ß-Sheet to Random Coil Transition in Self-Assembling Peptide Scaffolds Promotes Proteolytic Degradation.

One of the most desirable properties that biomaterials designed for tissue engineering or drug delivery applications should fulfill is biodegradation and resorption without toxicity. Therefore, there is an increasing interest in the development of biomaterials able to be enzymatically degraded once implanted at the injury site or once delivered to the target organ. In this paper, we demonstrate the protease sensitivity of self-assembling amphiphilic peptides, in particular, RAD16-I (AcN-RADARADARADARADA-CONH2), which contains four potential cleavage sites for trypsin. We detected that when subjected to thermal denaturation, the peptide secondary structure suffers a transition from ß-sheet to random coil. We also used Matrix-Assisted Laser Desorption/Ionization-Time-Of-Flight (MALDI-TOF) to detect the proteolytic breakdown products of samples subjected to incubation with trypsin as well as atomic force microscopy (AFM) to visualize the effect of the degradation on the nanofiber scaffold. Interestingly, thermally treated samples had a higher extent of degradation than non-denatured samples, suggesting that the transition from ß-sheet to random coil leaves the cleavage sites accessible and susceptible to protease degradation. These results indicate that the self-assembling peptide can be reduced to short peptide sequences and, subsequently, degraded to single amino acids, constituting a group of naturally biodegradable materials optimal for their application in tissue engineering and regenerative medicine.

Functionalized self-assembling peptide hydrogel enhance maintenance of hepatocyte activity in vitro.

There is a major challenge in maintaining functional hepatocytes in vivo as these cells rapidly lose their metabolic properties in culture. In this work we have developed a bioengineered platform that replaces the use of the collagen I--in the traditional culture sandwich technique--by a defined extracellular matrix analogue, the self-assembling peptide hydrogel RAD16-I functionalized with biologically active motifs. Thus, after examining side by side the two culture systems we have found that in both cases hepatocytes acquired similar parenchymal morphology, presence of functional bile canaliculi structures, CYP3A2 induction by dexamethasone, urea production, secretion of proteins such as apolipoprotein (class A1, E, J), alpha(1)-microglobulin, alpha(1)-macroglobulin, retinol binding protein, fibronectin, alpha(1)-inhibitor III and biotin-dependent carboxylases. Interestingly, by assessing in more detail some other hepatic markers, one of the functionalized matrix analogues--carrying the 67 kD laminin receptor ligand--enhanced the gene expression of albumin, HNF4-alpha, MDR2 and tyrosine aminotransferase. We conclude that the use of a synthetic culture system with designed matrix functionalization has the advantage in controlling specific cellular responses.

Early tissue patterning recreated by mouse embryonic fibroblasts in a three-dimensional environment.

Cellular self-organization studies have been mainly focused on models such as Volvox, the slime mold Dictyostelium discoideum, and animal (metazoan) embryos. Moreover, animal tissues undergoing regeneration also exhibit properties of embryonic systems such as the self-organization process that rebuilds tissue complexity and function. We speculated that the recreation in vitro of the biological, biophysical, and biomechanical conditions similar to those of a regenerative milieu could elicit the intrinsic capacity of differentiated cells to proceed to the development of a tissue-like structure. Here we show that, when primary mouse embryonic fibroblasts are cultured in a soft nanofiber scaffold, they establish a cellular network that causes an organized cell contraction,proliferation, and migration that ends in the formation of a symmetrically bilateral structure with a distinct central axis. A subset of mesodermal genes (brachyury, Sox9, Runx2) is upregulated during this morphogenetic process. The expression of brachyury was localized first at the central axis, extending then to both sides of the structure. The spontaneous formation of cartilage-like tissue mainly at the paraxial zone followed expression ofSox9 and Runx2. Because cellular self-organization is an intrinsic property of the tissues undergoing development,this model could lead to new ways to consider tissue engineering and regenerative medicine.

Interstitial fluid flow intensity modulates endothelial sprouting in restricted Src-activated cell clusters during capillary morphogenesis.

Development of tissues in vitro with dimensions larger than 150 to 200 microm requires the presence of a functional vascular network. Therefore, we have studied capillary morphogenesis under controlled biological and biophysical conditions with the aim of promoting vascular structures in tissue constructs. We and others have previously demonstrated that physiological values of interstitial fluid flow normal to an endothelial monolayer in combination with vascular endothelial growth factor play a critical role during capillary morphogenesis by promoting cell sprouting. In the present work, we studied the effect that a range of interstitial flow velocities (0-50 microm/min) has in promoting the amount, length, and branching of developing sprouts during capillary morphogenesis. The number of capillary-like structures developed from human umbilical vein endothelial cell monolayers across the interstitial flow values tested was not significantly affected. Instead, the length and branching degree of the sprouts presented a significant maximum at flow velocities of 10 to 20 microm/min. More-over, at these same flow values, the phosphorylation level of Src also showed its peak. We discovered that capillary morphogenesis is restricted to patches of Src-activated cells (phosphorylated Src (pSrc)) at the monolayer, suggesting that the transduction pathway in charge of sensing the mechanical stimulus induced by flow is promoting predetermined mechanically sensitive areas (pSrc) to undergo capillary morphogenesis

Osteogenic differentiation of mouse embryonic stem cells and mouse embryonic fibroblasts in a three-dimensional self-assembling peptide scaffold.

In the present work, we studied the differentiation capacity of mouse embryonic stem cells (mESCs) and mouse embryonic fibroblasts (MEFs) to differentiate into osteoblast-like cells in a 3-dimensional (3D) self-assembling peptide scaffold, a synthetic nanofiber biomaterial with potential applications in regenerative medicine. We demonstrated that 2D and 3D systems promoted differentiation of mESCs into cells with an osteoblast-like phenotype consisting of osteopontin and collagen I marker expression, as well as high alkaline phosphatase (ALP) activity and calcium phosphate deposits. In 3D cultures the frequency of appearance of embryonic stem cell-like colonies was substantially greater, suggesting that the 3D microenvironment promoted the generation of a stem cell-like niche that allows undifferentiated stem cell maintenance. On the other hand, after MEFs were cultured in the 3D system with their regular growth medium, but not in the 2D system, they expressed osteopontin, up-regulated metalloproteinase activities, and acquired a distinct phenotype consisting of small, elongated cells with remaining mitotic activity. Furthermore, only 3D MEF cultures underwent osteoblast differentiation after osteogenic induction, based on matrix mineralization, collagen I synthesis, ALP activity, and expression of the osteoblast transcription factor Runx2, suggesting that the 3D environment promotes differentiation of MEFs into osteoblast-like cells. We propose that the 3D system provides a unique microenvironment that promotes differentiation of mESCs and MEFs into osteoblast-like cells.

The effect of functionalized self-assembling peptide scaffolds on human aortic endothelial cell function.

A class of designed self-assembling peptide nanofiber scaffolds with more than 99% water content has been shown to be a good biological material for cell culture. Here, we report the functionalization of one of these peptide scaffolds, RAD16-I (AcN-RADARADARADARADA-CONH2), by direct solid phase synthesis extension at the amino terminal with three short-sequence motifs. These motifs are present in two major protein components of the basement membrane, laminin 1 (YIGSR, RYVVLPR) and collagen IV (TAGSCLRKFSTM). These motifs have been previously shown to promote specific biological activities including endothelial cell adhesion, spreading, and tubular formation. Therefore, the generic functionalized peptide developed was AcN-X-GG-RADARADARADARADA-CONH2 with each motif represented by "X". We show in this work that these tailor-made peptide scaffolds enhance the formation of confluent cell monolayers of human aortic endothelial cells (HAEC) in culture. Moreover, additional assays designed to evaluate endothelial cell function showed that HAEC monolayers obtained on these scaffolds not only maintained LDL uptake activity but also enhanced nitric oxide release and elevated laminin 1 and collagen IV deposition. These results suggest that this new scaffold provide a better physiological substrate for endothelial cell culture and suggest its further application for biomedical research, cancer biology and regenerative biology.