In vitro vascularization of tissue engineered constructs by non-viral delivery of pro-angiogenic genes

Vascularization is still one of the major challenges in tissue engineering. In the context of tissue regeneration, the formation of capillary-like structures is often triggered by the addition of growth factors which are associated with high cost, bolus release and short half-life. As an alternative to growth factors, we hypothesized that delivering genes-encoding angiogenic growth factors to cells in a scaffold microenvironment would lead to a controlled release of angiogenic proteins promoting vascularization, simultaneously offering structural support for new matrix deposition. Two non-viral vectors, chitosan (Ch) and polyethyleneimine (PEI), were tested to deliver plasmids encoding for vascular endothelial growth factor (pVEGF) and fibroblast growth factor-2 (pFGF2) to human dermal fibroblasts (hDFbs). hDFbs were successfully transfected with both Ch and PEI, without compromising the metabolic activity. Despite low transfection efficiency, superior VEGF and FGF-2 transgene expression was attained when pVEGF was delivered with PEI and when pFGF2 was delivered with Ch, impacting the formation of capillary-like structures by primary human dermal microvascular endothelial cells (hDMECs). Moreover, in a 3D microenvironment, when PEI-pVEGF and Ch-FGF2 were delivered to hDFbs, cells produced functional pro-angiogenic proteins which induced faster formation of capillary-like structures that were retained in vitro for longer time in a Matrigel assay. The dual combination of the plasmids resulted in a downregulation of the production of VEGF and an upregulation of FGF-2. The number of capillary-like segments obtained with this system was inferior to the delivery of plasmids individually but superior to what was observed with the non-transfected cells. This work confirmed that cell-laden scaffolds containing transfected cells offer a novel, selective and alternative approach to impact the vascularization during tissue regeneration. Moreover, this work provides a new platform for pathophysiology studies, models of disease, culture systems and drug screening

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

<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

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

Mechanomodulatory biomaterials prospects in scar prevention and treatment

Scarring is a major clinical issue that affects a considerable number of patients. The associated problems go beyond the loss of skin functionality, as scars bring aesthetic, psychological, and social difficulties. Therefore, new strategies are required to improve the process of healing and minimize scar formation. Research has highlighted the important role of mechanical forces in the process of skin tissue repair and scar formation, in addition to the chemical signalling. A more complete understanding of how engineered biomaterials can modulate these mechanical stimuli and modify the mechanotransduction signals in the wound microenvironment is expected to enable scar tissue reduction. The present review aims to provide an overview of our current understanding of skin biomechanics and mechanobiology underlying wound healing and scar formation, with an emphasis on the development of novel mechanomodulatory wound dressings with the capacity to offload mechanical tension in the wound environment. Furthermore, a broad overview of current challenges and future perspectives of promising mechanomodulatory biomaterials for this application are provided. Statement of significance: Scarring still is one of the biggest challenges in cutaneous wound healing. Beyond the loss of skin functionality, pathological scars, like keloids and hypertrophic, are associated to aesthetic, psychological, and social distress. Nonetheless, the understanding of the pathophysiology behind the formation of those scars remains elusive, which has in fact hindered the development of effective therapeutics. Therefore, in this review we provide an overview of our current understanding of skin biomechanics and mechanobiology underlying wound healing and scar formation, with an emphasis on the development of novel mechanomodulatory wound dressings with the capacity to offload mechanical tension in the wound environment

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

3D printed scaffolds incorporated with platelet-rich plasma show enhanced angiogenic potential while not inducing fibrosis

Successful therapeutic strategies for wound healing rely on proper vascularization while inhibiting fibrosis. However, scaffolds designed for skin tissue engineering generally lack the biochemical cues that can enhance their vascularization without inducing fibrosis. Therefore, the objective of this work is to incorporate platelet-rich plasma (PRP), a natural source of angiogenic growth factors, into a gelatin methacrylate (GelMA) hydrogel, yielding a bioink that can subsequently be used to 3D print a novel regenerative scaffold with defined architecture for skin wound healing. A PRP-activated bioink is successfully 3D printed, and the resulting scaffolds present similar structural, rheological, and mechanical properties compared to GelMA-only scaffolds. Furthermore, 3D printed PRP-activated scaffolds facilitate controlled release of PRP-derived growth factors for up to 14 days, presenting superior angiogenic potential in vitro (e.g., tubulogenesis assay) and in vivo (chick chorioallantoic membrane) compared to GelMA-only scaffolds, while not inducing a myofibroblastic phenotype in fibroblasts (e.g., α-smooth muscle actin expression). This disruptive technology offers the opportunity for a patient's autologous growth factors to be incorporated into a tailored 3D-printed scaffold in theatre prior to implantation, as part of a single-stage procedure, and has potential in other tissue engineering applications in which enhanced vascularization with limited fibrosis is desired.</p