Skin Regeneration Potential of A Pro Angiogenic Secretome Article Essay the assignment file would be uploaded, and the link for the original article also w

Skin Regeneration Potential of A Pro Angiogenic Secretome Article Essay the assignment file would be uploaded, and the link for the original article also will be provided.

taking the information from the summary in the assignment restate the ideas and go with a deeper thoughts and evaluation of the techniques discussed. please feel free to use external sources but cite them ( a credible source is not NewYork time or nay source with .com.

the idea here is to design a skin tissue or regenerate skin. I need to evaluate all the things used in the paper too. as stated, the paper should start with the assignment information and then get deeper whether from the original article or outside valid sources. please keep in mind to relate everything to tissue engineering, as in the paper I am trying to show what is the current states in tissue engineering in regard of skin making or regeneration. Try to avoid long introductions or conclusion because this assignment is a part in a different paper I am working on, so we would already have introduction at the beginning of the original paper. any further questions, please let me know.

* make sure to use the information from the homework and add more details from the original source provided (please note that I should have to submit any repeated sentences, means the information you would pull out of the assignment should not be copy paste, it needs to be paraphrased).

* make sure to have 1 or 2 external sources that talks about the same thing to pull some information out of them

* make sure to evaluate the techniques used

* make sure to provide all the challenges and negativities in the techniques

* make sure to provide alternative solutions if possible ( to the challenges)

* make sure to look for future development of the techniques.

* make sure all this is related to tissue engineering-skin

I am very tight with time and have tons of other projects to work, that is why I really appreciate that you would make a good effort into my paper. Thank you In the article, Robert et al., (2019) investigate whether conditioned medium from multipotent
stromal cells derived from skin is useful when restoring skin structures in mice. The results of
the experiment show that when secretome (a set of humans’ proteins) are derived from
multipotent stromal cells from human skin, it can be used to help repair the skin of lesions
because it has pro-angiogenic properties. The researchers used the triad of tissue engineering to
achieve the objectives of the study which are scaffold, regulatory signals, and cells. Stem cell
therapy that uses multipotent stromal cells (MSCs) has the ability to induce repair of skin when
someone has acute or chronic injuries. The researchers used the most common MSCs, which are
skin derived (SD) considering they are readily available. Previous studies have shown that
conditioned medium (CM) of MSCs is conductive for regeneration of skin. The authors wanted
to determine whether the CM from SD-MSCs can be used for skin regeneration or not.
First, the authors used natural and synthetic hydrogels considering that they have the
capacity to deliver entrapped bioactive molecules. Specifically, Robert et al., (2019) used
carrageenan’s (CGs) which are sulfated hydrophilic polysaccharides that are available in red
algae species and medicine. The authors used CG hydrogel and SD-MSCs to treat skin lesions.
They discovered that GG and SD-MSCs were able to successfully reduce inflamed skin. Enhance
recovery and, increase extracellular recovery deposition to the treated area. Robert et al. (2019)
used human skin fragments from a face lift to create the SD-MSCs.
The SD-MSCs cells were maintained in a regulatory signal of Dulbecco’s modified
Eagle’s medium (DMEM), which was digested with 12.5U/mL of filtered dispase. In order for
the researchers to collect a conditioned medium, the cells were washed using phosphate saline
and cultured in DMEM for 10 days. Prior to being cultured, the cells were tested and checked for
whether they were valid for phenotype changes.
To prepare the hydrogels and incorporate them with SD-MSC-CM, the authors used
kappa-type CG which is extracted from seaweed in Brazil. The CG hydrogels were sterilized
using steam power. Another type of scaffold used was PVA hydrogel. According to Robert et al.,
(2019) polyvinyl alcohol (PVA) is a common type of polymer that is used in wound dressing and
drug delivery. PVA’s features include water solubility, non- toxic, biodegradable, and
biocompatibility. PVA can form hydrogel by both physical and chemical crosslinking. The
authors used PVA due to its biocompatibility, which means that it causes conditioned medium to
be absorbed while maintaining its structural integrity.
The authors wanted to conduct the experiment in vitro. They needed human umbilical
vein endothelial cells in endothelia cell growth basal medium-2 which was also contained of
other growth factors. When the procedure was conducted in vivo, a mouse was used. Robert et
al., (2019) extracted human cells, SD-MSCs were put through PVA and CGs scaffold and
DMEM regulatory signals to conclude that SD-MSCs are useful for skin regeneration.
References
Robert, A. W., Azevedo Gomes, F., Rode, M. P., Marques da Silva, M., Veleirinho, M. B. D. R.,
Maraschin, M., … & Stimamiglio, M. A. (2019). The skin regeneration potential of a proangiogenic secretome from human skin-derived multipotent stromal cells. Journal of
tissue engineering, 10, 2041731419833391.
833391
research-article2019
TEJ0010.1177/2041731419833391Journal of Tissue EngineeringRobert et al.
Original Article
The skin regeneration potential of a
pro-angiogenic secretome from human
skin-derived multipotent stromal cells
Journal of Tissue Engineering
Volume 10: 1­–10
© The Author(s) 2019
Article reuse guidelines:
sagepub.com/journals-permissions
DOI: 10.1177/2041731419833391
https://doi.org/10.1177/2041731419833391
journals.sagepub.com/home/tej
Anny Waloski Robert1, Felipe Azevedo Gomes2,
Michele Patricia Rode2, Maiara Marques da Silva2,
Maria Beatriz da Rocha Veleirinho2, Marcelo Maraschin2,
Leila Hayashi2, Giordano Wosgrau Calloni2
and Marco Augusto Stimamiglio1
Abstract
Multipotent stromal cells stimulate skin regeneration after acute or chronic injuries. However, many stem cell therapy
protocols are limited by the elevated number of cells required and poor cell survival after transplantation. Considering
that the beneficial effects of multipotent stromal cells on wound healing are typically mediated by paracrine mechanisms,
we examined whether the conditioned medium from skin-derived multipotent stromal cells would be beneficial for
restoring the skin structure of mice after wounding. A proteomic characterization of skin-derived multipotent stromal
cell-conditioned medium was performed, and the angiogenic function of this secretome was investigated in vitro using an
endothelial cell tube formation assay. We then applied the skin-derived multipotent stromal cell-conditioned medium
directly to full-thickness excisional wounds or embedded it into carrageenan or poly(vinyl alcohol) hydrogels to monitor
tissue regeneration in mice. Biological processes related to wound healing and angiogenesis were highlighted by the
analysis of the skin-derived multipotent stromal cell secretome, and a pro-angiogenic capacity for promoting tubulelike structures was first confirmed in vitro. Skin wounds treated with skin-derived multipotent stromal cell-conditioned
medium also displayed increased angiogenesis, independently of the association of the conditioned medium with
hydrogels. However, improvements in wound closure and epidermis or decreased inflammatory cell presence were
not observed. Hence, the use of the secretome obtained from human skin-derived multipotent stromal cells may be a
potential strategy to aid the natural skin repair of full-thickness lesions mainly based on its pro-angiogenic properties.
Keywords
Secretome, skin-derived multipotent stromal cell, angiogenesis, wound healing
Date received: 10 September 2018; accepted: 26 January 2019
Introduction
The skin is the largest organ in human body, and it
performs important functions such as protection, hydration
maintenance, and thermoregulation. Superficial wounds
heal by a natural repairing process; however, repair of acute
or severe skin lesion in the dermal and epidermal layers is
challenging.1 Currently, the combinatorial use of threedimensional biodegradable scaffolds, cells, and their derivatives has become attractive approaches for treating skin
injuries.2,3
1Instituto
2Federal
Carlos Chagas, FIOCRUZ/PR, Curitiba, Brazil
University of Santa Catarina, Florianópolis, Brazil
Corresponding authors:
Giordano Wosgrau Calloni, Laboratory of Neural Crest Cells Plasticity
and Differentiation, Federal University of Santa Catarina, Florianópolis,
Santa Catarina, 88040-900, Brazil.
Email: giordano.calloni@ufsc.br
Marco Augusto Stimamiglio, Instituto Carlos Chagas, FIOCRUZ/PR, Rua
Professor Algacyr Munhoz Mader, 3775, 81350-010, Curitiba, Paraná, Brazil.
Email: marco.stimamiglio@fiocruz.br
Creative Commons Non Commercial CC BY-NC: This article is distributed under the terms of the Creative Commons
Attribution-NonCommercial 4.0 License (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial
use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and
Open Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage).
2
Thus, stem cell therapy, using multipotent stromal
cells (MSCs), has been shown to modulate wound
repair, inducing skin regeneration after acute or chronic
injuries.4,5 Among the sources of MSCs, skin-derived
MSCs (SD-MSCs) are an option, since they are accessible by minimally invasive procedures, such as facelifting surgery, also enabling autologous transplantation.
Notably, the beneficial effects of MSCs on wound healing are typically mediated by paracrine mechanisms.6,7
Thus, secreted factors obtained from cultured MSCs
have been used as substitutes for numerous cell-based
therapies.3
Previous studies have demonstrated that the conditioned
medium (CM) from MSCs enhances the regeneration of
skin lesions, as well as stimulates the migration of dermal
cells, such as fibroblasts and keratinocytes, and promotes
angiogenesis, both in vitro and in vivo.8–11 Considering that
the different tissue origins or culture conditions of MSCs
may alter the profile of the secreted proteins (cell
secretome), the CM derived from SD-MSCs (SD-MSC-CM)
could be a singular source for skin regeneration. We therefore decided to examine whether SD-MSC-CM could
be beneficial to the restoration of mouse skin structure
and function after wounding. However, in an attempt to
maintain the bioactive molecules of the CM at the wound
site, the use of biocompatible hydrogels as carriers is
desirable.12,13
Both natural hydrogels (e.g. alginate, chitosan, gelatin) and/or synthetic hydrogels (e.g. polyesters, polyacrylamide) are broadly used in tissue regeneration due
to their ability to locally deliver entrapped bioactive molecules.14 Carrageenans (CGs) are sulfated hydrophilic
polysaccharides obtained from different red algae species
and are extensively employed by the food industry15 and
in regenerative medicine as scaffolds and controlledrelease systems for pharmaceutical drug delivery,16
growth factors,17 and cells.18,19 Considering its potential,
we previously used CG hydrogels in association with
SD-MSC to treat skin lesions on a murine model of
wound healing.20 The combination of CG and SD-MSC
were able to reduce inflammation, accelerate recovery,
and increase the deposition of extracellular matrix at
wounded area.20 Similarly, polyvinyl alcohol (PVA)
hydrogel is one of the most commonly used synthetic
polymers for wound dressings and drug delivery.21,22
PVA is a water soluble, non-toxic, biodegradable, and
biocompatible polymer, which can form hydrogels via
chemical or physical crosslinking, and it is also widely
used in blends with other polymers and composites.23,24
PVA is also a highly biocompatible excipient that enables
CM absorption while maintaining structural integrity.25
Thus, the purpose of the present study was to evaluate
the potential use of human SD-MSC-CM associated with a
natural (CG) or a synthetic (PVA) polymer-based hydrogel
to heal cutaneous wounds in mice. We first performed a
Journal of Tissue Engineering
proteomic characterization of the SD-MSC-CM and examined its potential to stimulate angiogenesis in vitro. Then,
SD-MSC-CM was directly applied to the full thickness of
excisional wounds or embedded into CG or PVA hydrogels
to monitor tissue regeneration.
Materials and methods
Isolation and culture of human SD-MSCs
Human skin fragments from a face-lift were used to isolate SD-MSCs as previously reported.26 These protocols
were approved by the Human Ethics Committee of the
Federal University of Santa Catarina (protocol
46674215.7.0000.0121). Briefly, human skin samples
from three donors were maintained in Dulbecco’s
Modified Eagle’s Medium (DMEM), digested with
12.5 U/mL dispase (BD Bioscience, San Diego, CA,
USA), filtered through a 70-µm filter mesh and centrifuged at 300 g for 7 min. The cell pellet was suspended in
DMEM supplemented with 10% fetal bovine serum
(FBS) and cultivated at 37°C in a 5% CO2 incubator until
the preparation of the CM. The SD-MSCs used in the present study were previously characterized as MSCs.20,26
Preparation of the CM from SD-MSC cultures
SD-MSC cultures were maintained in DMEM supplemented
with 10% FBS, 1% penicillin/streptomycin, and 1%
l-glutamine until they reached 90% confluency. To collect the
CM, the SD-MSC cultures were washed with phosphate buffered saline (PBS) and cultured for 10 days in DMEM without
FBS. SD-MSC were tested to check viability and phenotypic
changes before and after cultures, remaining viable and phenotypically unchanged during the assays (Supplementary
Figure S1). After the media was collected, the samples were
filtered through a 0.22-µm filter mesh and concentrated using
centrifugal filter units with a 10-kDa cutoff (Merck Millipore,
Darmstadt, Germany). The concentrated CM samples were
stored at −80°C until further use. This procedure was performed with three distinct biological samples. The protein
concentrations of all samples were measured by the bicinchoninic acid (BCA) Protein Assay Kit (Thermo Fisher
Scientific, Waltham MA, USA). For functional assays, the
SD-MSC-CM samples were pooled, and each treatment was
performed with a total of 50 µg of protein.
Mass spectrometry and proteomic data
analysis of the CM
Twenty micrograms of protein from each CM sample
(three biological replicates and a technical replicate) were
separated by 10% Sodium Dodecyl Sulfate–Polyacrylamide
Gel Electrophoresis (SDS-PAGE). The gel lanes were
excised and sliced, and the proteins were subjected to
3
Robert et al.
in-gel tryptic digestion as previously described.27 Five
micrograms of extracted peptides were analyzed in triplicate by liquid chromatography–tandem mass spectrometry
(LC-MS/MS) by using the Thermo Scientific Easy-nLC
1000 system coupled to an LTQ Orbitrap XL ETD system
(Mass Spectrometry Facility RPT02H/Carlos Chagas
Institute—Fiocruz-Parana, Brazil).
Data analysis were initiated by removing proteins
identified “by site”, potential contaminants, and reverse
identifications. Then, for data interpretation, we considered only proteins with a minimum of two unique peptides that were identified in at least three samples. The
Gene Ontology (GO) analysis was performed with
g:Profiler bioinformatics toolkit (http://biit.cs.ut.ee/gprofiler/).28 The most relevant terms (p < 0.001) identified by g:Profiler were summarized by REVIGO (http://revigo. irb.hr/).29 Hydrogel preparations and SD-MSC-CM incorporation CG hydrogel was prepared and used as already standardized.20 Shortly, the kappa-type CG (extracted from Kappaphycus alvarezii seaweed cultivated on the island of Florianópolis, Brazil) was used at 2% (w/v) in ultra-pure water after heating at 60°C for 30 min under stirring. The CG hydrogels were sterilized by steam power for 30 min at 120°C, filtered through a 0.8-µm filter mesh, polymerized in Lab-Tek® plates (8-well chamber slides; Thermo Fisher Scientific, Waltham MA, USA) and chopped with a 6-mm biopsy punch after polymerization (proportional to the excisional wound area in the animal model). The CM embedding (≈100 µL sample equivalent to 50 µg of protein) into the CG hydrogels was performed in a laminar flow chamber at 40°C to avoid the denaturation of CM proteins. PVA hydrogel was prepared from suspensions of 20% (w/v; PVA Mw = 85,000–124,000; Merck KGaA, Darmstadt, Germany) in ultra-pure water incubated in a water bath at 85°C to 90°C under constant stirring. The obtained solution was placed in a glass Petri dish, autoclaved, and incubated at −20°C for 2 h to achieve polymerization in one cycle of freeze thawing as previously described.24 The PVA hydrogel was cut with a 6-mm sterile biopsy punch in a laminar flow chamber, and each cylinder of PVA hydrogel was incubated overnight with the CM solution (≈100 µL sample equivalent to 50 µg of protein), which was fully incorporated into the hydrogel. Human umbilical vein endothelial cell culture and in vitro tube formation assay Human umbilical vein endothelial cells (HUVECs) were cultured according to the manufacturer’s instructions (Lonza®, Basel, Switzerland; catalog number C2519A). Endothelial Cell Growth Basal Medium-2 (EBM-2) complete medium, supplemented with 5% FBS, human fibroblast growth factor b (hFGF-b), human epidermal growth factor (hEGF), human vascular endothelial cell growth factor (hVEGF), long R insulin-like growth factor-1 (R3-IGF-1), ascorbic acid, and hydrocortisone, was applied on expansion cultures. The cell cultures were maintained in a humidified atmosphere at 37ºC with 5% CO2. Tube formation assay using HUVECs was employed to verify the potential of CM to generate vessel-like structures in vitro. Briefly, 7 × 104 HUVECs/cm2 were cultured on Matrigel® (Corning, Inc., Corning, New York, USA), and for treatments with CM, the equivalent to 50 µg of protein was applied to 500 µL of EBM-2 medium without supplementation. Positive controls received EBM-2 fully supplemented medium and negative controls received EBM-2 without any supplementation. After 12 and 24 h of culture, five randomly selected regions of the culture wells were photographed to manually count meshes and nodes. In vivo cutaneous wound healing The animal procedures were approved by the Ethics Committee on Animal Research of Federal University of Santa Catarina, Brazil (protocol number PP00810). The mouse model of cutaneous wound healing was adapted from the previously described protocol.20 Briefly, C57BL/6 mice (4–5 months of age from both genders with body weights of 20–30 g) were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) and had their dorsal region shaved. Subsequently, a full-thickness excisional wound diameter of 6 mm was produced with a sterile biopsy punch in the animal dorsum.30 The mice were subdivided into six experimental groups: control (CT; n = 12), which did not receive any treatment; CG hydrogel (CG; n = 9); CG hydrogel-embedded with SD-MSC-CM (CG + CM; n = 10); PVA hydrogel (PVA; n = 10); PVA hydrogel-embedded with SD-MSC-CM (PVA + CM; n = 11); and SD-MSC-CM only (CM; n = 8). The Tegaderm® (3M, St. Paul, MN, USA) transparent film was used to dress the wounds with a silicone ring sutured around the wound boundary. At 3 and 14 days post-treatment, the wounds were photographed, and their diameters were measured with ImageJ software. Histopathological analysis On days 3 and 14 post-treatment, the animals in each group were euthanized, and wound tissue samples were collected and fixed with 4% paraformaldehyde. The samples were then dehydrated in increasing concentrations of ethanol (70%–100%), cleared in xylol, embedded in paraffin, and sectioned (4 µm) for hematoxylin–eosin (H&E) staining. Inflammatory cell infiltration was assessed by estimating the leukocyte density at the site of the lesion at 3 days posttreatment. The inflammatory infiltration was classified 4 Journal of Tissue Engineering Figure 1. Gene ontology (GO) analysis of (a) cellular components and (b) biological processes based on the proteins identified in the SD-MSC secretome. A REVIGO scatterplot shows representative clusters of the GO analysis performed with g:Profiler. The log10 p-value of each GO term after REVIGO analysis is plotted on the x-axis, while the frequency of GO term in the GO Annotation Database is displayed on the y-axis (log size). Bubble colors indicate log10 p-value (from smaller p-values on blue color to highest p-values on red color). into (1) low, (2) moderate, and (3) high levels of leukocyte density (represented in Supplementary Figure S2). Granulation tissue thickness, epidermal thickness, and uniformity were measured in H&E-stained sections from samples collected 14 days post-treatment. These measurements were performed using Zen software (Carl Zeiss Microscopy, Oberkochen, Germany) and analyzing five different sites of the wound. The epidermal uniformity parameter was obtained by considering the standard deviations of the thickness measurements. We also measured the density of blood vessels at the granulation tissue using ImageJ software. The analysis of blood vessels density was performed by counting the number of capillaries (containing a lumen with red blood cells) in each slide and calculating the mean number per animal. All analyzed images were obtained at 200× magnification. Statistical analysis Statistical analyses were performed using GraphPad Prism 7 software. Significant differences among treatments wer... Purchase answer to see full attachment