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.

Overexpression of OLIG2 and MYT1L Transcription Factors Enhance the Differentiation Potential of Human Mesenchymal Stem Cells into Oligodendrocytes.

BACKGROUND: Demyelinating diseases represent a broad spectrum of disorders and are characterized by the loss of specialized glial cells (oligodendrocytes), which eventually leads to neuronal degeneration. Stem cell-based regenerative approaches provide therapeutic options to regenerate demyelination-induced neurodegeneration. OBJECTIVES: The current study aims to explore the role of oligodendrocyte-specific transcription factors (OLIG2 and MYT1L) under suitable media composition to facilitate human umbilical-cord-derived mesenchymal stem cells (hUC-MSCs) differentiation toward oligodendrocyte for their potential use to treat demyelinating disorders. METHODOLOGY: hUC-MSCs were isolated, cultured, and characterized based on their morphological and phenotypic characteristics. hUC-MSCs were transfected with OLIG2 and MYT1L transcription factors individually and in synergistic (OLIG2 + MYT1L) groups using a lipofectamine-based transfection method and incubated under two different media compositions (normal and oligo induction media). Transfected hUC-MSCs were assessed for lineage specification and differentiation using qPCR. Differentiation was also analyzed via immunocytochemistry by determining the expression of oligodendrocyte-specific proteins. RESULTS: All the transfected groups showed significant upregulation of GFAP and OLIG2 with downregulation of NES, demonstrating the MSC commitment toward the glial lineage. Transfected groups also presented significant overexpression of oligodendrocyte-specific markers (SOX10, NKX2.2, GALC, CNP, CSPG4, MBP, and PLP1). Immunocytochemical analysis showed intense expression of OLIG2, MYT1L, and NG2 proteins in both normal and oligo induction media after 3 and 7 days. CONCLUSIONS: The study concludes that OLIG2 and MYT1L have the potential to differentiate hUC-MSCs into oligodendrocyte-like cells, which is greatly facilitated by the oligo induction medium. The study may serve as a promising cell-based therapeutic strategy against demyelination-induced neuronal degeneration.

Mesenchymal Stem Cell-Derived Extracellular Vesicles Protect Rat Nucleus Pulposus Cells from Oxidative Stress.

BACKGROUND: Oxidative stress (OS) is mainly associated with the pathogenesis of intervertebral disc (IVD) degeneration; it causes nucleus pulposus cells (NPCs) to undergo senescence and triggers autophagy and apoptosis. This study aims to evaluate the regeneration potential of extracellular vesicles (EVs) derived from human umbilical cord-mesenchymal stem cells (hUC-MSCs) in an in vitro rat NPC-induced OS model. DESIGN: NPCs were isolated from rat coccygeal discs, propagated, and characterized. OS was induced by hydrogen peroxide (H2O2), which is confirmed by 2,7-dichlorofluorescein diacetate (H2DCFDA) assay. EVs were isolated from hUC-MSCs and characterized by analyzing the vesicles using fluorescence microscope, scanning electron microscope (SEM), atomic force microscope (AFM), dynamic light scattering (DLS), and Western blot (WB). The in vitro effects of EVs on migration, uptake, and survival of NPCs were determined. RESULTS: SEM and AFM topographic images revealed the size distribution of EVs. The phenotypes of isolated EVs showed that the size of EVs was 403.3 ± 85.94 nm, and the zeta potential was -0.270 ± 4.02 mV. Protein expression analysis showed that EVs were positive for CD81 and annexin V. Treatment of NPCs with EVs reduced H2O2-induced OS as evidenced by a decrease in reactive oxygen species (ROS) levels. Co-culture of NPCs with DiI-labeled EVs showed the cellular internalization of EVs. In the scratch assay, EVs significantly increased NPC proliferation and migration toward the scratched area. Quantitative polymerase chain reaction analysis showed that EVs significantly reduced the expression of OS genes. CONCLUSION: EVs protected NPCs from H2O2-induced OS by reducing intracellular ROS generation and improved NPC proliferation and migration.

Osteochondral Tissue Engineering Dilemma: Scaffolding Trends in Regenerative Medicine.

Orthopedic surgeons face a lot of difficulties in managing and repairing osteochondral defects. Damaged articular cartilage and the subchondral bone underneath are both present in osteochondral defects. The demands of the bone, cartilage, and the contact between the bone and the cartilage must be taken into consideration while repairing an osteochondral defect. Only palliative, not curative, therapeutic interventions are now available for the healing of osteochondral abnormalities. With its ability to successfully rebuild bone, cartilage, and the junction between bone and cartilage, tissue engineering has been recognized as an effective substitute. In correlation, mechanical stress and physical processes are commonly applied to the osteochondral area. Therefore, the ability of chondrocytes and osteoblasts to regenerate is influenced by bioactive molecules and the physicochemical characteristics of the surrounding matrix. The treatment of osteochondral disorders is said to benefit from the use of stem cells as an alternative intervention. In the field of tissue engineering, various approaches have been used such as the direct implantation of scaffolding materials at the site of tissue injury in patients, either alone or loaded with cells and bioactive molecules at the target site to imitate the natural extracellular matrix. Despite the extensive use and advancements of tissue-engineered biomaterials such as natural and synthetic polymer-based scaffolds, their repair capacity is limited due to challenges in combating antigenicity, designed to simulate in vivo microenvironment, and conducting mechanical or metabolic characteristics comparable to native organs/tissues. This study explores numerous osteochondral tissue engineering methodologies focusing on scaffold design, material varieties, manufacturing techniques, and functional features. This review is focused on recent breakthroughs in bioactive scaffolds that aid osteogenic and chondrogenic differentiation for bone and cartilage repair. The topic will cover fundamental anatomy, osteochondral repair methodologies and obstacles, cell selection, biochemical variables, and bioactive materials, as well as the design and manufacture of bioactive scaffolds. Additionally, we focus on the concept and construction of decellularized scaffolds, and the fabrication of dECM scaffolds in tissue engineering from various skin, bone, nerve, heart tissue, lung, liver, and kidney, and their application in osteochondral regeneration.

Co-regulation of Sox9 and TGFß1 transcription factors in mesenchymal stem cells regenerated the intervertebral disc degeneration.

Background: Intervertebral disc (IVD) shows aging and degenerative changes earlier than any other body connective tissue. Its repair and regeneration provide a considerable challenge in regenerative medicine due to its high degree of infrastructure and mechanical complexity. Mesenchymal stem cells, due to their tissue resurfacing potential, represent many explanatory pathways to regenerate a tissue breakdown. Methods: This study was undertaken to evaluate the co-regulation of Sox9 and TGFß1 in differentiating human umbilical cord mesenchymal stem cells (hUC-MSC) into chondrocytes. The combinatorial impact of Sox9 and TGFß1 on hUC-MSCs was examined in vitro by gene expression and immunocytochemical staining. In in vivo, an animal model of IVD degeneration was established under a fluoroscopic guided system through needle puncture of the caudal disc. Normal and transfected MSCs were transplanted. Oxidative stress, pain, and inflammatory markers were evaluated by qPCR. Disc height index (DHI), water content, and gag content were analyzed. Histological examinations were performed to evaluate the degree of regeneration. Results: hUC-MSC transfected with Sox9+TGFß1 showed a noticeable morphological appearance of a chondrocyte, and highly expressed chondrogenic markers (aggrecan, Sox9, TGFß1, TGFß2, and type II collagens) after transfection. Histological observation demonstrated that cartilage regeneration, extracellular matrix synthesis, and collagen remodeling were significant upon staining with H&E, Alcian blue, and Masson's trichrome stain on day 14. Additionally, oxidative stress, pain, and inflammatory markers were positively downregulated in the animals transplanted with Sox9 and TGFß1 transfected MSCs. Conclusion: These findings indicate that the combinatorial effect of Sox9 and TGFß1 substantially accelerates the chondrogenesis in hUC-MSCs. Cartilage regeneration and matrix synthesis were significantly enhanced. Therefore, a synergistic effect of Sox9 and TGFß1 could be an immense therapeutic combination in the tissue engineering of cartilaginous joint bio-prostheses and a novel candidate for cartilage stabilization.

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.

Kisspeptin-10 induces dose dependent degeneration in prepubertal rat prostate gland.

BACKGROUND: Kisspeptin peptides mediate their actions through the GnRH loop system. How kisspeptins affect prostate gland in prepubertal male mammals remains elusive. METHODS: To address this kisspeptin was administered as subchronic (12 days) twice daily i.p. dose at three different dosage regimens: 10 pg, 1 ng and 1 µg, to prepubertal male Sprague-Dawley rats (PND 35). Control rats were maintained in parallel. At the end of the experiment prostate gland was dissected out and processed for light and electron microscopy. DNA damage was also estimated by DNA ladder assay and DNA fragmentation assay. RESULTS: Prostate weights decreased significantly (P < 0.05) at 1 µg treatment dose of kisspeptin. The epithelial height of secretory acini of prostate decreased at 10 pg (P < 0.05), 1 ng, and 1 µg doses (P < 0.001). Histomorphology and ultrastructure demonstrated, decrease in epithelial cell height, epithelial folding and dilatation of the organelles with kisspeptin treatment. Percent DNA damage to the prostatic tissue was 20.74 ± 2.18, 43.60 ± 2.39, and 58.18 ± 2.59 at 10 pg, 1 ng and 1 µg doses, respectively. CONCLUSION: The study reveals that continuous administration of kisspeptin does not lead to an early maturation but instead severe degeneration of prepubertal prostate gland. Wiley Periodicals, Inc.

Immature rat seminal vesicles show histomorphological and ultrastructural alterations following treatment with kisspeptin-10.

BACKGROUND: Degenerative effects of critical regulators of reproduction, the kisspeptin peptides, on cellular aspects of sexually immature male gonads are known but similar information on accessory sex glands remain elusive. METHODS: Prepubertal laboratory rats were injected kisspeptin-10 at three different dosage concentrations (10 pg, 1 ng and 1 microgram) for a period of continuous 12 days at the rate of two doses per day. Control rats were maintained in parallel. The day following the end of the experimental period, seminal vesicles were removed and processed for light and electron microscopic examination using the standard methods. DNA damage was estimated by DNA ladder assay and DNA fragmentation assay. RESULTS: The results demonstrated cellular degeneration. Epithelial cell height of seminal vesicles decreased significantly at all doses (P < 0.05). Marked decrease in epithelial folds was readily noticeable, while the lumen was dilated. Ultrastructural changes were characterized by dilatation of endoplasmic reticulum and Golgi complex, heterochromatization of nuclei, invagination of nuclear membranes and a decreased number of secretory granules. Percent DNA damage to the seminal vesicle was 19.54 +/- 1.98, 38.06 +/- 2.09 and 58.18 +/- 2.59 at 10 pg, 1 ng and 1 microgram doses respectively. CONCLUSION: The study reveals that continuous administration of kisspeptin does not lead to an early maturation but instead severe degeneration of sexually immature seminal vesicles.

Metabolic syndrome in school children of Dera Ismail Khan, Pakistan.

BACKGROUND: Childhood obesity has increased considerably in many regions of the world including Pakistan. The recent phenomenon of 'nutritional transition' with a westernisation of food so prevalent in developing countries, has caused a significant rise in obesity among population that were unaware of this problem in the recent past. The aim of this study was to find out the frequency of metabolic syndrome and cardiovascular risk factors in obese school children (6-11 years) in Dera Ismail Khan. METHODS: Eighty-six children were included in this study with 61 (70.94%) obese and 25 (29.06%) normal weight children. Obese children comprised of 34 (39.53%) boys and 27 (31.40%) girls. Normal weight children included 15 (17.44%) boys and 10 (11.63%) girls. They were selected among 1.336 children from 8 primary schools of Dera Ismail Khan city. Anthropometric parameters of each subject were recorded, BMI determined and body mass status calculated. Children were categorized by the presence or absence of Obesity. Blood Pressure was also measured. Non-fasting venous blood samples were taken, analysed for lipids; Triglycerides (TG), Cholesterol (TC); Lipoproteins: High and Low Density Lipoprotein-cholesterol (HDL-C, LDL-C) and Plasma Glucose Concentration (PGC). Metabolic syndrome was identified in the presence of > or = 3 of the followings with cut-off values: TG > 170 mg/dl, HDL-C < 35 mg/dl, WC > 71 cm, BP >120/80 mm Hg, PGC > 200 mg/dl. RESULTS: Metabolic syndrome was identified in 22.95% of the obese children. It was 19.67% and 3.27% in obese boys and girls respectively. Metabolic syndrome was not found in normal weight children. Clustering of cardiovascular factors was abundantly present in obese and rare in normal weight children.