Nanostructured polymer scaffolds for tissue engineering and regenerative medicine.

The structural features of tissue engineering scaffolds affect cell response and must be engineered to support cell adhesion, proliferation and differentiation. The scaffold acts as an interim synthetic extracellular matrix (ECM) that cells interact with prior to forming a new tissue. In this review, bone tissue engineering is used as the primary example for the sake of brevity. We focus on nanofibrous scaffolds and the incorporation of other components including other nanofeatures into the scaffold structure. Since the ECM is comprised in large part of collagen fibers, between 50 and 500 nm in diameter, well-designed nanofibrous scaffolds mimic this structure. Our group has developed a novel thermally induced phase separation (TIPS) process in which a solution of biodegradable polymer is cast into a porous scaffold, resulting in a nanofibrous pore-wall structure. These nanoscale fibers have a diameter (50-500 nm) comparable to those collagen fibers found in the ECM. This process can then be combined with a porogen leaching technique, also developed by our group, to engineer an interconnected pore structure that promotes cell migration and tissue ingrowth in three dimensions. To improve upon efforts to incorporate a ceramic component into polymer scaffolds by mixing, our group has also developed a technique where apatite crystals are grown onto biodegradable polymer scaffolds by soaking them in simulated body fluid (SBF). By changing the polymer used, the concentration of ions in the SBF and by varying the treatment time, the size and distribution of these crystals are varied. Work is currently being done to improve the distribution of these crystals throughout three-dimensional scaffolds and to create nanoscale apatite deposits that better mimic those found in the ECM. In both nanofibrous and composite scaffolds, cell adhesion, proliferation and differentiation improved when compared to control scaffolds. Additionally, composite scaffolds showed a decrease in incidence of apoptosis when compared to polymer control in bone tissue engineering. Nanoparticles have been integrated into the nanostructured scaffolds to deliver biologically active molecules such as growth and differentiation factors to regulate cell behavior for optimal tissue regeneration.

Confocal laser scanning microscopy as a tool for imaging cancellous bone.

Understanding the bimodal structure of cancellous bone is important for tissue engineering in order to more accurately fabricate scaffolds to promote bone ingrowth and vascularization in newly forming bone. In this study, confocal laser scanning microscopy (CLSM) was used to create detailed images of the bimodally porous intertrabecular space of defatted and deproteinized cancellous canine bone taken from the epiphysis of the humerus. The bimodal pore structure was imaged using both reflective and fluorescent modes in CLSM, resulting in four different, but complementary image types: (1) a Z-stack overlay, (2) a phi-Z scan, (3) a topographical map, and (4) a contour map. Submerging the bone in rhodamine B dye prior to fluorescent imaging enhanced the pore surface details, giving a more accurate pore size measurement. The average macropore diameter was found to be 260 +/- 97 microm while the average micropore diameter was 13 +/- 10 microm. When compared with common techniques, including microcomputed tomography, magnetic resonance imaging, scanning electron microscopy, and environmental scanning electron microscopy, for imaging cancellous bone, CLSM was found to be an effective tool, given its ability to nondestructively image the surface and near-surface pore structure.

Surface potential and osteoblast attraction to calcium phosphate compounds is affected by selected alkaline hydrolysis processing.

This study examines the link(s) between the suspension behavior of calcium deficient apatites (CDAs) and biphasic calcium phosphate (BCP), as measured by the zeta-potential, with respect to both whole bone and osteoblasts. CDA is fabricated by hydrolyzing an acidic CaP such as dicalcium diphosphate dihydrate (DCPD; CaHPO4.2H2O) and has a structure and composition close to bone apatite. Sintering CDA results in the formation of BCP ceramics consisting of mixtures of hydroxyapatite (HA) and beta-tricalcium phosphate (beta-TCP), with the HA/beta-TCP weight ratio proportional to the Ca/P ratio of CDA. The choice of the base for the DCPD hydrolysis allows various ionic partial substitution of the formed CDA. Na for Ca partial substitution is of interest because of the resulting improvement in mechanical properties of the resulting BCP ceramics and NH4OH was used as a negative control. The zeta-potential was measured for these materials and the stability of the ceramic to bone interaction calculated. zeta-potential values decrease for CDA(NH4OH) versus CDA(NaOH) and increase for BCP(NH4OH) versus BCP(NaOH). While results of these analyses indicate that NH4OH and NaOH processed CDA and BCP will likely yield osteoblast attachment in vivo, differences in the zeta-potentials may explain varying degrees of cell attachment.

Electrostatic interactions as a predictor for osteoblast attachment to biomaterials.

The present study utilizes zeta (zeta)-potential analysis as an indicator of bonding of osteoblasts and whole bone to various biomaterials. Common metal alloys (316L stainless steel, CoCrMo, and Ti6Al4V) and bioceramics (hydroxyapatite and beta-tricalcium phosphate) used in orthopedic applications were suspended in particulate form in physiologic saline, both as-received and supplemented with bovine serum albumin (BSA). Metal alloys were also treated with NaOH washing to study the effect of such a surface treatment on the zeta-potential. The NaOH wash was found to increase the zeta-potential for CoCrMo and Ti6Al4V, but there was a decrease in the magnitude of the zeta-potential for 316L stainless steel. When the metal alloy powders were suspended in BSA-supplemented physiologic saline, the zeta-potential as a function of pH increased, thereby increasing the electronegativity gap and increasing the propensity for bonding between each of the metal alloys and bone. This increase is likely due to matrix proteins in the BSA, which adsorb onto the metal alloy surfaces, promoting bone growth. With the addition of BSA to each bioceramic system, a uniform decrease in zeta-potential was observed. However, the electronegativity gap remained large in each case, maintaining the anticipation of bonding. zeta-Potential analysis is an effective predictor of biomaterial attraction to osteoblasts and bone, providing a useful in vitro method for predicting such interactions.