The role of bone marrow mesenchymal stromal cell derivatives in skin wound healing in diabetic mice

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Abstract

Mesenchymal stromal cells (MSCs) have shown to be a promising tool in cell therapies to treat different conditions. Several pre-clinical and clinical studies have proved that the transplantation of MSCs improves wound healing. Here, we compare the beneficial effects of mouse bone marrow-derived allogeneic MSCs (allo-mBM-MSCs) and their acelullar derivatives (allo-acd-mMSCs) on skin wound healing in Non-Obese Diabetic (NOD) mice. One dose of allo-mBM-MSCs (1×10 ⁶ cells) or one dose of allo-acd-mMSCs (1X) were intradermally injected around wounds in 8–10 week old female NOD mice. Wound healing was evaluated macroscopically (wound closure) every two days, and microscopically (reepithelialization, dermoepidermal junction, skin appendage regeneration, leukocyte infiltration, vascularization, granulation tissue formation, and density of collagen fibers in the dermis) after 16 days of MSC injection. In addition, we measured growth factors and specific proteins that were present in the allo-acd-mMSCs. Results showed significant differences in the wound healing kinetics of lesions that received allo-acd-mMSCs compared to lesions that received vehicle or allo-mBM-MSCs. In particular, mice treated with allo-acd-mMSCs reached significantly higher percentages of wound closure at day 4, 6 and 8, relative to the allo-mBM-MSCs and vehicle groups (p < 0.05), while wound closure percentages could not be statistically distinguished between the allo-mBM-MSCs and vehicle groups. Also, allo-acd-mMSCs had a greater influence in the skin would healing process. Specifically, they caused a less pronounced inflammatory severe response (p < 0.0001), more granulation tissue formation at an advanced stage (p < 0.0001), and higher density of collagen fibers (p < 0.05) compared to the other groups. Nevertheless, at day 16, both allo-mBM-MSCs and allo-acd-mMSCs revealed a higher effect on the recovery of the quality skin (continuous epidermis; regular dermoepidermal junction and skin appendages) relative to untreated lesions (p < 0.0001), but not between them. On the other hand, ELISA analyses indicated that the allo-acd-mMSCs contained growth factors and proteins relevant to wound healing such as IGF-1, KGF, HGF, VEGF, ANG-2, MMP-1, CoL-1 and PGE2. Compared to allo-acd-mMSCs, the administration of allo-mBM-MSCs is insufficient for wound healing in diabetic mice and delays the therapeutic effect, which maybe explained by the fact that trophic factors secreted by MSCs are critical for skin regeneration, and not the cells per se, suggesting that MSCs may require some time to secrete these factors after their administration.

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Exosomes released from human induced pluripotent stem cells-derived MSCs facilitate cutaneous wound healing by promoting collagen synthesis and angiogenesis

Jieyuan Zhang, Junjie Guan, Xin Niu … (2015)

Background Human induced pluripotent stem cell-derived mesenchymal stem cells (hiPSC-MSCs) have emerged as a promising alternative for stem cell transplantation therapy. Exosomes derived from mesenchymal stem cells (MSC-Exos) can play important roles in repairing injured tissues. However, to date, no reports have demonstrated the use of hiPSC-MSC-Exos in cutaneous wound healing, and little is known regarding their underlying mechanisms in tissue repair. Methods hiPSC-MSC-Exos were injected subcutaneously around wound sites in a rat model and the efficacy of hiPSC-MSC-Exos was assessed by measuring wound closure areas, by histological and immunofluorescence examinations. We also evaluated the in vitro effects of hiPSC-MSC-Exos on both the proliferation and migration of human dermal fibroblasts and human umbilical vein endothelial cells (HUVECs) by cell-counting and scratch assays, respectively. The effects of exosomes on fibroblast collagen and elastin secretion were studied in enzyme-linked immunosorbent assays and quantitative reverse-transcriptase–polymerase chain reaction (qRT-PCR). In vitro capillary network formation was determined in tube-formation assays. Results Transplanting hiPSC-MSC-Exos to wound sites resulted in accelerated re-epithelialization, reduced scar widths, and the promotion of collagen maturity. Moreover, hiPSC-MSC-Exos not only promoted the generation of newly formed vessels, but also accelerated their maturation in wound sites. We found that hiPSC-MSC-Exos stimulated the proliferation and migration of human dermal fibroblasts and HUVECs in a dose-dependent manner in vitro. Similarly, Type I, III collagen and elastin secretion and mRNA expression by fibroblasts and tube formation by HUVECs were also increased with increasing hiPSC-MSC-Exos concentrations. Conclusions Our findings suggest that hiPSC-MSC-Exos can facilitate cutaneous wound healing by promoting collagen synthesis and angiogenesis. These data provide the first evidence for the potential of hiPSC-MSC-Exos in treating cutaneous wounds.

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Growth kinetics, self-renewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation

Scott Bruder, Neelam Jaiswal, Stephen E. Haynesworth (1997)

Recent studies have demonstrated the existence of a subset of cells in human bone marrow capable of differentiating along multiple mesenchymal lineages. Not only do these mesenchymal stem cells (MSCs) possess multilineage developmental potential, but they may be cultured ex vivo for many passages without overt expression of a differentiated phenotype. The goals of the current study were to determine the growth kinetics, self-renewing capacity and the osteogenic potential of purified MSCs during extensive subcultivation and following cryopreservation. Primary cultures of MSCs were established from normal iliac crest bone marrow aspirates, an aliquot was cryopreserved and thawed, and then both frozen and unfrozen populations were subcultivated in parallel for as many as 15 passages. Cells derived from each passage were assayed for their kinetics of growth and their osteogenic potential in response to an osteoinductive medium containing dexamethasone. Spindle-shaped human MSCs in primary culture exhibit a lag phase of growth, followed by a log phase, finally resulting in a growth plateau state. Passaged cultures proceed through the same stages, however, the rate of growth in log phase and the final number of cells after a fixed period in culture diminishes as a function of continued passaging. The average number of population doublings for marrow-derived adult human MSCs was determined to be 38 +/- 4, at which time the cells finally became very broad and flattened before degenerating. The osteogenic potential of cells was conserved throughout every passage as evidenced by the significant increase in APase activity and formation of mineralized nodular aggregates. Furthermore, the process of cryopreserving and thawing the cells had no effect on either their growth or osteogenic differentiation. Importantly, these studies demonstrate that replicative senescence of MSCs is not a state of terminal differentiation since these cells remain capable of progressing through the osteogenic lineage. The use of population doubling potential as a measure of biological age suggests that MSCs are intermediately between embryonic and adult tissues, and as such, may provide an in situ source for mesenchymal progenitor cells throughout an adult's lifetime.

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Quantitative and reproducible murine model of excisional wound healing.

Robert D Galiano, Joseph Michaels, Michael Dobryansky … (2004)

The goal of animal wound healing models is to replicate human physiology and predict therapeutic outcomes. There is currently no model of wound healing in rodents that closely parallels human wound healing. Rodents are attractive candidates for wound healing studies because of their availability, low cost, and ease of handling. However, rodent models have been criticized because the major mechanism of wound closure is contraction, whereas in humans reepithelialization and granulation tissue formation are the major mechanisms involved. This article describes a novel model of wound healing in mice utilizing wound splinting that is accurate, reproducible, minimizes wound contraction, and allows wound healing to occur through the processes of granulation and reepithelialization. Our results show that splinted wounds have an increased amount of granulation tissue deposition as compared to controls, but the rate of reepithelialization is not affected. Thus, this model eliminates wound contraction and allows rodents' wounds to heal by epithelialization and granulation tissue formation. Given these analogies to human wound healing, we believe that this technique is a useful model for the study of wound healing mechanisms and for the evaluation of new therapeutic modalities.

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Author and article information

Contributors

Paolo Fiorina: Role: Editor

Journal

Journal ID (nlm-ta): PLoS One

Journal ID (iso-abbrev): PLoS ONE

Journal ID (publisher-id): plos

Journal ID (pmc): plosone

Title: PLoS ONE

Publisher: Public Library of Science (San Francisco, CA USA )

ISSN (Electronic): 1932-6203

Publication date (Electronic): 8 June 2017

Publication date Collection: 2017

Volume: 12

Issue: 6

Electronic Location Identifier: e0177533

Affiliations

[1 ]School of Medicine Clínica Alemana Universidad del Desarrollo, Lo Barnechea, Santiago, Chile

[2 ]Center for Regenerative Medicine, School of Medicine Clínica Alemana Universidad del Desarrollo, Lo Barnechea, Santiago, Chile

[3 ]Universidad Autónoma de Bucaramanga (UNAB), Bucaramanga, Colombia

[4 ]Production Unity of Advanced Therapy, Fundación Ofalmológica de Santander, Clínica Carlos Ardila Lulle (FOSCAL Internacional), Bucaramanga, Colombia

[5 ]Centro Oftalmológico Virgilio Galvis, Bucaramanga, Colombia

[6 ]Fundación Oftalmológica de Santander FOSCAL, Bucaramanga, Colombia

Children's Hospital Boston, UNITED STATES

Author notes

Competing Interests: The authors have declared that no competing interests exist.

Conceptualization: MLA-R.
Data curation: MLA-R TdM SB-B.
Formal analysis: MLA-R TdM SB-B.
Funding acquisition: MLA-R PC.
Investigation: MLA-R TdM SB-B.
Methodology: MLA-R PC.
Project administration: MLA-R.
Resources: MLA-R PC SB-B CLS VG.
Software: MLA-R TdM SB-B.
Supervision: MLA-R PC.
Validation: MLA-R TdM SB-B.
Visualization: MLA-R.
Writing – original draft: MLA-R.
Writing – review & editing: MLA-R TdM PC SB-B CLS VG.

* E-mail: martha.arango@ 123456foscal.com.co

Author information

Martha L. Arango-Rodríguez http://orcid.org/0000-0003-0409-1512

Article

Publisher ID: PONE-D-17-07431

DOI: 10.1371/journal.pone.0177533

PMC ID: 5464535

PubMed ID: 28594903

SO-VID: 4e583ee3-883b-4e17-b912-d327751e4f94

License:

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

History

Date received : 23 February 2017

Date accepted : 28 April 2017

Page count

Figures: 4, Tables: 0, Pages: 17

Funding

Funded by: Fondo Nacional de Desarrollo Científico y Tecnológico

Award ID: 11110009

Award Recipient :

ORCID: http://orcid.org/0000-0003-0409-1512

Martha L. Arango-Rodríguez

This work was supported by a grant from the Fondo Nacional de Desarrollo Científico y Tecnológico # 11110009 which was awarded to Martha L Arango Rodríguez. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Data Availability All relevant data are within the paper and its Supporting Information files.

The role of bone marrow mesenchymal stromal cell derivatives in skin wound healing in diabetic mice

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