Perivascular-like cells contribute to the stability of the vascular network of osteogenic tissue formed from cell sheet-based constructs

In recent years several studies have been supporting the existence of a close relationship in terms of function and progeny between Mesenchymal Stem Cells (MSCs) and Pericytes. This concept has opened new perspectives for the application of MSCs in Tissue Engineering (TE), with special interest for the pre-vascularization of cell dense constructs. In this work, cell sheet technology was used to create a scaffold-free construct composed of osteogenic, endothelial and perivascular-like (CD146+) cells for improved in vivo vessel formation, maturation and stability. The CD146 pericyte-associated phenotype was induced from human bone marrow mesenchymal stem cells (hBMSCs) by the supplementation of standard culture medium with TGF-b1. Co-cultured cell sheets were obtained by culturing perivascular-like (CD146+) cells and human umbilical vein endothelial cells (HUVECs) on an hBMSCs monolayer maintained in osteogenic medium for 7 days. The perivascular-like (CD146+) cells and the HUVECs migrated and organized over the collagen-rich osteogenic cell sheet, suggesting the existence of cross-talk involving the co-cultured cell types. Furthermore the presence of that particular ECM produced by the osteoblastic cells was shown to be the key regulator for the singular observed organization. The osteogenic and angiogenic character of the proposed constructs was assessed in vivo. Immunohistochemistry analysis of the explants revealed the integration of HUVECs with the host vasculature as well as the osteogenic potential of the created construct, by the expression of osteocalcin. Additionally, the analysis of the diameter of human CD146 positive blood vessels showed a higher mean vessel diameter for the co-cultured cell sheet condition, reinforcing the advantage of the proposed model regarding blood vessels maturation and stability and for the in vitro pre-vascularization of TE constructs.Funding provided by Fundacao para a Ciencia e a Tecnologia project Skingineering (PTDC/SAU-OSM/099422/2008). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

<i>In vitro</i> culture methodology to obtain a stacked co-cultured cell sheets (CS)-based model.

<p>hBMSCs were seeded and cultured for 7 days in osteogenic medium in thermoresponsive dishes. To obtain co-cultured CS, HUVECs and perivascular-like (CD146⁺) cells were cultured, at a ratio of 4∶1, on the osteogenic CS in M199 supplemented with osteogenic factors for further 7 days (experimental). Control homotypic osteogenic CS were maintained in osteogenic medium. At day 14, CS were retrieved from the thermoresponsive dishes by temperature decrease and the experimental model was built by stacking of a homotypic osteogenic CS onto the co-cultured CS using a poly(vinylidene difluoride) (PVDF) membrane.</p

Histological characterization of single and co-cultured cell sheets after 14 days in culture in osteogenic medium and after detachment by temperature decrease and contraction.

<p>Single osteogenic cell sheet derived from hBMSCs A) after H&E staining and immunostained for (B) osteocalcin and (C) type-I collagen; Co-cultured cell sheets after (E) H&E staining and immunostaining for (F) CD31, (G) CD146, (H) osteocalcin and (I) Type-I collagen. Identification of positive signal was determined in comparison to immunocytochemistry negative controls (D;J). * PVDF membrane used to protect cell sheet during processing.</p

Eumelanin-releasing spongy-like hydrogels for skin re-epithelialization purposes

Melanin function in the skin has been associated with pigmentation but other properties such as electrical conductance, photoprotection, and antioxidant and antimicrobial activity have also been recognized. Nonetheless, the use of melanin in a skin wound healing context has never been considered. In this sense, eumelanin particles with a typical round and nano-sized morphology and electrical conductivity of 2.09 × 10−8 S cm−1 were extracted from the ink of Sepia officinalis. The ability of primary human keratinocytes (hKCs) to phagocyte eumelanin, which was then accumulated in cytosolic vesicles and nuclei surroundings, was demonstrated. Keratinocyte viability and maturation was not affected by eumelanin contact, but at eumelanin amounts higher than 0.1 mg l−1 cell morphology was altered and cell proliferation was inhibited. A time and eumelanin amount-dependent reduction of reactive oxygen species (ROS) released by eumelanin-containing ultraviolet (UV)-irradiated keratinocytes was observed. Eumelanin-containing gellan gum (GG) spongy-like hydrogels allowed a sustained release of eumelanin in the range of 0.1 to 5 mg l−1, which was shown in vitro to not be harmful to hKCs, and the absence of a strong host reaction after subcutaneous implantation in mice. Herein, we propose spongy-like hydrogels as sustained release matrices of S. officinalis eumelanin for predicting a beneficial role in skin wound healing through a direct effect over keratinocytes.The authors would like to acknowledge Fundacao para a Ciencia e Tecnologia (FCT) for SFRH/BD/78025/2011 (LdS), SFRH/BPD/101886/2014 (RPP), SFRH/BPD/101952/2014 (TCS) and IF/01214/2014 (VC) grants, Mariana Cerqueira and Manuela Lago for their support on human primary keratinocytes cell culture, and Rui Fernandes from the Institute for Molecular and Cell Biology of the University of Porto for TEM analysis.info:eu-repo/semantics/publishedVersio

Angiogenic potential of the transplanted cell sheet-based constructs.

<p>Immunohistochemistry for (A;B) CD31 and (C–F) CD146 on (C;D) control and (E,F) experimental conditions at days 7 (C;E) and 21 (D;F) of implantation; (G–J) Immunostaining negative control of respective conditions. → negative blood vessels for CD146; ▸ positive blood vessel for CD146. (K) Human cells (green) detected using human-specific anti-mitochondria antibodies on the experimental condition 7 days after implantation. (L) Co-localization (yellow) of CD146 (red) and human-specific anti-mitochondria (green) revealed cellular assembling in a blood vessel-like structure (arrow) on the experimental condition 7 days after implantation. DAPI (blue) was used as nuclear staining. Representation of (M) the mean diameter and (N) the number of CD146 positive vessels present on control and experimental conditions at days 7 and 21 of implantation. *p≤0.05; **p≤0.01.</p

H&E staining and osteocalcin immunolocalization on explants retrieved 7 and 21 days after transplantation of cell sheet-based constructs.

<p>(A–D) H&E staining on (A;B )control and (C;D) experimental explants after 7 (A;C) and 21 days (B;D) of subcutaneous implantation showing their localization and morphology. (E–L) Immunolocalization of osteocalcin on (E;F) control and (G;H) experimental explants at 7 (E;G) and 21 (F;H) days of implantation revealing osteogenic commitment on both test conditions. (I–L) immunostaining negative control of respective E–H conditions.</p

Representative flow cytometry and immunocytochemistry analysis of human bone marrow derived cells at different passages and cultured with and without TGF-β1.

<p>(A) CD146 expression of bone marrow mononuclear fraction at isolation day; (B) CD146 expression on hBMSCs (P5) cultured in complete α-MEM; (C; D) CD146 expression analysis, by flow cytometry (C) and immunocytochemistry (green) (D), on hBMSCs (P5) cultured in complete α-MEM supplemented with 1 ng/mL TGF-β1 for 7 days; Evolution of cell morphology of hBMSCs (E) before and (F) after culture in α-MEM +1 ng/mL TGF-β1 for 7 days. For immunocytochemistry DAPI (blue) was used as nuclear staining. Right upper corner image in D represent a higher magnification.</p

Immunocytochemistry for CD146 expression and Dil-AcLDL uptake by hBMSCs monocultures and co-cultures with perivascular-like (CD146⁺) cells and HUVECs, after 14 days of culture.

<p>(A,B) Co-cultures on hBMSCs-derived osteogenic cells showing endothelial colonies (red) and elongated perivascular-like (CD146⁺) cells (green) interacting with HUVECs and with them-self (Arrow). (C) Co-cultures of HUVECs (red) and perivascular-like (CD146⁺) cells (green) on plastic adherent conditions showing random organization. (D) Confluent layer of hBMSCs-derived osteogenic cells lacking the expression of CD146. DAPI (blue) was used as nuclear staining.</p

Cell sheet engineering using the stromal vascular fraction of adipose tissue as a vascularization strategy

Current vascularization strategies for Tissue Engineering constructs, in particular cell sheet-based, are limited by time-consuming and expensive endothelial cell isolation and/or by the complexity of using extrinsic growth factors. Herein, we propose an alternative strategy using angiogenic cell sheets (CS) obtained from the stromal vascular fraction (SVF) of adipose tissue that can be incorporated into more complex constructs. Cells from the SVF were cultured in normoxic and hypoxic conditions for up to 8 days in the absence of extrinsic growth factors. Immunocytochemistry against CD31 and CD146 revealed spontaneous organization in capillary-like structures, more complex after hypoxic conditioning. Inhibition of HIF-1α pathway hindered capillary-like structure formation in SVF cells cultured in hypoxia, suggesting a role of HIF-1α. Moreover, hypoxic SVF cells showed a trend for increased secretion of angiogenic factors, which was reflected in increased network formation by endothelial cells cultured on matrigel using that conditioned medium. In vivo implantation of SVF CS in a mouse hind limb ischemia model revealed that hypoxia-conditioned CS led to improved restoration of blood flow. Both in vitro and in vivo data suggest that SVF CS can be used as simple and cost-efficient tools to promote functional vascularization of TE constructs.R.P. Pirraco contract financed by SFRH/BPD/101886/2014. B.S. Marques contract financed by SFRH/BPD/90533/2012. M.T. Cerqueira contract financed by SFRH/BPD/96611/2013. T.C. Santos contract financed by SFRH/BPD/101952/2014. Financial support by RL3-TECT-NORTE-01-0124-FEDER-000020 and the European Research Council Advanced Grant (ERC-2012-AdG_20120216- 321266) for the project ComplexiTE is also acknowledged.info:eu-repo/semantics/publishedVersio