Three-dimensional Stem Cell Aggregates for Cartilage Tissue Engineering

Osteoarthritis is the degradation of the knee cartilage within the joints that is often painful and can be debilitating and affect movement, thus impacting the overall quality of life. Current approved treatment methods include microfracture, autologous chondrocyte implantation (ACI) and osteochondral autograft transfer system (OATS). Tissue engineering approaches that are still under clinical trials include naturally derived or synthetic biocompatible scaffolds that possess good mechanical properties, or minimally manipulated devices like decellularized juvenile cartilage that are yet to demonstrate long-term benefits. Using the therapeutic potential of undifferentiated stem cells not only provides a sustainable and long-term approach but also promotes natural healing of the damages cartilage through biochemical cues. The current thesis work describes the evaluation of 3D stem cell aggregates for the regeneration of the osteochondral interface to overcome some of the limitations posed by the above surgical techniques. In vitro and in vivo studies were completed to evaluate the performance of aggregates in both a hydrogel system and a rat in vivo system. Compared to conventional cell suspensions, aggregates had improved cellular response, biochemical content and higher chondroinductive gene expression. Furthermore, the chondrogenic nature of the aggregate was next investigated as a consequence of cellular media priming and substrate coating. Specifically, ‘raw materials’ such as chondroitin sulfate and aggrecan in the culture medium outperformed growth factors in chondroinductive gene expression and collagen production, thus producing chondromimetic cellular aggregates. In vivo studies demonstrated the feasibility and efficacy of cellular aggregate technology with two different cell densities and combining them with fibrin at the defect site. In the Sprague-Dawley rat knee, implants with higher cell density aggregates received higher morphology scores, better immunohistochemistry staining, and demonstrated the best defect filling compared to the fibrin-alone group. Moreover, this thesis has taken the cell-aggregate technology from an in vitro proof of concept study to a translational in vivo animal model, thus opening new avenues of investigation for further refinement of the technology. The important next steps will be to characterize the mechanical properties of the scaffolds in an in vivo model, and further explore the regenerative-potential of the stem cell aggregates in a large animal model

Subcritical CO2 Sintering of Microspheres of Different Polymeric Materials to Fabricate Scaffolds for Tissue Engineering

The aim of this study was to use CO2 at sub-critical pressures as a tool to sinter 3D, macroporous, microsphere-based scaffolds for bone and cartilage Tissue Engineering Porous scaffolds composed of ~200 µm microspheres of either poly(lactic-co-glycolic acid) (PLGA) or polycaprolactone (PCL) were prepared using dense phase CO2 sintering, which were seeded with rat bone marrow mesenchymal stromal cells (rBMSCs), and exposed to either osteogenic (PLGA, PCL) or chondrogenic (PLGA) conditions for 6 weeks. Under osteogenic conditions, the PLGA constructs produced over an order of magnitude more calcium than the PCL constructs, whereas the PCL constructs had far superior mechanical and structural integrity (125 times stiffer than PLGA constructs) at week 6, along with twice the cell content of the PLGA constructs. Chondrogenic cell performance was limited in PLGA constructs, perhaps as a result of the polymer degradation rate being too high. The current study represents the first long-term culture of CO2-sintered microsphere-based scaffolds, and has established important thermodynamic differences in sintering between the selected formulations of PLGA and PCL, with the former requiring adjustment of pressure only, and the latter requiring the adjustment of both pressure and temperature. Based on more straightforward sintering conditions and more favorable cell performance, PLGA may be the material of choice for microspheres in a CO2 sintering application, although a different PLGA formulation with the encapsulation of growth factors, extracellular matrix-derived nanoparticles, and/or buffers in the microspheres may be advantageous for achieving a more superior cell performance than observed here

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

Tailoring of processing parameters for sintering microsphere-based scaffolds with dense phase carbon dioxide

Jeon, J. H., Bhamidipati, M., Sridharan, B., Scurto, A. M., Berkland, C. J. and Detamore, M. S. (2013), Tailoring of processing parameters for sintering microsphere-based scaffolds with dense-phase carbon dioxide. J. Biomed. Mater. Res., 101B: 330–337. doi:10.1002/jbm.b.32843Microsphere-based polymeric tissue-engineered scaffolds offer the advantage of shape-specific constructs with excellent spatiotemporal control and interconnected porous structures. The use of these highly versatile scaffolds requires a method to sinter the discrete microspheres together into a cohesive network, typically with the use of heat or organic solvents. We previously introduced subcritical CO2 as a sintering method for microsphere-based scaffolds; here we further explored the effect of processing parameters. Gaseous or subcritical CO2 was used for making the scaffolds, and various pressures, ratios of lactic acid to glycolic acid in poly(lactic acid-co-glycolic acid), and amounts of NaCl particles were explored. By changing these parameters, scaffolds with different mechanical properties and morphologies were prepared. The preferred range of applied subcritical CO2 was 15–25 bar. Scaffolds prepared at 25 bar with lower lactic acid ratios and without NaCl particles had a higher stiffness, while the constructs made at 15 bar, lower glycolic acid content, and with salt granules had lower elastic moduli. Human umbilical cord mesenchymal stromal cells (hUCMSCs) seeded on the scaffolds demonstrated that cells penetrate the scaffolds and remain viable. Overall, the study demonstrated the dependence of the optimal CO2 sintering parameters on the polymer and conditions, and identified desirable CO2 processing parameters to employ in the sintering of microsphere-based scaffolds as a more benign alternative to heat-sintering or solvent-based sintering methods

Portable microfluidic chip for detection of Escherichia coli in produce and blood

Pathogenic agents can lead to severe clinical outcomes such as food poisoning, infection of open wounds, particularly in burn injuries and sepsis. Rapid detection of these pathogens can monitor these infections in a timely manner improving clinical outcomes. Conventional bacterial detection methods, such as agar plate culture or polymerase chain reaction, are time-consuming and dependent on complex and expensive instruments, which are not suitable for point-of-care (POC) settings. Therefore, there is an unmet need to develop a simple, rapid method for detection of pathogens such as Escherichia coli. Here, we present an immunobased microchip technology that can rapidly detect and quantify bacterial presence in various sources including physiologically relevant buffer solution (phosphate buffered saline [PBS]), blood, milk, and spinach. The microchip showed reliable capture of E. coli in PBS with an efficiency of 71.8% ± 5% at concentrations ranging from 50 to 4,000 CFUs/mL via lipopolysaccharide binding protein. The limits of detection of the microchip for PBS, blood, milk, and spinach samples were 50, 50, 50, and 500 CFUs/mL, respectively. The presented technology can be broadly applied to other pathogens at the POC, enabling various applications including surveillance of food supply and monitoring of bacteriology in patients with burn wounds

Living Bacterial Sacrificial Porogens to Engineer Decellularized Porous Scaffolds

Decellularization and cellularization of organs have emerged as disruptive methods in tissue engineering and regenerative medicine. Porous hydrogel scaffolds have widespread applications in tissue engineering, regenerative medicine and drug discovery as viable tissue mimics. However, the existing hydrogel fabrication techniques suffer from limited control over pore interconnectivity, density and size, which leads to inefficient nutrient and oxygen transport to cells embedded in the scaffolds. Here, we demonstrated an innovative approach to develop a new platform for tissue engineered constructs using live bacteria as sacrificial porogens. E.coli were patterned and cultured in an interconnected three-dimensional (3D) hydrogel network. The growing bacteria created interconnected micropores and microchannels. Then, the scafold was decellularized, and bacteria were eliminated from the scaffold through lysing and washing steps. This 3D porous network method combined with bioprinting has the potential to be broadly applicable and compatible with tissue specific applications allowing seeding of stem cells and other cell types

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

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

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

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