Validation of self-collected buccal swab and saliva as a diagnostic tool for COVID-19.

BACKGROUND: Effective management of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires large-scale testing to identify and isolate infectious carriers. Self-administered buccal swab and saliva collection are convenient, painless, and safe alternatives to the current healthcare worker (HCW)-collected nasopharyngeal swab (NPS). METHODS: A cross-sectional single-centre study was conducted on 42 participants who had tested positive for SARS-CoV-2 via an NPS within the past 7 days. Real-time polymerase chain reaction (RT-PCR) was performed and cycle threshold (Ct) values were obtained for each test. The positive percent agreement (PPA), negative percent agreement (NPA), and overall agreement (OA) were calculated for the saliva samples and buccal swabs, and compared with NPS. RESULTS: Among the 42 participants, 73.8% (31/42) tested positive by any one of the three tests. With reference to NPS, the saliva test had PPA 66.7%, NPA 91.7%, and OA 69.0%; the buccal swab had PPA 56.7%, NPA 100%, and OA 73.8%. CONCLUSION: Self-collected saliva tests and buccal swabs showed only moderate agreement with HCW-collected NPS. Primary screening for SARS-CoV-2 may be performed with a saliva test or buccal swab, with a negative test warranting a confirmatory NPS to avoid false-negatives, minimize discomfort, and reduce the risk of spread to the community and HCWs.

Biomimetic fetal rotation bioreactor for engineering bone tissues-Effect of cyclic strains on upregulation of osteogenic gene expression.

Cells respond to physiological mechanical stresses especially during early fetal development. Adopting a biomimetic approach, it is necessary to develop bioreactor systems to explore the effects of physiologically relevant mechanical strains and shear stresses for functional tissue growth and development. This study introduces a multimodal bioreactor system that allows application of cyclic compressive strains on premature bone grafts that are cultured under biaxial rotation (chamber rotation about 2 axes) conditions for bone tissue engineering. The bioreactor is integrated with sensors for dissolved oxygen levels and pH that allow real-time, non-invasive monitoring of the culture parameters. Mesenchymal stem cells-seeded polycaprolactone-ß-tricalcium phosphate scaffolds were cultured in this bioreactor over 2 weeks in 4 different modes-static, cyclic compression, biaxial rotation, and multimodal (combination of cyclic compression and biaxial rotation). The multimodal culture resulted in 1.8-fold higher cellular proliferation in comparison with the static controls within the first week. Two weeks of culture in the multimodal bioreactor utilizing the combined effects of optimal fluid flow conditions and cyclic compression led to the upregulation of osteogenic genes alkaline phosphatase (3.2-fold), osteonectin (2.4-fold), osteocalcin (10-fold), and collagen type 1 α1 (2-fold) in comparison with static cultures. We report for the first time, the independent and combined effects of mechanical stimulation and biaxial rotation for bone tissue engineering using a bioreactor platform with non-invasive sensing modalities. The demonstrated results show leaning towards the futuristic vision of using a physiologically relevant bioreactor system for generation of autologous bone grafts for clinical implantation.

In vitro cyclic compressive loads potentiate early osteogenic events in engineered bone tissue.

Application of dynamic mechanical loads on bone and bone explants has been reported to enhance osteogenesis and mineralization. To date, published studies have incorporated a range of cyclic strains on 3D scaffolds and platforms to demonstrate the effect of mechanical loading on osteogenesis. However, most of the loading parameters used in these studies do not emulate the in vivo loading conditions. In addition, the scaffolds/platforms are not representative of the native osteoinductive environment of bone tissue and hence may not be entirely accurate to study the in vivo mechanical loading. We hypothesized that biomimicry of physiological loading will potentiate accelerated osteogenesis in bone grafts. In this study, we present a compression bioreactor system that applies cyclic compression to cellular grafts in a controlled manner. Polycaprolactone-ß Tricalcium Phosphate (PCL-TCP) scaffolds seeded with Mesenchymal Stem Cells (MSC) were cyclically compressed in bioreactor for a period of 4 weeks at 1 Hz and physiological strain value of 0.22% for 4 h per day. Gene expression studies revealed increased expressions of osteogenesis-related genes (Osteonectin and COL1A1) on day 7 of cyclic loading group relative to its static controls. Cyclic compression resulted in a 3.76-fold increase in the activity of Alkaline Phosphatase (ALP) on day 14 when compared to its static group (p < 0.001). In addition, calcium deposition of cyclic loading group was found to attain saturation on day 14 (1.96 fold higher than its static scaffolds). The results suggested that cyclic, physiological compression of stem cell-seeded scaffolds generated highly mineralized bone grafts. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2366-2375, 2017.

Development and Characterization of Organic Electronic Scaffolds for Bone Tissue Engineering.

Bones have been shown to exhibit piezoelectric properties, generating electrical potential upon mechanical deformation and responding to electrical stimulation with the generation of mechanical stress. Thus, the effects of electrical stimulation on bone tissue engineering have been extensively studied. However, in bone regeneration applications, only few studies have focused on the use of electroactive 3D biodegradable scaffolds at the interphase with stem cells. Here a method is described to combine the bone regeneration capabilities of 3D-printed macroporous medical grade polycaprolactone (PCL) scaffolds with the electrical and electrochemical capabilities of the conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT). PCL scaffolds have been highly effective in vivo as bone regeneration grafts, and PEDOT is a leading material in the field of organic bioelectronics, due to its stability, conformability, and biocompatibility. A protocol is reported for scaffolds functionalization with PEDOT, using vapor-phase polymerization, resulting in a conformal conducting layer. Scaffolds' porosity and mechanical stability, important for in vivo bone regeneration applications, are retained. Human fetal mesenchymal stem cells proliferation is assessed on the functionalized scaffolds, showing the cytocompatibility of the polymeric coating. Altogether, these results show the feasibility of the proposed approach to obtain electroactive scaffolds for electrical stimulation of stem cells for regenerative medicine.

Polysaccharide nanofibers with variable compliance for directing cell fate.

Cells perceive their microenvironment through physical and mechanical cues, such as extracellular matrix topography or stiffness. In this study, we developed a polysaccharide scaffold that can provide combined substrate topography and matrix compliance signals to direct cell fate. Pullulan/dextran (P/D) nanofibers were fabricated with variable stiffness by in situ crosslinking during electrospinning. By varying the chemical crosslinking content between 10, 12, 14, and 16%, (denoted as STMP10, STMP12, STMP14, and STMP16 respectively), scaffold mechanical stiffness was altered. We characterized substrate stiffness by various methods. Under hydrated conditions, atomic force microscopy and tensile tests of bulk scaffolds were conducted. Under dry conditions, tensile tests of scaffolds and single nanofibers were examined. In addition, we evaluated the efficacy of the scaffolds in directing stem cell differentiation. Using human first trimester mesenchymal stem cells (fMSCs) cultured on STMP14 P/D scaffolds (Young's modulus: 7.84 kPa) in serum-free neuronal differentiation medium exhibited greatest extent of differentiation. Cells showed morphological changes and significantly higher expression of motor neuron markers. Further analyses by western blotting also revealed the enhanced expression of choline acetyltransferase on STMP14 (7.84 kPa) and STMP16 (11.08 kPa) samples as compared to STMP12 (7.19 kPa). Taken together, this study demonstrates that the stiffness of P/D nanofibers can be altered by differential in situ crosslinking during electrospinning and suggests the feasibility of using such polysaccharide nanofibers in supporting fMSC neuronal commitment.

Electrospun nanofibers as a bioadhesive platform for capturing adherent leukemia cells.

This study investigated the adhesive behaviors of normal and abnormal hematopoietic cells on nanotopographical materials. Previously, electrospun nanofiber scaffolds (NFSs) were used to capture and expand hematopoietic stem cells in vitro; here, we demonstrate that NFS could also serve as a useful bioadhesive platform for capturing functionally adherent leukemia cells. Collagen-blended poly(d,l-lactide-co-glycolide) NFS enabled more rapid and efficient capture of K562 leukemia cells than tissue culture polystyrene surfaces with up to 70% improved adhesion and shorter time. Cellular extensions, stronger adhesion, and enhanced cell-cell interactions were observed in K562 cells captured on NFS. While NFS promoted hematopoietic progenitor cell proliferation, it inhibited leukemia cell proliferation and affected cell cycle status by shifting more cells toward the G0/G1 phase. The expression of α-integrins was equally high in both captured and uncaptured leukemia cell populations demonstrating no relation to its adhesive nature. Hematopoietic morphological signatures of NFS captured cells presented no impact on cell differentiation. We conclude that electrospun NFS serves as an excellent platform not only for capturing functionally adherent leukemia cells but also for studying the impact of niche-like structure in the nanoscale.

Biomimetic three-dimensional anisotropic geometries by uniaxial stretching of poly(ε-caprolactone) films: degradation and mesenchymal stem cell responses.

Geometric cues have been used for a variety of cell regulation and tissue regenerative applications. While the function of geometric cues is being recognized, their stability and degradation behaviors are not well known. Here, we studied the influence of degradation on uniaxial-stretch-induced poly(ε-caprolactone) (UX-PCL) ridge/groove arrays and further cellular responses. Results from accelerated hydrolysis in vitro showed that UX-PCL ridge/groove arrays followed a surface-controlled erosion, with an overall geometry remained even at ∼45% film weight loss. Compared to unstretched PCL flat surfaces and/or ridge/groove arrays, UX-PCL ridge/groove arrays achieved an enhanced morphological stability against degradation. Over the degradation period, UX-PCL ridge/groove arrays exhibited an "S-shape" behavior of film weight loss, and retained more stable surface hydrophilicity and higher film mechanical properties than those of unstretched PCL surfaces. Human mesenchymal stem cells (MSCs) aligned better toward UX-PCL ridge/groove arrays when the geometries were remained intact, and became sensitive with gradually declined nucleus alignment and elongation to the geometric degradation of ridges. We speculate that uniaxial stretching confers UX-PCL ridge/groove arrays with enhanced stability against degradation in erosive environment. This study provides insights of how degradation influences geometric cues and further cell responses, and has implications for the design of biomaterials with stability-enhanced geometric cues for long-term tissue regeneration.

Biocompatibility studies and characterization of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)/polycaprolactone blends.

Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) is a biocompatible and bioresorbable copolymer that has generated research interest as a bone scaffold material. However, its brittleness and degradation characteristics can be improved upon. We hypothesized that blending with medical-grade polycaprolactone (PCL) can improve degradation and mechanical characteristics. Here, we report the development of solvent-blended PHBHHx/PCL for application as a potential biomaterial for tissue engineering. Enhanced yield strength, yield strain and Young's modulus occurred at 30/70 blend when compared with PHBHHx and PCL. Polarized light microscopy demonstrated PHBHHx and PCL to exist as morphologically and optically distinct phases and, together with thermal analyses, revealed immiscibility. Hydrophilicity improved with the addition of PCL. Accelerated hydrolytic studies suggested predictable behavior of PHBHHx/PCL. Notably, 30/70 blend exhibited similar degradation behavior to PCL in terms of changes in crystallinity, molecular weight, morphology, and mass loss. Finally, human fetal mesenchymal stem cells (hfMSCs) were evaluated on PHBHHx/PCL using live/dead assay and results suggested encouraging hfMSC adhesion and proliferative capacity, with near-confluence occurring in PHBHHx and 30/70 blend after 5 days. Taken together, these are encouraging results for the further development of PHBHHx/PCL as a potential biomaterial for tissue engineering.

Contrasting effects of vasculogenic induction upon biaxial bioreactor stimulation of mesenchymal stem cells and endothelial progenitor cells cocultures in three-dimensional scaffolds under in vitro and in vivo paradigms for vascularized bone tissue engineering.

Clinical translation of bone tissue engineering approaches for fracture repair has been hampered by inadequate vascularization required for maintaining cell survival, skeletal regeneration, and remodeling. The potential of vasculature formation within tissue-engineered grafts depends on various factors, including an appropriate choice of scaffold and its microarchitectural design for the support of tissue ingrowth and vessel infiltration, vasculogenic potential of cell types and mechanostimulation on cells to enhance cytokine expression. Here, we demonstrated the effect of biomechanical stimulation on vasculogenic and bone-forming capacity of umbilical-cord-blood endothelial progenitor cells (UCB-EPC) and human fetal bone marrow-derived mesenchymal stem cell (hfMSC) seeded within macroporous scaffolds and cocultured dynamically in a biaxial bioreactor. Dynamically cultured EPC/hfMSC constructs generated greater mineralization and calcium deposition consistently over 14 days of culture (1.7-fold on day 14; p<0.05). However, in vitro vessel formation was not observed as compared to an extensive EPC-vessel network formed under static culture on day 7. Subsequent subcutaneous implantations in NOD/SCID mice showed 1.4-fold higher human:mouse cell chimerism (p<0.001), with a more even cellular distribution throughout the dynamically cultured scaffolds. In addition, there was earlier evidence of vessel infiltration into the scaffold and a trend toward increased ectopic bone formation, suggesting improved efficacy and cellular survival through early vascularization upon biomechanical stimulation. The integrative use of bioreactor culture systems with macroporous scaffolds and cocultured osteogenic and vasculogenic cells promotes maturation of EPC/hfMSC-scaffold grafts necessary for vascularized bone tissue engineering applications.

A generic micropatterning platform to direct human mesenchymal stem cells from different origins towards myogenic differentiation.

Human mesenchymal stem cells (MSCs) derived from various origins show varied differentiation capability. Recent work shows that cell shape manipulation via micropatterning can modulate the differentiation of bone-marrow-derived MSCs. Herein, the effect of micropatterning on the myogenesis of MSCs isolated from three different sources (bone marrow, fetal tissue, and adipose) is reported. All the well-aligned cells, regardless of source, predominantly commit to myogenic lineage, as shown by the significant upregulation of myogenic gene markers and positive myosin heavy chain staining. It is demonstrated that our novel micropattern can be used as a generic platform for inducing myogenesis of MSCs from different sources and may also have the potential to be extended to induce other lineage commitment.

In vitro angiogenesis assay for the study of cell-encapsulation therapy.

Cell encapsulation within alginate beads has potential as a sustained release system for delivering therapeutic agents in vivo while protecting encapsulated cells from the immune system. There is, however, no in vitro model for cell-encapsulation therapy that provides a suitable platform for quantitative assessment of physiological responses to secreted factors. Here we introduce a new microfluidic system specifically designed to evaluate and quantify the pro-angiogenic potential of factors secreted from human fetal lung fibroblasts encapsulated in beads on an intact endothelial cell monolayer. We confirmed that cell-encapsulating beads induced an angiogenic response in vitro, demonstrated by a strong correlation between the encapsulated cell density in the beads and the length of the vascular lumen formed in vitro. Conditions established by in vitro tests were then further shown to exert a pro-angiogenic response in vivo using a subcutaneous mouse model, forming an extensive network of functional luminal structures perfused with red blood cells.

A comparison of bioreactors for culture of fetal mesenchymal stem cells for bone tissue engineering.

Bioreactors provide a dynamic culture system for efficient exchange of nutrients and mechanical stimulus necessary for the generation of effective tissue engineered bone grafts (TEBG). We have shown that biaxial rotating (BXR) bioreactor-matured human fetal mesenchymal stem cell (hfMSC) mediated-TEBG can heal a rat critical sized femoral defect. However, it is not known whether optimal bioreactors exist for bone TE (BTE) applications. We systematically compared this BXR bioreactor with three most commonly used systems: Spinner Flask (SF), Perfusion and Rotating Wall Vessel (RWV) bioreactors, for their application in BTE. The BXR bioreactor achieved higher levels of cellularity and confluence (1.4-2.5x, p < 0.05) in large 785 mm(3) macroporous scaffolds not achieved in the other bioreactors operating in optimal settings. BXR bioreactor-treated scaffolds experienced earlier and more robust osteogenic differentiation on von Kossa staining, ALP induction (1.2-1.6×, p < 0.01) and calcium deposition (1.3-2.3×, p < 0.01). We developed a Micro CT quantification method which demonstrated homogenous distribution of hfMSC in BXR bioreactor-treated grafts, but not with the other three. BXR bioreactor enabled superior cellular proliferation, spatial distribution and osteogenic induction of hfMSC over other commonly used bioreactors. In addition, we developed and validated a non-invasive quantitative micro CT-based technique for analyzing neo-tissue formation and its spatial distribution within scaffolds.

Cartilage repair using hyaluronan hydrogel-encapsulated human embryonic stem cell-derived chondrogenic cells.

Human embryonic stem cells (hESCs) have the potential to offer a virtually unlimited source of chondrogenic cells for use in cartilage repair and regeneration. We have recently shown that expandable chondrogenic cells can be derived from hESCs under selective growth factor-responsive conditions. In this study, we explore the potential of these hESC-derived chondrogenic cells to produce an extracellular matrix (ECM)-enriched cartilaginous tissue construct when cultured in hyaluronic acid (HA)-based hydrogel, and further investigated the long-term reparative ability of the resulting hESC-derived chondrogenic cell-engineered cartilage (HCCEC) in an osteochondral defect model. We hypothesized that HCCEC can provide a functional template capable of undergoing orderly remodeling during the repair of critical-sized osteochondral defects (1.5 mm in diameter, 1 mm depth into the subchondral bone) in a rat model. In the process of repair, we observed an orderly spatial-temporal remodeling of HCCEC over 12 weeks into osteochondral tissue, with characteristic architectural features including a hyaline-like neocartilage layer with good surface regularity and complete integration with the adjacent host cartilage and a regenerated subchondral bone. By 12 weeks, the HCCEC-regenerated osteochondral tissue resembled closely that of age-matched unoperated native control, while only fibrous tissue filled in the control defects which left empty or treated with hydrogel alone. Here we demonstrate that transplanted hESC-derived chondrogenic cells maintain long-term viability with no evidence of tumorigenicity, providing a safe, highly-efficient and practical strategy of applying hESCs for cartilage tissue engineering.