Superporous, Semi-degradable Hydrogels for Cartilage Tissue Engineering +1Spiller, K L; 1Lowman A M

+1Drexel University, Philadelphia, PA, USA alowman@drexel.edu

INTRODUCTION: Repair of articular cartilage is limited because of a lack of access to the blood supply or to reparative stem cells. The use of biodegradable tissue engineering scaffolds to replace damaged cartilage has had some success, but the regenerated tissue has insufficient mechanical properties to support loads. Nondegradable hydrogels based on poly(vinyl-alcohol) (PVA) have been studied extensively as scaffolds for cartilage repair because of their similar mechanical and fluid transport properties. However, integration of PVA hydrogels with surrounding tissue is insufficient due to a lack of cell adhesion to the hydrophilic polymer matrix, limiting their feasibility as permanent implants. We hypothesized that blending a commonly used material known to be adhesive for cells, poly(lactic-co-glycolic acid) (PLGA), into the PVA hydrogel would increase cell migration, creating a simple method for making PVA hydrogels suitable as cartilage tissue engineering scaffolds. METHODS: Superporous hydrogels with degradable phases of 0, 30, and 60wt% PLGA with respect to the nondegradable phase of PVA were created through a modified double emulsion technique. A primary emulsion of PBS and PLGA dissolved in dichloromethane (DCM) was added to a 10wt% solution of PVA and 0.1wt% poly(vinyl pyrrolidone) for added stability, and stirred for 30 minutes before freezing in an acetone/dry ice bath. The volumes of DCM used were 10vol% for the hydrogels prepared with 0wt% PLGA and 35vol% for the hydrogels prepared with 30 or 60wt% PLGA. This double emulsion was then subjected to 6 cycles of freezing for 21hrs and thawing for 3hrs, creating a physically crosslinked hydrogel network around the organic phase. The resultant PLGA-PVA hydrogels were then dehydrated through an ethanol series and dried by critical point drying to retain their shape. Microcomputed tomography (µCT) and wet-mode environmental scanning electron microscopy (ESEM) were used to analyze the structure and porosity of the hydrogels over 7wks of swelling in phosphate buffered saline (PBS) at 37˚C. Five million immature porcine chondrocytes were seeded onto each cylindrical scaffold, about 10mm in diameter and 3mm in height. After 4 and 7 wks in standard chondrocyte culture conditions, the hydrogels were sectioned using a cryostat into 25µm sections and stained using hematoxylin and Safranin O to analyze cell and proteoglycan distribution and the depth of cell migration down into the hydrogels. The compressive moduli of the hydrogels were measured using uniaxial, unconfined compression. Porosity and cell migration were characterized as a function of PLGA content. Student’s t-test was used to analyze differences in porosity (n=3), compressive modulus (n=5), and depth of migration (n=3) (p<0.05). RESULTS: ESEM and 3D models generated from µCT revealed the interconnected porous structure of the semidegradable hydrogels (Fig 1).

Figure 1: Structure of the superporous, semi-degradable hydrogels.

The initial porosity of the hydrogels was around 60%, which did not change over the course of 7 weeks for the hydrogels without any PLGA, but increased to 75-80% porosity for the hydrogels made with PLGA, though the increases were not statistically significant (Fig 2).

Figure 2: Porosity of the hydrogels, determined using µCT, over the course of 7 weeks in physiological conditions. After 4 and 7 weeks of cell culture, the chondrocytes migrated less than 200µm in hydrogels prepared without PLGA, but they migrated over 1mm down into hydrogels prepared with 30 or 60wt% PLGA, travelling farther in hydrogels with higher PLGA content (Fig 3). Chondrocytes did not migrate farther after 7 weeks in culture than after 4 weeks.

Figure 3: Depth of migration, defined as the deepest section where clusters of cells were present. Histological analysis showed that the chondrocytes filled the pores of the hydrogels with deposited proteoglycans (Fig 4).

Figure 4: Cells (stained with hematoxylin) and proteoglycans (stained red with Safranin O) filled the pores of the hydrogels made with initial amounts of PLGA of (a) 0, (b) 30, and (c) 60wt% after 4 wks, and of (d) 0, (e), 30, and (f) 60wt% after 7 wks. Original magnification is 200x. The compressive moduli of the hydrogels were on the same order as healthy cartilage (6.93±1.75MPa), and were unaffected by the presence of cells, indicating that regenerated tissue did not fill all of the pores. Table 1: Compressive moduli of hydrogels.

Initial PLGA content

Comp. modulus after 4wks of swelling

Comp. modulus after 4wks in cell culture

0wt% 1.73±1.65 MPa 0.40±0.26 MPa 30wt% 0.37±0.27 MPa 0.31±0.20 MPa 60wt% 0.26±0.20 MPa 0.63±0.45 MPa

DISCUSSION: Hydrogels based on PVA were made porous and adhesive for chondrocytes through the simple blending of PLGA, using a novel technique based on a double emulsion. Porcine chondrocytes migrated through the highly interconnected porous network lined with PLGA to 1.4mm in the hydrogels with the highest amount of PLGA. This technology has the potential to finally make PVA hydrogels feasible for cartilage repair. Future work includes further enhancing cell migration through the use of sustained release of growth factors from the hydrogels.

Poster No. 1285 • 56th Annual Meeting of the Orthopaedic Research Society