Tunable Growth Factor Delivery from Injectable Hydrogels for Tissue Engineering

Current sustained delivery strategies of protein therapeutics are limited by the fragility of the protein, resulting in minimal quantities of bioactive protein delivered. In order to achieve prolonged release of bioactive protein, an affinity-based approach was designed which exploits the specific binding of the Src homology 3 (SH3) domain with short proline-rich peptides. Specifically, methyl cellulose was modified with SH3-binding peptides (MC-peptide) with either a weak affinity or strong affinity for SH3. The release profile of SH3-rhFGF2 fusion protein from hyaluronan MC-SH3 peptide (HAMC-peptide) hydrogels was investigated and compared to unmodified controls. SH3-rhFGF2 release from HAMC-peptide was extended to 10 days using peptides with different binding affinities compared to the 48 h release from unmodified HAMC. This system is capable of delivering additional proteins with tunable rates of release, while maintaining bioactivity, and thus is broadly applicable

Control Growth Factor Release Using a Self-Assembled [polycation∶heparin] Complex

The importance of growth factors has been recognized for over five decades; however their utilization in medicine has yet to be fully realized. This is because free growth factors have short half-lives in plasma, making direct injection inefficient. Many growth factors are anchored and protected by sulfated glycosaminoglycans in the body. We set out to explore the use of heparin, a well-characterized sulfated glycosaminoglycan, for the controlled release of fibroblast growth factor-2 (FGF-2). Heparin binds a multitude of growth factors and maintains their bioactivity for an extended period of time. We used a biocompatible polycation to precipitate out the [heparin∶FGF-2] complex from neutral buffer to form a release matrix. We can control the release rate of FGF-2 from the resultant matrix by altering the molecular weight of the polycation. The FGF-2 released from the delivery complex maintained its bioactivity and initiated cellular responses that were at least as potent as fresh bolus FGF-2 and fresh heparin stabilized FGF-2. This new delivery platform is not limited to FGF-2 but applicable to the large family of heparin-binding growth factors

The inhibitory effect of an RGD-human chitin-binding domain fusion protein on the adhesion of fibroblasts to reacetylated chitosan films

Biomaterials used for tissue engineering applications must provide a structural support for the tissue development and also actively interact with cells, promoting adhesion, proliferation, and differentiation. To achieve this goal, adhesion molecules may be used, such as the tripeptide Arg-Gly-Asp (RGD). A method based on the use of a carbohydrate-binding module, with affinity for chitin, was tested as an alternative approach to the chemical grafting of bioactive peptides. This approach would simultaneously allow the production of recombinant peptides (alternatively to peptide synthesis) and provide a simple way for the specific and strong adsorption of the peptides to the biomaterial. A fusion recombinant protein, containing the RGD sequence fused to a human chitin-binding module (ChBM), was expressed in E. coli. The adhesion of fibroblasts to reacetylated chitosan (RC) films was the model system selected to analyze the properties of the obtained proteins. Thus, the evaluation of cell attachment and proliferation on polystyrene surfaces and reacetylated chitosan films, coated with the recombinant proteins, was performed using mouse embryo fibroblasts 3T3. The results show that the recombinant proteins affect negatively fibroblasts anchorage to the materials surface, inhibiting its adhesion and proliferation. We also conclude that this negative effect is fundamentally due to the human chitin-binding domain.Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/27359/2006, POCTI/BIO/45356/200

Understanding the role of growth factors in modulating stem cell tenogenesis

Current treatments for tendon injuries often fail to fully restore joint biomechanics leading to the recurrence of symptoms, and thus resulting in a significant health problem with a relevant social impact worldwide. Cell-based approaches involving the use of stem cells might enable tailoring a successful tendon regeneration outcome. As growth factors (GFs) powerfully regulate the cell biological response, their exogenous addition can further stimulate stem cells into the tenogenic lineage, which might eventually depend on stem cells source. In the present study we investigate the tenogenic differentiation potential of human- amniotic fluid stem cells (hAFSCs) and adipose-derived stem cells (hASCs) with several GFs associated to tendon development and healing; namely, EGF, bFGF, PDGF-BB and TGF-β1. Stem cells response to biochemical stimuli was studied by screening of tendon-related genes (collagen type I, III, decorin, tenascin C and scleraxis) and proteins found in tendon extracellular matrix (ECM) (Collagen I, III, and Tenascin C). Despite the fact that GFs did not seem to influence the synthesis of tendon ECM proteins, EGF and bFGF influenced the expression of tendon-related genes in hAFSCs, while EGF and PDGF-BB stimulated the genetic expression in hASCs. Overall results on cellular alignment morphology, immunolocalization and PCR analysis indicated that both stem cell source can be biochemically induced towards tenogenic commitment, validating the potential of hASCs and hAFSCs for tendon regeneration strategies.Authors thank the Portuguese Foundation for Science and Technology (FCT) for the research project BIBS (PTDC/CVT/102972/2008) and for the post-doc fellowship grant: SFRH/BPD/86775/2012. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Three-Dimensional Cell and Tissue Patterning in a Strained Fibrin Gel System

Techniques developed for the in vitro reproduction of three-dimensional (3D) biomimetic tissue will be valuable for investigating changes in cell function in tissues and for fabricating cell/matrix composites for applications in tissue engineering techniques. In this study, we show that the simple application of a continuous strain to a fibrin gel facilitates the development of fibril alignment and bundle-like structures in the fibrin gel in the direction of the applied strain. Myoblasts cultured in this gel also exhibited well-aligned cell patterning in a direction parallel to the direction of the strain. Interestingly, the direction of cell proliferation was identical to that of cell alignment. Finally, the oriented cells formed linear groups that were aligned parallel to the direction of the strain and replicated the native skeletal muscle cell patterning. In addition, vein endothelial cells formed a linear, aligned vessel-like structure in this system. Thus, the system enables the in vitro reproduction of 3D aligned cell sets replicating biological tissue patterns

Human Neutrophil Elastase Responsive Delivery from Poly(ethylene glycol) Hydrogels

A novel enzyme-responsive hydrogel drug delivery system was developed with the potential to treat inflammation locally. Human neutrophil elastase (HNE) is a serine protease secreted by neutrophils which are the first cells recruited to inflammatory sites. We exploited this cell-secreted enzyme as a biological cue for controlled release. HNE sensitive peptide linkers were immobilized within poly(ethylene glycol) hydrogels using photopolymerization techniques. The kinetics of the enzyme reaction within the gel was tailored by varying the amino acid residues present in the P1 and P1 ′ substrate positions (immediately adjacent to cleavage location). A novel FRET-based hydrogel platform was designed to characterize the accessibility of the substrate within the cross-linked, macroscopic hydrogel. Lastly, a diffusion-reaction mathematical model with Michaelis-Menten kinetics was developed to predict the overall release profile and captured the initial 80 % of the experimentally observed release. The hydrogel platform presented shows highly controlled release kinetics with potential applications in cellular responsive drug delivery. 1

Carrageenan-based hydrogels for the controlled delivery of PDGF-BB in bone tissue engineering applications

One of the major drawbacks found in most bone tissue engineering approaches developed so far consists in the lack of strategies to promote vascularisation. Some studies have addressed different issues that may enhance vascularisation in tissue engineered constructs, most of them involving the use of growth factors (GFs) that are involved in the restitution of the vascularity in a damaged zone. The use of sustained delivery systems might also play an important role in the re-establishment of angiogenesis. In this study, !-carrageenan, a naturally occurring polymer, was used to develop hydrogel beads with the ability to incorporate GFs with the purpose of establishing an effective angiogenesis mechanism. Some processing parameters were studied and their influence on the final bead properties was evaluated. Platelet derived growth factor (PDGF-BB) was selected as the angiogenic factor to incorporate in the developed beads, and the results demonstrate the achievement of an efficient encapsulation and controlled release profile matching those usually required for the development of a fully functional vascular network. In general, the obtained results demonstrate the potential of these systems for bone tissue engineering applications.This work was supported by the European NoE EXPERTISSUES (NMP3-CT-2004-500283), the European STREP HIPPOCRATES (NMP3-CT-2003-505758), and by the Portuguese Foundation for Science and Technology (FCT) through the project PTDC/FIS/68517/2006 and through the V. Espirito Santo's Ph.D. grant (SFRH/BD/39486/2007)