pinecone/core-2020-05-10-deduplication huggingface dataset:
The secretome of bone marrow mesenchymal stem cells-conditioned media varies with time and drives a distinct effect on mature neurons and glial cells (primary cultures)

Transplantation of bone marrow mesenchymal stem cells (BM-MSCs) has been shown to ameliorate the injured central nervous system (CNS). Although these effects were initially attributed to the putative differentiation of MSCs towards the neural lineage, it is now known that most of them are mediated by the secretome. Up to now most in vitro reports have dealt with the effects of the secretome on neural stem cells and their differentiation. Consequently, there is a lack of information regarding the role of the secretome on the viability and survival of pre-existent matured differentiated cell populations. Moreover, it is also not known how the time points of conditioned media (CM) collection affect such parameters. In the present study, primary cultures of hippocampal neurons and glial cells were incubated with CM obtained from MSCs. To determine how the temporal profiles of CM collection impact on post-natal neurons and glial cells, we collected MSCs CM at 24, 48, 72 and 96 h of conditioning. MTS test revealed that for the hippocampal cultures the incubation with CM increased cell viability for all time points, with significant increases in the percentage of neurons in culture incubated with CM 24 h. For glial cells only the later time point of CM collection (96 h) increased cell viability. Fluorescence microscopy observations also revealed that CM 48 h and 72 h increased astrocytes percentages, while CM 24 h decreased microglial cell and oligodendrocytes values. These results revealed that post-natal neuronal and glial cells respond differently to MSCs CM; moreover, there are specific temporal variations in the composition of the CM of MSCs collected at different time points that trigger different effects on mature neurons and the distinct glial cell populations (astrocytes, oligodendrocytes and microglial cells).Fundação Calouste de Gulbenkia
Microchannel-patterned and heparin micro-contact-printed biodegradable composite membranes for tissue-engineering applications

Microchannel-patterned starch–poly(capro-lactone)/hydydroxyapatite (SPCL–HA) and starch–
poly(lactic acid) (SPLA) composite membranes were produced for use as a laminated tissueengineering
scaffold that incorporates both physical and biochemical patterns. For this purpose,
SPCL (30% starch) blended with inorganic hydroxyl apatite (50%) and SPLA (50% starch)
membranes were made with compressive moulding. Consequently, the microchannel structures
(width 102 μm, 174 μm intervals) were developed on the composite membranes by means of
micro-patterned metal mould(s) and hydraulic pressing. An elastomer poly(dimetylsiloxane) stamp
was used to transfer heparin as a biochemical cue over the microchannel surfaces by micro-contact
printing (μCP). Toluidine blue staining of developed capillaries and heparin μCP-coated membranes
showed that heparin was transferred predominantly over the microchannel surfaces. Fibroblast cell
culture over the microchannel-formed and heparin μCP-modified SPCL–HA and SPLA membranes
showed distinct growth patterns. In contrast to the uniform cell layer formed on unmodified
microchannels, the cells were bridging across the grooves of heparin-printed microchannels. At
extended culture periods, the heparin-printed microchannels were covered with a layer of fibroblast
cells without cellular ingrowths inside. This study indicated that the topographical pattern could
induce an organization of fibroblasts only with the biochemical cue and the cells’ functions can be
controlled spatially over the microchannels by using both cues.E. T. Baran thanks the Portuguese Foundation for Science and Technology (FCT) for providing a post-doctoral scholarship (No. SFRH/BPD/30768/2006). This work was partially supported by the FCT through funds from the POCTI and/or FEDER programmes and also by the European Union-funded STREP Project HIPPOCRATES (Grant No. NMP3-CT-2003-505758)
Osteochondral defects : present situation and tissue engineering approaches

Articular cartilage is often damaged due to trauma or degenerative diseases, resulting in severe
pain and disability. Most clinical approaches have been shown to have limited capacity to treat
cartilage lesions. Tissue engineering (TE) has been proposed as an alternative strategy to repair
cartilage. Cartilage defects often penetrate to the subchondral bone, or full-thickness defects are
also produced in some therapeutic procedures. Therefore, in TE strategies one should also consider
the need for a simultaneous regeneration of both cartilage and subchondral bone in situations
where osteochondral defects are present, or to provide an enhanced support for the cartilage
hybrid construct. In this review, different concepts related to TE in osteochondral regeneration
will be discussed. The focus is on the need to produce new biphasic scaffolds that will provide
differentiated and adequate conditions for guiding the growth of the two tissues, satisfying their
different biological and functional requirements
Rapid vascularization of starchâ  poly(caprolactone) in vivo by outgrowth endothelial cells in co-culture with primary osteoblasts

The successful integration of in vitro-generated tissues is dependent on adequate vascularization
in vivo. Human outgrowth endothelial cells (OECs) isolated from the mononuclear cell fraction of
peripheral blood represent a potent population of circulating endothelial progenitors that could
provide a cell source for rapid anastomosis and scaffold vascularization. Our previous work with
these cells in co-culture with primary human osteoblasts has demonstrated their potential to
form perfused vascular structures within a starch–poly(caprolactone) biomaterial in vivo. In the
present study, we demonstrate the ability of OECs to form perfused vascular structures as early
as 48 h following subcutaneous implantation of the biomaterial in vivo. The number of OECderived
vessels increased throughout the study, an effect that was independent of the OEC donor.
This finding of rapid and thorough OEC-mediated scaffold vascularization demonstrates the great
potential for OEC-based strategies to promote vascularization in tissue engineering. OECs have
the potential to contribute to host-derived scaffold vascularization, and formed vascular structures
at a similar density as those arising from the host. Additionally, immunohistochemical evidence
demonstrated the close interaction between OECs and the co-cultured osteoblasts. In addition to
the known paracrine activity osteoblasts have in modulating angiogenesis of co-cultured OECs, we
demonstrate the potential of osteoblasts to provide additional structural support for OEC-derived
vessels, perhaps acting in a pericyte-like role.The authors would like to thank Mrs B Pavic and Mrs U. Hilbig for their excellent technical assistance. This work was financially supported by grants from the European Commission (EXPERTISSUES Contract No. 500283-2) and the German Federal Ministry of Education and Research, BMBF (German-Chinese Cooperation in Regenerative Medicine; Contract No. 0315033)
Osteogenic differentiation of two distinct subpopulations of human adipose-derived stem cells : an in vitro and in vivo study

The first stem cells considered for the reconstruction of bone were bone marrow mesenchymal stem cells (BMSCs). Subsequently, cells with similar marker expression panel and differentiation potential were found in new sources of cells, such as adipose tissue. This source of stem cells has a promising future in tissue-engineering applications, considering the abundance of this tissue in the human body, the easy harvesting and the high number of stem cells that are available from such a small amount of tissue. The isolation of the adipose stem cells is generally performed by means of enzymatic digestion of the tissues, followed by a natural selection of the stem cells based on their capacity to adhere to the culture flasks, leading to a quite heterogeneous population. This constitutes a major drawback for the use of these cells, since the heterogeneity of the cell culture obtained can compromise their proliferation and differentiation potential. In the present study we have analysed the in vitro and in vivo behaviour of two selected subpopulations with high osteogenic potential. For this purpose, ASCsCD29+ and ASCs STRO−1+ subpopulations were isolated and in vitro cultured onto a biodegradable polymeric scaffold, using osteogenic medium, before implantation in a nude mice model. The biodegradable polymeric scaffold used is a fibre-mesh structure based on a blend of starch and polycaprolatone (SPCL) that has been successfully used in several bone
tissue-engineering studies. The implanted ASCs–scaffold constructs promoted the formation of new bone tissue in nude mice. However, the results obtained show differences in the behaviour of the
two ASCs subpopulations under study, particularly regarding their potential to differentiate into the osteogenic lineage, and allowed the identification of ASCs STRO−1+ as the best subpopulation for bone tissue-engineering applications
Chitosan – poly(butylene succinate) scaffolds and human bone marrow stromal cells induce bone repair in a mouse calvaria model

Tissue engineering sustains the need of a three-dimensional (3D) scaffold to promote the
regeneration of tissues in volume. Usually, scaffolds are seeded with an adequate cell population,
allowing their growth and maturation upon implantation in vivo. Previous studies obtained by our
group evidenced significant growth patterns and osteogenic differentiation of human bone marrow
mesenchymal stem cells (hBMSCs) when seeded and cultured on melt-based porous chitosan
fibre mesh scaffolds (cell constructs). Therefore, it is crucial to test the in vivo performance of
these in vitro 3D cell constructs. In this study, chitosan-based scaffolds were seeded and cultured
in vitro with hBMSCs for 3 weeks under osteogenic stimulation conditions and analysed for cell
adhesion, proliferation and differentiation. Implantation of 2 weeks precultured cell constructs in
osteogenic culture conditions was performed into critical cranial size defects in nude mice. The
objective of this study was to verify the scaffold integration and new bone formation. At 8 weeks
of implantation, scaffolds were harvested and prepared for micro-computed tomography (μCT)
analysis. Retrieved implants showed good integration with the surrounding tissue and significant
bone formation, more evident for the scaffolds cultured and implanted with human cells. The results
of this work demonstrated that chitosan-based scaffolds, besides supporting in vitro proliferation
and osteogenic differentiation of hBMSCs, induced bone formation in vivo. Thus, their osteogenic
potential in orthotopic location in immunodeficient mice was validated, evidencing good prospects
for their use in bone tissue-engineering therapies.Fundação para a Ciência e a Tecnologia (FCT
Biological responses to spider silk - antibiotic fusion protein

The development of a new generation of multifunctional biomaterials is a continual goal for the
field of materials science. The in vivo functional behaviour of a new fusion protein that combines
the mechanical properties of spider silk with the antimicrobial properties of hepcidin was addressed
in this study. This new chimeric protein, termed 6mer + hepcidin, fuses spider dragline consensus
sequences (6mer) and the antimicrobial peptide hepcidin, as we have recently described, with
retention of bactericidal activity and low cytotoxicity. In the present study, mouse subcutaneous
implants were studied to access the in vivo biological response to 6mer + hepcidin, which were
compared with controls of silk alone (6mer), polylactic–glycolic acid (PLGA) films and empty defects.
Along with visual observations, flow cytometry and histology analyses were used to determine the
number and type of inflammatory cells at the implantation site. The results show a mild to low
inflammatory reaction to the implanted materials and no apparent differences between the 6mer +
hepcidin films and the other experimental controls, demonstrating that the new fusion protein has
good in vivo biocompatibility, while maintaining antibiotic function.Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/28603/2006, Chimera project (No. PTDC/EBB-EBI/109093/2008), Proteo-Light (No. PTDC/FIS/68517/2006), NIH (Grant No. P41 EB002520) Tissue Engineering Resource Center; and the NIH (Grant Nos EB003210
and DE017207).European -  Projeto EXPERTISSUES (No. NMP3-
CT-2004-500283)Tissue Engineering Resource Center - Bolsa No. P41 EB002520, bolsa Nos EB003210
and DE01720
Synergistic effect of scaffold composition and dynamic culturing environment in multi-layered systems for bone tissue engineering

Bone extracellular matrix (ECM) is composed of mineralized collagen fibrils which support biological
apatite nucleation that participates in bone outstanding properties. Understanding and mimicking
bone morphological and physiological parameters at a biological scale is a major challenge in tissue
engineering scaffolding. Using emergent (nano)technologies scaffold designing may be critically
improved, enabling highly functional tissue substitutes for bone applications. This study aims to develop
novel biodegradable composite scaffolds of tricalcium phosphate (TCPs) and electrospun nanofibers
of poly(e-caprolactone) (PCL), combining TCPs osteoconductivity with PCL biocompatibility
and elasticity, mimicking bone structure and composition. We hypothesized that scaffolds with such
structure/composition would stimulate the proliferation and differentiation of bone marrow stromal
cells (BMSCs) towards the osteogenic phenotype. Composite scaffolds, developed by electrospining
using consecutive stacked layers of PCL and TCPs, were characterized by FTIR spectroscopy, X-Ray diffraction
and scanning electronic microscopy. Cellular behavior was assessed in goat BMSCs seeded
onto composite scaffolds and cultured in static or dynamic conditions, using basal or osteogenic media
during 7, 14 or 21 days. Cellular proliferation was quantified and osteogenic differentiation confirmed
by alkaline phosphatase activity, alizarin red staining and immunocytochemistry for osteocalcin and
collagen I. Results suggest that PCL-TCP scaffolds provide a 3D support for gBMSCs proliferation
and osteogenic differentiation with production of ECM. TCPs positively stimulate the osteogenic
process, especially under dynamic conditions, where PCL-TCP scaffolds are sufficient to promote
osteogenic differentiation even in basal medium conditions. The enhancement of the osteogenic
potential in dynamic conditions evidences the synergistic effect of scaffold composition and dynamic
stimulation in gBMSCs osteogenic differentiation.Fundação para a Ciência e a Tecnologia (FCT)European NoE EXPERTISSUES (NMP3-CT- 2004-500283
Hydrogels in acellular and cellular strategies for intervertebral disc regeneration

Low back pain is an extremely common illness syndrome that causes patient suffering and disability and
requires urgent solutions to improve the quality of life of these patients. Treatment options aimed to regenerate
the intervertebral disc (IVD) are still under development. The cellular complexity of IVD, and
consequently its fine regulatory system, makes it a challenge to the scientific community. Biomaterialsbased
therapies are the most interesting solutions to date, whereby tissue engineering and regenerative
medicine (TE&RM) strategies are included. By using such strategies, i.e., combining biomaterials, cells,
and biomolecules, the ultimate goal of reaching a complete integration between native and neo-tissue
can be achieved. Hydrogels are promising materials for restoring IVD, mainly nucleus pulposus (NP).
This study presents an overviewof the use of hydrogels in acellular and cellular strategies for intervertebral
disc regeneration. To better understand IVD and its functioning, this study will focus on several
aspects: anatomy, pathophysiology, cellular and biomolecular performance, intrinsic healing processes,
and current therapies. In addition, the application of hydrogels as NP substitutes will be addressed due
to their similarities to NP mechanical properties and extracellular matrix. These hydrogels can be used
in cellular strategies when combined with cells from different sources, or in acellular strategies by
performing the functionalization of the hydrogelswith biomolecules. In addition, a brief summary of therapies
based on simple injection for primary biological repair will be examined. Finally, special emphasis
will focus on reviewing original studies reporting on the use of autologous cells and biomolecules such
as platelet-rich plasma and their potential clinical applications.The authors would like to acknowledge the Portuguese Foundation for Science and Technology (FCT) through the POCTI and FEDER programs, including Project ProteoLight (Grant No. PTDC/FIS/68517/2006) for the provided funds. This work was also carried out under the scope of European Union-funded Collaborative Project Disc Regeneration (Grant No. NMP3-LA-2008-213904)
A miniaturized bioreactor system for the evaluation of cell interaction with designed substrates in perfusion culture

This is the peer reviewed version of the article, which has been published in final form at http://dx.doi.org/10.1002/term.510. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.In tissue engineering, the chemical and topographical cues within three-dimensional (3D) scaffolds\ud
are normally tested using static cell cultures but applied directly to tissue cultures in perfusion\ud
bioreactors. As human cells are very sensitive to the changes of culture environment, it is essential to\ud
evaluate the performance of any chemical, and topographical cues in a perfused environment before\ud
they are applied to tissue engineering. Thus the aim of this research was to bridge the gap between\ud
static and perfusion cultures by addressing the effect of perfusion on cell cultures within 3D\ud
scaffolds. For this we developed a scale down bioreactor system, which allows to evaluate the\ud
effectiveness of various chemical and topographical cues incorporated into our previously developed\ud
tubular ε-polycaprolactone scaffold under perfused conditions. Investigation of two exemplary cell\ud
types (fibroblasts and cortical astrocytes) using the miniaturized bioreactor indicated that: (1) quick\ud
and firm cell adhesion in 3D scaffold was critical for cell survival in perfusion culture compared\ud
with static culture, thus cell seeding procedures for static cultures might not be applicable. Therefore\ud
it was necessary to re-evaluate cell attachment on different surfaces under perfused conditions before\ud
a 3D scaffold was applied for tissue cultures, (2) continuous medium perfusion adversely influenced\ud
cell spread and survival, which could be balanced by intermittent perfusion, (3) micro-grooves still\ud
maintained its influences on cell alignment under perfused conditions, while medium perfusion\ud
demonstrated additional influence on fibroblast alignment but not on astrocyte alignment on grooved\ud
substrates. This research demonstrated that the mini-bioreactor system is crucial for the development of functional scaffolds with suitable chemical and topographical cues by bridging the gap between\ud
static culture and perfusion culture
Effect of monocytes/macrophages on the early osteogenic differentiation of hBMSCs

Heterotypic cell interactions are essential for the homeostasis of bone tissue, in particular the widely studied interaction between osteoblasts and osteoclasts. Closely related with osteoclasts are monocytes/macrophages. These have been shown to produce osteogenic factors, e.g. BMP-2, which plays a key role in bone metabolism. However, the mechanisms through which monocytes/macrophages interact with osteoblasts are still elusive. The aim of this work was to assess the influence of human peripheral blood monocytes/macrophages over the early osteogenic differentiation of human bone marrow stromal cells (hBMSCs) in the presence of dexamethasone-supplemented medium. The co-cultures were performed using porous transwells that allowed the interaction between both cell types through the production of paracrine factors. The potential effect of BMP-2 produced by monocytes/macrophages was addressed by adding an anti-BMP-2 antibody to the co-cultures. hBMSCs cultured in the presence of monocytes/macrophages had a higher proliferation rate than hBMSCs monocultures. The quantification of early osteogenic marker alkaline phosphatase (ALP) revealed higher activity of this enzyme in cells in the co-culture throughout the time of culture. Both of these effects were inhibited by adding an anti-BMP-2 antibody to the cultures. Moreover, qRTPCR for osteocalcin and osteopontin transcripts showed overexpression of both markers. Once again, the effect of monocytes/macrophages over hBMSC osteogenic differentiation was completely inhibited in the co-cultures by blocking BMP-2. The present report confirmed that monocytes/macrophages produce BMP-2, which promotes osteogenic differentiation and proliferation of hBMSCs cumulatively to dexamethasone-supplemented medium. This potentially implies that monocyte/macrophages play a stronger role in bone homeostasis than so far supposed
Effect of implantation on engineered skeletal muscle constructs

The development of engineered skeletal muscle would provide a viable tissue for replacement and repair of muscle damaged by disease or injury. Our current tissue‐engineering methods result in three‐dimensional (3D) muscle constructs that generate tension but do not advance phenotypically beyond neonatal characteristics. To develop to an adult phenotype, innervation and vascularization of the construct must occur. In this study, 3D muscle constructs were implanted into the hindlimb of a rat, along the sciatic nerve, with the sural nerve isolated, transected and sutured to the construct to encourage innervation. Aortic ring anchors were sutured to the tendons of the biceps femoris muscle so that the construct would move dynamically with the endogenous muscle. After 1 week  in vivo , the constructs were explanted, evaluated for force production and stained for muscle, nerve and collagen markers. Implanted muscle constructs showed a developing capillary system, an epimysium‐like outer layer of connective tissue and an increase in myofibre content. The beginning of  α ‐bungarotoxin clustering suggests that neuromuscular junctions (NMJs) could form on the implanted muscle, given more time  in vivo . Additionally, the constructs increased maximum isometric force from 192 ± 41 μN to 549 ± 103 μN (245% increase) compared to  in vitro  controls, which increased from 276 ± 23 μN to 329 ± 27μN (25% increase). These findings suggest that engineered muscle tissue survives 1 week of implantation and begins to develop the necessary interfaces needed to advance the phenotype toward adult muscle. However, in terms of force production, the muscle constructs need longer implantation times to fully develop an adult phenotype. Copyright © 2012 John Wiley & Sons, Ltd