Carbon fiber reinforced polymers for implantable medical devices.

Carbon fibers reinforced polymers (CFRPs) are prolifically finding applications in the medical field, moving beyond the aerospace and automotive industries. Owing to its high strength-to-weight ratio, lightness and radiolucency, CFRP-based materials are emerging to replace traditional metal-based medical implants. Numerous types of polymers matrices can be incorporated with carbon fiber using various manufacturing methods, creating composites with distinct properties. Thus, prior to biomedical application, comprehensive evaluation of material properties, biocompatibility and safety are of paramount importance. In this study, we systematically evaluated a series of novel CFRPs, aiming at analyzing biocompatibility for future development into medical implants or implantable drug delivery systems. These CFRPs were produced either via Carbon Fiber-Sheet Molding Compound or Fused Deposition Modelling-based additive manufacturing. Unlike conventional methods, both fabrication processes afford high production rates in a time-and cost-effective manner. Importantly, they offer rapid prototyping and customization in view of personalized medical devices. Here, we investigate the physicochemical and surface properties, material mutagenicity or cytotoxicity of 20 CFRPs, inclusive of 2 surface finishes, as well as acute and sub-chronic toxicity in mice and rabbits, respectively. We demonstrate that despite moderate in vitro physicochemical and surface changes over time, most of the CFRPs were non-mutagenic and non-cytotoxic, as well as biocompatible in small animal models. Future work will entail extensive material assessment in the context of orthopedic applications such as evaluating potential for osseointegration, and a chronic toxicity study in a larger animal model, pigs.

Enhanced In Vivo Vascularization of 3D-Printed Cell Encapsulation Device Using Platelet-Rich Plasma and Mesenchymal Stem Cells.

The current standard for cell encapsulation platforms is enveloping cells in semipermeable membranes that physically isolate transplanted cells from the host while allowing for oxygen and nutrient diffusion. However, long-term viability and function of encapsulated cells are compromised by insufficient oxygen and nutrient supply to the graft. To address this need, a strategy to achieve enhanced vascularization of a 3D-printed, polymeric cell encapsulation platform using platelet-rich plasma (PRP) and mesenchymal stem cells (MSCs) is investigated. The study is conducted in rats and, for clinical translation relevance, in nonhuman primates (NHP). Devices filled with PRP, MSCs, or vehicle hydrogel are subcutaneously implanted in rats and NHP and the amount and maturity of penetrating blood vessels assessed via histopathological analysis. In rats, MSCs drive the strongest angiogenic response at early time points, with the highest vessel density and endothelial nitric oxide synthase (eNOS) expression. In NHP, PRP and MSCs result in similar vessel densities but incorporation of PRP ensues higher levels of eNOS expression. Overall, enrichment with PRP and MSCs yields extensive, mature vascularization of subcutaneous cell encapsulation devices. It is postulated that the individual properties of PRP and MSCs can be leveraged in a synergistic approach for maximal vascularization of cell encapsulation platforms.

2-Hydroxypropyl-ß-cyclodextrin-enhanced pharmacokinetics of cabotegravir from a nanofluidic implant for HIV pre-exposure prophylaxis.

Preexposure prophylaxis (PrEP) with antiretrovirals (ARV) can prevent human immunodeficiency virus (HIV) transmission, but its efficacy is highly dependent on strict patient adherence to daily dosing regimen. Long-acting (LA) ARV formulations or delivery systems that reduce dosing frequency may increase adherence and thus PrEP efficacy. While cabotegravir (CAB) long-acting injectable (CAB LA), an integrase strand transfer inhibitor (INSTI), reduces dosing frequency to bimonthly injections, variable pharmacokinetics (PK) between patients and various adverse reactions necessitate improvement in delivery methods. Here we developed a subcutaneously implantable nanofluidic device for the sustained delivery of CAB formulated with 2-hydroxypropyl-ß-cyclodextrin (ßCAB) and examined the pharmacokinetics (PK) in Sprague-Dawley rats for 3 months in comparison to CAB. Our study demonstrated ßCAB treatment group maintained clinically-relevant plasma CAB concentrations 2 times above the protein-adjusted concentration that inhibits viral replication by 90% (2 × PA-IC90) and drug penetration into tissues relevant to HIV-1 transmission. Further, we successfully fitted plasma CAB concentrations into a PK model (R2 = 0.9999) and determined CAB apparent elimination half-life of 47 days. Overall, our data shows the potential of sustained release of ßCAB via a nanofluidic implant for long-term PrEP delivery, warranting further investigation for efficacy against HIV infections.

Production and transplantation of bioengineered lung into a large-animal model.

The inability to produce perfusable microvasculature networks capable of supporting tissue survival and of withstanding physiological pressures without leakage is a fundamental problem facing the field of tissue engineering. Microvasculature is critically important for production of bioengineered lung (BEL), which requires systemic circulation to support tissue survival and coordination of circulatory and respiratory systems to ensure proper gas exchange. To advance our understanding of vascularization after bioengineered organ transplantation, we produced and transplanted BEL without creation of a pulmonary artery anastomosis in a porcine model. A single pneumonectomy, performed 1 month before BEL implantation, provided the source of autologous cells used to bioengineer the organ on an acellular lung scaffold. During 30 days of bioreactor culture, we facilitated systemic vessel development using growth factor-loaded microparticles. We evaluated recipient survival, autograft (BEL) vascular and parenchymal tissue development, graft rejection, and microbiome reestablishment in autografted animals 10 hours, 2 weeks, 1 month, and 2 months after transplant. BEL became well vascularized as early as 2 weeks after transplant, and formation of alveolar tissue was observed in all animals (n = 4). There was no indication of transplant rejection. BEL continued to develop after transplant and did not require addition of exogenous growth factors to drive cell proliferation or lung and vascular tissue development. The sterile BEL was seeded and colonized by the bacterial community of the native lung.

Nanofluidic drug-eluting seed for sustained intratumoral immunotherapy in triple negative breast cancer.

Conventional systemic immunotherapy administration often results in insufficient anti-tumor immune response and adverse side effects. Delivering immunotherapeutics intratumorally could maximize tumor exposure, elicit efficient anti-tumor immune response, and minimize toxicity. To fulfill the unmet clinical need for sustained local drug delivery and to avoid repeated intratumoral injections, we developed a nanofluidic-based device for intratumoral drug delivery called the nanofluidic drug-eluting seed (NDES). The NDES is inserted intratumorally using a minimally invasive trocar method similar to brachytherapy seed insertion and offers a clinical advantage of drug elution. Drug diffusion from the NDES is regulated by physical and electrostatic nanoconfinement, thereby resulting in constant and sustained immunotherapeutic delivery without the need for injections or clinician intervention. In this study, the NDES was used to deliver immunotherapeutics intratumorally in the 4 T1 orthotopic murine mammary carcinoma model, which recapitulates triple negative breast cancer. We demonstrated that NDES-mediated intratumoral release of agonist monoclonal antibodies, OX40 and CD40, resulted in potentiation of local and systemic anti-tumor immune response and inhibition of tumor growth compared to control mice. Further, mice treated with NDES-CD40 demonstrated minimal liver damage compared to systemically treated mice. Collectively, our study highlights the NDES as an effective platform for sustained intratumoral immunotherapeutic delivery. The potential clinical impact is tremendous given that the NDES is applicable to a broad spectrum of drugs and solid tumors.

Transcutaneously refillable, 3D-printed biopolymeric encapsulation system for the transplantation of endocrine cells.

Autologous cell transplantation holds enormous promise to restore organ and tissue functions in the treatment of various pathologies including endocrine, cardiovascular, and neurological diseases among others. Even though immune rejection is circumvented with autologous transplantation, clinical adoption remains limited due to poor cell retention and survival. Cell transplant success requires homing to vascularized environment, cell engraftment and importantly, maintenance of inherent cell function. To address this need, we developed a three dimensional (3D) printed cell encapsulation device created with polylactic acid (PLA), termed neovascularized implantable cell homing and encapsulation (NICHE). In this paper, we present the development and systematic evaluation of the NICHE in vitro, and the in vivo validation with encapsulated testosterone-secreting Leydig cells in Rag1-/- castrated mice. Enhanced subcutaneous vascularization of NICHE via platelet-rich plasma (PRP) hydrogel coating and filling was demonstrated in vivo via a chorioallantoic membrane (CAM) assay as well as in mice. After establishment of a pre-vascularized bed within the NICHE, transcutaneously transplanted Leydig cells, maintained viability and robust testosterone secretion for the duration of the study. Immunohistochemical analysis revealed extensive Leydig cell colonization in the NICHE. Furthermore, transplanted cells achieved physiologic testosterone levels in castrated mice. The promising results provide a proof of concept for the NICHE as a viable platform technology for autologous cell transplantation for the treatment of a variety of diseases.

Giving new life to old lungs: methods to produce and assess whole human paediatric bioengineered lungs.

We report, for the first time, the development of an organ culture system and protocols to support recellularization of whole acellular (AC) human paediatric lung scaffolds. The protocol for paediatric lung recellularization was developed using human transformed or immortalized cell lines and single human AC lung scaffolds. Using these surrogate cell populations, we identified cell number requirements, cell type and order of cell installations, flow rates and bioreactor management methods necessary for bioengineering whole lungs. Following the development of appropriate cell installation protocols, paediatric AC scaffolds were recellularized using primary lung alveolar epithelial cells (AECs), vascular cells and tracheal/bronchial cells isolated from discarded human adult lungs. Bioengineered paediatric lungs were shown to contain well-developed vascular, respiratory epithelial and lung tissue, with evidence of alveolar-capillary junction formation. Types I and II AECs were found thoughout the paediatric lungs. Furthermore, surfactant protein-C and -D and collagen I were produced in the bioengineered lungs, which resulted in normal lung compliance measurements. Although this is a first step in the process of developing tissues for transplantation, this study demonstrates the feasibility of producing bioengineered lungs for clinical use. Copyright © 2016 John Wiley & Sons, Ltd.

Porcine acellular lung matrix for wound healing and abdominal wall reconstruction: A pilot study.

Surgical wound healing applications require bioprosthetics that promote cellular infiltration and vessel formation, metrics associated with increased mechanical strength and resistance to infection. Porcine acellular lung matrix is a novel tissue scaffold known to promote cell adherence while minimizing inflammatory reactions. In this study, we evaluate the capacity of porcine acellular lung matrix to sustain cellularization and neovascularization in a rat model of subcutaneous implantation and chronic hernia repair. We hypothesize that, compared to human acellular dermal matrix, porcine acellular lung matrix would promote greater cell infiltration and vessel formation. Following pneumonectomy, porcine lungs were processed and characterized histologically and by scanning electron microscopy to demonstrate efficacy of the decellularization. Using a rat model of subcutaneou implantation, porcine acellular lung matrices (n = 8) and human acellular dermal matrices (n = 8) were incubated in vivo for 6 weeks. To evaluate performance under mechanically stressed conditions, porcine acellular lung matrices (n = 7) and human acellular dermal matrices (n = 7) were implanted in a rat model of chronic ventral incisional hernia repair for 6 weeks. After 6 weeks, tissues were evaluated using hematoxylin and eosin and Masson's trichrome staining to quantify cell infiltration and vessel formation. Porcine acellular lung matrices were shown to be successfully decellularized. Following subcutaneous implantation, macroscopic vessel formation was evident. Porcine acellular lung matrices demonstrated sufficient incorporation and showed no evidence of mechanical failure after ventral hernia repair. Porcine acellular lung matrices demonstrated significantly greater cellular density and vessel formation when compared to human acellular dermal matrix. Vessel sizes were similar across all groups. Cell infiltration and vessel formation are well-characterized metrics of incorporation associated with improved surgical outcomes. Porcine acellular lung matrices are a novel class of acellular tissue scaffold. The increased cell and vessel density may promote long-term improved incorporation and mechanical properties. These findings may be due to the native lung scaffold architecture guiding cell migration and vessel formation. Porcine acellular lung matrices represent a new alternative for surgical wound healing applications where increased cell density and vessel formation are sought.

Enhanced gene delivery in porcine vasculature tissue following incorporation of adeno-associated virus nanoparticles into porous silicon microparticles.

There is an unmet clinical need to increase lung transplant successes, patient satisfaction and to improve mortality rates. We offer the development of a nanovector-based solution that will reduce the incidence of lung ischemic reperfusion injury (IRI) leading to graft organ failure through the successful ex vivo treatment of the lung prior to transplantation. The innovation is in the integrated application of our novel porous silicon (pSi) microparticles carrying adeno-associated virus (AAV) nanoparticles, and the use of our ex vivo lung perfusion/ventilation system for the modulation of pro-inflammatory cytokines initiated by ischemic pulmonary conditions prior to organ transplant that often lead to complications. Gene delivery of anti-inflammatory agents to combat the inflammatory cascade may be a promising approach to prevent IRI following lung transplantation. The rationale for the device is that the microparticle will deliver a large payload of virus to cells and serve to protect the AAV from immune recognition. The microparticle-nanoparticle hybrid device was tested both in vitro on cell monolayers and ex vivo using either porcine venous tissue or a pig lung transplantation model, which recapitulates pulmonary IRI that occurs clinically post-transplantation. Remarkably, loading AAV vectors into pSi microparticles increases gene delivery to otherwise non-permissive endothelial cells.

Adjuvant cationic liposomes presenting MPL and IL-12 induce cell death, suppress tumor growth, and alter the cellular phenotype of tumors in a murine model of breast cancer.

Dendritic cells (DC) process and present antigens to T lymphocytes, inducing potent immune responses when encountered in association with activating signals, such as pathogen-associated molecular patterns. Using the 4T1 murine model of breast cancer, cationic liposomes containing monophosphoryl lipid A (MPL) and interleukin (IL)-12 were administered by intratumoral injection. Combination multivalent presentation of the Toll-like receptor-4 ligand MPL and cytotoxic 1,2-dioleoyl-3-trmethylammonium-propane lipids induced cell death, decreased cellular proliferation, and increased serum levels of IL-1ß and tumor necrosis factor (TNF)-α. The addition of recombinant IL-12 further suppressed tumor growth and increased expression of IL-1ß, TNF-α, and interferon-γ. IL-12 also increased the percentage of cytolytic T cells, DC, and F4/80(+) macrophages in the tumor. While single agent therapy elevated levels of nitric oxide synthase 3-fold above basal levels in the tumor, combination therapy with MPL cationic liposomes and IL-12 stimulated a 7-fold increase, supporting the observed cell cycle arrest (loss of Ki-67 expression) and apoptosis (TUNEL positive). In mice bearing dual tumors, the growth of distal, untreated tumors mirrored that of liposome-treated tumors, supporting the presence of a systemic immune response.

Multivalent presentation of MPL by porous silicon microparticles favors T helper 1 polarization enhancing the anti-tumor efficacy of doxorubicin nanoliposomes.

Porous silicon (pSi) microparticles, in diverse sizes and shapes, can be functionalized to present pathogen-associated molecular patterns that activate dendritic cells. Intraperitoneal injection of MPL-adsorbed pSi microparticles, in contrast to free MPL, resulted in the induction of local inflammation, reflected in the recruitment of neutrophils, eosinophils and proinflammatory monocytes, and the depletion of resident macrophages and mast cells at the injection site. Injection of microparticle-bound MPL resulted in enhanced secretion of the T helper 1 associated cytokines IFN-γ and TNF-α by peritoneal exudate and lymph node cells in response to secondary stimuli while decreasing the anti-inflammatory cytokine IL-10. MPL-pSi microparticles independently exhibited anti-tumor effects and enhanced tumor suppression by low dose doxorubicin nanoliposomes. Intravascular injection of the MPL-bound microparticles increased serum IL-1ß levels, which was blocked by the IL-1 receptor antagonist Anakinra. The microparticles also potentiated tumor infiltration by dendritic cells, cytotoxic T lymphocytes, and F4/80+ macrophages, however, a specific reduction was observed in CD204+ macrophages.

Multipotent adult progenitor cells decrease cold ischemic injury in ex vivo perfused human lungs: an initial pilot and feasibility study.

BACKGROUND: Primary graft dysfunction (PGD) is a significant cause of early morbidity and mortality following lung transplantation. Improved organ preservation techniques will decrease ischemia-reperfusion injury (IRI) contributing to PGD. Adult bone marrow-derived adherent stem cells, including mesenchymal stromal (stem) cells (MSCs) and multipotent adult progenitor cells (MAPCs), have potent anti-inflammatory actions, and we thus postulated that intratracheal MAPC administration during donor lung processing would decrease IRI. The goal of the study was therefore to determine if intratracheal MAPC instillation would decrease lung injury and inflammation in an ex vivo human lung explant model of prolonged cold storage and subsequent reperfusion. METHODS: Four donor lungs not utilized for transplant underwent 8 h of cold storage (4°C). Following rewarming for approximately 30 min, non-HLA-matched allogeneic MAPCs (1 × 10(7) MAPCs/lung) were bronchoscopically instilled into the left lower lobe (LLL) and vehicle comparably instilled into the right lower lobe (RLL). The lungs were then perfused and mechanically ventilated for 4 h and subsequently assessed for histologic injury and for inflammatory markers in bronchoalveolar lavage fluid (BALF) and lung tissue. RESULTS: All LLLs consistently demonstrated a significant decrease in histologic and BALF inflammation compared to vehicle-treated RLLs. CONCLUSIONS: These initial pilot studies suggest that use of non-HLA-matched allogeneic MAPCs during donor lung processing can decrease markers of cold ischemia-induced lung injury.