Search

Summary: Background: Clodronate is prescribed to performance horses with lameness. Despite its clinical popularity, little research has been done to understand the effects of clodronate in the horse. Objectives: Our objective was to determine if a single treatment with clodronate at the clinically approved dose altered bone remodelling, bone cell recruitment or lameness in the horse. Study design: Twelve university‐owned equestrian team competition horses with a history of forelimb lameness due to navicular syndrome were randomised to receive either 1.4 mg/kg clodronate (CLOD n = 6) or an equivalent volume of LRS (CONT; n = 6) in a blinded manner. Methods: Blood was evaluated weekly for 8 weeks before and after drug administration (clodronate or placebo) for bone turnover markers CTX‐I and osteocalcin. Lameness evaluations were performed to assess for change in lameness 1 week before and 1, 2, 3 and 8 weeks after drug administration. Coach questionnaires were performed to assess for change in ridden performance 1, 2, 3 and 8 weeks after drug administration. Bone cell recruitment was evaluated in vitro 2 weeks before and after drug administration. Results: There were no differences in in vitro bone cell recruitment from whole bone marrow or in bone turnover markers CTX‐I or osteocalcin. A small but significant decrease in forelimb lameness was detected in CLOD treated horses 1 week after treatment (P = 0.005). There were no significant differences in hindlimb lameness. Coaches identified an improvement in performance significantly more often in CLOD vs. CONT (P = 0.01) at week 8. Main limitations: Two CONT horses received intra‐articular anti‐inflammatory medication after treatment, which may have altered lameness results. Conclusions: A single dose of clodronate appears to reduce lameness without producing detectable effects on bone turnover markers. Due to the long half‐life of a bisphosphonate drug, the effect of multiple doses on bone remodelling and lameness should be investigated. The Summary is available in Portuguese ‐ see Supporting Information [ABSTRACT FROM AUTHOR]

Background: Liposomal anthracyclines (such as pegylated liposomal doxorubicin (PLD) or HER2-targeted liposomal doxorubicin (MM-302)) are being used and/or evaluated for the clinical management of breast cancer, but responses vary from patient to patient. It is hypothesized that variability in the deposition of liposomal therapeutics within tumors leads to differences in drug exposure, thereby directly influencing tumor response. We have developed MM-DX-929, a novel 64Cu-liposomal PET imaging agent, as a clinically-implementable tool to investigate whether image-based quantification of liposome deposition in tumors can predict treatment response to liposomal chemotherapeutics, including PLD, MM-302 and/or liposomal irinotecan (MM-398). Objectives: Our primary objective is to demonstrate that the extent of tumor uptake of MM-DX-929 is predictive of tumor response to MM-302 in preclinical breast cancer xenograft models. A secondary objective is to enable clinical translation of MM-DX-929 to enable incorporation into existing therapeutic trials. Methods: Mice bearing BT474-M3 mammary and subcutaneous tumors were injected intravenously with MM-DX-929 prior to dosing with MM-302. PET/CT imaging was performed at 16h post MM-DX-929 injection, and tumor uptake was determined. Response to treatment was quantified as tumor volume changes measured over a 2-month period by MRI. Results: Tumor deposition of MM-DX-929 administration correlated well with treatment response to MM-302 (Spearman correlation coefficient of -0.891 and a p-value of 0.0004). MM-DX-929 accumulation in tumors prior to the start of the MM-302 treatment successfully predicted improved tumor growth inhibition following MM-302 treatment. Conclusion: These findings support trials of MM-DX-929 as a predictive imaging agent to select patients who are most likely to respond to liposomal therapies. The clinical development of MM-DX-929 for identification of breast cancer patients likely to respond to liposomal therapeutics is currently being pursued. [ABSTRACT FROM AUTHOR]

Background: Tissue non-specific alkaline phosphatase (TNSALP; encoded by the ALPL gene) has a critical role in the postnatal regulation of phosphate homeostasis, yet how TNSALP activity and expression are regulated during pregnancy remain largely unknown. This study tested the hypothesis that progesterone (P4) and/or interferon tau (IFNT) regulate TNSALP activity during pregnancy in sheep., Methods: In Exp. 1, ewes were bred and received daily intramuscular injections of either corn oil vehicle (CO) or 25 mg progesterone in CO (P4) for the first 8 days of pregnancy and were hysterectomized on either Day 9, 12, or 125 of gestation. In Exp. 2, ewes were fitted with intrauterine catheters on Day 7 of the estrous cycle and received daily intramuscular injections of 50 mg P4 in CO and/or 75 mg progesterone receptor antagonist (RU486) in CO from Days 8 to 15, and twice daily intrauterine injections of either control proteins (CX) or IFNT (25 µg/uterine horn/d) from Days 11 to 15 (treatment groups: P4 + CX; P4 + IFNT; RU486 + P4 + CX; and RU486 + P4 + IFNT) and were hysterectomized on Day 16., Results: In Exp. 1, endometria from ewes administered P4 had greater expression of ALPL mRNA than ewes administered CO on Day 12. TNSALP activity appeared greater in the epithelia, stratum compactum stroma, and endothelium of the blood vessels in the endometrium and myometrium from ewes administered P4 than ewes administered CO on Day 12. On Day 125, TNSALP activity localized to uterine epithelial and endothelial cells, independent of P4 treatment. TNSALP activity in placentomes appeared greater in P4 treated ewes and was detected in endothelial cells and caruncular tissue in P4 treated but not CO treated ewes. In Exp. 2, endometrial homogenates from ewes administered RU486 + P4 + CX had lower TNSALP activity those for P4 + CX and P4 + IFNT ewes. Immunoreactive TNSALP protein appeared greater in the mid- and deep-glandular epithelia in RU486 + P4 + CX treated ewes as compared to the other treatment groups. Enzymatic activity appeared greater on the apical surface of the deep glandular epithelia in endometria from ewes treated with RU486 + P4 + CX compared to the other treatment groups., Conclusions: These results suggest that P4, but not IFNT, regulates the expression and activity of TNSALP in utero-placental tissues and has the potential to contribute to the regulation of phosphate availability that is critical for conceptus development during pregnancy., (© 2024. The Author(s).)

Tissue-nonspecific alkaline phosphatase (TNSALP; encoded by ALPL gene) has a critical role in the regulation of phosphate homeostasis postnatally. However, the utero-placental expression of TNSALP and the role in phosphate transport in pregnancy is poorly understood. Estrous cycles of ewes were synchronized, and ewes were euthanized and hysterectomized on Days 1, 9, or 14 of the estrous cycle or bred to fertile rams and euthanized and hysterectomized on Days 9, 12, 17, 30, 50, 70, 90, 110, or 125 of pregnancy. The expression of ALPL mRNA, immunolocalization of TNSALP protein, and quantification and localization of TNSALP enzymatic activity was performed on ovine endometria and placentomes. Day of the estrous cycle did not alter ALPL mRNA expression or enzymatic activity of TNSALP. TNSALP protein localized to uterine epithelial and stromal cells, blood vessels, myometrium, caruncular, and cotyledonary stroma. TNSALP activity was localized to uterine epithelia, blood vessels, caruncular stroma (from Day 70 of gestation), and the apical surface of chorionic epithelia (from Day 50 of gestation). TNSALP protein and activity localized to the apical surface of uterine epithelia during the estrous cycle and in early pregnancy. Endometrial TNSALP enzymatic activity was downregulated on Days 17 and 30 of gestation (P < 0.05). Expression of ALPL mRNA decreased in late gestation in endometria and placentomes (P < 0.05). TNSALP activity peaked in placentomes on Days 70 and 90 of gestation. Collectively, these results suggest a potential role of TNSALP in the regulation of phosphate transport and homeostasis at the maternal-conceptus interface in ruminants., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Estimation of the hip joint center in ovine biomechanical analysis is often overlooked or estimated using a marker on the greater trochanter which can result in large errors that propagate through subsequent analyses. The purpose of this study was to develop a novel method of estimating the hip joint centers in sheep to facilitate more accurate analysis of ovine biomechanics. CT scans from 16 sheep of varying ages, weight, sex, and phenotypes were acquired and the data was used to calculate the known hip joint center by sphere fitting the femoral head. Anatomical measurements and additional subject information were used to create a variety of regression models to estimate the hip joint centers in absence of CT data. The best regression equation created utilized markers placed on the tuber coxae and tuber ischii of the pelvis and resulted in a mean 3D Euclidean distance error of 6.43 ± 2.22 mm (mean ± standard deviation) between the known and estimated hip joint center. The regression models produced allow for more detailed, accurate and robust analysis of sheep biomechanics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Humans and mice have the ability to regenerate the distal digit tip, the terminal phalanx (P3) in response to amputation. What distinguishes P3 regeneration from regenerative failure is formation of the blastema, a proliferative structure that undergoes morphogenesis to regenerate the amputated tissues. P3 regeneration is characterised by the phases of inflammation, tissue histolysis and expansive bone degradation with simultaneous blastema formation, wound closure and finally blastemal differentiation to restore the amputated structures. While each regenerating digit faithfully progresses through all phases of regeneration, phase progression has traditionally been delineated by time, that is, days postamputation (DPA), yet there is widespread variability in the timing of the individual phases. To diminish variability between digits during tissue histolysis and blastema formation, we have established an in-vivo method using microcomputed tomography (micro CT) scanning to identify five distinct stages of the early regeneration response based on anatomical changes of the digit stump. We report that categorising the initial phases of digit regeneration by stage rather than time greatly diminishes the variability between digits with respect to changes in bone volume and length. Also, stages correlate with the levels of cell proliferation, osteoclast recruitment and osteoprogenitor cell recruitment. Importantly, micro CT staging provides a means to estimate open versus closed digit wounds. We demonstrate two spatially distinct and stage specific bone repair/regeneration responses that occur during P3 regeneration. Collectively, these studies showcase the utility of micro CT imaging to infer the composition of radiolucent soft tissues during P3 blastema formation. Specifically, the staging system identifies the onset of cell proliferation, osteoclastogenesis, osteoprogenitor recruitment, the spatial initiation of de novo bone formation and epidermal closure., (© 2022 The Wound Healing Society.)

C-type natriuretic peptide (CNP) activation of guanylyl cyclase-B (GC-B) catalyzes the synthesis of cGMP in chondrocytes and osteoblasts. Elevated cGMP stimulates long bone growth, and inactivating mutations in CNP or GC-B reduce cGMP, which causes dwarfism. GC-B 7E/7E mice that express a GC-B mutant that cannot be inactivated by dephosphorylation exhibit increased CNP-dependent GC-B activity, which increases bone length, as well as bone mass and strength. Importantly, how GC-B increases bone mass is not known. Here, we injected 12-week-old, wild type mice once daily for 28 days with or without BMN-111 (Vosoritide), a proteolytically resistant CNP analog. We found that BMN-111 treated mice had elevated levels of osteocalcin and collagen 1 C-terminal telopeptide (CTX) as well as increased osteoblasts and osteoclasts. In BMN-111 injected mice, tibial mRNAs for Rank ligand and osteoprotegrin were increased and decreased, respectively, whereas sclerostin mRNA was elevated 400-fold, consistent with increased osteoclast activity and decreased osteoblast activity. Mineral apposition rates and trabecular bone mass were not elevated in response to BMN-111. Because 9-week-old male GC-B 7E/7E mice have increased bone mass but do not exhibit increased mineral apposition rates, we examined 4-week-old male GC-B 7E/7E mice and found that these animals had increased serum osteocalcin, but not CTX. Importantly, tibias from these mice had 37% more osteoblasts, 26% fewer osteoclasts as well as 36% and 40% higher mineral apposition and bone formation rates, respectively. We conclude that GC-B-dependent bone formation is coupled to an early juvenile process that requires both increased osteoblasts and decreased osteoclasts., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Hypophosphatasia (HPP) is the inherited error-of-metabolism caused by mutations in ALPL, reducing the function of tissue-nonspecific alkaline phosphatase (TNAP/TNALP/TNSALP). HPP is characterized by defective skeletal and dental mineralization and is categorized into several clinical subtypes based on age of onset and severity of manifestations, though premature tooth loss from acellular cementum defects is common across most HPP subtypes. Genotype-phenotype associations and mechanisms underlying musculoskeletal, dental, and other defects remain poorly characterized. Murine models that have provided significant insights into HPP pathophysiology also carry limitations including monophyodont dentition, lack of osteonal remodeling of cortical bone, and differing patterns of skeletal growth. To address this, we generated the first gene-edited large-animal model of HPP in sheep via CRISPR/Cas9-mediated knock-in of a missense mutation (c.1077C>G; p.I359M) associated with skeletal and dental manifestations in humans. We hypothesized that this HPP sheep model would recapitulate the human dentoalveolar manifestations of HPP. Compared to wild-type (WT), compound heterozygous (cHet) sheep with one null allele and the other with the targeted mutant allele exhibited the most severe alveolar bone, acellular cementum, and dentin hypomineralization defects. Sheep homozygous for the mutant allele (Hom) showed alveolar bone and hypomineralization effects and trends in dentin and cementum, whereas sheep heterozygous (Het) for the mutation did not exhibit significant effects. Important insights gained include existence of early alveolar bone defects that may contribute to tooth loss in HPP, observation of severe mantle dentin hypomineralization in an HPP animal model, association of cementum hypoplasia with genotype, and correlation of dentoalveolar defects with alkaline phosphatase (ALP) levels. The sheep model of HPP faithfully recapitulated dentoalveolar defects reported in individuals with HPP, providing a new translational model for studies into etiopathology and novel therapies of this disorder, as well as proof-of-principle that genetically engineered large sheep models can replicate human dentoalveolar disorders. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR)., (© 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).)

Individuals with Down syndrome (DS), the result of trisomy of human chromosome Hsa21 (Ts21), present with an array of skeletal abnormalities typified by altered craniofacial features, short stature and low bone mineral density (BMD). While bone deficits progress with age in both sexes, low bone mass is more pronounced in DS men than women and osteopenia appears earlier. In the current study, the reproductive hormone status (FSH, LH, testosterone) of 17 DS patients (males, ages range 19-52 years) was measured. Although testosterone was consistently low, the hypothalamic-pituitary-gonadal axis was intact with corresponding rises in FSH and LH. To provide further insight into the heterogeneity of the bone mass in DS, the skeletal phenotypes of three of the most used murine DS models, Ts65Dn (Ts65), TC1, and Dp16(Yey1) (Dp16) were characterized and contrasted. Evaluation of the bone phenotype of both male and female 3-month-old Dp16 mice demonstrated sexual dimorphism, with low bone mass apparent in males, as it is in Ts65, but not in female Dp16. In contrast, male TC1 mice had no apparent bone phenotype. To determine whether low bone mass in DS impacted fracture healing, fractures of the middle phalanx (P2) digits were generated in both male and female Dp16 mice at 15 weeks of age, an age where the sexually dimorphic low BMD persisted. Fracture healing was assessed via in vivo microCT over (13 weeks) 93 days post fracture (DPF). At 93 DPF, 0 % of DS male (n = 12) or female (n = 8) fractures healed, compared to 50 % of the male (n = 28) or female (n = 8) WT littermate fractures. MicroCT revealed periosteal unbridged mineralized callus formation across the fracture gap in Dp16 mice, which was confirmed by subsequent histology. These studies provide the first direct evidence of significantly impaired fracture healing in the setting of DS., (Copyright © 2022 Elsevier Inc. All rights reserved.)

This study aimed to determine whether the acceleration of conceptus development induced by the administration of exogenous progesterone (P4) during the preimplantation period of pregnancy alters calcium, phosphate, and vitamin D signaling at the maternal-conceptus interface. Suffolk ewes (n = 48) were mated to fertile rams and received daily intramuscular injections of either corn oil (CO) vehicle or 25 mg of progesterone in CO (P4) for the first 8 days of pregnancy and hysterectomized on either Day 9 (CO, n = 5; P4, n = 6), 12 (CO, n = 9; P4, n = 4) or 125 (CO, n = 14; P4, n = 10) of gestation. The expression of S100A12 (P < 0.05) and fibroblast growth factor receptor (FGFR2) (P < 0.01) messenger RNAs (mRNAs) was lower in endometria from P4-treated ewes on Day 12. The expression of ADAM10 (P < 0.05) mRNA was greater in endometria from P4-treated ewes on Day 125. The expression of ADAM10 (P < 0.01), FGFR2 (P < 0.05), solute carrier (SLC)20A1 (P < 0.05), TRPV5 (P < 0.05), and TRPV6 (P < 0.01) mRNAs was greater, but KL mRNA expression was lower (P < 0.05) in placentomes from P4-treated ewes at Day 125. There was lower endometrial and greater placentomal expression of mRNAs involved in mineral metabolism and transport in twin compared to singleton pregnancies. Further, the expression of mRNAs involved in mineral metabolism and transport was greater in P4-treated twin placentomes. KL, FGF23, vitamin D receptor (VDR), S100A9, S100A12, S100G, and CYP27B1 proteins were immunolocalized in endometria and placentomes. Exogenous P4 in early pregnancy altered the expression of regulators of calcium, phosphate, and vitamin D on Day 125 of pregnancy indicating a novel effect of P4 on mineral transport at the maternal-conceptus interface., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

It is long-established that innervation-dependent production of neurotrophic factors is required for blastema formation and epimorphic regeneration of appendages in fish and amphibians. The regenerating mouse digit tip and the human fingertip are mammalian models for epimorphic regeneration, and limb denervation in mice inhibits this response. A complicating issue of limb denervation studies in terrestrial vertebrates is that the experimental models also cause severe paralysis therefore impairing appendage use and diminishing mechanical loading of the denervated tissues. Thus, it is unclear whether the limb denervation impairs regeneration via loss of neurotrophic signaling or loss of mechanical load, or both. Herein, we developed a novel surgical procedure in which individual digits were specifically denervated without impairing ambulation and mechanical loading. We demonstrate that digit specific denervation does not inhibit but attenuates digit tip regeneration, in part due to a delay in wound healing. However, treating denervated digits with a wound dressing that enhances closure results in a partial rescue of the regeneration response. Contrary to the current understanding of mammalian epimorphic regeneration, these studies demonstrate that mouse digit tip regeneration is not peripheral nerve dependent, an observation that should inform continued mammalian regenerative medicine approaches., Competing Interests: Declaration of competing interest The authors have no conflicts of interest or competing interests to declare., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Given recent reports of expression of postnatal mineral transport regulators at the maternal-conceptus interface during the peri-implantation period, this study tested the hypothesis that progesterone (P4) and interferon tau (IFNT) regulate phosphate, calcium, and vitamin D signaling in the ovine endometrium. Mature Rambouillet ewes (n = 24) were surgically fitted with intrauterine catheters on day 7 of the estrous cycle. Ewes received daily intramuscular injections of 50 mg of P4 in corn oil vehicle and 75 mg of progesterone receptor antagonist (RU486) in corn oil from days 8 to 15, and twice-daily intrauterine injections of either control proteins (CX) or IFNT (25 μg/uterine horn/day) from days 11 to 15 resulting in four treatment groups: P4 + CX; P4 + IFNT; RU486 + P4 + CX; and RU486 + P4 + IFNT. On day 16, ewes were hysterectomized. RU486 + P4 + CX treated ewes had lower concentrations of 25 (OH) D in plasma than P4 + CX treated ewes (P < 0.05). Endometria from ewes treated with IFNT had greater expression of FGF23 (P < 0.01), S100A9 (P < 0.05), and S100A12 (P = 0.05) mRNAs and lower expression of ADAM10 mRNA (P < 0.01) than of ewes treated with CX proteins. Expression of FGF23 mRNA was greater in endometria of ewes that received RU486 + P4 + IFNT than in ewes that received RU486 + P4 + CX (hormone × protein interaction, P < 0.05). The expression of S100G mRNA was greater in endometria of ewes that received P4 + IFNT compared to ewes that received RU486 + P4 + IFNT (P < 0.05; hormone × protein interaction, P < 0.01). These data implicate P4 and IFNT in the regulation of phosphate, calcium, and vitamin D signaling during the peri-implantation period of pregnancy and provide a platform for continued mechanistic investigations., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Structural regeneration of amputated appendages by blastema-mediated, epimorphic regeneration is a process whose mechanisms are beginning to be employed for inducing regeneration. While epimorphic regeneration is classically studied in non-amniote vertebrates such as salamanders, mammals also possess a limited ability for epimorphic regeneration, best exemplified by the regeneration of the distal mouse digit tip. A fundamental, but still unresolved question is whether epimorphic regeneration and blastema formation is exhaustible, similar to the finite limits of stem-cell mediated tissue regeneration., Methods: In this study, distal mouse digits were amputated, allowed to regenerate and then repeatedly amputated. To quantify the extent and patterning of the regenerated digit, the digit bone as the most prominent regenerating element in the mouse digit was followed by in vivo µCT., Results: Analyses revealed that digit regeneration is indeed progressively attenuated, beginning after the second regeneration cycle, but that the pattern is faithfully restored until the end of the fourth regeneration cycle. Surprisingly, when unamputated digits in the vicinity of repeatedly amputated digits were themselves amputated, these new amputations also exhibited a similarly attenuated regeneration response, suggesting a systemic component to the amputation injury response., Conclusions: In sum, these data suggest that epimorphic regeneration in mammals is finite and due to the exhaustion of the proliferation and differentiation capacity of the blastema cell source., (© 2022. The Author(s).)

Amputation of the mouse digit tip results in blastema-mediated regeneration. In this model, new bone regenerates de novo to lengthen the amputated stump bone, resulting in a functional replacement of the terminal phalangeal element along with associated non-skeletal tissues. Physiological examples of bone repair, such as distraction osteogenesis and fracture repair, are well known to require mechanical loading. However, the role of mechanical loading during mammalian digit tip regeneration is unknown. In this study, we demonstrate that reducing mechanical loading inhibits blastema formation by attenuating bone resorption and wound closure, resulting in the complete inhibition of digit regeneration. Mechanical unloading effects on wound healing and regeneration are completely reversible when mechanical loading is restored. Mechanical unloading after blastema formation results in a reduced rate of de novo bone formation, demonstrating mechanical load dependence of the bone regenerative response. Moreover, enhancing the wound-healing response of mechanically unloaded digits with the cyanoacrylate tissue adhesive Dermabond improves wound closure and partially rescues digit tip regeneration. Taken together, these results demonstrate that mammalian digit tip regeneration is mechanical load-dependent. Given that human fingertip regeneration shares many characteristics with the mouse digit tip, these results identify mechanical load as a previously unappreciated requirement for de novo bone regeneration in humans. © 2021 American Society for Bone and Mineral Research (ASBMR)., (© 2021 American Society for Bone and Mineral Research (ASBMR).)

After spinal cord injury (SCI), 80% of individuals are diagnosed with osteopenia or osteoporosis. The dramatic loss of bone after SCI increases the potential for fractures 100-fold, with post-fracture complications occurring in 54% of cases. With the age of new SCI injuries increasing, we hypothesized that a SCI-induced reduction in weight bearing could further exacerbate age-induced bone loss. To test this, young (2-3 months) and old (20-30 months) male and female mice were given a moderate spinal contusion injury (T9-T10), and recovery was assessed for 28 days (BMS, rearing counts, distance traveled). Tibial trabecular bone volume was measured after 28 days with ex vivo microCT. While BMS scores did not differ across groups, older subjects travelled less in the open field and there was a decrease in rearing with age and SCI. As expected, aging decreased trabecular bone volume and cortical thickness in both old male and female mice. SCI alone also reduced trabecular bone volume in young mice, but did not have an additional effect beyond the age-dependent decrease in trabecular and cortical bone volume seen in both sexes. Interestingly, both rearing and total activity correlated with decreased bone volume. These data underscore the importance of load and use on bone mass. While partial weight-bearing does not stabilize/reverse bone loss in humans, our data suggest that therapies that simulate complete loading may be effective after SCI.

Amputation injuries in mammals are typically non-regenerative; however, joint regeneration is stimulated by BMP9 treatment, indicating the presence of latent articular chondrocyte progenitor cells. BMP9 induces a battery of chondrogenic genes in vivo, and a similar response is observed in cultures of amputation wound cells. Extended cultures of BMP9-treated cells results in differentiation of hyaline cartilage, and single cell RNAseq analysis identified wound fibroblasts as BMP9 responsive. This culture model was used to identify a BMP9-responsive adult fibroblast cell line and a culture strategy was developed to engineer hyaline cartilage for engraftment into an acutely damaged joint. Transplanted hyaline cartilage survived engraftment and maintained a hyaline cartilage phenotype, but did not form mature articular cartilage. In addition, individual hypertrophic chondrocytes were identified in some samples, indicating that the acute joint injury site can promote osteogenic progression of engrafted hyaline cartilage. The findings identify fibroblasts as a cell source for engineering articular cartilage and establish a novel experimental strategy that bridges the gap between regeneration biology and regenerative medicine., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Normal calcium and bone homeostasis in the adult is virtually fully explained by the interactions of several key regulatory hormones, including parathyroid hormone, 1,25 dihydroxy vitamin D3, fibroblast growth factor-23, calcitonin, and sex steroids (estradiol and testosterone). In utero, bone and mineral metabolism is regulated differently from the adult. During development, it is the placenta and not the fetal kidneys, intestines, or skeleton that is the primary source of minerals for the fetus. The placenta is able to meet the almost inexhaustible needs of the fetus for minerals by actively driving the transport of calcium and phosphorus from the maternal circulation to the growing fetus. These fundamentally important minerals are maintained in the fetal circulation at higher concentrations than those in maternal blood. Maintenance of these inordinately higher fetal levels is necessary for the developing skeleton to accrue sufficient minerals by term. Importantly, in livestock species, prenatal mineralization of the skeleton is crucial for the high levels of offspring activity soon after birth. Calcium is required for mineralization, as well as a plethora of other physiological functions. Placental calcium and phosphate transport are regulated by several mechanisms that are discussed in this review. It is clear that phosphate and calcium metabolism is intimately interrelated and, therefore, placental transport of these minerals cannot be considered in isolation., (© 2022. Springer Nature Switzerland AG.)

Mineralization of the fetal mammalian skeleton requires a hypercalcemic gradient across the placenta from mother to fetus. However, the mechanisms responsible for maintaining the placental transport of calcium remain poorly understood. This study aimed to identify calcium and vitamin D regulatory pathway components in ovine endometria and placentae across gestation. Suffolk ewes were bred with fertile rams upon detection of estrus (Day 0). On Days 9, 12, 17, 30, 70, 90, 110, and 125 of pregnancy (n=3-14/Day), ewes were euthanized and hysterectomized. Calcium abundance was influenced by gestational day in uterine flushings and allantoic fluid (P<0.05). The expression of S100G, S100A9, S100A12, ATP2B3, ATP2B4, TRPV5, TRPV6, CYP11A1, CYP2R1, CYP24, and VDR mRNAs known to be involved in calcium binding, calcium transport, and vitamin D metabolism were quantified by qPCR. Mediators of calcium and vitamin D signaling were expressed by Day 17 conceptus tissue, and endometria and placentae across gestation. Gestational day influenced the expression of S100G, S100A9, S100A12, TRPV6, VDR, and CYP24 mRNAs in endometria and placentae (P<0.05). Gestational day influenced endometrial expression of ATP2B3, and placental expression of TRPV5, ATP2B4, and CYP11A1 (P<0.05). VDR protein localized to the endoderm and trophectoderm (Day 17 conceptus) and was expressed in endometria and placentae throughout gestation. The observed spatiotemporal profile suggests a potential role of calcium and vitamin D in the establishment of pregnancy and regulation of fetal and placental growth, providing a platform for further mechanistic investigation., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Appropriate mineralization of the fetal skeleton requires an excess of phosphate in the fetus compared to the mother. However, mechanisms for placental phosphate transport are poorly understood. This study aimed to identify phosphate regulatory pathways in ovine endometria and placentae throughout gestation. Suffolk ewes were bred with fertile rams upon visual detection of estrus (Day 0). On Days 9, 12, 17, 30, 70, 90, 110, and 125 of pregnancy (n = 3-14/Day), ewes were euthanized and hysterectomized. Phosphate abundance varied across gestational days in uterine flushings, allantoic fluid, and homogenized endometria and placentae (P < 0.05). The expression of mRNAs for sodium-dependent phosphate transporters (SLC20A1 and SLC20A2) and klotho signaling mediators (FGF7, FGF21, FGF23, FGFR1-4, KL, KLB, ADAM10, and ADAM17) were quantified by qPCR. Day 17 conceptus tissue expressed SLC20A1, SLC20A2, KLB, FGF7, FGF21, FGF23, FGFR1, and FGFR2 mRNAs. Both sodium-dependent phosphate transporters and klotho signaling mediators were expressed in endometria and placentae throughout gestation. Gestational day influenced the expression of SLC20A1, ADAM10, ADAM17, FGF21, FGFR1, and FGFR3 mRNAs in both endometria and placentae (P < 0.05). Gestational day influenced endometrial expression of FGF7 (P < 0.001), and placental expression of FGF23 (P < 0.05). Immunohistochemistry confirmed that both FGF23 and KL proteins were expressed in endometria and placentae throughout gestation. The observed spatiotemporal profile of KL-FGF signaling suggests a potential role in the establishment of pregnancy and regulation of fetal growth. This study provides a platform for further mechanistic investigation into the role for KL-FGF signaling in the regulation of phosphate transport at the ovine maternal-conceptus interface., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Bisphosphonates (BPs) are characterized by their ability to bind strongly to bone mineral and inhibit bone resorption. However, BPs exert a wide range of pharmacological activities beyond the inhibition of bone resorption, including the inhibition of cancer cell metastases and angiogenesis and the inhibition of proliferation and apoptosis in vitro. Additionally, the inhibition of matrix metalloproteinase activity, altered cytokine and growth factor expression, as well as reductions in parameters of pain have also been reported. In humans, clinical BP use has transformed the treatment of post-menopausal osteoporosis, rare bone diseases such as osteogenesis imperfecta, as well as multiple myeloma and metastatic breast and prostate cancer, albeit not without infrequent but significant adverse events. Despite the well-characterized health benefits of BP use in humans, the evidence-base for the therapeutic efficacy of BPs in veterinary medicine is, by comparison, limited. Notwithstanding, BPs are used widely in small animal veterinary practice for the medical management of hyperparathyroidism, idiopathic hypercalcemia in cats, as well as for the palliative care of bone tumors which are common in dogs, and in particular, primary bone tumors such as osteosarcoma. Palliative BP treatment has also recently increased in veterinary oncology to alleviate tumor-associated bone pain. In equine veterinary practice, non-nitrogen-containing BPs are FDA-approved to control clinical signs associated with navicular syndrome in adult horses. However, there are growing concerns regarding the off-label use of BPs in juvenile horses. Here we discuss the current understanding of the strengths, weaknesses and current controversies surrounding BP use in veterinary medicine to highlight the future utility of these potentially beneficial drugs., (Copyright © 2020. Published by Elsevier Inc.)

C-type natriuretic peptide (CNP) activation of guanylyl cyclase (GC)-B, also known as NPR2, stimulates cGMP synthesis and bone elongation. CNP activation requires the phosphorylation of multiple GC-B residues and dephosphorylation inactivates the receptor. GC-B 7E/7E knockin mice, expressing a glutamate-substituted, "pseudophosphorylated," form of GC-B, exhibit increased CNP-dependent GC activity. Since mutations that constitutively activate GC-B in the absence of CNP result in low bone mineral density in humans, we determined the skeletal phenotype of 9-week old male GC-B 7E/7E mice. Unexpectedly, GC-B 7E/7E mice have significantly greater tibial and L5 vertebral trabecular bone volume fraction, tibial trabecular number, and tibial bone mineral density. Cortical cross-sectional area, cortical thickness, periosteal diameter and cortical cross-sectional moment of inertia were also significantly increased in GC-B 7E/7E tibiae. Three-point bending measurements demonstrated that the mutant tibias and femurs had greater ultimate load, stiffness, energy to ultimate load, and energy to failure. No differences in microhardness indicated similar bone quality at the tissue level between the mutant and wildtype bones. Procollagen 1 N-terminal propeptide and osteocalcin were elevated in serum, and osteoblast number per bone perimeter and osteoid width per bone perimeter were elevated in tibias from the mutant mice. In contrast to mutations that constitutively activate GC-B, we report that mutations that enhance GC-B activity only in the presence of its natural ligand, increase bone mass, bone strength, and the number of active osteoblasts at the bone surface., Competing Interests: Declaration of competing interest The authors have declared that no conflict of interest exists., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Osteoarticular disease is a frequent complication of human brucellosis. Vaccination remains a critical component of brucellosis control, but there are currently no vaccines for use in humans, and no in vitro models for assessing the safety of candidate vaccines in reference to the development of bone lesions currently exist. While the effect of Brucella infection on osteoblasts has been extensively evaluated, little is known about the consequences of osteoclast infection. Murine bone marrow-derived macrophages were derived into mature osteoclasts and infected with B. abortus 2308, the vaccine strain S19, and attenuated mutants S19 vjbR and B. abortus ΔvirB2 While B. abortus 2308 and S19 replicated inside mature osteoclasts, the attenuated mutants were progressively killed, behavior that mimics infection kinetics in macrophages. Interestingly, B. abortus 2308 impaired the growth of osteoclasts without reducing resorptive activity, while osteoclasts infected with B. abortus S19 and S19 vjbR were significantly larger and exhibited enhanced resorption. None of the Brucella strains induced apoptosis or stimulated nitric oxide or lactose dehydrogenase production in mature osteoclasts. Finally, infection of macrophages or osteoclast precursors with B. abortus 2308 resulted in generation of smaller osteoclasts with decreased resorptive activity. Overall, Brucella exhibits similar growth characteristics in mature osteoclasts compared to the primary target cell, the macrophage, but is able to impair the maturation and alter the resorptive capacity of these cells. These results suggest that osteoclasts play an important role in osteoarticular brucellosis and could serve as a useful in vitro model for both analyzing host-pathogen interactions and assessing vaccine safety., (Copyright © 2020 American Society for Microbiology.)

The availability of tools to accurately replicate the clinical phenotype of rare human diseases is a key step toward improved understanding of disease progression and the development of more effective therapeutics. We successfully generated the first large animal model of a rare human bone disease, hypophosphatasia (HPP) using CRISPR/Cas9 to introduce a single point mutation in the tissue nonspecific alkaline phosphatase (TNSALP) gene (ALPL) (1077 C > G) in sheep. HPP is a rare inherited disorder of mineral metabolism that affects bone and tooth development, and is associated with muscle weakness. Compared to wild-type (WT) controls, HPP sheep have reduced serum alkaline phosphatase activity, decreased tail vertebral bone size, and metaphyseal flaring, consistent with the mineralization deficits observed in human HPP patients. Computed tomography revealed short roots and thin dentin in incisors, and reduced mandibular bone in HPP vs. WT sheep, accurately replicating odonto-HPP. Skeletal muscle biopsies revealed aberrant fiber size and disorganized mitochondrial cristae structure in HPP vs. WT sheep. These genetically engineered sheep accurately phenocopy human HPP and provide a novel large animal platform for the longitudinal study of HPP progression, as well as other rare human bone diseases.

Deposition of liposomal drugs into solid tumors is a potentially rate-limiting step for drug delivery and has substantial variability that may influence probability of response. Tumor deposition is a shared mechanism for liposomal therapeutics such that a single companion diagnostic agent may have utility in predicting response to multiple nanomedicines. Methods: We describe the development, characterization and preclinical proof-of-concept of the positron emission tomography (PET) agent, MM-DX-929, a drug-free untargeted 100 nm PEGylated liposome stably entrapping a chelated complex of 4-DEAP-ATSC and 64 Cu (copper-64). MM-DX-929 is designed to mimic the biodistribution of similarly sized therapeutic agents and enable quantification of deposition in solid tumors. Results: MM-DX-929 demonstrated sufficient in vitro and in vivo stability with PET images accurately reflecting the disposition of liposome nanoparticles over the time scale of imaging. MM-DX-929 is also representative of the tumor deposition and intratumoral distribution of three different liposomal drugs, including targeted liposomes and those with different degrees of PEGylation. Furthermore, stratification using a single pre-treatment MM-DX-929 PET assessment of tumor deposition demonstrated that tumors with high MM-DX-929 deposition predicted significantly greater anti-tumor activity after multi-cycle treatments with different liposomal drugs. In contrast, MM-DX-929 tumor deposition was not prognostic in untreated tumor-bearing xenografts, nor predictive in animals treated with small molecule chemotherapeutics. Conclusions: These data illustrate the potential of MM-DX-929 PET as a companion diagnostic strategy to prospectively select patients likely to respond to liposomal drugs or nanomedicines of similar molecular size., Competing Interests: Competing Interests: Merrimack employees receive salaries and stock options from Merrimack Pharmaceuticals. This study was solely funded by Merrimack Pharmaceuticals. Post-doc salaries were in-part funded through the MITACS Accelerate and Elevate Postdoctoral Fellowship Programs, with financial contribution from Merrimack Pharmaceuticals and the Canadian Federal and the Ontario Provincial governments. Authors affiliated with Merrimack Pharmaceuticals as indicated were employees of Merrimack Pharmaceuticals at the time of study. Jinzi Zheng is the Lead Investigator of an industry-sponsored research agreement funded by Merrimack Pharmaceuticals.

The Leprecan protein family which includes the prolyl 3-hydroxylase enzymes (P3H1, P3H2, and P3H3), the closely related cartilage-associated protein (CRTAP), and SC65 (Synaptonemal complex 65, aka P3H4, LEPREL4), is involved in the post-translational modification of fibrillar collagens. Mutations in CRTAP , P3H1 and P3H2 cause human genetic diseases. We recently showed that SC65 forms a stable complex in the endoplasmic reticulum with P3H3 and lysyl hydroxylase 1 and that loss of this complex leads to defective collagen lysyl hydroxylation and causes low bone mass and skin fragility. Interestingly, SC65 was initially described as a synaptonemal complex-associated protein, suggesting a potential additional role in germline cells. In the present study, we describe the expression of SC65, CRTAP and other Leprecan proteins in postnatal mouse reproductive organs. We detect SC65 expression in peritubular cells of testis up to 4 weeks of age but not in cells within seminiferous tubules, while its expression is maintained in ovarian follicles until adulthood. Similar to bone and skin, SC65 and P3H3 are also tightly co-expressed in testis and ovary. Moreover, we show that CRTAP, a protein normally involved in collagen prolyl 3-hydroxylation, is highly expressed in follicles and stroma of the ovary and in testes interstitial cells at 4 weeks of age, germline cells and mature sperm. Importantly, CrtapKO mice have a mild but significant increase in morphologically abnormal mature sperm (17% increase compared to WT). These data suggest a role for the Leprecans in the post-translational modification of collagens expressed in the stroma of the reproductive organs. While we could not confirm that SC65 is part of the synaptonemal complex, the expression of CRTAP in the seminiferous tubules and in mature sperm suggest a role in the testis germ cell lineage and sperm morphogenesis., Competing Interests: Conflict of interest All authors declare no conflicts of interest in this paper.

Down syndrome (DS), characterized by trisomy of human chromosome 21, is associated with a variety of endocrine disorders as well as profound skeletal abnormalities. The low bone mass phenotype in DS is defined by low bone turnover due to decreased osteoclast and osteoblast activity, decreasing the utility of antiresorptive agents in people with DS. Sclerostin antibody (SclAb) is a therapeutic candidate currently being evaluated as a bone anabolic agent. Scl, the product of the sclerostin gene ( SOST ), inhibits bone formation through its inhibition of Wnt signaling. SclAb increases bone mass by suppressing the action of the endogenous inhibitor of bone formation, Scl. To examine the effects of SclAb on the DS bone phenotype, 8-week-old male wild-type (WT) andTs65Dn DS mice were treated with 4 weekly iv injections of 100 mg/kg SclAb. Dual-energy X-ray absorptiometry (DXA), microCT, and dynamic histomorphometry analyses revealed that SclAb had a significant anabolic effect on both age-matched WT littermate controls and Ts65Dn DS mice that was osteoblast mediated, without significant changes in osteoclast parameters. SclAb treatment significantly increased both cortical and trabecular bone mass at multiple sites; SclAb treatment resulted in the normalization of Ts65Dn bone mineral density (BMD) to WT levels in the proximal tibia, distal femur, and whole body. Ex vivo bone marrow cultures demonstrated that SclAb increased the recruitment of the mesenchymal progenitors into the osteoblast lineage, as indicated by increased alkaline phosphatase-positive colonies, with no effect on osteoclast differentiation. Together, in the setting of a murine model of DS and decreased bone turnover, SclAb had a potent anabolic effect. SclAb stimulated bone formation and increased osteoblastogenesis without affecting osteoclastogenesis or bone resorption. These data suggest that SclAb is a promising new therapy to improve bone mass and reduce fracture risk in the face of the low bone mass and turnover prevalent in the DS population., (© 2017 The Authors published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.)