Development of Brain-Derived Bioscaffolds for Neural Progenitor Cell Culture.

Biomaterials derived from brain extracellular matrix (ECM) have the potential to promote neural tissue regeneration by providing instructive cues that can direct cell survival, proliferation, and differentiation. This study focused on the development and characterization of microcarriers derived from decellularized brain tissue (DBT) as a platform for neural progenitor cell culture. First, a novel detergent-free decellularization protocol was established that effectively reduced the cellular content of porcine and rat brains, with a >97% decrease in the dsDNA content, while preserving collagens (COLs) and glycosaminoglycans (GAGs). Next, electrospraying methods were applied to generate ECM-derived microcarriers incorporating the porcine DBT that were stable without chemical cross-linking, along with control microcarriers fabricated from commercially sourced bovine tendon COL. The DBT microcarriers were structurally and biomechanically similar to the COL microcarriers, but compositionally distinct, containing a broader range of COL types and higher sulfated GAG content. Finally, we compared the growth, phenotype, and neurotrophic factor gene expression levels of rat brain-derived progenitor cells (BDPCs) cultured on the DBT or COL microcarriers within spinner flask bioreactors over 2 weeks. Both microcarrier types supported BDPC attachment and expansion, with immunofluorescence staining results suggesting that the culture conditions promoted BDPC differentiation toward the oligodendrocyte lineage, which may be favorable for cell therapies targeting remyelination. Overall, our findings support the further investigation of the ECM-derived microcarriers as a platform for neural cell derivation for applications in regenerative medicine.

Probing the effects of matrix-derived microcarrier composition on human adipose-derived stromal cells cultured dynamically within spinner flask bioreactors.

Recognizing the cell-instructive capacity of the extracellular matrix (ECM), this study investigated the effects of expanding human adipose-derived stromal cells (hASCs) on ECM-derived microcarriers fabricated from decellularized adipose tissue (DAT) or decellularized cartilage tissue (DCT) within spinner flask bioreactors. Protocols were established for decellularizing porcine auricular cartilage and electrospraying methods were used to generate microcarriers comprised exclusively of DAT or DCT, which were compositionally distinct, but had matching Young's moduli. Both microcarrier types supported hASC attachment and growth over 14 days within a low-shear spinner culture system, with a significantly higher cell density observed on the DCT microcarriers at 7 and 14 days. Irrespective of the ECM source, dynamic culture on the microcarriers altered the expression of genes and proteins associated with cell adhesion and ECM remodeling. Label-free mass spectrometry analysis showed upregulation of proteins associated with cartilage development and ECM in the hASCs expanded on the DCT microcarriers. Based on Luminex analysis, the hASCs expanded on the DCT microcarriers secreted significantly higher levels of IL-8 and PDGFAA, supporting that the ECM source can modulate hASC paracrine factor secretion. Finally, the hASCs expanded on the microcarriers were extracted for analysis of adipogenic and chondrogenic differentiation relative to baseline controls. The microcarrier-cultured hASCs showed enhanced intracellular lipid accumulation at 7 days post-induction of adipogenic differentiation. In the chondrogenic studies, a low level of differentiation was observed in all groups. Future studies are warranted using alternative cell sources with greater chondrogenic potential to further assess the chondro-inductive properties of the DCT microcarriers.

Development of 2-D and 3-D culture platforms derived from decellularized nucleus pulposus.

Bioscaffolds derived from the extracellular matrix (ECM) have shown the capacity to promote regeneration by providing tissue-specific biological instructive cues that can enhance cell survival and direct lineage-specific differentiation. This study focused on the development and characterization of two-dimensional (2-D) and three-dimensional (3-D) cell culture platforms incorporating decellularized nucleus pulposus (DNP). First, a detergent-free protocol was developed for decellularizing bovine nucleus pulposus (NP) tissues that was effective at removing cellular content while preserving key ECM constituents including collagens, glycosaminoglycans, and the cell-adhesive glycoproteins laminin and fibronectin. Next, novel 2-D coatings were generated using the DNP or commercially-sourced bovine collagen type I (COL) as a non-tissue-specific control. In addition, cryo-milled DNP or COL particles were incorporated within methacrylated chondroitin sulphate (MCS) hydrogels as a 3-D cell culture platform for exploring the effects of ECM particle composition. Culture studies showed that the 2-D coatings derived from the DNP could support cell attachment and growth, but did not maintain or rescue the phenotype of primary bovine NP cells, which de-differentiated when serially passaged in monolayer culture. Similarly, while bovine NP cells remained highly viable following encapsulation and 14 days of culture within the hydrogel composites, the incorporation of DNP particles within the MCS hydrogels was insufficient to maintain or rescue changes in NP phenotype associated with extended in vitro culture based on gene expression patterns. Overall, DNP produced with our new decellularization protocol was successfully applied to generate both 2-D and 3-D bioscaffolds; however, further studies are required to assess if these platforms can be combined with additional components of the endogenous NP microenvironment to stimulate regeneration or lineage-specific cell differentiation.

Leveraging a multidimensional linguistic analysis of constructed responses produced by college readers.

The goal of this study was to assess the relationships between computational approaches to analyzing constructed responses made during reading and individual differences in the foundational skills of reading in college readers. We also explored if these relationships were consistent across texts and samples collected at different institutions and texts. The study made use of archival data that involved college participants who produced typed constructed responses under thinking aloud instructions reading history and science texts. They also took assessments of vocabulary knowledge and proficiency in comprehension. The protocols were analyzed to assess two different ways to determine their cohesion. One approach involved assessing how readers established connections with themselves (i.e., to other constructed responses they produced). The other approach involved assessing connections between the constructed responses and the texts that were read. Additionally, the comparisons were made by assessing both lexical (i.e., word matching) and semantic (i.e., high dimensional semantic spaces) comparisons. The result showed that both approaches for analyzing cohesion and making the comparisons were correlated with vocabulary knowledge and comprehension proficiency. The implications of the results for theory and practice are discussed.

Utility of severity assessment tools in COVID-19 pneumonia: a multicentre observational study.

BACKGROUND: Severity scores in pneumonia and sepsis are being applied to SARS-CoV-2 infection. We aimed to assess whether these severity scores are accurate predictors of early adverse outcomes in COVID-19. METHODS: We conducted a multicentre observational study of hospitalised SARS-CoV-2 infection. We assessed risk scores (CURB65, qSOFA, Lac-CURB65, MuLBSTA and NEWS2) in relation to admission to intensive care or death within 7 days of admission, defined as early severe adverse events (ESAE). The 4C Mortality Score was also assessed in a sub-cohort of patients. FINDINGS: In 2,387 participants, the overall mortality was 18%. In all scores examined, increasing score was associated with increased risk of ESAE. Area under the curve (AUC) to predict ESAE for CURB65, qSOFA, Lac-CURB65, MuLBSTA and NEWS2 were 0.61, 0.62, 0.59, 0.59 and 0.68, respectively. AUC to predict ESAE was 0.60 with ISARIC 4C Mortality Score. CONCLUSION: None of the scores examined accurately predicted ESAE in SARS-CoV-2 infection. Non-validated scores should not be used to inform clinical decision making in COVID-19.

The efficacy of fluoride varnish vs a filled resin sealant for preventing white spot lesions during orthodontic treatment.

OBJECTIVES: To compare the efficacy of casein phosphopeptide (CPP)-amorphous calcium phosphate (ACP) MI Varnish (GC America, Inc, Alsip, IL) and ProSeal (Reliance Orthodontic Products, Itasca, IL) sealant in preventing the development of white spot lesions (WSLs) in orthodontic patients. MATERIALS AND METHODS: This prospective randomized clinical trial included 40 orthodontic patients 12-17 years of age. One group had sealants placed on their anterior maxillary teeth, with reapplications every 3 months. The other group had MI Varnish applied every 4-6 weeks. WSL formation and oral hygiene were evaluated at the initial appointment before bonding (T1) and 12 months later (T2). Standardized digital photographs were analyzed using the enamel decalcification index (EDI). Statistical comparisons were made using independent and paired-sample t-tests as well as chi-square tests. RESULTS: In this trial, 43% of patients and 15% of teeth developed new WSLs. Lateral incisors showed the highest incidence of decalcification and WSL formation. WSL formation and EDI score increases during treatment were significantly greater in the gingival region than in the mesial, distal, or incisal regions. Of the EDI scores at T2, 93.8% were 0 and 5.5% were 1. Poor oral hygiene at T2 showed a high positive predictive value (76%) for the development of WSLs. There were no statistically significant between-group differences for the development of WSLs. CONCLUSIONS: MI Varnish and ProSeal sealant provided similar levels of protection during the first 12 months of fixed orthodontic treatment. The severity of the WSLs that developed was minimal. WSLs were most likely to develop on lateral incisors and in the gingival regions of teeth, especially among patients with poorer oral hygiene.

Development and characterization of matrix-derived microcarriers from decellularized tissues using electrospraying techniques.

Stirred bioreactor systems integrating microcarriers represent a promising approach for therapeutic cell manufacturing. While a variety of microcarriers are commercially available, current options do not integrate the tissue-specific composition of the extracellular matrix (ECM), which can play critical roles in directing cell function. The current study sought to generate microcarriers comprised exclusively of ECM from multiple tissue sources. More specifically, porcine decellularized dermis, porcine decellularized myocardium, and human decellularized adipose tissue were digested with α-amylase to obtain ECM suspensions that could be electrosprayed into liquid nitrogen to generate 3D microcarriers that were stable over a range of ECM concentrations without the need for chemical crosslinking or other additives. Characterization studies confirmed that all three microcarrier types had similar soft and compliant mechanical properties and were of a similar size range, but that their composition varied depending on the native tissue source. In vivo testing in immunocompetent mice revealed that the microcarriers integrated into the host tissues, supporting the infiltration of host cells including macrophages and endothelial cells at 2 weeks post-implantation. In vitro cell culture studies validated that the novel microcarriers supported the attachment of tissue-specific stromal cell populations under dynamic culture conditions within spinner flasks, with a significant increase in live cell numbers observed over 1 week on the dermal- and adipose-derived microcarriers. Overall, the findings demonstrate the versatility of the electrospraying methods and support the further development of the microcarriers as cell culture and delivery platforms.

Pannexin 3 deletion reduces fat accumulation and inflammation in a sex-specific manner.

BACKGROUND: Pannexin 3 (PANX3) is a channel-forming glycoprotein that enables nutrient-induced inflammation in vitro, and genetic linkage data suggest that it regulates body mass index. Here, we characterized inflammatory and metabolic parameters in global Panx3 knockout (KO) mice in the context of forced treadmill running (FEX) and high-fat diet (HFD). METHODS: C57BL/6N (WT) and KO mice were randomized to either a FEX running protocol or no running (SED) from 24 until 30 weeks of age. Body weight was measured biweekly, and body composition was measured at 24 and 30 weeks of age. Male WT and KO mice were fed a HFD from 12 to 28 weeks of age. Metabolic organs were analyzed for a panel of inflammatory markers and PANX3 expression. RESULTS: In females there were no significant differences in body composition between genotypes, which could be due to the lack of PANX3 expression in female white adipose tissue, while male KOs fed a chow diet had lower body weight and lower fat mass at 24 and 30 weeks of age, which was reduced to the same extent as 6 weeks of FEX in WT mice. In addition, male KO mice exhibited significantly lower expression of multiple pro-inflammatory genes in white adipose tissue compared to WT mice. While on a HFD body weight differences were insignificant, multiple inflammatory genes were significantly different in quadriceps muscle and white adipose tissue resulting in a more anti-inflammatory phenotype in KO mice compared to WT. The lower fat mass in male KO mice may be due to significantly fewer adipocytes in their subcutaneous fat compared to WT mice. Mechanistically, adipose stromal cells (ASCs) cultured from KO mice grow significantly slower than WT ASCs. CONCLUSION: PANX3 is expressed in male adult mouse adipose tissue and may regulate adipocyte numbers, influencing fat accumulation and inflammation.

The pig as a model system for investigating the recruitment and contribution of myofibroblasts in skin healing.

In the skin-healing field, porcine models are regarded as a useful analogue for human skin due to their numerous anatomical and physiological similarities. Despite the widespread use of porcine models in skin healing studies, the initial origin, recruitment and transition of fibroblasts to matrix-secreting contractile myofibroblasts are not well defined for this model. In this review, we discuss the merit of the pig as an animal for studying myofibroblast origin, as well as the challenges associated with assessing their contributions to skin healing. Although a variety of wound types (incisional, partial thickness, full thickness, burns) have been investigated in pigs in attempts to mimic diverse injuries in humans, direct comparison of human healing profiles with regards to myofibroblasts shows evident differences. Following injury in porcine models, which often employ juvenile animals, myofibroblasts are described in the developing granulation tissue at 4 days, peaking at Days 7-14, and persisting at 60 days post-wounding, although variations are evident depending on the specific pig breed. In human wounds, the presence of myofibroblasts is variable and does not correlate with the age of the wound or clinical contraction. Our comparison of porcine myofibroblast-mediated healing processes with those in humans suggests that further validation of the pig model is essential. Moreover, we identify several limitations evident in experimental design that need to be better controlled, and standardisation of methodologies would be beneficial for the comparison and interpretation of results. In particular, we discuss anatomical location of the wounds, their size and depth, as well as the healing microenvironment (wet vs. moist vs. dry) in pigs and how this could influence myofibroblast recruitment. In summary, although a widespread model used in the skin healing field, further research is required to validate pigs as a useful analogue for human healing with regards to myofibroblasts.

Effect of inflammatory bowel disease treatments on patients with diabetes mellitus.

As medical care progresses and the number of patients with chronic conditions increases there is the inevitable challenge of managing patients with multiple co-morbidities. Inflammatory bowel disease (IBD) is an umbrella term for are inflammatory conditions affecting the gastrointestinal tract, the two most common forms being Ulcerative Colitis and Crohn's disease. These diseases, usually diagnosed in young adults, exhibit a relapsing and remitting course and usually require long-term treatment. IBD can be treated with a number of topical and systemic treatments. We conducted a review of the current published evidence for the effects these medications can have on diabetes mellitus (DM) and glycaemic control. Searches were conducted on medline and embase with a timeframe from 1947 (the date from which studies on embase are recorded) to November 2020. Suitable publications were selected and reviewed. Current evidence of the impact of aminosalicylates, corticosteroids, thiopurines, and biologic agents was reviewed. Though there was limited evidence for certain agents, IBD medications have been shown to have an effect of DM and these effects should be considered in managing patients with dual pathologies. The effects of steroids on blood sugar control is well documented, but consideration of other agents is also important. In patients requiring steroids for Ulcerative Colitis, locally acting steroid agents delivered rectally may be preferred to minimise side effects in those with distal bowel Ulcerative Colitis. A switch to other agents should be considered as soon as possible in people with diabetes to limit the impact on glycaemic control. 5-aminosalicylates appear to play a role in the reduction of hemoglobin A1c (HbA1c), although the literature suggests these may be falsely low readings. Consequently, monitoring of people with diabetes on these agents may require daily monitoring of capillary blood sugars rather than relying simply on HbA1c; for example fructosamine performed 3-6 monthly, although this risks missing the rise in readings. There is only limited evidence of the effects of thiopurines on diabetes and further investigation is needed into the possible relationship between them. However, given the current available evidence it may be preferable to commence patients with diabetes on thiopurines as soon as possible, whilst also monitoring for side effects such as pancreatitis. There appears to be more evidence supporting a link between tumor necrosis factor-α inhibitors and DM. Both infliximab and adalimumab have evidence suggesting that both can cause reduced blood sugar levels. Further studies on the effects of the various biological agents mentioned are required alongside any novel biologic therapy and the impact of dual biologic therapy in the future.

Lineage tracing of Foxd1-expressing embryonic progenitors to assess the role of divergent embryonic lineages on adult dermal fibroblast function.

Recent studies have highlighted the functional diversity of dermal fibroblast populations in health and disease, with part of this diversity linked to fibroblast lineage and embryonic origin. Fibroblasts derived from foxd1-expressing progenitors contribute to the myofibroblast populations present in lung and kidney fibrosis in mice but have not been investigated in the context of dermal wound repair. Using a Cre/Lox system to genetically track populations derived from foxd1-expressing progenitors, lineage-positive fibroblasts were identified as a subset of the dermal fibroblast population. During development, lineage-positive cells were most abundant within the dorsal embryonic tissues, contributing to the developing dermal fibroblast population, and remaining in this niche into adulthood. In adult mice, assessment of fibrosis-related gene expression in lineage-positive and lineage-negative populations isolated from wounded and unwounded dorsal skin was performed, identifying an enrichment of transcripts associated with matrix synthesis and remodeling in the lineage-positive populations. Using a novel excisional wound model, ventral skin healed with a greatly reduced frequency of foxd1 lineage-positive cells. This work supports that the embryonic origin of fibroblasts is an important predictor of fibroblast function, but also highlights that within disparate regions, fibroblasts of different lineages likely undergo convergent differentiation contributing to phenotypic similarities.

Modular cell-assembled adipose matrix-derived bead foams as a mesenchymal stromal cell delivery platform for soft tissue regeneration.

With the goal of establishing a new clinically-relevant bioscaffold format to enable the delivery of high densities of human adipose-derived stromal cells (ASCs) for applications in soft tissue regeneration, a novel "cell-assembly" method was developed to generate robust 3-D scaffolds comprised of fused networks of decellularized adipose tissue (DAT)-derived beads. In vitro studies confirmed that the assembly process was mediated by remodelling of the extracellular matrix by the seeded ASCs, which were well distributed throughout the scaffolds and remained highly viable after 8 days in culture. The ASC density, sulphated glycosaminoglycan content and scaffold stability were enhanced under culture conditions that included growth factor preconditioning. In vivo testing was performed to compare ASCs delivered within the cell-assembled DAT bead foams to an equivalent number of ASCs delivered on a previously-established pre-assembled DAT bead foam platform in a subcutaneous implant model in athymic nude mice. Scaffolds were fabricated with human ASCs engineered to stably co-express firefly luciferase and tdTomato to enable long-term cell tracking. Longitudinal bioluminescence imaging showed a significantly stronger signal associated with viable human ASCs at timepoints up to 7 days in the cell-assembled scaffolds, although both implant groups were found to retain similar densities of human ASCs at 28 days. Notably, the infiltration of CD31+ murine endothelial cells was enhanced in the cell-assembled implants at 28 days. Moreover, microcomputed tomography angiography revealed that there was a marked reduction in vascular permeability in the cell-assembled group, indicating that the developing vascular network was more stable in the new scaffold format. Overall, the novel cell-assembled DAT bead foams represent a promising platform to harness the pro-regenerative paracrine functionality of human ASCs and warrant further investigation as a clinically-translational approach for volume augmentation.

Decellularized adipose tissue scaffolds guide hematopoietic differentiation and stimulate vascular regeneration in a hindlimb ischemia model.

Cellular therapies to stimulate therapeutic angiogenesis in individuals with critical limb ischemia (CLI) remain under intense investigation. In this context, the efficacy of cell therapy is dependent on the survival, biodistribution, and pro-angiogenic paracrine signaling of the cells transplanted. Hematopoietic progenitor cells (HPC) purified from human umbilical cord blood using high aldehyde dehydrogenase-activity (ALDHhi cells) and expanded ex vivo, represent a heterogeneous mixture of progenitor cells previously shown to support limb revascularization in mouse models of CLI. The objectives of this study were to investigate the utility of bioscaffolds derived from human decellularized adipose tissue (DAT) to guide the differentiation of seeded HPC in vitro and harness the pro-angiogenic capacity of HPC at the site of ischemia after implantation in vivo. Probing whether the DAT scaffolds altered HPC differentiation, label-free quantitative mass spectrometry and flow cytometric phenotype analyses indicated that culturing the HPC on the DAT scaffolds supported their differentiation towards the pro-angiogenic monocyte/macrophage lineage at the expense of megakaryopoiesis. Moreover, implantation of HPC in DAT scaffolds within a unilateral hindlimb ischemia model in NOD/SCID mice increased cell retention at the site of ischemia relative to intramuscular injection, and accelerated the recovery of limb perfusion, improved functional limb use and augmented CD31+ capillary density when compared to DAT implantation alone or saline-injected controls. Collectively, these data indicate that cell-instructive DAT scaffolds can direct therapeutic HPC differentiation towards the monocyte/macrophage lineage and represent a promising delivery platform for improving the efficacy of cell therapies for CLI.

Preconditioning Human Adipose-Derived Stromal Cells on Decellularized Adipose Tissue Scaffolds Within a Perfusion Bioreactor Modulates Cell Phenotype and Promotes a Pro-regenerative Host Response.

Cell-based therapies involving the delivery of adipose-derived stromal cells (ASCs) on decellularized adipose tissue (DAT) scaffolds are a promising approach for soft tissue augmentation and reconstruction. Our lab has recently shown that culturing human ASCs on DAT scaffolds within a perfusion bioreactor prior to implantation can enhance their capacity to stimulate in vivo adipose tissue regeneration. Building from this previous work, the current study investigated the effects of bioreactor preconditioning on the ASC phenotype and secretory profile in vitro, as well as host cell recruitment following implantation in an athymic nude mouse model. Immunohistochemical analyses indicated that culturing within the bioreactor increased the percentage of ASCs co-expressing inducible nitric oxide synthase (iNOS) and arginase-1 (Arg-1), as well as tumor necrosis factor-alpha (TNF-α) and interleukin-10 (IL-10), within the peripheral regions of the DAT relative to statically cultured controls. In addition, bioreactor culture altered the expression levels of a range of immunomodulatory factors in the ASC-seeded DAT. In vivo testing revealed that culturing the ASCs on the DAT within the perfusion bioreactor prior to implantation enhanced the infiltration of host CD31+ endothelial cells and CD26+ cells into the DAT implants, but did not alter CD45+F4/80+CD68+ macrophage recruitment. However, a higher fraction of the CD45+ cell population expressed the pro-regenerative macrophage marker CD163 in the bioreactor group, which may have contributed to enhanced remodeling of the scaffolds into host-derived adipose tissue. Overall, the findings support that bioreactor preconditioning can augment the capacity of human ASCs to stimulate regeneration through paracrine-mediated mechanisms.

Adipose Stromal Cells Enhance Decellularized Adipose Tissue Remodeling Through Multimodal Mechanisms.

Decellularized adipose tissue (DAT) scaffolds represent a promising cell-instructive platform for soft tissue engineering. While recent work has highlighted that mesenchymal stromal cells, including adipose-derived stromal cells (ASCs), can be combined with decellularized scaffolds to augment tissue regeneration, the mechanisms involved require further study. The objective of this work was to probe the roles of syngeneic donor ASCs and host-derived macrophages in tissue remodeling of DAT scaffolds within an immunocompetent mouse model. Dual transgenic reporter mouse strains were employed to track and characterize the donor ASCs and host macrophages within the DAT implants. More specifically, ASCs isolated from dsRed mice were seeded on DAT scaffolds, and the seeded and unseeded control scaffolds were implanted subcutaneously into MacGreen transgenic mice for up to 8 weeks. ASC seeding was shown to augment cell infiltration into the DAT implants at 8 weeks, and this was linked to significantly enhanced angiogenesis relative to the unseeded controls. Immunohistochemical staining demonstrated long-term retention of the syngeneic donor ASCs over the duration of the 8-week study, providing evidence that the DAT scaffolds are a cell-supportive delivery platform. Notably, newly formed adipocytes within the DAT implants were not dsRed+, indicating that the donor ASCs supported fat formation through indirect mechanisms. Immunohistochemical tracking of host macrophages through costaining for enhanced green fluorescent protein with the macrophage marker Iba1 revealed that ASC seeding significantly increased the number of infiltrating macrophages within the DAT implants at 3 weeks, while the fraction of macrophages relative to the total cellular infiltrate was similar between the groups at 1, 3, and 8 weeks. Consistent with the tissue remodeling response that was observed, western blotting demonstrated that there was significantly augmented expression of CD163 and CD206, markers of constructive M2-like macrophages, within the ASC-seeded DAT implants. Overall, our results demonstrate that exogenous ASCs enhance tissue regeneration within DAT scaffolds indirectly through multimodal mechanisms that include host cell recruitment and immunomodulation. These data provide further evidence to support the use of decellularized scaffolds as a delivery platform for ASCs in tissue engineering.

Perfusion bioreactor culture of human adipose-derived stromal cells on decellularized adipose tissue scaffolds enhances in vivo adipose tissue regeneration.

Tissue-engineering approaches hold promise to address the need in plastic and reconstructive surgery for new therapies that promote stable adipose tissue regeneration. Previous studies have demonstrated the potential of combining decellularized adipose tissue (DAT) scaffolds with adipose-derived stromal cells (ASCs) for volume augmentation applications. With the goal of enhancing in vivo angiogenesis and adipogenesis, this study evaluated the effects of culturing human ASCs on DAT scaffolds within a perfusion bioreactor. Using this system, the impact of both dynamic culture and hypoxic preconditioning were explored in vitro and in vivo. Initial studies compared the effects of 14 days of culture within the perfusion bioreactor under hypoxia (2% O2 ) or normoxia (~20% O2 ) on human ASC expansion and expression of hypoxia inducible factor-1 alpha (HIF-1α) in vitro relative to static cultured controls. The findings indicated that culturing within the bioreactor under hypoxia significantly increased ASC proliferation on the DAT, with a higher cell density observed in the scaffold periphery. Subsequent characterization in a subcutaneous implant model in athymic nude mice revealed that in vivo angiogenesis and adipogenesis were markedly enhanced when the ASCs were cultured on the DAT within the perfusion bioreactor under hypoxia for 14 days prior to implantation relative to the other culture conditions, as well as freshly seeded and unseeded DAT control groups. Overall, dynamic culture within the perfusion bioreactor system under hypoxia represents a promising approach for preconditioning ASCs on DAT scaffolds to enhance their capacity to stimulate angiogenesis and host-derived adipose tissue regeneration.

Culture on Tissue-Specific Coatings Derived from α-Amylase-Digested Decellularized Adipose Tissue Enhances the Proliferation and Adipogenic Differentiation of Human Adipose-Derived Stromal Cells.

While extracellular matrix (ECM)-derived coatings have the potential to direct the response of cell populations in culture, there is a need to investigate the effects of ECM sourcing and processing on substrate bioactivity. To develop improved cell culture models for studying adipogenesis, the current study examines the proliferation and adipogenic differentiation of human adipose-derived stem/stromal cells (ASCs) on a range of ECM-derived coatings. Human decellularized adipose tissue (DAT) and commercially available bovine tendon collagen (COL) are digested with α-amylase or pepsin to prepare the coatings. Physical characterization demonstrates that α-amylase digestion generates softer, thicker, and more stable coatings, with a fibrous tissue-like ultrastructure that is lost in the pepsin-digested thin films. ASCs cultured on the α-amylase-digested ECM have a more spindle-shaped morphology, and proliferation is significantly enhanced on the α-amylase-digested DAT coatings. Further, the α-amylase-digested DAT provides a more pro-adipogenic microenvironment, based on higher levels of adipogenic gene expression, glycerol-3-phosphate dehydrogenase (GPDH) enzyme activity, and perilipin staining. Overall, this study supports α-amylase digestion as a new approach for generating bioactive ECM-derived coatings, and demonstrates tissue-specific bioactivity using adipose-derived ECM to enhance ASC proliferation and adipogenic differentiation.

Adult MTM1-related myopathy carriers: Classification based on deep phenotyping.

OBJECTIVE: To better characterize adult myotubularin 1 (MTM1)-related myopathy carriers and recommend a phenotypic classification. METHODS: This cohort study was performed at the NIH Clinical Center. Participants were required to carry a confirmed MTM1 mutation and were recruited via the Congenital Muscle Disease International Registry (n = 8), a traveling local clinic of the Neuromuscular and Neurogenetic Disorders of Childhood Section, National Institute of Neurological Disorders and Stroke, NIH and Cure CMD (n = 1), and direct physician referral (n = 1). Neuromuscular examinations, muscle MRI, dynamic breathing MRI, cardiac MRI, pulmonary function tests (PFTs), physical therapy assessments including the Motor Function Measure 32 (MFM-32) scale, and X chromosome inactivation (XCI) studies were performed. RESULTS: Phenotypic categories were proposed based on ambulatory status and muscle weakness. Carriers were categorized as severe (nonambulatory; n = 1), moderate (minimal independent ambulation/assisted ambulation; n = 3), mild (independent ambulation but with evidence of muscle weakness; n = 4), and nonmanifesting (no evidence of muscle weakness; n = 2). Carriers with more severe muscle weakness exhibited greater degrees of respiratory insufficiency and abnormal signal on muscle imaging. Skeletal asymmetries were evident in both manifesting and nonmanifesting carriers. Skewed XCI did not explain phenotypic severity. CONCLUSION: This work illustrates the phenotypic range of MTM1-related myopathy carriers in adulthood and recommends a phenotypic classification. This classification, defined by ambulatory status and muscle weakness, is supported by muscle MRI, PFT, and MFM-32 scale composite score findings, which may serve as markers of disease progression and outcome measures in future gene therapy or other clinical trials.

Clinical Evaluation of Melorheostosis in the Context of a Natural History Clinical Study.

Melorheostosis is a rare dysostosis involving cortical bone overgrowth that affects the appendicular skeleton. Patients present with pain, deformities, contractures, range of motion limitation(s), and limb swelling. It has been described in children as well as adults. We recently identified somatic mosaicism for gain-of-function mutations in MAP2K1 in patients with melorheostosis. Despite these advances in genetic understanding, there are no effective therapies or clinical guidelines to help clinicians and patients in disease management. In a study to better characterize the clinical and genetic aspects of the disease, we recruited 30 adults with a radiographic appearance of melorheostosis and corresponding increased uptake on 18F-NaF positron emission tomography (PET)/CT. Patients underwent physical exam, imaging studies, and laboratory assessment. All patients underwent nerve conduction studies and ultrasound imaging of the nerve in the anatomic distribution of melorheostosis. We found sensory deficits in approximately 77% of patients, with evidence of focal nerve entrapment in five patients. All patients reported pain; 53% of patients had changes in skin overlying the affected bone. No significant laboratory abnormalities were noted. Our findings suggest that patients with melorheostosis may benefit from a multidisciplinary team of dermatologists, neurologists, orthopedic surgeons, pain and palliative care specialists, and physical medicine and rehabilitation specialists. Future studies focused on disease management are needed. © 2019 The Authors. JBMR Plus Published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Time to endoscopy for acute upper gastrointestinal bleeding: Results from a prospective multicentre trainee-led audit.

Background: Endoscopy within 24 h of admission (early endoscopy) is a quality standard in acute upper gastrointestinal bleeding (AUGIB). We aimed to audit time to endoscopy outcomes and identify factors affecting delayed endoscopy (>24 h of admission). Methods: This prospective multicentre audit enrolled patients admitted with AUGIB who underwent inpatient endoscopy between November and December 2017. Analyses were performed to identify factors associated with delayed endoscopy, and to compare patient outcomes, including length of stay and mortality rates, between early and delayed endoscopy groups. Results: Across 348 patients from 20 centres, the median time to endoscopy was 21.2 h (IQR 12.0-35.7), comprising median admission to referral and referral to endoscopy times of 8.1 h (IQR 3.7-18.1) and 6.7 h (IQR 3.0-23.1), respectively. Early endoscopy was achieved in 58.9%, although this varied by centre (range: 31.0-87.5%, p = 0.002). On multivariable analysis, lower Glasgow-Blatchford score, delayed referral, admissions between 7:00 and 19:00 hours or via the emergency department were independent predictors of delayed endoscopy. Early endoscopy was associated with reduced length of stay (median difference 1 d; p = 0.004), but not 30-d mortality (p = 0.344). Conclusions: The majority of centres did not meet national standards for time to endoscopy. Strategic initiatives involving acute care services may be necessary to improve this outcome.