Multipotent mesenchymal stromal cells (MSCs) hold tremendous promise for tissue engineering and regenerative medicine, yet with so many sources of MSCs, what are the primary criteria for selecting leading candidates? Ideally, the cells will be multipotent, inexpensive, lack donor site morbidity, donor materials should be readily available in large numbers, immunocompatible, politically benign and expandable in vitro for several passages. Bone marrow MSCs do not meet all of these criteria and neither do embryonic stem cells. However, a promising new cell source is emerging in tissue engineering that appears to meet these criteria: MSCs derived from Wharton's jelly of umbilical cord MSCs. Exposed to appropriate conditions, umbilical cord MSCs can differentiate in vitro along several cell lineages such as the chondrocyte, osteoblast, adipocyte, myocyte, neuronal, pancreatic or hepatocyte lineages. In animal models, umbilical cord MSCs have demonstrated in vivo differentiation ability and promising immunocompatibility with host organs/tissues, even in xenotransplantation. In this article, we address their cellular characteristics, multipotent differentiation ability and potential for tissue engineering with an emphasis on musculoskeletal tissue engineering. Keywords differentiation; musculoskeletal; regenerative medicine; umbilical cord stroma An ideal cell source for tissue engineering should satisfy requirements including ease of access, sufficient cell number and immunocompatibility. Mature autologous cells have commonly been used in previous studies, owing to the advantages of already being differentiated and immunocompatible. In contrast to mature autologous cells, embryonic stem cells (ESCs) are a major area of contemporary research interest, and offer significant †

Breathing is a natural function that most of us do not even think about, but for those who suffer from disease or damage of the trachea, the obstruction of breathing can mean severe restrictions to quality of life or may even be fatal. Replacement and reconstruction of the trachea is one of the most difficult procedures in otolaryngology/head and neck surgery, and also one of the most vital. Previous reviews have focused primarily on clinical perspectives or instead on engineering strategies. However, the current review endeavors to bridge this gap by evaluating engineering approaches in a practical clinical context. For example, although contemporary approaches often include in vitro bioreactor pre-culture, or sub-cutaneous in vivo conditioning, the limitations they present in terms of regulatory approval, cost, additional surgery, and/or risk of infection challenge engineers to develop the next generation of biodegradable/resorbable biomaterials that can be directly implanted in situ. Essentially, the functionality of the replacement is the most important requirement. It must be the correct shape and size, achieve an airtight fit, resist collapse as it is replaced by new tissue, and be non-immunogenic. As we look to the future, there will be no one-size-fits-all solution.

Drug-induced liver injury (DILI) and drug-drug interactions (DDIs) are concerns when developing safe and efficacious compounds. We have developed an automated multiplex assay to detect hepatotoxicity (i.e., ATP depletion) and metabolism (i.e., cytochrome P450 1A [CYP1A] and cytochrome P450 3A4 [CYP3A4] enzyme activity) in two-dimensional (2D) and three-dimensional (3D) cell cultures. HepaRG cells were cultured in our proprietary micromold plates and produced spheroids. HepaRG cells, in 2D or 3D, expressed liver-specific proteins throughout the culture period, although 3D cultures consistently exhibited higher albumin secretion and CYP1A/CYP3A4 enzyme activity than 2D cultures. Once the spheroid hepatic quality was assessed, 2D and 3D HepaRGs were challenged to a panel of DILI-and CYP-inducing compounds for 7 days. The 3D HepaRG model had a 70% sensitivity to liver toxins at 7 days, while the 2D model had a 60% sensitivity. In both the 2D and 3D HepaRG models, 83% of compounds were predicted to be CYP inducers after 7 days of compound exposure. Combined, our results demonstrate that an automated multiplexed liver spheroid system is a promising cell-based method to evaluate DILI and DDI for early-stage drug discovery.

Tracheal stenosis caused by congenital anomalies, tumors, trauma, or intubation-related damage can cause severe breathing issues, diminishing the quality of life, and potentially becoming fatal. Current treatment methods include laryngotracheal reconstruction or slide tracheoplasty. Laryngotracheal reconstruction utilizes rib cartilage harvested from the patient, requiring a second surgical site. Slide tracheoplasty involves a complex surgical procedure to splay open the trachea and reconnect both segments to widen the lumen. A clear need exists for new and innovative approaches that can be easily adopted by surgeons, and to avoid harvesting autologous tissue from the patient. This study evaluated the use of an electrospun patch, consisting of randomly layered polycaprolactone (PCL) nanofibers enveloping 3D-printed PCL rings, to create a mechanically robust, suturable, air-tight, and bioresorbable graft for the treatment of tracheal defects. The study design incorporated two distinct uses of PCL: electrospun fibers to promote tissue integration, while remaining air-tight when wet, and 3D-printed rings to hold the airway open and provide external support and protection during the healing process. Electrospun, reinforced tracheal patches were evaluated in an ovine model, in which all sheep survived for 10 weeks, although an overgrowth of fibrous tissue surrounding the patch was observed to significantly narrow the airway. Minimal tissue integration of the surrounding tissue and the electrospun fibers suggested the need for further improvement. Potential areas for further improvement include a faster degradation rate, agents to increase cellular adhesion, and/or an antibacterial coating to reduce the initial bacterial load.

Tracheal stenosis can become a fatal condition, and current treatments include augmentation of the airway with autologous tissue. A tissue-engineered approach would not require a donor source, while providing an implant that meets both surgeons' and patients' needs. A fibrous, polymeric scaffold organized in gradient bilayers of polycaprolactone (PCL) and poly-lactic-co-glycolic acid (PLGA) with 3D printed structural ring supports, inspired by the native trachea rings, could meet this need. The purpose of the current study was to characterize the tracheal scaffolds with mechanical testing models to determine the design most suitable for maintaining a patent airway. Degradation over 12 weeks revealed that scaffolds with the 3D printed rings had superior properties in tensile and radial compression, with at least a three fold improvement and 8.5-fold improvement, respectively, relative to the other scaffold groups. The ringed scaffolds produced tensile moduli, radial compressive forces, and burst pressures similar to or exceeding physiological forces and native tissue data. Scaffolds with a thicker PCL component had better suture retention and tube flattening recovery properties, with the monolayer of PCL (PCL-only group) exhibiting a 2.3-fold increase in suture retention strength (SRS). Tracheal scaffolds with ring reinforcements have improved mechanical properties, while the fibrous component increased porosity and cell infiltration potential. These scaffolds may be used to treat various trachea defects (patch or circumferential) and have the potential to be employed in other tissue engineering applications.

Cell microencapsulation is a rapidly expanding field with broad potential for stem cell therapies and tissue engineering research. Traditional alginate microspheres suffer from poor biocompatibility, and microencapsulation of more advanced hydrogels is challenging due to their slower gelation rates. We have developed a novel, noncytotoxic, nonemulsion-based method to produce hydrogel microspheres compatible with a wide variety of materials, called core-shell spherification (CSS). Fabrication of microspheres by CSS derived from two slow-hardening hydrogels, hyaluronic acid (HA) and polyethylene glycol diacrylate (PEGDA), was characterized. HA microspheres were manufactured with two different crosslinking methods: thiolation and methacrylation. Microspheres of methacrylated HA (MeHA) had the greatest swelling ratio, the largest average diameter, and the lowest diffusion barrier. In contrast, PEGDA microspheres had the smallest diameters, the lowest swelling ratio, and the highest diffusion barrier, while microspheres of thiolated HA had characteristics that were in between the other two groups. To test the ability of the hydrogels to protect cells, while promoting function, diabetic NOD mice received intraperitoneal injections of PEGDA or MeHA microencapsulated canine islets. PEGDA microspheres reversed diabetes for the length of the study (up to 16 weeks). In contrast, islets encapsulated in MeHA microspheres at the same dose restored normoglycemia, but only transiently (3-4 weeks). Nonencapsulated canine islet transplanted at the same dose did not restore normoglycemia for any length of time. In conclusion, CSS provides a nontoxic microencapsulation procedure compatible with various hydrogel types.

Tracheal stenosis is a life-threatening disease and current treatments include surgical reconstruction with autologous rib cartilage and the highly complex slide tracheoplasty surgical technique. We propose using a sustainable implant, composed of a tunable, fibrous scaffold with encapsulated chondrogenic growth factor (transforming growth factor-beta3 [TGF-β3]) or seeded allogeneic rabbit bone marrow mesenchymal stromal cells (BMSCs). In vivo functionality of these constructs was determined by implanting them in induced tracheal defects in rabbits for 6 or 12 weeks. The scaffolds maintained functional airways in a majority of the cases, with the BMSC-seeded group having an improved survival rate and the Scaffold-only group having a higher occurrence of more patent airways as determined by microcomputed tomography. The BMSC group had a greater accumulation of inflammatory cells over the graft, while also exhibiting normal epithelium, subepithelium, and cartilage formation. Overall, it was concluded that a simple, acellular scaffold is a viable option for tracheal tissue engineering, with the intraoperative addition of cells being an optional variation to the scaffolds.

The field of cell therapy has blossomed, providing exciting new options for treating a variety of diseases. While few cell therapy products have US FDA approval, there are thousands of cell treatments at various stages of development, pointing to a potential revolutionary shift in patient care. The expanding number and nature of cellular therapies necessitate greater standardization. Several international organizations are collaborating to pursue some level of global standardization, especially concerning cell banking. However, less harmonization surrounds assays used for critical quality characterization including: identity, purity, safety and potency. Frequently, there is divergence regarding the terms describing the characterization assays across regulatory authorities and guidances. This review summarizes the critical quality assays currently used for different categories of cell therapies. Areas of harmonization and an absence of standardization are highlighted. We propose potential solutions to facilitate harmonization of critical quality characterization assays and the language used to describe them.