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.

Distinguishing the meaning making processes of survivors of suicide loss: An expansion of the meaning of loss codebook.

Suicide loss represents particularly a difficult form of bereavement due to the challenges that volitional death poses to survivors. Understanding these challenges requires recognition of the idiosyncratic processes of meaning reconstruction for this specific group of grievers. The current study investigates such processes in survivors of suicide loss (SOSL) by utilizing the Meaning of Loss Codebook (MLC) to analyze the narratives of eight SOSL. The findings contribute to a broader understanding of meaning making following suicide, strengthen the validity of the MLC by demonstrating its appropriateness for SOSL, and illuminate unique challenges faced by SOSL, resulting in the proposal of supplemental MLC codes. Research and clinical implications are discussed.

Disease spread through contiguity and axonal tracts in primary lateral sclerosis.

Our goal in this report was to determine whether symptom progression in primary lateral sclerosis (PLS) was consistent with disease spread through axonal pathways or contiguous cortical regions. The date of symptom onset in each limb and cranial region was obtained from 45 PLS patient charts. Each appearance of symptoms in a new body region was classified as axonal, contiguous, possibly contiguous, or unrelated, according to whether the somatotopic representations were adjacent in the cortex. Of 152 spread events, the first spread event was equally divided between axonal (22) and contiguous (23), but the majority of subsequent spread events were classified as contiguous. Symptom progression in PLS patients is consistent with disease spread along axonal tracts and by local cortical spread. Both were equally likely for the first spread event, but local cortical spread was predominant thereafter, suggesting that late degeneration does not advance through long axonal tracts.

Characterization of biologically active insulin-loaded alginate microparticles prepared by spray drying.

BACKGROUND: Spray drying has been used as a means to encapsulate therapeutics in polymeric matrices to improve stability and alter pharmacokinetics. This research aims to characterize alginate microparticles formed by spray drying to encapsulate insulin for therapeutic delivery applications. METHODS: Particle size was characterized by laser diffraction spectroscopy, morphology by scanning electron microscopy, and protein and polymer distribution by confocal laser scanning microscopy. In addition, particle fines collected from the spray-dryer exhaust unit were characterized for size and morphology. The insulin encapsulation efficiency (EE) was determined after particle dissolution through quantification by spectrophotometric analysis. An in-vitro bioassay involving stimulation of rat L6 myoblasts was developed to confirm the bioactivity of released insulin. RESULTS: Mean diameter of the product was 2.1 ± 0.3 µm. Larger particles appeared spherical, with some smaller particles presenting surface topography variability and divoting. Protein EE was 38.2% ± 9.5%, with confocal microscopy showing the protein and polymer concentrated at the surface of larger particles, but more evenly distributed throughout smaller particles. A bioassay for the in-vitro quantification of insulin bioactivity was developed by calibrating the ratio of phosphorylated to total cellular protein kinase B (PKB; also known as AKT). in insulin-stimulated rat L6 myoblasts. Insulin released from the particles was 88% ± 15% bioactive, showing that spray drying had minimal impact on protein structure. CONCLUSION: Spray drying was effective in producing microparticles containing bioactive insulin. Future studies will focus on the improvement of the EE and particle uniformity with the aim of developing this technology further for the encapsulation and delivery of peptide or protein-based therapeutics.

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.

The effect of serial passaging on the proliferation and differentiation of bovine adipose-derived stem cells.

Adipose-derived stem cells (ASCs) represent an excellent cell source for the development of regenerative therapies for a broad variety of tissue disorders. Commonly, in vitro expansion is necessary to obtain sufficient cell populations for research purposes and clinical applications. Although it has been demonstrated that human ASCs can maintain their adipogenic, chondrogenic and osteogenic potential in long-term culture (up to 15 passages), it is not guaranteed that a satisfactory level of differentiation is achievable in later passages. In this study, we investigated the self-renewal and multilineage differentiation capacity of bovine ASCs, isolated from the interdigital fat pad, and explored how serial passaging influences the cells. A proliferation study examined the changes in growth kinetics from passage 1 to 5, and multilineage (adipogenesis, chondrogenesis and osteogenesis) differentiation studies were conducted to compare the potential between passage 2 (P2) and passage 5 (P5). From the proliferation study, a statistically significant change in the doubling time did not appear until P5. In the differentiation study, both P2 and P5 ASCs could be stimulated to undergo multilineage differentiation under specific culturing conditions. However, adipogenic and chondrogenic cultures showed significantly lower levels of differentiation in the P5-induced cultures. In contrast, P5-induced osteogenic cultures had higher alkaline phosphatase enzyme activity than P2-induced cultures, suggesting an increase in the osteogenic response with serial passaging. Overall, bovine ASCs are capable of self-renewal and multilineage differentiation; however, long-term in vitro expansion has a negative effect on adipogenic and chondrogenic differentiation, while potentially favoring osteogenesis.

Co-delivery of adipose-derived stem cells and growth factor-loaded microspheres in RGD-grafted N-methacrylate glycol chitosan gels for focal chondral repair.

The coencapsulation of growth factor-loaded microspheres with adipose-derived stem cells (ASCs) within a hydrogel matrix was studied as a potential means to enhance ASC chondrogenesis in the development of a cell-based therapeutic strategy for the regeneration of partial thickness chondral defects. A photopolymerizable N-methacrylate glycol chitosan (MGC) was employed to form an in situ gel used to encapsulate microspheres loaded with bone morphogenetic protein 6 (BMP-6) and transforming growth factor-ß3 (TGF-ß3) with human ASCs. ASC viability and retention were enhanced when the Young's modulus of the MGC ranged between 225 and 380 kPa. Grafting an RGD-containing peptide onto the MGC backbone (RGD-MGC) improved ASC viability within the gels, remaining at greater than 90% over 14 days in culture. The effects of BMP-6 and TGF-ß3 released from the polymer microspheres on ASC chondrogenesis were assessed, and the level of differentiation was compared to ASCs in control gels containing nongrowth factor-loaded microspheres cultured with and without the growth factors supplied in the medium. There was enhanced expression of chondrogenic markers at earlier time points when the ASCs were induced with the sustained and local release of BMP-6 and TGF-ß3 from the microspheres. More specifically, the normalized glycosaminoglycan and collagen type II protein expression levels were significantly higher than in the controls. In addition, the ratio of collagen type II to type I was significantly higher in the microsphere delivery group and increased over time. End-point RT-PCR analysis supported that there was a more rapid induction and enhancement of ASC chondrogenesis in the controlled release group. Interestingly, in all of the assays, there was evidence of chondrogenic differentiation when the ASCs were cultured in the gels in the absence of growth factor stimulation. Overall, the co-delivery of growth-factor-loaded microspheres and ASCs in RGD-modified MGC gels successfully induced ASC chondrogenesis and is a promising strategy for cartilage repair.

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.

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 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.

Current thoughts on chronic cerebrospinal venous insufficiency in multiple sclerosis.

PURPOSE OF REVIEW: To review the most current literature relating to chronic cerebrospinal venous insufficiency (CCSVI). RECENT FINDINGS: Recently, a vascular phenomenon known as CCSVI was proposed to be the cause of multiple sclerosis (MS). Its prevalence reportedly reflected 100% sensitivity and specificity for the disease. The authors went on to perform invasive procedures for the treatment of CCSVI with questionable success. Since then, many have tried to duplicate the data outlined in the original studies, but none have achieved remotely similar outcomes. Furthermore, there is conflicting information regarding the safety of invasive treatment for CCSVI. SUMMARY: MS is a complex and devastating disease with imperfect treatment strategies. Further research is needed to determine what role, if any, venous insufficiency plays in the course of MS and also the most appropriate treatment course based on those results.

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.

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.

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.

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.

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.