Development of a bioreactor for in-vitro compression cycling of tissue engineered meniscal implants.

Injuries to the meniscus are common and can impair physical activities. Bioprinted meniscal tissue offers an attractive alternative to donor tissue for meniscal repair but achieving the strength of native tissue is a challenge. Here we report the development of a tissue engineering bioreactor designed to apply repetitive force which may lead to an increase in the compressive modulus and durability of bioprinted meniscal tissues. The modular bioreactor system is composed of a sterilizable tissue culture vessel together with a dock that applies and measures mechanical force. The culture vessel allows for simultaneous compression cycling of two anatomically sized menisci. Using a hybrid linear actuator with a stepper motor, the dock can apply up to 300 N of force at speeds up to 20 mm/s, corresponding to the upper limits of anatomical force and motion in the knee. An interchangeable 22 N load cell was mated between the culture vessel and the dock to log changes in force. Both the culture vessel and dock are maintained in a standard cell culture incubator to provide heat and CO2, while the dock is powered and controlled externally using a step motor drive and customized software.

Additive Manufacturing for Fabrication of Point-of-Care Therapies in Austere Environments.

INTRODUCTION: Known as the "golden hour," survival of most critically injured patients is highly dependent on providing the required treatment within the first hour of injury. Recent technological advances in additive manufacturing (also known as three-dimensional [3D] printing) allow for austere deployment and point-of-care rapid fabrication of a variety of medical supplies, including human tissues and bioactive bandages, in prolonged field care scenarios. In this pilot project, our aim was to investigate the ability to 3D print a range of potential biomedical supplies and solutions in an austere field environment. MATERIALS AND METHODS: We specifically designed and fabricated novel surgical tools, bioactive bandages, objects (screw and anatomic models), and human meniscal tissue in an austere African desert environment. A total of seven packages were sent using a commercial carrier directly to the end destination. A multi-tool ruggedized 3D printer was used as the manufacturing platform for all objects fabricated downrange. Human mesenchymal stem cells were shipped for 3D bioprinting of human menisci and bioactive bandages. Design and fabrication for all 3D-printed products utilized computer-aided design (CAD) tools. RESULTS: Initial shipment from a single U.S. site to the sub-Saharan Africa location was relatively prompt, taking an average of 4.7 days to deliver three test packages. However, the actual delivery of the seven packages from Orlando, FL, to the same sub-Saharan Africa site took an average of 16 days (range 7-23 days). The ruggedized printer successfully fabricated relevant medical supplies using biocompatible filament, bioink hydrogels, and stem cell-loaded bioinks. This prototype did not, however, have the capacity to provide a sterile environment. A multi-material complete bandage was 3D printed using polyamide polyolefin and cellulose, live cells, neomycin salve, and adhesive. The bandage, wound covering backing, and adhesive backing print took under 2 min to 3D print. Surgical instrument CAD files were based on commercially available medical-grade stainless-steel instruments. The screw CAD file was downloaded from the NIH 3D Print Exchange website. The prints of the two surgical tools and screw using thermoplastic material were successful. Menisci, relatively complex forms of the cartilage, were 3D bioprinted with a gel that held their form well after printing and were then solidified slightly using a cross-linking solution. After 2 min of solidification, it was possible to remove and handle the menisci. CONCLUSION: The current and future challenges of prolonged field care need to be addressed with new techniques, training, and technology. Ruggedized, deployable 3D printers allow for the direct fabrication of medical tools, supplies, and biological solutions for austere use. Delivery of packages can vary, and attention to routes and location is key, especially for transit of time-sensitive perishable supplies such as live cells. The significance of this study provides the real possibility to 3D print "just-in-time" medical solutions tailored to the need of an individual service member in any environment. This is a potentially exciting opportunity to bring critical products to the war front.

Meniscal Salvage: Where We Are Today.

The menisci are fibrocartilaginous semilunar structures in the knee that provide load support. Injury to the meniscus alters its load sharing and biomechanical profile. Knee arthroscopy with meniscus débridement is the most common orthopaedic surgical procedure done in the United States. The current goals of meniscal surgery are to preserve native meniscal tissue and maintain structural integrity. Meniscal preservation is critical to maintain the normal mechanics and homeostasis of the knee; however, it is not always feasible because of the structure's poor blood supply and often requires removal of irreparable tissue with meniscectomy. Efforts have increasingly focused on the promotion of meniscal healing and the replacement of damaged menisci with allografts, scaffolds, meniscal implants, or substitutes. The purpose of this article was to review current and future meniscal salvage treatments such as meniscus transplant, synthetic arthroplasty, and possible bioprinted meniscus to allow patients to maintain quality of life, limit pain, and delay osteoarthritis.

A review of strategies for development of tissue engineered meniscal implants.

The meniscus is a key stabilizing tissue of the knee that facilitates proper tracking and movement of the knee joint and absorbs stresses related to physical activity. This review article describes the biology, structure, and functions of the human knee meniscus, common tears and repair approaches, and current research and development approaches using modern methods to fabricate a scaffold or tissue engineered meniscal replacement. Meniscal tears are quite common, often resulting from sports or physical training, though injury can result without specific contact during normal physical activity such as bending or squatting. Meniscal injuries often require surgical intervention to repair, restore basic functionality and relieve pain, and severe damage may warrant reconstruction using allograft transplants or commercial implant devices. Ongoing research is attempting to develop alternative scaffold and tissue engineered devices using modern fabrication techniques including three-dimensional (3D) printing which can fabricate a patient-specific meniscus replacement. An ideal meniscal substitute should have mechanical properties that are close to that of natural human meniscus, and also be easily adapted for surgical procedures and fixation. A better understanding of the organization and structure of the meniscus as well as its potential points of failure will lead to improved design approaches to generate a suitable and functional replacement.

3D Bioprinting and Its Application to Military Medicine.

INTRODUCTION: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Flow-Through Optical Chromatography in Combination with Confocal Raman Microspectroscopy: A Novel Label-Free Approach To Detect Responses of Live Macrophages to Environmental Stimuli.

Flow-through optical chromatography (FT-OC), an advanced mode of optical chromatography, achieved baseline separation of a mixture of silica microparticles (SiO2, 1.00 and 2.50 µm) and a mixture of polystyrene microparticles (PS, 1.00, 2.00, and 3.00 µm) based on particle size. Comparisons made between experimentally determined velocities for the microparticles and theoretically derived velocities from Mie theory and Stokes' law validated the data collection setup and the data analysis for FT-OC. A population shift in live macrophages (cell line IC-21, ATCC TIB-186) responding to environmental stimuli was sensitively detected by FT-OC. The average velocity of macrophages stressed by nutritional deprivation was decreased considerably together with a small but statistically significant increase in cell size. Mie scattering calculations demonstrated that the small increase in cell size of macrophages stressed by nutritional deprivation was not entirely responsible for this decrease. Confocal fluorescence microscopy and atomic force microscopy (AFM) studies revealed morphological changes of macrophages induced by nutritional deprivation, and these changes were more likely responsible for the decrease in average velocity detected by FT-OC. Confocal Raman microspectroscopy was used to shed light upon biochemical transformations of macrophages suffering from nutritional deprivation.

Mechanotransduction of vocal fold fibroblasts and mesenchymal stromal cells in the context of the vocal fold mechanome.

The design of cell-based therapies for vocal fold tissue engineering requires an understanding of how cells adapt to the dynamic mechanical forces found in the larynx. Our objective was to compare mechanotransductive processes in therapeutic cell candidates (mesenchymal stromal cells from adipose tissue and bone marrow, AT-MSC and BM-MSC) to native cells (vocal fold fibroblasts-VFF) in the context of vibratory strain. A bioreactor was used to expose VFF, AT-MSC, and BM-MSC to axial tensile strain and vibration at human physiological levels. Microarray, an empirical Bayes statistical approach, and geneset enrichment analysis were used to identify significant mechanotransductive pathways associated with the three cell types and three mechanical conditions. Two databases (Gene Ontology, Kyoto Encyclopedia of Genes and Genomes) were used for enrichment analyses. VFF shared more mechanotransductive pathways with BM-MSC than with AT-MSC. Gene expression that appeared to distinguish the vibratory strain condition from polystyrene condition for these two cells types related to integrin activation, focal adhesions, and lamellipodia activity, suggesting that vibratory strain may be associated with cytoarchitectural rearrangement, cell reorientation, and extracellular matrix remodeling. In response to vibration and tensile stress, BM-MSC better mimicked VFF mechanotransduction than AT-MSC, providing support for the consideration of BM-MSC as a cell therapy for vocal fold tissue engineering. Future research is needed to better understand the sorts of physical adaptations that are afforded to vocal fold tissue as a result of focal adhesions, integrins, and lamellipodia, and how these adaptations could be exploited for tissue engineering.

Gene Expression Changes in Long-Term In Vitro Human Blood-Brain Barrier Models and Their Dependence on a Transwell Scaffold Material.

Disruption of the blood-brain barrier (BBB) is the hallmark of many neurovascular disorders, making it a critically important focus for therapeutic options. However, testing the effects of either drugs or pathological agents is difficult due to the potentially damaging consequences of altering the normal brain microenvironment. Recently, in vitro coculture tissue models have been developed as an alternative to animal testing. Despite low cost, these platforms use synthetic scaffolds which prevent normal barrier architecture, cellular crosstalk, and tissue remodeling. We created a biodegradable electrospun gelatin mat "biopaper" (BP) as a scaffold material for an endothelial/astrocyte coculture model allowing cell-cell contact and crosstalk. To compare the BP and traditional models, we investigated the expression of 27 genes involved in BBB permeability, cellular function, and endothelial junctions at different time points. Gene expression levels demonstrated higher expression of transcripts involved in endothelial junction formation, including TJP2 and CDH5, in the BP model. The traditional model had higher expression of genes associated with extracellular matrix-associated proteins, including SPARC and COL4A1. Overall, the results demonstrate that the BP coculture model is more representative of a healthy BBB state, though both models have advantages that may be useful in disease modeling.

Biomechanical Screening of Cell Therapies for Vocal Fold Scar.

Candidate cell sources for vocal fold scar treatment include mesenchymal stromal cells from bone marrow (BM-MSC) and adipose tissue (AT-MSC). Mechanosensitivity of MSC can alter highly relevant aspects of their behavior, yet virtually nothing is known about how MSC might respond to the dynamic mechanical environment of the larynx. Our objective was to evaluate MSC as a potential cell source for vocal fold tissue engineering in a mechanically relevant context. A vibratory strain bioreactor and cDNA microarray were used to evaluate the similarity of AT-MSC and BM-MSC to the native cell source, vocal fold fibroblasts (VFF). Posterior probabilities for each of the microarray transcripts fitting into specific expression patterns were calculated, and the data were analyzed for Gene Ontology (GO) enrichment. Significant wound healing and cell differentiation GO terms are reported. In addition, proliferation and apoptosis were evaluated with immunohistochemistry. Results revealed that VFF shared more GO terms related to epithelial development, extracellular matrix (ECM) remodeling, growth factor activity, and immune response with BM-MSC than with AT-MSC. Similarity in glycosaminoglycan and proteoglycan activity dominated the ECM analysis. Analysis of GO terms relating to MSC differentiation toward osteogenic, adipogenic, and chondrogenic lineages revealed that BM-MSC expressed fewer osteogenesis GO terms in the vibrated and scaffold-only conditions compared to polystyrene. We did not evaluate if vibrated BM-MSC recover osteogenic expression markers when returned to polystyrene culture. Immunostaining for Ki67 and cleaved caspase 3 did not vary with cell type or mechanical condition. We conclude that VFF may have a more similar wound healing capacity to BM-MSC than to AT-MSC in response to short-term vibratory strain. Furthermore, BM-MSC appear to lose osteogenic potential in the vibrated and scaffold-only conditions compared to polystyrene, potentially attenuating the risk of osteogenesis for in vivo applications.

Electrospun fiber constructs for vocal fold tissue engineering: effects of alignment and elastomeric polypeptide coating.

Vocal fold lamina propria extracellular matrix (ECM) is highly aligned and when injured, becomes disorganized with loss of the tissue's critical biomechanical properties. This study examines the effects of electrospun fiber scaffold architecture and elastin-like polypeptide (ELP4) coating on human vocal fold fibroblast (HVFF) behavior for applications toward tissue engineering the vocal fold lamina propria. Electrospun Tecoflex™ scaffolds were made with aligned and unaligned fibers, and were characterized using scanning electron microscopy and uniaxial tensile testing. ELP4 was successfully adsorbed onto the scaffolds; HVFFs were seeded and their viability, proliferation, morphology and gene expression were characterized. Aligned and unaligned scaffolds had initial elastic moduli of ∼14 MPa, ∼5 MPa and ∼0.3 MPa, ∼0.6 MPa in the preferred and cross-preferred directions, respectively. Scaffold topography had an effect on the orientation of the cells, with HVFFs seeded on aligned scaffolds having a significantly different (p<0.001) angle of orientation than HVFFs cultured on unaligned scaffolds. This same effect and significant difference (p<0.001) was seen on aligned and unaligned scaffolds coated with ELP4. Scaffold alignment and ELP4 coating impacted ECM gene expression. ELP4 coating, and aligned scaffolds upregulated elastin synthesis when tested on day 7 without a concomitant upregulation of collagen III synthesis. Collectively, results indicate that aligned electrospun scaffolds and ELP4 coating are promising candidates in the development of biodegradeable vocal fold lamina propria constructs.

Formulation and characterization of a porous, elastomeric biomaterial for vocal fold tissue engineering research.

OBJECTIVE: Biomaterials able to mimic the mechanical properties of vocal fold tissue may be particularly useful for furnishing a 3-dimensional microenvironment allowing for in vitro investigation of cell and molecular responses to vibration. Motivated by the dearth of biomaterials available for use in an in vitro model for vocal fold tissue, we investigated polyether polyurethane (PEU) matrices, which are porous, mechanically tunable biomaterials that are inexpensive and require only standard laboratory equipment for fabrication. METHODS: Rheology, dynamic mechanical analysis, and scanning electron microscopy were performed on PEU matrices at 5%, 10%, and 20% w/v mass concentrations. RESULTS: For 5%, 10%, and 20% w/v concentrations, shear storage moduli were 2 kPa, 3.4 kPa, and 6 kPa, respectively, with shear loss moduli being 0.2 kPa, 0.38 kPa, and 0.62 kPa, respectively. Storage moduli responded to applied frequency as a linear function. Mercury intrusion porosimetry revealed that all 3 mass concentrations of PEU have a similar overall percentage porosity but differ in pore architecture. CONCLUSION: Twenty-µm diameter pores are ideal for cell seeding, and a range of mechanical properties indicates that the lower [corrected] mass concentration PEU formulations are best suited for mimicking the viscoelastic properties of vocal fold tissue for in vitro research.

Hyaluronic acid hydrogels for vocal fold wound healing.

The unique vibrational properties inherent to the human vocal fold have a significant detrimental impact on wound healing and scar formation. Hydrogels have taken prominence as a tissue engineered strategy to restore normal vocal structure and function as cellularity is low. The frequent vibrational and shear forces applied to, and present in this connective tissue make mechanical properties of such hydrogels a priority in this active area of research. Hyaluronic acid has been chemically modified in a variety of ways to address cell function while maintaining desirable tissue mechanical properties. These various modifications have had mixed results when injected in vivo typically resulting in better biomechanical function but not necessarily with a concomitant decrease in tissue fibrosis. Recent work has focused on seeding mesenchymal progenitor cells within 3D architecture of crosslinked hydrogels. The data from these studies demonstrate that this approach has a positive effect on cells in both early and late wound healing, but little work has been done regarding the biomechanical effects of these treatments. This paper provides an overview of the various hyaluronic acid derivatives, their crosslinking agents, and their effect when implanted into the vocal folds of various animal models.

The response of vocal fold fibroblasts and mesenchymal stromal cells to vibration.

Illumination of cellular changes caused by mechanical forces present within the laryngeal microenvironment may well guide strategies for tissue engineering the vocal fold lamina propria. The purpose of this study was to compare the response of human vocal fold fibroblasts (hVFF) and bone marrow mesenchymal stem cells (BM-MSC) to vibratory stimulus. In order to study these effects, a bioreactor capable of vibrating two cell seeded substrates was developed. The cell seeded substrates contact each other as a result of the sinusoidal frequency, producing a motion similar to the movement of true vocal folds. Utilizing this bioreactor, hVFF and BM-MSC were subjected to 200 Hz vibration and 20% strain for 8 hours. Immunohistochemistry (Ki-67 and TUNEL) was performed to examine cell proliferation and apoptosis respectively, while semi-quantitative RT-PCR was used to assess extracellular matrix related gene expression. HVFF significantly proliferated (p = 0.011) when subjected to 200 Hz vibration and 20% strain, while BM-MSC did not (p = 1.0). A statistically significant increase in apoptosis of BM-MSC (p = 0.0402) was observed under the experimental conditions; however high cell viability (96%) was maintained. HVFF did not have significantly altered apoptosis (p = 0.7849) when subjected to vibration and strain. Semi-quantitative RT-PCR results show no significant differences in expression levels of collagen I (BM-MSC p = 0.1951, hVFF p = v0.3629), fibronectin (BM-MSC p = 0.1951, hVFF p = 0.2513), and TGF-ß1 (BM-MSC p = 0.2534, hVFF p = 0.6029) between vibratory and static conditions in either cell type. Finally, smooth muscle actin mRNA was not present in either vibrated or static samples, indicating that no myofibroblast differentiation occurred for either cell type. Together, these results demonstrate that BM-MSC may be a suitable alternative to hVFF for vocal fold tissue engineering. Further investigation into a larger number of gene markers, protein levels, increased number of donors and vibratory conditions are warranted.