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Coatings Releasing the Relaxin Peptide Analogue B7-33 Reduce Fibrotic Encapsulation
Abstract
The development of antifibrotic materials and coatings that can resist the foreign body response (FBR) continues to present a major hurdle in the advancement of current and next generation implantable medical devices, biosensors and cell therapies. From an implant perspective, the most important issue associated with the FBR is the prolonged inflammatory response leading to a collagenous capsule that ultimately blocks mass transport and communication between the implant and the surrounding tissue. Up to now, most attempts to reduce the capsule thickness have focused on providing surface coatings that reduce protein fouling and cell attachment. Here, we present an approach that is based on the sustained release of a peptide drug interfering with the FBR. In this study, the biodegradable polymer poly(lactic-co-glycolic) acid (PLGA) was used as a coating releasing the relaxin peptide analogue B7 33, which has been demonstrated to reduce organ fibrosis in animal models. While in vitro protein quantification was used to demonstrate controlled release of the antifibrotic peptide B7-33 from PLGA coatings, an in vitro reporter cell assay was used to demonstrate that B7 33 retains activity against the relaxin family peptide receptor 1 (RXFP1). Subcutaneous implantation of PLGA coated polypropylene samples in mice with and without the peptide demonstrated a marked reduction in capsule thickness (49.2 %) over a 6-week period. It is expected that this novel approach will open the door to a range of new and improved implantable medical devices.
... For ma tion around a PLGA coated polypropy lene im plant. Adapted with per mis sion from Welch et al. [2] Copy right 2019 Amer i can Chem i cal So ci ety. ...
... His tor ically, the most im por tant ad vances in re duc ing the FBR of im planted bio ma te ri als have arisen from com bi na tion strate gies em ploy ing mate ri als and bioac tive com po nents that mod u late both me chan i cal and bio chem i cal cues that drive cell be hav iour ( Fig. 4 [15][16][17][31][32][33][34][35][36][37][38][39][40][41][42]). Sig nif i cant im prove ments have also re sulted from in cor po rat ing advances in or gan fi bro sis drug re search, by com bin ing low FBR ma teri als with an tifi brotic small mol e cules [2,17,36,38]. ...
... A re cent ar ti cle also demon strated the elu tion of an an tifi brotic pep tide, B7-33 [91], de rived from hu man re laxin from a PLGA coating to re duce fi brotic en cap su la tion [2]. This method was used to en dow a polypropy lene model im plant with a PLGA+B7-33 coat ing that re duced the fi brotic cap sule thick ness by 50% af ter 6 weeks in vivo in mice. ...
A broad range of medical devices initiate an immune reaction known as the foreign body response (FBR) upon implantation. Here, collagen deposition at the surface of the implant occurs as a result of the FBR, ultimately leading to fibrous encapsulation and, in many cases, reduced function or failure of the device. Despite significant efforts, the prevention of fibrotic encapsulation has not been realized at this point in time. However, many next-generation medical technologies including cellular therapies, sensors and devices depend on the ability to modulate and control the FBR. For these technologies to become viable, significant advances must be made in understanding the underlying mechanism of this response as well as in the methods modulating this response. In this review, we highlight recent advances in the development of materials and coatings providing a reduced FBR and emphasize key characteristics of high-performing approaches. We also provide a detailed overview of the state-of-the-art in strategies relying on controlled drug release, the surface display of bioactive signals, materials-based approaches, and combinations of these approaches. Finally, we offer perspectives on future directions in this field.
... B7-33 consists of residues 7-29 of the B chain of H2 relaxin ( Figure 1B,C), with the addition of a KRSL sequence from positions 30-33 of the B1-33 isoform of H2 relaxin, which dramatically improves solubility over B7-29 [13]. B7-33, being a single-chain peptide, is far easier and cheaper to produce than the parent peptide, making it an excellent scaffold for modification with the aim of improving its pharmacological properties as an anti-fibrotic therapeutic [13] and a component of anti-fibrotic device coating [16]. While B7-33 has poor affinity and cAMP potency in HEK cells overexpressing RXFP1 (HEK-RXFP1), it retains the known beneficial effects of H2 relaxin (e.g., anti-fibrotic, vasodilatory, anti-inflammatory, and anti-hypertrophy) [13,17,18]. ...
Human relaxin-2 (H2 relaxin) is a peptide hormone with potent vasodilatory and anti-fibrotic effects, which is of interest for the treatment of heart failure and fibrosis. H2 relaxin binds to the Relaxin Family Peptide Receptor 1 (RXFP1). Native H2 relaxin is a two-chain, three-disulfide-bond-containing peptide, which is unstable in human serum and difficult to synthesize efficiently. In 2016, our group developed B7-33, a single-chain peptide derived from the B-chain of H2 relaxin. B7-33 demonstrated poor affinity and potency in HEK cells overexpressing RXFP1; however, it displayed equivalent potency to H2 relaxin in fibroblasts natively expressing RXFP1, where it also demonstrated the anti-fibrotic effects of the native hormone. B7-33 reversed organ fibrosis in numerous pre-clinical animal studies. Here, we detail our efforts towards a minimal H2 relaxin scaffold and attempts to improve scaffold activity through Aib substitution and hydrocarbon stapling to re-create the peptide helicity present in the native H2 relaxin.
... The most significant progress in decreasing the FBR of implanted biomaterials has been made through combined strategies using materials with bioactive components to modulate both mechanical and biochemical cues driving cell behaviours [123]. ...
The usage of bone substitute granule materials has improved the clinical results of alveolar bone deficiencies treatment and thus broadened applications in implant dentistry. However, because of the complicated mechanisms controlling the foreign body response (FBR), no perfect solution can avoid the fibrotic encapsulation of materials till now, which may impair the results of bone regeneration, even cause the implant materials rejection. Recently, the concept of "osteoimmunology" has been stressed. The outcomes of bone regeneration are proved to be related to the bio-physicochemical properties of biomaterials, which allow them to regulate the biological behaviors of both innate and adaptive immune cells. With the development of single cell transcriptome, the truly heterogeneity of osteo-immune cells has been clarifying, which is helpful to overcome the limitations of traditional M1/M2 macrophage nomenclature and drive the advancements of particulate biomaterials applications. This review aims at introducing the mechanisms of optimal osseointegration regulated by immune systems and provides feasible strategies for the design of next generation "osteoimmune-smart" particulate bone substitute materials in dental clinic.
... In agreement with the concept that the presence of M2 macrophages is associated with a better in vivo biointegration [41] , we observed here that biointegration was optimal for all four LMWHs, as no sign of any fibrous capsule formation was evidenced after 3 weeks ( Fig. 6 b). These data contrast with the development of fibrous capsules with most implanted biomaterials [42][43][44][45] , such as PEG hydrogels recognized as gold standard biomaterials [46] . Interestingly, the hydrogel invasion by macrophages that remain with a proinflammatory M1 phenotype after 3 weeks may be suggestive of biomaterials promoting the formation of a thick fibrous capsule, as mentioned in many articles and reviews. ...
Hydrogels used in regenerative medicine are often designed to allow cellular infiltration, degradation, and neovascularization. Low molecular weight hydrogels (LMWHs), formed by self-assembly via non-covalent interactions, are gaining significant interest because they are soft, easy to use and injectable. We propose LMWHs as suitable body implant materials that can stimulate tissue regeneration. We produced four new LMWHs with molecular entities containing nucleic acid and lipid building blocks and analyzed the foreign body response upon subcutaneous implantation into mice. Despite being infiltrated with macrophages, none of the hydrogels triggered detrimental inflammatory responses. Most macrophages present in the hydrogel-surrounding tissue acquired an immuno-modulatory rather than inflammatory phenotype. Concomitantly, no fibrotic capsule was formed after three weeks. Our glyconucleolipid LMWHs exhibited different degradation kinetics in vivo and in vitro. LMWHs with high angiogenic properties in vivo, were found to release glyconucleoside (glucose covalently linked to thymidine via a triazole moiety) as a common by-product of in vitro LMWH degradation. Chemically synthesized glyconucleoside exhibited angiogenic properties in vitro in scratch assays with monolayers of human endothelial cells and in vivo using the chick chorioallantoic membrane assay. Collectively, LMWHs hold promise as efficient scaffolds for various regenerative applications by displaying good biointegration without causing fibrosis, and by promoting angiogenesis through the release of a pro-angiogenic degradation product.
Statement of Significance
The main limitations of biomaterials developed in the field of tissue engineering remains their biocompatibility and vascularisation properties. In this context, we developed injectable Low Molecular Weight Hydrogels (LMWH) exhibiting thixotropic (reversible gelation) and thermal reversible properties. LMWH having injectability is of great advantage since it allows for their delivery without wounding the surrounding tissues.
The resulting gels aim at forming scaffolds that the host cells colonize without major inflammation, and that won't be insulated by a strong fibrosis reaction. Importantly, their molecular degradation releases a product (a glycosyl-nucleoside conjugate) promoting angiogenesis. In this sense, these LMWH represent an important advance in the development of biomaterials promoting tissue regeneration.
Nanotechnology is essential in the development of advanced technologies for medicine and healthcare. Advances depend on the ability to study these materials and nano-biotechnologies using advanced analytical techniques. Providing a comprehensive overview of analytical techniques for applications in biomedical nanotechnology at both the fundamental and applied level, this book provides a broad perspective of the development of analytical methods involved in materials science and electronics. It highlights the fundamentals and systematic developments of techniques to achieve better characterization, rapid diagnostics, cost-effective and user-friendly approaches, and state-of-the-art methodologies for personalized health management, and explains how this fundamental knowledge can be translated into applications in nano-enabled biomedical research. The multidisciplinary readership includes chemists, biologists, physicists, materials scientists, engineers and information technologists.Key features• Provides a comprehensive overview of analytical techniques for applications in biomedical nanotechnology at both the fundamental and applied level.• Explains how fundamental knowledge of the techniques can be translated to applications in nano-enabled biomedical research.• Covers state-of-art analytical techniques for biomedical nanotechnology.• Includes the prospects and challenges with each technique, including potential solutions.• Provides a multidisciplinary view of interest to chemists, biologists, physicists, materials scientists, engineers and information technologists.
Over the last few decades, magnetic nanostructures have been providing the platform to develop magnetically guided systems for biomedical nanotechnology and applications such as biosensors, nanomedicine, targeted drug delivery, etc. Magnetic nanoparticles especially super-paramagnetic iron oxide nanoparticles (SPIONs) have gained more importance in biomedical applications. This chapter discusses the biomedical uses of magnetic nanoparticles. The use of magnetic nanoparticles in hyperthermia, drug delivery, imaging tools, MRI, electrochemical sensors, cell and gene therapy for cancer treatment are discussed after reviewing some of the basic principles of magnetism. Finally, a summary of the magnetic nanostructure based biomedical nanotechnology is presented by examining current possibilities in this area, especially the huge hurdles faced when implementing laboratory-tested technologies.
After implantation of a biomaterial, both the host immune system and properties of the material determine the local immune response. Through triggering or modulating the local immune response, materials can be designed towards a desired direction of promoting tissue repair or regeneration. High-throughput sequencing technologies such as single-cell RNA sequencing (scRNA-seq) emerging as a powerful tool for dissecting the immune micro-environment around biomaterials, have not been fully utilized in the field of soft tissue regeneration. In this review, we first discussed the procedures of foreign body reaction in brief. Then, we summarized the influences that physical and chemical modulation of biomaterials have on cell behaviors in the micro-environment. Finally, we discussed the application of scRNA-seq in probing the scaffold immune micro-environment and provided some reference to designing immunomodulatory biomaterials. The foreign body response consists of a series of biological reactions. Immunomodulatory materials regulate immune cell activation and polarization, mediate divergent local immune micro-environments and possess different tissue engineering functions. The manipulation of physical and chemical properties of scaffolds can modulate local immune responses, resulting in different outcomes of fibrosis or tissue regeneration. With the advancement of technology, emerging techniques such as scRNA-seq provide an unprecedented understanding of immune cell heterogeneity and plasticity in a scaffold-induced immune micro-environment at high resolution. The in-depth understanding of the interaction between scaffolds and the host immune system helps to provide clues for the design of biomaterials to optimize regeneration and promote a pro-regenerative local immune micro-environment.
Liver metastasis is common in patients with pancreatic cancer (PC) and is the leading cause of death associated with PC. Liver fibrosis induced by activated hepatic stellate cells (HSCs) creates a favorable metastatic microenvironment that promotes metastasis growth. B7‐33, a therapeutic peptide (relaxin analog) that targets relaxin family peptide receptors on activated HSCs, inhibits the pSMAD2/3 signaling pathway and weakens the fibrogenic properties of activated HSCs. However, the short half‐life and highly conserved nature of the B7‐33 sequence limit its application in vivo. Here, B7‐33 is modified with the cRGD sequence, which does not affect the efficacy of B7‐33 and allows B7‐33 to assemble into vascular endothelial cell membrane‐derived vesicles by specifically interacting with integrin αvβ3. These rationally designed vesicles (B7‐33‐HNPs) are able to prolong the half‐life of B7‐33 in vivo and accumulate in the liver to reverse HSCs activation. Moreover, B7‐33‐HNPs prevent the formation and growth of liver metastases in a mouse model of metastatic PC. This study proposes a feasible strategy for building a therapeutic peptide delivery system through specific interactions, serving as a reference for preventing liver metastasis of PC through the regulation of HSCs.
Fibrosis refers to the scarring and hardening of tissues, which results from a failed immune system-coordinated wound healing response to chronic organ injury and which manifests from the aberrant accumulation of various extracellular matrix components (ECM), primarily collagen. Despite being a hallmark of prolonged tissue damage and related dysfunction, and commonly associated with high morbidity and mortality, there are currently no effective cures for its regression. An emerging therapy that meets several criteria of an effective anti-fibrotic treatment, is the recombinant drug-based form of the human hormone, relaxin (also referred to as serelaxin, which is bioactive in several other species). This review outlines the broad anti-fibrotic and related organ-protective roles of relaxin, mainly from studies conducted in preclinical models of ageing and fibrotic disease, including its ability to ameliorate several aspects of fibrosis progression and maturation, from immune cell infiltration, pro-inflammatory and pro-fibrotic cytokine secretion, oxidative stress, organ hypertrophy, cell apoptosis, myofibroblast differentiation and ECM production, to its ability to facilitate established ECM degradation. Studies that have compared and/or combined these therapeutic effects of relaxin with currently standard of care medication have also been discussed, along with the main challenges that have hindered the translation of the anti-fibrotic efficacy of relaxin to the clinic. The review then outlines the future directions as to where scientists and several pharmaceutical companies that have recognized the therapeutic potential of relaxin are working towards, to progress its development as a treatment for human patients suffering from various fibrotic diseases.
Foreign‐body response caused by implanted biomaterials seriously impedes the function of implants and is a major obstacle to the development of implantable biomaterials and medical devices. Recent advances in implantable biomaterials and medical devices have provided strategies to resist the foreign‐body response. In this review, the mechanism of the foreign‐body response and conventional strategies to mitigate foreign‐body response is briefly introduced. Then, three types of promising foreign‐body response resisting materials are focused and the advantages, characteristics, and applications of each material are discussed. Finally, prospects are put forward for future development of foreign‐body response resisting materials and current challenges that require in‐depth study. Normal implants frequently induce the foreign‐body response (FBR), including four stages: protein adsorption, acute inflammation, chronic inflammation, and collagen encapsulation. Fortunately, three classes of promising anti‐FBR materials including zwitterionic materials (e.g., poly(carboxybetaine methacrylate)), modified alginate, and poly‐β‐homoserine were recently studied to show great potential for a variety of applications such as artificial vitreous, sensor coatings, and cell encapsulation therapies.
XPS is widely used to identify and quantify the elements present at the surface of polymeric materials. The energy distribution of photoelectrons emitted from these elements contains information about their chemical state, potentially allowing the analyst to identify and quantify specific functional groups. These functional groups may originate from the synthesis and processing of the polymers, from postsynthetic modifications such as surface grafting, or indeed may be unrelated to the polymer (additives and contaminants). Extracting reliable and meaningful information from XPS data is not trivial and relies on careful and appropriate experimentation, including experimental design, sample preparation, data collection, data processing, and data interpretation. Here, the authors outline some of these challenges when performing XPS analysis of polymers and provide practical examples to follow. This guide will cover all relevant aspects over the course of a typical experiment, including tips and considerations when designing the experiment, sample preparation, charge neutralization, x-ray induced sample damage, depth profiling, data analysis and interpretation, and, finally, reporting of results. Many of these topics are more widely applicable to insulating organic materials, and the recommendations of this guide will help to ensure that data is collected and interpreted using current best practices.
An excessive foreign body response (FBR) has contributed to the adverse events associated with polypropylene mesh usage for augmenting pelvic organ prolapse surgery. Consequently, current biomaterial research considers the critical role of the FBR and now focuses on developing better biocompatible biomaterials rather than using inert implants to improve the clinical outcomes of their use. Tissue engineering approaches using mesenchymal stem cells (MSCs) have improved outcomes over traditional implants in other biological systems through their interaction with macrophages, the main cellular player in the FBR. The unique angiogenic, immunomodulatory and regenerative properties of MSCs have a direct impact on the FBR following biomaterial implantation. In this review, we focus on key aspects of the FBR to tissue-engineered MSC-based implants for supporting pelvic organs and beyond. We also discuss the immunomodulatory effects of the recently discovered endometrial MSCs on the macrophage response to new biomaterials designed for use in pelvic floor reconstructive surgery. We conclude with a focus on considerations in biomaterial design that take into account the FBR and will likely influence the development of the next generation of biomaterials for gynaecological applications.
Implantable medical devices have revolutionized modern medicine. However, immune-mediated foreign body response (FBR) to the materials of these devices can limit their function or even induce failure. Here we describe long-term controlled-release formulations for local anti-inflammatory release through the development of compact, solvent-free crystals. The compact lattice structure of these crystals allows for very slow, surface dissolution and high drug density. These formulations suppress FBR in both rodents and non-human primates for at least 1.3 years and 6 months, respectively. Formulations inhibited fibrosis across multiple implant sites—subcutaneous, intraperitoneal and intramuscular. In particular, incorporation of GW2580, a colony stimulating factor 1 receptor inhibitor, into a range of devices, including human islet microencapsulation systems, electrode-based continuous glucose-sensing monitors and muscle-stimulating devices, inhibits fibrosis, thereby allowing for extended function. We believe that local, long-term controlled release with the crystal formulations described here enhances and extends function in a range of medical devices and provides a generalized solution to the local immune response to implanted biomaterials.
The transplantation of pancreatic islet cells could restore glycaemic control in patients with type 1 diabetes. Microspheres for islet encapsulation have enabled long-term glycaemic control in rodent models of diabetes; however, humans transplanted with equivalent microsphere formulations have experienced only transient islet graft function owing to a vigorous foreign-body response (FBR), to pericapsular fibrotic overgrowth (PFO) and, in upright bipedal species, to the sedimentation of the microspheres within the peritoneal cavity. Here, we report the results of the testing in non-human primate (NHP) models of seven alginate formulations that were efficacious in rodents, including three that led to transient islet graft function in clinical trials. All formulations elicited significant FBR and PFO 1 month post implantation; however, three chemically modified, immune-modulating alginate formulations elicited a reduced FBR. In conjunction with a minimally invasive transplantation technique into the bursa omentalis of NHPs, the most promising chemically modified alginate derivative (Z1-Y15) protected viable and glucose-responsive allogeneic islets for 4 months without the need for immunosuppression. Chemically modified alginate formulations may enable the long-term transplantation of islets for the correction of insulin deficiency.
Patients admitted for acute heart failure (AHF) experience high rates of in-hospital and post-discharge morbidity and mortality despite current therapies. Serelaxin is recombinant human relaxin-2, a hormone with vasodilatory and end-organ protective effects believed to play a central role in the cardiovascular and renal adaptations of human pregnancy. In the phase 3 RELAX-AHF trial, serelaxin met its primary endpoint of improving dyspnoea through day 5 in patients admitted for AHF. Compared to placebo, serelaxin also reduced worsening heart failure (WHF) by 47% through day 5 and both all-cause and cardiovascular mortality by 37% through day 180. RELAX-AHF-2 ( ClinicalTrials.gov NCT01870778) is designed to confirm serelaxin's effect on these clinical outcomes. RELAX-AHF-2 is a multicentre, randomized, double-blind, placebo-controlled, event-driven, phase 3 trial enrolling ∼6800 patients hospitalized for AHF with dyspnoea, congestion on chest radiograph, increased natriuretic peptide levels, mild-to-moderate renal insufficiency, and systolic blood pressure ≥125 mmHg. Patients are randomized within 16 h of presentation to 48 h intravenous infusions of serelaxin (30 µg/kg/day) or placebo, both in addition to standard of care treatments. The primary objectives are to demonstrate that serelaxin is superior to placebo in reducing: (i) 180 day cardiovascular death, and (ii) occurrence of WHF through day 5. Key secondary endpoints include 180 day all-cause mortality, composite of 180 day combined cardiovascular mortality or heart failure/renal failure rehospitalization, and in-hospital length of stay during index AHF. The results from RELAX-AHF-2 will provide data on the potential beneficial effect of serelaxin on cardiovascular mortality and WHF in selected patients with AHF.
Host recognition and immune-mediated foreign body response to biomaterials can compromise the performance of implanted medical devices. To identify key cell and cytokine targets, here we perform in-depth systems analysis of innate and adaptive immune system responses to implanted biomaterials in rodents and non-human primates. While macrophages are indispensable to the fibrotic cascade, surprisingly neutrophils and complement are not. Macrophages, via CXCL13, lead to downstream B cell recruitment, which further potentiated fibrosis, as confirmed by B cell knockout and CXCL13 neutralization. Interestingly, colony stimulating factor-1 receptor (CSF1R) is significantly increased following implantation of multiple biomaterial classes: ceramic, polymer and hydrogel. Its inhibition, like macrophage depletion, leads to complete loss of fibrosis, but spares other macrophage functions such as wound healing, reactive oxygen species production and phagocytosis. Our results indicate that targeting CSF1R may allow for a more selective method of fibrosis inhibition, and improve biomaterial biocompatibility without the need for broad immunosuppression.
Human gene-2 relaxin (H2 relaxin) is a pleiotropic hormone with powerful vasodilatory and anti-fibrotic properties which has led to its clinical evaluation and provisional FDA approval as a treatment for acute heart failure. The diverse effects of H2 relaxin are mediated via its cognate G protein coupled-receptor (GPCR), Relaxin Family Peptide Receptor (RXFP1), leading to stimulation of a combination of cell signalling pathways that includes cyclic adenosine monophosphate (cAMP) and extracellular-signal-regulated kinases (ERK)1/2. However, its complex two-chain (A and B), disulfide-rich insulin-like structure is a limitation to its facile preparation, availability and affordability. Furthermore, its strong activation of cAMP signaling is likely responsible for reported detrimental tumor-promoting actions that may preclude long-term use of this drug for treating human disease. Here we report the design and synthesis of a H2 relaxin B-chain-only analogue, B7-33, which was shown to bind to RXFP1 and preferentially activate the pERK pathway over cAMP in cells that endogenously expressed RXFP1. Thus, B7-33 represents the first functionally selective agonist of the complex GPCR, RXFP1. Importantly, this small peptide agonist prevented or reversed organ fibrosis and dysfunction in three pre-clinical rodent models of heart or lung disease with similar potency to H2 relaxin. The molecular mechanism behind the strong anti-fibrotic actions of B7-33 involved its activation of RXFP1-angiotensin II type 2 receptor heterodimers that induced selective downstream signaling of pERK1/2 and the collagen-degrading enzyme, matrix metalloproteinase (MMP)-2. Furthermore, in contrast to H2 relaxin, B7-33 did not promote prostate tumor growth in vivo. Our results represent the first known example of the minimisation of a two-chain cyclic insulin-like peptide to a single-chain linear peptide that retains potent beneficial agonistic effects.
The transplantation of glucose-responsive, insulin-producing cells offers the potential for restoring glycemic control in individuals with diabetes. Pancreas transplantation and the infusion of cadaveric islets are currently implemented clinically, but these approaches are limited by the adverse effects of immunosuppressive therapy over the lifetime of the recipient and the limited supply of donor tissue. The latter concern may be addressed by recently described glucose-responsive mature beta cells that are derived from human embryonic stem cells (referred to as SC-β cells), which may represent an unlimited source of human cells for pancreas replacement therapy. Strategies to address the immunosuppression concerns include immunoisolation of insulin-producing cells with porous biomaterials that function as an immune barrier. However, clinical implementation has been challenging because of host immune responses to the implant materials. Here we report the first long-term glycemic correction of a diabetic, immunocompetent animal model using human SC-β cells. SC-β cells were encapsulated with alginate derivatives capable of mitigating foreign-body responses in vivo and implanted into the intraperitoneal space of C57BL/6J mice treated with streptozotocin, which is an animal model for chemically induced type 1 diabetes. These implants induced glycemic correction without any immunosuppression until their removal at 174 d after implantation. Human C-peptide concentrations and in vivo glucose responsiveness demonstrated therapeutically relevant glycemic control. Implants retrieved after 174 d contained viable insulin-producing cells.
The foreign body response is an immune-mediated reaction that can lead to the failure of implanted medical devices and discomfort for the recipient. There is a critical need for biomaterials that overcome this key challenge in the development of medical devices. Here we use a combinatorial approach for covalent chemical modification to generate a large library of variants of one of the most widely used hydrogel biomaterials, alginate. We evaluated the materials in vivo and identified three triazole-containing analogs that substantially reduce foreign body reactions in both rodents and, for at least 6 months, in non-human primates. The distribution of the triazole modification creates a unique hydrogel surface that inhibits recognition by macrophages and fibrous deposition. In addition to the utility of the compounds reported here, our approach may enable the discovery of other materials that mitigate the foreign body response.
Surface resistance to nonspecific protein adsorption, cell/bacterial adhesion, and biofilm formation is critical for the development and performance of biomedical and analytical devices. Significant needs and efforts have been made in the development of biocompatible and bioactive materials for antifouling surfaces, but much of the work retains an empirical flavor due to the complexity of experiments and the lack of robust theoretical models. In this review, two major classes of nonfouling materials (i.e. hydrophilic and zwitterionic materials) and associated basic nonfouling mechanisms and practical examples are discussed. Highly hydrated chemical groups with optimized physical properties of the surface, along with appropriate surface coating methods, are the keys to developing effective and stable nonfouling materials for long-term biomedical applications. The zwitterionic polymers are promising nonfouling biomaterials due to the simplicity of synthesis, ease of applicability, abundance of raw materials, and availability of functional groups.
H2 relaxin is a peptide hormone associated with a number of therapeutically relevant physiological effects, including regulation of collagen metabolism and multiple vascular control pathways. It is currently in phase III clinical trials for the treatment of acute heart failure due to its ability to induce vasodilation and influence renal function. It comprises 53 amino acids and is characterized by two separate polypeptide chains (A-B) that are cross-linked by three disulfide bonds. This size and complex structure represents a considerable challenge for the chemical synthesis of H2 relaxin, a major limiting factor for the exploration of modifications and derivatizations of this peptide, to optimize effect and drug-like characteristics. To address this issue, we describe the solid phase peptide synthesis and structural and functional evaluation of 24 analogues of H2 relaxin with truncations at the termini of its peptide chains. We show that it is possible to significantly truncate both the N and C termini of the B-chain while still retaining potent biological activity. This suggests that these regions are not critical for interactions with the H2 relaxin receptor, RXFP1. In contrast, truncations do reduce the activity of H2 relaxin for the related receptor RXFP2 by improving RXFP1 selectivity. In addition to new mechanistic insights into the function of H2 relaxin, this study identifies a critical active core with 38 amino acids. This minimized core shows similar antifibrotic activity as native H2 relaxin when tested in human BJ3 cells and thus represents an attractive receptor-selective lead for the development of novel relaxin therapeutics.
The level at which implanted sensors and sampling devices maintain their calibration is an important research area. In this work, microdialysis probes with identical geometry and different membranes, polycarbonate/polyether (PC) or polyethersulfone (PES), were used with internal standards (Vitamin B(12) (MW 1355), antipyrine (MW 188) and 2-deoxyglucose (2-DG, MW 164)) and endogenous glucose to investigate changes in their long-term calibration after implantation into the subcutaneous space of Sprague-Dawley rats. Histological analysis confirmed an inflammatory response to the microdialysis probes and the presence of a collagen capsule. The membrane extraction efficiency (percentage delivered to the tissue space) for antipyrine and 2-DG was not altered throughout the implant lifetime for either PC- or PES membranes. Yet, Vitamin B(12) extraction efficiency and collected glucose concentrations decreased during the implant lifetime. Antipyrine was administered i.v. and its concentrations obtained in both PC- and PES-membrane probes were significantly reduced between the implant day and seven (PC) or 10 (PES) days post-implantation suggesting that solute supply is critical for in vivo extraction efficiency. For the low molecular weight solutes such as antipyrine and glucose, localized delivery is not affected by the foreign body reaction, but recovery is significantly reduced. For Vitamin B(12), a larger solute, the fibrotic capsule formed around the probe significantly restricts diffusion from the implanted microdialysis probes.
Relaxin (RLX) is a peptide hormone with known antifibrotic properties. However, its significance in the lung and its role as a therapeutic agent against diseases characterized by pulmonary fibrosis are yet to be established. In this study, we examined age-related structural and functional changes in the lung of relaxin-deficient mice. Lung tissues of male and female RLX knockout (-/-) and RLX wild-type (+/+) mice at various ages were analyzed for changes in collagen expression and content. We demonstrate an age-related progression of lung fibrosis in RLX -/- mice with significantly increased tissue wet weight, collagen content and concentration, alveolar congestion, and bronchiole epithelium thickening. The increased fibrosis was associated with significantly altered peak expiratory flow and lung recoil (lung function) in RLX -/- mice. Treatment of RLX -/- mice with relaxin in early and developed stages of fibrosis resulted in the reversal of collagen deposition. Organ bath studies showed that precontracted lung strips relaxed in the presence of relaxin. Together, these data indicate that relaxin may provide a means to regulate excessive collagen deposition in diseased states characterized by pulmonary fibrosis.
The relaxin and insulin-like peptide 3 receptors, LGR7 and LGR8, respectively, are unique members of the leucine-rich repeat-containing G-protein-coupled receptor (LGR) family, because they possess an N-terminal motif with homology to the low density lipoprotein class A (LDLa) modules. By characterizing several LGR7 and LGR8 splice variants, we have revealed that the LDLa module directs ligand-activated cAMP signaling. The LGR8-short variant encodes an LGR8 receptor lacking the LDLa module, whereas LGR7-truncate, LGR7-truncate-2, and LGR7-truncate-3 all encode truncated secreted proteins retaining the LGR7 LDLa module. LGR8-short and an engineered LGR7 variant missing its LDLa module, LGR7-short, bound to their respective ligands with high affinity but lost their ability to signal via stimulation of intracellular cAMP accumulation. Conversely, secreted LGR7-truncate protein with the LDLa module was able to block relaxin-induced LGR7 cAMP signaling and did so without compromising the ability of LGR7 to bind to relaxin or be expressed on the cell membrane. Although the LDLa module of LGR7 was N-glycosylated at position Asn-14, an LGR7 N14Q mutant retained relaxin binding affinity and cAMP signaling, implying that glycosylation is not essential for optimal LDLa function. Using real-time PCR, the expression of mouse LGR7-truncate was detected to be high in, and specific to, the uterus of pregnant mice. The differential expression and evolutionary conservation of LGR7-truncate further suggests that it may also play an important role in vivo. This study highlights the essential role of the LDLa module in LGR7 and LGR8 function and introduces a novel model of GPCR regulation.
Many biomedical devices benefit from antibiofouling coatings, which can reduce biointerfacial interactions such as protein adsorption and cell attachment. In this study, we synthesized zwitterionic copolymers consisting of sulfobetaine methacrylate (SB) and 2-aminoethyl methacrylate (AE) via free radical polymerization and combined these copolymers in solution with aminomalononitrile (AMN) to form zwitterionic coatings in an autopolymerization process. The successful deposition of coatings containing different SB/AE ratios was demonstrated by X-ray photoelectron spectroscopy (XPS). The one-step surface modification process was carried out on polydimethylsiloxane (PDMS), tissue culture polystyrene (TCPS) and gold substrates, demonstrating that this method can be transferred to different substrate materials. The ability of optimized coatings to reduce serum protein adsorption was demonstrated by quartz crystal microbalance (QCM) measurements while the ability to resist cell attachment for 24 h was demonstrated using L929 mouse fibroblasts. The stability of the coating under physiological conditions was investigated and resistance to cell attachment was maintained over a period of 45 days. Furthermore, the resistance of the copolymer coating to cell attachment was maintained after both ethylene oxide sterilization and autoclaving. Finally copolymer modified PDMS samples were investigated in regard to their ability to reduce the foreign body response in vivo. Here, a significant reduction in the capsule thickness (approximately 50%) was observed in nude mice after 2 weeks and 4 weeks. We expect that the one-step, facile and versatile surface modification strategy discussed here will find applications in biomedical device applications.
Fibrosis is an inadequate response to tissue stress with very few therapeutic options to prevent its progression to organ dysfunction. There is an urgent need to identify drugs with a therapeutic potential for fibrosis, either by designing and developing new compounds or by repurposing drugs already in clinical use which were developed for other indications. In this Perspective, we summarize some pathways and biological targets involved in fibrosis development and maintenance, focusing on common mechanisms between organs and diseases. We review the therapeutic agents under experimental development, clinical trials or in clinical use for the treatment of fibrotic disorders, evaluating the reasons for the discrepancies observed between preclinical and clinical results. We also discuss the improvement that we envision in the development of therapeutic molecules able to achieve improved potential for treatment, including indirect modulators, targeting approaches or drug combinations.
Reducing the foreign body response (FBR) to implanted biomaterials will enhance their performance in tissue engineering. Poly(ethylene glycol) (PEG) hydrogels are increasingly popular for this application due to their low cost, ease of use, and the ability to tune their compliance via molecular weight and crosslinking densities. PEG hydrogels can elicit chronic inflammation in vivo, but recent evidence has suggested that extremely hydrophilic, zwitterionic materials and particles can evade the immune system. To combine the advantages of PEG-based hydrogels with the hydrophilicity of zwitterions, we synthesized hydrogels with co-monomers PEG and the zwitterion phosphorylcholine (PC). Recent evidence suggests that stiff hydrogels elicit increased immune cell adhesion to hydrogels, which we attempted to reduce by increasing hydrogel hydrophilicity. Surprisingly, hydrogels with the highest amount of zwitterionic co-monomer elicited the highest FBR we observed. Lowering the hydrogel modulus (165 kPa to 3 kPa), or PC content (20 wt% to 0 wt%), mitigated this effect. A high density of macrophages was found at the surface of implants associated with a high FBR, and mass spectrometry analysis of the proteins adsorbed to these gels implicated extracellular matrix, immune response, and cell adhesion protein categories as drivers of macrophage recruitment to these hydrogels. Overall, we show that modulus regulates macrophage adhesion to zwitterionic-PEG hydrogels, and demonstrate that chemical modifications to hydrogels should be studied in parallel with their physical properties to optimize implant design.
The peptide relaxin was first identified as an important circulating hormone during pregnancy over 90 years ago. Research over many years defined the numerous biological roles that relaxin plays throughout pregnancy in many mammalian species. These important biological actions have led to the testing of relaxin as a therapeutic agent for a number of indications. The discovery of the relaxin receptor, RXFP1, in 2002 facilitated the better understanding of the cellular targets of relaxin, its mechanism of action and enabled the development of relaxin mimetics and screening for small molecule agonists. Additionally, the rapid expansion of the genome databases and bioinformatics tools has significantly advanced our understanding of the evolution of the relaxin/RXFP1 signaling system. It is now clear that the relaxin-RXFP1 signaling axis is far more ancient than previously appreciated with important roles for invertebrate relaxin-like peptides in reproductive and non-reproductive functions. This review summarizes these advances as well as developments in drug targeting of RXFP1. Hence the complex mode of activation of RXFP1 is discussed as is the discovery and development of a peptide mimetic and small molecule agonist. Detailed signaling studies are summarized which highlight the cell specific signaling of a peptide mimetic and biased signaling of a small molecule agonist. These studies highlight the complexities of targeting peptide GPCRs such as RXFP1.
Tissues stiffen during aging and during the pathological progression of cancer, fibrosis, and cardiovascular disease. Extracellular matrix stiffness is emerging as a prominent mechanical cue that precedes disease and drives its progression by altering cellular behaviors. Targeting extracellular matrix mechanics, by preventing or reversing tissue stiffening or interrupting the cellular response, is a therapeutic approach with clinical potential. Major drivers of changes to the mechanical properties of the extracellular matrix include phenotypically converted myofibroblasts, transforming growth factor β (TGFβ), and matrix cross-linking. Potential pharmacological interventions to overcome extracellular matrix stiffening are emerging clinically. Aside from targeting stiffening directly, alternative approaches to mitigate the effects of increased matrix stiffness aim to identify and inhibit the downstream cellular response to matrix stiffness. Therapeutic interventions that target tissue stiffening are discussed in the context of their limitations, preclinical drug development efforts, and clinical trials.
Tissue damage and inflammation are important triggers for regeneration and fibrosis. Tissue damage not only induces inflammation in general, it also determines the type and polarization of inflammation by recruiting and activating a variety of different cells types of the innate and adaptive immune system. This review focuses on the pathways leading from tissue damage to inflammation, from inflammation to fibrosis and from fibrosis to function. It covers the pro- and antifibrotic properties of immunological mediators released from T cells, monocytes/macrophages, innate lymphoid cells, basophils and eosinophils and takes into account that extracellular matrix proteins are not only produced by mesenchymal fibroblasts but also by infiltrating hematopoietic cells. The special requirements for activation and recruitment of these so called fibrocytes are also summarized.
The human relaxin peptide family consists of seven cystine-rich peptides, four of which are known to signal through relaxin family peptide receptors, RXFP1-4. As these peptides play a vital role physiologically and in various diseases, they are of considerable importance for drug discovery and development. Detailed structure-activity relationship (SAR) studies towards understanding the role of important residues in each of these peptides have been reported over the years and utilized for the design of antagonists and minimized agonist variants. This review summarizes the current knowledge of the SAR of human relaxin 2 (H2 relaxin), human relaxin 3 (H3 relaxin), human insulin-like peptide 3 (INSL3) and human insulin-like peptide 5 (INSL5).
Linked articles:
This article is part of a themed section on Recent Progress in the Understanding of Relaxin Family Peptides and their Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.10/issuetoc.
The healing process after implantation of biomaterials involves the interaction of many contributing factors. Besides their in vivo functionality, biomaterials also require characteristics that allow their integration into the designated tissue without eliciting an overshooting foreign body reaction. The targeted design of biomaterials with these features, thus, needs understanding of the molecular mechanisms of the foreign body reaction. Much effort has been put into research on the interaction of engineered materials and the host tissue. This elucidated many aspects of the five foreign body reaction phases, i.e. protein adsorption, acute inflammation, chronic inflammation, foreign body giant cell formation and fibrous capsule formation. However, in practice, it is still difficult to predict the response against a newly designed biomaterial purely based on the knowledge of its physical-chemical surface features. This insufficient knowledge leads to a high number of factors potentially influencing the foreign body reaction, which have to be analyzed in complex animal experiments including appropriate data-based sample sizes. This review is focused on the current knowledge on the general mechanisms of the foreign body reaction against biomaterials and the influence of biomaterial surface topography and chemical and physical features on the quality and quantity of the reaction. This article is protected by copyright. All rights reserved.
Fibrosis refers to the hardening or scarring of tissues that usually results from aberrant wound healing in response to organ injury, and its manifestations in various organs have collectively been estimated to contribute to around 45-50% of deaths in the Western world. Despite this, there is currently no effective cure for the tissue structural and functional damage induced by fibrosis-related disorders. Relaxin meets several criteria of an effective anti-fibrotic based on its specific ability to inhibit pro-fibrotic cytokine and/or growth factor-mediated, but not normal/unstimulated, fibroblast proliferation, differentiation and matrix production. Furthermore, relaxin augments matrix degradation through its ability to up-regulate the release and activation of various matrix-degrading matrix metalloproteinases and/or being able to down-regulate tissue inhibitor of metalloproteinase activity. Relaxin can also indirectly suppress fibrosis through its other well-known (anti-inflammatory, antioxidant, anti-hypertrophic, anti-apoptotic, angiogenic, wound healing and vasodilator) properties. This review will outline the organ-specific and general anti-fibrotic significance of exogenously administered relaxin and its mechanisms of action that have been documented in various non-reproductive organs such as the cardiovascular system, kidney, lung, liver, skin and tendons. In addition, it will outline the influence of sex on relaxin's anti-fibrotic actions, highlighting its potential as an emerging anti-fibrotic therapeutic.
Linked articles:
This article is part of a themed section on Recent Progress in the Understanding of Relaxin Family Peptides and their Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.10/issuetoc.
Polymer brushes have been utilized as a proper model interface for the investigation of wetting, lubrication, hydrated chain dimension and the structure of hydrated water at hydrated polymer interfaces. Zwitterionic polyelectrolyte-tethered surfaces represent significant anti-fouling materials for bio-macromolecules, marine fouling organisms and pipeline foulants. Systematic anti-fouling tests for various polymer brushes were performed to demonstrate the critical factors for universal anti-fouling properties. In this focused review, material design concepts and recent progress on anti-fouling polymer materials are reviewed, and our recent research on anti-fouling studies using polymer brushes is provided.
Continuous glucose monitoring (CGM) sensors are often advocated as a clinical solution to improve long-term glycemic control in the context of diabetes. Subcutaneous sensor inflammatory response, fouling and fibrous encapsulation resulting from the host foreign body response (FBR) reduce sensor sensitivity to glucose, eventually resulting in sensor performance compromise and device failure. Several combination device strategies load CGM sensors with drug payloads that release locally to tissue sites to mitigate FBR-mediated sensor failure. In this study, the mast cell-targeting tyrosine kinase inhibitor, masitinib, was released from degradable polymer microspheres delivered from the surfaces of FDA-approved human commercial CGM needle-type implanted sensors in a rodent subcutaneous test bed. By targeting the mast cell c-Kit receptor and inhibiting mast cell activation and degranulation, local masitinib penetration around the CGM to several hundred microns sought to reduce sensor fibrosis to extend CGM functional lifetimes in subcutaneous sites. Drug-releasing and control CGM implants were compared in murine percutaneous implant sites for 21 days using direct-wire continuous glucose reporting. Drug-releasing implants exhibited no significant difference in CGM fibrosis at implant sites but showed relatively stable continuous sensor responses over the study period compared to blank microsphere control CGM implants.
Dexamethasone-releasing PLGA poly(lactic-co-glycolic acid) microsphere/PVA (polyvinyl alcohol) hydrogel composite coatings have been shown to prevent the foreign body reaction (FBR) to subcutaneous implants in small and large animal models. Such coatings were developed to extend the lifetime of implantable biosensors. However, long-term exposure of tissue to low levels of dexamethasone results in a reduction in blood vessel density due to the anti-angiogenic effect of dexamethasone. This mild effect, while not threatening to the subject's health, may interfere with analyte detection and the sensor response time over the long-term. The present work is focused on the development of coatings that deliver combinations of three tissue response modifiers (TRMs): dexamethasone, VEGF (vascular endothelial growth factor) and PDGF (platelet derived growth factor). Dexamethasone, VEGF and PDGF prevent the FBR, increase angiogenesis and promote blood vessel maturation (which increases blood flow), respectively. To minimize any potential interference among these three TRMs (for example, PDGF increases fibrosis), the relative doses of dexamethasone, VEGF and PDGF were adjusted. It was determined that: a) all three TRMs are required for maximum promotion of angiogenesis, blood vessel maturation and prevention of the FBR; b) VEGF has to be administered at higher doses than PDGF; c) an increase in dexamethasone dosing must be accompanied by a proportional increase in growth factor dosing; and d) modification of the TRM ratio can achieve a constant capillary density throughout the implantation period which is important for applications such as biosensors to maintain sensitivity and a stable sensor baseline. Moreover, an osmosis-driven process for encapsulation of proteins in PLGA microspheres that showed low burst release was developed.
Copyright © 2015. Published by Elsevier B.V.
The influence of electrostatic interactions and/or acylation on release of charged (“sticky”) agents from biodegradable polymer matrices was systematically characterized. We hypothesized that release of peptides with positive charge would be hindered from negatively charged poly(lactic-co-glycolic acid) (PLGA) microparticles. Thus, we investigated release of peptides with different degrees of positive charge from several PLGA microparticle formulations, with different molecular weights and/or end groups (acid- or ester-terminated). Indeed, release studies revealed distinct inverse correlations between the amount of positive charge on peptides and their release rates from each PLGA microparticle formulation. Furthermore, we examined the case of peptides with net charge that changes from negative to positive within the pH range observed in degrading microparticles. These charge changing peptides displayed counterintuitive release kinetics, initially releasing faster from slower degrading (less acidic) microparticles, and releasing slower from the faster degrading (more acidic) microparticles. Importantly, trends between agent charge and release rates for model peptides also translated to larger, therapeutically relevant proteins and oligonucleotides. The results of these studies may improve future design of controlled release systems for numerous therapeutic biomolecules exhibiting positive charge, ultimately reducing time-consuming and costly trial and error iterations of such formulations.
Heart failure due to Myocardial Infarction (MI) remains the leading cause of death worldwide due to the inability of myocardial tissue to regenerate following infarction. Current therapies could only retard the progression of disease but fails to bring functional improvement and cardiac regeneration. The present study analyze the potentials of Poly(glycerol sebacate)/Fibrinogen (PGS/Fib) core/shell fibers as a structural support and initial entrapment of cells in an in vivo porcine model using echocardiography, histology and immunohistochemistry. The echocardiography results showed that the increased ejection fraction (EF) in PGS/Fib/VEGF/Cells compared to MI controls. The percentage increase in the End Diastolic Volume (EDV) dimension from post-MI period to 4 weeks follow-up was the least in PGS/Fib/VEGF/Cells groups compared to MI and cell control group proving that the PGS/Fib/VEGF/Cells group restored the left ventricle (LV) function after MI, evident from the improvement in EF and prevention of LV enlargement. Further, immunohistochemistry results demonstrated that the most of transplanted MSCs within the PGS/Fib/VEGF scaffolds expressed cardiac marker proteins troponin and actinin and endothelial cell marker protein CD31 indicating differentiation of human bone marrow mesenchymal stem cells (MSCs) into cardiac cells and endothelial cells. The developed nanofibrous cardiac patch PGS/Fib/VEGF/Cells provides both functional and structural integrity to the infarcted myocardium and also serves as a suitable matrix for the entrapment of MSCs in clinical applications for cardiac tissue engineering.
Mast cells are recognized for their functional role in wound healing, allergic and inflammatory responses, host responses that are frequently detrimental to implanted biomaterials if extended beyond acute reactivity. These tissue reactions are especially impacting to the performance of sensing implants such as continuous glucose monitoring (CGM) devices. Our hypothesis that effective blockade of mast cell activity around implants could alter the host foreign body response (FBR) and enhance the in vivo lifetime of these implantable devices motivated this study. Stem cell factor (SCF) and its ligand c-KIT receptor are critically important for mast cell survival, differentiation, and degranulation. Therefore, a mast cell-deficient sash mouse model was used to assess mast cell relationships to the in vivo performance of CGM implants. Additionally, local delivery of a tyrosine kinase inhibitor (TKI) that inhibits c-KIT activity was also used to evaluate the role of mast cells in modulating the FBR. Model sensor implants comprising polyester fibers coated with a rapidly dissolving polymer coating containing drug-releasing degradable microspheres were implanted subcutaneously in sash mice for various time points, and the FBR was evaluated for chronic inflammation and fibrous capsule formation around the implants. No significant differences were observed in the foreign body capsule formation between control and drug-releasing implant groups in mast cell-deficient mice. However, fibrous encapsulation was significantly greater around the drug-releasing implants in sash mice compared to drug-releasing implants in wild-type (e.g., mast cell competent) mice. These results provide insights into the role of mast cells in the FBR, suggesting that mast cell deficiency provides alternative pathways for host inflammatory responses to implanted biomaterials.
The host foreign body response (FBR) adversely effects the performance of numerous implanted biomaterials especially biosensors, including clinically popular glucose-monitoring sensors. Reactive formation of a fibrous capsule around implanted sensors hinders the transport of essential analytes to the sensor from the surrounding tissue, resulting in loss of glucose response sensitivity and eventual sensor failure. Several strategies have sought to mitigate the foreign body response's effects on CGM sensors through the use of local delivery of pharmaceuticals and biomolecules with limited success. This study describes release of a tyrosine kinase inhibitor - masitinib - from the sensor implant to target tissue resident mast cells as key mediators of the FBR. Model implants are coated with a composite polymer hydrophilic matrix that rapidly dissolves upon tissue implantation to deposit slower-degrading polymer microparticles containing masitinib. Matrix dissolution limits coating interference with sensor function while establishing a local controlled-release delivery depot formulation to alter implant tissue pharmacology and addressing the FBR. Drug efficacy was evaluated in a murine subcutaneous pocket implant model. Drug release extends to more than 30 days in vitro. The resulting FBR in vivo, evaluated by implant capsule thickness and inflammatory cell densities at 14, 21, and 28 days, displays statistically significant reduction in capsule thickness around masitinib-releasing implant sites compared to control implant sites.
A simple, inexpensive and versatile custom built reactor for experimental plasma deposition and surface treatment is presented. The two outstanding features are a compact web conveyancing system with a web speed that is variable in the range 0.15–6 m min−1, and a defined gas flow path. The web transport accepts rolls containing of the order of 100 m of flexible thin substrate and is reversible in order to enable multistep plasma processing without breaking the vacuum. The reactor provides stable and uniform plasma conditions for extended coating of thin polymer films by plasma polymerization.
The performance of implantable biomedical devices is impeded by the foreign-body reaction, which results in formation of a dense collagenous capsule that blocks mass transport and/or electric communication between the implant and the body. No known materials or coatings can completely prevent capsule formation. Here we demonstrate that ultra-low-fouling zwitterionic hydrogels can resist the formation of a capsule for at least 3 months after subcutaneous implantation in mice. Zwitterionic hydrogels also promote angiogenesis in surrounding tissue, perhaps owing to the presence of macrophages exhibiting phenotypes associated with anti-inflammatory, pro-healing functions. Thus, zwitterionic hydrogels may be useful in a broad range of applications, including generation of biocompatible implantable medical devices and tissue scaffolds.
Application of implantable glucose biosensors for "real-time" monitoring is reliant on controlling the negative tissue reaction at the sensor tissue interphase. A novel polymer coating consisting of poly(lactic-co-glycolic) acid (PLGA) microsphere dispersed in poly(vinyl alcohol) (PVA) hydrogels was evaluated in combination with dummy sensors as a "smart" drug eluting biocompatible coating for implantable biosensors to prevent the foreign body response, and thus enhance sensor performance in vivo. The polymeric microspheres slowly release tissue-modifying drugs at the implantation sites to control the inflammation and fibrous encapsulation, while the hydrogel allows rapid analyte diffusion to the sensing elements. Dummy sensors with identical dimensions to that of the functional glucose sensors (0.5 x 0.5 x 5 mm) were coated with the PLGA/PVA composites using a mold fabrication process. Both normal and diabetic rats were used in the current study to investigate the effect of the diabetic state on tissue sensor interactions. It was evident that the PLGA/PVA hydrogel composite was able to form a uniform coating around the dummy sensor and stayed intact throughout the course of the study (one month). Tissue samples containing dummy sensors that were coated with dexamethasone free composites exhibited acute and chronic inflammation as well as fibrous encapsulation in both normal and diabetic rats. However, the diabetic rats exhibited decreased intensity and delayed onset of the foreign body response following implantation of drug free dummy sensors in comparion to those of normal rats. On the other hand, tissues containing dummy sensors that were coated with dexamethasone containing composites remained normal (i.e. similar to untreated tissues), with no inflammatory reaction or fibrous encapsulation occurring over the one-month period in both the normal and diabetic rats. The feasibility of utilizing PLGA microsphere/PVA hydrogel composites as coatings for implantable biosensors was demonstrated. This polymeric composite is an innovative approach to control the foreign body reaction at the tissue-device interface to prolong biosensor lifetime.
Background:
Serelaxin, recombinant human relaxin-2, is a vasoactive peptide hormone with many biological and haemodynamic effects. In a pilot study, serelaxin was safe and well tolerated with positive clinical outcome signals in patients with acute heart failure. The RELAX-AHF trial tested the hypothesis that serelaxin-treated patients would have greater dyspnoea relief compared with patients treated with standard care and placebo.
Methods:
RELAX-AHF was an international, double-blind, placebo-controlled trial, enrolling patients admitted to hospital for acute heart failure who were randomly assigned (1:1) via a central randomisation scheme blocked by study centre to standard care plus 48-h intravenous infusions of placebo or serelaxin (30 μg/kg per day) within 16 h from presentation. All patients had dyspnoea, congestion on chest radiograph, increased brain natriuretic peptide (BNP) or N-terminal prohormone of BNP, mild-to-moderate renal insufficiency, and systolic blood pressure greater than 125 mm Hg. Patients, personnel administering study drug, and those undertaking study-related assessments were masked to treatment assignment. The primary endpoints evaluating dyspnoea improvement were change from baseline in the visual analogue scale area under the curve (VAS AUC) to day 5 and the proportion of patients with moderate or marked dyspnoea improvement measured by Likert scale during the first 24 h, both analysed by intention to treat. This trial is registered at ClinicalTrials.gov, NCT00520806.
Findings:
1161 patients were randomly assigned to serelaxin (n=581) or placebo (n=580). Serelaxin improved the VAS AUC primary dyspnoea endpoint (448 mm × h, 95% CI 120-775; p=0·007) compared with placebo, but had no significant effect on the other primary endpoint (Likert scale; placebo, 150 patients [26%]; serelaxin, 156 [27%]; p=0·70). No significant effects were recorded for the secondary endpoints of cardiovascular death or readmission to hospital for heart failure or renal failure (placebo, 75 events [60-day Kaplan-Meier estimate, 13·0%]; serelaxin, 76 events [13·2%]; hazard ratio [HR] 1·02 [0·74-1·41], p=0·89] or days alive out of the hospital up to day 60 (placebo, 47·7 [SD 12·1] days; serelaxin, 48·3 [11·6]; p=0·37). Serelaxin treatment was associated with significant reductions of other prespecified additional endpoints, including fewer deaths at day 180 (placebo, 65 deaths; serelaxin, 42; HR 0·63, 95% CI 0·42-0·93; p=0·019).
Interpretation:
Treatment of acute heart failure with serelaxin was associated with dyspnoea relief and improvement in other clinical outcomes, but had no effect on readmission to hospital. Serelaxin treatment was well tolerated and safe, supported by the reduced 180-day mortality.
Funding:
Corthera, a Novartis affiliate company.
Mass spectrometry has played a key role in characterizing the primary structure of native and recombinant relaxin, a peptide hormone that induces ripening of the cervix prior to childbirth. The peptide is composed of two chains, A and B, and is formed from a single-chain prohormone, as is insulin. Aside from conserved cysteines, though, it has little sequence homology with insulin. Due to the small amounts of native peptide initially available ( < 10 pmol), traditional techniques could not provide information on the blocked A-chain sequence, on the carboxy-terminal sequences, nor on other possible post-translational modifications. Mass measurements by fast atom bombardment (FAB) were made on reduced human relaxin isolated from corpora lutea. The detection limit by FAB for reduced relaxin was 500 fmol. The B-chain was four amino acids shorter than expected from comparison of the previously known cDNA sequence with homologous rat and porcine sequences. The A-chain, as predicted, was 24 amino acids in length and had a pyroglutamic acid residue on the amino-terminus. The purified samples were homogeneous with no other post-translational modifications. The recombinant relaxin molecule was also extensively characterized by mass spectrometry. In addition to the intact molecule, all tryptic peptides were characterized by FAB. A capillary high-performance liquid chromatography continuous-flow FAB system, developed for high-sensitivity peptide mapping, aided in these analyses. Finally, the three disulfide bonds were shown by tandem mass spectrometry to match those of insulin.
The pharmacokinetics of recombinant human relaxin (rhRlx) after intravenous (iv) bolus administration and the absorption of rhRlx after intracervical or intravaginal administration were determined in nonpregnant women. The study was conducted in two parts. In part I, 25 women received 0.01 mg/kg rhRlx iv. After a minimum 7-day washout period, these women were dosed intracervically (n = 10) or intravaginally (n = 15) with 0.75 or 1.5 mg rhRlx, respectively, in 3% methylcellulose gel. Part II was a double-blind, randomized, three-way crossover study in 26 women. At 1-month intervals, each woman received one of three intravaginal treatments consisting of 0 (placebo), 1, or 6 mg rhRlx in 3% methylcellulose gel. The serum concentrations of relaxin following iv administration were described as the sum of three exponentials. The mean (±SD) initial, intermediate, and terminal half-lives were 0.09 ± 0.04, 0.72 ± 0.11, and 4.6 ± 1.2 hr, respectively. Most of the area under the curve was associated with the intermediate half-life. The weight-normalized clearance was 170 ± 50 mL/hr/kg. The observed peak concentration was 98 ± 29 ng/mL, and the weight-normalized initial volume of distribution was 78 ± 40 mL/kg, which is approximately equivalent to the serum volume. If central compartment elimination was assumed, the volume of distribution at steady state (V
ss/W) was 280 ± 100 mL/kg, which is approximately equivalent to extracellular fluid volume. V
ss/W could be as large as 1300 ± 400 mL/kg without this assumption. After intravaginal administration of the placebo gel, endogenous relaxin concentrations were evident (i.e., ≥20 pg/mL) in 9 of the 26 women (maximum concentrations, 23–234 pg/mL). A similar proportion of women (approximately 35–40%) exhibited measurable serum concentrations of relaxin following intravaginal rhRlx treatment; this proportion increased to 90% following intracervical rhRlx treatment. For both routes of administration, the maximum serum concentrations of relaxin were usually within the range of values observed for endogenous relaxin, suggesting that the absorption of rhRlx was minimal.
A library of cationic polymers, poly(beta-amino alcohols) with a great chemical diversity are synthesized using combinatorial polymerization. These polymers, when immobilized on a surface, drastically affect the behavior of monocyte/macrophage cells in vitro and early inflammatory reactions in vivo. Certain polymers are found capable of mitigating the foreign-body responses.
Oxidative reactions play important roles in a variety of biochemical events ranging from normal metabolism to aging and disease processes. Proteins represent major targets for modification in these reactions, and identification of sites and structures of modifications may lead to mechanistic understanding and approaches for prevention. In this Account, the utility of mass spectrometry and its advantages are described for the identification of oxidative modifications to protein targets. A variety of examples are provided to illustrate how modifications are accurately identified and quantitated using modern methods of ionization coupled with HPLC and tandem mass spectrometry.
The effects of pinning density, chain length, and 'cloud point' (CP) versus non-CP grafting conditions have been studied on the ability of polyethylene glycol (PEG) layers to minimize adsorption from a multicomponent (lysozyme, human serum albumin (HSA), IgG and lactoferrin) protein solution. Methoxy-terminated aldehyde-PEG (M-PEG, MW 5000) and dialdehyde-PEG (PEG(ald)2, MW 3400) were grafted by reductive amination onto two surfaces of different amine group density, generated by radiofrequency glow discharge (r.f.g.d.) deposition of n-heptylamine (HA) (low density) or allylamine (AlA) (high density) r.f.g.d. polymer layers. The PEG graft density was varied also by increasing the temperature and salt (K2SO4) content of the grafting solution; it reached a maximum at the CP of the PEGs. The CP reaction conditions were critical for producing PEG layers capable of minimizing protein adsorption. X-ray photoelectron spectroscopy (XPS) showed that under these conditions, PEG(ald)2 produced a thick linear PEG layer, most likely by aldol condensation. Protein adsorption was assessed using XPS and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF-MS) in the surface mode (Surface-MALDI-MS). Coatings grafted at non-CP conditions showed some protein adsorption, as did the HA/M-PEG layer grafted at the CP. On the other hand, no protein adsorption was detected on the HA/PEG(ald)2, AlA/M-PEG, and AlA/PEG(ald)2 surfaces when grafted at the CP. Thus, the effects of pinning density and chain length are interrelated, but the key factor is optimization of PEG chain density by use of the CP conditions, provided that a sufficient density of pinning sites exists.
Since its discovery as a reproductive hormone 80 years ago, relaxin has been implicated in a number of pregnancy-related functions involving extracellular matrix (ECM) turnover and collagen degradation. It is now becoming evident that relaxin's ability to reduce matrix synthesis and increase ECM degradation has important implications in several nonreproductive organs, including the heart, lung, kidney, liver and skin. The identification of relaxin and RXFP1 (Relaxin family peptide receptor-1) mRNA and/or binding sites in cells or vessels of these nonreproductive tissues, has confirmed them as targets for relaxin binding and activity. Recent studies on Rln1 and Rxfp1 gene-knockout mice have established relaxin as an important naturally occurring and protective moderator of collagen turnover, leading to improved organ structure and function. Furthermore, through its ability to regulate the ECM and in particular, collagen at multiple levels, relaxin has emerged as a potent anti-fibrotic therapy, with rapid-occurring efficacy. It not only prevents fibrogenesis, but also reduces established scarring (fibrosis), which is a leading cause of organ failure and affects several tissues regardless of etiology. This chapter will summarize these coherent findings as a means of highlighting the significance and therapeutic potential of relaxin.
Fibrosis is defined by the overgrowth, hardening, and/or scarring of various tissues and is attributed to excess deposition of extracellular matrix components including collagen. Fibrosis is the end result of chronic inflammatory reactions induced by a variety of stimuli including persistent infections, autoimmune reactions, allergic responses, chemical insults, radiation, and tissue injury. Although current treatments for fibrotic diseases such as idiopathic pulmonary fibrosis, liver cirrhosis, systemic sclerosis, progressive kidney disease, and cardiovascular fibrosis typically target the inflammatory response, there is accumulating evidence that the mechanisms driving fibrogenesis are distinct from those regulating inflammation. In fact, some studies have suggested that ongoing inflammation is needed to reverse established and progressive fibrosis. The key cellular mediator of fibrosis is the myofibroblast, which when activated serves as the primary collagen-producing cell. Myofibroblasts are generated from a variety of sources including resident mesenchymal cells, epithelial and endothelial cells in processes termed epithelial/endothelial-mesenchymal (EMT/EndMT) transition, as well as from circulating fibroblast-like cells called fibrocytes that are derived from bone-marrow stem cells. Myofibroblasts are activated by a variety of mechanisms, including paracrine signals derived from lymphocytes and macrophages, autocrine factors secreted by myofibroblasts, and pathogen-associated molecular patterns (PAMPS) produced by pathogenic organisms that interact with pattern recognition receptors (i.e. TLRs) on fibroblasts. Cytokines (IL-13, IL-21, TGF-beta1), chemokines (MCP-1, MIP-1beta), angiogenic factors (VEGF), growth factors (PDGF), peroxisome proliferator-activated receptors (PPARs), acute phase proteins (SAP), caspases, and components of the renin-angiotensin-aldosterone system (ANG II) have been identified as important regulators of fibrosis and are being investigated as potential targets of antifibrotic drugs. This review explores our current understanding of the cellular and molecular mechanisms of fibrogenesis.