CHME - Bioseparations Biomaterials Group Project F

Project F

Role of matrix architecture
Our research seeks to better understand how mechanical factors and their interaction with scaffold/matrix design parameters influence cellular activity in biomimetic matrices. The goal is to obtain the following: (1) a correlation between fabrication technique, materials property, and cellular-response and, (2) to ascertain if nanofibrous scaffolds impact cellular orientation, ultrastructure of deposited collagen and biosynthetic activity of seeded chondrocytes. Development of a scaffold with aligned nano- or sub-micron fibers that can support chondrogenesis would be a significant advancement not only in terms of the quantity of neocartilage produced, but also in terms of the ability of that tissue to integrate with the host matrix.

My research has included the evaluation of the material and cell-responsive property of nanofibrillar scaffolds made from natural polymers (chitosan and gelatin) and blends of natural and synthetic polymers (PLA grafted chitosan) [Subramanian et al., J.Biomat.Sci.Polymer Ed. 1-3, 2005 and Skotak et al., Biomacromolecules, 9(7), 1902-1908,2008]. In the context of tissue engineering the effect of micro-topography as afforded by scaffold morphology is an important design parameter. Specifically, the technique of electrospinning has extensively been employed to generate scaffolds with nanoscale topography and there are abundant examples in the literature on the ability of nanofibrous scaffolds to support cell growth and proliferation. However, the mechanisms by which cells adapt and respond to nanoscale topography remains unclear and is thus worthy of investigation.

In our work, we have varied the nanoscale topography by varying the fiber diameter and we have evaluated the effect of fiber diameter on gene regulation and biosynthetic activity of chondrocytes on nanofibrous scaffolds. To assess the ability of the nanofibrous scaffolds to permit or promote chondrogenic redifferentiation, chondrogenesis was assessed by RT-PCR for cartilage-specific gene markers and expression was normalized to GADPH, a housekeeping gene. We have analyzed the expression of select cartilage specific genes at day 14 of human articular chondrocytes cultured on TCP, on scaffolds prepared by freeze-drying and lyophilization and chitosan nanofibrous (NF) scaffolds (shown in figure 1). The nanofibrous scaffolds were able to sustain differentiation on the basis of the mRNA expression levels of collagen type II and aggrecans (GAG). These preliminary results indicate that biological activities of chondrocytes are at least partially dependent on the architecture of the scaffolds, and that nanofibrous chitosan scaffolds may act as biologically preferred substrates.

Project F Diagram
Project F Diagram
Figure 1: Collagen-I ratio by RT-PCR analysis

In a parallel project, hybrid- or grafted-polymeric systems like PLA-grafted chitosan fibers were developed, in an effort to find a substrate with tunable biomaterial properties [Skotak et al., Biomacromolecules, 9(7), 1902-1908,2008]. This general synthetic route rendered functionalized chitosan soluble in a broad range of organic solvents, facilitating formation of ultrafine fibers via electrospinning. Electrospun L-lactide modified chitosan fibers are promising, as a scaffolding material for tissue engineering applications.

In a separate experiment, creep indentation tests were conducted to determine the aggregate modulus, shear modulus, and permeability of hydrated seeded scaffolds at various cultures’ time periods and reported in Table 1. As noted in Table 1, a wide variability was observed; however, values in the range of articular cartilage were also noted. The overall favorable response of chondrocytes to the nanofibrous scaffolds indicates they are promising candidates for further studies on an in-vitro cartilage model and as substrates for the cell expansion application in the field of cartilage regenerative medicine (Figure-2).

Project F Diagram
Figure 2: Confocal images of chondrocytes stained with DAPI and Eosin cultured for 8 and 15 days (A, C and B, D, respectively) on gelatin electrospun scaffolds cross-linked with 0.1% (A, B) and 1.0% (C, D) of glutaraldehyde. Bovine cartilage chondrocytes are presented for comparison (E). Scale bar (50µm) is the same for all images. Cell density measurement (F) indicates statistical difference (p > 0.05) between scaffolds cross-linked with 0.1% GA cultured for 7 days and remaining specimen. Samples marked with asterisk were not statistically different (p < 0.05). Estimated cell density for bovine cartilage (E) is 1103±260 mm-2.
Project F Diagram