Immune profiling of adeno-associated virus response identifies B cell-specific targets that enable vector re-administration in mice.

Adeno-associated virus (AAV) vector-based gene therapies can be applied to a wide range of diseases. AAV expression can last for months to years, but vector re-administration may be necessary to achieve life-long treatment. Unfortunately, immune responses against these vectors are potentiated after the first administration, preventing the clinical use of repeated administration of AAVs. Reducing the immune response against AAVs while minimizing broad immunosuppression would improve gene delivery efficiency and long-term safety. In this study, we quantified the contributions of multiple immune system components of the anti-AAV response in mice. We identified B-cell-mediated immunity as a critical component preventing vector re-administration. Additionally, we found that IgG depletion alone was insufficient to enable re-administration, suggesting IgM antibodies play an important role in the immune response against AAV. Further, we found that AAV-mediated transduction is improved in µMT mice that lack functional IgM heavy chains and cannot form mature B-cells relative to wild-type mice. Combined, our results suggest that B-cells, including non-class switched B-cells, are a potential target for therapeutics enabling AAV re-administration. Our results also suggest that the µMT mice are a potentially useful experimental model for gene delivery studies since they allow repeated dosing for more efficient gene delivery from AAVs.

Reducing Peri-implant Capsule Thickness in Submuscular Rodent Model of Breast Reconstruction With Delayed Radiotherapy.

INTRODUCTION: Capsular contracture remains the most common complication following device-based breast reconstruction, occurring in up to 50% of women who also undergo adjuvant radiotherapy either before or after device-based reconstruction. While certain risk factors for capsular contracture have been identified, there remains no clinically effective method of prevention. The purpose of the present study is to determine the effect of coating the implant with the novel small molecule Met-Z2-Y12, with and without delayed, targeted radiotherapy, on capsule thickness and morphologic change around smooth silicone implants placed under the latissimus dorsi in a rodent model. METHODS: Twenty-four female Sprague Dawley rats each had 2 mL smooth round silicone breast implants implanted bilaterally under the latissimus dorsi muscle. Twelve received uncoated implants and twelve received implants coated with Met-Z2-Y12. Half of the animals from each group received targeted radiotherapy (20 Gray) on postoperative day ten. At three and 6 months after implantation, the tissue surrounding the implants was harvested for analysis of capsular histology including capsule thickness. Additionally, microCT scans were qualitatively analyzed for morphologic change. RESULTS: Capsules surrounding Met-Z2-Y12-coated implants were significantly thinner (P = 0.006). The greatest difference in capsule thickness was seen in the irradiated 6-month groups, where mean capsule thickness was 79.1 ± 27.3 µm for uncoated versus 50.9 ± 9.6 µm for Met-Z2-Y12-coated implants (P = 0.038). At the time of explant, there were no capsular morphologic differences between the groups either grossly or per microCT. CONCLUSIONS: Met-Z2-Y12 coating of smooth silicone breast implants significantly reduces capsule thickness in a rodent model of submuscular breast reconstruction with delayed radiotherapy.

Colony stimulating factor-1 receptor is a central component of the foreign body response to biomaterial implants in rodents and non-human primates.

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.

Size- and shape-dependent foreign body immune response to materials implanted in rodents and non-human primates.

The efficacy of implanted biomedical devices is often compromised by host recognition and subsequent foreign body responses. Here, we demonstrate the role of the geometry of implanted materials on their biocompatibility in vivo. In rodent and non-human primate animal models, implanted spheres 1.5 mm and above in diameter across a broad spectrum of materials, including hydrogels, ceramics, metals and plastics, significantly abrogated foreign body reactions and fibrosis when compared with smaller spheres. We also show that for encapsulated rat pancreatic islet cells transplanted into streptozotocin-treated diabetic C57BL/6 mice, islets prepared in 1.5-mm alginate capsules were able to restore blood-glucose control for up to 180 days, a period more than five times longer than for transplanted grafts encapsulated within conventionally sized 0.5-mm alginate capsules. Our findings suggest that the in vivo biocompatibility of biomedical devices can be significantly improved simply by tuning their spherical dimensions.

Injectable and Glucose-Responsive Hydrogels Based on Boronic Acid-Glucose Complexation.

Injectable hydrogels have been widely used for a number of biomedical applications. Here, we report a new strategy to form an injectable and glucose-responsive hydrogel using the boronic acid-glucose complexation. The ratio of boronic acid and glucose functional groups is critical for hydrogel formation. In our system, polymers with 10-60% boronic acid, with the balance being glucose-modified, are favorable to form hydrogels. These hydrogels are shear-thinning and self-healing, recovering from shear-induced flow to a gel state within seconds. More importantly, these polymers displayed glucose-responsive release of an encapsulated model drug. The hydrogel reported here is an injectable and glucose-responsive hydrogel constructed from the complexation of boronic acid and glucose within a single component polymeric material.

Development of a 3D printed perfusable in vitro blood-brain barrier model for use as a scalable screening tool.

Despite recent technological advances in drug discovery, the success rate for neurotherapeutics remains alarmingly low compared to treatments for other areas of the body. One of the biggest challenges for delivering therapeutics to the central nervous system (CNS) is the presence of the blood-brain barrier (BBB). In vitro blood-brain barrier models with high predictability are essential to aid in designing parameters for new therapeutics, assess their ability to cross the BBB, and investigate therapeutic strategies that can be employed to enhance transport. Here, we demonstrate the development of a 3D printable hydrogel blood-brain barrier model that mimics the cellular composition and structure of the blood-brain barrier with human brain endothelial cells lining the surface, pericytes in direct contact with the endothelial cells on the abluminal side of the endothelium, and astrocytes in the surrounding printed bulk matrix. We introduce a simple, static printed hemi-cylinder model to determine design parameters such as media selection, co-culture ratios, and cell incorporation timing in a resource-conservative and high-throughput manner. Presence of cellular adhesion junction, VE-Cadherin, efflux transporters, P-glycoprotein (P-gp) and Breast cancer resistance protein (BCRP), and receptor-mediated transporters, Transferrin receptor (TfR) and low-density lipoprotein receptor-related protein 1 (LRP1) were confirmed via immunostaining demonstrating the ability of this model for screening in therapeutic strategies that rely on these transport systems. Design parameters determined in the hemi-cylinder model were translated to a more complex, perfusable vessel model to demonstrate its utility for determining barrier function and assessing permeability to model therapeutic compounds. This 3D-printed blood-brain barrier model represents one of the first uses of projection stereolithography to fabricate a perfusable blood-brain barrier model, enabling the patterning of complex vessel geometries and precise arrangement of cell populations. This model demonstrates potential as a new platform to investigate the delivery of neurotherapeutic compounds and drug delivery strategies through the blood-brain barrier, providing a useful in vitro screening tool in central nervous system drug discovery and development.

Persistent tailoring of MSC activation through genetic priming.

Mesenchymal stem/stromal cells (MSCs) are an attractive platform for cell therapy due to their safety profile and unique ability to secrete broad arrays of immunomodulatory and regenerative molecules. Yet, MSCs are well known to require preconditioning or priming to boost their therapeutic efficacy. Current priming methods offer limited control over MSC activation, yield transient effects, and often induce expression of pro-inflammatory effectors that can potentiate immunogenicity. Here, we describe a 'genetic priming' method that can both selectively and sustainably boost MSC potency via the controlled expression of the inflammatory-stimulus-responsive transcription factor IRF1 (interferon response factor 1). MSCs engineered to hyper-express IRF1 recapitulate many core responses that are accessed by biochemical priming using the proinflammatory cytokine interferon-γ (IFNγ). This includes the upregulation of anti-inflammatory effector molecules and the potentiation of MSC capacities to suppress T cell activation. However, we show that IRF1-mediated genetic priming is much more persistent than biochemical priming and can circumvent IFNγ-dependent expression of immunogenic MHC class II molecules. Together, the ability to sustainably activate and selectively tailor MSC priming responses creates the possibility of programming MSC activation more comprehensively for therapeutic applications.

Ultrasonographic Identification of Shell Surface Types in Commercially Available Silicone Gel-Filled Breast Implants.

BACKGROUND: Breast implant safety issues have resulted in the need for global product recalls and medical device tracing. Conventional methods of breast implant tracing, have to date proven to be unsuccessful. This study aims to evaluate the effectiveness of high-resolution ultrasound (HRUS) screening in identifying implanted breast devices. METHODS: Data from 113 female patients undergoing preoperative ultrasound screening for secondary breast surgery between 2019 and 2022 was prospectively reviewed to evaluate the effectiveness of HRUS imaging with the aid of a sonographic surface catalog to identify the surface and brand type of implanted breast devices. To corroborate the findings and assess the reproducibility of the approach, further evaluations were replicated in New Zealand white rabbits and compared with the results found in humans. RESULTS: In the human recipients, implant surface and brand types were correctly identified by ultrasound imaging in 99% (112 of 113) and 96% (69 of 72) of the cases, either consultation-only or revision, respectively. This constituted an overall success rate of 98% (181 of 185). Furthermore, in a corroborating New Zealand white rabbit model where full-scale commercial implants were introduced and monitored over many months, from the total 28 analyzed, the surface was accurately identified in a total of 27 cases (the one failure being before generation of a sonograph surface catalogue), demonstrating an overall success rate of 96.4%. CONCLUSION: HRUS is, therefore, a valid and first-hand tool for breast implant imaging that can correctly evaluate both surface type and brand type alongside other variables such as implant placement, positioning, flipping, or rupture. CLINICAL RELEVANCE STATEMENT: HRUS is a valid and first-hand tool for the identification and traceability of breast implants that evaluates surface type and brand type. This low-cost, accessible, and reproducible practice provides patients with peace of mind and surgeons with a promising diagnostic tool.

Enhanced dynamic covalent chemistry for the controlled release of small molecules and biologics from a nanofibrous peptide hydrogel platform.

Maintaining safe and potent pharmaceutical drug levels is often challenging. Multidomain peptides (MDPs) assemble into supramolecular hydrogels with a well-defined, highly porous nanostructure that makes them attractive for drug delivery, yet their ability to extend release is typically limited by rapid drug diffusion. To overcome this challenge, we developed self-assembling boronate ester release (SABER) MDPs capable of engaging in dynamic covalent bonding with payloads containing boronic acids (BAs). As examples, we demonstrate that SABER hydrogels can prolong the release of five BA-containing small-molecule drugs as well as BA-modified insulin and antibodies. Pharmacokinetic studies revealed that SABER hydrogels extended the therapeutic effect of ganfeborole from days to weeks, preventing Mycobacterium tuberculosis growth better than repeated oral administration in an infection model. Similarly, SABER hydrogels extended insulin activity, maintaining normoglycemia for six days in diabetic mice after a single injection. These results suggest that SABER hydrogels present broad potential for clinical translation.

In vivo safety evaluation of polyarginine coated magnetic nanovectors.

Safety and efficacy are of critical importance to any nanomaterial-based diagnostic and therapy. The innocuity and functionality of a nanomaterial in vivo is largely dependent on the physicochemical properties of the material, particularly its surface coating. Here, we evaluated the influence of polycationic coating on the efficacy, clearance organ uptake, and safety of magnetic nanovectors designed for siRNA delivery. Polyethylene glycol (PEG) coated superparamagnetic iron oxide nanoparticles (NPs) of 12 nm in core diameter were modified with a polycationic coating of either poly-l-arginine (pArg) or polyethylenimine (PEI) and further covalently functionalized with siRNA oligonucleotides. The produced NP-pArg-siRNA and NP-PEI-siRNA nanovectors were similar in hydrodynamic size (21 and 22 nm, respectively) but significantly differed in zeta potentials (+2.1 mV and +29.8 mV, respectively). Fluorescence quantification assays revealed that the NP-pArg-siRNA nanovector was 3-fold more potent than NP-PEI-siRNA in delivering siRNA and 1.8-fold more effective in gene silencing when tested in rat C6 glioblastoma cells. In vivo, both nanovector formulations were similarly taken up by the spleen and liver as determined by histopathological and hemopathological assays. However, PEI coated nanovectors elicited severe hemoincompatibility and damage to the liver and spleen, while pArg coated nanovectors were found to be safe and tolerable. Combined, our findings suggest that polycationic coatings of pArg were more effective and safer than commonly used PEI coatings for preparation of nanovectors. The NP-pArg-siRNA nanovector formulation developed here shows great potential for in vivo based biomedical applications.

Localized immunomodulation technologies to enable cellular and organoid transplantation.

Localized immunomodulation technologies are rapidly emerging as a new modality with the potential to revolutionize transplantation of cells and organs. In the past decade, cell-based immunomodulation therapies saw clinical success in the treatment of cancer and autoimmune diseases. In this review, we describe recent advances in engineering solutions for the development of localized immunomodulation techniques focusing on cellular and organoid transplantation. We begin by describing cell transplantation and highlighting notable clinical successes, particularly in the areas of stem cell therapy, chimeric antigen receptor (CAR)-T cell therapy, and islet transplantation. Next, we detail recent preclinical studies centered on genome editing and biomaterials to enhance localized immunomodulation. We close by discussing future opportunities to improve clinical and commercial success using these approaches to facilitate long-term immunomodulation technologies.

An Antifibrotic Breast Implant Surface Coating Significantly Reduces Periprosthetic Capsule Formation.

BACKGROUND: The body responds to prosthetic materials with an inflammatory foreign body response and deposition of a fibrous capsule, which may be deleterious to the function of the device and cause significant discomfort for the patient. Capsular contracture (CC) is the most common complication of aesthetic and reconstructive breast surgery. The source of significant patient morbidity, it can result in pain, suboptimal aesthetic outcomes, implant failure, and increased costs. The underlying mechanism remains unknown. Treatment is limited to reoperation and capsule excision, but recurrence rates remain high. In this study, the authors altered the surface chemistry of silicone implants with a proprietary anti-inflammatory coating to reduce capsule formation. METHODS: Silicone implants were coated with Met-Z2-Y12, a biocompatible, anti-inflammatory surface modification. Uncoated and Met-Z2-Y12-coated implants were implanted in C57BL/6 mice. After 21, 90, or 180 days, periprosthetic tissue was removed for histologic analysis. RESULTS: The authors compared mean capsule thickness at three time points. At 21, 90, and 180 days, there was a statistically significant reduction in capsule thickness of Met-Z2-Y12-coated implants compared with uncoated implants ( P < 0.05). CONCLUSIONS: Coating the surface of silicone implants with Met-Z2-Y12 significantly reduced acute and chronic capsule formation in a mouse model for implant-based breast augmentation and reconstruction. As capsule formation obligatorily precedes CC, these results suggest contracture itself may be significantly attenuated. Furthermore, as periprosthetic capsule formation is a complication without anatomical boundaries, this chemistry may have additional applications beyond breast implants, to a myriad of other implantable medical devices. CLINICAL RELEVANCE STATEMENT: Coating of the silicone implant surface with Met-Z2-Y12 alters the periprosthetic capsule architecture and significantly reduces capsule thickness for at least 6 months postoperatively in a murine model. This is a promising step forward in the development of a therapy to prevent capsular contracture.

Hydrogel-encapsulation to enhance bacterial diagnosis of colon inflammation.

Bacteria can be genetically programmed to sense and report the presence of disease biomarkers in the gastrointestinal (GI) tract. However, diagnostic bacteria are typically delivered via oral administration of liquid cultures, resulting in poor survival and high dispersal in vivo. These limitations confound recovery and analysis of engineered bacteria from GI or stool samples. Here, we demonstrate that encapsulating bacteria inside of alginate core-shell particles enables robust survival, containment, and diagnostic function in vivo. We demonstrate these benefits by encapsulating a strain engineered to report the presence of the biomarker thiosulfate via fluorescent protein expression in order to diagnose dextran sodium sulfate-induced colitis in rats. Hydrogel-encapsulated bacteria engineered to sense and respond to physiological stimuli should enable minimally invasive monitoring of a wide range of diseases and have applications as next-generation smart therapeutics.

Perfusable cell-laden matrices to guide patterning of vascularization in vivo.

The survival and function of transplanted tissue engineered constructs and organs require a functional vascular network. In the body, blood vessels are organized into distinct patterns that enable optimal nutrient delivery and oxygen exchange. Mimicking these same patterns in engineered tissue matrices is a critical challenge for cell and tissue transplantation. Here, we leverage bioprinting to assemble endothelial cells in to organized networks of large (>100 µm) diameter blood vessel grafts to enable spatial control of vessel formation in vivo. Acellular PEG/GelMA matrices with perfusable channels were bioprinted and laminar flow was confirmed within patterned channels, beneficial for channel endothelialization and consistent wall shear stress for endothelial maturation. Next, human umbilical vein endothelial cells (HUVECs) were seeded within the patterned channel and maintained under perfusion culture for multiple days, leading to cell-cell coordination within the construct in vitro. HUVEC and human mesenchymal stromal cells (hMSCs) were additionally added to bulk matrix to further stimulate anastomosis of our bioprinted vascular grafts in vivo. Among multiple candidate matrix designs, the greatest degree of biomaterial vascularization in vivo was seen within matrices fabricated with HUVECs and hMSCs encapsulated within the bulk matrix and HUVECs lining the walls of the patterned channels, dubbed design M-C_E. For this lead design, vasculature was detected within the endothelialized, perfusable matrix channels as early as two weeks and αSMA+ CD31+ vessels greater than 100 µm in diameter had formed by eight weeks, resulting in durable and mature vasculature. Notably, vascularization occurred within the endothelialized, bioprinted channels of the matrix, demonstrating the ability of bioprinted perfusable structures to guide vascularization patterns in vivo. The ability to influence vascular patterning in vivo can contribute to the future development of vascularized tissues and organs.

Development of an automated biomaterial platform to study mosquito feeding behavior.

Mosquitoes carry a number of deadly pathogens that are transmitted while feeding on blood through the skin, and studying mosquito feeding behavior could elucidate countermeasures to mitigate biting. Although this type of research has existed for decades, there has yet to be a compelling example of a controlled environment to test the impact of multiple variables on mosquito feeding behavior. In this study, we leveraged uniformly bioprinted vascularized skin mimics to create a mosquito feeding platform with independently tunable feeding sites. Our platform allows us to observe mosquito feeding behavior and collect video data for 30-45 min. We maximized throughput by developing a highly accurate computer vision model (mean average precision: 92.5%) that automatically processes videos and increases measurement objectivity. This model enables assessment of critical factors such as feeding and activity around feeding sites, and we used it to evaluate the repellent effect of DEET and oil of lemon eucalyptus-based repellents. We validated that both repellents effectively repel mosquitoes in laboratory settings (0% feeding in experimental groups, 13.8% feeding in control group, p < 0.0001), suggesting our platform's use as a repellent screening assay in the future. The platform is scalable, compact, and reduces dependence on vertebrate hosts in mosquito research.

Screening hydrogels for antifibrotic properties by implanting cellularly barcoded alginates in mice and a non-human primate.

Screening implantable biomaterials for antifibrotic properties is constrained by the need for in vivo testing. Here we show that the throughput of in vivo screening can be increased by cellularly barcoding a chemically modified combinatorial library of hydrogel formulations. The method involves the implantation of a mixture of alginate formulations, each barcoded with human umbilical vein endothelial cells from different donors, and the association of the identity and performance of each formulation by genotyping single nucleotide polymorphisms of the cells via next-generation sequencing. We used the method to screen 20 alginate formulations in a single mouse and 100 alginate formulations in a single non-human primate, and identified three lead hydrogel formulations with antifibrotic properties. Encapsulating human islets with one of the formulations led to long-term glycaemic control in a mouse model of diabetes, and coating medical-grade catheters with the other two formulations prevented fibrotic overgrowth. High-throughput screening of barcoded biomaterials in vivo may help identify formulations that enhance the long-term performance of medical devices and of biomaterial-encapsulated therapeutic cells.

Lipid Deposition Profiles Influence Foreign Body Responses.

Fibrosis remains a significant cause of failure in implanted biomedical devices and early absorption of proteins on implant surfaces has been shown to be a key instigating factor. However, lipids can also regulate immune activity and their presence may also contribute to biomaterial-induced foreign body responses (FBR) and fibrosis. Here it is demonstrated that the surface presentation of lipids on implant affects FBR by influencing reactions of immune cells to materials as well as their resultant inflammatory/suppressive polarization. Time-of-flight secondary ion mass spectroscopy (ToF-SIMS) is employed to characterize lipid deposition on implants that are surface-modified chemically with immunomodulatory small molecules. Multiple immunosuppressive phospholipids (phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin) are all found to deposit preferentially on implants with anti-FBR surface modifications in mice. Significantly, a set of 11 fatty acids is enriched on unmodified implanted devices that failed in both mice and humans, highlighting relevance across species. Phospholipid deposition is also found to upregulate the transcription of anti-inflammatory genes in murine macrophages, while fatty acid deposition stimulated the expression of pro-inflammatory genes. These results provide further insights into how to improve the design of biomaterials and medical devices to mitigate biomaterial material-induced FBR and fibrosis.

Identification of a humanized mouse model for functional testing of immune-mediated biomaterial foreign body response.

Biomedical devices comprise a major component of modern medicine, however immune-mediated fibrosis and rejection can limit their function over time. Here, we describe a humanized mouse model that recapitulates fibrosis following biomaterial implantation. Cellular and cytokine responses to multiple biomaterials were evaluated across different implant sites. Human innate immune macrophages were verified as essential to biomaterial rejection in this model and were capable of cross-talk with mouse fibroblasts for collagen matrix deposition. Cytokine and cytokine receptor array analysis confirmed core signaling in the fibrotic cascade. Foreign body giant cell formation, often unobserved in mice, was also prominent. Last, high-resolution microscopy coupled with multiplexed antibody capture digital profiling analysis supplied spatial resolution of rejection responses. This model enables the study of human immune cell-mediated fibrosis and interactions with implanted biomaterials and devices.