Notch signaling enhances bone regeneration in the zebrafish mandible.

Loss or damage to the mandible caused by trauma, treatment of oral malignancies, and other diseases is treated using bone-grafting techniques that suffer from numerous shortcomings and contraindications. Zebrafish naturally heal large injuries to mandibular bone, offering an opportunity to understand how to boost intrinsic healing potential. Using a novel her6:mCherry Notch reporter, we show that canonical Notch signaling is induced during the initial stages of cartilage callus formation in both mesenchymal cells and chondrocytes following surgical mandibulectomy. We also show that modulation of Notch signaling during the initial post-operative period results in lasting changes to regenerate bone quantity one month later. Pharmacological inhibition of Notch signaling reduces the size of the cartilage callus and delays its conversion into bone, resulting in non-union. Conversely, conditional transgenic activation of Notch signaling accelerates conversion of the cartilage callus into bone, improving bone healing. Given the conserved functions of this pathway in bone repair across vertebrates, we propose that targeted activation of Notch signaling during the early phases of bone healing in mammals may both augment the size of the initial callus and boost its ossification into reparative bone.

Vertebral pattern and morphology is determined during embryonic segmentation.

BACKGROUND: The segmented nature of the adult vertebral column is based on segmentation of the paraxial mesoderm during early embryogenesis. Disruptions to embryonic segmentation, whether caused by genetic lesions or environmental stress, result in adult vertebral pathologies. However, the mechanisms linking embryonic segmentation and the details of adult vertebral morphology are poorly understood. RESULTS: We induced border defects using two approaches in zebrafish: heat stress and misregulation of embryonic segmentation genes tbx6, mesp-ba, and ripply1. We assayed vertebral length, regularity, and polarity using microscopic and radiological imaging. In population studies, we find a correlation between specific embryonic border defects and specific vertebral defects, and within individual fish, we trace specific adult vertebral defects to specific embryonic border defects. CONCLUSIONS: Our data reveal that transient disruptions of embryonic segment border formation led to significant vertebral anomalies that persist through adulthood. The spacing of embryonic borders controls the length of the vertebra. The positions of embryonic borders control the positions of ribs and arches. Embryonic borders underlie fusions and divisions between adjacent spines and ribs. These data suggest that segment borders have a dominant role in vertebral development.

Local versus systemic control of bone and skeletal muscle mass by components of the transforming growth factor-ß signaling pathway.

Skeletal muscle and bone homeostasis are regulated by members of the myostatin/GDF-11/activin branch of the transforming growth factor-ß superfamily, which share many regulatory components, including inhibitory extracellular binding proteins and receptors that mediate signaling. Here, we present the results of genetic studies demonstrating a critical role for the binding protein follistatin (FST) in regulating both skeletal muscle and bone. Using an allelic series corresponding to varying expression levels of endogenous Fst, we show that FST acts in an exquisitely dose-dependent manner to regulate both muscle mass and bone density. Moreover, by employing a genetic strategy to target Fst expression only in the posterior (caudal) region of the animal, we show that the effects of Fst loss are mostly restricted to the posterior region, implying that locally produced FST plays a much more important role than circulating FST with respect to regulation of muscle and bone. Finally, we show that targeting receptors for these ligands specifically in osteoblasts leads to dramatic increases in bone mass, with trabecular bone volume fraction being increased by 12- to 13-fold and bone mineral density being increased by 8- to 9-fold in humeri, femurs, and lumbar vertebrae. These findings demonstrate that bone, like muscle, has an enormous inherent capacity for growth that is normally kept in check by this signaling system and suggest that the extent to which this regulatory mechanism may be used throughout the body to regulate tissue mass may be more significant than previously appreciated.

Functional redundancy of type I and type II receptors in the regulation of skeletal muscle growth by myostatin and activin A.

Myostatin (MSTN) is a transforming growth factor-ß (TGF-ß) family member that normally acts to limit muscle growth. The function of MSTN is partially redundant with that of another TGF-ß family member, activin A. MSTN and activin A are capable of signaling through a complex of type II and type I receptors. Here, we investigated the roles of two type II receptors (ACVR2 and ACVR2B) and two type I receptors (ALK4 and ALK5) in the regulation of muscle mass by these ligands by genetically targeting these receptors either alone or in combination specifically in myofibers in mice. We show that targeting signaling in myofibers is sufficient to cause significant increases in muscle mass, showing that myofibers are the direct target for signaling by these ligands in the regulation of muscle growth. Moreover, we show that there is functional redundancy between the two type II receptors as well as between the two type I receptors and that all four type II/type I receptor combinations are utilized in vivo. Targeting signaling specifically in myofibers also led to reductions in overall body fat content and improved glucose metabolism in mice fed either regular chow or a high-fat diet, demonstrating that these metabolic effects are the result of enhanced muscling. We observed no effect, however, on either bone density or muscle regeneration in mice in which signaling was targeted in myofibers. The latter finding implies that MSTN likely signals to other cells, such as satellite cells, in addition to myofibers to regulate muscle homeostasis.

Targeting myostatin/activin A protects against skeletal muscle and bone loss during spaceflight.

Among the physiological consequences of extended spaceflight are loss of skeletal muscle and bone mass. One signaling pathway that plays an important role in maintaining muscle and bone homeostasis is that regulated by the secreted signaling proteins, myostatin (MSTN) and activin A. Here, we used both genetic and pharmacological approaches to investigate the effect of targeting MSTN/activin A signaling in mice that were sent to the International Space Station. Wild type mice lost significant muscle and bone mass during the 33 d spent in microgravity. Muscle weights of Mstn-/- mice, which are about twice those of wild type mice, were largely maintained during spaceflight. Systemic inhibition of MSTN/activin A signaling using a soluble form of the activin type IIB receptor (ACVR2B), which can bind each of these ligands, led to dramatic increases in both muscle and bone mass, with effects being comparable in ground and flight mice. Exposure to microgravity and treatment with the soluble receptor each led to alterations in numerous signaling pathways, which were reflected in changes in levels of key signaling components in the blood as well as their RNA expression levels in muscle and bone. These findings have implications for therapeutic strategies to combat the concomitant muscle and bone loss occurring in people afflicted with disuse atrophy on Earth as well as in astronauts in space, especially during prolonged missions.

Contextual Regulation of Skeletal Physiology by Notch Signaling.

PURPOSE OF REVIEW: This article reviews the past 2 years of research on Notch signaling as it relates to bone physiology, with the goal of reconciling seemingly discrepant findings and identifying fruitful areas of potential future research. RECENT FINDINGS: Conditional animal models and high-throughput omics have contributed to a greater understanding of the context-dependent role of Notch signaling in bone. However, significant gaps remain in our understanding of how spatiotemporal context and epigenetic state dictate downstream Notch phenotypes. Biphasic activation of Notch signaling orchestrates progression of mesenchymal progenitor cells through the osteoblast lineage, but there is a limited understanding of ligand- and receptor-specific functions. Paracrine Notch signaling through non-osteoblastic cell types contributes additional layers of complexity, and we anticipate impactful future work related to the integration of these cell types and signaling mechanisms.

Tenogenesis of bone marrow-, adipose-, and tendon-derived stem cells in a dynamic bioreactor.

Tendons are frequently damaged and fail to regenerate, leading to pain, loss of function, and reduced quality of life. Mesenchymal stem cells (MSCs) possess clinically useful tissue-regenerative properties and have been exploited for use in tendon tissue engineering and cell therapy. However, MSCs exhibit phenotypic heterogeneity based on the donor tissue used, and the efficacy of cell-based treatment modalities may be improved by optimizing cell source based on relative differentiation capacity. Equine MSCs were isolated from bone marrow (BM), adipose (AD), and tendon (TN), expanded in monolayer prior to seeding on decellularized tendon scaffolds (DTS), and cell-laden constructs were placed in a bioreactor designed to mimic the biophysical environment of the tendon. It was hypothesized that TN MSCs would differentiate toward a tendon cell phenotype better than BM and AD MSCs in response to a conditioning period involving cyclic mechanical stimulation for 1 hour per day at 3% strain and 0.33 Hz. All cell types integrated into DTS adopted an elongated morphology similar to tenocytes, expressed tendon marker genes, and improved tissue mechanical properties after 11 days. TN MSCs expressed the greatest levels of scleraxis, collagen type-I, and cartilage oligomeric matrix protein. Major histocompatibility class-II protein mRNA expression was not detected in any of the MSC types, suggesting low immunogenicity for allogeneic transplantation. The results suggest that TN MSCs are the ideal cell type for regenerative medicine therapies for tendinopathies, exhibiting the most mature tendon-like phenotype in vitro. When TN MSCs are unavailable, BM or AD MSCs may serve as robust alternatives.

Microangiographic Comparison of the Effects of 3 Loop Pulley and 6 Strand Savage Tenorrhaphy Techniques on Equine Superficial Digital Flexor Tendon.

OBJECTIVE: The 6-strand Savage (SSS) tenorrhaphy pattern is biomechanically superior to the commonly employed 3-loop pulley (3LP); however, its effects on intrinsic tendon vasculature remain unknown. The objective of this study was to compare perfusion of intrinsic vasculature of the equine superficial digital flexor tendon (SDFT) after 3LP and SSS tenorrhaphies. We hypothesized that the SSS technique would significantly decrease vascular perfusion compared to the 3LP technique. STUDY DESIGN: Ex vivo, randomized, paired design. ANIMALS: Horses (n = 9) METHODS: Under general anesthesia, 9 pairs of forelimb SDFT were transected. Two tendons served as baseline control, the remainder had either SSS or 3LP tenorrhaphy performed. Horses were heparinized, euthanatized, and forelimbs perfused with barium sulfate solution were then fixed with formalin under tension. Tendons were transected every 5 mm and microangiographic images obtained. Microvascular analysis of sections proximal to, throughout, and distal to the tenorrhaphy was completed using a custom macro. Differences in vascular count were assessed using MANOVA. RESULTS: A significant reduction in the number of perfused vessels was seen for SSS compared with 3LP at 2 locations within the tenorrhaphy (P = .039 and P = .009). The SSS technique took on average 4.7 ± 0.9 times longer to place. CONCLUSIONS: The SSS technique causes an acute reduction in tendon perfusion compared to the 3LP, which may limit its clinical use. Further research is required to elucidate the clinical significance of this difference.

Genetically engineered zebrafish as models of skeletal development and regeneration.

Zebrafish (Danio rerio) are aquatic vertebrates with significant homology to their terrestrial counterparts. While zebrafish have a centuries-long track record in developmental and regenerative biology, their utility has grown exponentially with the onset of modern genetics. This is exemplified in studies focused on skeletal development and repair. Herein, the numerous contributions of zebrafish to our understanding of the basic science of cartilage, bone, tendon/ligament, and other skeletal tissues are described, with a particular focus on applications to development and regeneration. We summarize the genetic strengths that have made the zebrafish a powerful model to understand skeletal biology. We also highlight the large body of existing tools and techniques available to understand skeletal development and repair in the zebrafish and introduce emerging methods that will aid in novel discoveries in skeletal biology. Finally, we review the unique contributions of zebrafish to our understanding of regeneration and highlight diverse routes of repair in different contexts of injury. We conclude that zebrafish will continue to fill a niche of increasing breadth and depth in the study of basic cellular mechanisms of skeletal biology.

Wnt-associated adult stem cell marker Lgr6 is required for osteogenesis and fracture healing.

Despite the remarkable regenerative capacity of skeletal tissues, nonunion of bone and failure of fractures to heal properly presents a significant clinical concern. Stem and progenitor cells are present in bone and become activated following injury; thus, elucidating mechanisms that promote adult stem cell-mediated healing is important. Wnt-associated adult stem marker Lgr6 is implicated in the regeneration of tissues with well-defined stem cell niches in stem cell-reliant organs. Here, we demonstrate that Lgr6 is dynamically expressed in osteoprogenitors in response to fracture injury. We used an Lgr6-null mouse model and found that Lgr6 expression is necessary for maintaining bone volume and efficient postnatal bone regeneration in adult mice. Skeletal progenitors isolated from Lgr6-null mice have reduced colony-forming potential and reduced osteogenic differentiation capacity due to attenuated cWnt signaling. Lgr6-null mice consist of a lower proportion of self-renewing stem cells. In response to fracture injury, Lgr6-null mice have a deficiency in the proliferation of periosteal progenitors and reduced ALP activity. Further, analysis of the bone regeneration phase and remodeling phase of fracture healing in Lgr6-null mice showed impaired endochondral ossification and decreased mineralization. We propose that in contrast to not being required for successful skeletal development, Lgr6-positive cells have a direct role in endochondral bone repair.

Functional replacement of myostatin with GDF-11 in the germline of mice.

BACKGROUND: Myostatin (MSTN) is a transforming growth factor-ß superfamily member that acts as a major regulator of skeletal muscle mass. GDF-11, which is highly related to MSTN, plays multiple roles during embryonic development, including regulating development of the axial skeleton, kidneys, nervous system, and pancreas. As MSTN and GDF-11 share a high degree of amino acid sequence identity, behave virtually identically in cell culture assays, and utilize similar regulatory and signaling components, a critical question is whether their distinct biological functions result from inherent differences in their abilities to interact with specific regulatory and signaling components or whether their distinct biological functions mainly reflect their differing temporal and spatial patterns of expression. METHODS: We generated and characterized mice in which we precisely replaced in the germline the portion of the Mstn gene encoding the mature C-terminal peptide with the corresponding region of Gdf11. RESULTS: In mice homozygous for the knock-in allele, all of the circulating MSTN protein was replaced with GDF-11, resulting in ~ 30-40-fold increased levels of circulating GDF-11. Male mice homozygous for the knock-in allele had slightly decreased muscle weights, slightly increased weight gain in response to a high-fat diet, slightly increased plasma cholesterol and HDL levels, and significantly decreased bone density and bone mass, whereas female mice were mostly unaffected. CONCLUSIONS: GDF-11 appears to be capable of nearly completely functionally replacing MSTN in the control of muscle mass. The developmental and physiological consequences of replacing MSTN with GDF-11 are strikingly limited.

miR-433-3p suppresses bone formation and mRNAs critical for osteoblast function in mice.

MicroRNAs (miRNAs) are key posttranscriptional regulators of osteoblastic commitment and differentiation. miR-433-3p was previously shown to target Runt-related transcription factor 2 (Runx2) and to be repressed by bone morphogenetic protein (BMP) signaling. Here, we show that miR-433-3p is progressively decreased during osteoblastic differentiation of primary mouse bone marrow stromal cells in vitro, and we confirm its negative regulation of this process. Although repressors of osteoblastic differentiation often promote adipogenesis, inhibition of miR-433-3p did not affect adipocyte differentiation in vitro. Multiple pathways regulate osteogenesis. Using luciferase-3' untranslated region (UTR) reporter assays, five novel miR-433-3p targets involved in parathyroid hormone (PTH), mitogen-activated protein kinase (MAPK), Wnt, and glucocorticoid signaling pathways were validated. We show that Creb1 is a miR-433-3p target, and this transcription factor mediates key signaling downstream of PTH receptor activation. We also show that miR-433-3p targets hydroxysteroid 11-ß dehydrogenase 1 (Hsd11b1), the enzyme that locally converts inactive glucocorticoids to their active form. miR-433-3p dampens glucocorticoid signaling, and targeting of Hsd11b1 could contribute to this phenomenon. Moreover, miR-433-3p targets R-spondin 3 (Rspo3), a leucine-rich repeat-containing G-protein coupled receptor (LGR) ligand that enhances Wnt signaling. Notably, Wnt canonical signaling is also blunted by miR-433-3p activity. In vivo, expression of a miR-433-3p inhibitor or tough decoy in the osteoblastic lineage increased trabecular bone volume. Mice expressing the miR-433-3p tough decoy displayed increased bone formation without alterations in osteoblast or osteoclast numbers or surface, indicating that miR-433-3p decreases osteoblast activity. Overall, we showed that miR-433-3p is a negative regulator of bone formation in vivo, targeting key bone-anabolic pathways including those involved in PTH signaling, Wnt, and endogenous glucocorticoids. Local delivery of miR-433-3p inhibitor could present a strategy for the management of bone loss disorders and bone defect repair. © 2021 American Society for Bone and Mineral Research (ASBMR).

Silver-doped bioactive glass particles for in vivo bone tissue regeneration and enhanced methicillin-resistant Staphylococcus aureus (MRSA) inhibition.

Infection is a significant risk factor for failed healing of bone and other tissues. We have developed a sol-gel (solution-gelation) derived bioactive glass doped with silver ions (Ag-BG), tailored to provide non-cytotoxic antibacterial activity while significantly enhancing osteoblast-lineage cell growth in vitro and bone regeneration in vivo. Our objective was to engineer a biomaterial that combats bacterial infection while maintaining the capability to promote bone growth. We observed that Ag-BG inhibits bacterial growth and potentiates the efficacy of conventional antibiotic treatment. Ag-BG microparticles enhance cell proliferation and osteogenic differentiation in human bone marrow stromal cells (hBMSC) in vitro. Moreover, in vivo tests using a calvarial defect model in mice demonstrated that Ag-BG microparticles induce bone regeneration. This novel system with dual biological and advanced antibacterial properties is a promising therapeutic for combating resistant bacteria while triggering new bone formation.

Skeletal screening IMPC/KOMP using µCT and computer automated cryohistology: Application to the Efna4 KO mouse line.

The IMPC/KOMP program provides the opportunity to screen mice harboring well defined gene-inactivation mutations in a uniform genetic background. The program performs a global tissue phenotyping survey that includes skeletal x-rays and bone density measurements. Because of the relative insensitivity of the two screening tests for detecting variance in bone architecture, we initiated a secondary screen based on µCT and a cryohistolomorphological workflow that was performed on the femur and vertebral compartments on 220 randomly selected knockouts (KOs) and 36 control bone samples over a 2 1/2 year collection period provided by one of the production/phenotyping centers. The performance of the screening protocol was designed to balance throughput and cost versus sensitivity and informativeness such that the output would be of value to the skeletal biology community. Here we report the reliability of this screening protocol to establish criteria for control skeletal variance at the architectural, dynamic and cellular histomorphometric level. Unexpected properties of the control population include unusually high variance in BV/TV in male femurs and greater bone formation and bone turnover rates in the female femur and vertebral trabeculae bone compartments. However, the manner for maintaining bone formation differed between these two bone sites. The vertebral compartment relies on maintaining a greater number of bone forming surfaces while the femoral compartment utilized more matrix production per cell. The comparison of the architectural properties obtained by µCT and histomorphology revealed significant differences in values for BV/TV, Tb.Th and Tb.N which is attributable to sampling density of the two methods. However, as a screening tool, expressing the ratio of KO to the control line as obtained by either method was remarkably similar. It identified KOs with significant variance which led to a more detailed histological analysis. Our findings are exemplified by the Efna4 KO, in which a high BV/TV was identified by µCT and confirmed by histomorphometry in the femur but not in the vertebral compartment. Dynamic labeling showed a marked increase in BFR which was attributable to increased labeling surfaces. Cellular analysis confirmed partitioning of osteoblast to labeling surfaces and a marked decrease in osteoclastic activity on both labeling and quiescent surfaces. This pattern of increased bone modeling would not be expected based on prior studies of the Ephrin-Ephrin receptor signaling pathways between osteoblasts and osteoclasts. Overall, our findings underscore why unbiased screening is needed because it can reveal unknown or unanticipated genes that impact skeletal variation.

A thermally stable form of bacterial cocaine esterase: a potential therapeutic agent for treatment of cocaine abuse.

Rhodococcal cocaine esterase (CocE) is an attractive potential treatment for both cocaine overdose and cocaine addiction. CocE directly degrades cocaine into inactive products, whereas traditional small-molecule approaches require blockade of the inhibitory action of cocaine on a diverse array of monoamine transporters and ion channels. The usefulness of wild-type (wt) cocaine esterase is hampered by its inactivation at 37 degrees C. Herein, we characterize the most thermostable form of this enzyme to date, CocE-L169K/G173Q. In vitro kinetic analyses reveal that CocE-L169K/G173Q displays a half-life of 2.9 days at 37 degrees C, which represents a 340-fold improvement over wt and is 15-fold greater than previously reported mutants. Crystallographic analyses of CocE-L169K/G173Q, determined at 1.6-A resolution, suggest that stabilization involves enhanced domain-domain interactions involving van der Waals interactions and hydrogen bonding. In vivo rodent studies reveal that intravenous pretreatment with CocE-L169K/G173Q in mice provides protection from cocaine-induced lethality for longer time periods before cocaine administration than wt CocE. Furthermore, intravenous administration (pretreatment) of CocE-L169K/G173Q prevents self-administration of cocaine in a time-dependent manner. Termination of the in vivo effects of CoCE seems to be dependent on, but not proportional to, its clearance from plasma as its half-life is approximately 2.3 h and similar to that of wt CocE (2.2 h). Taken together these data suggest that CocE-L169K/G173Q possesses many of the properties of a biological therapeutic for treating cocaine abuse but requires additional development to improve its serum half-life.

CTRP3 Regulates Endochondral Ossification and Bone Remodeling During Fracture Healing.

C1q/TNF-related protein 3 (CTRP3) is a cytokine known to regulate a variety of metabolic processes. Though previously undescribed in the context of bone regeneration, high throughput gene expression experiments in mice identified CTRP3 as one of the most highly upregulated genes in fracture callus tissue. Hypothesizing a positive regulatory role for CTRP3 in bone regeneration, we phenotyped skeletal development and fracture healing in CTRP3 knockout (KO) and CTRP3 overexpressing transgenic (TG) mice relative to wild-type (WT) control animals. CTRP3 KO mice experienced delayed endochondral fracture healing, resulting in abnormal mineral distribution, the presence of periosteal marrow compartments, and a nonunion-like state. Decreased osteoclast number was also observed in CTRP3 KO mice, whereas CTRP3 TG mice underwent accelerated callus remodeling. Gene expression profiling revealed a broad impact on osteoblast/osteoclast lineage commitment and metabolism, including arrested progression toward mature skeletal lineages in the KO group. A single systemic injection of CTRP3 protein at the time of fracture was insufficient to phenocopy the chronic TG healing response in WT mice. By associating CTRP3 levels with fracture healing progression, these data identify a novel protein family with potential therapeutic and diagnostic value. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:00-19966, 2020.

Helical conformation of the SEVI precursor peptide PAP248-286, a dramatic enhancer of HIV infectivity, promotes lipid aggregation and fusion.

In previous in vivo studies, amyloid fibers formed from a peptide ubiquitous in human seminal fluid (semen-derived enhancer of viral infection (SEVI)) were found to dramatically enhance the infectivity of the HIV virus (3-5 orders of magnitude by some measures). To complement those studies, we performed in vitro assays of PAP(248-286), the most active precursor to SEVI, and other polycationic polymers to investigate the physical mechanisms by which the PAP(248-286) promotes the interaction with lipid bilayers. At acidic (but not at neutral) pH, freshly dissolved PAP(248-286) catalyzes the formation of large lipid flocculates in a variety of membrane compositions, which may be linked to the promotion of convective transport in the vaginal environment rather than transport by a random Brownian motion. Furthermore, PAP(248-286) is itself fusiogenic and weakens the integrity of the membrane in such a way that may promote fusion by the HIV gp41 protein. An alpha-helical conformation of PAP(248-286), lying parallel to the membrane surface, is implicated in promoting bridging interactions between membranes by the screening of the electrostatic repulsion that occurs when two membranes are brought into close contact. This suggests that nonspecific binding of monomeric or small oligomeric forms of SEVI in a helical conformation to lipid membranes may be an additional mechanism by which SEVI enhances the infectivity of the HIV virus.

Implicating candidate genes at GWAS signals by leveraging topologically associating domains.

Genome-wide association studies (GWAS) have contributed significantly to the understanding of complex disease genetics. However, GWAS only report association signals and do not necessarily identify culprit genes. As most signals occur in non-coding regions of the genome, it is often challenging to assign genomic variants to the underlying causal mechanism(s). Topologically associating domains (TADs) are primarily cell-type-independent genomic regions that define interactome boundaries and can aid in the designation of limits within which an association most likely impacts gene function. We describe and validate a computational method that uses the genic content of TADs to prioritize candidate genes. Our method, called 'TAD_Pathways', performs a Gene Ontology (GO) analysis over genes that reside within TAD boundaries corresponding to GWAS signals for a given trait or disease. Applying our pipeline to the bone mineral density (BMD) GWAS catalog, we identify 'Skeletal System Development' (Benjamini-Hochberg adjusted P=1.02x10-5) as the top-ranked pathway. In many cases, our method implicated a gene other than the nearest gene. Our molecular experiments describe a novel example: ACP2, implicated near the canonical 'ARHGAP1' locus. We found ACP2 to be an important regulator of osteoblast metabolism, whereas ARHGAP1 was not supported. Our results via BMD, for example, demonstrate how basic principles of three-dimensional genome organization can define biologically informed association windows.

Intraoperative delivery of the Notch ligand Jagged-1 regenerates appendicular and craniofacial bone defects.

Each year, 33% of US citizens suffer from a musculoskeletal condition that requires medical intervention, with direct medical costs approaching $1 trillion USD per year. Despite the ubiquity of skeletal dysfunction, there are currently limited safe and efficacious bone growth factors in clinical use. Notch is a cell-cell communication pathway that regulates self-renewal and differentiation within the mesenchymal/osteoblast lineage. The principal Notch ligand in bone, Jagged-1, is a potent osteoinductive protein that positively regulates post-traumatic bone healing in animals. This report describes the temporal regulation of Notch during intramembranous bone formation using marrow ablation as a model system and demonstrates decreased bone formation following disruption of Jagged-1 in mesenchymal progenitor cells. Notch gain-of-function using recombinant Jagged-1 protein on collagen scaffolds promotes healing of craniofacial (calvarial) and appendicular (femoral) surgical defects in both mice and rats. Localized delivery of Jagged-1 promotes bone apposition and defect healing, while avoiding the diffuse bone hypertrophy characteristic of the clinically problematic bone morphogenetic proteins. It is concluded that Jagged-1 is a bone-anabolic agent with therapeutic potential for regenerating traumatic or congenital bone defects.

Tendon Differentiation on Decellularized Extracellular Matrix Under Cyclic Loading.

Tendon bioreactors combine cells, scaffold, and mechanical stimulation to drive tissue neogenesis ex vivo. Faithful recapitulation of the native tendon microenvironment is essential for stimulating graft maturation or modeling tendon biology. As the mediator between cells and mechanical stimulation, the properties of a scaffold constitute perhaps the most essential elements in a bioreactor system. One method of achieving native scaffold properties is to process tendon allograft in a manner that removes cells without modifying structure and function: "decellularization." This chapter describes (1) production of tendon scaffolds derived from native extracellular matrix, (2) preparation of cell-laden scaffolds prior to bioreactor culture, and (3) tissue processing post-harvest for gene expression analysis. These methods may be applied for a variety of applications including graft production, cell priming prior to transplantation and basic investigations of tendon cell biology.