Development of a UV crosslinked biodegradable hydrogel containing adipose derived stem cells to promote vascularization for skin wounds and tissue engineering

WOS: 000399256500014PubMed ID: 28343005The aim of this study was to design a dermal substitute containing adipose derived stem cells (ADSC) that can be used to improve the regeneration of skin on difficult wound beds by stimulating rapid neovascularization. This was achieved by first synthesizing methacrylated gelatin (GeIMA) and methacrylated hyaluronic acid (HAMA) precursors which could be stored at -80 degrees C after lyophilisation. Polymer precursors were then dissolved in media (in 15:1 ratio), ADSCs added together with the photoinitiator and crosslinked with 40 s of W. Hydrogels degraded by 50% over 3 weeks in an in vitro environment. ADSC loaded hydrogels could be easily handled with forceps (compressive modulus was 6 kPa). Transparency of the gel would allow a full field-of-view of a wound site. The hydrogels provided a suitable microenvironment for ADSC proliferation as shown by the filopodia observed in confocal micrographs. In vivo studies demonstrated that stem cell loaded hydrogels increased vascularization by up to 3 fold compared to their cell free counterparts. In conclusion, GelMA/HAMA hydrogels loaded with ADSC showed the desired proliferative and angiogenic properties essential to promote angiogenesis for wound healing and improving survival of tissue engineered skin. (C) 2017 Elsevier Ltd. All rights reserved.Middle East Technical University Center of Excellence in Biomaterials and Tissue Engineering (BIOMATEN); TUBITAKTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [2211-C, 2214-A]; European Association of Urology Scholarship Programme (EUSP); Rosetrees TrustRosetrees Trust; Urology Foundation; Rosetrees TrustRosetrees Trust [M537]The authors acknowledge Middle East Technical University Center of Excellence in Biomaterials and Tissue Engineering (BIOMATEN) for the use of the facilities and for the financial support, and METU Central Laboratory for SEM analysis. The authors also acknowledge TUBITAK 2211-C and 2214-A scholarships and Menekse Ermis Sen, MD, PhD, for statistical analysis. Naside Mangir was funded by the European Association of Urology Scholarship Programme (EUSP), The Rosetrees Trust and The Urology Foundation. We thank Dr. Sabiniano Roman for providing the cultured ADSC used in this study

Nuclear targeting peptide-modified, DOX-loaded, PHBV nanoparticles enhance drug efficacy by targeting to Saos-2 cell nuclear membranes

WOS: 000424943700004PubMed ID: 29297759The aim of this study was to target nano sized (266 +/- 25nm diameter) poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) particles carrying Doxorubicin (DOX), an anticancer agent, to human osteosarcoma cells (Saos-2). A nuclear targeting molecule (Nuclear Localization Signal, NLS), a 17 a.a. peptide, was attached onto the doxorubicin loaded nanoparticles. NLS conjugated nanoparticles surrounded the cell nuclei, but did not penetrate them. Free doxorubicinand doxorubicin loadednanoparticles entered the cytoplasm and were evenly distributed within the cytoplasm. The localization of the NLS-targetedparticles around the nuclear membrane caused a significantly higher decrease in the cancer cell numbers due to apoptosis or necrosis than the untargeted and free doxorubicin formulations showing the importance of targeting the nanoparticles to the nuclear membrane in the treatment of cancer.Middle East Technical University Center of Excellence in Biomaterials and Tissue Engineering (BIOMATEN)This work was financially supported by Middle East Technical University Center of Excellence in Biomaterials and Tissue Engineering (BIOMATEN)

An estradiol releasing, proangiogenic hydrogel as a candidate material for use in soft tissue interposition

Introduction and Objectives Soft tissue interposition (STI) using local and/or regional flaps is often necessary in urogenital reconstruction to stimulate wound healing and prevent recurrence. Harvesting STI flaps can cause donor site morbidity and may not be available in some patients. In this study, we designed estradiol (E2) releasing hydrogel that could be used as an alternative to a STI flap and to investigate its ability to stimulate tissue production and angiogenesis. Materials and Methods A hydrogel was constructed by crosslinking a solution of estradiol, methacrylated gelatin (15%, w/v), and methacrylated hyaluronic acid (1%, w/v). The release of estradiol was measured using a UV-spectrophotometer (lambda(max) = 220 nm). Angiogenesis was evaluated by an ex ovo chicken embryo chorioallantoic membrane (CAM) assay. Results Estradiol was gradually released from the hydrogel over 21 days. The hydrogels could be easily manipulated with surgical forceps without any deformation. The hydrogels significantly increased collagen production of human dermal fibroblasts (HDFs). Scanning electron microscopic examination demonstrated that HDFs produced significantly more extracellular matrix (ECM) on estradiol releasing hydrogels compared with the controls. Estradiol releasing hydrogels doubled the number of blood vessels growing toward the hydrogel compared with the controls (vasculogenic index, 59.6 [+/- 6.4] and 25.6 [+/- 4.0], respectively; [P < 0.05]). Conclusion We present a proangiogenic, degradable hydrogel that can be used as an off-the-shelf available substitute to traditional STI flaps. This is achieved by using estradiol as a potent stimulator of new tissue production and new blood vessel formation

An estradiol releasing, proangiogenic hydrogel as a candidate material for use in soft tissue interposition

Introduction and Objectives Soft tissue interposition (STI) using local and/or regional flaps is often necessary in urogenital reconstruction to stimulate wound healing and prevent recurrence. Harvesting STI flaps can cause donor site morbidity and may not be available in some patients. In this study, we designed estradiol (E2) releasing hydrogel that could be used as an alternative to a STI flap and to investigate its ability to stimulate tissue production and angiogenesis. Materials and Methods A hydrogel was constructed by crosslinking a solution of estradiol, methacrylated gelatin (15%, w/v), and methacrylated hyaluronic acid (1%, w/v). The release of estradiol was measured using a UV-spectrophotometer (lambda(max) = 220 nm). Angiogenesis was evaluated by an ex ovo chicken embryo chorioallantoic membrane (CAM) assay. Results Estradiol was gradually released from the hydrogel over 21 days. The hydrogels could be easily manipulated with surgical forceps without any deformation. The hydrogels significantly increased collagen production of human dermal fibroblasts (HDFs). Scanning electron microscopic examination demonstrated that HDFs produced significantly more extracellular matrix (ECM) on estradiol releasing hydrogels compared with the controls. Estradiol releasing hydrogels doubled the number of blood vessels growing toward the hydrogel compared with the controls (vasculogenic index, 59.6 [+/- 6.4] and 25.6 [+/- 4.0], respectively; [P < 0.05]). Conclusion We present a proangiogenic, degradable hydrogel that can be used as an off-the-shelf available substitute to traditional STI flaps. This is achieved by using estradiol as a potent stimulator of new tissue production and new blood vessel formation

Biocompatibility of Dead Sea Water and retinyl palmitate carrying poly(3-hydroxybutyrate-co-3-hydroxyvalerate) micro/nanoparticles designed for transdermal skin therapy

WOS: 000360829900001In this study, novel drug carriers were developed for the treatment of skin conditions such as psoriasis, aging, or ultraviolet damage using micro/nanocapsules and micro/nanospheres of poly(3-hydroxybutyrate-co-3-hydroxyvalerate). The sizes of the particles were in the micron range and were loaded with retinyl palmitate and Dead Sea Water. In some tests, MgCl2 was used as a substitute for Dead Sea Water for accurate determination of released ions of Dead Sea Water. Encapsulation efficiency and loading of water-soluble excipients Dead Sea Water and MgCl2 were almost eight times lower than the hydrophobic compound retinyl palmitate. The particles were not cytotoxic as determined with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test using L929 mouse fibroblasts, BALB/3T3 mouse embryo fibroblasts, and HaCaT human keratinocytes. Ames test showed that the carriers were not genotoxic. The particles penetrated the membrane of human osteosarcoma cells Saos 2 and accumulated in their cytoplasm. No reactive oxygen species production could be detected which indicated low or no inflammatory response toward the particles. In the tests with intact human skin, 1.2% of the retinyl palmitate-loaded poly(3-hydroxybutyrate-co-3-hydroxyvalerate) particles penetrated into the human skin, but when the skin was without stratum corneum and increased to 6.9%. In conclusion, these carriers have shown a significant potential as topical drug delivery systems in the personalized treatment of skin diseases because their contents could be modified according to a patient's needs and several drugs could be loaded in one type of microparticle, or several populations, each carrying a different drug, can be used in the treatment.FP7-NMP "SkinTreat" project [213202-2]; METUMiddle East Technical University [BAP-07-02-2012-101-91]The research was performed within the framework of the FP7-NMP "SkinTreat" project theme under grant agreement no. 213202-2. The authors also acknowledge the support of METU through the project BAP-07-02-2012-101-91

Fate of Poly-3-Hydroxybutyrate-co3-Hydroxyvalerate on Skin

Трансдермальные системы доставки лекарственных средств становятся всё более совершенными. Однако самым большим препятствием для разработки систем доставки лекарственных средств является наличие практически непроницаемого верхнего рогового слоя эпидермиса. Материалы, проникающие через кожу, должны иметь низкий молекулярный вес и быть липофильными. Существует много способов преодолеть барьер рогового слоя, и один из перспективных методов – использование микро- и наночастиц. Это исследование было направлено на разработку трёх различных микро- и наноразмерных носителей, изучение их проникновения в кожу и оценку их эффективности в коже. Были сконструированы полимерные микро/нанокапсулы из поли(3-гидроксибутирата/3-гидроксивалерата) (ПГБВ) (ГВ 5 мол. %) с флуоресцентным красителем Nile Red. Трансдермальное проникновение микро/нанокапсул из ПГБВ было изучено in vivo, на мышах. Согласно анализу размера частиц, проведённому с помощью анализатора Master Sizer и системы измерения дзета-потенциала, микро/ нанокапсулы имели диаметр 1,9 мкм, 426 нм и 166 нм. Частицы наносили на здоровую кожу спины мышей линии BALB/c. Чтобы оценить проникновение частиц, определяли присутствие ПГБВ на коже методом хромато-масс-спектрометрии. Морфология исследовалась с помощью растрового электронного микроскопа. Результаты хромато-масс-спектрометрии показали, что проникновение капсул через кожу зависело от размера частиц, несмотря на труднопроницаемый верхний слой кожи. Однако гистологический поперечный разрез показал, что частицы не могли проникнуть через неповреждённую кожу; роговой слой эпидермиса явно препятствовал переносу полимерных частиц. Данное исследование продемонстрировало, что, изменяя размер наночастиц, несущих лекарственные средства, можно регулировать глубину проникновения частиц и, следовательно, осуществлять целевую доставку лекарственных средств.Transdermal drug delivery systems have become increasingly sophisticated over time. However, the greatest limitation for developing an effective drug delivery system is the highly impermeable outermost layer of the skin called the stratum corneum. Therefore, materials penetrating the skin must be of low molecular weight, and lipophilic. There are many techniques to safely pass the stratum corneum and one of the promising method of transdermal drug delivery is the use of micro and nano sized particles. The aim of this study was to develop three different micro and nano sized carriers to study their skin penetration and to judge their effectiveness within the skin. Polymeric micro/ nanocapsules carrying a fluorescent dye Nile Red, were prepared using poly(3-hydroxybutyrate-co 3-hydroxyvalerate) (PHBV) (5 %mol. hydroxyvalerate). The in vivo transdermal permeation of PHBV micro/nanoparticles were studied using a mouse model. According to the particle size analysis with Master Sizer and Zeta Potential Measurement System, the PHBV micro/nanocapsules were 1.9 µm, 426 nm and 166 nm in diameter. The particles were applied to healthy skin of the dorsal region of BALB/c mice. Penetration of the particles was determined by GC-MS analysis of the skin for PHBV. Scanning electron microscope (SEM) imaging was used to study their morphology. GC-MS results showed that the capsules penetrated into the skin in relation with their particle size, despite the highly impermeable outer skin layer. However, histology cross-section revealed that uncompromised skin could not penetrate; the transport of the polymeric particle was clearly impeded by the stratum corneum. It was thus shown in this study that control of penetration depth, and therefore, the target size within into the skin is possible by varying the size of the drug carrying nanocapsules

Cell Aggregate Assembly through Microengineering for Functional Tissue Emergence

International audienceCompared to cell suspensions or monolayers, 3D cell aggregates provide cellular interactions organized in space and heterogeneity that better resume the real organization of native tissues. They represent powerful tools to narrow down the gap between in vitro and in vivo models, thanks to their self-evolving capabilities. Recent strategies have demonstrated their potential as building blocks to generate microtissues. Developing specific methodologies capable of organizing these cell aggregates into 3D architectures and environments has become essential to convert them into functional microtissues adapted for regenerative medicine or pharmaceutical screening purposes. Although the techniques for producing individual cell aggregates have been on the market for over a decade, the methodology for engineering functional tissues starting from them is still a young and quickly evolving field of research. In this review, we first present a panorama of emerging cell aggregates microfabrication and assembly technologies. We further discuss the perspectives opened in the establishment of functional tissues with a specific focus on controlled architecture and heterogeneity to favor cell differentiation and proliferation

Fate of Poly-3-Hydroxybutyrate-co3-Hydroxyvalerate on Skin

Трансдермальные системы доставки лекарственных средств становятся всё более совершенными. Однако самым большим препятствием для разработки систем доставки лекарственных средств является наличие практически непроницаемого верхнего рогового слоя эпидермиса. Материалы, проникающие через кожу, должны иметь низкий молекулярный вес и быть липофильными. Существует много способов преодолеть барьер рогового слоя, и один из перспективных методов – использование микро- и наночастиц. Это исследование было направлено на разработку трёх различных микро- и наноразмерных носителей, изучение их проникновения в кожу и оценку их эффективности в коже. Были сконструированы полимерные микро/нанокапсулы из поли(3-гидроксибутирата/3-гидроксивалерата) (ПГБВ) (ГВ 5 мол. %) с флуоресцентным красителем Nile Red. Трансдермальное проникновение микро/нанокапсул из ПГБВ было изучено in vivo, на мышах. Согласно анализу размера частиц, проведённому с помощью анализатора Master Sizer и системы измерения дзета-потенциала, микро/ нанокапсулы имели диаметр 1,9 мкм, 426 нм и 166 нм. Частицы наносили на здоровую кожу спины мышей линии BALB/c. Чтобы оценить проникновение частиц, определяли присутствие ПГБВ на коже методом хромато-масс-спектрометрии. Морфология исследовалась с помощью растрового электронного микроскопа. Результаты хромато-масс-спектрометрии показали, что проникновение капсул через кожу зависело от размера частиц, несмотря на труднопроницаемый верхний слой кожи. Однако гистологический поперечный разрез показал, что частицы не могли проникнуть через неповреждённую кожу; роговой слой эпидермиса явно препятствовал переносу полимерных частиц. Данное исследование продемонстрировало, что, изменяя размер наночастиц, несущих лекарственные средства, можно регулировать глубину проникновения частиц и, следовательно, осуществлять целевую доставку лекарственных средств.Transdermal drug delivery systems have become increasingly sophisticated over time. However, the greatest limitation for developing an effective drug delivery system is the highly impermeable outermost layer of the skin called the stratum corneum. Therefore, materials penetrating the skin must be of low molecular weight, and lipophilic. There are many techniques to safely pass the stratum corneum and one of the promising method of transdermal drug delivery is the use of micro and nano sized particles. The aim of this study was to develop three different micro and nano sized carriers to study their skin penetration and to judge their effectiveness within the skin. Polymeric micro/ nanocapsules carrying a fluorescent dye Nile Red, were prepared using poly(3-hydroxybutyrate-co 3-hydroxyvalerate) (PHBV) (5 %mol. hydroxyvalerate). The in vivo transdermal permeation of PHBV micro/nanoparticles were studied using a mouse model. According to the particle size analysis with Master Sizer and Zeta Potential Measurement System, the PHBV micro/nanocapsules were 1.9 µm, 426 nm and 166 nm in diameter. The particles were applied to healthy skin of the dorsal region of BALB/c mice. Penetration of the particles was determined by GC-MS analysis of the skin for PHBV. Scanning electron microscope (SEM) imaging was used to study their morphology. GC-MS results showed that the capsules penetrated into the skin in relation with their particle size, despite the highly impermeable outer skin layer. However, histology cross-section revealed that uncompromised skin could not penetrate; the transport of the polymeric particle was clearly impeded by the stratum corneum. It was thus shown in this study that control of penetration depth, and therefore, the target size within into the skin is possible by varying the size of the drug carrying nanocapsules

Scalable Generation of Pre‐Vascularized and Functional Human Beige Adipose Organoids

International audienceObesity and type 2 diabetes are becoming a global sociobiomedical burden. Beige adipocytes are emerging as key inducible actors and putative relevant therapeutic targets for improving metabolic health. However, in vitro models of human beige adipose tissue are currently lacking and hinder research into this cell type and biotherapy development. Unlike traditional bottom-up engineering approaches that aim to generate building blocks, here a scalable system is proposed to generate pre-vascularized and functional human beige adipose tissue organoids using the human stromal vascular fraction of white adipose tissue as a source of adipose and endothelial progenitors. This engineered method uses a defined biomechanical and chemical environment using tumor growth factor β (TGFβ) pathway inhibition and specific gelatin methacryloyl (GelMA) embedding parameters to promote the self-organization of spheroids in GelMA hydrogel, facilitating beige adipogenesis and vascularization. The resulting vascularized organoids display key features of native beige adipose tissue including inducible Uncoupling Protein-1 (UCP1) expression, increased uncoupled mitochondrial respiration, and batokines secretion. The controlled assembly of spheroids allows to translate organoid morphogenesis to a macroscopic scale, generating vascularized centimeter-scale beige adipose micro-tissues. This approach represents a significant advancement in developing in vitro human beige adipose tissue models and facilitates broad applications ranging from basic research to biotherapies