Use of growth factors as biochemical molecules to elicit cellular differentiation is a common strategy in tissue engineering. However, limitations associated with growth factors, such as short half‐life, high effective physiological doses, and high costs, have prompted the search for growth factor alternatives, such as growth factor mimics and other proteins. This work explores the use of insulin protein as a biochemical factor to aid in tendon healing and differentiation of cells on a biomimetic electrospun micro‐nanostructured scaffold. Dose response studies were conducted using human mesenchymal stem cells (MSCs) in basal media supplemented with varied insulin concentrations. A dose of 100‐ng/mL insulin showed increased expression of tendon markers. Synthetic‐natural blends of various ratios of polycaprolactone (PCL) and cellulose acetate (CA) were used to fabricate micro‐nanofibers to balance physicochemical properties of the scaffolds in terms of mechanical strength, hydrophilicity, and insulin delivery. A 75:25 ratio of PCL:CA was found to be optimal in promoting cellular attachment and insulin immobilization. Insulin immobilized fiber matrices also showed increased expression of tendon phenotypic markers by MSCs similar to findings with insulin supplemented media, indicating preservation of insulin bioactivity. Insulin functionalized scaffolds may have potential applications in tendon healing and regeneration.
In this paper, we report on a novel biocompatible micromechanical bioreactor (actuator and sensor) designed for the in situ manipulation and characterization of live microtissues. The purpose of this study was to develop and validate an application-targeted sterile bioreactor that is accessible, inexpensive, adjustable, and easily fabricated. Our method relies on a simple polydimethylsiloxane (PDMS) molding technique for fabrication and is compatible with commonly-used laboratory equipment and materials. Our unique design includes a flexible thin membrane that allows for the transfer of an external actuation into the PDMS beam-based actuator and sensor placed inside a conventional 35 mm cell culture Petri dish. Through computational analysis followed by experimental testing, we demonstrated its functionality, accuracy, sensitivity, and tunable operating range. Through time-course testing, the actuator delivered strains of over 20% to biodegradable electrospun poly (D, L-lactide-co-glycolide) (PLGA) 85:15 non-aligned nanofibers (~91 µm thick). At the same time, the sensor was able to characterize time-course changes in Young’s modulus (down to 10–150 kPa), induced by an application of isopropyl alcohol (IPA). Furthermore, the actuator delivered strains of up to 4% to PDMS monolayers (~30 µm thick), simultaneously characterizing their elastic modulus up to ~2.2 MPa. The platform repeatedly applied dynamic (0.23 Hz) tensile stimuli to live Human Dermal Fibroblast (HDF) cells for 12 hours (h) and recorded the cellular reorientation towards two angle regimes, with averages of −58.85° and +56.02°. The device biocompatibility with live cells was demonstrated for one week, with no signs of cytotoxicity. We can conclude that our PDMS bioreactor is advantageous for low-cost tissue/cell culture micromanipulation studies involving mechanical actuation and characterization. Our device eliminates the need for an expensive experimental setup for cell micromanipulation, increasing the ease of live-cell manipulation studies by providing an affordable way of conducting high-throughput experiments without the need to open the Petri dish, reducing manual handling, cross-contamination, supplies, and costs. The device design, material, and methods allow the user to define the operational range based on their targeted samples/application.
Approximately half of annual musculoskeletal injuries in the US involve tendon tears. The naturally hypocellular and hypovascular tendon environment makes tendons injury-prone and heal slowly. Tendon tissue engineering strategies often use biomimetic scaffolds combined with bioactive factors and/or cells to enhance healing. FDA-approved growth factors to promote tendon healing are lacking, which highlights the need for safe and effective bioactive factors. Our previous work evaluated insulin as a bioactive factor and identified an optimal dose to promote in vitro mesenchymal stem cell survival, division, and tenogenesis. The present work evaluates the ability of insulin-functionalized electrospun nanofiber matrices with or without mesenchymal stem cells to enhance tendon repair in a rat Achilles injury model. Electrospun nanofiber matrices were functionalized with insulin, cultured with or without mesenchymal stem cells, and sutured to transected Achilles tendons in rats. We analyzed rat tendons 4 and 8 weeks after surgery for the tendon morphology, collagen production, and mechanical properties. Bioactive insulin-functionalized fiber matrices with mesenchymal stem cells resulted in significantly increased collagen I and III at 4 and 8 weeks postsurgery. Additionally, these matrices supported highly aligned collagen fibrils in the regenerated tendon tissue at 8 weeks. However, treatment- and control-regenerated tissues had similar tensile properties at 8 weeks, which were less than that of the native Achilles tendon. Our preliminary results establish the benefits of insulin-functionalized fiber matrices in promoting higher levels of collagen synthesis and alignment needed for functional recovery of tendon repair.
Damage to intervertebral discs (IVD) can lead to chronic pain and disability, and no current treatments can fully restore their function. Some non-surgical treatments have shown promise; however, these approaches are generally limited by burst release and poor localization of diverse molecules. In this proof-of-concept study, we developed a nanoparticle (NP) delivery system to efficiently deliver high- and low-solubility drug molecules. Nanoparticles of cellulose acetate and polycaprolactone-polyethylene glycol conjugated with 1-oxo-1H-pyrido [2,1-b][1,3]benzoxazole-3-carboxylic acid (PBC), a novel fluorescent dye, were prepared by the oil-in-water emulsion. Two drugs, a water insoluble indomethacin (IND) and a water soluble 4-aminopyridine (4-AP), were used to study their release patterns. Electron microscopy confirmed the spherical nature and rough surface of nanoparticles. The particle size analysis revealed a hydrodynamic radius ranging ~150–162 nm based on dynamic light scattering. Zeta potential increased with PBC conjugation implying their enhanced stability. IND encapsulation efficiency was almost 3-fold higher than 4-AP, with release lasting up to 4 days, signifying enhanced solubility, while the release of 4-AP continued for up to 7 days. Nanoparticles and their drug formulations did not show any apparent cytotoxicity and were taken up by human IVD nucleus pulposus cells. When injected into coccygeal mouse IVDs in vivo, the nanoparticles remained within the nucleus pulposus cells and the injection site of the nucleus pulposus and annulus fibrosus of the IVD. These fluorescent nano-formulations may serve as a platform technology to deliver therapeutic agents to IVDs and other tissues that require localized drug injections.
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