Carlos E. Semino

Self-assembling peptide hydrogel fosters chondrocyte extracellular matrix production and cell division: Implications for cartilage tissue repair

Emerging medical technologies for effective and lasting repair of articular cartilage include delivery of cells or cell-seeded scaffolds to a defect site to initiate de novo tissue regeneration. Biocompatible scaffolds assist in providing a template for cell distribution and extracellular matrix (ECM) accumulation in a three-dimensional geometry. A major challenge in choosing an appropriate scaffold for cartilage repair is the identification of a material that can simultaneously stimulate high rates of cell division and high rates of cell synthesis of phenotypically specific ECM macromolecules until repair evolves into steady-state tissue maintenance. We have devised a self-assembling peptide hydrogel scaffold for cartilage repair and developed a method to encapsulate chondrocytes within the peptide hydrogel. During 4 weeks of culture in vitro, chondrocytes seeded within the peptide hydrogel retained their morphology and developed a cartilage-like ECM rich in proteoglycans and type II collagen, indicative of a stable chondrocyte phenotype. Time-dependent accumulation of this ECM was paralleled by increases in material stiffness, indicative of deposition of mechanically functional neo-tissue. Taken together, these results demonstrate the potential of a self-assembling peptide hydrogel as a scaffold for the synthesis and accumulation of a true cartilage-like ECM within a three-dimensional cell culture for cartilage tissue repair.

Morphogenetic and regulatory mechanisms during developmental chondrogenesis: new paradigms for cartilage tissue engineering.

Cartilage is the first skeletal tissue to be formed during embryogenesis leading to the creation of all mature cartilages and bones, with the exception of the flat bones in the skull. Therefore, errors occurring during the process of chondrogenesis, the formation of cartilage, often lead to severe skeletal malformations such as dysplasias. There are hundreds of skeletal dysplasias, and the molecular genetic etiology of some remains more elusive than of others. Many efforts have aimed at understanding the morphogenetic event of chondrogenesis in normal individuals, of which the main morphogenetic and regulatory mechanisms will be reviewed here. For instance, many signaling molecules that guide chondrogenesis--for example, transforming growth factor-beta, bone morphogenetic proteins, fibroblast growth factors, and Wnts, as well as transcriptional regulators such as the Sox family--have already been identified. Moreover, extracellular matrix components also play an important role in this developmental event, as evidenced by the promotion of the chondrogenic potential of chondroprogenitor cells caused by collagen II and proteoglycans like versican. The growing evidence of the elements that control chondrogenesis and the increasing number of different sources of progenitor cells will, hopefully, help to create tissue engineering platforms that could overcome many developmental or degenerative diseases associated with cartilage defects.

Cloning and expression of chitinases of Entamoebae

Entamoeba histolytica (Eh) and Entamoeba dispar (Ed) are protozoan parasites that infect hundreds of millions of persons. In the colonic lumen, amebae form chitin-walled cysts, the infectious stage of the parasite. Entamoeba invadens (Ei), which infects reptiles and is a model for amebic encystation, produces chitin synthase and chitinase during encystation. Ei cysts formation is blocked by the chitinase-inhibitor allosamidin. Here molecular cloning techniques were used to identify homologous genes of Eh, Ed, and Ei that encode chitinases (EC 3.2.1.14). The Eh gene (Eh cht1) predicts a 507-amino acid (aa) enzyme, which has 93 and 74% positional identities with Ed and Ei chitinases, respectively. The Entamoeba chitinases have signal sequences, followed by acidic and hydrophilic sequences composed of multiple tandemly arranged 7-aa repeats (Eh and Ed) or repeats varying in length (Ei). The aa compositions of the chitinase repeats are similar to those of the repeats of the Eh and Ed Ser-rich proteins. The COOH-terminus of each chitinase has a catalytic domain, which resembles those of Brugia malayi (33% positional identity) and Manduca sexta (29%). Recombinant entamoeba chitinases are precipitated by chitin and show chitinase activity with chitooligosacharide substrates. Consistent with previous biochemical data, chitinase mRNAs are absent in Ei trophozoites and accumulate to maximal levels in Ei encysting for 48 h.

CLONING AND EXPRESSION OF TWO CHITIN DEACETYLASE GENES OF SACCHAROMYCES CEREVISIAE

Chitin deacetylase (EC 3.5.1.41), which hydrolyses the N‐acetamido groups of N‐acetyl‐d‐glucosamine residues in chitin, has been demonstrated in crude extracts from sporulating Saccharomyces cerevisiae. Two S. cerevisiae open reading frames (ORFs), identified by the Yeast Genome Project, have protein sequence homology to a chitin deacetylase from Mucor rouxii. Northern blot hybridizations show each ORF was transcribed in diploid cells after transfer to sporulation medium and prior to formation of asci. Each ORF was cloned in a vector under transcriptional control of the GAL 1, 10 promoter and introduced back into haploid strains of S. cerevisiae. Chitin deacetylase activity was detected by in vitro assays from vegetative cells grown in galactose. Chemical analysis of these cells also demonstrated the synthesis of chitosam in vivo. Both recombinant chitin deacetylases showed similar qualitative and quantitative activities toward chitooligosaccharides in vitro. A diploid strain deleted of both ORFs, when sporulated, did not show deacetylase activity. The mutant spores were hypersensitive to lytic enzymes (Glusulase or Zymolyase).

Bioengineering 3D environments for cancer models.

Tumor development is a dynamic process where cancer cells differentiate, proliferate and migrate interacting among each other and with the surrounding matrix in a three-dimensional (3D) context. Interestingly, the process follows patterns similar to those involved in early tissue formation by accessing specific genetic programs to grow and disseminate. Thus, the complex biological mechanisms driving tumor progression cannot easily be recreated in the laboratory. Yet, essential tumor stages, including epithelial-mesenchymal transition (EMT), tumor-induced angiogenesis and metastasis, urgently need more realistic models in order to unravel the underlying molecular and cellular mechanisms that govern them. The latest implementation of successful 3D models is having a positive impact on the fight against cancer by obtaining more predictive systems for pre-clinical research, therapeutic drug screening, and early cancer diagnosis. In this review we explore the latest advances and challenges in tumor tissue engineering, by accessing knowledge and tools from cancer biology, material science and bioengineering.

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), α1‐microglobulin, α1‐macroglobulin, retinol binding protein, fibronectin, α1‐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‐α, 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.

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

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.

The effect of self-assembling peptide nanofiber scaffolds on mouse embryonic fibroblast implantation and proliferation.

Development of new materials for tissue engineering can be facilitated by the capacity to efficiently monitor in vivo the survival, proliferation and differentiation behaviour of cells implanted in different target tissues. We present here the application of a previously developed platform that allows to monitor in real time the survival and proliferative behaviour of implanted cells in two anatomical sites: subcutaneous and intramuscular. Basically, the system is based on the use of a non-invasive bioluminescence imaging (BLI) technique to detect luciferase expressing C57BL/6 cells, mouse embryonic fibroblasts, seeded in two sets of scaffolds: 1, a RAD16-I self-assembling peptide nanofiber matrix and 2, a composite consisted of the same RAD16-I nanofibers contained into a microporous biorubber scaffold. Interestingly, our results indicated considerable differences in the behaviour of implanted cells in each scaffold type. We observed that the self-assembling peptide scaffold alone foster cell survival and promotes cell proliferation where the composite scaffold not. Since self-assembling peptide scaffolds presents value stiffness proximal to the implanted tissues it is suggestive to think that harder materials will provide a physical constriction for cells to proliferate as well as mechanical discontinuity. We therefore propose that it is important to close match the implantation environment with the cell/material constructs in order to obtain the best response of the cells, illustrating the convenience of this strategy for the development of new tissue engineering platforms.

In Vitro Fungicidal Photodynamic Effect of Hypericin on Candida Species

Hypericin is a natural photosensitizer considered for the new generation of photodynamic therapy (PDT) drugs. The aim of this study was to evaluate the in vitro fungicidal effect of hypericin PDT on various Candida spp., assessing its photocytotoxicity to keratinocytes (HaCaT) and dermal fibroblasts (hNDF) to determine possible side effects. A 3 log fungicidal effect was observed at 0.5 McFarland for two Candida albicans strains, Candida parapsilosis and Candida krusei with hypericin concentrations of 0.625, 1.25, 2.5 and 40 μm, respectively, at a fluence of 18 J cm−2 (LED lamp emitting at 602 ± 10 nm). To obtain a 6 log reduction, significantly higher hypericin concentrations and light doses were needed (C. albicans 5 μm, C. parapsilosis 320 μM and C. krusei 320 μM; light dose 37 J cm−2). Keratinocytes and fibroblasts can be preserved by keeping the hypericin concentration below 1 μm and the light dose below 37 J cm−2. C. albicans appears to be suitable for treatment with hypericin PDT without significant damage to cutaneous cells.