Sperm are allogenic to the female genital tract; however, oviducts provide optimal conditions for survival and capacitation of these non-self cells until fertilization.Recently, we showed that oviduct-conditioned media and prostaglandinE2 (PGE2) suppress sperm phagocytosis by polymorphonuclearneutrophils (PMNs) under physiological conditions. We hypothesized that sperm binding to bovine oviduct epithelial cells (BOECs) could change the local innate immunity via PGE2. As the first step to obtain basic information, sub-confluent BOEC monolayers were co-cultured with swim-up sperm for 2 h. BOECs with viable bound sperm were cultured for an additional 3, 6, 12, or 24 h. Then, we confirmedthe impact of the sperm-BOECbinding on both BOECs and PMN gene expression. Immunohistochemistry revealed that BOECs strongly express TGFB1 and IL10 in the oviduct. Sperm binding to BOECs in culture induced the antiinflammatorycytokines (TGFB1 and IL10) and PGE2 production by BOECs. Exogenous PGE2 in vitro suppressed pro-inflammatorycytokine expression (TNF and IL1B) in BOECs. Moreover,pre-exposure of PMNs to BOEC-conditioned media suppressed the TNF expression, but the BOEC media co-cultured with sperm stimulated PMNs to express TGFB1 and IL10, with increasing PGE2 secretion. Of note, exogenous PGE2 led PMNs in vitro to decrease their TNF expression and increase anti-inflammatory cytokines expression. Our findings strongly suggest that BOECs provide an anti-inflammatoryenvironment under physiological conditions and the sperm-BOECbinding furtherstrengthens this milieu thus suppresses PMNs in the bovine oviduct. PGE2 is likely to drive this stable anti-inflammatoryenvironment in the oviduct.

Sperm are allogenic to the female genital tract; however, oviducts provide optimal conditions for survival and capacitation of these non-self cells until fertilization.Recently, we showed that oviduct-conditioned media and prostaglandinE2 (PGE2) suppress sperm phagocytosis by polymorphonuclearneutrophils (PMNs) under physiological conditions. We hypothesized that sperm binding to bovine oviduct epithelial cells (BOECs) could change the local innate immunity via PGE2. As the first step to obtain basic information, sub-confluent BOEC monolayers were co-cultured with swim-up sperm for 2 h. BOECs with viable bound sperm were cultured for an additional 3, 6, 12, or 24 h. Then, we confirmedthe impact of the sperm-BOECbinding on both BOECs and PMN gene expression. Immunohistochemistry revealed that BOECs strongly express TGFB1 and IL10 in the oviduct. Sperm binding to BOECs in culture induced the antiinflammatorycytokines (TGFB1 and IL10) and PGE2 production by BOECs. Exogenous PGE2 in vitro suppressed pro-inflammatorycytokine expression (TNF and IL1B) in BOECs. Moreover,pre-exposure of PMNs to BOEC-conditioned media suppressed the TNF expression, but the BOEC media co-cultured with sperm stimulated PMNs to express TGFB1 and IL10, with increasing PGE2 secretion. Of note, exogenous PGE2 led PMNs in vitro to decrease their TNF expression and increase anti-inflammatory cytokines expression. Our findings strongly suggest that BOECs provide an anti-inflammatoryenvironment under physiological conditions and the sperm-BOECbinding furtherstrengthens this milieu thus suppresses PMNs in the bovine oviduct. PGE2 is likely to drive this stable anti-inflammatoryenvironment in the oviduct.

Sperm are allogenic to the female genital tract; however, oviducts provide optimal conditions for survival and capacitation of these non-self cells until fertilization.Recently, we showed that oviduct-conditioned media and prostaglandinE2 (PGE2) suppress sperm phagocytosis by polymorphonuclearneutrophils (PMNs) under physiological conditions. We hypothesized that sperm binding to bovine oviduct epithelial cells (BOECs) could change the local innate immunity via PGE2. As the first step to obtain basic information, sub-confluent BOEC monolayers were co-cultured with swim-up sperm for 2 h. BOECs with viable bound sperm were cultured for an additional 3, 6, 12, or 24 h. Then, we confirmedthe impact of the sperm-BOECbinding on both BOECs and PMN gene expression. Immunohistochemistry revealed that BOECs strongly express TGFB1 and IL10 in the oviduct. Sperm binding to BOECs in culture induced the antiinflammatorycytokines (TGFB1 and IL10) and PGE2 production by BOECs. Exogenous PGE2 in vitro suppressed pro-inflammatorycytokine expression (TNF and IL1B) in BOECs. Moreover,pre-exposure of PMNs to BOEC-conditioned media suppressed the TNF expression, but the BOEC media co-cultured with sperm stimulated PMNs to express TGFB1 and IL10, with increasing PGE2 secretion. Of note, exogenous PGE2 led PMNs in vitro to decrease their TNF expression and increase anti-inflammatory cytokines expression. Our findings strongly suggest that BOECs provide an anti-inflammatoryenvironment under physiological conditions and the sperm-BOECbinding furtherstrengthens this milieu thus suppresses PMNs in the bovine oviduct. PGE2 is likely to drive this stable anti-inflammatoryenvironment in the oviduct.

Objective: The aim of this study was to monitor the ovarian response and conception rate following estrous synchronization using CIDR, Ovsynch and double prostaglandin F2
protocols in Egyptian buffalo heifers. Material and methods: A total of 80 cyclic buffalo heifers were divided into four equal groups: CIDR (intravaginal progesterone releasing device, EAZI-BREEDTM CIDR®), Ovsynch (GnRH, PGF2
, GnRH injections), PGF (double PGF2
doses) and control. Timed artificial insemination (TAI) was performed in all heifers. All animals were examined using ultrasound and blood samples were collected for measurement of progesterone. Results: A new follicular wave occurred earlier in the Ovsynch and PGF groups than in the CIDR group (p 0.05). The mean diameter of the ovulatory follicle was smaller in the CIDR group than in the Ovsynch and PGF groups (p 0.05). The ovulation rate was 100% in the CIDR group, 75% in the Ovsynch group and 70% in the PGF group. In the control group a lower pregnancy rate (20%) was determined in than in the CIDR (35%), Ovsynch (40%) and PGF (35%) groups. Progesterone concentrations were numerically higher in pregnant heifers of the CIDR group but the difference was non-significantly compared to the Ovsynch, PGF and control groups (p > 0.05). Conclusion and clinical relevance: EAZIBREEDTM CIDR®, Ovsynch-based TAI and PGF protocols were effective in synchronizing oestrus and resulted in nearly similar pregnancy rates in Egyptian buffalo heifers.

Abstract Research on hydroxyapatite is experiencing a remarkable increase during the recent years due to its potential biological and biomedical applications. Herein, we have synthesized nano-hydroxyapatite (HAp) by solid state reaction (SSR), wherein recycled eggshell bio-waste as Ca precursor on processing is reacted with synthetic ammonium dihydrogen orthophosphate to form hydroxyapatite (SSHAp). The solid state synthesis reaction is studied by thermogravimetric analysis. The morphology and crystal structure of SSHAp nanoparticles is characterized and found to be spherical and free of aggregation with average crystallite size ∼ 10 nm. The SSHAp nanoparticles show large surface area (9.64 m2g−1). Furthermore, the invitro bioactivity and osteogenic activity of SSHAp is investigated. The osteogenic activity is evaluated by incubating the SSHAp with hFOB cells. The SSHAp is observed to promote appreciable cell attachment and proliferation unlike in control material. While hFOB cells is found to express higher level of osteocalcin, osteopontin, collagen I, osteonectin, BMP-2 and GAPDH on the SSHAp bone graft. The results show that SSHAp nanoparticles graft is bioactive, nontoxic and osteogenic, and has the potential to promote bone therapy applications. The HAp is synthesized using recycled bio-waste contributing to green chemistry, addressing the environmental concerns of recycle and reuse.

Abstract Research on hydroxyapatite is experiencing a remarkable increase during the recent years due to its potential biological and biomedical applications. Herein, we have synthesized nano-hydroxyapatite (HAp) by solid state reaction (SSR), wherein recycled eggshell bio-waste as Ca precursor on processing is reacted with synthetic ammonium dihydrogen orthophosphate to form hydroxyapatite (SSHAp). The solid state synthesis reaction is studied by thermogravimetric analysis. The morphology and crystal structure of SSHAp nanoparticles is characterized and found to be spherical and free of aggregation with average crystallite size ∼ 10 nm. The SSHAp nanoparticles show large surface area (9.64 m2g−1). Furthermore, the invitro bioactivity and osteogenic activity of SSHAp is investigated. The osteogenic activity is evaluated by incubating the SSHAp with hFOB cells. The SSHAp is observed to promote appreciable cell attachment and proliferation unlike in control material. While hFOB cells is found to express higher level of osteocalcin, osteopontin, collagen I, osteonectin, BMP-2 and GAPDH on the SSHAp bone graft. The results show that SSHAp nanoparticles graft is bioactive, nontoxic and osteogenic, and has the potential to promote bone therapy applications. The HAp is synthesized using recycled bio-waste contributing to green chemistry, addressing the environmental concerns of recycle and reuse.

Abstract Biomaterials based on seeding of cells on decellularized scaffolds have gained increasing interest in the last few years and suggested to serve as an alternative approach to bioengineer artificial organs and tissues for transplantation. The reaction of the host toward the decellularized scaffold and transplanted cells depends on the biocompatibility of the construct. Before proceeding to the clinical application step of decellularized scaffolds, it is greatly important to apply a number of biocompatibility tests in vitro and in vivo. This review describes the different methodology involved in cytotoxicity, pathogenicity, immunogenicity and biodegradability testing for evaluating the biocompatibility of various decellularized matrices obtained from human or animals.

Abstract Biomaterials based on seeding of cells on decellularized scaffolds have gained increasing interest in the last few years and suggested to serve as an alternative approach to bioengineer artificial organs and tissues for transplantation. The reaction of the host toward the decellularized scaffold and transplanted cells depends on the biocompatibility of the construct. Before proceeding to the clinical application step of decellularized scaffolds, it is greatly important to apply a number of biocompatibility tests in vitro and in vivo. This review describes the different methodology involved in cytotoxicity, pathogenicity, immunogenicity and biodegradability testing for evaluating the biocompatibility of various decellularized matrices obtained from human or animals.

Abstract Whole organ decellularization is a cell removal process that creates a natural extracellular matrix for use in transplantation. A lack of an intact endothelial layer in the vascular network of decellularized organs results in blood clotting even with anti-coagulation treatment. Furthermore, shear stress caused by blood flow may affect reseeded parenchymal cells. We hypothesized that a heparin-gelatin mixture (HG) can act as an antithrombotic coating reagent and induce attachment and migration of endothelial cells (ECs) on vascular wall surfaces within decellularized livers, with subsequent parenchymal cell function enhancement. Portal vein (PV) perfusion was performed for right lateral lobe decellularization of porcine livers. We tested if HG-precoating of isolated decellularized PV could increase EC attachment and migration. Additionally, we coated PV and hepatic artery walls in decellularized liver with HG, and then repopulated it with ECs and maintained it under vascular flow in a bioreactor for 10days. Re-endothelialized scaffolds were perfused with porcine blood for thrombogenicity evaluation. We then co-cultured hepatocellular carcinoma (HepG2) cells and ECs to evaluate the effect of endothelialization on parenchymal cells. Finally, we transplanted these scaffolds heterotopically in pigs. HG improved ECs' ability to migrate and adhere to vessel discs. ECs efficiently covered the vascular compartments within decellularized scaffolds and maintained function and proliferation after HG-precoating. No thrombosis was observed after 24h blood perfusion in HG-precoated scaffolds, indicating an efficiently endothelialized vascular tree. HepG2 cells displayed a higher function in scaffolds endothelialized after HG-precoating compared to uncoated scaffolds in vitro and after in vivo transplantation. Our results lay the groundwork for engineering human-sized whole-liver scaffolds for clinical applications. STATEMENT OF SIGNIFICANCE: A major obstacle to successful organ bioengineering is vasculature reconstruction to avoid thrombosis and deliver nutrients through blood to the whole scaffold after in vivo transplantation. Although many attempts have been made to construct endothelial cell layers on the vascular network within decellularized organs, complete coverage has not be achieved. Here, we describe an effective approach for endothelial cell seeding to reconstruct a patent vascular tree within decellularized livers by coating the vasculature using heparin-gelatin mixture. Our results have demonstrate that enhancement of endothelial cell attachment by heparin-gelatin treatment could improve vascular patency and parenchymal cell function in vitro and in vivo. These results represent a significant advancement toward bioengineering functional liver tissue that maintains vascular patency for transplantation.

Abstract Whole organ decellularization is a cell removal process that creates a natural extracellular matrix for use in transplantation. A lack of an intact endothelial layer in the vascular network of decellularized organs results in blood clotting even with anti-coagulation treatment. Furthermore, shear stress caused by blood flow may affect reseeded parenchymal cells. We hypothesized that a heparin-gelatin mixture (HG) can act as an antithrombotic coating reagent and induce attachment and migration of endothelial cells (ECs) on vascular wall surfaces within decellularized livers, with subsequent parenchymal cell function enhancement. Portal vein (PV) perfusion was performed for right lateral lobe decellularization of porcine livers. We tested if HG-precoating of isolated decellularized PV could increase EC attachment and migration. Additionally, we coated PV and hepatic artery walls in decellularized liver with HG, and then repopulated it with ECs and maintained it under vascular flow in a bioreactor for 10days. Re-endothelialized scaffolds were perfused with porcine blood for thrombogenicity evaluation. We then co-cultured hepatocellular carcinoma (HepG2) cells and ECs to evaluate the effect of endothelialization on parenchymal cells. Finally, we transplanted these scaffolds heterotopically in pigs. HG improved ECs' ability to migrate and adhere to vessel discs. ECs efficiently covered the vascular compartments within decellularized scaffolds and maintained function and proliferation after HG-precoating. No thrombosis was observed after 24h blood perfusion in HG-precoated scaffolds, indicating an efficiently endothelialized vascular tree. HepG2 cells displayed a higher function in scaffolds endothelialized after HG-precoating compared to uncoated scaffolds in vitro and after in vivo transplantation. Our results lay the groundwork for engineering human-sized whole-liver scaffolds for clinical applications. STATEMENT OF SIGNIFICANCE: A major obstacle to successful organ bioengineering is vasculature reconstruction to avoid thrombosis and deliver nutrients through blood to the whole scaffold after in vivo transplantation. Although many attempts have been made to construct endothelial cell layers on the vascular network within decellularized organs, complete coverage has not be achieved. Here, we describe an effective approach for endothelial cell seeding to reconstruct a patent vascular tree within decellularized livers by coating the vasculature using heparin-gelatin mixture. Our results have demonstrate that enhancement of endothelial cell attachment by heparin-gelatin treatment could improve vascular patency and parenchymal cell function in vitro and in vivo. These results represent a significant advancement toward bioengineering functional liver tissue that maintains vascular patency for transplantation.