Robust Colonic Epithelial Regeneration and Amelioration of Colitis via FZD-Specific Activation of Wnt Signaling.

BACKGROUND AND AIMS: Current management of inflammatory bowel disease leaves a clear unmet need to treat the severe epithelial damage. Modulation of Wnt signaling might present an opportunity to achieve histological remission and mucosal healing when treating IBD. Exogenous R-spondin, which amplifies Wnt signals by maintaining cell surface expression of Frizzled (Fzd) and low-density lipoprotein receptor-related protein receptors, not only helps repair intestine epithelial damage, but also induces hyperplasia of normal epithelium. Wnt signaling may also be modulated with the recently developed Wnt mimetics, recombinant antibody-based molecules mimicking endogenous Wnts. METHODS: We first compared the epithelial healing effects of RSPO2 and a Wnt mimetic with broad Fzd specificity in an acute dextran sulfate sodium mouse colitis model. Guided by Fzd expression patterns in the colon epithelium, we also examined the effects of Wnt mimetics with subfamily Fzd specificities. RESULTS: In the DSS model, Wnt mimetics repaired damaged colon epithelium and reduced disease activity and inflammation and had no apparent effect on uninjured tissue. We further identified that the FZD5/8 and LRP6 receptor-specific Wnt mimetic, SZN-1326-p, was associated with the robust repair effect. Through a range of approaches including single-cell transcriptome analyses, we demonstrated that SZN-1326-p directly impacted epithelial cells, driving transient expansion of stem and progenitor cells, promoting differentiation of epithelial cells, histologically restoring the damaged epithelium, and secondarily to epithelial repair, reducing inflammation. CONCLUSIONS: It is feasible to design Wnt mimetics such as SZN-1326-p that impact damaged intestine epithelium specifically and restore its physiological functions, an approach that holds promise for treating epithelial damage in inflammatory bowel disease.

Tissue Engineered Neurovascularization Strategies for Craniofacial Tissue Regeneration.

Craniofacial tissue injuries, diseases, and defects, including those within bone, dental, and periodontal tissues and salivary glands, impact an estimated 1 billion patients globally. Craniofacial tissue dysfunction significantly reduces quality of life, and successful repair of damaged tissues remains a significant challenge. Blood vessels and nerves are colocalized within craniofacial tissues and act synergistically during tissue regeneration. Therefore, the success of craniofacial regenerative approaches is predicated on successful recruitment, regeneration, or integration of both vascularization and innervation. Tissue engineering strategies have been widely used to encourage vascularization and, more recently, to improve innervation through host tissue recruitment or prevascularization/innervation of engineered tissues. However, current scaffold designs and cell or growth factor delivery approaches often fail to synergistically coordinate both vascularization and innervation to orchestrate successful tissue regeneration. Additionally, tissue engineering approaches are typically investigated separately for vascularization and innervation. Since both tissues act in concert to improve craniofacial tissue regeneration outcomes, a revised approach for development of engineered materials is required. This review aims to provide an overview of neurovascularization in craniofacial tissues and strategies to target either process thus far. Finally, key design principles are described for engineering approaches that will support both vascularization and innervation for successful craniofacial tissue regeneration.

Macrophage depletion increases target specificity of bone-targeted nanoparticles.

Despite efforts to achieve tissue selectivity, the majority of systemically administered drug delivery systems (DDSs) are cleared by the mononuclear phagocyte system (MPS) before reaching target tissues regardless of disease or injury pathology. Previously, we showed that while tartrate-resistant acid phosphatase (TRAP) binding peptide (TBP)-targeted polymeric nanoparticles (TBP-NP) delivering a bone regenerative Wnt agonist improved NP fracture accumulation and expedited healing compared with controls, there was also significant MPS accumulation. Here we show that TBP-NPs are taken up by liver, spleen, lung, and bone marrow macrophages (Mϕ), with 76 ± 4%, 49 ± 11%, 27 ± 9%, and 92 ± 5% of tissue-specific Mϕ positive for NP, respectively. Clodronate liposomes (CLO) significantly depleted liver and spleen Mϕ, resulting in 1.8-fold and 3-fold lower liver and spleen and 1.3-fold and 1.6-fold greater fracture and naïve femur accumulation of TBP-NP. Interestingly, depletion and saturation of MPS using 10-fold greater TBP-NP doses also resulted in significantly higher TBP-NP accumulation at lungs and kidneys, potentially through compensatory clearance mechanisms. The higher NP dose resulted in greater TBP-NP accumulation at naïve bone tissue; however, other MPS tissues (i.e., heart and lungs) exhibited greater TBP-NP accumulation, suggesting uptake by other cell types. Most importantly, neither Mϕ depletion nor saturation strategies improved fracture site selectivity of TBP-NPs, possibly due to a reduction of Mϕ-derived osteoclasts, which deposit the TRAP epitope. Altogether, these data support that MPS-mediated clearance is a key obstacle in robust and selective fracture accumulation for systemically administered bone-targeted DDS and motivates the development of more sophisticated approaches to further improve fracture selectivity of DDS.

Development of selective bispecific Wnt mimetics for bone loss and repair.

The Wnt signaling pathway is intricately connected with bone mass regulation in humans and rodent models. We designed an antibody-based platform that generates potent and selective Wnt mimetics. Using this platform, we engineer bi-specific Wnt mimetics that target Frizzled and low-density lipoprotein receptor-related proteins and evaluate their effects on bone accrual in murine models. These synthetic Wnt agonists induce rapid and robust bone building effects, and correct bone mass deficiency and bone defects in various disease models, including osteoporosis, aging, and long bone fracture. Furthermore, when these Wnt agonists are combined with antiresorptive bisphosphonates or anti-sclerostin antibody therapies, additional bone accrual/maintenance effects are observed compared to monotherapy, which could benefit individuals with severe and/or acute bone-building deficiencies. Our data support the continued development of Wnt mimetics for the treatment of diseases of low bone mineral density, including osteoporosis.

Delivery of RNAi-Based Therapeutics for Bone Regeneration.

PURPOSE OF REVIEW: The clinical significance, target pathways, recent successes, and challenges that preclude translation of RNAi bone regenerative approaches are overviewed. RECENT FINDINGS: RNA interference (RNAi) is a promising new therapeutic approach for bone regeneration by stimulating or inhibiting critical signaling pathways. However, RNAi suffers from significant delivery challenges. These challenges include avoiding nuclease degradation, achieving bone tissue targeting, and reaching the cytoplasm for mRNA inhibition. Many drug delivery systems have overcome stability and intracellular localization challenges but suffer from protein adsorption that results in clearance of up to 99% of injected dosages, thus severely limiting drug delivery efficacy. While RNAi has myriad promising attributes for use in bone regenerative applications, delivery challenges continue to plague translation. Thus, a focus on drug delivery system development is critical to provide greater delivery efficiency and bone targeting to reap the promise of RNAi.

In Vivo Translation of Peptide-Targeted Drug Delivery Systems Discovered by Phage Display.

Therapeutic compounds with narrow therapeutic windows and significant systemic side effects benefit from targeted drug delivery strategies. Peptide-protein interactions are often exploited for targeting, with phage display a primary method to identify high-affinity peptide ligands that bind cell surface and matrix bound receptors preferentially expressed in target tissues. After isolating and sequencing high-binding phages, peptides are easily synthesized and chemically modified for incorporation into drug delivery systems, including peptide-drug conjugates, polymers, and nanoparticles. This review describes the phage display methodology to identify targeting peptide sequences, strategies to functionalize drug carriers with phage-derived peptides, specific examples of drug carriers with in vivo translation, and limitations and future applications of phage display to drug delivery.

Development of controlled drug delivery systems for bone fracture-targeted therapeutic delivery: A review.

Impaired fracture healing is a major clinical problem that can lead to patient disability, prolonged hospitalization, and significant financial burden. Although the majority of fractures heal using standard clinical practices, approximately 10% suffer from delayed unions or non-unions. A wide range of factors contribute to the risk for nonunions including internal factors, such as patient age, gender, and comorbidities, and external factors, such as the location and extent of injury. Current clinical approaches to treat nonunions include bone grafts and low-intensity pulsed ultrasound (LIPUS), which realizes clinical success only to select patients due to limitations including donor morbidities (grafts) and necessity of fracture reduction (LIPUS), respectively. To date, therapeutic approaches for bone regeneration rely heavily on protein-based growth factors such as INFUSE, an FDA-approved scaffold for delivery of bone morphogenetic protein 2 (BMP-2). Small molecule modulators and RNAi therapeutics are under development to circumvent challenges associated with traditional growth factors. While preclinical studies has shown promise, drug delivery has become a major hurdle stalling clinical translation. Therefore, this review overviews current therapies employed to stimulate fracture healing pre-clinically and clinically, including a focus on drug delivery systems for growth factors, parathyroid hormone (PTH), small molecules, and RNAi therapeutics, as well as recent advances and future promise of fracture-targeted drug delivery.

Multivalent Presentation of Peptide Targeting Groups Alters Polymer Biodistribution to Target Tissues.

Drug delivery to bone is challenging, whereby drug distribution is commonly <1% of injected dose, despite development of several bone-targeted drug delivery systems specific to hydroxyapatite. These bone-targeted drug delivery systems still suffer from poor target cell localization within bone, as at any given time overall bone volume is far greater than acutely remodeling bone volume, which harbors relevant cell targets (osteoclasts or osteoblasts). Thus, there exists a need to target bone-acting drugs specifically to sites of bone remodeling. To address this need, this study synthesized oligo(ethylene glycol) copolymers based on a peptide with high affinity to tartrate-resistant acid phosphatase (TRAP), an enzyme deposited by osteoclasts during the bone resorption phase of bone remodeling, which provides greater specificity relevant for bone cell drugging. Gradient and random peptide orientations, as well as polymer molecular weights, were investigated. TRAP-targeted, high molecular weight (Mn) random copolymers exhibited superior accumulation in remodeling bone, where fracture accumulation was observed for at least 1 week and accounted for 14% of tissue distribution. Intermediate and low Mn random copolymer accumulation was lower, indicating residence time depends on Mn. High Mn gradient polymers were cleared, with only 2% persisting at fractures after 1 week, suggesting TRAP binding depends on peptide density. Peptide density and Mn are easily modified in this versatile targeting platform, which can be applied to a range of bone drug delivery applications.

Fracture-Targeted Delivery of ß-Catenin Agonists via Peptide-Functionalized Nanoparticles Augments Fracture Healing.

Despite several decades of progress, bone-specific drug delivery is still a major challenge. Current bone-acting drugs require high-dose systemic administration which decreases therapeutic efficacy and increases off-target tissue effects. Here, a bone-targeted nanoparticle (NP) delivery system for a ß-catenin agonist, 3-amino-6-(4-((4-methylpiperazin-1-yl)sulfonyl)phenyl)-N-(pyridin-3-yl)pyrazine-2-carboxamide, a glycogen synthase kinase 3 beta (GSK-3ß) inhibitor, was developed to enhance fracture healing. The GSK-3ß inhibitor loading capacity was found to be 15 wt % within highly stable poly(styrene-alt-maleic anhydride)-b-poly(styrene) NPs, resulting in ∼50 nm particles with ∼ -30 mV surface charge. A peptide with high affinity for tartrate-resistant acid phosphatase (TRAP), a protein deposited by osteoclasts on bone resorptive surfaces, was introduced to the NP corona to achieve preferential delivery to fractured bone. Targeted NPs showed improved pharmacokinetic profiles with greater accumulation at fractured bone, accompanied by significant uptake in regenerative cell types (mesenchymal stem cells (MSCs) and osteoblasts). MSCs treated with drug-loaded NPs in vitro exhibited 2-fold greater ß-catenin signaling than free drug that was sustained for 5 days. To verify similar activity in vivo, TOPGAL reporter mice bearing fractures were treated with targeted GSK-3ß inhibitor-loaded NPs. Robust ß-galactosidase activity was observed in fracture callus and periosteum treated with targeted carriers versus controls, indicating potent ß-catenin activation during the healing process. Enhanced bone formation and microarchitecture were observed in mice treated with GSK-3ß inhibitor delivered via TRAP-binding peptide-targeted NPs. Specifically, increased bone bridging, ∼4-fold greater torsional rigidity, and greater volumes of newly deposited bone were observed 28 days after treatment, indicating expedited fracture healing.

Local and targeted drug delivery for bone regeneration.

While experimental bone regeneration approaches commonly employ cells, technological hurdles prevent translation of these therapies. Alternatively, emulating the spatiotemporal cascade of endogenous factors through controlled drug delivery may provide superior bone regenerative approaches. Surgically placed drug depots have clinical indications. Additionally, noninvasive systemic delivery can be used as needed for poorly healing bone injuries. However, a major hurdle for systemic delivery is poor bone biodistribution of drugs. Thus, peptides, aptamers, and phosphate-rich compounds with specificity toward proteins, cells, and molecules within the regenerative bone microenvironment may enable the design of targeted carriers with bone biodistribution greater than that achieved by drug alone. These carriers, combined with osteoregenerative drugs and/or stimuli-sensitive linkers, may enhance bone regeneration while minimizing off-target tissue effects.

Educating nurses in Rhode Island: a lot of diversity in a little place.

Faculty at a baccalaureate nursing program at a state college discuss a multifaceted approach to meeting the needs of a very diverse student population. These students reflect the changing demographics of the state and their education contributes to providing a racially, ethnically, and culturally diverse nursing workforce. The program addresses multiple issues which may impede academic performance. These measures have enhanced and promoted graduates' success on NCLEX-RN. Creative and varied strategies are presented and their implementation and evaluation described.

The effects of aerial spraying with Bacillus thuringiensis Kurstaki on children with asthma.

OBJECTIVE: To determine if aerially spraying a biological pesticide was associated with an increase in the symptoms or change in the Peak Expiratory Flow Rate of children with asthma. METHODS: A pre/post matched pairs cohort design was used. Children living in the spray zone were matched with children outside of the spray zone. Peak Expiratory Flow Rates, asthma symptoms and non-asthma symptoms were recorded in diaries. RESULTS: There were no differences in asthma symptom scores between subjects and controls, neither before nor after the spray; nor were there significant changes in Peak Expiratory Flow Rates for subjects after the spray period. CONCLUSIONS: No evidence of adverse effects from the use of the biological pesticide was found. We believe that this is the first paper to address the issue of whether or not aerial spraying with Btk has a harmful effect on children with asthma.