Comparative evaluation of fracture healing potential of differentiated and undifferentiated guinea pig and canine bone marrow-derived mesenchymal stem cells in a guinea pig model.

BACKGROUND AND AIM: This work was conducted to compare the therapeutic potential of undifferentiated and osteogenic differentiated canine (xenogeneic) and guinea pig (allogeneic) BMSCs in fracture healing using guinea pig as a model. MATERIALS AND METHODS: A well-characterized homogenous population of third passage mesenchymal stem cells of bone marrow origin was used in all the experiments. MSCs from both the species, i.e., canine and guinea pigs, were differentiated and characterized. Expression of MHC I and II along with co-stimulatory molecules was assessed based on relative mRNA expression. The osteogenic differentiated and undifferentiated MSCs from both species were used for evaluating fracture healing in the guinea pig model. The healing potential was assessed based on radiographic, histopathology, and clinical observations. RESULTS: BMSCs from both species expressed MSC surface antigens and successfully differentiated to osteogenic, chondrogenic, and adipogenic lineages. The mRNA expression of class I and II MHC molecules in all the three lineages showed no significant (p > 0.05) differences after differentiating to adipogenic, chondrogenic, and osteogenic lineages. Radiographic and clinical examination revealed that MSCs therapy significantly improved bone fracture healing with a non-significant (p > 0.05) difference between differentiated and undifferentiated BMSCs. In addition, allogeneic MSCs therapy performed better than xenogeneic therapy. CONCLUSION: MSCs remained hypo immunogenic after differentiation and have comparable fracture healing potential though allogeneic MSCs have better therapeutic potential than xenogenic MSCs.

Smad4 and γ-secretase knock-down effect on osteogenic differentiation mediated via Runx2 in canine mesenchymal stem cells.

Cell lineage determination during mesenchymal stem cell (MSCs) differentiation is a highly orchestrated process involving diverse signaling pathways and distinct classes of regulatory molecules. Bone morphogenetic protein (BMP) signaling positively influence the osteoblast lineage determination, whereas the Notch signaling may have a dimorphic action. Effective regenerative therapy for repairing bone defects requires ample knowledge of the signaling pathways responsible for the differentiation of MSCs. To elucidate the signaling pathways that drives canine bone-marrow derived MSCs towards osteogenic lineage, the current work was focused on BMP and Notch signaling. Target genes of Runx2, Smad4 and γ-secretase were silenced by short hairpin RNA (shRNA) in canine MSCs. Evaluation of the effect of gene silencing on in-vitro osteogenic differentiation potential was done by quantitative polymerase chain reaction (qPCR) for osteoblastic markers (Osteocalcin and Osteopontin) and Alizarin red S staining for the extracellular deposition of calcium. Silencing of Runx2 significantly reduced the osteocalcin and osteopontin gene expression while a similar trend was observed in the case of smad 4 silencing and their combination groups, but there was no difference found in Hey 1 expression. Runx2 and Smad4 silencing groups showed very less positive staining with Alizarin red S staining, whereas knockdown of γ-secretase and its combination groups showed reverse results as that of Runx2 and Smad4. Runx2 plays an indispensable part in directing the canine mesenchymal stem cells towards osteogenic lineage. Also, Smad-mediated BMP signaling induced the osteoblast-specific gene expression, whereas the notch pathway negatively regulated the osteogenic differentiation of canine MSCs.

Expression profile of adhesion molecules in blastocyst vis-a-vis uterine epithelial cells.

Models using in vitro produced buffalo embryos and in vitro cultured uterine epithelial cells (UECs) may be useful in understanding the intricacies of embryo-uterine cross talk. In the present study, buffalo UECs were obtained from slaughterhouse derived non-gravid uterus. UECs monolayer was treated with steroids (10pg/ml estradiol for 24h and 3.14 ng/ml progesterone for another 5 days). In vitro produced buffalo blastocysts were co-cultured over steroid treated UECs monolayer and at 72 h of co-culture, embryo attachment rate was higher in UECs treated with steroids (71.86% vs. 26.55%) while no attachment was observed on plastic surface. Naturally hatched or assisted hatched blastocysts were co-cultured over UECs monolayer treated with 3.14ng/ml progesterone (P4), or without any treatment for 72 h and the effect of co-culture on the expression profile of adhesion related biomolecules was analyed in UECs and blastocysts. Cultured UECs and blastocysts cultured in embryo culture media were considered as control. It was observed that the expression of MUC1 in UECs was significantly (p < 0.05) higher in control group than treatment groups. The relative mRNA abundance of integrins and osteopontin was significantly (p < 0.05) higher in UECs and blastocysts of treatment groups than control group. Expression of IFN-τ was significantly higher (p < 0.05) in embryos co-cultured with UECs than other treatment groups. It can be concluded that P4 supplementation is required for the modulation of adhesion molecules and co-culture of blastocysts and UECs together affect the expression of adhesion molecules both in blastocyts and in UECs.

Progesterone modulates adhesion molecules in uterine epithelial cells and in vitro embryo production in buffalo.

This study was undertaken to evaluate the role of progesterone (P4) in modulation of the expression profile of adhesion-related molecules in uterine epithelial cells (UECs) and in vitro blastocyst production in buffalo. UECs were isolated from slaughterhouse-derived uteri by enzymatic treatment, and cells were characterized by immunocytochemistry (ICC) and PCR assays. The well-characterized UECs were exposed to different concentrations of P4 (0, 0.314, 3.14 and 6.28 ng/ml) along with the basal level of oestradiol for 6 days. Thereafter, the relative mRNA expression of different biomolecules such as mucin 1 (MUC1), osteopontin, integrin alpha (α3, α6 and αV) and beta (ß1 and ß3) subunits, progesterone receptor (PR) and oestrogen receptor, was evaluated. Further, day 2 post-insemination embryos were cultured in mSOF supplemented with or without P4. UECs were found positive for cytokeratin expression and negative for vimentin expression. Progesterone treatment significantly enhanced the mRNA expression of most of the transcripts compared with the control group, and correspondingly, the immunofluorescence depicted higher protein expression of all these molecules. Further, the long-term exposure of UECs to P4 downregulated the expression of PR and, concomitantly, MUC1. Progesterone supplementation to embryo culture medium significantly (p < .05) improved the blastocyst rate. The study demonstrates the role of P4 hormone in modulation of the expression of early implantation-related biomolecules in uterine epithelial cells; hence, adequate level of steroids is crucial for normal embryo development and its implantation.

Effect of cryopreservation on therapeutic potential of canine bone marrow derived mesenchymal stem cells augmented mesh scaffold for wound healing in guinea pig.

The objective of this study was to compare the therapeutic potential of canine bone marrow derived mesenchymal stem cells (BM MSCs) augmented mesh scaffold for wound healing potential in guinea pig before and after cryopreservation. Bone marrow aspirate was obtained from healthy dogs and culture was expanded in vitro. MSCs augmented mesh scaffold were cryopreserved for 30 days and then used for therapeutic purposes. Both fresh and frozen thaw MSCs augmented mesh scaffold along with fresh MSCs were used for therapeutic purposes in guinea pig. No significant (P > 0.05) difference was observed in population doubling time (PDT) among fresh and frozen thawed BM MSCs. Both fresh and frozen thawed BM MSCs expressed cell surface markers (CD73, CD90, and CD105), and did not express CD34 as was confirmed by Immunocytochemistry and Real-Time Polymerase Chain Reaction. The fresh and frozen thawed BM MSCs successfully differentiated into osteogenic, chondrogenic and adipogenic lineages. Therapeutic results revealed that the percent wound contraction on day 14 was more than 65 % for the mesh augmented with MSCs as well as freshly injected MSCs group as against 33-34 % in the control group. Healed wound quality parameters viz. surface epithelium, neovascularization, and collagen characteristics were better for the mesh augmented with MSCs as well as freshly injected MSCs group compared to the control group. No significant difference was noted among fresh and frozen thawed BM MSCs group and fresh MSCs injected group. Thus, it is concluded from this study that canine BM MSCs augmented mesh scaffold both fresh and frozen thaw can be used for quality wound healing.