Cellular Constructs Research Articles

Viability of Quercetin-Induced Dental Pulp Stem Cells in Forming Living Cellular Constructs for Soft Tissue Augmentation.

Autogenous gingival grafts used for root coverage or gingival augmentation procedures often result in donor site morbidity. Living cellular constructs as an exogenous alternative have been proven to be associated with lower morbidity. With the available background information, the present study aims to assess if quercetin-induced living cell constructs, derived from dental pulp stem cells, have the potential to be applied as a tool for soft tissue augmentation. The characterized dental pulp stem cells (positive for CD73, CD90, and negative for CD34, HLA-DR) were expanded in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10 mM quercetin. The handling properties of the quercetin-induced dental pulp stem cell constructs were assessed by visual, and tactile sensation. A microscopic characterization using hematoxylin and eosin staining, and qRT-PCR-based analysis for stemness-associated genes (OCT4, NANOG, SOX2, and cMyc) was also performed. Dental pulp stem cells without quercetin administration were used as the control. Dental pulp stem cell constructs induced by quercetin easily detached from the surface of the plate, whereas there was no formation in the control cells. It was also simple to transfer the induced cellular construct on the flattened surface. Microscopic characterization of the constructs showed cells embedded in a tissue matrix. Quercetin also increased the expression of stemness-related genes. The use of quercetin-induced DPSC living constructs for soft tissue augmentation could provide an alternative to autogenous soft tissue grafts to lower patient morbidity and improve esthetic outcomes.

Demineralized Dentin Matrix Particle-Based Bio-Ink for Patient-Specific Shaped 3D Dental Tissue Regeneration.

Demineralized dentin matrix (DDM)-based materials have been actively developed and are well-known for their excellent performance in dental tissue regeneration. However, DDM-based bio-ink suitable for fabrication of engineered dental tissues that are patient-specific in terms of shape and size, has not yet been developed. In this study, we developed a DDM particle-based bio-ink (DDMp bio-ink) with enhanced three-dimensional (3D) printability. The bio-ink was prepared by mixing DDM particles and a fibrinogen–gelatin mixture homogeneously. The effects of DDMp concentration on the 3D printability of the bio-ink and dental cell compatibility were investigated. As the DDMp concentration increased, the viscosity and shear thinning behavior of the bio-ink improved gradually, which led to the improvement of the ink’s 3D printability. The higher the DDMp content, the better were the printing resolution and stacking ability of the 3D printing. The printable minimum line width of 10% w/v DDMp bio-ink was approximately 252 μm, whereas the fibrinogen–gelatin mixture was approximately 363 μm. The ink’s cytocompatibility test with dental pulp stem cells (DPSCs) exhibited greater than 95% cell viability. In addition, as the DDMp concentration increased, odontogenic differentiation of DPSCs was significantly enhanced. Finally, we demonstrated that cellular constructs with 3D patient-specific shapes and clinically relevant sizes could be fabricated through co-printing of polycaprolactone and DPSC-laden DDMp bio-ink.

Bioelectrical interfaces with cortical spheroids in three-dimensions

Objective. Three-dimensional (3D) neuronal spheroid culture serves as a powerful model system for the investigation of neurological disorders and drug discovery. The success of such a model system requires techniques that enable high-resolution functional readout across the entire spheroid. Conventional microelectrode arrays and implantable neural probes cannot monitor the electrophysiology (ephys) activity across the entire native 3D geometry of the cellular construct. Approach. Here, we demonstrate a 3D self-rolled biosensor array (3D-SR-BA) integrated with a 3D cortical spheroid culture for simultaneous in vitro ephys recording, functional Ca2+ imaging, while monitoring the effect of drugs. We have also developed a signal processing pipeline to detect neural firings with high spatiotemporal resolution from the ephys recordings based on established spike sorting methods. Main results. The 3D-SR-BAs cortical spheroid interface provides a stable, high sensitivity recording of neural action potentials (<50 µV peak-to-peak amplitude). The 3D-SR-BA is demonstrated as a potential drug screening platform through the investigation of the neural response to the excitatory neurotransmitter glutamate. Upon addition of glutamate, the neural firing rates increased notably corresponding well with the functional Ca2+ imaging. Significance. Our entire system, including the 3D-SR-BA integrated with neuronal spheroid culture, enables simultaneous ephys recording and functional Ca2+ imaging with high spatiotemporal resolution in conjunction with chemical stimulation. We demonstrate a powerful toolset for future studies of tissue development, disease progression, and drug testing and screening, especially when combined with native spheroid cultures directly extracted from humans.

Coaxial Alginate Hydrogels: From Self-Assembled 3D Cellular Constructs to Long-Term Storage.

Alginate as a versatile naturally occurring biomaterial has found widespread use in the biomedical field due to its unique features such as biocompatibility and biodegradability. The ability of its semipermeable hydrogels to provide a favourable microenvironment for clinically relevant cells made alginate encapsulation a leading technology for immunoisolation, 3D culture, cryopreservation as well as cell and drug delivery. The aim of this work is the evaluation of structural properties and swelling behaviour of the core-shell capsules for the encapsulation of multipotent stromal cells (MSCs), their 3D culture and cryopreservation using slow freezing. The cells were encapsulated in core-shell capsules using coaxial electrospraying, cultured for 35 days and cryopreserved. Cell viability, metabolic activity and cell–cell interactions were analysed. Cryopreservation of MSCs-laden core-shell capsules was performed according to parameters pre-selected on cell-free capsules. The results suggest that core-shell capsules produced from the low viscosity high-G alginate are superior to high-M ones in terms of stability during in vitro culture, as well as to solid beads in terms of promoting formation of viable self-assembled cellular structures and maintenance of MSCs functionality on a long-term basis. The application of 0.3 M sucrose demonstrated a beneficial effect on the integrity of capsules and viability of formed 3D cell assemblies, as compared to 10% dimethyl sulfoxide (DMSO) alone. The proposed workflow from the preparation of core-shell capsules with self-assembled cellular structures to the cryopreservation appears to be a promising strategy for their off-the-shelf availability.

Evaluation of Marine Agarose Biomaterials for Tissue Engineering Applications.

Five agarose types (D1LE, D2LE, LM, MS8 and D5) were evaluated in tissue engineering and compared for the first time using an array of analysis methods. Acellular and cellular constructs were generated from 0.3–3%, and their biomechanical properties, in vivo biocompatibility (as determined by LIVE/DEAD, WST-1 and DNA release, with n = 6 per sample) and in vivo biocompatibility (by hematological and biochemical analyses and histology, with n = 4 animals per agarose type) were analyzed. Results revealed that the biomechanical properties of each hydrogel were related to the agarose concentration (p < 0.001). Regarding the agarose type, the highest (p < 0.001) Young modulus, stress at fracture and break load were D1LE, D2LE and D5, whereas the strain at fracture was higher in D5 and MS8 at 3% (p < 0.05). All agaroses showed high biocompatibility on human skin cells, especially in indirect contact, with a correlation with agarose concentration (p = 0.0074 for LIVE/DEAD and p = 0.0014 for WST-1) and type, although cell function tended to decrease in direct contact with highly concentrated agaroses. All agaroses were safe in vivo, with no systemic effects as determined by hematological and biochemical analysis and histology of major organs. Locally, implants were partially encapsulated and a pro-regenerative response with abundant M2-type macrophages was found. In summary, we may state that all these agarose types can be safely used in tissue engineering and that the biomechanical properties and biocompatibility were strongly associated to the agarose concentration in the hydrogel and partially associated to the agarose type. These results open the door to the generation of specific agarose-based hydrogels for definite clinical applications such as the human skin, cornea or oral mucosa.

Heterocellular spheroids of the neurovascular blood-brain barrier as a platform for personalized nanoneuromedicine

SummaryNanoneuromedicine investigates nanotechnology to target the brain and treat neurological diseases. In this work, we biofabricated heterocellular spheroids comprising human brain microvascular endothelial cells, brain vascular pericytes and astrocytes combined with primary cortical neurons and microglia isolated from neonate rats. The structure and function are characterized by confocal laser scanning and light sheet fluorescence microscopy, electron microscopy, western blotting, and RNA sequencing. The spheroid bulk is formed by neural cells and microglia and the surface by endothelial cells and they upregulate key structural and functional proteins of the blood-brain barrier. These cellular constructs are utilized to preliminary screen the permeability of polymeric, metallic, and ceramic nanoparticles (NPs). Findings reveal that penetration and distribution patterns depend on the NP type and that microglia would play a key role in this pathway, highlighting the promise of this platform to investigate the interaction of different nanomaterials with the central nervous system in nanomedicine, nanosafety and nanotoxicology.

PLGA Multiplex Membrane Platform for Disease Modelling and Testing of Therapeutic Compounds.

A proper validation of an engineered brain microenvironment requires a trade of between the complexity of a cellular construct within the in vitro platform and the simple implementation of the investigational tool. The present work aims to accomplish this challenging balance by setting up an innovative membrane platform that represents a good compromise between a proper mimicked brain tissue analogue combined with an easily accessible and implemented membrane system. Another key aspect of the in vitro modelling disease is the identification of a precise phenotypic onset as a definite hallmark of the pathology that needs to be recapitulated within the implemented membrane system. On the basis of these assumptions, we propose a multiplex membrane system in which the recapitulation of specific neuro-pathological onsets related to Alzheimer’s disease pathologies, namely oxidative stress and β-amyloid1–42 toxicity, allowed us to test the neuroprotective effects of trans-crocetin on damaged neurons. The proposed multiplex membrane platform is therefore quite a versatile tool that allows the integration of neuronal pathological events in combination with the testing of new molecules. The present paper explores the use of this alternative methodology, which, relying on membrane technology approach, allows us to study the basic physiological and pathological behaviour of differentiated neuronal cells, as well as their changing behaviour, in response to new potential therapeutic treatment.

Pulsed laser assisted high-throughput intracellular delivery in hanging drop based three dimensional cancer spheroids.

Targeted intracellular delivery of biomolecules and therapeutic cargo enables the controlled manipulation of cellular processes. Laser-based optoporation has emerged as a versatile, non-invasive technique that employs light-based transient physical disruption of the cell membrane and achieves high transfection efficiency with low cell damage. Testing of the delivery efficiency of optoporation-based techniques has been conducted on single cells in monolayers, but its applicability in three-dimensional (3D) cell clusters/spheroids has not been explored. Cancer cells grown as 3D tumor spheroids are widely used in anti-cancer drug screening and can be potentially employed for testing delivery efficiency. Towards this goal, we demonstrated the optoporation-based high-throughput intracellular delivery of a model fluorescent cargo (propidium iodide, PI) within 3D SiHa human cervical cancer spheroids. To enable this technique, nano-spiked core-shell gold-coated polystyrene nanoparticles (ns-AuNPs) with a high surface-to-volume ratio were fabricated. ns-AuNPs exhibited high electric field enhancement and highly localized heating at an excitation wavelength of 680 nm. ns-AuNPs were co-incubated with cancer cells within hanging droplets to enable the rapid aggregation and assembly of spheroids. Nanosecond pulsed-laser excitation at the optimized values of laser fluence (45 mJ cm-2), pulse frequency (10 Hz), laser exposure time (30 s), and ns-AuNP concentration (5 × 1010 particles per ml) resulted in the successful delivery of PI dye into cancer cells. This technique ensured high delivery efficiency (89.6 ± 2.8%) while maintaining high cellular viability (97.4 ± 0.4%), thereby validating the applicability of this technique for intracellular delivery. The optoporation-based strategy can enable high-throughput single cell manipulation, is scalable towards larger 3D tissue constructs, and may provide translational benefits for the delivery of anti-cancer therapeutics to tumors.

Bioinspired Device Improves The Cardiogenic Potential of Cardiac Progenitor Cells.

Objective Functional cardiac tissue engineering holds promise as a candidate approach for myocardial infarction. Tissue engineering has emerged to generate functional tissue constructs and provide an alternative means to repair and regenerate damaged heart tissues. Materials and Methods In this experimental study, we fabricated a composite polycaprolactone (PCL)/gelatine electrospun scaffold with aligned nanofibres. The electrospinning parameters and optimum proportion of the PCL/ gelatine were tested to design a scaffold with aligned and homogenized nanofibres. Using scanning electron microscopy (SEM) and mechanophysical testes, the PCL/gelatine composite scaffold with a ratio of 70:30 was selected. In order to simulate cardiac contraction, a developed mechanical loading device (MLD) was used to apply a mechanical stress with specific frequency and tensile rate to cardiac progenitor cells (CPCs) in the direction of the aligned nanofibres. Cell metabolic determination of CPCs was performed using real-time polymerase chain reaction(RT-PCR). Results Physicochemical and mechanical characterization showed that the PCL/gelatine composite scaffold with a ratio of 70:30 was the best sample. In vitro analysis showed that the scaffold supported active metabolism and proliferation of CPCs, and the generation of uniform cellular constructs after five days. Real-time PCR analysis revealed elevated expressions of the specific genes for synchronizing beating cells (MYH-6, TTN and CX-43) on the dynamic scaffolds compared to the control sample with a static culture system. ConclusionOur study provides a robust platform for generation of synchronized beating cells on a nanofibre patch that can be used in cardiac tissue engineering applications in the near future.

3D bioprinting of bicellular liver lobule-mimetic structures via microextrusion of cellulose nanocrystal-incorporated shear-thinning bioink

3D bioprinting of living cellular constructs with heterogeneity in cell types and extra cellular matrices (ECMs) matching those of biological tissues remains challenging. Here, we demonstrate that, through bioink material design, microextrusion-based (ME) bioprinting techniques have the potential to address this challenge. A new bioink employing alginate (1%), cellulose nanocrystal (CNC) (3%), and gelatin methacryloyl (GelMA) (5%) (namely 135ACG hybrid ink) was formulated for the direct printing of cell-laden and acellular architectures. The 135ACG ink displayed excellent shear-thinning behavior and solid-like properties, leading to high printability without cell damage. After crosslinking, the ACG gel can also provide a stiff ECM ideal for stromal cell growth. By controlling the degree of substitution and polymer concentration, a GelMA (4%) bioink was designed to encapsulate hepatoma cells (hepG2), as GelMA gel possesses the desired low mechanical stiffness matching that of human liver tissue. Four different versions of to-scale liver lobule-mimetic constructs were fabricated via ME bioprinting, with precise positioning of two different cell types (NIH/3T3 and hepG2) embedded in matching ECMs (135ACG and GelMA, respectively). The four versions allowed us to exam effects of mechanical cues and intercellular interactions on cell behaviors. Fibroblasts thrived in stiff 135ACG matrix and aligned at the 135ACG/GelMA boundary due to durotaxis, while hepG2 formed spheroids exclusively in the soft GelMA matrix. Elevated albumin production was observed in the bicellular 3D co-culture of hepG2 and NIH/3T3, both with and without direct intercellular contact, indicating that improved hepatic cell function can be attributed to soluble chemical factors. Overall, our results showed that complex constructs with multiple cell types and varying ECMs can be bioprinted and potentially useful for both fundamental biomedical research and translational tissue engineering.

Assessment of human ovarian follicular fluid derived mesenchymal stem cells in chitosan/PCL/Zn scaffold for bone tissue regeneration

Bone tissue engineering compasses the use of mesenchymal stem cells (MSCs) along with engineered biomaterial construct to augment bone regeneration. Till now, MSCs were isolated from various sources and used in cellular constructs. For the first time, in this study, MSCs were isolated from human Ovarian Follicular Fluid (OFF) and characterized by CD 44+ and CD 105+ markers via confocal microscopy and flow cytometry. Additionally, MSCs stemness, proliferation and colony-forming unit ability, multi-lineage differentiation potential were also studied. To test its suitability for bone tissue engineering applications, we grew the MSCs with the conditioned medium obtained from biocomposite scaffold by fusing a natural polymer, Chitosan (CS) and a synthetic polymer, Polycaprolactone (PCL) and the scaffold were coated with Zinc divalent ions to impart osteogenic properties. The physico-chemical characterization of scaffold, such as FTIR, XRD, and SEM studies was carried out. The biological characterization showed that the scaffolds were compatible with MSCs and promoted osteoblast differentiation which was confirmed at both cellular and molecular levels. The cellular construct increased calcium deposition, analyzed by alizarin red staining and ALP activity at cellular level. At the molecular level, the osteoblast markers expression such as Runx2 and type 1 collagen mRNAs, and osteonectin (ON) and osteocalcin (OC) secretory proteins were increased in the presence of scaffold. Overall, the current study recommends that MSCs can be easily obtained from human waste OFF, and grown in standard in vitro conditions. Successful growth of such MSCs with CS/PCL/Zn scaffold opens new avenues in utilizing the cell source for bone tissue engineering.

Emulating Human Tissues and Organs: A Bioprinting Perspective Toward Personalized Medicine.

The lack of in vitro tissue and organ models capable of mimicking human physiology severely hinders the development and clinical translation of therapies and drugs with higher in vivo efficacy. Bioprinting allow us to fill this gap and generate 3D tissue analogues with complex functional and structural organization through the precise spatial positioning of multiple materials and cells. In this review, we report the latest developments in terms of bioprinting technologies for the manufacturing of cellular constructs with particular emphasis on material extrusion, jetting, and vat photopolymerization. We then describe the different base polymers employed in the formulation of bioinks for bioprinting and examine the strategies used to tailor their properties according to both processability and tissue maturation requirements. By relating function to organization in human development, we examine the potential of pluripotent stem cells in the context of bioprinting toward a new generation of tissue models for personalized medicine. We also highlight the most relevant attempts to engineer artificial models for the study of human organogenesis, disease, and drug screening. Finally, we discuss the most pressing challenges, opportunities, and future prospects in the field of bioprinting for tissue engineering (TE) and regenerative medicine (RM).

Treatment of venous leg ulcers using bilayered living cellular construct.

Background: Venous leg ulcers (VLUs) present a significant economic burden on the US healthcare system and payers (US$14.9 billion). Aim: To evaluate the quality of life (QoL) of patients with VLUs; to analyze the limitations of standard of care (SOC) for VLUs; and to explain how using bilayered living cellular construct (BLCC) with SOC for treatment of VLUs can help heal more VLUs faster (than using SOC alone) as well as help improve QoL and help reduce the burden on the US healthcare system and payers. Materials & methods: This is a review study. The search was conducted in February 2020 by way of electronic databases to find relevant articles that provided information related to QoL of patients with VLUs, limitations of SOC for VLUs and economic analyses of using BLCC for treatment of VLUs. Results: VLUs impact patients' physical, functional and psychological status and reduce QoL. A total75% of VLU patients who used SOC alone failed to achieve healing in a timely fashion, which led to increased healthcare costs and healthcare resource utilization. Although the upfront cost is high, the greater effectiveness of BLCC offsets the added cost of the product during the time period of the studies. Therefore, BLCC helps to improve the QoL of VLU patients. As an example, for every 100 VLU patients in a healthcare plan, the use of BLCC can create cost savings of US$1,349,829.51. Conclusion: Payers' coverage of BLCC results in reduction of the overall medical cost for treating VLU patients.

Effects of Irgacure 2959 and lithium phenyl-2,4,6-trimethylbenzoylphosphinate on cell viability, physical properties, and microstructure in 3D bioprinting of vascular-like constructs

Photocrosslinkable polymers such as gelatin methacrylate (GelMA) have various 3D bioprinting applications. These polymers crosslink upon exposure to UV irradiation with the existence of an appropriate photoinitiator. Two photoinitiators, Irgacure 2959 and lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) are commonly used. This study systematically investigates the effects of photoinitiator types on the cell viability, physical properties, and microstructure in 3D bioprinting of GelMA-based cellular constructs. The main conclusions are: (1) during the 3D bioprinting, the cell viability generally decreases as the photoinitiator concentration and printing time increase using both Irgacure 2959 and LAP. At the low photoinitiator concentrations (such as 0.3% and 0.5% (w/v)), the overall cell viability is good within the printing time of 60 min using both Irgacure 2959 and LAP. However, at the high photoinitiator concentrations (such as 0.7% and 0.9% (w/v)), the overall cell viability using LAP is much higher than that using Irgacure 2959 within the printing time of 60 min; (2) after the 3D bioprinting, the photoinitiator types, either Irgacure 2959 or LAP, have negligible effects on the post-printing cell viability after crosslinking; (3) after the 3D bioprinting, GelMA samples cured with Irgacure 2959 have slightly larger pore size, faster degradation rate, and greater swelling ratio compared to those cured with LAP; (4) 3D GelMA-based vascular-like constructs have been fabricated using dynamic optical projection stereolithography, and the measured dimensions have been compared with the designed dimensions showing good shape fidelity.

Gingival phenotype modification therapies on natural teeth: A network meta-analysis.

The periodontal phenotype consists of the bone morphotype, the keratinized tissue (KT), and gingival thickness (GT). The latter two components, overlying the bone, constitute the gingival phenotype. Several techniques have been proposed for enhancing or augmenting KT or GT. However, how phenotype modification therapy (PMT) affects periodontal health and whether the obtained outcomes are maintained over time have not been elucidated. The aim of the present review was to summarize the available evidence in regard to the utilized approaches for gingival PMT and assess their comparative efficacy in augmenting KT, GT and in improving periodontal health using autogenous, allogenic, and xenogeneic grafting approaches. A detailed systematic search was performed to identify eligible randomized clinical trials (RCTs) reporting on the changes in GT and KT (primary outcomes). The selected articles were segregated into the type of approach based on having performed a root coverage, or non-root coverage procedure. A network meta-analysis (NMA) was conducted for each approach to assess and compare the outcomes among different treatment arms for the primary outcomes. A total of 105 eligible RCTs were included. 95 pertaining to root coverage (3,539 treated gingival recessions [GRs]), and 10 for non-root coverage procedures (699 total treated sites). The analysis on root coverage procedures showed that all investigated techniques (the acellular dermal matrix [ADM], collagen matrix [CM], connective tissue graft [CTG]) are able to significantly increase the GT, compared with treatment with flap alone. However, KT was only significantly increased with the use of CTG or ADM. Early post-treatment GT was found to inversely predict future GR. For non-root coverage procedures, only the changes in KT could be analyzed; all investigated treatment groups (ADM, CM, free gingival graft [FGG], living cellular construct [LCC], in combination with an apically positioned flap [APF]), resulted in significantly more KT than treatment with APF alone. Additionally, the augmented GT was shown to be sustained, and KT displayed an incremental increase over time. Within its limitations, it was observed that any graft material was able to significantly enhance GT, while KT in root coverage procedures was significantly enhanced with CTG and ADM, and in non-root coverage procedures, with ADM, CM, FGG, and LCC compared with APF alone. The autogenous soft tissue graft (CTG/FGG) proved to be superior in all comparisons for both outcomes of GT and KT.

3D Bioprinting and Its Application to Military Medicine.

Traditionally, tissue engineering techniques have largely focused on 2D cell culture models-monolayers of immortalized or primary cells growing on tissue culture plastic. Although these techniques have proven useful in research, they often lack physiological validity, because of the absence of fundamental tissue properties, such as multicellular organization, specialized extracellular matrix structures, and molecular or force gradients essential to proper physiological function. More recent advances in 3D cell culture methods have facilitated the development of more complex physiological models and tissue constructs; however, these often rely on self-organization of cells (bottom-up design), and the range of tissue construct size and complexity generated by these methods remains relatively limited. By borrowing from advances in the additive manufacturing field, 3D bioprinting techniques are enabling top-down design and fabrication of cellular constructs with controlled sizing, spacing, and chemical functionality. The high degree of control over engineered tissue architecture, previously unavailable to researchers, enables the generation of more complex, physiologically relevant 3D tissue constructs. Three main 3D bioprinting techniques are reviewed-extrusion, droplet-based, and laser-assisted bioprinting techniques are among the more robust 3D bioprinting techniques, each with its own strengths and weaknesses. High complexity tissue constructs created through 3D bioprinting are opening up new avenues in tissue engineering, regenerative medicine, and physiological model systems for researchers in the military medicine community. Recent primary literature and reviews were selected to provide a broad overview of the field of 3D bioprinting and illustrate techniques and examples of 3D bioprinting relevant to military medicine. References were selected to illustrate specific examples of advances and potential military medicine applications in the 3D bioprinting field, rather than to serve as a comprehensive review. Three classes of 3D bioprinting techniques were reviewed: extrusion, droplet-based, and laser-assisted bioprinting. Advantages, disadvantages, important considerations, and constraints of each technique were discussed. Examples from the primary literature were given to illustrate the techniques. Relevant applications of 3D bioprinting to military medicine, namely tissue engineering/regenerative medicine and new models of physiological systems, are discussed in the context of advancing military medicine. 3D bioprinting is a rapidly evolving field that provides researchers the ability to build tissue constructs that are more complex and physiologically relevant than traditional 2D culture methods. Advances in bioprinting techniques, bioink formulation, and cell culture methods are being translated into new paradigms in tissue engineering and physiological system modeling, advancing the state of the art, and increasing construct availability to the military medicine research community.

Tissue Engineering: Synthetic Biology and Tissue Engineering: Toward Fabrication of Complex and Smart Cellular Constructs (Adv. Funct. Mater. 26/2020)

In article number 1909882, Samad Ahadian, Leonardo Morsut, Ali Khademhosseini, and co-workers highlight the great potential of tissue engineering approaches and synthetic biology principles to fabricate biological tissues and cellular constructs in a biomimetic and precise manner via the manipulation of cell behavior and function. Advanced biomaterials and biofabrication tools can also be employed to improve the functionality of engineered tissues for therapeutic and in vitro applications.

Label-Free Magnetic-Field-Assisted Assembly of Layer-on-Layer Cellular Structures.

Controlled cell assembly is essential for fabricating in vitro 3D models that mimic the physiology of in vivo cellular architectures. Whereas tissue engineering techniques often rely on intrusive magnetic nanoparticles placed in cells and hydrogel encapsulation of cells to produce multilayered cellular constructs, we describe a high-throughput, label-free, and scaffold-free magnetic field-guided technique that assembles cells into a layered aggregate. An inhomogeneous magnetic field influences the diamagnetic cells suspended in a paramagnetic culture medium. Driven by the magnetic susceptibility difference and the field gradient, the cells are displaced toward the region of lowest field strength. Two cell lines are used to demonstrate the sequential assembly of layer-on-layer aggregates in microwells within 6 h. The effect of magnet size on the assembly dynamics is characterized and a microwell size criterion for the highest cell aggregation provided. Label-free magnetic-field-assisted assembly is relevant for on-demand scalable biofabrication of complex layered structures. Potential applications include drug discovery, developmental biology, lab-on-chip devices, and cancer research.

PIT14 COMPARATIVE EFFECTIVENESS OF A BILAYERED LIVING CELLULAR CONSTRUCT AND A DEHYDRATED HUMAN AMNION/CHORION MEMBRANE FOR THE TREATMENT OF PRESSURE ULCERS

Using deidentified, patient data from a wound-specific electronic medical record (WoundExpert, Net Health, PA, US), we compared the effectiveness of a bilayered living cellular construct (BLCC) to a dehydrated human amnion/chorion membrane (dHACM) for the treatment of pressure ulcers (PRUs) in a retrospective comparative effectiveness (CER) study. PRUs over anatomical locations including sacrum, coccyx, greater trochanter, ischial tuberosity, calcaneus, and lateral malleolus were included. Data from 1,189 refractory PRUs, Stages II–IV, with surface areas between 1 and 200 cm2 in size, treated between July 2014 and October 2019 at 188 wound care facilities across the US were analyzed. All treated PRUs that closed by <40% within 28 days of the first treatment application were included. Unadjusted time to event analyses were performed by the method of Kaplan-Meier (K-M), and adjusted analyses were performed using Cox’s proportional hazards regression (Cox). Patient baseline demographics and wound characteristics were comparable between groups. Cox derived estimates of wound closure for BLCC (728 wounds) were significantly greater (p=0.0001; Wald Test) by weeks 12 (36% vs. 27%), 18 (51% vs. 39%), and 24 (59% vs. 47%) respectively, compared with dHACM (461 wounds). BLCC treatment significantly reduced the median time to wound closure by 21%, achieving healing 4.4 weeks sooner (17.0 vs. 21.4 weeks, p=0.0001; Wald Test). Cox regression analysis adjusting for multiple covariates including ulcer area, depth, and duration showed that treatment with BLCC increased the probability of healing by 38% compared with dHACM (Hazard Ratio=1.38 [95% confidence interval (1.17, 1.62)], p=0.0001). These real-world data demonstrate that BLCC significantly improved wound closure compared to dHACM for the treatment of PRUs. The improvements in the probability, speed and the incidence of wound closure in VLUs treated with BLCC suggest a greater clinical benefit as well as a potential cost savings benefit.

PCV1 A RETROSPECTIVE, COMPARATIVE EFFECTIVENESS ANALYSIS OF A BILAYERED LIVING CELLULAR CONSTRUCT AND AN ACELLULAR FETAL BOVINE COLLAGEN DRESSING FOR THE TREATMENT OF VENOUS LEG ULCERS

In a real-world, retrospective, comparative effectiveness research (CER) study design we evaluated a large venous leg ulcer (VLU) patient population comparing clinical outcomes using a bilayered living cellular construct (BLCC) versus an acellular fetal bovine collagen dressing (FBCD). Electronic medical records (WoundExpert, Net Health, PA, US) used by wound care facilities across the US were the source of these retrospective data. Data collected between 1/2015 and 1/2019 on 6,916 patients (9,552 VLUs) were analyzed. Ulcers 1-40 cm2 that closed ≤40% in the 4 weeks prior to treatment were included. Patients with no baseline wound measurements or follow-up visits were excluded. Evaluations were performed on 6,648 BLCC patients (9,270 VLUs), and 268 FBCD patients (282 VLUs). At baseline, median areas were 6.8 and 7.1 cm2, and median durations were 5.1 and 5.5 months, for BLCC and FBCD, respectively. A Cox analysis adjusting for key covariates including ulcer size and duration was conducted. The median time to closure was significantly faster for BLCC (18 weeks) when compared to FBCD (25 weeks), representing a 28% reduction (p=0.003). The frequency of wound closure for BLCC was significantly greater compared to FBCD by week 12 (35 vs 29%), 24 (54 vs 47%), and 36 (69 vs 52%) (p=0.003). Analyses of two de-identified insurance claims databases showed that VLU treatment costs were directly proportional to the frequency of and time to healing. Patients with non-healed VLUs had 78% more hospitalization days, 50% more emergency department visits, 27% more outpatient office visits, and 60% more days of home healthcare. Total healthcare costs were $537/week higher for patients with open VLUs. These data show that BLCC significantly improved the incidence and decreased the time to wound closure compared to FBCD suggesting that BLCC may provide significant cost savings for the treatment of VLUs.