Vascularization in bone tissue engineering : physiology, current strategies, major hurdles and future challenges

The lack of a functional vascular supply has, to a large extent, hampered the whole range of clinical applications of ‘successful’ laboratory-based bone tissue engineering strategies. To the present, grafts have been dependent on post-implant vascularization, which jeopardizes graft integration and often leads to its failure. For this reason, the development of strategies that could effectively induce the establishment of a microcirculation in the engineered constructs has become a major goal for the tissue engineering research community. This review addresses the role and importance of the development of a vascular network in bone tissue engineering and provides an overview of the most up to date research efforts to develop such a network.M. I. Santos would like to acknowledge the Portuguese Foundation for Science and Technology (FCT) for her Ph.D. grant (SFRH/BD/13428/2003). This work was partially supported by FCT through funds from POCTI and/or FEDER programs and by the European Union funded STREP Project HIPPOCRATES (NMP3-CT-2003-505758). This work was carried out under the scope of the European NoE EXPERTISSUES (NMP3-CT-2004-500283). The authors also acknowledge Gabriela A. Silva for critically reading the manuscript

SP-Sephadex equilibrium chromatography of bradykinin and related peptides: Application to trypsin-treated human plasma

An analytical method is deseribed for the separation of bradykinin, Lys-bradykinin, and Met-Lys-bradykinin by equilibrium chromatography on SP-Sephadex C-25 eluted in 0.02 Tris-HCl buffer, pH 8.10, 0.12 NaCl. A second elution buffer, 0.02 Tris-HCl buffer, pH 7.70, 0.06 NaCl, serves as a second parameter for the identification of bradykinin and also separates the hormone from plasma bradykinin-potentiating peptides. Ten to one-hundred nanomoles of each peptide can be recovered in high yields, identified by elution position, and measured by bioassay with the isolated guinea pig ileum. The identification of bradykinin as the peptide released by trypsin acting on acid-denatured plasma is documented as an illustration of the method

Design of nano- and microfiber combined scaffolds by electrospinning of collagen onto starch-based fiber meshes : a man-made equivalent of natural extracellular matrix

Mimicking the structural organization and biologic function of natural extracellular matrix has been one of the main goals of tissue engineering. Nevertheless, the majority of scaffolding materials for bone regeneration highlights biochemical functionality in detriment of mechanical properties. In this work we present a rather innovative construct that combines in the same structure electrospun type I collagen nanofibers with starchbased microfibers. These combined structures were obtained by a two-step methodology and structurally consist in a type I collagen nano-network incorporated on a macro starch-based support. The morphology of the developed structures was assessed by several microscopy techniques and the collagenous nature of the nanonetwork was confirmed by immunohistochemistry. In addition, and especially regarding the requirements of large bone defects, we also successfully introduced the concept of layer by layer, as a way to produce thicker structures. In an attempt to recreate bone microenvironment, the design and biochemical composition of the combined structures also envisioned bone-forming cells and endothelial cells (ECs). The inclusion of a type I collagen nano-network induced a stretched morphology and improved the metabolic activity of osteoblasts. Regarding ECs, the presence of type I collagen on the combined structures provided adhesive support and obviated the need of precoating with fibronectin. It was also importantly observed that ECs on the nano-network organized into circular structures, a three-dimensional arrangement distinct from that observed for osteoblasts and resembling the microcappillary-like organizations formed during angiogenesis. By providing simultaneously physical and chemical cues for cells, the herein-proposed combined structures hold a great potential in bone regeneration as a man-made equivalent of extracellular matrixK. Tuzlakoglu and M. I. Santos thank the Portuguese Foundation for Science and Technology for their Ph.D. scholarship (SFRH/BD/8502/2002 and SFRH/BD/13428/2003). This work was partially supported by FCT Foundation for Science and Technology, through funds from the POCTI and/or FEDER programs and by the European Union funded STREP Project HIPPOCRATES (NMP3-CT-2003-505758). This work was carried out under the scope of the European NoE EXPERTISSUES (NMP3-CT-2004-500283). Work developed under the cooperation agreement between UM-3B's research group and the Hospital de S. Marcos, Braga. The authors thank to L. Goreti Pinto for her help on confocal microscopy studies

Crosstalk between osteoblasts and endothelial cells co-cultured on a polycaprolactone-starch scaffold and the in vitro development of vascularization

The reconstruction of bone defects based on cell-seeded constructs requires a functional microvasculature that meets the metabolic demands of the engineered tissue. Therefore, strategies that augment neovascularization need to be identified. We propose an in vitro strategy consisting of the simultaneous culture of osteoblasts and endothelial cells on a starch-based scaffold for the formation of pre-vascular structures, with the final aim of accelerating the establishment of a vascular bed in the implanted construct. Human dermal microvascular endothelial cells (HDMECs) were co-cultured with human osteoblasts (hOBs) on a 3D starch-based scaffold and after 21 days of culture HDMEC aligned and organized into microcapillary-like structures. These vascular-like structures evolved from a cord-like configuration to a more complex branched morphology, had a lumen and stained in the perivascular region for type IV collagen. Genetic profiling of 84 osteogenesis-related genes was performed on coculture vs. monoculture. Osteoblasts in co-culture showed a significant up-regulation of type I collagen and immunohistochemistry revealed that the scaffold was filled with a dense matrix stained for type I collagen. In direct contact with HDMEC hOBs secreted higher amounts of VEGF in relation to monoculture and the highest peak in the release profile correlated with the formation of microcapillary-like structures. The heterotypic communication between the two cell types was also assured by direct cell– cell contact as shown by the expression of the gap junction connexin 43. In summary, by making use of heterotypic cellular crosstalk this co-culture system is a strategy to form vascular-like structures in vitro on a 3D scaffold.M.I. Santos would like to acknowledge the Portuguese Foundation for Science and Technology (FCT) for her PhD scholarship (SFRH/BD/13428/2003). This work was partially supported by FCT through funds from POCTI and/or FEDER programs and by the European Union funded STREP Project HIPPOCRATES (NMP3-CT-2003-505758). This work was carried out under the scope of the European NOE EXPERTISSUES (NMP3-CT-2004-500283)

Strategies for the hypothermic preservation of cell sheets of human adipose stem cells

Cell Sheet (CS) Engineering is a regenerative medicine strategy proposed for the treatment of injured or diseased organs and tissues. In fact, several clinical trials are underway using CS-based methodologies. However, the clinical application of such cell-based methodologies poses several challenges related with the preservation of CS structure and function from the fabrication site to the bedside. Pausing cells at hypothermic temperatures has been suggested as a valuable method for short-term cell preservation. In this study, we tested the efficiency of two preservation strategies, one using culture medium supplementation with Rokepie and the other using the preservation solution Hypothermosol, in preserving human adipose stromal/stem cells (hASC) CS-like confluent cultures at 4°C, during 3 and 7 days. Both preservation strategies demonstrated excellent ability to preserve cell function during the first 3 days in hypothermia, as demonstrated by metabolic activity results and assessment of extracellular matrix integrity and differentiation potential. At the end of the 7th day of hypothermic incubation, the decrease in cell metabolic activity was more evident for all conditions. Nonetheless, hASC incubated with Rokepie and Hypothermosol retained a higher metabolic activity and extracellular matrix integrity in comparison with unsupplemented cells. Differentiation results for the later time point showed that supplementation with both Rokepie and Hypothermosol rescued adipogenic differentiation potential but only Rokepie was able to preserve hASC osteogenic potential.This work was supported by: SF-R by PhD grant PD/BD/135252/2017; AFC by contract financed by SFRH/BPD/109595/2015; MTC by NORTE-01-0145-FEDER-000021 and RPP by IF/ 00347/2015, all supported by Fundac¸ão para a Ciencia e Tecnologia (FCT). This work was supported by LA ICVS/3B’s project UID/Multi/ 50026/2013 (POCI-01-0145-FEDER-007038), financed by FCT and Fundo Europeu de Desenvolvimento Regional (FEDER), as well as Gene2Skin Project (H2020-TWINN-2015-692221). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Antimicrobial properties of membrane-active dodecapeptides derived from MSI-78

Antimicrobial peptides (AMPs) are a class of broad-spectrum antibiotics known by their ability to disrupt bacterial membranes and their low tendency to induce bacterial resistance, arising as excellent candidates to fight bacterial infections. In this study we aimed at designing short 12-mer AMPs, derived from a highly effective and broad spectrum synthetic AMP, MSI-78 (22 residues), by truncating this peptide at the N- and/or C-termini while spanning its entire sequence with 1 amino add (aa) shifts. These designed peptides were evaluated regarding antimicrobial activity against selected gram-positive Staphylococcus strains and the gram-negative Pseudomonas aeruginosa (P. aeruginosa). The short 12-mer peptide CEM1 (GIGMFLKKAKICF) was identified as an excellent candidate to fight P. aeruginosa infections as it displays antimicrobial activity against this strain and selectivity, with negligible toxicity to mammalian cells even at high concentrations. However, in general most of the short 12-mer peptides tested showed a reduction in antimicrobial activity, an effect that was more pronounced for gram-positive Staphylococcus strains. Interestingly, CEM1 and a highly similar peptide differing by only one aa-shift (CEM2: IGKFLKKAKICFG), showed a remarkably contrasting AMP activity. These two peptides were chosen for a more detailed study regarding their mechanism of action, using several biophysical assays and simple membrane models that mimic the mammalian and bacterial lipid composition. We confirmed the correlation between peptide helicity and antimicrobial activity and propose a mechanism of action based on the disruption of the bacterial membrane permeability barrier

Endothelial cell colonization and angiogenic potential of combined nano- and micro-fibrous scaffolds for bone tissue engineering

Presently the majority of tissue engineering approaches aimed at regenerating bone relies only on postimplantation vascularization. Strategies that include seeding endothelial cells (ECs) on biomaterials and promoting their adhesion, migration and functionality might be a solution for the formation of vascularized bone. Nano/micro-fiber-combined scaffolds have an innovative structure, inspired by extracellular matrix (ECM) that combines a nano-network, aimed to promote cell adhesion, with a micro-fiber mesh that provides the mechanical support. In this work we addressed the influence of this nano-network on growth pattern, morphology, inflammatory expression profile, expression of structural proteins, homotypic interactions and angiogenic potential of human EC cultured on a scaffold made of a blend of starch and poly(caprolactone). The nano-network allowed cells to span between individual micro-fibers and influenced cell morphology. Furthermore, on nano-fibers as well as on micro-fibers ECs maintained the physiological expression pattern of the structural protein vimentin and PECAM-1 between adjacent cells. In addition, ECs growing on the nano/micro-fiber-combined scaffold were sensitive to pro-inflammatory stimulus. Under pro-angiogenic conditions in vitro, the ECM-like nano-network provided the structural and organizational stability for ECs’ migration and organization into capillary-like structures. The architecture of nano/micro-fiber-combined scaffolds elicited and guided the 3D distribution of ECs without compromising the structural requirements for bone regeneration.M.I. Santos would like to acknowledge the Portuguese Foundation for Science and Technology (FCT) for her PhD scholarship (SFRH/BD/13428/2003). This work was partially supported by FCT through funds from POCTI and/or FEDER programs and by the European Union funded STREP Project HIPPOCRATES (NMP3-CT-2003-505758). This work was carried out under the scope of the European NoE EXPERTISSUES (NMP3-CT-2004-500283)

Análise da prontidão para o tratamento em alcoolistas em um centro de tratamento

The aim of this research was to verify the readiness for the treatment in alcoholics who were taking part of a therapeutical intervention, following the Minnesota Model. A sample of 25 moderate and severe level alcoholists took part on this research. They ranged from 23 to 60 years of age, both male and female. The scale SADD and SOCRATES were applied during the first interview, in the beginning of the treatment. Three additional interviews were performed in the middle, at the end and one month after the treatment, to appraise the readiness for the treatment through SOCRATES scale. The results showed that, although there had been 64% of adhesion to treatment, there was not reduction of ambivalence of the alcoholics. There has been significant correlation between the SOCRATES scale factors. This research points to the need of using motivating techniques to reduce the ambivalence, in order to increase the adhesion and to prevent relapse.O objetivo desta pesquisa foi verificar a prontidão para o tratamento em alcoolistas participantes de uma intervenção terapêutica, seguindo o Modelo Minnesota. Participaram desta pesquisa 25 alcoolistas, de grau moderado e grave, com idade entre 23 a 60 anos, de ambos os sexos. Foram aplicadas as escalas SADD e SOCRATES na primeira entrevista, no início do tratamento. Três entrevistas adicionais realizadas no meio, no final e um mês após o tratamento, avaliaram a prontidão para o tratamento por meio da escala SOCRATES. Os resultados mostraram que, embora tenha havido 64% de adesão, não houve diminuição da ambivalência dos alcoolistas. Houve correlação significativa entre os fatores da escala SOCRATES. Esta pesquisa aponta para a necessidade de se utilizar técnicas motivacionais para a diminuição da ambivalência, visando o aumento na adesão e prevenção de recaída

A 17-mer Membrane-Active MSI-78 Derivative with Improved Selectivity toward Bacterial Cells

Antimicrobial peptides are widely recognized as an excellent alternative to conventional antibiotics. MSI-78, a highly effective and broad spectrum AMP, is one of the most promising AMPs for clinical application. In this study, we have designed shorter derivatives of MSI-78 with the aim of improving selectivity while maintaining antimicrobial activity. Shorter 17-mer derivatives were created by truncating MSI-78 at the N- and/or C-termini, while spanning MSI-78 sequence. Despite the truncations made, we found a 17-mer peptide, MSI-78(4-20) (KFLKKAKKFGKAFVKIL), which was demonstrated to be as effective as MSI-78 against the Gram-positive Staphylococcus strains tested and the Gram-negative Pseudomonas aeruginosa. This shorter derivative is more selective toward bacterial cells as it was less toxic to erythrocytes than MSI-78, representing an improved version of the lead peptide. Biophysical studies support a mechanism of action for MSI-78(4-20) based on the disruption of the bacterial membrane permeability barrier, which in turn leads to loss of membrane integrity and ultimately to cell death. These features point to a mechanism of action similar to the one described for the lead peptide MSI-78