While the benefits of both hydrogels and drug delivery to enhance wound healing have been demonstrated, the highly hydrophilic nature of hydrogels creates challenges with respect to the effective ...loading and delivery of hydrophobic drugs beneficial to wound healing. Herein, we utilize pressurized gas expanded liquid (PGX) technology to produce very high surface area (~200 m2/g) alginate scaffolds and describe a method for loading the scaffolds with ibuprofen (via adsorptive precipitation) and crosslinking them (via calcium chelation) to create a hydrogel suitable for wound treatment and hydrophobic drug delivery. The high surface area of the PGX-processed alginate scaffold facilitates >8 wt% loading of ibuprofen into the scaffold and controlled in vitro ibuprofen release over 12–24 h. In vivo burn wound healing assays demonstrate significantly accelerated healing with ibuprofen-loaded PGX-alginate/calcium scaffolds relative to both hydrogel-only and untreated controls, demonstrating the combined benefits of ibuprofen delivery to suppress inflammation as well as the capacity of the PGX-alginate/calcium hydrogel to maintain wound hydration and facilitate continuous calcium release to the wound. The use of PGX technology to produce highly porous scaffolds with increased surface areas, followed by adsorptive precipitation of a hydrophobic drug onto the scaffolds, offers a highly scalable method of creating medicated wound dressings with high drug loadings.
While medicated hydrogel-based wound dressings offer clear advantages in accelerating wound healing, the inherent incompatibility between conventional hydrogels and many poorly water-soluble drugs of relevance in wound healing remains a challenge. Herein, we leveraged supercritical fluids-based strategies to both process and subsequently impregnate alginate, followed by post-crosslinking to form a hydrogel, to create a very high surface area alginate hydrogel scaffold loaded with high hydrophobic drug contents (here, >8 wt% ibuprofen) without the need for any pore-forming additives. The impregnated scaffolds significantly accelerated burn wound healing while also promoting regeneration of the native skin morphology. We anticipate this approach can be leveraged to load clinically-relevant and highly bioavailable dosages of hydrophobic drugs in hydrogels for a broad range of potential applications.
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Nemaline myopathies (NM) are a group of congenital myopathies that lead to muscle weakness and dysfunction. While 13 genes have been identified to cause NM, over 50% of these genetic defects are due ...to mutations in nebulin (NEB) and skeletal muscle actin (ACTA1), which are genes required for normal assembly and function of the thin filament. NM can be distinguished on muscle biopsies due to the presence of nemaline rods, which are thought to be aggregates of the dysfunctional protein. Mutations in ACTA1 have been associated with more severe clinical disease and muscle weakness. However, the cellular pathogenesis linking ACTA1 gene mutations to muscle weakness are unclear To evaluate cellular disease phenotypes, iPSC-derived skeletal myocytes (iSkM) harboring an ACTA1 H40Y point mutation were used to model NM in skeletal muscle. These were generated by Crispr-Cas9, and include one non-affected healthy control (C) and 2 NM iPSC clone lines, therefore representing isogenic controls. Fully differentiated iSkM were characterized to confirm myogenic status and subject to assays to evaluate nemaline rod formation, mitochondrial membrane potential, mitochondrial permeability transition pore (mPTP) formation, superoxide production, ATP/ADP/phosphate levels and lactate dehydrogenase release. C- and NM-iSkM demonstrated myogenic commitment as evidenced by mRNA expression of Pax3, Pax7, MyoD, Myf5 and Myogenin; and protein expression of Pax4, Pax7, MyoD and MF20. No nemaline rods were observed with immunofluorescent staining of NM-iSkM for ACTA1 or ACTN2, and these mRNA transcript and protein levels were comparable to C-iSkM. Mitochondrial function was altered in NM, as evidenced by decreased cellular ATP levels and altered mitochondrial membrane potential. Oxidative stress induction revealed the mitochondrial phenotype, as evidenced by collapsed mitochondrial membrane potential, early formation of the mPTP and increased superoxide production. Early mPTP formation was rescued with the addition of ATP to media. Together, these findings suggest that mitochondrial dysfunction and oxidative stress are disease phenotypes in the in vitro model of ACTA1 nemaline myopathy, and that modulation of ATP levels was sufficient to protect NM-iSkM mitochondria from stress-induced injury. Importantly, the nemaline rod phenotype was absent in our in vitro model of NM. We conclude that this in vitro model has the potential to recapitulate human NM disease phenotypes, and warrants further study.
•Directed differentiation of ACTA1 H40Y nemaline myopathy (NM) iPSCs into skeletal myotubes.•Nemaline myopathy iPSC-derived skeletal myocytes (NM-iSkM) do not display nemaline rods.•NM-iSkM show mitochondrial dysfunction under conditions of stress.•NM-iSkM are vulnerable to oxidative stress.
Nemaline myopathy (NM) is a genetically and clinically heterogeneous disease that is diagnosed on the basis of the presence of nemaline rods on skeletal muscle biopsy. Although NM has typically been ...classified by causative genes, disease severity or prognosis cannot be predicted. The common pathologic end point of nemaline rods (despite diverse genetic causes) and an unexplained range of muscle weakness suggest that shared secondary processes contribute to the pathogenesis of NM. We speculated that these processes could be identified through a proteome-wide interrogation using a mouse model of severe NM in combination with pathway validation and structural/functional analyses. A proteomic analysis was performed using skeletal muscle tissue from the Neb conditional knockout mouse model compared with its wild-type counterpart to identify pathophysiologically relevant biological processes that might impact disease severity or provide new treatment targets. A differential expression analysis and Ingenuity Pathway Core Analysis predicted perturbations in several cellular processes, including mitochondrial dysfunction and changes in energetic metabolism and stress-related pathways. Subsequent structural and functional studies demonstrated abnormal mitochondrial distribution, decreased mitochondrial respiratory function, an increase in mitochondrial transmembrane potential, and extremely low ATP content in Neb conditional knockout muscles relative to wild type. Overall, the findings of these studies support a role for severe mitochondrial dysfunction as a novel contributor to muscle weakness in NM.
ACTA1 encodes skeletal muscle-specific α-actin, which polymerizes to form the thin filament of the sarcomere. Mutations in ACTA1 are responsible for approximately 30% of nemaline myopathy (NM) cases. ...Previous studies of weakness in NM have focused on muscle structure and contractility, but genetic issues alone do not explain the phenotypic heterogeneity observed in patients with NM or NM mouse models. To identify additional biological processes related to NM phenotypic severity, proteomic analysis was performed using muscle protein isolates from wild-type mice in comparison to moderately affected knock-in (KI) Acta1H40Y and the minimally affected transgenic (Tg) ACTA1D286G NM mice. This analysis revealed abnormalities in mitochondrial function and stress-related pathways in both mouse models, supporting an in-depth assessment of mitochondrial biology. Interestingly, evaluating each model in comparison to its wild-type counterpart identified different degrees of mitochondrial abnormality that correlated well with the phenotypic severity of the mouse model. Muscle histology, mitochondrial respiration, electron transport chain function, and mitochondrial transmembrane potential were all normal or minimally affected in the TgACTA1D286G mouse model. In contrast, the more severely affected KI.Acta1H40Y mice displayed significant abnormalities in relation to muscle histology, mitochondrial respirometry, ATP, ADP, and phosphate content, and mitochondrial transmembrane potential. These findings suggest that abnormal energy metabolism is related to symptomatic severity in NM and may constitute a contributor to phenotypic variability and a novel treatment target.
We previously demonstrated that a depletion of regulatory T (Treg) cells in Lyme arthritis-resistant C57BL/6 mice leads to pathological changes in the tibiotarsal joints following infection with
. ...Here, we assessed the effects of Treg cells on the response to
infection in BALB/c mice, which exhibit infection-dose-dependent disease and a different sequence of immune events than C57BL/6 mice. The depletion of Treg cells prior to infection with 1 × 10
, but not 5 × 10
, organisms led to increased swelling of the tibiotarsal joints. However, Treg cell depletion did not significantly affect the development of histopathology at these low doses of infection. BALB/c mice depleted of Treg cells before infection with 1 × 10
spirochetes harbored a higher borrelial load in the hearts and exhibited higher levels of serum interleukin-10 five weeks later. These results indicate that Treg cells regulate certain aspects of the response to
in a mouse strain that may display a range of disease severities. As the presentation of Lyme disease may vary among humans, it is necessary to consider multiple animal models to obtain a complete picture of the various means by which Treg cells affect the host response to
.
Loss‐of‐function mutations in the deoxyguanosine kinase (DGUOK) gene result in a mitochondrial DNA (mtDNA) depletion syndrome. DGUOK plays an important role in converting deoxyribonucleosides to ...deoxyribonucleoside monophosphates via the salvage pathway for mtDNA synthesis. DGUOK deficiency manifests predominantly in the liver; the most common cause of death is liver failure within the first year of life and no therapeutic options are currently available. in vitro supplementation with deoxyguanosine or deoxyguanosine monophosphate (dGMP) were reported to rescue mtDNA depletion in DGUOK‐deficient, patient‐derived fibroblasts and myoblasts. CERC‐913, a novel ProTide prodrug of dGMP, was designed to bypass defective DGUOK while improving permeability and stability relative to nucleoside monophosphates. To evaluate CERC‐913 for its ability to rescue mtDNA depletion, we developed a primary hepatocyte culture model using liver tissue from DGUOK‐deficient rats. DGUOK knockout rat hepatocyte cultures exhibit severely reduced mtDNA copy number (~10%) relative to wild type by qPCR and mtDNA content remains stable for up to 8 days in culture. CERC‐913 increased mtDNA content in DGUOK‐deficient hepatocytes up to 2.4‐fold after 4 days of treatment in a dose‐dependent fashion, which was significantly more effective than dGMP at similar concentrations. These early results suggest primary hepatocyte culture is a useful model for the study of mtDNA depletion syndromes and that CERC‐913 treatment can improve mtDNA content in this model.
Biomarkers are clinically relevant markers of pathological processes or predictors of response to therapeutic treatments that can be accurately and reliably measured in the body or its products. In ...neuromuscular diseases (NMDs), biomarkers can be particularly important due to challenges with diagnosis, predicting disease behavior, and developing treatment strategies. Congenital muscle diseases (CMDs) are a subgroup of NMDs with known genetic causes that can further be separated into mutations that affect muscle membrane stability/repair (muscular dystrophies) or function (congenital myopathies). Currently, when a patient is suspected of having a CMD, serum biomarkers can be used to verify that an underlying muscle condition exists. However, serum biomarkers have shown to be variable and unreliable as they are confounded by environmental factors.This dissertation attempts to identify novel biomarkers in one congenital myopathy (Nemaline myopathy (NM)) and one muscular dystrophy (Duchenne muscular dystrophy (DMD)). In both NM and DMD, primary disease mechanisms resulting from causative mutations have been well-studied but have not elucidated reliable biomarkers indicative of disease severity or treatment response. Secondary perturbations in response to primary disease mechanisms often contribute to disease severity and can be used to identify new disease markers but this has yet to be fully accomplished in NM and DMD. Therefore, it is essential that novel reliable biomarkers of NM- and DMD-related secondary disease processes are discovered to aid in prognostic capabilities, treatment target identification, and/or treatment evaluation. Here, we utilized a parallel omics screening and pathway validation approach in NM and DMD models to identify perturbed secondary processes that may contribute to disease state and determine if molecules in these pathways can be considered as candidate biomarkers. We hypothesize that candidate biomarkers identified by omics screening and validation are indicative of secondary pathophysiological processes that contribute to disease severity in muscle disorders. Understanding mechanisms contributing to disease severity, uncovering potential treatment targets, and elucidating ways in which to best evaluate treatments for these disorders will accelerate treatment development currently lacking for this patient population.
We previously demonstrated that a depletion of regulatory T (Treg) cells in Lyme arthritis-resistant C57BL/6 mice leads to pathological changes in the tibiotarsal joints following infection with ...Borrelia burgdorferi. Here, we assessed the effects of Treg cells on the response to B. burgdorferi infection in BALB/c mice, which exhibit infection-dose-dependent disease and a different sequence of immune events than C57BL/6 mice. The depletion of Treg cells prior to infection with 1 × 10sup.2 , but not 5 × 10sup.3 , organisms led to increased swelling of the tibiotarsal joints. However, Treg cell depletion did not significantly affect the development of histopathology at these low doses of infection. BALB/c mice depleted of Treg cells before infection with 1 × 10sup.3 spirochetes harbored a higher borrelial load in the hearts and exhibited higher levels of serum interleukin-10 five weeks later. These results indicate that Treg cells regulate certain aspects of the response to B. burgdorferi in a mouse strain that may display a range of disease severities. As the presentation of Lyme disease may vary among humans, it is necessary to consider multiple animal models to obtain a complete picture of the various means by which Treg cells affect the host response to B. burgdorferi.
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