M. T. Cerqueira

An investigation of the potential application of chitosan/aloe-based membranes for regenerative medicine

A significant number of therapeutics derived from natural polymers and plants have been developed to replace or to be used in conjunction with existing dressing products. The use of the therapeutic properties of aloe vera could be very useful in the creation of active wound dressing materials. The present work was undertaken to examine issues concerning structural features, topography, enzymatic degradation behavior, antibacterial activity and cellular response of chitosan/aloe vera-based membranes. The chitosan/aloe vera-based membranes that were developed displayed satisfactory degradation, roughness, wettability and mechanical properties. A higher antibacterial potency was displayed by the blended membranes. Moreover, in vitro assays demonstrated that these blended membranes have good cell compatibility with primary human dermal fibroblasts. The chitosan/aloe vera-based membranes might be promising wound dressing materials.

Human Adipose Stem Cells Cell Sheet Constructs Impact Epidermal Morphogenesis in Full-Thickness Excisional Wounds

Among the wide range of strategies to target skin repair/regeneration, tissue engineering (TE) with stem cells at the forefront, remains as the most promising route. Cell sheet (CS) engineering is herein proposed, taking advantage of particular cell-cell and cell-extracellular matrix (ECM) interactions and subsequent cellular milieu, to create 3D TE constructs to promote full-thickness skin wound regeneration. Human adipose derived stem cells (hASCs) CS were obtained within five days using both thermoresponsive and standard cell culture surfaces. hASCs-based constructs were then built by superimposing three CS and transplanted into full-thickness excisional mice skin wounds with delayed healing. Constructs obtained using thermoresponsive surfaces were more stable than the ones from standard cell culture surfaces due to the natural adhesive character of the respective CS. Both CS-generating strategies lead to prolonged hASCs engraftment, although no transdifferentiation phenomena were observed. Moreover, our findings suggest that the transplanted hASCs might be promoting neotissue vascularization and extensively influencing epidermal morphogenesis, mainly through paracrine actions with the resident cells. The thicker epidermis, with a higher degree of maturation characterized by the presence of rete ridges-like structures, as well as a significant number of hair follicles observed after transplantation of the constructs combining the CS obtained from the thermoresponsive surfaces, reinforced the assumptions of the influence of the transplanted hASCs and the importance of the higher stability of these constructs promoted by cohesive cell-cell and cell-ECM interactions. Overall, this study confirmed the potential of hASCs CS-based constructs to treat full-thickness excisional skin wounds and that their fabrication conditions impact different aspects of skin regeneration, such as neovascularisation, but mainly epidermal morphogenesis.

The use of ionic liquids in the processing of chitosan/silk hydrogels for biomedical applications

Natural polymers are adequate renewable resources for the processability of well-defined architectures for several applications. Combinations of polysaccharides and proteins may mimic the naturally occurring environment of certain tissues. The main goal of this work renders the application of green chemistry principles, namely the use of ionic liquids (ILs) and biorenewable sources, such as chitosan (CHT) and silk fibroin (SF), to process new hydrogel-based constructs. Although the solubilization of both materials in ILs has been studied individually, this work reports, for the first time, the role of ILs as solvent, for the production of hydrogels from blends of chitosan and silk fibroin (CSF). These systems offer the advantage of being homogeneous and presenting easy and short dissolution time of both biomacromolecules. Moreover, the use of chitosan obtained from α- and β-chitin allowed the production of blended hydrogels with distinct physical–chemical properties. In vitro assays demonstrated that these hydrogels supported the adhesion and growth of primary human dermal fibroblasts. Taken these properties together, the CSF hydrogels might be promising biomaterials to be explored for skin tissue engineering approaches.

Expression, purification and osteogenic bioactivity of recombinant human BMP-4, -9, -10, -11 and -14.

Bone morphogenetic proteins (BMPs) are cytokines from the TGF-beta superfamily, with important roles during embryonic development and in the induction of bone and cartilage tissue differentiation in the adult body. In this contribution, we report the expression of recombinant human BMP-4, BMP-9, BMP-10, BMP-11 (or growth differentiation factor-11, GDF-11) and BMP-14 (GDF-5), using Escherichia coli pET-25b vector. BMPs were overexpressed, purified by affinity his-tag chromatography and shown to induce the expression of early markers of bone differentiation (e.g. smad-1, smad-5, runx2/cbfa1, dlx5, osterix, osteopontin, bone sialoprotein and alkaline phosphatase) in C2C12 cells and in human adipose stem cells. The described approach is a promising method for producing large amounts of different recombinant BMPs that show potential for novel biomedical applications.

Gellan Gum-Hyaluronic Acid Spongy-like Hydrogels and Cells from Adipose Tissue Synergize Promoting Neoskin Vascularization

Currently available substitutes for skin wound healing often result in the formation of nonfunctional neotissue. Thus, urgent care is still needed to promote an effective and complete regeneration. To meet this need, we proposed the assembling of a construct that takes advantage of cell-adhesive gellan gum-hyaluronic acid (GG-HA) spongy-like hydrogels and a powerful cell-machinery obtained from adipose tissue, human adipose stem cells (hASCs), and microvascular endothelial cells (hAMECs). In addition to a cell-adhesive character, GG-HA spongy-like hydrogels overpass limitations of traditional hydrogels, such as reduced physical stability and limited manipulation, due to improved microstructural arrangement characterized by pore wall thickening and increased mean pore size. The proposed constructs combining cellular mediators of the healing process within the spongy-like hydrogels that intend to recapitulate skin matrix aim to promote neoskin vascularization. Stable and off-the-shelf dried GG-HA polymeric networks, rapidly rehydrated at the time of cell seeding then depicting features of both sponges and hydrogels, enabled the natural cell entrapment/encapsulation and attachment supported by cell-polymer interactions. Upon transplantation into mice full-thickness excisional wounds, GG-HA spongy-like hydrogels absorbed the early inflammatory cell infiltrate and led to the formation of a dense granulation tissue. Consequently, spongy-like hydrogel degradation was observed, and progressive wound closure, re-epithelialization, and matrix remodelling was improved in relation to the control condition. More importantly, GG-HA spongy-like hydrogels promoted a superior neovascularization, which was enhanced in the presence of human hAMECs, also found in the formed neovessels. These observations highlight the successful integration of a valuable matrix and prevascularization cues to target angiogenesis/neovascularization in skin full-thickness excisional wounds.

Cell sheet technology-driven re-epithelialization and neovascularization of skin wounds

Skin regeneration remains a challenge, requiring a well-orchestrated interplay of cell-cell and cell-matrix signalling. Cell sheet (CS) engineering, which has the major advantage of allowing the retrieval of the intact cell layers along with their naturally organized extracellular matrix (ECM), has been poorly explored for the purpose of creating skin substitutes and skin regeneration. This work proposes the use of CS technology to engineer cellular constructs based on human keratinocytes (hKC), key players in wound re-epithelialization, dermal fibroblasts (hDFb), responsible for ECM remodelling, and dermal microvascular endothelial cells (hDMEC), part of the dermal vascular network and modulators of angiogenesis. Homotypic and heterotypic three-dimensional (3-D) CS-based constructs were developed simultaneously to target wound re-vascularization and re-epithelialization. After implantation of the constructs in murine full-thickness wounds, human cells were engrafted into the host wound bed and were present in the neotissue formed up to 14 days post-implantation. Different outcomes were obtained by varying the composition and organization of the 3-D constructs. Both hKC and hDMEC significantly contributed to re-epithelialization by promoting rapid wound closure and early epithelial coverage. Moreover, a significant increase in the density of vessels at day 7 and the incorporation of hDMEC in the neoformed vasculature confirmed its role over neotissue vacularization. As a whole, the obtained results confirmed that the proposed 3-D CS-based constructs provided the necessary cell machinery, when in a specific microenvironment, guiding both re-vascularization and re-epithelialization. Although dependent on the nature of the constructs, the results obtained sustain the hypothesis that different CS-based constructs lead to improved skin healing.

Semipermeable Capsules Wrapping a Multifunctional and Self-regulated Co-culture Microenvironment for Osteogenic Differentiation.

A new concept of semipermeable reservoirs containing co-cultures of cells and supporting microparticles is presented, inspired by the multi-phenotypic cellular environment of bone. Based on the deconstruction of the “stem cell niche”, the developed capsules are designed to drive a self-regulated osteogenesis. PLLA microparticles functionalized with collagen I, and a co-culture of adipose stem (ASCs) and endothelial (ECs) cells are immobilized in spherical liquified capsules. The capsules are coated with multilayers of poly(L-lysine), alginate, and chitosan nano-assembled through layer-by-layer. Capsules encapsulating ASCs alone or in a co-culture with ECs are cultured in endothelial medium with or without osteogenic differentiation factors. Results show that osteogenesis is enhanced by the co-encapsulation, which occurs even in the absence of differentiation factors. These findings are supported by an increased ALP activity and matrix mineralization, osteopontin detection, and the up regulation of BMP-2, RUNX2 and BSP. The liquified co-capsules also act as a VEGF and BMP-2 cytokines release system. The proposed liquified capsules might be a valuable injectable self-regulated system for bone regeneration employing highly translational cell sources.

Engineering cell-adhesive gellan gum spongy-like hydrogels for regenerative medicine purposes.

The similarity between the extracellular matrix of soft tissue and hydrogels, characterized by high-water-content viscoelastic polymeric networks, has been sustaining the advancement of hydrogels for tissue engineering and regenerative medicine (TERM) purposes. Current research on hydrogels has focused on introducing cell-adhesive peptides to promote cell adhesion and spreading, a critical applicability limitation. Here we report the development of gellan gum (GG) spongy-like hydrogels with ameliorated mechanical performance and flexibility in relation to hydrogels, using a simple and cost-effective method. Most importantly, these materials allow the entrapment of different cell types representing mesenchymal, epidermal and osteoblastic phenotypes that spread within the three-dimensional microstructure. This effect was associated with microstructural rearrangements characterized by pore wall thickening and pore size augmentation, and lower water content than precursor hydrogels. These properties significantly affected protein adsorption once cell adhesion was inhibited in the absence of serum. Spongy-like hydrogels are not adhesive for endothelial cells; however, this issue was surpassed by a pre-incubation with a cell-adhesive protein, as demonstrated for other substrates but not for traditional hydrogels. The proposed cell-compatible GG-based structures avoid time-consuming and expensive strategies that have been used to include cell-adhesive features in traditional hydrogels. This, associated with their off-the-shelf availability in an intermediary dried state, represents unique and highly relevant features for diverse TERM applications.

Poly(hydroxybutyrate-co-hydroxyvalerate) bilayer skin tissue engineering constructs with improved epidermal rearrangement

Bilayer skin substitutes constitute an attractive strategy towards improved skin wound healing. Therefore, solvent casting and freeze-drying methodologies are used to produce polyhydroxybutyrate-co-hydroxyvalerate (PHBV) thin nanoporous membranes and 3D porous scaffolds that are combined in bilayer structures to recreate the epidermal and dermal layers, respectively. The combination of these methodologies allow attaining a bilayer structure with a high water retention capability and adequate mechanical properties, susceptible to enzymes degradative action. Cultures established with human keratinocytes (hKC) and dermal fibroblasts (hDFb) confirm the suitability of the PHBV structures to support cell adhesion and proliferation. Nonetheless, when co-cultured under defined conditions, hKC are able to grow and rearrange in a multilayer structure with proliferative cells in the basal layer, and cells expressing a terminal differentiation marker in the upper layer. Therefore, PHBV bilayer structures demonstrate properties that favor skin cells performance, thus representing a promising strategy to improve wound healing.

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 8days 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. STATEMENT OF SIGNIFICANCE Neovascularization after implantation is a major obstacle for producing clinically viable cell sheet-based tissue engineered constructs. Strategies using endothelial cells and extrinsic angiogenic growth factors are expensive and time consuming and may raise concerns of tumorigenicity. In this manuscript, we describe a simplified approach using angiogenic cell sheets fabricated from the stromal vascular fraction of adipose tissue. The strong angiogenic behavior of these cell sheets, achieved without the use of external growth factors, was further stimulated by low oxygen culture. When implanted in an in vivo model of hind limb ischemia, the angiogenic cell sheets contributed to blood flux recovery. These cell sheets can therefore be used as a straightforward tool to increase the neovascularization of cell sheet-based thick constructs.