Prevascularized spongy-like hydrogels maintain their angiogenic potential after prolonged hypothermic storage.

The chronic shortage of organs and tissues for transplantation represents a dramatic burden on healthcare systems worldwide. Tissue engineering offers a potential solution to address these shortages, but several challenges remain, with prevascularization being a critical factor for in vivo survival and integration of tissue engineering products. Concurrently, a different challenge hindering the clinical implementation of such products, regards their efficient preservation from the fabrication site to the bedside. Hypothermia has emerged as a potential solution for this issue due to its milder effects on biologic systems in comparison with other cold preservation methodologies. Its impact on prevascularization, however, has not been well studied. In this work, 3D prevascularized constructs were fabricated using adipose-derived stromal vascular fraction cells and preserved at 4 °C using Hypothermosol or basal culture media (α-MEM). Hypothermosol efficiently preserved the structural and cellular integrity of prevascular networks as compared to constructs before preservation. In contrast, the use of α-MEM led to a clear reduction in prevascular structures, with concurrent induction of high levels of apoptosis and autophagy at the cellular level. In vivo evaluation using a chorioallantoic membrane model demonstrated that, in opposition to α-MEM, Hypothermosol preservation retained the angiogenic potential of constructs before preservation by recruiting a similar number of blood vessels from the host and presenting similar integration with host tissue. These results emphasize the need of studying the impact of preservation techniques on key properties of tissue engineering constructs such as prevascularization, in order to validate and streamline their clinical application.

Stromal Vascular Fraction Obtained From Subcutaneous Adipose Tissue: Ex-Obese and Older Population as Main Clinical Targets.

INTRODUCTION: Human adipose tissue contains a heterogeneous and synergistic mixture of cells called stromal vascular fraction (SVF) with highly proliferative and angiogenic properties, conferring promising applicability in the field of regenerative medicine. This study aims to investigate if age, body mass index (BMI), history of obesity and massive weight loss, and harvest site are related to SVF cell marker expression. METHODS: A total of 26 samples of subcutaneous adipose tissue were harvested from patients admitted to the Plastic and Reconstructive department in University Hospital Center of São João, Porto, Portugal, for body contouring surgery. The percentage of cells expressing CD31, CD34, CD45, CD73, CD90, and CD105 was assessed and compared with patient's age, BMI, history of obesity and massive weight loss (ex-obese group), and harvest site. RESULTS: In the ex-obese group, a significantly higher number of cells expressing CD90 (P = 0.002) was found. BMI, harvest site, and age appear to have no association with SVF subpopulations. CONCLUSIONS: This study suggests that ex-obese patients have a higher percentage of SVF cells expressing CD90, which correlates with higher proliferative and angiogenic rates. The effect of former obesity and massive weight loss on the expression of CD90 is a new and relevant finding because it makes this population a suitable candidate for reconstructive and aesthetic surgery and other fields of regenerative medicine. The use of SVF appears also promising in older patients because no negative correlation between increasing age and different cell markers expression was found.

Spongy-like hydrogels prevascularization with the adipose tissue vascular fraction delays cutaneous wound healing by sustaining inflammatory cell influx.

In vitro prevascularization is one of the most explored approaches to foster engineered tissue vascularization. We previously demonstrated a benefit in tissue neovascularization by using integrin-specific biomaterials prevascularized by stromal vascular fraction (SVF) cells, which triggered vasculogenesis in the absence of extrinsic growth factors. SVF cells are also associated to biological processes important in cutaneous wound healing. Thus, we aimed to investigate whether in vitro construct prevascularization with SVF accelerates the healing cascade by fostering early vascularization vis-à-vis SVF seeding prior to implantation. Prevascularized constructs delayed re-epithelization of full-thickness mice wounds compared to both non-prevascularized and control (no SVF) groups. Our results suggest this delay is due to a persistent inflammation as indicated by a significantly lower M2(CD163+)/M1(CD86+) macrophage subtype ratio. Moreover, a slower transition from the inflammatory to the proliferative phase of the healing was confirmed by reduced extracellular matrix deposition and increased presence of thick collagen fibers from early time-points, suggesting the prevalence of fiber crosslinking in relation to neodeposition. Overall, while prevascularization potentiates inflammatory cell influx, which negatively impacts the cutaneous wound healing cascade, an effective wound healing was guaranteed in non-prevascularized SVF cell-containing spongy-like hydrogels confirming that the SVF can have enhanced efficacy.

Growth Factor-Free Vascularization of Marine-Origin Collagen Sponges Using Cryopreserved Stromal Vascular Fractions from Human Adipose Tissue.

The successful integration of transplanted three-dimensional tissue engineering (TE) constructs depends greatly on their rapid vascularization. Therefore, it is essential to address this vascularization issue in the initial design of constructs for perfused tissues. Two of the most important variables in this regard are scaffold composition and cell sourcing. Collagens with marine origins overcome some issues associated with mammal-derived collagen while maintaining their advantages in terms of biocompatibility. Concurrently, the freshly isolated stromal vascular fraction (SVF) of adipose tissue has been proposed as an advantageous cell fraction for vascularization purposes due to its highly angiogenic properties, allowing extrinsic angiogenic growth factor-free vascularization strategies for TE applications. In this study, we aimed at understanding whether marine collagen 3D matrices could support cryopreserved human SVF in maintaining intrinsic angiogenic properties observed for fresh SVF. For this, cryopreserved human SVF was seeded on blue shark collagen sponges and cultured up to 7 days in a basal medium. The secretome profile of several angiogenesis-related factors was studied throughout culture times and correlated with the expression pattern of CD31 and CD146, which showed the formation of a prevascular network. Upon in ovo implantation, increased vessel recruitment was observed in prevascularized sponges when compared with sponges without SVF cells. Immunohistochemistry for CD31 demonstrated the improved integration of prevascularized sponges within chick chorioalantoic membrane (CAM) tissues, while in situ hybridization showed human cells lining blood vessels. These results demonstrate the potential of using cryopreserved SVF combined with marine collagen as a streamlined approach to improve the vascularization of TE constructs.

Integrin-specific hydrogels for growth factor-free vasculogenesis.

Integrin-binding biomaterials have been extensively evaluated for their capacity to enable de novo formation of capillary-like structures/vessels, ultimately supporting neovascularization in vivo. Yet, the role of integrins as vascular initiators in engineered materials is still not well understood. Here, we show that αvß3 integrin-specific 3D matrices were able to retain PECAM1+ cells from the stromal vascular fraction (SVF) of adipose tissue, triggering vasculogenesis in vitro in the absence of extrinsic growth factors. Our results suggest that αvß3-RGD-driven signaling in the formation of capillary-like structures prevents the activation of the caspase 8 pathway and activates the FAK/paxillin pathway, both responsible for endothelial cells (ECs) survival and migration. We also show that prevascularized αvß3 integrin-specific constructs inosculate with the host vascular system fostering in vivo neovascularization. Overall, this work demonstrates the ability of the biomaterial to trigger vasculogenesis in an integrin-specific manner, by activating essential pathways for EC survival and migration within a self-regulatory growth factor microenvironment. This strategy represents an improvement to current vascularization routes for Tissue Engineering constructs, potentially enhancing their clinical applicability.

Mineralized collagen as a bioactive ink to support encapsulation of human adipose stem cells: A step towards the future of bone regeneration.

Bioprinting - printing with incorporated living cells - has earned special attention on tissue engineering approaches, aiming to closer reproduce the 3D microenvironment of the target tissue. However, it raises extra complexity related to the need to use cell-friendly printing conditions that still comply with material printing fidelity. Inspired by the composite nano structural organization of mineralized tissues, this work reports the efficiency of the chemical approach followed to in situ mineralize blue shark skin collagen, at a nano scale level, to ultimately produce stable inks. The influence of initial cellular density was evaluated by assessing three different concentrations (2.5, 5 and 7.5 × 106 cells·ml-1) of human adipose stem cells (hASC), with the higher density of encapsulated cells presenting improved viability in a long culture term. Immunodetection of osteogenic-related markers, like RUNX2 and osteopontin, 21 days after cell culture in basal conditions confirmed the potential of the ink to be applied for osteogenic purposes, which may be associated with the success of the cell-to-ink interaction and the Ca2+ ions released from the co-precipitated hydroxyapatite. A combination of mineralized shark collagen, alginate and hASC is thus proposed as a bioactive bioink with potential properties for regeneration of bone tissue.

Long-term and short-term preservation strategies for tissue engineering and regenerative medicine products: state of the art and emerging trends.

There is an ever-growing need of human tissues and organs for transplantation. However, the availability of such tissues and organs is insufficient by a large margin, which is a huge medical and societal problem. Tissue engineering and regenerative medicine (TERM) represent potential solutions to this issue and have therefore been attracting increased interest from researchers and clinicians alike. But the successful large-scale clinical deployment of TERM products critically depends on the development of efficient preservation methodologies. The existing preservation approaches such as slow freezing, vitrification, dry state preservation, and hypothermic and normothermic storage all have issues that somehow limit the biomedical applications of TERM products. In this review, the principles and application of these approaches will be summarized, highlighting their advantages and limitations in the context of TERM products preservation.

Correction: Strategies for the hypothermic preservation of cell sheets of human adipose stem cells.

[This corrects the article DOI: 10.1371/journal.pone.0222597.].

Prionace glauca skin collagen bioengineered constructs as a promising approach to trigger cartilage regeneration.

Representing a strategy of marine by-products valorization, based on isolation of biocompounds and assessment of biomedical applicability, the potential of blue shark (Prionace glauca (PG)) skin collagen to induce chondrogenic differentiation of human adipose stem cells (hASC) was investigated, with and without exogenous stimulation. For that, a cryogelation method was applied to produce highly interconnected porous 3-dimensional (3D) constructs made of collagen and collagen:hyaluronic acid (20:1). In vitro studies reveal that hASC adhere abundantly to the constructs which then suggests the early chondrogenic differentiation of those cells. These findings are supported by the mRNA expression encoding chondrogenic-related markers like Coll II and Sox-9 that are markedly upregulated at an early stage for both conditions, with and without exogenous stimulation. The introduction of hyaluronic acid (Hya) seems to play a crucial role at later time points, as shown by the evident immunodetection of aggrecan (ACAN), even without exogenous stimulation. It is hypothesized that the PG collagen itself can support chondrogenic differentiation at early time points, but exogenous stimulation is required to ensure phenotype maintenance. The present work highlights the relevance of using blue shark collagen biopolymer as a building block to produce highly effective temporary matrices for cartilage applications.

Strategies for the hypothermic preservation of cell sheets of human adipose stem cells.

Cell Sheet (CS) Engineering is a regenerative medicine strategy proposed for the treatment of injured or diseased organs and tissues. In fact, several clinical trials are underway using CS-based methodologies. However, the clinical application of such cell-based methodologies poses several challenges related with the preservation of CS structure and function from the fabrication site to the bedside. Pausing cells at hypothermic temperatures has been suggested as a valuable method for short-term cell preservation. In this study, we tested the efficiency of two preservation strategies, one using culture medium supplementation with Rokepie and the other using the preservation solution Hypothermosol, in preserving human adipose stromal/stem cells (hASC) CS-like confluent cultures at 4°C, during 3 and 7 days. Both preservation strategies demonstrated excellent ability to preserve cell function during the first 3 days in hypothermia, as demonstrated by metabolic activity results and assessment of extracellular matrix integrity and differentiation potential. At the end of the 7th day of hypothermic incubation, the decrease in cell metabolic activity was more evident for all conditions. Nonetheless, hASC incubated with Rokepie and Hypothermosol retained a higher metabolic activity and extracellular matrix integrity in comparison with unsupplemented cells. Differentiation results for the later time point showed that supplementation with both Rokepie and Hypothermosol rescued adipogenic differentiation potential but only Rokepie was able to preserve hASC osteogenic potential.