No abstract

PC12 cells form aggregates when suspended within three-dimensional, self-assembled, type I collagen gels; these aggregates increase in size over time. In addition, when the cells are cultured in the presence of nerve growth factor, they express neurites, which extend through the three-dimensional matrix. In this report, the roles of fibronectin, laminin and nerve growth factor in PC12 cell aggregation and neurite growth following suspension in collagen matrices were evaluated. Single cells and small clusters of cells were suspended in collagen gels; the kinetics of aggregation were determined by measurement of the projected area of each aggregate, and neurite lengths were determined by measurement of end-to-end distance. Fibronectin and laminin inhibited the aggregation of PC12 cells at 50 micrograms/ml, and fibronectin, but not laminin, inhibited the growth of neurites at 100 micrograms/ml. In the absence of serum, the aggregation of cells cultured with nerve growth factor was almost completely inhibited, but the average neurite length was unaffected. In the presence of nerve growth factor, the extent of cell aggregation could not be explained simply by an increase in cell number, suggesting the presence of two separate mechanisms for aggregate growth: one dependent on cell motility and another dependent on cell proliferation.

Components of the extracellular matrix are believed to guide both nerve cells and neurites to their targets during embryogenesis and, therefore, might be useful for controlling regeneration of nervous tissue in adults. To study the influence of extracellular conditions on neurite outgrowth and cell motility, PC12 cells were suspended in three-dimensional gels containing (i) collagen (0.4 to 2 mg/mL), (ii) collagen (1 mg/mL) with added fibronectin or laminin (1 to 100 mug/mL), and (iii) agarose (7 mg/mL) with added collagen (0.001 to 1 mg/mL). Neurite outgrwoth was stimulated with nerve growth factor (NGF) and both the extent of neurite outgrowth ad cell aggregation were quantitated over 10 to 12 days in culture. The extent of neurite outgrowth was greatest at the lowest collagen concentration tested (0.4 mg/mL) and decreased with increasing concentration. The addition of laminin or fibronectin altered the extent of neurite outgrowth in collagen gels, but the differences were small. Although no neurite growth was observed in pure agarose gels, considerable neurite outgrowth occurred with the addition of small amounts (>/=0.01 mg/mL) of collagen. Mean aggregate size increased more quickly in gels with lower concentrations of collagen. For cells in 1.0 mg/mL collagen, a four- to fivefold increase in aggregate volume was seen between days 2 and 10 o the culture period, whereas the increase in DNA content during this same period was less than twofold, suggesting that the cells were aggregating, not multiplying. These results suggest that the composition of the matrix supporting nerve cells has a significant effect on both neurite outgrowth and cell motility. (c) 1994 John Wiley & Sons, Inc.

We have studied the release of nerve growth factor (NGF), a protein under consideration for treatment of Alzheimer's Disease, from polymer matrices and microspheres to characterize the stability of NGF, the dynamics of NGF release, and the distribution of NGF within the brain interstitium. Poly(ethylene-co-vinyl acetate) (EVAc) disks and poly(L-lactic acid) (PLA) microspheres were formed by codispersing NGF with one of a variety of molecules. The mass of mouse NGF (mNGF) detected following release from EVAc disks into buffered saline varied five-fold over the range of codispersants studied, with carboxymethyldextran providing optimal release, while the mass of recombinant human NGF (rhNGF) released varied four-fold from both EVAc disks and PLA microspheres, with albumin and carboxymethyldextran providing optimal release. Variation of the codispersant species significantly affected NGF release into buffered saline; it also had a noticeable, but small, effect of the amount of NGF found in the brain tissue following implantation of a polymer device. To improve NGF retention in tissue, NGF was conjugated to 70 000 molecular weight dextran and incorporated into a polymeric device. The distribution of NGF was enhanced by conjugation; comparison of NGF concentrations in the brain to a mathematical model of diffusion and elimination suggested that the elimination rate of NGF-dextran conjugate in the tissue was over seven times slower than the elimination rate of NGF. These results indicate that variation of the properties of the controlled release system may be useful in regulating the time course of NGF delivery to tissue, and that modification of the NGF itself can improve penetration and retention in the brain.

Local delivery of protein agents is potentially important in many tissue engineering systems. In this report, we evaluate an experimental system for measuring the rate of nerve growth factor (NGF) transport and biological activity within a 3-dimensional, tissue-like environment. Fetal brain cells or PC12 cells were suspended throughout collagen gel cultures; controlled-release matrices were used to control the spatial and temporal pattern of NGF release. Experimentally measured concentration profiles were compared to profiles predicted by a mathematical model encompassing diffusion and first-order elimination. Our results suggest that NGF moves through gels by diffusion while being eliminated at a rate that depends on cell density. Since diffusion and elimination also govern protein transport in brain tissue, the collagen gel serves as a model system that replicates the main features of transport in the brain and, therefore, can be used to identify new strategies that enhance NGF distribution in the central nervous system. As an example of the utility of this biophysical model, we demonstrate that implantation of multiple controlled-release matrices can broaden NGF distribution in gel cultures; this broadening was accompanied by a significant increase in cellular biological activity. This approach may be useful in customizing NGF distribution throughout degenerating or damaged central nervous system tissue while minimizing toxicity to surrounding healthy tissue.