Bone Marrow-Derived Mesenchymal Stem Cell-Laden Nanocomposite Scaffolds Enhance Bone Regeneration in Rabbit Critical-Size Segmental Bone Defect Model.

Bone regeneration poses a significant challenge in the field of tissue engineering, prompting ongoing research to explore innovative strategies for effective bone healing. The integration of stem cells and nanomaterial scaffolds has emerged as a promising approach, offering the potential to enhance regenerative outcomes. This study focuses on the application of a stem cell-laden nanomaterial scaffold designed for bone regeneration in rabbits. The in vivo study was conducted on thirty-six healthy skeletally mature New Zealand white rabbits that were randomly allocated into six groups. Group A was considered the control, wherein a 15 mm critical-sized defect was created and left as such without any treatment. In group B, this defect was filled with a polycaprolactone-hydroxyapatite (PCL + HAP) scaffold, whereas in group C, a PCL + HAP-carboxylated multiwalled carbon nanotube (PCL + HAP + MWCNT-COOH) scaffold was used. In group D, a PCL + HAP + MWCNT-COOH scaffold was used with local injection of bone morphogenetic protein-2 (BMP-2) on postoperative days 30, 45, and 60. The rabbit bone marrow-derived mesenchymal stem cells (rBMSCs) were seeded onto the PCL + HAP + MWCNT-COOH scaffold by the centrifugal method. In group E, an rBMSC-seeded PCL + HAP + MWCNT-COOH scaffold was used along with the local injection of rBMSC on postoperative days 7, 14, and 21. For group F, in addition to the treatment given to group E, BMP-2 was administered locally on postoperative days 30, 45, and 60. Gross observations, radiological observation, scanning electron microscopic assessment, and histological evaluation study showed that group F displayed the best healing properties, followed by group E, group D, group C, and B. Group A showed no healing with ends blunting minimal fibrous tissue. Incorporating growth factor BMP-2 in tissue-engineered rBMSC-loaded nanocomposite PCL + HAP + MWCNT-COOH construct can augment the osteoinductive and osteoconductive properties, thereby enhancing the healing in a critical-sized bone defect. This novel stem cell composite could prove worthy in the treatment of non-union and delayed union fractures in the near future.

Canine umbilical cord tissue derived mesenchymal stem cells naturally express mRNAs of some antimicrobial peptides.

Antimicrobial peptides (AMPs) are naturally produced by all living organisms at a constitutive rate. They represent the first line of active defence systems against invading microorganisms, helping in innate immunity. Besides their therapeutic applications, great attention has also been given to the mesenchymal stem cells (MSCs) due to their antimicrobial activities. The study aimed to observe the mRNA expression profile of few antimicrobial peptides (AMPs) in canine MSCs during standard in vitro culture. MSCs were isolated from canine umbilical cord tissue, propagated and characterized by morphology, surface markers and tri-lineage differentiation capability. The mRNA expression of eleven commonly known antimicrobial peptides was checked by Reverse Transcriptase PCR. It has been found for the first time that canine MSCs naturally express the mRNAs of AMPs like C-X-C motif chemokine ligand 8 (CXCL8), Elafin (PI3), Hepcidin (HAMP), Lipocalin 2 (LCN2) and Secretory leukocyte protease inhibitor (SLPI). However, their expressions at protein level and, relation with antimicrobial effect of canine MSCs need to be explored.

Substance P, a Neuropeptide, Promotes Wound Healing via Neurokinin-1 Receptor.

Substance P (SP), an endogenous neuropeptide, mediates intracellular signaling, mainly through a tachykinin receptor. The tachykinin receptors family consists of neurokinin-1 (NK-1), neurokinin-2 (NK-2), and neurokinin-3 receptors. Our previous studies on SP have shown its wound healing potentials. But the exact mechanism of wound healing by SP is not exactly known. In view of this, the present study was aimed at evaluating the in vitro wound healing effect of SP alone and in the presence of NK-1, NK-2, and both receptor antagonists. Scratch assay, transwell assay, and tumor growth factor-beta 1 (TGF-ß1) assay were performed on buffalo fetal fibroblast culture. The cotreatment of fibroblast cultures with SP alone during the 24 h caused the significant proliferation and migrations of cells in both horizontal and vertical directions. The SP in the presence of spantide II (NK-1 antagonist) failed to stimulate this migration. The treatment of cells with SP in the presence of NK-2 antagonist treatment also showed a significant reduction of migration of cells with respect to SP treatment alone. The SP in the presence of both NK-1 and NK-2 antagonists failed to stimulate the horizontal migration of cells and most of the ineffectiveness of SP was observed in this combination. The TGF-ß1 levels were significantly higher in the supernatants of cells that were exposed to SP alone. All other treatments have significantly lower TGF-ß1 levels than SP alone treatment. It is concluded that different actions on fibroblast cells by SP were mainly mediated through the NK-1 receptor.

Nano-depletion of morbid spermatozoa up-regulate Ca2+ channel, depolarization of membrane potential and fertility in buffalo.

Despite recent advances in technique of spermatozoa cryopreservation, there are still ejaculates present that fail to meet strict quality standard; mainly due to detrimental effect of imbalance of free radicals. The omnipresence of dead/defective spermatozoa in ejaculates of eutherian species is a major source of excessive free radicals. Though sperm-selection techniques, as well as addition of antioxidants addressed the problem to a certain extent, the major source of free radicals in the semen remained, causing much damage. This study attempts to remove dead/damaged spermatozoa using negative fertility-marker. The effect is unraveled by Hypo-osmotic (HOS), and fluorescein-conjugated Pisum sativum agglutinin (FITC-PSA) assay, further confirmed by Ca2+-regulating mechanisms and depolarization of sperm membrane potential, reduction in concentration of free radicals and finally by in vitro fertility assay. The study involved functionalization of iron oxide nanoparticles (IONPs) with silane followed by bio-conjugation with anti-ubiquitin antibodies. The nano-purification of semen using anti-ubiquitin conjugated iron oxide nanoparticles (IONPs) (antibody concentrations 0.5, 1.0 and 2.0 µg/ml) was attempted. The efficiency of nano-purification was 18.1%-43.8% in the study. The results revealed greater (P ≤ 0.05) spermatozoa population with intact plasma membrane, acrosome integrity, high mitochondrial membrane potential and pattern-F (least intracellular Ca2+), evidence of low lipid peroxidation and higher total antioxidant capacity in nano-purified groups. More number of spermatozoa were bound to zona pellucida of matured oocytes from nano-depleted than non-depleted group. The findings demonstrate antibody concentration of 1.0 µg/ml bio-conjugated with IONPs as most efficient in enriching the ejaculate with functional spermatozoa with the highest percentage of zona binding.

Microarray analysis and PCR validation of genes associated with facultative parthenogenesis in Meleagris gallopavo (Turkey).

A cDNA microarray containing 43,661 differentially expressed genes was carried out on the blastoderm of fertilized and facultative parthenogenic turkey embryos at different hours of development. The total number of up-regulated (UR) and down-regulated (DR) genes at 0, 12, and 24 h of development were 725 and 1436, 942 and 599, and 589 and 1044, respectively. Common genes between 0 and 12 h, 12 and 24 h, and 0 and 24 h were 55, 67, and 110, respectively. The proportion of genes showing above 50-fold UR and DR at 0, 12, and 24 h of development were 2.0% and 1.5%, 0.5% and 1.2%, and 0.2% and 1.1%, respectively. Eight UR genes were validated (APOA1, THRAP3, ARL14EP, PSAP, MOG, MYBPC2, MTIF3 and EDG4) and relative expression of six of them was significantly higher (P ≤ 0.05) in parthenogenic embryos, while two genes showed non-significant (P ≥ 0.05) variation. The expression of BCL11A, PRP4B, TCP1, and TPI1 genes was significantly (P ≤ 0.05) DR in parthenotes in the micro-array study, while the TCP1 gene was up-regulated, and there was no variation in TPI1 gene expression in the PCR validation study. In conclusion, our findings demonstrate differential expression of a large number of genes in parthenotes at different stages of embryo development compared to fertilized embryos. Up-regulation of APOA1, MYBPC2, TCP1, and THRAP3 genes, suggest their crucial role in spontaneous facultative parthenogenic development in turkey birds.

Optimization of dissolved oxygen levels in extender prevents development of cryocapacitation like changes, oxidative stress and augments zona binding capacity of crossbred bull spermatozoa.

The present study was undertaken to determine the efficacy of partial deoxygenation of extender at constant temperature (35°C) in freezability of crossbred bull semen. The dissolved oxygen (DO) levels were reduced by the use of newly developed technique of nitrogen effervescence at a flow rate of 2-3 bubbles per second. Four different levels of oxygen in semen extender, that is 11.7, 2, 4 and 8 ppm as control (Group-I), Group-II, Group-III and Group-IV, respectively, were used to assess the effect of partial deoxygenation on semen quality parameters. The 4 ppm level of DO resulted in higher (p < 0.05) progressive motility in comparison with non-treated group at post-thaw stage, whereas reduction up to 2 ppm resulted in drastic fall in motility. Oxidative stress status revealed low superoxide dismutase (SOD) and total antioxidant capacity (TAC) in Group-II, whereas higher (p < 0.05) SOD and TAC activities were observed in Group-III in comparison with non-treated group at pre-freeze and post-thaw stages. The sperm-zona binding at 4 ppm level of DO was significantly higher than control group, 2 and 8 ppm levels of DO. In conclusion, reduction of DO in the extender up to 4 ppm reduced oxidative stress and improved in vitro fertility of crossbred bull spermatozoa.

Thin films of functionalized carbon nanotubes support long-term maintenance and cardio-neuronal differentiation of canine induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) are a promising cell source for regenerative medicine. However, their feeder-free maintenance in undifferentiated states remains challenging. In recent past extensive studies have been directed using pristine or functionalized carbon nanotube in tissue engineering. Here we proposed thin films of functionalized carbon nanotubes (OH-single-walled CNTs [SWCNTs] and OH-multiwalled CNTs [MWCNTs]), as alternatives for the feeder-free in vitro culture of canine iPSCs (ciPSCs), considered as the cellular model. The ciPSC colonies could maintain their dome-shaped compactness and other characteristics when propagated on CNT films. Concomitantly, high cell viability and upregulation of pluripotency-associated genes and cell adhesion molecules were observed, further supported by molecular docking. Moreover, CNTs did not have profound toxic effects compared to feeder cultures as evident by cytocompatibility studies. Further, cardiac and neuronal differentiation of ciPSCs was induced on these films to determine their influence on the differentiation process. The cells retained differentiation potential and the nanotopographical features of the substrates provided positive cues to enhance differentiation to both lineages as evident by immunocytochemical staining and marker gene expression. Overall, OH-SWCNT provided better cues, maintained pluripotency, and induced the differentiation of ciPSCs. These results indicate that OH-functionalized CNT films could be used as alternatives for the feeder-free maintenance of ciPSCs towards prospective utilization in regenerative medicine.

In vitro propagation and cardiac differentiation of canine induced pluripotent stem cells on carbon nanotube substrates.

Induced pluripotent stem cells (iPSCs) have attracted an interest for personalized cell based therapy along with various other applications. There have been few studies that effective nanomaterial based scaffolds act as alternative to the commonly used feeder dependent in vitro maintenance of iPSCs. The present study provides the fundamental information on ex vivo behavior of canine iPSC (ciPSCs) maintained on carboxylic acid (COOH) functionalized single-walled carbon nanotubes (COOH-SWCNTs) and multi-walled carbon nanotubes (COOH-MWCNTs) substrates. Here in we evaluated the comparative colony morphology, propagation, characterization, cytocompatibility and differentiation capability of ciPSC cultured on MEF feeder taken as control, and COOH-SWCNTs and COOH-MWCNTs substrates. We observed a healthy growth of ciPSCs on both the types of carbon nanotubes (CNTs) similar to feeder. The ciPSC colonies grown on both CNTs were positive for alkaline phosphatase staining and expressed pluripotent markers with notable significance. Further, the ciPSC colonies grew on these CNTs retained the in vitro differentiation ability into three germ layers as well as cardiac cell. Cytotoxicity analysis revealed that (COOH) functionalized CNTs provided a culture condition of low cytotoxicity. The results of the present study indicated that (COOH) functionalized CNTs could be used as xeno-free substrate to support the maintenance of iPSCs.

Synthesis of iron oxide nanoparticles-antiubiquitin antibodies conjugates for depletion of dead/damaged spermatozoa from buffalo (Bubalus bubalis) semen.

The synthesis of iron oxide nanoparticles (IONPs)-antiubiquitin antibodies (Abs) complex for depletion of dead/damaged spermatozoa from buffalo semen was done. The IONPs synthesized were round in shape with size of 12.09 ± 0.91 nm. At the end of the two-step functionalization, that is, silanization and pegylation of bare IONPs and bioconjugation of functionalized IOPNs, particles with the sizes of 19.15 ± 1.46, 20.72 ± 0.95, and 73.01 ± 7.56 nm, respectively, were obtained. Twenty-four semen samples from four bulls with mean individual progressive motility (%) and sperm concentration (million/mL) of 77.1 ± 0.9 and 1,321.2 ± 84.7, respectively, were divided into Group I (control), and treatment groups viz. Groups II, III, and IV; with each group containing 150 ± 25 million dead/damaged spermatozoa. The IONPs-Abs complex was added at the ratio of 1:1 (0.5 µg/mL), 1:2 (1.0 µg/mL), and 1:4 (2.0 µg/mL), respectively, in the Groups II, III, and IV. The mean efficiency (%) of nanopurification was estimated to be greater in nanopurified semen with the increasing doses of the IONPs-Abs complex. A reduction of 29.3 ± 6.4%, 48.4 ± 5.3%, and 55.4 ± 4.4% in dead/damaged spermatozoa following nanopurification in Groups II, III, and IV, respectively, was observed. The study shows that in-house synthesized IONPs-Abs complex can be successfully used to deplete dead/damaged spermatozoa from buffalo semen with improvement in quality.

Nano-purification of raw semen minimises oxidative stress with improvement in post-thaw quality of buffalo spermatozoa.

The study consisted of application of anti-ubiquitin antibodies (Abs)-coated iron oxide-nanoparticles (IONPs) for minimisation of oxidative stress to contemporary live spermatozoa from the raw semen. Round-shaped IONPs (12.09 ± 0.91 nm) after two-stage functionalisation (silanisation and pegylation) were conjugated with Abs. Four aliquots from each of the 24 ejaculates (4 buffalo bulls) formed Control (Group I) and treatment (II, III and IV) groups; each containing 150 ± 25 million dead/damaged spermatozoa. IONPs-Abs complex were added at ratio of 1:1 (0.5 µg/ml), 1:2 (1.0 µg/ml) and 1:4 (2.0 µg/ml), respectively, in Groups II, III and IV. The semen quality parameters showed improvement at lag-stage (post-nano-purification before processing for cryopreservation). The mean post-thaw motility (%) in Group IV was found to be greater (p < .05) than Group I. Moreover, the overall DNA integrity (%) at post-thaw stage was improved in the nano-purified semen samples. The value of malondialdehyde was greater (p < .001) in Group I than Groups II, III and IV. The mean total antioxidant capacity and superoxide dismutase (U/mg protein) activity values in Group IV was greater (p < .05) than Group I. The study results show that IONPs conjugated with anti-ubiquitin Abs at 2.0 µg/ml can be an effective dose for depletion of dead/damaged spermatozoa from buffalo ejaculates to minimise oxidative stress.

Early growth response gene mediates in VEGF and FGF signaling as dissected by CRISPR in corpus luteum of water buffalo.

The EGR family comprises of EGR 1, EGR 2, EGR 3 and EGR 4 which are involved in the transactivation of several genes. A broad range of extracellular stimuli by growth factors is capable of activating EGR mediated transactivation of genes involved in angiogenesis and cell proliferation. However, their role in controlling VEGF A and FGF 2 signaling in the CL of water buffalo is not known. The present study was conducted to understand the role of EGR mediated regulation of VEGF A and FGF 2 signaling in buffalo luteal cells. Towards this goal, luteal cells were cultured and treated with VEGF A and FGF 2 and the mRNA expression pattern of EGR family members were documented. The EGR 1 message was found to be up-regulated in luteal cells of buffalo at 72 hours of culture. The functional validation of EGR 1 gene was accomplished by knocking out (KO) of EGR 1 in cultured luteal cells by CRISPR/Cas9 mediated gene editing technology. The EGR 1 KO cells were then cultured and stimulated with VEGF A and FGF 2. It was observed that VEGF A and FGF 2 induced angiogenesis, cell proliferation and steroidogenesis in wild type luteal cells, whereas the response of the growth factors was attenuated in the EGR 1 KO cells. Taken together our study provides evidence convincingly that both VEGF and FGF mediate their biological action through a common intermediate, EGR 1, to regulate corpus luteum function of buffalo.

Exogenous cholesterol prevents cryocapacitation-like changes, membrane fluidity, and enhances in vitro fertility in bubaline spermatozoa.

A study was conducted to determine the optimum dosage of the exogenous cholesterol-loaded cyclodextrins (CLC) to get maximum cryoprotection for bubaline spermatozoa. In the present study, 120 × 106 spermatozoa were incubated in 2, 3 and 4 mg of CLC as grouped as Gr II, III and IV, respectively, and sperm progressive motility, intracellular Ca2+ , capacitation status by protein tyrosine phosphorylation (PTP) assay and zona binding per cent (ZBP) and cleavage rate (CR) of the cryopreserved buffalo spermatozoa by in vitro fertility assay were assessed in comparison with an untreated control group (Gr I). Results revealed that there was a significant (p < .05) linear decrease in percentage of sperm population with higher intracellular Ca2+ and percentage of sperm population with medium or high capacitated by PTP in CLC treated from 2 to 3 mg and then increased to 4 mg/120 × 106 spermatozoa whereas sperm progressive motility, percentage of sperm population with low capacitated, ZBP and CR were increased significantly (p < .05) in sperm population treated from 2 to 3 mg CLC and then decreased to 4 mg/120 × 106 spermatozoa. The study has clearly indicated that CLC at 3 mg/120 × 106 spermatozoa has maximum beneficial effects in protection of sperm progressive motility, membrane fluidity (low intracellular Ca2+ ); prevention of cryocapacitation (low capacitation pattern in immunolocalization) and enhancement of in vitro ZBP and CR. Post-thaw motility of the CLC-treated sperm has shown positively significant (p < .05) correlation with sperm population with low intracellular Ca2+ , low capacitated sperm population, ZBP and CR, whereas it was negatively (p < .05) correlated with sperm population with high intracellular Ca2+ , medium or high capacitated sperm. The present study has revealed for the first time that incubation of spermatozoa with CLC of higher dose (>3 mg/120 × 106 spermatozoa) had adverse effects on sperm cryopreservation, although incubation of sperm with 3 mg/120 million prior to processing had minimised the freezing-thawing-associated damages in bubaline species.

Post-calving umbilical cord tissue offcut: A potential source for the isolation of bovine mesenchymal stem cells.

BACKGROUND AND AIM: Veterinary health care is an emergent area in animal sciences and innovative therapeutic approaches happen to be imperative in the present days. In view of the importance of cattle health and production, it is necessary to take up contemporary approach of stem cell therapy in this sector also. This study aimed to standardize an explant culture method of bovine umbilical tissue offcut to isolate mesenchymal stem cells (MSCs) because considerable efforts are required for ensuring easy accessibility and availability of MSCs in bulk quantity, as well as in establishing and characterizing the cell lines. MATERIALS AND METHODS: The umbilical cord (UC) tissue matrix offcut was collected after calving. A simplified in vitro cell isolation technique was followed to collect the emerged out cells from the explants of UC. Further, we expanded these isolated cells in vitro, observed its growth kinetics, and characterized to confirm as per the criterion of bovine MSCs. RESULTS: A considerable exponential growth rate of the UC-derived cells was noticed. In addition to their confirmation as MSCs, the cells also exhibited plastic adherent property and maintained the spindle-shaped morphology throughout the in vitro culture. The cultured cells were found positive MSC-specific surface markers CD105, CD90, and CD73 and were negative for hematopoietic cell marker CD45. Cytochemical studies revealed the ability of the cells to differentiate into osteogenic, chondrogenic, and adipogenic lineages. CONCLUSION: This simplified method of isolation and culture of bovine multipotent MSCs from the UC offcut collected after calving could be extrapolated for the greater availability of the cells for prospective therapeutic applications.

In vitro proliferation and differentiation of canine bone marrow derived mesenchymal stem cells over hydroxyl functionalized CNT substrates.

Nanotopography of culture substrate acts as a positive cue in cell-biomaterial based tissue regeneration. Considering the potentiality of carbon nanotubes (CNTs) this study was designed to evaluate its two functionalized form by an in vitro culture condition using canine mesenchymal stem cells as cellular model. Cells were isolated and its behaviour, proliferation and differentiation processes were elucidated onto CNT substrates. Beside the variations in cellular behaviour it was remarkably noted that even though proliferation was reduced but osteogenic and chondrogenic differentiation was enhanced over multi-walled CNTs, whereas neuronal differentiation was better supported by single walled CNTs as evidenced by our cytochemical, immunocytochemical, gene expression and flow cytometry assays. The former one was noticed more cytocompatible by our different apoptosis studies. The outcome of these experiments collectively indicated that hydroxylated functionalized CNTs could be a potential scaffold constituent for future experimentations as well as for the application in regenerative medicine.

Transcriptional Regulation of Thrombospondins and Its Functional Validation through CRISPR/Cas9 Mediated Gene Editing in Corpus Luteum of Water Buffalo (Bubalus Bubalis).

BACKGROUND/AIMS: Thrombospondins (TSPs) are large multi-modular proteins, identified as natural angiogenesis inhibitors that exert their activity by binding to CD36 and CD47 receptors. The anti-angiogenic effect of TSPs in luteal regression of water buffalo has not been addressed. The present study characterized the expression pattern and localization of TSPs and their receptors in ovarian corpus luteum during different stages of development in buffalo. This study also elucidated the effect of exogenous Thrombospondin1 (TSP1) or the knocking out of the endogenous protein on luteal cell viability and function. Further, the in vitro transcriptional interaction of TSP1 with hormones, LH, PGF2α and angiogenic growth factors, VEGF and FGF2 were also evaluated. METHODS: First, the CLs were classified into four groups based on macroscopic observation and progesterone concentration. mRNA expression of examined factors was measured by qPCR, localization by immunoblotting and immunohistochemistry. TSP1 was knocked out (KO) in cultured luteal cells isolated from late luteal stage CLs (day 1116) by CRISPR/Cas9 mediated gene editing technology in order to functionally validate the TSP1 gene. Isolated cells from late stage CLs were also stimulated with different doses of TSP1, LH, PGF2α, VEGF and FGF2 for various time intervals to determine transcriptional regulation of thrombospondins. RESULTS: mRNA expression of TSPs and their receptors were found to be significantly higher in late and regressed stage of CL as compared to other groups which was consistent with the findings of immunoblotting and immunolocalization experiments. It was observed that TSP1 induced apoptosis, down regulated angiogenic growth factors, VEGF and FGF2 and attenuated progesterone production in cultured luteal cells. However, knocking out of endogenous TSP1 with CRISPR/Cas9 system improved the viability of luteal cells, progesterone synthesis and upregulated the expression of VEGF and FGF2 in the KO luteal cells. PGF2α induced the upregulation of TSPs and Caspase 3 transcripts, whereas treatment with LH and angiogenic growth factors (VEGF and FGF2) down regulated the TSP system in luteal cells. CONCLUSION: Collectively, these data provide evidence that thrombospondins along with their receptors are expressed at varying levels in different stages of CL progression with maximum expression during the late and regressing stages. These results are consistent with the hypothesis that thrombospondins stimulated by PGF2α plays an essential modulatory role in bringing about structural and functional luteolysis in buffalo.

Preparation of NGF encapsulated chitosan nanoparticles and its evaluation on neuronal differentiation potentiality of canine mesenchymal stem cells.

Sustained and controlled release of neurotrophic factors in target tissue through nanomaterial based delivery system could be a better strategy for nerve tissue regeneration. The present study aims to prepare the nerve growth factor (NGF) encapsulated chitosan nanoparticles (NGF-CNPs) and its evaluation on neuronal differentiation potentiality of canine bone marrow derived mesenchymal stem cells (cBM-MSCs). The NGF-CNPs were prepared by ionotropic gelation method with tripolyphosphate (TPP) as an ionic cross-linking agent. Observations on physiochemical properties displayed the size of nanoparticles as 80-90 nm with positive zeta potential as well as an ionic interaction between NGF and nanoparticle. NGF loading efficiency was found to be 61% while its sustained release was observed by an in vitro release kinetics study. These nanoparticles were found to be cytocompatible to cBM-MSCs when supplemented at a concentration upto 4 mg/ml in culture media. The NGF-CNP supplemented culture media was able to transdifferentiate the preinduced cBM-MSCs into neurons in a better way than unbound NGF supplementation. Further, it was also noticed that NGF-CNPs were able to transdifferentiate cBM-MSCs without any chemical based preinduction. In conclusion, our findings propose that NGF-CNPs are capable of releasing bioactive NGF with the ability to transdifferentiate mesenchymal stem cells into neurons, suggesting its potential future application in nerve tissue regeneration.

Functionalized carbon nanotubes as suitable scaffold materials for proliferation and differentiation of canine mesenchymal stem cells.

In the field of regenerative medicine, numerous potential applications of mesenchymal stem cells (MSCs) can be envisaged, due to their ability to differentiate into a range of tissues on the basis of the substrate on which they grow. With the advances in nanotechnology, carbon nanotubes (CNTs) have been widely explored for use as cell culture substrate in tissue engineering applications. In this study, canine bone marrow-derived MSCs were considered as the cellular model for an in vitro study to elucidate the collective cellular processes, using three different varieties of thin films of functionalized carbon nanotubes (COOH-single-walled CNTs [SWCNTs], COOH-multiwalled CNTs [MWCNTs] and polyethylene glycol [PEG]-SWCNTs), which were spray dried onto preheated cover slips. Cells spread out better on the CNT films, resulting in higher cell surface area and occurrence of filopodia, with parallel orientation of stress fiber bundles. Canine MSCs proliferated at a slower rate on all types of CNT substrates compared to the control, but no decline in cell number was noticed during the study period. Expression of apoptosis-associated genes decreased on the CNT substrates as time progressed. On flow cytometry after AnnexinV-fluorescein isothiocyanate/propidium iodide (PI) staining, total number of apoptotic and necrotic cells remained lower in COOH-functionalized films compared to PEG-functionalized ones. Collectively, these results indicate that COOH-MWCNT substrate provided an environment of low cytotoxicity. Canine MSCs were further induced to differentiate along osteogenic, chondrogenic, and neuronal lineages by culturing under specific differentiation conditions. The cytochemical and immunocytochemical staining results, as well as the expression of the bone marker genes, led us to hypothesize that the COOH-MWCNT substrate acted as a better cue, accelerating the osteogenic differentiation process. However, while chondrogenesis was promoted by COOH-SWCNT, neuronal differentiation was promoted by both COOH-SWNCT and COOH-MWCNT. Taken together, these findings suggest that COOH-functionalized CNTs represent a promising scaffold component for future utilization in the selective differentiation of canine MSCs in regenerative medicine.

Proliferation of canine bone marrow derived mesenchymal stem cells on different nanomaterial based thin film scaffolds.

Stem cell niche research uses nanotechnologies to mimic the extra-cellular microenvironment to promote proliferation and differentiation. The aim of designing different scaffolds is to simulate the best structural and environmental pattern for extracellular matrix. This experiment was designed to study the proliferative behaviour of canine bone marrow deriver mesenchymal stem cells (MSCs) on different nanomaterial based thin film scaffolds of carbon nanotubes (CNT), chitosan and poly ε-caprolactone. Similar number of cells was seeded on the scaffolds and standard cell culture flask, taken as control. Cells were maintained on DMEM media and relative number of metabolically active cells was determined by MTT assay up to day six of culture. Cells proliferated on control and all the scaffolds as the days progressed. Although proliferation rate was slow but no decline of cell number was noticed on the scaffolds during the study period. Initially, the cell proliferation was lower on CNT but as time progressed no significant difference was observed compared to control. The result indicated that nanomaterial based scaffolds reduce the proliferation rate of canine MSCs. However, canine MSCs adapted and proliferated better on CNT substrate in vitro and may be used as a scaffold component in canine tissue engineering in future.

Valproic acid assisted reprogramming of fibroblasts for generation of pluripotent stem cells in buffalo (Bubalus bubalis).

Generation of pluripotent stem cells by reprogramming somatic cells of quality animals has numerous potential applications in agricultural and biomedical sciences. Unfortunately, till now, reprogramming of buffalo fetal fibroblast cells (bFFs) has been very ineffient despite intensive efforts. Here, we attempted to enhance reprogramming efficiency by using the HDAC inhibitor valproic acid (VPA) in bFFs transfected with pLentG-KOSM pseudo virus carrying mouse specific pluripotent genes. FACS analysis revealed that VPA treatment significantly increased (p < 0.05) GFP+ cells in comparison to VPA untreated control. Further, among different concentrations, 1.5 mM VPA was found to be optimal, increasing about 5 fold GFP+ cells and 2.5-fold GFP+ colonies with significantly (P < 0.05) larger size as compared to control. These colonies were further propagated and characterised. The colonies displayed embryonic stem cell (ESC)-like morphology, normal karyotype, and were positive for alkaline phosphatase staining as well as immune-positive for the ESC specific markers Oct4, Nanog, SSEA1, TRA-1-60 and TRA-1-81. The primary colonies revealed significantly higher (P < 0.05) expression of pluripotent genes than control, which declined gradually on subsequent passages. The reprogrammed cells readily formed embryoid bodies in vitro and cells of all three germ layers. These results indicated that VPA treatment of viral transducted cells can improve the generation of induced pluripotent stem cells and help their long term maintenance in buffalo.

Expression and functional role of Bone Morphogenetic Proteins (BMPs) in cyclical corpus luteum in buffalo (Bubalus bubalis).

The role of growth factors in the modulation of ovarian function is an interesting area of research in reproductive biology. Recently, we have shown the expression and role of IGF, EGF, VEGF and FGF in the follicle and CL. Here, we report the presence of Bone Morphogenetic Proteins (BMPs) and their functional receptors in the corpus luteum (CL) of buffalo. The bubaline CL was classified into four stages according to the morphology and progesterone (P4) concentration. The qPCR, immunoblot and immunohistochemistry studies revealed that BMP2 and BMP Receptors (BMPR1A, BMPR1B and BMPR2) were significantly upregulated during the mid stage whereas BMP4 and BMP7 were upregulated during the early stage of CL (P<0.05). Studies on primary luteal cell culture (LCC) using mid CL showed a significant time and concentration dependent effect of BMP4 and BMP7 (P<0.05). At 100ngml-1, the BMPs maximally stimulated the transcripts of StAR, CYP11A1 and 3ßHSD that paralleled with P4 accretion in the media (P<0.05). Further, the BMP4 as well as BMP7 upregulated the transcripts of PCNA and downregulated CASPASE3 in the LCC at the same concentration (P<0.05). Though the combined effect of BMP4 and 7 was significantly higher (P<0.05) than that of individual one, it was not additive. In conclusion, the expression of BMPs and their receptors were dependent on the stages of CL in the buffalo. Treatment of LCC with BMPs in vitro confirmed the presence of functional receptors that stimulated the P4 production and luteal cell survival. Moreover, the results support the concept that the upregulation of P4 and its biosynthetic pathway enzymes such as CYP11A1, StAR and 3ßHSD in the CL is likely due to the autocrine and /or paracrine effects of BMP4 and BMP7 under physiological milieu.