Thermal conditioning improves quality and speed of keratinocyte sheet production for burn wound treatment

BACKGROUND AIMS Cultured patient-specific keratinocyte sheets have been used clinically since the 1970s for the treatment of large severe burns. However, despite significant developments in recent years, successful and sustainable treatment is still a challenge. Reliable, high-quality grafts with faster availability and a flexible time window for transplantation are required to improve clinical outcomes. METHODS Keratinocytes are usually grown in vitro at 37°C. Given the large temperature differences in native skin tissue, the aim of the authors' study was to investigate thermal conditioning of keratinocyte sheet production. Therefore, the influence of 31°C, 33°C and 37°C on cell expansion and differentiation in terms of proliferation and sheet formation efficacy was investigated. In addition, the thermal effect on the biological status and thus the quality of the graft was assessed on the basis of the release of wound healing-related biofactors in various stages of graft development. RESULTS The authors demonstrated that temperature is a decisive factor in the production of human keratinocyte sheets. By using specific temperature ranges, the authors have succeeded in optimizing the individual manufacturing steps. During the cell expansion phase, cultivation at 37°C was most effective. After 6 days of culture at 37°C, three times and six times higher numbers of viable cells were obtained compared with 33°C and 31°C. During the cell differentiation and sheet formation phase, however, the cells benefited from a mildly hypothermic temperature of 33°C. Keratinocytes showed increased differentiation potential and formed better epidermal structures, which led to faster biomechanical sheet stability at day 18. In addition, a cultivation temperature of 33°C resulted in a longer lasting and higher secretion of the investigated immunomodulatory, anti-inflammatory, angiogenic and pro-inflammatory biofactors. CONCLUSIONS These results show that by using specific temperature ranges, it is possible to accelerate the large-scale production of cultivated keratinocyte sheets while at the same time improving quality. Cultivated keratinocyte sheets are available as early as 18 days post-biopsy and at any time for 7 days thereafter, which increases the flexibility of the process for surgeons and patients alike. These findings will help to provide better clinical outcomes, with an increased take rate in severe burn patients

Puncturing of lyophilized tissue engineered vascular matrices enhances the efficiency of their recellularization

Data on in vitro engineered "off the shelf" matrices support the concept of endogenous cellular repopulation driving the graft's remodeling via immune-mediated response. This seems important to further accelerate the cell reconstitution and may play a crucial role when mononuclear cells are used. Nevertheless, studies on decellularized xenogeneic grafts showed only limited host cell repopulation post-implantation. This study aims at a systematic comparison of reseeding methods (dripping, injection, bathing in a cell suspension and combined puncturing-dripping method) to define the most efficient technique enhancing recellularization of tissue engineered vascular matrices (patches, vessels, small diameter and standard size valves) prior implantation. The constructs were analyzed histologically, biochemically and biomechanically. Various preconditioning treatments (wet, lyophilized and air-dried) combined with reseeding methods demonstrated the highest cell loading efficiency, despite applied crimping and flow stress, of lyophilization followed by puncturing-dripping technique. This novel seeding method allows for an efficient, time saving graft reseeding that can be used within a one-step cardiovascular clinical intervention. STATEMENT OF SIGNIFICANCE The concept of living tissue engineered, self-repairing, autologous cardiovascular replacements, was proposed alternatively to existing synthetic/xenogeneic prostheses. Recent studies in animal models demonstrate faster in vivo recellularization after grafts pre-seeding with cells prior implantation. Pre-seeded cells hold either, the ability to differentiate directionally or attract host cells, crucial for graft integration and remodeling. It is unclear, however, how efficient the pre-loading is and how well cells withstand the flow. The study presents a systematic overview on cell loading techniques of different cardiovascular constructs, tested under static and dynamic conditions. Comparison illustrates a significantly higher efficiency of cells loading in lyophilized tissues punctured before their standard seeding. This technique may beneficially accelerate remodeling of cardiovascular grafts in further in vivo studies

Self-actualization at work. How can the reward system of a company support employees in their self-actualization? A strategic approach for the case of VIŠE.

This research examines the role of self-actualization at work. Following the theory of Maslow’s hierarchy of needs, self-actualization is a goal that all human beings strive to achieve. Through the work field of VIŠE, which proves to be profound and consequently wants to build on current findings, self-actualization is an integral part of the company’s operations. For this reason, it is researched how self-actualization can be made tangible to employees using the reward system. In developing a method that uses the hierarchy of needs as a proceeding, reward types were assigned to each pyramid stage. The research was conducted both quantitative and qualitatively through a cross-industry questionnaire. The results present reward elements that make employees feel supported in their self- actualization at work. The quantitative results show a preference for extrinsic rewards, whereas the qualitative results present a great desire for intrinsic rewards. A balance can be achieved by involving each employee's perception, which has proven to be very important. Respectively, the inclusion of different rewards, both extrinsic and intrinsic, transmits important values into employees' lives and facilitates self-actualization at work

Heart Valve Replacements with Regenerative Capacity

The incidence of severe valvular dysfunctions (e.g., stenosis and insufficiency) is increasing, leading to over 300,000 valves implanted worldwide yearly. Clinically used heart valve replacements lack the capacity to grow, inherently requiring repetitive and high-risk surgical interventions during childhood. The aim of this review is to present how different tissue engineering strategies can overcome these limitations, providing innovative valve replacements that proved to be able to integrate and remodel in pre-clinical experiments and to have promising results in clinical studies. Upon description of the different types of heart valve tissue engineering (e.g., in vitro, in situ, in vivo, and the pre-seeding approach) we focus on the clinical translation of this technology. In particular, we will deepen the many technical, clinical, and regulatory aspects that need to be solved to endure the clinical adaptation and the commercialization of these promising regenerative valves

Adipose tissue-derived stem cells in regenerative medicine

\u3cp\u3eIn regenerative medicine, adult stem cells are the most promising cell types for cell-based therapies. As a new source for multipotent stem cells, human adipose tissue has been introduced. These so called adipose tissue-derived stem cells (ADSCs) are considered to be ideal for application in regenerative therapies. Their main advantage over mesenchymal stem cells derived from other sources, e.g. from bone marrow, is that they can be easily and repeatable harvested using minimally invasive techniques with low morbidity. ADSCs are multipotent and can differentiate into various cell types of the tri-germ lineages, including e.g. osteocytes, adipocytes, neural cells, vascular endothelial cells, cardiomyocytes, pancreatic β-cells, and hepatocytes. Interestingly, ADSCs are characterized by immunosuppressive properties and low immunogenicity. Their secretion of trophic factors enforces the therapeutic and regenerative outcome in a wide range of applications. Taken together, these particular attributes of ADSCs make them highly relevant for clinical applications. Consequently, the therapeutic potential of ADSCs is enormous. Therefore, this review will provide a brief overview of the possible therapeutic applications of ADSCs with regard to their differentiation potential into the tri-germ lineages. Moreover, the relevant advancements made in the field, regulatory aspects as well as other challenges and obstacles will be highlighted.\u3c/p\u3

Heart valve replacements with regenerative capacity

\u3cp\u3eThe incidence of severe valvular dysfunctions (e.g., stenosis and insufficiency) is increasing, leading to over 300,000 valves implanted worldwide yearly. Clinically used heart valve replacements lack the capacity to grow, inherently requiring repetitive and high-risk surgical interventions during childhood. The aim of this review is to present how different tissue engineering strategies can overcome these limitations, providing innovative valve replacements that proved to be able to integrate and remodel in pre-clinical experiments and to have promising results in clinical studies. Upon description of the different types of heart valve tissue engineering (e.g., in vitro, in situ, in vivo, and the pre-seeding approach) we focus on the clinical translation of this technology. In particular, we will deepen the many technical, clinical, and regulatory aspects that need to be solved to endure the clinical adaptation and the commercialization of these promising regenerative valves.\u3c/p\u3

Decellularized homologous tissue-engineered heart valves as off-the-shelf alternatives to xeno- and homografts

Decellularized xenogenic or allogenic heart valves have been used as starter matrix for tissue-engineering of valve replacements with (pre-)clinical promising results. However, xenografts are associated with the risk of immunogenic reactions or disease transmission and availability of homografts is limited. Alternatively, biodegradable synthetic materials have been used to successfully create tissue-engineered heart valves (TEHV). However, such TEHV are associated with substantial technological and logistical complexity and have not yet entered clinical use. Here, decellularized TEHV, based on biodegradable synthetic materials and homologous cells, are introduced as an alternative starter matrix for guided tissue regeneration. Decellularization of TEHV did not alter the collagen structure or tissue strength and favored valve performance when compared to their cell-populated counterparts. Storage of the decellularized TEHV up to 18 months did not alter valve tissue properties. Reseeding the decellularized valves with mesenchymal stem cells was demonstrated feasible with minimal damage to the reseeded valve when trans-apical valve delivery was simulated. In conclusion, decellularization of in-vitro grown TEHV provides largely available off-the-shelf homologous scaffolds suitable for reseeding with autologous cells and trans-apical valve delivery