Engineered Cartilage on Chitosan Calcium Phosphate Scaffolds for Osteochondral Defects

Articular cartilage provides an almost frictionless surface for the articulating ends of the bone. Cartilage functions to lubricate and transmit compressive forces resulting from joint loading and impact. If the cartilage is damaged, through traumatic injury or disease, it lacks the ability of self-repairing as the tissue lacks vascular system. If the injuries to articular cartilage are left untreated, they may progress to Osteoarthritis. Osteoarthritis, a degenerative disease, is one of the leading disabilities in the United States. Tissue engineering has the potential to regenerate healthy hyaline cartilage, which can alleviate pain and restore the functions of normal tissue. This study explores the production of engineered cartilage on top of composite calcium phosphate scaffold. The current research is related to a biphasic approach to cartilage tissue engineering — in which one layer supports to form subchondral bone (osteogenesis) and another supports cartilage formation (chondrogenesis). Chondrocyte and bone marrow-derived stem cell attachment to chitosan will be investigated for producing a bilayered construct for osteochondral repair. The main objectives of my research include the following: attachment and proliferation of human mesenchymal stem cells on chitosan calcium phosphate scaffolds, techniques to create a biphasic construct, the effect of coating chitosan calcium phosphate scaffolds with type I collagen and determining the ideal bead size for making chitosan calcium phosphate scaffolds

Engineered Cartilage on Chitosan Calcium Phosphate Scaffolds for Osteochondral Defects

Articular cartilage provides an almost frictionless surface for the articulating ends of the bone. Cartilage functions to lubricate and transmit compressive forces resulting from joint loading and impact. If the cartilage is damaged, through traumatic injury or disease, it lacks the ability of self-repairing as the tissue lacks vascular system. If the injuries to articular cartilage are left untreated, they may progress to Osteoarthritis. Osteoarthritis, a degenerative disease, is one of the leading disabilities in the United States. Tissue engineering has the potential to regenerate healthy hyaline cartilage, which can alleviate pain and restore the functions of normal tissue. This study explores the production of engineered cartilage on top of composite calcium phosphate scaffold. The current research is related to a biphasic approach to cartilage tissue engineering — in which one layer supports to form subchondral bone (osteogenesis) and another supports cartilage formation (chondrogenesis). Chondrocyte and bone marrow-derived stem cell attachment to chitosan will be investigated for producing a bilayered construct for osteochondral repair. The main objectives of my research include the following: attachment and proliferation of human mesenchymal stem cells on chitosan calcium phosphate scaffolds, techniques to create a biphasic construct, the effect of coating chitosan calcium phosphate scaffolds with type I collagen and determining the ideal bead size for making chitosan calcium phosphate scaffolds

Coatings on Mammalian Cells: Interfacing Cells with Their Environment

The research community is intent on harnessing increasingly complex biological building blocks. At present, cells represent a highly functional component for integration into higher order systems. In this review, we discuss the current application space for cellular coating technologies and emphasize the relationship between the target application and coating design. We also discuss how the cell and the coating interact in common analytical techniques, and where caution must be exercised in the interpretation of results. Finally, we look ahead at emerging application areas that are ideal for innovation in cellular coatings. In all, cellular coatings leverage the machinery unique to specific cell types, and the opportunities derived from these hybrid assemblies have yet to be fully realized

A Novel Through-Thickness Perfusion Bioreactor for the Generation of Scaffold-Free Tissue Engineered Cartilage

The objective of this study was to characterize our designed through-thickness perfusion bioreactor which could generate large scaffold-free tissue engineered cartilage constructs. The hypothesis being that through-thickness perfusion could accelerate maturation of scaffold-free tissue engineered cartilage, grown in transwell culture inserts large enough to repair typical size chondral lesions in the human knee. Internal cell culture media temperature and pH were examined over time, upon implementation of the bioreactor perfusion system inside a CO2 incubator, to ensure adequate regulation conducive to cell viability. Results indicate that temperature and pH both equilibrate within approximately 3 h. The bioreactor was tested for its efficacy to support formation of 4.5 cm2 constructs by porcine neonatal chondrocytes. Tests were conducted under three conditions: immediate perfusion with flow from bottom to top, immediate perfusion with media flow from top to bottom, and bottom to top perfusion after four weeks of static culture, giving the cells time to self-aggregate into a consolidated construct prior to perfusion. The best cell culture results were obtained when perfusion was delayed for four weeks relative to the immediate perfusion of the other methods, and this should be further investigated

A Novel Through-Thickness Perfusion Bioreactor for the Generation of Scaffold-Free Tissue Engineered Cartilage

The objective of this study was to characterize our designed through-thickness perfusion bioreactor which could generate large scaffold-free tissue engineered cartilage constructs. The hypothesis being that through-thickness perfusion could accelerate maturation of scaffold-free tissue engineered cartilage, grown in transwell culture inserts large enough to repair typical size chondral lesions in the human knee. Internal cell culture media temperature and pH were examined over time, upon implementation of the bioreactor perfusion system inside a CO2 incubator, to ensure adequate regulation conducive to cell viability. Results indicate that temperature and pH both equilibrate within approximately 3 h. The bioreactor was tested for its efficacy to support formation of 4.5 cm2 constructs by porcine neonatal chondrocytes. Tests were conducted under three conditions: immediate perfusion with flow from bottom to top, immediate perfusion with media flow from top to bottom, and bottom to top perfusion after four weeks of static culture, giving the cells time to self-aggregate into a consolidated construct prior to perfusion. The best cell culture results were obtained when perfusion was delayed for four weeks relative to the immediate perfusion of the other methods, and this should be further investigated

Coatings on mammalian cells: interfacing cells with their environment

Abstract The research community is intent on harnessing increasingly complex biological building blocks. At present, cells represent a highly functional component for integration into higher order systems. In this review, we discuss the current application space for cellular coating technologies and emphasize the relationship between the target application and coating design. We also discuss how the cell and the coating interact in common analytical techniques, and where caution must be exercised in the interpretation of results. Finally, we look ahead at emerging application areas that are ideal for innovation in cellular coatings. In all, cellular coatings leverage the machinery unique to specific cell types, and the opportunities derived from these hybrid assemblies have yet to be fully realized

Comparison of eosin and fluorescein conjugates for the photoinitiation of cell-compatible polymer coatings

<div><p>Targeted photopolymerization is the basis for multiple diagnostic and cell encapsulation technologies. While eosin is used in conjunction with tertiary amines as a water-soluble photoinitiation system, eosin is not widely sold as a conjugate with antibodies and other targeting biomolecules. Here we evaluate the utility of fluorescein-labeled bioconjugates to photopolymerize targeted coatings on live cells. We show that although fluorescein conjugates absorb approximately 50% less light energy than eosin in matched photopolymerization experiments using a 530 nm LED lamp, appreciable polymer thicknesses can still be formed in cell compatible environments with fluorescein photosensitization. At low photoinitiator density, eosin allows more sensitive initiation of gelation. However at higher functionalization densities, the thickness of fluorescein polymer films begins to rival that of eosin. Commercial fluorescein-conjugated antibodies are also capable of generating conformal, protective coatings on mammalian cells with similar viability and encapsulation efficiency as eosin systems.</p></div

Analysis of photoinitiator absorbance spectral overlap.

<p>Overlap of (a) SA-FITC and (b) SA-EITC with a Thorlabs collimated LED emitting green light. Lamp spectra obtained from manufacturer.</p

Comparison of polymer gelation thickness vs. photoinitiator density with cell compatible and unconstrained formulations and FITC photoinitiation.

<p>All samples were irradiated with 20mW/cm² of green light (530 nm LED lamp, ThorLabs) for 10 minutes. Cell compatible = 420 mM PEG-diacrylate, 21 mM triethanol amine, 35 mM vinyl pyrrolidone, in phosphate buffered media (pH = 7.5). Unconstrained = 420 mM PEG-diacrylate, 210 mM triethanol amine, 35 mM vinyl pyrrolidone in deionized water.</p