding MSC chondrogenesis which is critical for engineering zonally differentiated AC.