Pigs are emerging as important large animal models for biomedical research, and they may represent a source of organs for xenotransplantation. The MHC is pivotal to the function of the immune system ...in health and disease, and it is particularly important in infection and transplant rejection. Pigs deficient in class I MHC could serve as important reagents to study viral immunity as well as allograft and xenograft rejection. In this study, we report the creation and characterization of class I MHC knockout pigs using the Cas9 nuclease and guide RNAs. Pig fetal fibroblasts were genetically engineered using Cas9 and guide RNAs, and class I MHC(-) cells were then used as nuclear donors for somatic cell nuclear transfer. We produced three piglets devoid of all cell surface class I proteins. Although these animals have reduced levels of CD4(-)CD8(+) T cells in peripheral blood, the pigs appear healthy and are developing normally. These pigs are a promising reagent for immunological research.
The consequences of SARS-CoV-2 infection on the musculoskeletal system represent a dangerous knowledge gap. Aging patients are at added risk for SARS-CoV-2 infection; therefore, a greater ...understanding of the resulting musculoskeletal sequelae of SARS-CoV-2 infection may help guide clinical strategies. This study examined fundamental bone parameters among mice treated with escalating viral loads. Male C57BL/6J (WT, n = 17) and B6.Cg-Tg(K18-ACE2)2Prlmn/J mice (K18-hACE2 transgenic mice, n = 21) expressing human ACE2 (TG) were divided into eight groups (n = 4–6/group) and subjected to intranasal dosing of 0, 1 × 103, 1 × 104, and 1 × 105 PFU (plaque forming units) of human SARS-CoV-2. Animal health was assessed daily by veterinary staff using established and validated scoring criteria (activity, posture, body condition scores and body weight). We report here that mock and WT infected mice were healthy and completed the study, surviving until 12–14 days post infection (dpi). In contrast, the TG mice infected with 1 × 105 PFU all experienced severe health declines that necessitated early euthanasia (6–7 dpi). For TG mice infected with 1 × 104 PFU, 2 mice were also euthanized after 7 dpi, while 3 mice showed signs of moderate disease at day 6 dpi, but recovered fully by day 11 dpi. Four of the 5 TG mice that were infected with 1 × 103 PFU remained healthy throughout the study. This suggests that our study mimics what is seen during human disease, where some patients develop severe disease resulting in death, while others have moderate to severe disease but recover, and others are asymptomatic. At necropsy, femurs were extracted and analyzed by μCT. No difference was found in μCT determined bone parameters among the WT groups. There was, however, a significant 24.4% decrease in trabecular bone volume fraction (p = 0.0009), 19.0% decrease in trabecular number (p = 0.004), 6.2% decrease in trabecular thickness (p = 0.04), and a 9.8% increase in trabecular separation (p = 0.04) among surviving TG mice receiving any viral load compared to non-infected controls. No differences in cortical bone parameters were detected. TRAP staining revealed surviving infected mice had a significant 64% increase in osteoclast number, a 27% increase in osteoclast surface, and a 38% increase in osteoclasts per bone surface. While more studies are needed to investigate the long-term consequences of SARS-CoV-2 infection on skeletal health, this study demonstrates a significant reduction in several bone parameters and corresponding robust increases in osteoclast number observed within 2 weeks post-infection in surviving asymptomatic and moderately affected mice.
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•Mice surviving SARS-CoV-2 infection exhibit bone loss 12–14 days post-infection.•Mice surviving SARS-CoV-2 infection exhibit increases in TRAP+ osteoclasts.•Bone loss and elevated osteoclasts are seen in mice with no clinical signs of illness.
Autosomal dominant osteopetrosis type II (ADO2) is a heritable bone disease of impaired osteoclastic bone resorption caused by missense mutations in the chloride channel 7 (CLCN7) gene. Clinical ...features of ADO2 include fractures, osteomyelitis of jaw, vision loss, and in severe cases, bone marrow failure. Currently, there is no effective therapy for ADO2, and patients usually receive symptomatic treatments. Theoretically, bone marrow transplantation (BMT), which is commonly used in recessive osteopetrosis, could be used to treat ADO2, although the frequency of complications related to BMT is quite high. We created an ADO2 knock‐in (p.G213R mutation) mouse model on the 129 genetic background, and their phenotypes mimic the human disease of ADO2. To test whether BMT could restore osteoclast function and rescue the bone phenotypes in ADO2 mice, we transplanted bone marrow cells from 6–8 weeks old male WT donor mice into recipient female ADO2 mice. Also, to determine whether age at the time of transplant may play a role in transplant success, we performed BMT in young (12‐week‐old) and old (9‐month‐old) ADO2 mice. Our data indicate that ADO2 mice transplanted with WT marrow achieved more than 90% engraftment up to 6 months post‐transplantation at both young and old ages. The in‐vivo DXA data revealed that young ADO2 mice transplanted with WT marrow had significantly lower whole body and spine areal bone mineral density (aBMD) at month 6 post‐transplantation compared to the ADO2 control mice. The old ADO2 mice also displayed significantly lower whole body, femur, and spine aBMD at months 4 and 5 post‐transplantation compared to the age‐matched control mice. The in‐vivo micro‐CT data showed that ADO2 experimental mice transplanted with WT marrow had significantly lower BV/TV at months 2 and 4 post‐transplantation compared to the ADO2 control mice at a young age. In contrast, ADO2 control and experimental mice displayed similar BV/TV values for all post‐transplantation time points at old age. In addition, serum CTX was significantly higher at month 2 post‐transplantation in both young and old ADO2 experimental mice compared to the ADO2 control mice. Serum P1NP levels in young ADO2 experimental mice were significantly higher at baseline and month 2 post‐transplantation compared to the ADO2 control mice. These data suggest that BMT may provide, at least, some beneficial effect at both young and adult ages.
Type 2 diabetes (T2D) results in physiological and structural changes in bone, contributing to poor fracture healing. T2D compromises microvascular performance, which can negatively impact bone ...regeneration as angiogenesis is required for new bone formation. We examined the effects of bone morphogenetic protein‐2 (BMP‐2) administered locally at the time of femoral segmental bone defect (SBD) surgery, and its angiogenic impacts on endothelial cells (ECs) isolated from the ipsilateral or contralateral tibia in T2D mice. Male C57BL/6 mice were fed either a low‐fat diet (LFD) or high‐fat diet (HFD) starting at 8 weeks. After 12 weeks, the T2D phenotype in HFD mice was confirmed via glucose and insulin tolerance testing and echoMRI, and all mice underwent SBD surgery. Mice were treated with BMP‐2 (5 µg) or saline at the time of surgery. Three weeks postsurgery, bone marrow ECs were isolated from ipsilateral and contralateral tibias, and proliferation, angiogenic potential, and gene expression of the cells was analyzed. BMP‐2 treatment increased EC proliferation by two fold compared with saline in LFD contralateral tibia ECs, but no changes were seen in surgical tibia EC proliferation. BMP‐2 treatment enhanced vessel‐like structure formation in HFD mice whereas, the opposite was observed in LFD mice. Still, in BMP‐2 treated LFD mice, ipsilateral tibia ECs increased expression of CD31, FLT‐1, ANGPT1, and ANGPT2. These data suggest that the modulating effects of T2D and BMP‐2 on the microenvironment of bone marrow ECs may differentially influence angiogenic properties at the fractured limb versus the contralateral limb.
Angiogenesis is a vital process during the regeneration of bone tissue. The aim of this study was to investigate angiogenesis at the fracture site as well as at distal locations from obesity-induced ...type 2 diabetic mice that were treated with bone morphogenetic protein-2 (BMP-2, local administration at the time of surgery) to heal a femoral critical sized defect (CSD) or saline as a control. Mice were fed a high fat diet (HFD) to induce a type 2 diabetic-like phenotype while low fat diet (LFD) animals served as controls. Endothelial cells (ECs) were isolated from the lungs (LECs) and bone marrow (BMECs) 3 weeks post-surgery, and the fractured femurs were also examined. Our studies demonstrate that local administration of BMP-2 at the fracture site in a CSD model results in complete bone healing within 3 weeks for all HFD mice and 66.7% of LFD mice, whereas those treated with saline remain unhealed. At the fracture site, vessel parameters and adipocyte numbers were significantly increased in BMP-2 treated femurs, irrespective of diet. At distal sites, LEC and BMEC proliferation was not altered by diet or BMP-2 treatment. HFD increased the tube formation ability of both LECs and BMECs. Interestingly, BMP-2 treatment at the time of surgery reduced tube formation in LECs and humeri BMECs. However, migration of BMECs from HFD mice treated with BMP-2 was increased compared to BMECs from HFD mice treated with saline. BMP-2 treatment significantly increased the expression of CD31, FLT-1, and ANGPT2 in LECs and BMECs in LFD mice, but reduced the expression of these same genes in HFD mice. To date, this is the first study that depicts the systemic influence of fracture surgery and local BMP-2 treatment on the proliferation and angiogenic potential of ECs derived from the bone marrow and lungs.
•Mice fed HFD for 12 weeks acquired a T2D-like metabolic phenotype.•BMP-2 treatment enhanced fracture healing in HFD mice 3 weeks post-surgery.•BMP-2 promotes vessel and adipocyte formation in the fracture callus.•ECs isolated from lungs and bone marrow of HFD mice have enhanced tube formation.•BMP-2 treatment in vivo 3 weeks prior to EC isolation inhibits tube formation in vitro.
With an aging world population, there is an increased risk of fracture and impaired healing. One contributing factor may be aging‐associated decreases in vascular function; thus, enhancing ...angiogenesis could improve fracture healing. Both bone morphogenetic protein 2 (BMP‐2) and thrombopoietin (TPO) have pro‐angiogenic effects. The aim of this study was to investigate the effects of treatment with BMP‐2 or TPO on the in vitro angiogenic and proliferative potential of endothelial cells (ECs) isolated from lungs (LECs) or bone marrow (BMECs) of young (3‐4 months) and old (22‐24 months), male and female, C57BL/6J mice. Cell proliferation, vessel‐like structure formation, migration, and gene expression were used to evaluate angiogenic properties. In vitro characterization of ECs generally showed impaired vessel‐like structure formation and proliferation in old ECs compared to young ECs, but improved migration characteristics in old BMECs. Differential sex‐based angiogenic responses were observed, especially with respect to drug treatments and gene expression. Importantly, these studies suggest that NTN1, ROBO2, and SLIT3, along with angiogenic markers (CD31, FLT‐1, ANGPT1, and ANGP2) differentially regulate EC proliferation and functional outcomes based on treatment, sex, and age. Furthermore, treatment of old ECs with TPO typically improved vessel‐like structure parameters, but impaired migration. Thus, TPO may serve as an alternative treatment to BMP‐2 for fracture healing in aging owing to improved angiogenesis and fracture healing, and the lack of side effects associated with BMP‐2.
Bony union is a primary predictor of outcome after surgical fixation of long bone fractures. Murine models offer many advantages in assessing bony healing due to their low costs and small size. ...However, current fracture recovery investigations in mice frequently rely on animal sacrifice and costly analyses. The modified Radiographic Union Score for Tibia fractures (mRUST) scoring system is a validated metric for evaluating bony healing in humans utilizing plain radiographs, which are relatively inexpensive and do not require animal sacrifice. However, its use has not been well established in murine models. The aim of this study was to characterize the longitudinal course of mRUST and compare mRUST to other conventional murine fracture analyses. 158 mice underwent surgically created midshaft femur fractures. Mice were evaluated after fracture creation and at 7, 10, 14, 17, 21, 24, 28, 35, and 42 days post-injury. mRUST scoring of plain radiographs was performed by three orthopaedic surgeons in a randomized, blinded fashion. Interrater correlations were calculated. Micro-computed tomography (μCT) was analyzed for tissue mineral density (TMD), total callus volume (TV), bone volume (BV), trabecular thickness, trabecular number, and trabecular separation. Histomorphometry measures of total callus area, cartilage area, fibrous tissue area, and bone area were performed in a blinded fashion. Ultimate torque, stiffness, toughness, and twist to failure were calculated from torque-twist curves. A sigmoidal log-logistic curve fit was generated for mRUST scores over time which shows mRUST scores of 4 to 6 at 7 days post-injury that improve to plateaus of 14 to 16 by 24 days post-injury. mRUST interrater correlations at each timepoint ranged from 0.51 to 0.86, indicating substantial agreement. mRUST scores correlated well with biomechanical, histomorphometry, and μCT parameters, such as ultimate torque (r=0.46, p<0.0001), manual stiffness (r=0.51, p<0.0001), bone percentage based on histomorphometry (r=0.86, p<0.0001), cartilage percentage (r=-0.87, p<0.0001), tissue mineral density (r=0.83, p<0.0001), BV/TV based on μCT (r=0.65, p<0.0001), and trabecular thickness (r=0.78, p<0.0001), among others. These data demonstrate that mRUST is reliable, trends temporally, and correlates to standard measures of murine fracture healing. Compared to other measures, mRUST is more cost-effective and non-terminal. The mRUST log-logistic curve could be used to characterize differences in fracture healing trajectory between experimental groups, enabling high-throughput analysis.
Angiogenesis is important for successful fracture repair. Aging negatively affects the number and activity of endothelial cells (ECs) and subsequently leads to impaired bone healing. We previously ...showed that implantation of lung-derived endothelial cells (LECs) improved fracture healing in rats. In this study, we characterized and compared neonatal lung and bone marrow-derived endothelial cells (neonatal LECs and neonatal BMECs) and further asses3sed if implantation of neonatal BMECs could enhance bone healing in both young and aged mice. We assessed neonatal EC tube formation, proliferation, and wound migration ability
in vitro
in ECs isolated from the bone marrow and lungs of neonatal mice. The
in vitro
studies demonstrated that both neonatal LECs and neonatal BMECs exhibited EC traits. To test the function of neonatal ECs
in vivo
, we created a femoral fracture in young and aged mice and implanted a collagen sponge to deliver neonatal BMECs at the fracture site. In the mouse fracture model, endochondral ossification was delayed in aged control mice compared to young controls. Neonatal BMECs significantly improved endochondral bone formation only in aged mice. These data suggest BMECs have potential to enhance aged bone healing. Compared to LECs, BMECs are more feasible for translational cell therapy and clinical applications in bone repair. Future studies are needed to examine the fate and function of BMECs implanted into the fracture sites.
Our efforts to prevent and treat breast cancer are significantly impeded by a lack of knowledge of the biology and developmental genetics of the normal mammary gland. In order to provide the ...specimens that will facilitate such an understanding, The Susan G. Komen for the Cure Tissue Bank at the IU Simon Cancer Center (KTB) was established. The KTB is, to our knowledge, the only biorepository in the world prospectively established to collect normal, healthy breast tissue from volunteer donors. As a first initiative toward a molecular understanding of the biology and developmental genetics of the normal mammary gland, the effect of the menstrual cycle and hormonal contraceptives on DNA expression in the normal breast epithelium was examined.
Using normal breast tissue from 20 premenopausal donors to KTB, the changes in the mRNA of the normal breast epithelium as a function of phase of the menstrual cycle and hormonal contraception were assayed using next-generation whole transcriptome sequencing (RNA-Seq).
In total, 255 genes representing 1.4% of all genes were deemed to have statistically significant differential expression between the two phases of the menstrual cycle. The overwhelming majority (221; 87%) of the genes have higher expression during the luteal phase. These data provide important insights into the processes occurring during each phase of the menstrual cycle. There was only a single gene significantly differentially expressed when comparing the epithelium of women using hormonal contraception to those in the luteal phase.
We have taken advantage of a unique research resource, the KTB, to complete the first-ever next-generation transcriptome sequencing of the epithelial compartment of 20 normal human breast specimens. This work has produced a comprehensive catalog of the differences in the expression of protein-coding genes as a function of the phase of the menstrual cycle. These data constitute the beginning of a reference data set of the normal mammary gland, which can be consulted for comparison with data developed from malignant specimens, or to mine the effects of the hormonal flux that occurs during the menstrual cycle.
Megakaryocytes (MKs) support bone formation by stimulating osteoblasts (OBs) and inhibiting osteoclasts (OCs). Aging results in higher bone resorption, leading to bone loss. Whereas previous studies ...showed the effects of aging on MK-mediated bone formation, the effects of aging on MK-mediated OC formation is poorly understood. Here we examined the effect of thrombopoietin (TPO) and MK-derived conditioned media (CM) from young (3-4 months) and aged (22-25 months) mice on OC precursors. Our findings showed that aging significantly increased OC formation in vitro. Moreover, the expression of the TPO receptor, Mpl, and circulating TPO levels were elevated in the bone marrow cavity. We previously showed that MKs from young mice secrete factors that inhibit OC differentiation. However, rather than inhibiting OC development, we found that MKs from aged mice promote OC formation. Interestingly, these age-related changes in MK functionality were only observed using female MKs, potentially implicating the sex steroid, estrogen, in signaling. Further, RANKL expression was highly elevated in aged MKs suggesting MK-derived RANKL signaling may promote osteoclastogenesis in aging. Taken together, these data suggest that modulation in TPO-Mpl expression in bone marrow and age-related changes in the MK secretome promote osteoclastogenesis to impact skeletal aging.
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