Combination of Adipose-Derived Stromal/Stem Cells and Decellularized Adipose Tissue Hydrogel for Osteogenic Applications.

Adipose-derived stromal/stem cells (ASCs) and decellularized adipose tissue (DAT) are adipose tissue products obtained from individuals undergoing fat removal procedures like liposuction, lipectomy, or breast reduction. DAT hydrogel is prepared by removing the cells from the adipose tissue and digesting it to form a liquid material that forms a gel at physiological temperature. ASCs seeded on DAT have displayed osteogenic potential in vitro and in animal models of bone defects. Herein, we describe the methods for preparing DAT hydrogel, ASC seeding in DAT hydrogel, osteogenic differentiation of ASCs, creation of critical-sized femur defect model in mice, its treatment with ASC-DAT hydrogel, and analyses.

Preparation of Decellularized Amniotic Membrane and Adipose-Derived Stromal/Stem Cell Seeding.

Amniotic membrane, being part of the placenta, is discarded as medical waste after childbirth. It can be decellularized to convert it into an acellular material while retaining the extracellular matrix. Such amniotic membrane grafts support stem cell adhesion, growth, and proliferation. These properties make it a useful candidate to be used as a bio-scaffold in regenerative medicine. This chapter describes a method for the decellularization of the amniotic membrane. Furthermore, the method for seeding adipose-derived stem cells on the decellularized amniotic membrane is described.

3D bio scaffold support osteogenic differentiation of mesenchymal stem cells.

The regeneration of osteochondral lesions by tissue engineering techniques is challenging due to the lack of physicochemical characteristics and dual-lineage (osteogenesis and chondrogenesis). A scaffold with better mechanical properties and dual lineage capability is required for the regeneration of osteochondral defects. In this study, a hydrogel prepared from decellularized human umbilical cord tissue was developed and evaluated for osteochondral regeneration. Mesenchymal stem cells (MSCs) isolated from the umbilical cord were seeded with hydrogel for 28 days, and cell-hydrogel composites were cultured in basal and osteogenic media. Alizarin red staining, quantitative polymerase chain reaction, and immunofluorescent staining were used to confirm that the hydrogel was biocompatible and capable of inducing osteogenic differentiation in umbilical cord-derived MSCs. The findings demonstrate that human MSCs differentiated into an osteogenic lineage following 28 days of cultivation in basal and osteoinductive media. The expression was higher in the cell-hydrogel composites cultured in osteoinductive media, as evidenced by increased levels of messenger RNA and protein expression of osteogenic markers as compared to basal media cultured cell-hydrogel composites. Additionally, calcium deposits were also observed, which provide additional evidence of osteogenic differentiation. The findings demonstrate that the hydrogel is biocompatible with MSCs and possesses osteoinductive capability in vitro. It may be potentially useful for osteochondral regeneration.

Assessment of the proteome profile of decellularized human amniotic membrane and its biocompatibility with umbilical cord-derived mesenchymal stem cells.

Extracellular matrix-based bio-scaffolds are useful for tissue engineering as they retain the unique structural, mechanical, and physiological microenvironment of the tissue thus facilitating cellular attachment and matrix activities. However, considering its potential, a comprehensive understanding of the protein profile remains elusive. Herein, we evaluate the impact of decellularization on the human amniotic membrane (hAM) based on its proteome profile, physicochemical features, as well as the attachment, viability, and proliferation of umbilical cord-derived mesenchymal stem cells (hUC-MSC). Proteome profiles of decellularized hAM (D-hAM) were compared with hAM, and gene ontology (GO) enrichment analysis was performed. Proteomic data revealed that D-hAM retained a total of 249 proteins, predominantly comprised of extracellular matrix proteins including collagens (collagen I, collagen IV, collagen VI, collagen VII, and collagen XII), proteoglycans (biglycan, decorin, lumican, mimecan, and versican), glycoproteins (dermatopontin, fibrinogen, fibrillin, laminin, and vitronectin), and growth factors including transforming growth factor beta (TGF-ß) and fibroblast growth factor (FGF) while eliminated most of the intracellular proteins. Scanning electron microscopy was used to analyze the epithelial and basal surfaces of D-hAM. The D-hAM displayed variability in fibril morphology and porosity as compared with hAM, showing loosely packed collagen fibers and prominent large pore areas on the basal side of D-hAM. Both sides of D-hAM supported the growth and proliferation of hUC-MSC. Comparative investigations, however, demonstrated that the basal side of D-hAM displayed higher hUC-MSC proliferation than the epithelial side. These findings highlight the importance of understanding the micro-environmental differences between the two sides of D-hAM while optimizing cell-based therapeutic applications.

Biomechanical and Biological Characterization of XGel, a Human-Derived Hydrogel for Stem Cell Expansion and Tissue Engineering.

Hydrogels are 3D scaffolds used as alternatives to in vivo models for disease modeling and delivery of cells and drugs. Existing hydrogel classifications include synthetic, recombinant, chemically defined, plant- or animal-based, and tissue-derived matrices. There is a need for materials that can support both human tissue modeling and clinically relevant applications requiring stiffness tunability. Human-derived hydrogels are not only clinically relevant, but they also minimize the use of animal models for pre-clinical studies. This study aims to characterize XGel, a new human-derived hydrogel as an alternative to current murine-derived and synthetic recombinant hydrogels that features unique physiochemical, biochemical, and biological properties that support adipocyte and bone differentiation. Rheology studies determine the viscosity, stiffness, and gelation features of XGel. Quantitative studies for quality control support consistency in the protein content between lots. Proteomics studies reveal that XGel is predominantly composed of extracellular matrix proteins, including fibrillin, collagens I-VI, and fibronectin. Electron microscopy of the hydrogel provides phenotypic characteristics in terms of porosity and fiber size. The hydrogel demonstrates biocompatibility as a coating material and as a 3D scaffold for the growth of multiple cell types. The results provide insight into the biological compatibility of this human-derived hydrogel for tissue engineering.

Minocycline pre-treatment up-regulates antioxidant enzymes and enhances the regenerative potential of MSCs in rat myocardial infarction model.

Objectives: To determine the effect of the pre-treatment of mesenchymal stem cells (MSCs) with minocycline on the expression of antioxidant genes and cardiac repair post myocardial infarction (MI) in rats. METHODS: Rat bone marrow derived MSCs were used in the study. Cytotoxicity of minocycline in MSCs was determined using JC1 assay to identify a safe drug dose for further experiments. The MSCs were pre-treated with 1.0 µM minocycline for 24 hours and then treated with hydrogen peroxide (H2O2), after that mRNA was isolated and the expression levels of antioxidant genes including peroxiredoxin, glutathione peroxidase, and superoxide dismutase were determined. Finally, minocycline pre-treated MSCs were used to treat rats induced with MI by the ligation of left anterior descending coronary artery. The cardiac function was evaluated at two and four weeks post MI using echocardiography. RESULTS: At 1.0 µM concentration, minocycline was found to be safe for MSCs and used for subsequent experiments. Minocycline pre-treatment was found to up regulate several antioxidant genes in oxidatively stressed MSCs. Furthermore, minocycline pre-treated MSCs displayed greater improvement in cardiac left ventricular function at two and four-weeks post MI as compared to untreated rats. CONCLUSIONS: Pre-treatment of MSCs with minocycline enhances the expression of antioxidant genes and promotes their capability to repair cardiac function after MI.

The Emerging Use of ASC/Scaffold Composites for the Regeneration of Osteochondral Defects.

Articular cartilage is composed of chondrocytes surrounded by a porous permeable extracellular matrix. It has a limited spontaneous healing capability post-injury which, if left untreated, can result in severe osteochondral disease. Currently, osteochondral (OC) defects are treated by bone marrow stimulation, artificial joint replacement, or transplantation of bone, cartilage, and periosteum, while autologous osteochondral transplantation is also an option; it carries the risk of donor site damage and is limited only to the treatment of small defects. Allografts may be used for larger defects; however, they have the potential to elicit an immune response. A possible alternative solution to treat osteochondral diseases involves the use of stromal/stem cells. Human adipose-derived stromal/stem cells (ASCs) can differentiate into cartilage and bone cells. The ASC can be combined with both natural and synthetic scaffolds to support cell delivery, growth, proliferation, migration, and differentiation. Combinations of both types of scaffolds along with ASCs and/or growth factors have shown promising results for the treatment of OC defects based on in vitro and in vivo experiments. Indeed, these findings have translated to several active clinical trials testing the use of ASC-scaffold composites on human subjects. The current review critically examines the literature describing ASC-scaffold composites as a potential alternative to conventional therapies for OC tissue regeneration.

The role of MEK1/2 and MEK5 in melatonin-mediated actions on osteoblastogenesis, osteoclastogenesis, bone microarchitecture, biomechanics, and bone formation.

Melatonin, the primary hormone involved in circadian entrainment, plays a significant role in bone physiology. This study aimed to assess the role of MEK1/2 and MEK5 in melatonin-mediated actions in mouse and human mesenchymal stem cells (MSCs) and on bone using small-molecule inhibitors and CRISPR/Cas9 knockout approaches. Consistent with in vitro studies performed in mMSCs and hMSCs, nightly (25 mg/kg, i.p., 45 days) injections with PD184352 (MEK1/2 inhibitor) or Bix02189 (MEK5 inhibitor) or SC-1-151 (MEK1/2/5 inhibitor) demonstrated that MEK1/2 and MEK5 were the primary drivers underlying melatonin's actions on bone density, microarchitecture (i.e., trabecular number, separation, and connectivity density), and bone mechanical properties (i.e., ultimate stress) through increases in osteogenic (RUNX2, BMP-2, FRA-1, OPG) expression and decreases in PPARγ. Furthermore, CRISPR/Cas9 knockout of MEK1 or MEK5 in mMSCs seeded on PLGA scaffolds and placed into critical-size calvarial defects in Balb(c) mice (male and female) revealed that treatment with melatonin (15 mg/L; p.o., nightly, 90 days) mediates sex-specific actions of MEK1 and MEK5 in new bone formation. This study is the first to demonstrate a role for MEK1/2 and MEK5 in modulating melatonin-mediated actions on bone formation in vivo and in a sex-specific manner.

Decellularized Human Umbilical Tissue-Derived Hydrogels Promote Proliferation and Chondrogenic Differentiation of Mesenchymal Stem Cells.

Tissue engineering is a promising approach for the repair and regeneration of cartilaginous tissue. Appropriate three-dimensional scaffolding materials that mimic cartilage are ideal for the repair of chondral defects. The emerging decellularized tissue-based scaffolds have the potential to provide essential biochemical signals and structural integrity, which mimics the natural tissue environment and directs cellular fate. Umbilical cord-derived hydrogels function as 3D scaffolding material, which support adherence, proliferation, migration, and differentiation of cells due to their similar biochemical composition to cartilage. Therefore, the present study aimed to establish a protocol for the formulation of a hydrogel from decellularized human umbilical cord (DUC) tissue, and assess its application in the proliferation and differentiation of UC-MSCs along chondrogenic lineage. The results showed that the umbilical cord was efficiently decellularized. Subsequently, DUC hydrogel was prepared, and in vitro chondral differentiation of MSCs seeded on the scaffold was determined. The developed protocol efficiently removed the cellular and nuclear content while retaining the extracellular matrix (ECM). DUC tissue, pre-gel, and hydrogels were evaluated by FTIR spectroscopy, which confirmed the gelation from pre-gel to hydrogel. SEM analysis revealed the fibril morphology and porosity of the DUC hydrogel. Calcein AM and Alamar blue assays confirmed the MSC survival, attachment, and proliferation in the DUC hydrogels. Following seeding of UC-MSCs in the hydrogels, they were cultured in stromal or chondrogenic media for 28 days, and the expression of chondrogenic marker genes including TGF-ß1, BMP2, SOX-9, SIX-1, GDF-5, and AGGRECAN was significantly increased (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001). Moreover, the hydrogel concentration was found to significantly affect the expression of chondrogenic marker genes. The overall results indicate that the DUC-hydrogel is compatible with MSCs and supports their chondrogenic differentiation in vitro.

Breast Cancer Reconstruction: Design Criteria for a Humanized Microphysiological System.

International regulatory agencies such as the Food and Drug Administration have mandated that the scientific community develop humanized microphysiological systems (MPS) as an in vitro alternative to animal models in the near future. While the breast cancer research community has long appreciated the importance of three-dimensional growth dynamics in their experimental models, there are remaining obstacles preventing a full conversion to humanized MPS for drug discovery and pathophysiological studies. This perspective evaluates the current status of human tissue-derived cells and scaffolds as building blocks for an "idealized" breast cancer MPS based on bioengineering design principles. It considers the utility of adipose tissue as a potential source of endothelial, lymphohematopoietic, and stromal cells for the support of breast cancer epithelial cells. The relative merits of potential MPS scaffolds derived from adipose tissue, blood components, and synthetic biomaterials is evaluated relative to the current "gold standard" material, Matrigel, a murine chondrosarcoma-derived basement membrane-enriched hydrogel. The advantages and limitations of a humanized breast cancer MPS are discussed in the context of in-process and destructive read-out assays. Impact statement Regulatory authorities have highlighted microphysiological systems as an emerging tool in breast cancer research. This has been led by calls for more predictive human models and reduced animal experimentation. This perspective describes how human-derived cells, extracellular matrices, and hydrogels will provide the building blocks to create breast cancer models that accurately reflect diversity at multiple levels, that is, patient ethnicity, pathophysiology, and metabolic status.

Adipose-derived cells: building blocks of three-dimensional microphysiological systems.

Microphysiological systems (MPS) created with human-derived cells and biomaterial scaffolds offer a potential in vitro alternative to in vivo animal models. The adoption of three-dimensional MPS models has economic, ethical, regulatory, and scientific implications for the fields of regenerative medicine, metabolism/obesity, oncology, and pharmaceutical drug discovery. Key opinion leaders acknowledge that MPS tools are uniquely positioned to aid in the objective to reduce, refine, and eventually replace animal experimentation while improving the accuracy of the finding's clinical translation. Adipose tissue has proven to be an accessible and available source of human-derived stromal vascular fraction (SVF) cells, a heterogeneous population available at point of care, and adipose-derived stromal/stem cells, a relatively homogeneous population requiring plastic adherence and culture expansion of the SVF cells. The adipose-derived stromal/stem cells or SVF cells, in combination with human tissue or synthetic biomaterial scaffolds, can be maintained for extended culture periods as three-dimensional MPS models under angiogenic, stromal, adipogenic, or osteogenic conditions. This review highlights recent literature relating to the versatile use of adipose-derived cells as fundamental components of three-dimensional MPS models for discovery research and development. In this context, it compares the merits and limitations of the adipose-derived stromal/stem cells relative to SVF cell models and considers the likely directions that this emerging field of scientific discovery will take in the near future.

Adipose-Derived Stem Cells from Obese Donors Polarize Macrophages and Microglia toward a Pro-Inflammatory Phenotype.

Macrophages and microglia represent the primary phagocytes and first line of defense in the peripheral and central immune systems. They activate and polarize into a spectrum of pro- and anti-inflammatory phenotypes in response to various stimuli. This activation is tightly regulated to balance the appropriate immune response with tissue repair and homeostasis. Disruption of this balance results in inflammatory disease states and tissue damage. Adipose stem cells (ASCs) have great therapeutic potential because of the potent immunomodulatory capabilities which induce the polarization of microglia and macrophages to the anti-inflammatory, M2, phenotype. In this study, we examined the effects of donor heterogeneity on ASC function. Specifically, we investigated the impact of donor obesity on ASC stemness and immunomodulatory abilities. Our findings revealed that ASCs from obese donors (ObASCs) exhibited reduced stem cell characteristics when compared to ASCs from lean donors (LnASCs). We also found that ObASCs promote a pro-inflammatory phenotype in murine macrophage and microglial cells, as indicated by the upregulated expression of pro-inflammatory genes, increased nitric oxide pathway activity, and impaired phagocytosis and migration. These findings highlight the importance of considering individual donor characteristics such as obesity when selecting donors and cells for use in ASC therapeutic applications and regenerative medicine.

Clinical Translational Potential in Skin Wound Regeneration for Adipose-Derived, Blood-Derived, and Cellulose Materials: Cells, Exosomes, and Hydrogels.

Acute and chronic skin wounds due to burns, pressure injuries, and trauma represent a substantial challenge to healthcare delivery with particular impacts on geriatric, paraplegic, and quadriplegic demographics worldwide. Nevertheless, the current standard of care relies extensively on preventive measures to mitigate pressure injury, surgical debridement, skin flap procedures, and negative pressure wound vacuum measures. This article highlights the potential of adipose-, blood-, and cellulose-derived products (cells, decellularized matrices and scaffolds, and exosome and secretome factors) as a means to address this unmet medical need. The current status of this research area is evaluated and discussed in the context of promising avenues for future discovery.

Characterization and Proteomic Analysis of Decellularized Adipose Tissue Hydrogels Derived from Lean and Overweight/Obese Human Donors.

While decellularized adipose tissue (DAT) has potential as an "off-the-shelf" biomaterial product for regenerative medicine, it remains to be determined if donor-source body mass index (BMI) impacts the functionality of DAT. This study set out to comparatively characterize lean versus overweight/obese-donor derived DAT hydrogel based on proteome and to analyze their respective effects on adipose stromal/stem cell (ASC) viability, and differentiation in vitro. Decellularized adipose tissue from lean (lDAT) and overweight/obese (oDAT) donors is produced and characterized. Variability in the fibril microstructures is found, with dense fibrotic fiber clusters and large pore area uniquely present in the oDAT samples. Proteomic analysis reveals that lDAT contains a greater proportion of enriched extracellular proteins and a smaller proportion of enriched intracellular proteins relative to oDAT. Biocompatibility studies show that ASCs cultured in lDAT and oDAT hydrogels remain viable. The adipogenic and osteogenic differentiation capability of ASCs seeded in lDAT and oDAT hydrogels is confirmed by an upregulation in marker gene expression and phenotypic analysis. In conclusion, this study establishes that DAT hydrogels derived from lean and overweight/obese adipose donors present similar physicochemical profiles with some distinctive features while comparably supporting the viability and adipogenic differentiation of ASCs in vitro.

Decellularized Adipose Tissue: Biochemical Composition, in vivo Analysis and Potential Clinical Applications.

Decellularized tissues are gaining popularity as scaffolds for tissue engineering; they allow cell attachment, proliferation, differentiation, and are non-immunogenic. Adipose tissue is an abundant resource that can be decellularized and converted in to a bio-scaffold. Several methods have been developed for adipose tissue decellularization, typically starting with freeze thaw cycles, followed by washes with hypotonic/hypertonic sodium chloride solution, isopropanol, detergent (SDS, SDC and Triton X-100) and trypsin digestion. After decellularization, decellularized adipose tissue (DAT) can be converted into a powder, solution, foam, or sheet to allow for convenient subcutaneous implantation or to repair external injuries. Additionally, DAT bio-ink can be used to 3D print structures that closely resemble physiological tissues and organs. Proteomic analysis of DAT reveals that it is composed of collagens (I, III, IV, VI and VII), glycosaminoglycans, laminin, elastin, and fibronectin. It has also been found to retain growth factors like VEGF and bFGF after decellularization. DAT inherently promotes adipogenesis when seeded with adipose stem cells in vitro, and when DAT is implanted subcutaneously it is capable of recruiting host stem cells and forming adipose tissue in rodents. Furthermore, DAT has promoted healing in rat models of full-thickness skin wounds and peripheral nerve injury. These findings suggest that DAT is a promising candidate for repair of soft tissue defects, and is suitable for breast reconstruction post-mastectomy, wound healing, and adipose tissue regeneration. Moreover, since DAT's form and stiffness can be altered by physicochemical manipulation, it may prove suitable for engineering of additional soft and hard tissues.

Decellularized Adipose Tissue Hydrogel Promotes Bone Regeneration in Critical-Sized Mouse Femoral Defect Model.

Critical-sized bone defects fail to heal and often cause non-union. Standard treatments employ autologous bone grafting, which can cause donor tissue loss/pain. Although several scaffold types can enhance bone regeneration, multiple factors limit their level of success. To address this issue, this study evaluated a novel decellularized human adipose tissue (DAT) hydrogel as an alternative. In this study, DAT hydrogel alone, or in combination with adipose-derived stromal/stem cells (ASC), osteo-induced ASCs (OIASC), and hydroxyapatite were tested for their ability to mediate repair of a critical-sized (3 mm) femoral defect created in C57BL/6 mice. Micro-computed tomography results showed that all DAT hydrogel treated groups significantly enhanced bone regeneration, with OIASC + hydroxyapatite treated group displaying the most robust bone regeneration. Histological analyses revealed that all treatments resulted in significantly higher tissue areas with the relative mineralized tissue area significantly increased at 12 weeks; however, cartilaginous content was lowest among treatment groups with OIASC. Immunohistochemical analyses showed that DAT hydrogel enhanced collagen I and osteopontin expression, while the addition of OIASCs to the hydrogel reduced collagen II levels. Thus, DAT hydrogel promotes bone regeneration in a critical-sized femoral defect model that is further enhanced in the presence of OIASCs and hydroxyapatite.

Human Platelet Lysate as a Functional Substitute for Fetal Bovine Serum in the Culture of Human Adipose Derived Stromal/Stem Cells.

INTRODUCTION: Adipose derived stromal/stem cells (ASCs) hold potential as cell therapeutics for a wide range of disease states; however, many expansion protocols rely on the use of fetal bovine serum (FBS) as a cell culture nutrient supplement. The current study explores the substitution of lysates from expired human platelets (HPLs) as an FBS substitute. METHODS: Expired human platelets from an authorized blood center were lysed by freeze/thawing and used to examine human ASCs with respect to proliferation using hematocytometer cell counts, colony forming unit-fibroblast (CFU-F) frequency, surface immunophenotype by flow cytometry, and tri-lineage (adipocyte, chondrocyte, osteoblast) differentiation potential by histochemical staining. RESULTS: The proliferation assays demonstrated that HPLs supported ASC proliferation in a concentration dependent manner, reaching levels that exceeded that observed in the presence of 10% FBS. The concentration of 0.75% HPLs was equivalent to 10% FBS when utilized in cell culture media with respect to proliferation, immunophenotype, and CFU-F frequency. When added to osteogenic, adipogenic, and chondrogenic differentiation media, both supplements showed appropriate differentiation by staining. CONCLUSION: HPLs is an effective substitute for FBS in the culture, expansion and differentiation of human ASCs suitable for pre-clinical studies; however, additional assays and analyses will be necessary to validate HPLs for clinical applications and regulatory approval.

Beyond the Present Constraints That Prevent a Wide Spread of Tissue Engineering and Regenerative Medicine Approaches.

Despite the success of tissue engineered medical products (TEMPs) in preclinical translational research, very few have had success in the clinical market place. This gap, referred to as the "valley of death" is due to the large number of ventures that failed to attract or retain investor funding, promotion, and clinical acceptance of their products. This loss can be attributed to a focus on a bench to bedside flow of ideas and technology, which does not account for the multitude of adoption, commercial, and regulatory constraints. The implementation of an alternative bedside to bench and back again approach permits investigators to focus on a specific unmet clinical need, defining crucial translation related questions early in the research process. Investigators often fail to accurately identify critical clinical adoption criteria due to their focus on improved patient outcomes. Other adoption criteria (such as price, time, ethical concerns, and place in the workflow) can cause a product to fail despite improved patient outcomes. By applying simplified business principles such as the build-measure-learn loop and the business model canvas to early-stage research projects, investigators can narrow in on appropriate research topics and define design constraints. Additionally, 86% of all clinical trials fail to result in Federal Drug Administration approval, resulting in significant economic burdens. On the reverse side, approval through the European Medical Agency is widely considered to be more direct but has its challenges. The Committee for Advanced Therapies within the European Medical Agency has received 22 market authorization applications for advanced therapy medicinal products, of which only 10 received authorization. A thorough understanding of the various regulatory pathways permits investigators to plan for future regulatory obstacles and potentially increase their chances of success. By utilizing a bedside to bench and back again approach, investigators can improve the odds that their research will have a meaningful clinical impact.

Human Adipose-Derived Hydrogel Characterization Based on In Vitro ASC Biocompatibility and Differentiation.

Hydrogels serve as three-dimensional scaffolds whose composition can be customized to allow attachment and proliferation of several different cell types. Extracellular matrix-derived hydrogels are considered close replicates of the tissue microenvironment. They can serve as scaffolds for in vitro tissue engineering and are a useful tool to study cell-scaffold interaction. The aim of the present study was to analyze the effect of adipose-derived stromal/stem cells (ASCs) and decellularized adipose tissue-derived (DAT) hydrogel interaction on ASC morphology, proliferation, differentiation, and DAT hydrogel microstructure. First, the ASCs were characterized using flow cytometry, adipogenic/osteogenic differentiation, colony-forming unit fibroblast assay and doubling time. The viability and proliferation assays showed that ASCs seeded in DAT hydrogel at different concentrations and cultured for 21 days remained viable and displayed proliferation. ASCs were seeded on DAT hydrogel and cultured in stromal, adipogenic, or osteogenic media for 14 or 28 days. The analysis of adipogenic differentiation demonstrated the upregulation of adipogenic marker genes and accumulation of oil droplets in the cells. Osteogenic differentiation demonstrated the upregulation of osteogenic marker genes and mineral deposition in the DAT hydrogel. The analysis of DAT hydrogel fiber metrics revealed that ASC seeding, and differentiation altered both the diameter and arrangement of fibers in the matrix. Matrix metalloproteinase-2 (MMP-2) activity was assessed to determine the possible mechanism for DAT hydrogel remodeling. MMP-2 activity was observed in all ASC seeded samples, with the osteogenic samples displaying the highest MMP-2 activity. These findings indicate that DAT hydrogel is a cytocompatible scaffold that supports the adipogenic and osteogenic differentiation of ASCs. Furthermore, the attachment of ASCs and differentiation along adipogenic and osteogenic lineages remodels the microstructure of DAT hydrogel.

Evaluation of the effect of natural peptide 'Urocortin' on corticotrophin releasing factor (CRF) receptor expression in ND7/23 cells

CRF receptors are involved in the stress management of the cells and are believed to have a cytoprotective role in the body. CRF receptors have been reported to be potential drug targets for the treatment of neurodegenerative disorders. The cell line used in the study is ND7/23 (mouse neuroblastoma and rat dorsal root ganglion neuron hybridoma). The aim of the study was to confirm the expression of CRF receptors in ND7/23 cells and to determine if urocortin (Ucn) can enhance the expression of CRF receptors. ND7/23 cells were cultured in RPMI 1640 media and cells grown after the second passage were used for the experiments. RNA was extracted from the cells and amplified by RT-PCR to confirm the presence of CRF receptors. The cells were then subjected to oxidative stress by hydrogen peroxide (0.00375%) and divided into two groups i.e. control and Ucn (10-8 μM) treated. Later RNA was extracted from both group of cells and PCR was performed. Finally, densitometry analysis was conducted on the agarose gel to determine the quantity of PCR product formed. PCR experiment confirmed the expression of both CRF-R1 and CRF-R2 in the cell line, but CRF-R1 was found to be expressed more strongly. Densitometry analysis of the PCR product and calculation of the relative expression of CRF receptors indicated a higher level of expression of CRF receptors in samples treated with Ucn as compared to those that were kept untreated. The results indicate that Ucn may be useful for the management of neuro-degenerative disorders and further studies may be carried out to establish its use as a therapeutic agent.

Receptores de CRF estão envolvidos na gestão do estresse das células e são acreditados para ter um papel de cito-proteção no organismo. Os receptores do CRF têm sido relatados como alvos potenciais de fármacos para o tratamento de doenças neurodegenerativas. A linhagem celular utilizada no estudo é ND7/23 (neuroblastoma de camundongo e hibridoma de raíz dorsal do neurônio ganglionar de rato). O objetivo do estudo foi confirmar o que a expressão de receptores de CRF em células ND7/23 determinar se urocortina (Ucn) pode aumentar a expressão de receptores de CRF. Cultivaram-se células ND7/23 em meio RPMI 1640 e as células que cresceram após a segunda passagem foram usadas para os experimentos. O RNA foi extraído células e amplificado por RT-PCR para confirmar a presença de receptores de CRF. As células foram, então, submetidas a estresse oxidativo por peróxido de hidrogênio (0.00375 %) e divididas em dois grupos, ou seja, controle e tratadas com UCN (10-8 µM). Em seguida, o RNA foi extraído de ambos os grupo de células e realizou-se o PCR. Finalmente, realizou-se análise densitométrica em gel de agarose para determinar a quantidade de produto formado por PCR. O PCR confirmou a expressão de CRF-R1 e CRF-R2 na linhagem celular, mas o CRF-R1 expresso mais fortemente. A análise densitométrica do produto de PCR e o cálculo da expressão relativa de receptores de CRF indicaram um nível mais elevado de expressão de receptores de CRF em amostras tratadas com Ucn, em comparação com aqueles sem tratamento. Os resultados indicam que a Ucn pode ser útil no tratamento de doenças neurodegenerativas e mais estudos podem ser realizados para estabelecer seu uso como agente terapêutico.