Inductive tissue engineering with protein and DNA-releasing scaffolds

Abstract

Cellular differentiation, organization, proliferation and apoptosis are determined by a combination of an intrinsic genetic program, matrix/substrate interactions, and extracellular cues received from the local microenvironment. These molecular cues come in the form of soluble (e.g. cytokines) and insoluble (e.g. ECM proteins) factors, as well as signals from surrounding cells that can promote specific cellular processes leading to tissue formation or regeneration. Recent developments in the field of tissue engineering have employed biomaterials to present these cues, providing powerful tools to investigate the cellular processes involved in tissue development, or to devise therapeutic strategies based on cell replacement or tissue regeneration. These inductive scaffolds utilize natural and/or synthetic biomaterials fabricated into three-dimensional structures. This review summarizes the use of scaffolds in the dual role of structural support for cell growth and vehicle for controlled release of tissue inductive factors, or DNA encoding for these factors. The confluence of molecular and cell biology, materials science and engineering provides the tools to create controllable microenvironments that mimic natural developmental processes and direct tissue formation for experimental and therapeutic applications.

Fig. 1. Release mechanisms for protein and DNA

(A) Inductive factors (red circles) may be embedded or encapsulated within hydrogels or microspheres, which may, in turn, be used to form 3-D scaffolds that are capable of releasing factors at the site of implantation. The highest factor concentration exists within the scaffold, with lower concentrations found within the surrounding tissue. Delivered factors target a variety of different cell populations (e.g., myocytes, neurons, fibroblasts or osteoblasts) for various applications (e.g., muscle, nerve, bone, wound healing). (B) Substrate immobilization is characterized by factors being bound to a biomaterial. The interaction between the factor and biomaterial can be (top): (1) k_on ≫ k_off, such that the factor is effectively bound irreversibly; (2) k_on ≈ k_off, where the factor associates and dissociates from the surface at roughly equal rates; and (3) k_on ≪ k_off, such that the factor is loaded onto the biomaterial and dissociates to serve as a delivery vehicle. Cells interact with the immobilized factors upon interaction with the biomaterial (bottom).

Fig. 2. Design parameters for controlled release systems

(A) Local factor concentration (top) can be customized to provide for high to low levels of factor released in the vicinity of the delivery device (represented by blue circles). Release profiles (bottom) can be designed to allow for short- (1) or long-term (2) release that slowly decays over time, or for a burst release (3) that then decays. Alternatively, DNA delivery can result in long-term protein production (4). Tailoring the release profile based on the degradation and clearance rates of the local environment can sustain therapeutic protein levels. Importantly, excess factor may produce undesirable side effects (e.g., toxicity), whereas insufficient protein will not produce the desired effect. (B) Concentration gradients of soluble factors can induce a variety of cellular processes, including cellular differentiation, orientation and migration. Parts of (B) adapted from ref. . (C) Multiple factors may be delivered simultaneously with variable kinetics to take advantage of their synergistic effects. In the case of proteins (e.g., growth factors), factor effect may be mediated by receptor binding and subsequent cellular internalization. Alternatively, DNA must be internalized and successfully transported to the nucleus for expression. Protein graphic prepared using MOLMOL. DNA graphic used with permission. (D) Spatial patterning of factors on biomaterial substrates can lead to selective cell adhesion or orientation. Photomicrographs reprinted from ref. , Copyright 2005, with permission from Elsevier.