My research interests include tissue engineering, microfluidics, nanometer-scale integrated systems, biology of single mammalian embryos, embryonic and adult stem cells, drug delivery, remote sensing of embryo metabolism and embryonic health, NMR imaging, microcalorimetry and MEMS actuators.
The main objective of an embryo transfer (ET) program is to increase the genetic value of the off... more The main objective of an embryo transfer (ET) program is to increase the genetic value of the offspring produced in a given herd. Nutrition, management, and efficiency in the detection of oestrus are among the factors that affect the use of this technology. Protocols that induce oestrus or ovulation allow practitioners to transfer embryos in a predetermined time without oestrus detection (Bó et al. 2012 Reprod. Fertil. Dev. 24, 272-277; https://doi.org/10.1071/RD11918). Although these protocols have been performed adequately for several years, recent attention has been directed to the effect of oestrus expression and circulating oestradiol concentrations during the growth of the preovulatory follicle on embryo development and pregnancy. Oestrus expression is positively associated with high pregnancies per ET (P/ET) and reduced pregnancy losses in recipients receiving invitro-produced (IVP) bovine embryos (Bó and Cedeño 2018 Anim. Reprod. 15, 224-230; https://doi.org/10.21451/1984-31...
124 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1997.Results from embryo cultur... more 124 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1997.Results from embryo culture experiments indicated: (1) bovine serum albumin (BSA) supplementation improved developmental rates of four- and eight-cell Meishan embryos to the blastocyst stage, (2) fetal bovine serum supplementation improved hatching rates of later stage Meishan embryos, (3) fatty acid-free BSA did not improve developmental rates of early stage embryos, (4) more Meishan than Yorkshire one-cell embryos developed to the eight-cell and compact morula stages following 96 hours of culture, (5) more Yorkshire than Meishan two-cell embryos developed to the eight-cell stage following 96 hours of culture and (6) modified Whitten's medium +1.5% BSA was the most efficient medium for culture of one-cell embryos to the blastocyst stage. Results from activation and culture of in vivo matured oocytes indicated: (1) ethanol had no effect on activation rates, (2) cold shock, sham enucleation, electroactivation and electroactivation + culture with cycloheximide improved activation rates, and (3) electroactivation + culture with cycloheximide was the most efficient treatment for production of pronuclear stage cytoplasm. Results from NT experiments indicated that neither enucleated, unactivated oocytes nor enucleated, activated oocytes promoted development of NT embryos constructed from porcine ES cells beyond the four-cell stage. However, enucleated zygotes supported development of NT embryos beyond the four-cell stage to the compact morula stage. These results provide preliminary evidence for in vitro development of NT embryos produced from porcine ES cells.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD
The exact nature of the interaction(s) between cAMP and calcium-sensitive phospholipid-dependent ... more The exact nature of the interaction(s) between cAMP and calcium-sensitive phospholipid-dependent protein kinase-C effector pathways is not well understood in many tissues, including the ovary. In the present work we have evaluated the ability of protein kinase-C to modulate receptor-and nonreceptor-mediated cAMP generation in acute suspension cultures of swine luteal cells. Cells were exposed to LH (1 micrograms/ml), forskolin (100 microM), cholera toxin (1 microgram/ml), pertussis toxin (100 ng/ml), and/or phorbol ester [12-O-tetradecanoylphorbol-13-acetate (TPA)] for 0-90 min. TPA had no effect on basal cAMP accumulation, but increased (P less than 0.05) LH-, forskolin-, and cholera toxin-activated cAMP formation, with maximal facilitation at 30, 45, and 60 min, respectively. This facilitative effect was robust, as it could be demonstrated in both the presence and absence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.5 mM). TPA increased dose-dependent LH (0.1-1 microgram/ml)-, forskolin (3-300 microM-, and cholera toxin (0.3-10 microgram/ml)-stimulated cAMP accumulation. TPA induced a dose-dependent (0.3-30 ng/ml) increase in cAMP accumulation when incubated with the half-maximally effective (ED50) and maximally effective doses of LH (0.8 and 1 microgram/ml, respectively), forskolin (10 and 300 microM), and cholera toxin (0.2 and 3 micrograms/ml). TPA had an ED50 for this functional activation of 6.1 (67% confidence interval, 4.4-9.7) nM. The stimulatory effect of TPA could be mimicked by two synthetic diacylglycerols, 1,2-Dioctanoylglycerol and 1-oleoyl-2-acetylglycerol, but not by inactive phorbol esters. In addition, TPA augmented the stimulatory effect of pertussis toxin when combined with maximally effective doses of LH, forskolin, and cholera toxin. The stimulatory action of TPA on cAMP production was limited to endogenous cellular adenylyl cyclase. Bacterially derived adenylyl cyclase toxin isolated from Bordetella pertussis resulted in a dose-dependent increase in cAMP formation over 60 min, which was not facilitated by phorbol ester. We conclude that stimulatory coupling exists between the calcium-dependent protein kinase-C and cAMP-generating systems in swine luteal cells. This stimulatory coupling is enacted in part at the levels of both the guanine binding and the catalytic subunits of adenylyl cyclase.
In PubMed, it is possible to find more than 40,000 papers on embryo evaluation in various species... more In PubMed, it is possible to find more than 40,000 papers on embryo evaluation in various species. However, there is no consensus or gold standard method on how to assess their developmental potential. In assisted reproduction the evaluation "problem" is not only limited to embryos but involves the gametes as well. This manuscript provides an overview of some possible applications of label-free microscopy, in particular we describe the potential of the holographic microscopy in the IVF lab. We describe the positive aspects of several currently available microscopy label-free systems. In conclusion, we believe that a next generation of microscopy able to give objective markers for gamete and embryo quality is around the corner.
The advent of colour Doppler ultrasound has allowed evaluation of blood flow to bodily tissues. T... more The advent of colour Doppler ultrasound has allowed evaluation of blood flow to bodily tissues. This novel technology is being tested as a tool to improve efficiency in a variety of livestock reproduction programs. The objective of the present study was to evaluate the relationship between blood flow to the ovarian corpus luteum (CL), imaged via colour Doppler ultrasound, and pregnancy rate in crossbred dairy recipients for embryo transfer (ET). Oestrous cycles of Bos indicus×Bos taurus dairy heifers (n=90, 16-24 months of age) were synchronized for embryo transfer on Days 7-8. Immediately before ET, heifers were palpated for the presence of ovarian CL and CL papillae. Presence of CL, CL papillae, and CL lacunae were confirmed via transrectal B-mode ultrasound (Ibex EVO I, E. I. Medical Imaging). Transrectal colour Doppler ultrasound (Ibex EVO I, E. I. Medical Imaging) was used to evaluate blood flow to the CL. Invitro-produced (IVP) blastocysts (qualities I and II) were transferred to the uterine horn ipsilateral to the CL. All palpation, imaging, and embryo transfer was performed by a single technician. Pregnancy was determined via transrectal ultrasound 24-32 days post-embryo transfer (31-39 days of embryo age). Blood flow to the CL was split into three categories: high, median, and low. Presence or absence of two additional CL structures, CL papillae and lacunae, were recorded. Fisher's exact test was used to evaluate data. Statistical differences were considered significant at P<0.05. The overall pregnancy rate was determined to be 40%, 36 out of 90. Pregnancy rates were similar in high (33.9%, 20 of 59), median (59.09%, 13 of 22), and low (33.33%, 3 of 9) blood flow categories. Consistent with previous studies, absence of CL papillae and presence of CL lacunae at the time of ET did not affect pregnancy rate. Doppler blood flow, papillae, and lacunae were also evaluated together in each possible combination. Only one outlier was present; all other differences were not significant. The highest pregnancy rate (66.67%, 10 of 15) was observed in recipients with CLs with median Doppler blood flow, a palpable CL papillae, and no CL lacunae. A larger sample size is required to determine the accuracy of this measurement. In conclusion, recipients with high to low CL blood flow are suitable for IVP embryo transfer programs. Alone, colour Doppler imaging of the CL does not predict pregnancy rate.
Bovine embryos were produced in vitro using a 2 x 2 design of modified medium (KSOM or SOF) and o... more Bovine embryos were produced in vitro using a 2 x 2 design of modified medium (KSOM or SOF) and oxygen concentration (5% or 20%). Day 7 blastocysts were transferred in bulk (n = 11, on average) to recipient heifers and recovered non-surgically at Day 14. In two replications of a Latin square, eight heifers received embryos from each combination of factors. Recovered embryos were evaluated for trophoblast length and width, as well as the presence and diameter of an embryonic disc (ED). An ED was detected in a higher percentage of embryos that had been cultured in KSOM than SOF (72% v. 46%, respectively; P < 0.05). The aim of a second series of experiments was to associate Day 14 morphology with subsequent developmental capacity. In vitro-produced blastocysts were transferred (n = 17-20) on Day 7 to each of eight heifers and recovered at Day 14. Thirty-eight blastocysts were retransferred to heifers following morphological evaluation. Embryos in which an ED with no signs of degeneration had been detected maintained more pregnancies than other embryos in which an ED had either shown signs of degeneration or had not been detected (5/8 v. 2/30, respectively; P < 0.01). Further investigation into ED integrity at the elongating stage may contribute to our understanding of pregnancy establishment and maintenance.
The ability to identify embryos that have the highest developmental potential from a cohort would... more The ability to identify embryos that have the highest developmental potential from a cohort would significantly increase the chances of achieving pregnancy. Metabolic analysis is a well-established analytical approach in biological systems. Starting from this idea, we chose to use high-resolution nuclear magnetic resonance ( 1 H-NMR) spectroscopy. The aim of this study was to determine if it is possible to select viable embryos after 48 h of culture using metabolic activity as the parameter. We evaluated embryo metabolism after the first 48 h of culture and compared the activity of cleaved embryos that became blastocysts to cleaved embryos that did not develop to blastocysts, and in vitro fertilized (IVF) blastocysts and parthenogenetic-activated (PA) blastocysts. Our results show that citrate, pyruvate, myo-inositol and lysine have great impact on predicting embryo development. When we compared IVF and PA blastocysts, we found that acetate and phenylalanine concentrations are excellent parameters for evaluating blastocyst quality. Combining all these results, we were able to create a formula that predicts zygote development after 2 days of culture. In conclusion, we found that it is possible predict the future development of in vitro produced bovine embryos after only 2 days of culture using 1 H-NMR.
Tissue engineering offers a viable alternative to bone grafts in repairing large bone defects. Th... more Tissue engineering offers a viable alternative to bone grafts in repairing large bone defects. This involves using scaffolds of various sizes and shapes that contain stem cells and other osteoinductive molecules. The aim of this project was to evaluate the effects of vitamin K in osteogenic medium and its effect on the differentiation of adipose-derived stem cells (ASC) into osteoblasts. Vitamin K has been shown to increase bone mineral density by acting as a coenzyme in the γ-carboxylation of osteocalcin, a protein involved in bone mineralization (Weber 2001 Nutrition 11–12, 1024). Our hypothesis was that the presence of vitamin K in the osteogenic medium would positively influence the number of osteoblastic nodules formed. Swine ASC were isolated as described (Monaco et al. 2009 Open Tissue Eng. Regen. Med. J. 2, 20–33). The ASC were divided into 7 different treatments: 5 concentrations of vitamin K in the osteogenic medium (10, 50 100, 500, 1000 nM) plus 2 control treatments (ost...
Large bone defects present a tremendous challenge to the treating surgeon. Tissue engineering usi... more Large bone defects present a tremendous challenge to the treating surgeon. Tissue engineering using scaffolds of various sizes and shapes that contain stem cells and other osteoinductive molecules offer a potential solution to this difficult problem. The aim of this project was to evaluate if the osteogenic medium infused with vitamin B12 influences the differentiation of adipose-derived stem cells (ASC) into osteoblasts. Vitamin B12 has been shown to have a stimulatory effect on osteoclastogenesis in vitro (Vaes et al. 2009 Calcified Tissue Int. 84, 413-422). Our hypothesis was that the presence of vitamin B12 in the osteogenic medium would positively influence the number of osteoblastic nodules formed. Swine ASC were isolated as described (Monaco et al. 2009 Open Tissue Eng. Regen. Med. J. 2, 20-33). The ASC were divided in 8 different treatments: 8 concentrations of vitamin B12 in the osteogenic medium (0.1, 0.2, 1, 2, 10, and 20 μM) plus 2 control treatments (osteogenic medium w...
The main objective of an embryo transfer (ET) program is to increase the genetic value of the off... more The main objective of an embryo transfer (ET) program is to increase the genetic value of the offspring produced in a given herd. Nutrition, management, and efficiency in the detection of oestrus are among the factors that affect the use of this technology. Protocols that induce oestrus or ovulation allow practitioners to transfer embryos in a predetermined time without oestrus detection (Bó et al. 2012 Reprod. Fertil. Dev. 24, 272-277; https://doi.org/10.1071/RD11918). Although these protocols have been performed adequately for several years, recent attention has been directed to the effect of oestrus expression and circulating oestradiol concentrations during the growth of the preovulatory follicle on embryo development and pregnancy. Oestrus expression is positively associated with high pregnancies per ET (P/ET) and reduced pregnancy losses in recipients receiving invitro-produced (IVP) bovine embryos (Bó and Cedeño 2018 Anim. Reprod. 15, 224-230; https://doi.org/10.21451/1984-31...
124 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1997.Results from embryo cultur... more 124 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1997.Results from embryo culture experiments indicated: (1) bovine serum albumin (BSA) supplementation improved developmental rates of four- and eight-cell Meishan embryos to the blastocyst stage, (2) fetal bovine serum supplementation improved hatching rates of later stage Meishan embryos, (3) fatty acid-free BSA did not improve developmental rates of early stage embryos, (4) more Meishan than Yorkshire one-cell embryos developed to the eight-cell and compact morula stages following 96 hours of culture, (5) more Yorkshire than Meishan two-cell embryos developed to the eight-cell stage following 96 hours of culture and (6) modified Whitten's medium +1.5% BSA was the most efficient medium for culture of one-cell embryos to the blastocyst stage. Results from activation and culture of in vivo matured oocytes indicated: (1) ethanol had no effect on activation rates, (2) cold shock, sham enucleation, electroactivation and electroactivation + culture with cycloheximide improved activation rates, and (3) electroactivation + culture with cycloheximide was the most efficient treatment for production of pronuclear stage cytoplasm. Results from NT experiments indicated that neither enucleated, unactivated oocytes nor enucleated, activated oocytes promoted development of NT embryos constructed from porcine ES cells beyond the four-cell stage. However, enucleated zygotes supported development of NT embryos beyond the four-cell stage to the compact morula stage. These results provide preliminary evidence for in vitro development of NT embryos produced from porcine ES cells.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD
The exact nature of the interaction(s) between cAMP and calcium-sensitive phospholipid-dependent ... more The exact nature of the interaction(s) between cAMP and calcium-sensitive phospholipid-dependent protein kinase-C effector pathways is not well understood in many tissues, including the ovary. In the present work we have evaluated the ability of protein kinase-C to modulate receptor-and nonreceptor-mediated cAMP generation in acute suspension cultures of swine luteal cells. Cells were exposed to LH (1 micrograms/ml), forskolin (100 microM), cholera toxin (1 microgram/ml), pertussis toxin (100 ng/ml), and/or phorbol ester [12-O-tetradecanoylphorbol-13-acetate (TPA)] for 0-90 min. TPA had no effect on basal cAMP accumulation, but increased (P less than 0.05) LH-, forskolin-, and cholera toxin-activated cAMP formation, with maximal facilitation at 30, 45, and 60 min, respectively. This facilitative effect was robust, as it could be demonstrated in both the presence and absence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.5 mM). TPA increased dose-dependent LH (0.1-1 microgram/ml)-, forskolin (3-300 microM-, and cholera toxin (0.3-10 microgram/ml)-stimulated cAMP accumulation. TPA induced a dose-dependent (0.3-30 ng/ml) increase in cAMP accumulation when incubated with the half-maximally effective (ED50) and maximally effective doses of LH (0.8 and 1 microgram/ml, respectively), forskolin (10 and 300 microM), and cholera toxin (0.2 and 3 micrograms/ml). TPA had an ED50 for this functional activation of 6.1 (67% confidence interval, 4.4-9.7) nM. The stimulatory effect of TPA could be mimicked by two synthetic diacylglycerols, 1,2-Dioctanoylglycerol and 1-oleoyl-2-acetylglycerol, but not by inactive phorbol esters. In addition, TPA augmented the stimulatory effect of pertussis toxin when combined with maximally effective doses of LH, forskolin, and cholera toxin. The stimulatory action of TPA on cAMP production was limited to endogenous cellular adenylyl cyclase. Bacterially derived adenylyl cyclase toxin isolated from Bordetella pertussis resulted in a dose-dependent increase in cAMP formation over 60 min, which was not facilitated by phorbol ester. We conclude that stimulatory coupling exists between the calcium-dependent protein kinase-C and cAMP-generating systems in swine luteal cells. This stimulatory coupling is enacted in part at the levels of both the guanine binding and the catalytic subunits of adenylyl cyclase.
In PubMed, it is possible to find more than 40,000 papers on embryo evaluation in various species... more In PubMed, it is possible to find more than 40,000 papers on embryo evaluation in various species. However, there is no consensus or gold standard method on how to assess their developmental potential. In assisted reproduction the evaluation "problem" is not only limited to embryos but involves the gametes as well. This manuscript provides an overview of some possible applications of label-free microscopy, in particular we describe the potential of the holographic microscopy in the IVF lab. We describe the positive aspects of several currently available microscopy label-free systems. In conclusion, we believe that a next generation of microscopy able to give objective markers for gamete and embryo quality is around the corner.
The advent of colour Doppler ultrasound has allowed evaluation of blood flow to bodily tissues. T... more The advent of colour Doppler ultrasound has allowed evaluation of blood flow to bodily tissues. This novel technology is being tested as a tool to improve efficiency in a variety of livestock reproduction programs. The objective of the present study was to evaluate the relationship between blood flow to the ovarian corpus luteum (CL), imaged via colour Doppler ultrasound, and pregnancy rate in crossbred dairy recipients for embryo transfer (ET). Oestrous cycles of Bos indicus×Bos taurus dairy heifers (n=90, 16-24 months of age) were synchronized for embryo transfer on Days 7-8. Immediately before ET, heifers were palpated for the presence of ovarian CL and CL papillae. Presence of CL, CL papillae, and CL lacunae were confirmed via transrectal B-mode ultrasound (Ibex EVO I, E. I. Medical Imaging). Transrectal colour Doppler ultrasound (Ibex EVO I, E. I. Medical Imaging) was used to evaluate blood flow to the CL. Invitro-produced (IVP) blastocysts (qualities I and II) were transferred to the uterine horn ipsilateral to the CL. All palpation, imaging, and embryo transfer was performed by a single technician. Pregnancy was determined via transrectal ultrasound 24-32 days post-embryo transfer (31-39 days of embryo age). Blood flow to the CL was split into three categories: high, median, and low. Presence or absence of two additional CL structures, CL papillae and lacunae, were recorded. Fisher's exact test was used to evaluate data. Statistical differences were considered significant at P<0.05. The overall pregnancy rate was determined to be 40%, 36 out of 90. Pregnancy rates were similar in high (33.9%, 20 of 59), median (59.09%, 13 of 22), and low (33.33%, 3 of 9) blood flow categories. Consistent with previous studies, absence of CL papillae and presence of CL lacunae at the time of ET did not affect pregnancy rate. Doppler blood flow, papillae, and lacunae were also evaluated together in each possible combination. Only one outlier was present; all other differences were not significant. The highest pregnancy rate (66.67%, 10 of 15) was observed in recipients with CLs with median Doppler blood flow, a palpable CL papillae, and no CL lacunae. A larger sample size is required to determine the accuracy of this measurement. In conclusion, recipients with high to low CL blood flow are suitable for IVP embryo transfer programs. Alone, colour Doppler imaging of the CL does not predict pregnancy rate.
Bovine embryos were produced in vitro using a 2 x 2 design of modified medium (KSOM or SOF) and o... more Bovine embryos were produced in vitro using a 2 x 2 design of modified medium (KSOM or SOF) and oxygen concentration (5% or 20%). Day 7 blastocysts were transferred in bulk (n = 11, on average) to recipient heifers and recovered non-surgically at Day 14. In two replications of a Latin square, eight heifers received embryos from each combination of factors. Recovered embryos were evaluated for trophoblast length and width, as well as the presence and diameter of an embryonic disc (ED). An ED was detected in a higher percentage of embryos that had been cultured in KSOM than SOF (72% v. 46%, respectively; P < 0.05). The aim of a second series of experiments was to associate Day 14 morphology with subsequent developmental capacity. In vitro-produced blastocysts were transferred (n = 17-20) on Day 7 to each of eight heifers and recovered at Day 14. Thirty-eight blastocysts were retransferred to heifers following morphological evaluation. Embryos in which an ED with no signs of degeneration had been detected maintained more pregnancies than other embryos in which an ED had either shown signs of degeneration or had not been detected (5/8 v. 2/30, respectively; P < 0.01). Further investigation into ED integrity at the elongating stage may contribute to our understanding of pregnancy establishment and maintenance.
The ability to identify embryos that have the highest developmental potential from a cohort would... more The ability to identify embryos that have the highest developmental potential from a cohort would significantly increase the chances of achieving pregnancy. Metabolic analysis is a well-established analytical approach in biological systems. Starting from this idea, we chose to use high-resolution nuclear magnetic resonance ( 1 H-NMR) spectroscopy. The aim of this study was to determine if it is possible to select viable embryos after 48 h of culture using metabolic activity as the parameter. We evaluated embryo metabolism after the first 48 h of culture and compared the activity of cleaved embryos that became blastocysts to cleaved embryos that did not develop to blastocysts, and in vitro fertilized (IVF) blastocysts and parthenogenetic-activated (PA) blastocysts. Our results show that citrate, pyruvate, myo-inositol and lysine have great impact on predicting embryo development. When we compared IVF and PA blastocysts, we found that acetate and phenylalanine concentrations are excellent parameters for evaluating blastocyst quality. Combining all these results, we were able to create a formula that predicts zygote development after 2 days of culture. In conclusion, we found that it is possible predict the future development of in vitro produced bovine embryos after only 2 days of culture using 1 H-NMR.
Tissue engineering offers a viable alternative to bone grafts in repairing large bone defects. Th... more Tissue engineering offers a viable alternative to bone grafts in repairing large bone defects. This involves using scaffolds of various sizes and shapes that contain stem cells and other osteoinductive molecules. The aim of this project was to evaluate the effects of vitamin K in osteogenic medium and its effect on the differentiation of adipose-derived stem cells (ASC) into osteoblasts. Vitamin K has been shown to increase bone mineral density by acting as a coenzyme in the γ-carboxylation of osteocalcin, a protein involved in bone mineralization (Weber 2001 Nutrition 11–12, 1024). Our hypothesis was that the presence of vitamin K in the osteogenic medium would positively influence the number of osteoblastic nodules formed. Swine ASC were isolated as described (Monaco et al. 2009 Open Tissue Eng. Regen. Med. J. 2, 20–33). The ASC were divided into 7 different treatments: 5 concentrations of vitamin K in the osteogenic medium (10, 50 100, 500, 1000 nM) plus 2 control treatments (ost...
Large bone defects present a tremendous challenge to the treating surgeon. Tissue engineering usi... more Large bone defects present a tremendous challenge to the treating surgeon. Tissue engineering using scaffolds of various sizes and shapes that contain stem cells and other osteoinductive molecules offer a potential solution to this difficult problem. The aim of this project was to evaluate if the osteogenic medium infused with vitamin B12 influences the differentiation of adipose-derived stem cells (ASC) into osteoblasts. Vitamin B12 has been shown to have a stimulatory effect on osteoclastogenesis in vitro (Vaes et al. 2009 Calcified Tissue Int. 84, 413-422). Our hypothesis was that the presence of vitamin B12 in the osteogenic medium would positively influence the number of osteoblastic nodules formed. Swine ASC were isolated as described (Monaco et al. 2009 Open Tissue Eng. Regen. Med. J. 2, 20-33). The ASC were divided in 8 different treatments: 8 concentrations of vitamin B12 in the osteogenic medium (0.1, 0.2, 1, 2, 10, and 20 μM) plus 2 control treatments (osteogenic medium w...
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