Hybrid Shear-thinning Hydrogel Integrating Hyaluronic Acid with ROS-Responsive Nanoparticles.

Nanoparticle (NP) supra-assembly offers unique opportunities to tune macroscopic hydrogels' mechanical strength, material degradation, and drug delivery properties. Here, synthetic, reactive oxygen species (ROS)-responsive NPs are physically crosslinked with hyaluronic acid (HA) through guest-host chemistry to create shear-thinning NP/HA hydrogels. A library of triblock copolymers composed of poly(propylene sulfide)-bl-poly(N,N-dimethylacrylamide)-bl-poly(N,N-dimethylacrylamide-co-N-(1-adamantyl)acrylamide) are synthesized with varied triblock architectures and adamantane grafting densities and then self-assembled into NPs displaying adamantane on their corona. Self-assembled NPs are mixed with ß-cyclodextrin grafted HA to yield eighteen NP/HA hydrogel formulations. The NP/HA hydrogel platform demonstrates superior mechanical strength to HA-only hydrogels, susceptibility to oxidative/enzymatic degradation, and inherent cell-protective, antioxidant function. The performance of NP/HA hydrogels is shown to be affected by triblock architecture, guest/host grafting densities, and HA composition. In particular, the length of the hydrophilic second block and adamantane grafting density of self-assembled NPs significantly impacts hydrogel mechanical properties and shear-thinning behavior, while ROS-reactivity of poly(propylene sulfide) protects cells from cytotoxic ROS and reduces oxidative degradation of HA compared to HA-only hydrogels. This work provides insight into polymer structure-function considerations for designing hybrid NP/HA hydrogels and identifies antioxidant, shear-thinning hydrogels as promising injectable delivery platforms for small molecule drugs and therapeutic cells.

Newly Developed Di-Block Copolymer-Based Cell Membrane Stabilizers Protect Mouse Coronary Artery Endothelial Cells against Hypoxia/Reoxygenation Injury.

Reperfusion after ischemia causes additional cellular damage, known as reperfusion injury, for which there is still no effective remedy. Poloxamer (P)188, a tri-block copolymer-based cell membrane stabilizer (CCMS), has been shown to provide protection against hypoxia/reoxygenation (HR) injury in various models by reducing membrane leakage and apoptosis and improving mitochondrial function. Interestingly, substituting one of its hydrophilic poly-ethylene oxide (PEO) blocks with a (t)ert-butyl terminus added to the hydrophobic poly-propylene oxide (PPO) block yields a di-block compound (PEO-PPOt) that interacts better with the cell membrane lipid bi-layer and exhibits greater cellular protection than the gold standard tri-block P188 (PEO75-PPO30-PEO75). For this study, we custom-made three different new di-blocks (PEO113-PPO10t, PEO226-PPO18t and PEO113-PPO20t) to systemically examine the effects of the length of each polymer block on cellular protection in comparison to P188. Cellular protection was assessed by cell viability, lactate dehydrogenase release, and uptake of FM1-43 in mouse artery endothelial cells (ECs) following HR injury. We found that di-block CCMS were able to provide the same or better EC protection than P188. Our study provides the first direct evidence that custom-made di-block CCMS can be superior to P188 in improving EC membrane protection, raising their potential in treating cardiac reperfusion injury.

Morphometric analysis of foramen ovale, foramen spinosum, and foramen rotundum of human skull using computed tomography scan: a cross-sectional study.

There is limited literature of objective assessments of foramina of skull base using computed tomography (CT) scan. This study was carried out to analyze the dimensions of foramen ovale (FO), foramen spinosum (FS), and foramen rotundum (FR) using CT scan imaging of the human skull and their associations with sex, age, and laterality of the body. Materials and methods: A cross-sectional study was carried out in the Department of Radiodiagnosis and Imaging at BP Koirala Institute of Health Sciences (BPKIHS), Nepal using a purposive sampling method. We included 96 adult patients (≥18 years) who underwent CT scan of the head for any clinical indications. All those participants below 18 years, inadequate visualization or erosions of skull base foramina, and/or not consenting were excluded. Appropriate statistical calculations were done using the statistical package for social sciences (SPSS), version 21. The P-value of less than 0.05 was considered statistically significant. Results: The mean length, width, and area of FO was 7.79±1.10 mm, 3.68±0.64 mm, and 22.80±6.18 mm2, respectively. The mean length, width, and area of FS was 2.38±0.36 mm, 1.94±0.30 mm, and 3.69±0.95 mm2, respectively. Similarly, the mean height, width, and area of FR was 2.41±0.49 mm, 2.40±0.55 mm, and 4.58±1.49 mm2, respectively. The male participants had statistically significant higher mean dimensions of FO and FS (P<0.05) than the female participants. There were statistically insignificant correlations of dimensions of these foramina with age and between the left and right side of each foraminal dimensions (P>0.05). Conclusions: The sex-based difference in dimensions of FO and FS should be clinically considered in evaluating the pathology of these foramina. However, further studies using objective assessment of foraminal dimensions are required to draw obvious inferences.

Nanoparticle-mediated transgene expression of insulin-like growth factor 1 in the growth restricted guinea pig placenta increases placenta nutrient transporter expression and fetal glucose concentrations.

Fetal growth restriction (FGR) significantly contributes to neonatal and perinatal morbidity and mortality. Currently, there are no effective treatment options for FGR during pregnancy. We have developed a nanoparticle gene therapy targeting the placenta to increase expression of human insulin-like growth factor 1 (hIGF1) to correct fetal growth trajectories. Using the maternal nutrient restriction guinea pig model of FGR, an ultrasound-guided, intraplacental injection of nonviral, polymer-based hIGF1 nanoparticle containing plasmid with the hIGF1 gene and placenta-specific Cyp19a1 promotor was administered at mid-pregnancy. Sustained hIGF1 expression was confirmed in the placenta 5 days after treatment. Whilst increased hIGF1 did not change fetal weight, circulating fetal glucose concentration were 33%-67% higher. This was associated with increased expression of glucose and amino acid transporters in the placenta. Additionally, hIGF1 nanoparticle treatment increased the fetal capillary volume density in the placenta, and reduced interhaemal distance between maternal and fetal circulation. Overall, our findings, that trophoblast-specific increased expression of hIGF1 results in changes to glucose transporter expression and increases fetal glucose concentrations within a short time period, highlights the translational potential this treatment could have in correcting impaired placental nutrient transport in human pregnancies complicated by FGR.

Effect of Mycobacterium w on IL-6 and Oxygen Requirement in COVID-19.

The efficacy and safety of heat-killed Mycobacterium w (Mw) in severe COVID-19 were evaluated. Twenty-five hospitalized patients (mean age, 52.9 ± 13.1 years) with severe COVID-19 and having multiple comorbidities were intradermally injected with 0.3 mL of Mw daily for three consecutive days. Changes in leukocyte and platelet counts; C-reactive protein (CRP), interleukin-6 (IL-6), serum creatinine, and liver enzyme levels; and oxygen saturation were compared before and after treatment. An ordinal scale assessed the clinical response. There were significant improvements in the IL-6 level and oxygen saturation following treatment (p < 0.001). There were marked improvements in the platelet count, CRP level, serum aspartate transaminase level, and ordinal scale score. Eighty percent of patients who were on oxygen support were successfully shifted to room air within 5.6 days of treatment and discharged. No systemic adverse events were noted. Thus, Mw treatment could be a promising therapeutic modality in severe COVID-19.

Reactive oxygen species-degradable polythioketal urethane foam dressings to promote porcine skin wound repair.

Porous, resorbable biomaterials can serve as temporary scaffolds that support cell infiltration, tissue formation, and remodeling of nonhealing skin wounds. Synthetic biomaterials are less expensive to manufacture than biologic dressings and can achieve a broader range of physiochemical properties, but opportunities remain to tailor these materials for ideal host immune and regenerative responses. Polyesters are a well-established class of synthetic biomaterials; however, acidic degradation products released by their hydrolysis can cause poorly controlled autocatalytic degradation. Here, we systemically explored reactive oxygen species (ROS)-degradable polythioketal (PTK) urethane (UR) foams with varied hydrophilicity for skin wound healing. The most hydrophilic PTK-UR variant, with seven ethylene glycol (EG7) repeats flanking each side of a thioketal bond, exhibited the highest ROS reactivity and promoted optimal tissue infiltration, extracellular matrix (ECM) deposition, and reepithelialization in porcine skin wounds. EG7 induced lower foreign body response, greater recruitment of regenerative immune cell populations, and resolution of type 1 inflammation compared to more hydrophobic PTK-UR scaffolds. Porcine wounds treated with EG7 PTK-UR foams had greater ECM production, vascularization, and resolution of proinflammatory immune cells compared to polyester UR foam-based NovoSorb Biodegradable Temporizing Matrix (BTM)-treated wounds and greater early vascular perfusion and similar wound resurfacing relative to clinical gold standard Integra Bilayer Wound Matrix (BWM). In a porcine ischemic flap excisional wound model, EG7 PTK-UR treatment led to higher wound healing scores driven by lower inflammation and higher reepithelialization compared to NovoSorb BTM. PTK-UR foams warrant further investigation as synthetic biomaterials for wound healing applications.

Top-Down Fabricated microPlates for Prolonged, Intra-articular Matrix Metalloproteinase 13 siRNA Nanocarrier Delivery to Reduce Post-traumatic Osteoarthritis.

Post-traumatic osteoarthritis (PTOA) associated with joint injury triggers a degenerative cycle of matrix destruction and inflammatory signaling, leading to pain and loss of function. Here, prolonged RNA interference (RNAi) of matrix metalloproteinase 13 (MMP13) is tested as a PTOA disease modifying therapy. MMP13 is upregulated in PTOA and degrades the key cartilage structural protein type II collagen. Short interfering RNA (siRNA) loaded nanoparticles (siNPs) were encapsulated in shape-defined poly(lactic-co-glycolic acid) (PLGA) based microPlates (µPLs) to formulate siNP-µPLs that maintained siNPs in the joint significantly longer than delivery of free siNPs. Treatment with siNP-µPLs against MMP13 (siMMP13-µPLs) in a mechanical load-induced mouse model of PTOA maintained potent (65-75%) MMP13 gene expression knockdown and reduced MMP13 protein production in joint tissues throughout a 28-day study. MMP13 silencing reduced PTOA articular cartilage degradation/fibrillation, meniscal deterioration, synovial hyperplasia, osteophytes, and pro-inflammatory gene expression, supporting the therapeutic potential of long-lasting siMMP13-µPL therapy for PTOA.

Diflunisal-loaded poly(propylene sulfide) nanoparticles decrease S. aureus-mediated bone destruction during osteomyelitis.

Osteomyelitis is a debilitating infection of bone that results in substantial morbidity. Staphylococcus aureus is the most commonly isolated pathogen causing bone infections and features an arsenal of virulence factors that contribute to bone destruction and counteract immune responses. We previously demonstrated that diflunisal, a nonsteroidal anti-inflammatory drug, decreases S. aureus-induced bone destruction during osteomyelitis when delivered locally from a resorbable drug delivery depot. However, local diflunisal therapy was complicated by bacterial colonization of the depot's surface, highlighting a common pitfall of devices for local drug delivery to infected tissue. It is, therefore, critical to develop an alternative drug delivery method for diflunisal to successfully repurpose this drug as an antivirulence therapy for osteomyelitis. We hypothesized that a nanoparticle-based parenteral delivery strategy would provide a method for delivering diflunisal to infected tissue while circumventing the complications associated with local delivery. In this study, we demonstrate that poly(propylene sulfide) (PPS) nanoparticles accumulate at the infectious focus in a murine model of staphylococcal osteomyelitis and are capable of efficaciously delivering diflunisal to infected bone. Moreover, diflunisal-loaded PPS nanoparticles effectively decrease S. aureus-mediated bone destruction, establishing the feasibility of systemic delivery of an antivirulence compound to mitigate bone pathology during osteomyelitis.

Enhanced stem cell retention and antioxidative protection with injectable, ROS-degradable PEG hydrogels.

Poly(ethylene glycol) (PEG) hydrogels crosslinked with enzyme-cleavable peptides are promising biodegradable vehicles for therapeutic cell delivery. However, peptide synthesis at the level required for bulk biomaterial manufacturing is costly, and fabrication of hydrogels from scalable, low-cost synthetic precursors while supporting cell-specific degradation remains a challenge. Reactive oxygen species (ROS) are cell-generated signaling molecules that can also be used as a trigger to mediate specific in vivo degradation of biomaterials. Here, PEG-based hydrogels crosslinked with ROS-degradable poly(thioketal) (PTK) polymers were successfully synthesized via thiol-maleimide chemistry and employed as a cell-degradable, antioxidative stem cell delivery platform. PTK hydrogels were mechanically robust and underwent ROS-mediated, dose-dependent degradation in vitro, while promoting robust cellular infiltration, tissue regeneration, and bioresorption in vivo. Moreover, these ROS-sensitive materials successfully encapsulated mesenchymal stem cells (MSCs) and maintained over 40% more viable cells than gold-standard hydrogels crosslinked with enzymatically-degradable peptides. The higher cellular survival in PTK-based gels was associated with the antioxidative function of the ROS-sensitive crosslinker, which scavenged free radicals and protected encapsulated MSCs from cytotoxic doses of ROS. Improved MSC viability was also observed in vivo as MSCs delivered within injectable PTK hydrogels maintained significantly more viability over 11 days compared against cells delivered within gels crosslinked with either a PEG-only control polymer or a gold-standard enzymatically-degradable peptide. Together, this study establishes a new paradigm for scalable creation and application of cell-degradable hydrogels, particularly for cell delivery applications.

Tuning Ligand Density To Optimize Pharmacokinetics of Targeted Nanoparticles for Dual Protection against Tumor-Induced Bone Destruction.

Breast cancer patients are at high risk for bone metastasis. Metastatic bone disease is a major clinical problem that leads to a reduction in mobility, increased risk of pathologic fracture, severe bone pain, and other skeletal-related events. The transcription factor Gli2 drives expression of parathyroid hormone-related protein (PTHrP), which activates osteoclast-mediated bone destruction, and previous studies showed that Gli2 genetic repression in bone-metastatic tumor cells significantly reduces tumor-induced bone destruction. Small molecule inhibitors of Gli2 have been identified; however, the lipophilicity and poor pharmacokinetic profile of these compounds have precluded their success in vivo. In this study, we designed a bone-targeted nanoparticle (BTNP) comprising an amphiphilic diblock copolymer of poly[(propylene sulfide)-block-(alendronate acrylamide-co-N,N-dimethylacrylamide)] [PPS-b-P(Aln-co-DMA)] to encapsulate and preferentially deliver a small molecule Gli2 inhibitor, GANT58, to bone-associated tumors. The mol % of the bisphosphonate Aln in the hydrophilic polymer block was varied in order to optimize BTNP targeting to tumor-associated bone by a combination of nonspecific tumor accumulation (presumably through the enhanced permeation and retention effect) and active bone binding. Although 100% functionalization with Aln created BTNPs with strong bone binding, these BTNPs had highly negative zeta-potential, resulting in shorter circulation time, greater liver uptake, and less distribution to metastatic tumors in bone. However, 10 mol % of Aln in the hydrophilic block generated a formulation with a favorable balance of systemic pharmacokinetics and bone binding, providing the highest bone/liver biodistribution ratio among formulations tested. In an intracardiac tumor cell injection model of breast cancer bone metastasis, treatment with the lead candidate GANT58-BTNP formulation decreased tumor-associated bone lesion area 3-fold and increased bone volume fraction in the tibiae of the mice 2.5-fold. Aln conferred bone targeting to the GANT58-BTNPs, which increased GANT58 concentration in the tumor-associated bone relative to untargeted NPs, and also provided benefit through the direct antiresorptive therapeutic function of Aln. The dual benefit of the Aln in the BTNPs was supported by the observations that drug-free Aln-containing BTNPs improved bone volume fraction in bone-tumor-bearing mice, while GANT58-BTNPs created better therapeutic outcomes than both unloaded BTNPs and GANT58-loaded untargeted NPs. These findings suggest GANT58-BTNPs have potential to potently inhibit tumor-driven osteoclast activation and resultant bone destruction in patients with bone-associated tumor metastases.

Correlation of the changing trends of red cell distribution width and serum lactate as a prognostic factor in sepsis and septic shock.

BACKGROUND AND AIMS: Various biomarkers are used for predicting outcome from sepsis and septic shock but single value doesn't give clear-cut picture. Changing trends of serum lactate and red cell distribution width (RDW) gives more accurate information of patient outcome. So, aim of this prospective observational study was to identify the correlation, for initial and changing trend of blood lactate level and RDW, with 28-day mortality in sepsis and septic shock. MATERIAL AND METHODS: Patient who fulfills the criteria of sepsis and septic shock, according to the consensus conference published in 2016, were included in this study. All patients were resuscitated and managed according to institutional protocol for sepsis and septic shock. Serum lactate and RDW was obtained from arterial blood gas and complete blood count, respectively. Serum lactate and RDW were recorded at 0 h, 6 h, 24 h, day 2, day 3, day 7, week 2, and week 3. Mean between two groups were compared with student t-test. Pearson and Spearman correlation coefficient was used for establishing correlation between two continuous data. P value < 0.05 indicates significant difference between two groups. RESULTS: There is positive correlation between serum lactate and RDW at all-time point in non-survival group while negative correlation was found in survival group except on day1 and 2. CONCLUSION: Changing trends of serum lactate and RDW can be used as a prognostic marker in patient of sepsis and septic shock.

Functional comparison of beating cardiomyocytes differentiated from umbilical cord-derived mesenchymal/stromal stem cells and human foreskin-derived induced pluripotent stem cells.

In recent years, stem cell-based therapies shown to have promising effects on the clinical management of ischemic heart disease. Moreover, stem cells differentiation into cardiomyocytes (CMs) can overcome the cell source limitations. The current research involves the isolation and expansion of mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs), their differentiation into CMs and subsequent construction of tissue-engineered myocardium supported by random and aligned polycaprolactone (PCL) nanofibrous matrices (av. dia: 350-850 nm). Umbilical cord matrix (UCM)-derived MSCs were isolated successfully by routine enzymatic digestion and a nonenzymatic explant culture method and characterized by their morphology, differentiation into different lineages, and surface marker expression. Treatment of UCM-derived MSCs with 5-azacytidine (5 µM) induced their differentiation into putative cardiac cells, as revealed by the expression of cardiac-specific troponin T (cTnT), smooth muscles actin, myogenin (MYOG), smoothelin, cardiac α-actin genes and cTnT, α-actinin proteins by RT-PCR and immunocytochemistry, respectively. However, no beating cells were observed in differentiated MSCs. On the other hand, adult human foreskin-derived iPSCs cultured on Matrigel™-coated aligned PCL nanofibrous matrices showed anisotropic behavior along the PCL nanofibers and, upon differentiation, expressed cardiac-specific cTnT (23.34 vs. 32.55%) proteins and showed more synchronized beating than those differentiated on Matrigel™-coated tissue culture coated polystyrene surfaces. Moreover, aligned PCL nanofibers are able to promote cells orientation parallel to the fibers, thus providing an effective way to control anisotropic nature under in vitro condition.

Systemic delivery of a Gli inhibitor via polymeric nanocarriers inhibits tumor-induced bone disease.

Solid tumors frequently metastasize to bone and induce bone destruction leading to severe pain, fractures, and other skeletal-related events (SREs). Osteoclast inhibitors such as bisphosphonates delay SREs but do not prevent skeletal complications or improve overall survival. Because bisphosphonates can cause adverse side effects and are contraindicated for some patients, we sought an alternative therapy to reduce tumor-associated bone destruction. Our previous studies identified the transcription factor Gli2 as a key regulator of parathyroid hormone-related protein (PTHrP), which is produced by bone metastatic tumor cells to promote osteoclast-mediated bone destruction. In this study, we tested the treatment effect of a Gli antagonist GANT58, which inhibits Gli2 nuclear translocation and PTHrP expression in tumor cells. In initial testing, GANT58 did not have efficacy in vivo due to its low water solubility and poor bioavailability. We therefore developed a micellar nanoparticle (NP) to encapsulate and colloidally stabilize GANT58, providing a fully aqueous, intravenously injectable formulation based on the polymer poly(propylene sulfide)135-b-poly[(oligoethylene glycol)9 methyl ether acrylate]17 (PPS135-b-POEGA17). POEGA forms the hydrophilic NP surface while PPS forms the hydrophobic NP core that sequesters GANT58. In response to reactive oxygen species (ROS), PPS becomes hydrophilic and degrades to enable drug release. In an intratibial model of breast cancer bone metastasis, treatment with GANT58-NPs decreased bone lesion area by 49% (p<.01) and lesion number by 38% (p<.05) and resulted in a 2.5-fold increase in trabecular bone volume (p<.001). Similar results were observed in intracardiac and intratibial models of breast and lung cancer bone metastasis, respectively. Importantly, GANT58-NPs reduced tumor cell proliferation but did not alter mesenchymal stem cell proliferation or osteoblast mineralization in vitro, nor was there evidence of cytotoxicity after repeated in vivo treatment. Thus, inhibition of Gli2 using GANT58-NPs is a potential therapy to reduce bone destruction that should be considered for further testing and development toward clinical translation.

Drug-Free ROS Sponge Polymeric Microspheres Reduce Tissue Damage from Ischemic and Mechanical Injury.

The inherent antioxidant function of poly(propylene sulfide) (PPS) microspheres (MS) was dissected for different reactive oxygen species (ROS), and therapeutic benefits of PPS-MS were explored in models of diabetic peripheral arterial disease (PAD) and mechanically induced post-traumatic osteoarthritis (PTOA). PPS-MS (∼1 µm diameter) significantly scavenged hydrogen peroxide (H2O2), hypochlorite, and peroxynitrite but not superoxide in vitro in cell-free and cell-based assays. Elevated ROS levels (specifically H2O2) were confirmed in both a mouse model of diabetic PAD and in a mouse model of PTOA, with greater than 5- and 2-fold increases in H2O2, respectively. PPS-MS treatment functionally improved recovery from hind limb ischemia based on ∼15-25% increases in hemoglobin saturation and perfusion in the footpads as well as earlier remodeling of vessels in the proximal limb. In the PTOA model, PPS-MS reduced matrix metalloproteinase (MMP) activity by 30% and mitigated the resultant articular cartilage damage. These results suggest that local delivery of PPS-MS at sites of injury-induced inflammation improves the vascular response to ischemic injury in the setting of chronic hyperglycemia and reduces articular cartilage destruction following joint trauma. These results motivate further exploration of PPS as a stand-alone, locally sustained antioxidant therapy and as a material for microsphere-based, sustained local drug delivery to inflamed tissues at risk of ROS damage.

Recent strategies to design vascular theranostic nanoparticles.

Vascular disease is a leading cause of death and disability worldwide. Current surgical intervention and treatment options for vascular diseases have exhibited limited long-term success, emphasizing the need to develop advanced treatment paradigms for early detection and more effective treatment of dysfunctional cells in a specific blood vessel lesion. Advances in targeted nanoparticles mediating cargo delivery enables more robust prevention, screening, diagnosis, and treatment of vascular disorders. In particular, nanotheranostics integrates diagnostic imaging and therapeutic function into a single agent, and is an emerging platform towards more effective and localized vascular treatment. This review article highlights recent advances and current challenges associated with the utilization of targeted nanoparticles for real-time diagnosis and treatment of vascular diseases. Given recent developments, nanotheranostics offers great potential to serve as an effective platform for targeted, localized, and personalized vascular treatment.

Tunable Surface Repellency Maintains Stemness and Redox Capacity of Human Mesenchymal Stem Cells.

Human bone marrow derived mesenchymal stem cells (hMSCs) hold great promise for regenerative medicine due to their multipotent differentiation capacity and immunomodulatory capabilities. Substantial research has elucidated mechanisms by which extracellular cues regulate hMSC fate decisions, but considerably less work has addressed how material properties can be leveraged to maintain undifferentiated stem cells. Here, we show that synthetic culture substrates designed to exhibit moderate cell-repellency promote high stemness and low oxidative stress-two indicators of naïve, healthy stem cells-in commercial and patient-derived hMSCs. Furthermore, the material-mediated effect on cell behavior can be tuned by altering the molar percentage (mol %) and/or chain length of poly(ethylene glycol) (PEG), the repellant block linked to hydrophobic poly(ε-caprolactone) (PCL) in the copolymer backbone. Nano- and angstrom-scale characterization of the cell-material interface reveals that PEG interrupts the adhesive PCL domains in a chain-length-dependent manner; this prevents hMSCs from forming mature focal adhesions and subsequently promotes cell-cell adhesions that require connexin-43. This study is the first to demonstrate that intrinsic properties of synthetic materials can be tuned to regulate the stemness and redox capacity of hMSCs and provides new insight for designing highly scalable, programmable culture platforms for clinical translation.

Reactive Oxygen Species Shielding Hydrogel for the Delivery of Adherent and Nonadherent Therapeutic Cell Types.

Cell therapies suffer from poor survival post-transplant due to placement into hostile implant sites characterized by host immune response and innate production of high levels of reactive oxygen species (ROS). We hypothesized that cellular encapsulation within an injectable, antioxidant hydrogel would improve viability of cells exposed to high oxidative stress. To test this hypothesis, we applied a dual thermo- and ROS-responsive hydrogel comprising the ABC triblock polymer poly[(propylene sulfide)-block-(N,N-dimethyl acrylamide)-block-(N-isopropylacrylamide)] (PPS135-b-PDMA152-b-PNIPAAM225, PDN). The PPS chemistry reacts irreversibly with ROS such as hydrogen peroxide (H2O2), imparting inherent antioxidant properties to the system. Here, PDN hydrogels were successfully integrated with type 1 collagen to form ROS-protective, composite hydrogels amenable to spreading and growth of adherent cell types such as mesenchymal stem cells (MSCs). It was also shown that, using a control hydrogel substituting nonreactive polycaprolactone in place of PPS, the ROS-reactive PPS chemistry is directly responsible for PDN hydrogel cytoprotection of both MSCs and insulin-producing ß-cell pseudo-islets against H2O2 toxicity. In sum, these results establish the potential of cytoprotective, thermogelling PDN biomaterials for injectable delivery of cell therapies.

Thermogelling, ABC Triblock Copolymer Platform for Resorbable Hydrogels with Tunable, Degradation-Mediated Drug Release.

Clinical application of injectable, thermoresponsive hydrogels is hindered by lack of degradability and controlled drug release. To overcome these challenges, a family of thermoresponsive, ABC triblock polymer-based hydrogels has been engineered to degrade and release drug cargo through either oxidative or hydrolytic/enzymatic mechanisms dictated by the "A" block composition. Three ABC triblock copolymers are synthesized with varying "A" blocks, including oxidation-sensitive poly(propylene sulfide), slow hydrolytically/enzymatically degradable poly(ε-caprolactone), and fast hydrolytically/enzymatically degradable poly(D,L-lactide-co-glycolide), forming the respective formulations PPS135-b-PDMA152-b-PNIPAAM225 (PDN), PCL85-b-PDMA150-b-PNIPAAM150 (CDN), and PLGA60-b-PDMA148-b-PNIPAAM152 (LGDN). For all three polymers, hydrophilic poly(N,N-dimethylacrylamide) and thermally responsive poly(N-isopropylacrylamide) comprise the "B" and "C" blocks, respectively. These copolymers form micelles in aqueous solutions at ambient temperature that can be preloaded with small molecule drugs. These solutions quickly transition into hydrogels upon heating to 37 °C, forming a supra-assembly of physically crosslinked, drug-loaded micelles. PDN hydrogels are selectively degraded under oxidative conditions while CDN and LGDN hydrogels are inert to oxidation but show differential rates of hydrolytic/enzymatic decomposition. All three hydrogels are cytocompatible in vitro and in vivo, and drug-loaded hydrogels demonstrate differential release kinetics in vivo corresponding with their specific degradation mechanism. These collective data highlight the potential cell and drug delivery use of this tunable class of ABC triblock polymer thermogels.

Gradient release of cardiac morphogens by photo-responsive polymer micelles for gradient-mediated variation of embryoid body differentiation.

Retinoic acid (RA) is a well-known morphogen in human development. However, how an RA gradient distribution influences cardiac development remains obscure due to the lack of appropriate experimental apparatus. To address this issue, a polymeric micelle system with covalently attached RA was engineered to deliver gradient quantities of RA upon photo-irradiation. A photo-degradable polymeric nanoparticle (NP) composed of an amphiphilic methoxy(polyethylene glycol)-b-poly(ε-caprolactone)-co-poly(azido-ε-caprolactone-g-ortho nitrobenzyl retinoic ester) copolymer was synthesized, and hanging RA was covalently attached through a photo-sensitive o-nitrobenzyl (ONB) linker. The ONB linker was efficiently cleaved when exposed to a light (365 nm)-gradient, and the consequent gradient release of RA from the micelles was demonstrated. The efficacy of the photo-gradient-mediated RA release was validated across different concentrations of polymer micelles over varied irradiation periods. It was confirmed that polymer micelles demonstrated minimal cytotoxicity when exposed to mouse embryoid bodies (EBs). Finally, when the photo-gradient release of polymer micelles was applied, GFP-cardiac troponin T reporter mouse EBs demonstrated a concurrent gradient-like pattern of cardiac differentiation, verifying the utility of our novel photo-gradient approach to study morphogen gradients not only for cardiac development but also for other potential biological microenvironments subject to morphogen presentation with highly defined spatial and temporal geometries.

Oxidatively Degradable Poly(thioketal urethane)/Ceramic Composite Bone Cements with Bone-Like Strength.

Synthetic bone cements are commonly used in orthopaedic procedures to aid in bone regeneration following trauma or disease. Polymeric cements like PMMA provide the mechanical strength necessary for orthopaedic applications, but they are not resorbable and do not integrate with host bone. Ceramic cements have a chemical composition similar to that of bone, but their brittle mechanical properties limit their use in weight-bearing applications. In this study, we designed oxidatively degradable, polymeric bone cements with mechanical properties suitable for bone tissue engineering applications. We synthesized a novel thioketal (TK) diol, which was crosslinked with a lysine triisocyanate (LTI) prepolymer to create hydrolytically stable poly(thioketal urethane)s (PTKUR) that degrade in the oxidative environment associated with bone defects. PTKUR films were hydrolytically stable for up to 6 months, but degraded rapidly (<1 week) under simulated oxidative conditions in vitro. When combined with ceramic micro- or nanoparticles, PTKUR cements exhibited working times comparable to calcium phosphate cements and strengths exceeding those of trabecular bone. PTKUR/ceramic composite cements supported appositional bone growth and integrated with host bone near the bone-cement interface at 6 and 12 weeks post-implantation in rabbit femoral condyle plug defects. Histological evidence of osteoclast-mediated resorption of the cements was observed at 6 and 12 weeks. These findings demonstrate that a PTKUR bone cement with bone-like strength can be selectively resorbed by cells involved in bone remodeling, and thus represent an important initial step toward the development of resorbable bone cements for weight-bearing applications.