Large-scale and cost-effective production of recombinant human serum albumin (rHSA) in transgenic Bombyx mori cocoons.

HSA is considered a versatile natural cargo carrier with multiple bio-functions and applications. However, insufficient supply of HSA has limited widespread use. Although various recombinant expression systems had been applied to produce the rHSA to overcome the limited resource, cost-effective and large scale production of rHSA remains a challenge. Herein, we provide a strategy for the large-scale and cost-effective production of rHSA in cocoons of transgenic silkworms, achieving a final 13.54 ± 1.34 g/kg of rHSA yield in cocoons. rHSA was efficiently synthesized and stable over the long-term in the cocoons at room temperature. Artificial control of silk crystal structure during silk spinning significantly facilitated rHSA extraction and purification, with 99.69 ± 0.33 % purity and a productivity of 8.06 ± 0.17 g rHSA from 1 kg cocoons. The rHSA had the same secondary structure to natural HSA, along with effective drug binding capacity, biocompatibility, and bio-safe. The rHSA was successfully evaluated as a potential substitute in serum-free cell culture. These findings suggest the silkworm bioreactor is promising for large-scale and cost-effective production of high quality rHSA to meet the increased worldwide demand.

Entomoculture: A Preliminary Techno-Economic Assessment.

Cultured meat, or the practice of growing meat from cell culture, has been experiencing rapid advances in research and technology as the field of biotechnology attempts to answer the call to fight climate change and feed a growing global population. A major hurdle for cell-based meat products entering the market in the near-future is their price. The complex production facilities required to make such products will require advanced bioreactor systems, resources such as energy and water, and a skilled labor force, among other factors. The use of insect cells in this process is hypothesized to address some of these costs due to the characteristics that make them more resilient in cell culture when compared to traditional livestock-derived cells. To address the potential for cost savings by utilizing insect cells in the cultivation of protein-enriched foods, here we utilized a techno-economic assessment model. Three different insect cell lines were used in the model. The results indicate that insect cell lines offer potential to significantly reduce the cost per kilogram of cell cultivated meat, along with further opportunities to optimize production processes through technological advances and scaling.

Silk Hydrogel-Mediated Delivery of Bone Morphogenetic Protein 7 Directly to Subcutaneous White Adipose Tissue Increases Browning and Energy Expenditure.

Objective: Increasing the mass and/or activity of brown adipose tissue (BAT) is one promising avenue for treating obesity and related metabolic conditions, given that BAT has a high potential for energy expenditure and is capable of improving glucose and lipid homeostasis. BAT occurs either in discrete "classical" depots, or interspersed in white adipose tissue (WAT), termed "inducible/recruitable" BAT, or 'beige/brite' adipocytes. We and others have demonstrated that bone morphogenetic protein 7 (BMP7) induces brown adipogenesis in committed and uncommitted progenitor cells, resulting in increased energy expenditure and reduced weight gain in mice. BMP7 is therefore a reliable growth factor to induce browning of WAT. Methods: In this study, we sought to deliver BMP7 specifically to subcutaneous (sc)WAT in order to induce tissue-resident progenitor cells to differentiate into energy-expending recruitable brown adipocytes, without off-target effects like bone formation, which can occur when BMPs are in the presence of bone progenitor cells (outside of WAT). BMP7 delivery directly to WAT may also promote tissue innervation, or directly activate mitochondrial activity in brown adipocytes, as we have demonstrated previously. We utilized silk protein in the form of an injectable hydrogel carrying BMP7. Silk scaffolds are useful for in vivo delivery of substances due to favorable material properties, including controlled release of therapeutic proteins in an active form, biocompatibility with minimal immunogenic response, and prior FDA approval for some medical materials. For this study, the silk was engineered to meet desirable release kinetics for BMP7 in order to mimic our prior in vitro brown adipocyte differentiation studies. Fluorescently-labeled silk hydrogel loaded with BMP7 was directly injected into WAT through the skin and monitored by non-invasive in vivo whole body imaging, including in UCP1-luciferase reporter mice, thereby enabling an approach that is translatable to humans. Results: Injection of the BMP7-loaded silk hydrogels into the subcutaneous WAT of mice resulted in "browning", including the development of multilocular, uncoupling protein 1 (UCP1)-positive brown adipocytes, and an increase in whole-body energy expenditure and skin temperature. In diet-induced obese mice, BMP7-loaded silk delivery to subcutaneous WAT resulted in less weight gain, reduced circulating glucose and lower respiratory exchange ratio (RER). Conclusions: In summary, BMP7 delivery via silk scaffolds directly into scWAT is a novel translational approach to increase browning and energy expenditure, and represents a potential therapeutic avenue for delivering substances directly to adipose depots in pursuit of metabolic treatments.

Switch rates and total cost associated with apremilast and biologics in biologic-naive patients with psoriatic arthritis.

Aim: Real-world treatment data for psoriatic arthritis are limited. We evaluated switch rates, adherence, and costs for patients initiating apremilast versus tumor necrosis factor inhibitor (TNFi) and interleukin inhibitor (ILi) among biologic-naive psoriatic arthritis patients. Materials & methods: This retrospective analysis used IBM MarketScan claims data to assess treatment switches, adherence and costs. Results: Twelve-month switch rates were significantly lower for apremilast versus TNFi (15.5% vs 26.6%; p < 0.0001) and similar to ILi (15.5% vs 14.0%; p = 0.71). Apremilast initiators had lower total costs versus TNFi and ILi (US$39,854 vs US$57,243 and US$65,687; p < 0.05) and adherence was slightly lower versus TNFi and higher versus ILi. Conclusion: Biologic-naive apremilast initiators had lower switch rates versus TNFi initiators and lower total costs versus TNFi or ILi initiators.

Switch Rates and Total Cost of Care Associated with Apremilast and Biologic Therapies in Biologic-Naive Patients with Plaque Psoriasis.

PURPOSE: Compare treatment switching rates and costs among biologic-naive psoriasis patients initiating apremilast or biologics. METHODS: This retrospective claims analysis used IBM MarketScan Commercial and Medicare Supplemental databases to identify patients who initiated apremilast or a biologic (ie, tumor necrosis factor [TNF] or interleukin [IL] inhibitor) for psoriasis treatment between January 1, 2015, and December 31, 2016. A 1:1 propensity score matching was used to adjust for possible selection bias and maximize the number of patients available for analysis. Treatment switching, days to switch, and healthcare costs were assessed at 12 months. T-test and chi-square test were used to evaluate differences between cohorts for continuous and categorical variables as appropriate; Wilcoxon rank-sum tests were used to assess cost differences. RESULTS: In total, 88,025 patients newly initiated apremilast, a TNF inhibitor, or an IL inhibitor. After inclusion/exclusion criteria were applied and patients were propensity score matched, 1645 (apremilast), 1207 (TNF inhibitor), and 438 (IL inhibitor) patients were included in this analysis. Twelve-month switch rates were significantly lower for apremilast initiators compared with TNF inhibitor initiators (14% vs 25%; p<0.01) and comparable to IL inhibitors (14% vs 11%; p>0.05). No statistical difference was observed in days to switch at 12 months for any treatment group. Total healthcare costs were lower for apremilast initiators compared with TNF and IL inhibitor initiators ($34,028 vs $55,973 and $64,430; p<0.0001). Per-patient per-month (PPPM) costs were significantly lower for apremilast initiators compared with TNF inhibitor and IL inhibitor initiators ($2834 vs $4662 and $5366; p<0.0001). CONCLUSION: Over a 12-month follow-up, biologic-naive psoriasis patients initiating apremilast had significantly lower switching rates compared with patients on TNF inhibitors and similar rates as patients on IL inhibitors. PPPM and total healthcare costs were significantly lower for patients initiating apremilast vs TNF or IL inhibitors, primarily due to lower pharmacy costs.

Assessment of Enrichment of Human Mesenchymal Stem Cells Based on Plasma and Mitochondrial Membrane Potentials.

Background: Human mesenchymal stem cells (hMSCs) are utilized preclinically and clinically as a candidate cell therapy for a wide range of inflammatory and degenerative diseases. Despite promising results in early clinical trials, consistent outcomes with hMSC-based therapies have proven elusive in many of these applications. In this work, we attempt to address this limitation through the design of a stem cell therapy to enrich hMSCs for desired electrical and ionic properties with enhanced stemness and immunomodulatory/regenerative capacity. Materials and Methods: In this study, we sought to develop initial protocols to achieve electrically enriched hMSCs (EE-hMSCs) with distinct electrical states and assess the potential relationship with respect to hMSC state and function. We sorted hMSCs based on fluorescence intensity of tetramethylrhodamine ethyl ester (TMRE) and investigated phenotypic differences between the sorted populations. Results: Subpopulations of EE-hMSCs exhibit differential expression of genes associated with senescence, stemness, immunomodulation, and autophagy. EE-hMSCs with low levels of TMRE, indicative of depolarized membrane potential, have reduced mRNA expression of senescence-associated markers, and increased mRNA expression of autophagy and immunomodulatory markers relative to EE-hMSCs with high levels of TMRE (hyperpolarized). Conclusions : This work suggests that the utilization of EE-hMSCs may provide a novel strategy for cell therapies, enabling live cell enrichment for distinct phenotypes that can be exploited for different therapeutic outcomes.

Preclinical assessment of resorbable silk splints for the treatment of pediatric tracheomalacia.

OBJECTIVE: Tracheomalacia is characterized by weakness of the tracheal wall resulting in dynamic airway collapse during respiration; severe cases often require surgical intervention. Off-label external splinting with degradable implants has been reported in humans; however, there remains a need to develop splints with tunable mechanical properties and degradation profiles for the pediatric population. The objective of this pilot study is to assess the safety and efficacy of silk fibroin-based splints in a clinically relevant preclinical model of tracheomalacia. METHODS: Silk splints were evaluated in a surgically induced model of severe tracheomalacia in N = 3 New Zealand white rabbits for 17, 24, and 31 days. An image-based assay was developed to quantify the dynamic change in airway area during spontaneous respiration, and histopathology was used to study the surrounding tissue response. RESULTS: The average change in area in the native trachea was 23% during spontaneous respiration; surgically induced tracheomalacia resulted in a significant increase to 86% (P < 0.001). The average change in airway area after splint placement was reduced at all terminal time points (17, 24, and 31 days postimplantation), indicating a clinical improvement, and was not statistically different than the native trachea. Histopathology showed a localized inflammatory reaction characterized by neutrophils, eosinophils, and mononuclear cells, with early signs suggestive of fibrosis at the splint and tissue interface. CONCLUSION: This pilot study indicates that silk fibroin splints are well tolerated and efficacious in a rabbit model of severe tracheomalacia, with marked reduction in airway collapse following implantation and good tolerability over the studied time course. LEVEL OF EVIDENCE: NA Laryngoscope, 129:2189-2194, 2019.

Assessment of Multipotent Mesenchymal Stromal Cells in Bone Marrow Aspirate From Human Calcaneus.

Bone marrow aspirates (BMAs), owing to their innate osteogenic potential, are well-documented supplements to osteoconductive and/or osteoinductive materials. The calcaneal body provides foot and ankle surgeons a convenient harvest site with low morbidity and minimal cost. In the present study, we sought to identify and characterize multipotent mesenchymal stromal cells (MSCs) in BMAs harvested from the human calcaneal body. Ten healthy patients aged 18 to 65 years were enrolled in the present study. BMAs were harvested from the patients without any reported postoperative complications related to the harvest. Cells isolated from all the aspirates were adherent to culture plates and expressed positive MSC surface markers (CD105, CD90, and CD73) and a low level of negative MSC markers (CD34 and CD45). The cells maintained the ability to proliferate and differentiate into cells of mesenchymal lineages. The BMAs from the human calcaneal body offer a healthy source of multipotent MSCs.

Silk fibroin conduits: a cellular and functional assessment of peripheral nerve repair.

Silk fibroin conduits were designed with appropriate porosity for peripheral nerve repair. The aim of this work was to use these conduits to examine cell inflammatory responses and functional recovery in a sciatic nerve defect model. A total of 45 randomized Lewis rats were used to create an 8-mm defect bridged by a silk guide, commercial collagen guide, or an autograft. After 1, 4, and 8 weeks, macrophage recruitment, percentage of newly formed collagen, number of myelinated axons, and gastrocnemius muscle mass were evaluated. Following 8 weeks, ED1+ cells in autograft and silk conduits decreased to <1% and 17% of week 1 values, respectively. Collagen formation revealed no difference for all measured time points, suggesting a similar foreign body response. Myelinated axon counts within the silk guide revealed a greater number of proximal spouts and distal connections than collagen guides. Gastrocnemius weights demonstrated a 27% decrease between silk and autografts after 8 weeks. This study demonstrates that, in addition to tailorable degradation rates, our silk conduits possess a favorable immunogenicity and remyelination capacity for nerve repair.