Thermal inkjet bioprinting drastically alters cell phenotype.

Since the first description of inkjet bioprinting of cells in 2003, quantifying the input and measuring the output of the printers has been the hallmark of the field of bioprinting, as it is virtually impossible to characterize cells that are inside the printing orifices or extrusion needles. We will describe here some recent discoveries of cell behavior due to inkjet bioprinting. Primary and immortalized adult dermal fibroblasts were expanded for 2-3 passages upon receiving. The cells were harvested, resuspended in PBS, and bioprinted into a 96-well plate with pluriSTEM media. Cells were then transferred either into precoated 96-well plates or 20µl drops were pipetted for hanging drop culture. IPC differentiation protocols were applied and the induction was begun approximately 45 min after printing. When differentiating aggregates, the initiation happened 45 min after the aggregates were transferred into the 96 wells. Standard immunostaining and RNA sequencing (RNA-Seq) were used to analyze the cell phenotypes. Preliminary results indicate that all cells expressed the three pluripotency markers oct-4, nanog, and sox-2. After applying a cardiomyocyte differentiation protocol, the cells stained positively for troponin-3. The cells also elongated and became more cardiomyocyte-like in their morphology. We analyzed bulk RNA seq data and our preliminary results show upregulation of some genes that have been implicated as stem cell markers: EPCAM, LEFTY1, ZFP42, and TEX19. In addition, differential expression of genes associated with pluripotency-relevant pathways shows some pathways are off like the MAPK/p38, MAPK/JNK1-3 which is expected for a pluripotent state. We also have data supporting the activation of the hippo pathway with transcriptional co-activator with PDZ binding motif (TAZ) highly upregulated and yes-associated protein staining the cell body. In addition, GSK3B is off and TGFB1, LIF/PIK3, and AKT1 are on as expected for pluripotency. Examining the gene network of upregulated genes, one can clearly distinguish the pivotal role of FOS, FOXO1, and PIK3 all related to pluripotency. Bioprinted fibroblasts will at least temporarily adopt a more primitive or dedifferentiated state, reminiscent of pluripotency. While immunochemistry shows the classic transcription factors required for pluripotency, gene expression shows a more nuanced picture of the transformations that occur upon printing. Understanding these transformations, even if temporary will be crucial when trying to build tissues using bioprinting technologies.

Repairing Volumetric Muscle Loss in the Ovine Peroneus Tertius Following a 6-Month Recovery.

Tissue-engineered skeletal muscle is a promising novel therapy for the treatment of volumetric muscle loss (VML). Our laboratory has developed tissue-engineered skeletal muscle units (SMUs) and engineered neural conduits (ENCs), and modularly scaled them to clinically relevant sizes for the treatment of VML in a large animal (sheep) model. In a previous study, we evaluated the effects of the SMUs and ENCs in treating a 30% VML injury in the ovine peroneus tertius muscle after a 3-month recovery period. The goal of the current study was to expand on our 3-month study and evaluate the SMUs and ENCs in restoring muscle function after a 6-month recovery period. Six months after implantation, we found that the repair groups with the SMU (VML+SMU and VML+SMU+ENC) restored muscle mass to a level that was statistically indistinguishable from the uninjured contralateral muscle. In contrast, the muscle mass in the VML-Only group was significantly less than groups repaired with an SMU. Following the 6-month recovery from VML, the maximum tetanic force was significantly lower for all VML injured groups compared with the uninjured contralateral muscle. However, we did demonstrate the ability of our ENCs to effectively regenerate nerve between the distal stump of the native nerve and the repair site in 14 of the 15 animals studied. Impact Statement Volumetric muscle loss (VML) is a clinically relevant problem for which current treatment options are lacking and for which tissue-engineered skeletal muscle presents a promising novel therapeutic option. However, the fabrication of tissues of clinically relevant sizes is necessary for advancement of the technology to the clinic. This study aimed to evaluate the efficacy of our scaled-up tissue-engineered skeletal muscle to treat VML in a large animal (sheep) model after a 6-month recovery.

A tissue engineering approach for repairing craniofacial volumetric muscle loss in a sheep following a 2, 4, and 6-month recovery.

Volumetric muscle loss (VML) is the loss of skeletal muscle that results in significant and persistent impairment of function. The unique characteristics of craniofacial muscle compared trunk and limb skeletal muscle, including differences in gene expression, satellite cell phenotype, and regenerative capacity, suggest that VML injuries may affect craniofacial muscle more severely. However, despite these notable differences, there are currently no animal models of craniofacial VML. In a previous sheep hindlimb VML study, we showed that our lab's tissue engineered skeletal muscle units (SMUs) were able to restore muscle force production to a level that was statistically indistinguishable from the uninjured contralateral muscle. Thus, the goals of this study were to: 1) develop a model of craniofacial VML in a large animal model and 2) to evaluate the efficacy of our SMUs in repairing a 30% VML in the ovine zygomaticus major muscle. Overall, there was no significant difference in functional recovery between the SMU-treated group and the unrepaired control. Despite the use of the same injury and repair model used in our previous study, results showed differences in pathophysiology between craniofacial and hindlimb VML. Specifically, the craniofacial model was affected by concomitant denervation and ischemia injuries that were not exhibited in the hindlimb model. While clinically realistic, the additional ischemia and denervation likely created an injury that was too severe for our SMUs to repair. This study highlights the importance of balancing the use of a clinically realistic model while also maintaining control over variables related to the severity of the injury. These variables include the volume of muscle removed, the location of the VML injury, and the geometry of the injury, as these affect both the muscle's ability to self-regenerate as well as the probability of success of the treatment.

A 30% Volumetric Muscle Loss Does Not Result in Sustained Functional Deficits after a 90-Day Recovery in Rats.

Volumetric muscle loss (VML) is defined as the loss of skeletal muscle tissue which exceeds the body's repair capabilities leading to sustained functional deficits over time. Some etiologies leading to VML include traumatic injuries, congenital diseases, and degenerative myopathies. Currently, the lack of standardized animal models prevents an appropriate estimation of the severity of injury capable of exceeding self-regeneration. Recent work in our laboratory has shown that a 30% VML does not create a sustained functional loss in rats after 3 months. Therefore, the purpose of this study was to evaluate the percentage threshold of muscle loss that results in permanent functional deficits. We surgically created models of 30, 40, and 50% VML injuries in the tibialis anterior (TA) of rats, and subsequently evaluated TA function and structure after a 90-day recovery period. TA muscle force production was measured in situ by stimulating the sciatic nerve to obtain a maximum tetanic force. Results revealed that the maximum force produced by rats with a 30% VML was not significantly different from the uninjured muscle, while the maximum force of the 40% and 50% VML groups was significantly lower in comparison to the uninjured muscle. Overall, this study further supports our observations, suggesting that a 30% VML rat model is not suitable for VML studies. Thus, increasing VML percentages might provide an improved standardized and clinically relevant model for VML that produces a long-term deficit in muscle self-regeneration, while providing a strong base for future tissue engineering techniques in medicine.

Repairing Volumetric Muscle Loss in the Ovine Peroneus Tertius Following a 3-Month Recovery.

Much effort has been made to fabricate engineered tissues on a scale that is clinically relevant to humans; however, scale-up remains one of the most significant technological challenges of tissue engineering to date. To address this limitation, our laboratory has developed tissue-engineered skeletal muscle units (SMUs) and engineered neural conduits (ENCs), and modularly scaled them to clinically relevant sizes for the treatment of volumetric muscle loss (VML). The goal of this study was to evaluate the SMUs and ENCs in vitro, and to test the efficacy of our SMUs and ENCs in restoring muscle function in a clinically relevant large animal (sheep) model. The animals received a 30% VML injury to the peroneus tertius muscle and were allowed to recover for 3 months. The animals were divided into three experimental groups: VML injury without a repair (VML only), repair with an SMU (VML+SMU), or repair with an SMU and ENC (VML+SMU+ENC). We evaluated the SMUs before implantation and found that our single scaled-up SMUs were characterized by the presence of contracting myotubes, linearly aligned extracellular matrix proteins, and Pax7+ satellite cells. Three months after implantation, we found that the repair groups (VML+SMU and VML+SMU+ENC) had restored muscle mass and tetanic force production to a level that was statistically indistinguishable from the uninjured contralateral muscle after 3 months in vivo. Furthermore, we demonstrated the ability of our ENCs to effectively bridge the gap between native nerve and the repair site by eliciting a muscle contraction through direct electrical stimulation of the re-routed nerve. Impact statement The fabrication of tissues of clinically relevant sizes is one of the largest obstacles preventing engineered tissues from achieving widespread use in the clinic. This study aimed to combat this limitation by developing a fabrication method to scale-up tissue-engineered skeletal muscle for the treatment of volumetric muscle loss in a large animal (sheep) model and evaluating the efficacy of the tissue-engineered constructs after a 3-month recovery.

A Comparison of Ovine Facial and Limb Muscle as a Primary Cell Source for Engineered Skeletal Muscle.

Volumetric muscle loss (VML) contributes to the number of soft tissue injuries that necessitate reconstructive surgery, but treatment options are often limited by tissue availability and donor site morbidity. To combat these issues, our laboratory has developed scaffold-free tissue-engineered skeletal muscle units (SMUs) as a novel treatment for VML injuries. Recently, we have begun experiments addressing VML in facial muscle, and the optimal starting cell population for engineered skeletal muscle tissue for this application may not be cells derived from hindlimb muscles due to reported heterogeneity of cell populations. Thus, the purpose of this study was to compare SMUs fabricated from both craniofacial and hindlimb sources to determine which cell source is best suited for the engineering of skeletal muscle. Herein, we assessed the development, structure, and function of SMUs derived from four muscle sources, including two hindlimb muscles (i.e., soleus and semimembranosus [SM]) and two craniofacial muscles (i.e., zygomaticus major and masseter). Overall, the zygomaticus major exhibited the least efficient digestion, and SMUs fabricated from this muscle exhibited the least aligned myosin heavy chain staining and consequently, the lowest average force production. Conversely, the SM muscle exhibited the most efficient digestion and the highest number of myotubes/mm2; however, the SM, masseter, and soleus groups were roughly equivalent in terms of force production and histological structure. Impact Statement An empirical comparison of the development, structure, and function of engineered skeletal muscle tissue fabricated from different muscles, including both craniofacial and hindlimb sources, will not only provide insight into innate regenerative mechanisms of skeletal muscle but also will give our team and other researchers the information necessary to determine which cell sources are best suited for the skeletal muscle tissue engineering.

The Maturation of Tissue-Engineered Skeletal Muscle Units following 28-Day Ectopic Implantation in a Rat.

Volumetric muscle loss (VML) is a loss of skeletal muscle that results in a sustained impairment of function and is often accompanied by physical deformity. To address the need for more innovative repair options, our laboratory has developed scaffold-free, multiphasic tissue-engineered skeletal muscle units (SMUs) to treat VML injuries. In our previous work, using the concept of the "body as a bioreactor", we have shown that implantation promotes the maturation of our SMUs beyond what is possible in vitro. Thus, in this study we sought to better understand the effect of implantation on the maturation of our SMUs, including the effects of implantation on SMU force production and cellular remodeling. We used an ectopic implantation so that we could more easily dissect the implanted tissues post-recovery and measure the force contribution of the SMU alone and compare it to pre-implantation values. This study also aimed to scale up the size of our SMUs to enable the replacement of larger volumes of muscle in our future VML studies. Overall, implantation resulted in extensive maturation of the SMUs, as characterized by an increase in force production, substantial integration with native tissue, innervation, vascularization, and the development of structural organization similar to native tissue.

Effects of Dexamethasone Dose and Timing on Tissue-Engineered Skeletal Muscle Units.

Our lab showed that administration of dexamethasone (DEX) stimulated myogenesis and resulted in advanced structure in our engineered skeletal muscle units (SMU). While administration of 25 nM DEX resulted in the most advanced structure, 10 nM dosing resulted in the greatest force production. We hypothesized that administration of 25 nM DEX during the entire fabrication process was toxic to the cells and that administration of DEX at precise time points during myogenesis would result in SMU with a more advanced structure and function. Thus, we fabricated SMU with 25 nM DEX administered at early proliferation (days 0-4), late proliferation (days 3-5), and early differentiation (days 5-7) stages of myogenesis and compared them to SMU treated with 10 nM DEX (days 0-16). Cell proliferation was measured with a BrdU assay (day 4) and myogenesis was examined by immunostaining for MyoD (day 4), myogenin (day 7), and α-actinin (day 11). Following SMU formation, isometric tetanic force production was measured. An analysis of cell proliferation indicated that 25 nM DEX administered at early proliferation (days 0-4) provided 21.5% greater myogenic proliferation than 10 nM DEX (days 0-4). In addition, 25 nM DEX administered at early differentiation (days 5-7) showed the highest density of myogenin-positive cells, demonstrating the greatest improvement in differentiation of myoblasts. However, the most advanced sarcomeric structure and the highest force production were exhibited with sustained administration of 10 nM DEX (days 0-16). In conclusion, alteration of the timing of 25 nM DEX administration did not enhance the structure or function of our SMU. SMU were optimally fabricated with sustained administration of 10 nM DEX.

A Transgenic tdTomato Rat for Cell Migration and Tissue Engineering Applications.

The growing deficit in suitable tissues for patients awaiting organ transplants demonstrates the clinical need for engineered tissues as alternative graft sources. Demonstrating safety and efficacy by tracking the migration and fate of implanted cells is a key consideration required for approval of promising engineered tissues. Cells from transgenic animals that express green fluorescent protein (GFP) are commonly used for this purpose. However, GFP can create difficulties in practice due to high levels of green autofluorescence in many musculoskeletal tissues. Tandem-dimer tomato (tdTomato) is a stable, robust red fluorescent protein that is nearly threefold brighter than GFP. Our objective was to create a line of transgenic rats that ubiquitously express tdTomato in all cells, driven by the human ubiquitin C promoter. We sought to determine the rats' utility in tissue engineering applications by fabricating engineered skeletal muscle units (SMUs) from isolated muscle-derived tdTomato cells. These tdTomato SMUs were implanted into a volumetric muscle loss (VML) defect of the tibialis anterior muscle in a rat ubiquitously expressing GFP. We also evaluated a novel method for modularly combining individual SMUs to create a larger engineered tissue. Following a recovery period of 28 days, we found that implantation of the modular SMU led to a significant decrease in the size of the remaining VML deficit. Histological analysis of explanted tissues demonstrated both tdTomato and GFP expression in the repair site, indicating involvement of both implanted and host cells in the regeneration process. These results demonstrate the successful generation of a tdTomato transgenic rat, and the use of these rats in tissue engineering and cell migration applications. Furthermore, this study successfully validated a method for scaling engineered tissues to larger sizes, a factor that will be important for repairing volumetric injuries in more clinically relevant models.

Ceftazidime/Avibactam and Ceftolozane/Tazobactam: Novel Therapy for Multidrug Resistant Gram Negative Infections in Children.

The rise in Multidrug-resistant (MDR) infections has become a significant problem in both the developing countries and in the United States (U.S.). Specifically, MDR gram-negative infections are emerging, affecting not only adults but children as well. The specific gram-negative organisms that have been most concerning within the pediatric population include MDR P. aeruginosa, Enterobacteriaceae, and Acinetobacter spp. The increase in antimicrobial resistance rates is associated with various mechanisms with one of the most common being the production of beta-lactamases. Both Ceftazidime/Avibactam (CZA) and Ceftolozane/Tazobactam (C/T) are two recently approved antibiotics in the U.S. While both of these agents are inhibitors of beta-lactamase enzymes, there are differences between them that are important to understand. At this time, the data in children for these agents are extremely limited. The aim of this review is to describe the characteristics of these agents and their potential uses in pediatric patients.

Measurement of ATP-Induced Membrane Potential Changes in IVD cells.

Extracellular adenosine-5'-triphosphate (ATP) triggers biological responses in a wide variety of cells and tissues and activates signaling cascades that affect cell membrane potential and excitability. It has been demonstrated that compressive loading promotes ATP production and release by intervertebral disc (IVD) cells, while a high level of extracellular ATP accumulates in the nucleus pulposus (NP) of the IVD. In this study, a noninvasive system was developed to measure ATP-induced changes in the membrane potential of porcine IVD cells using the potential sensitive dye di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate (di-8-ANEPPS).The responses of NP and annulus fibrosus (AF) cells to ATP were examined in monolayer and 3-dimensional cultures. It was found that the pattern and magnitude of membrane potential change in IVD cells induced by extracellular ATP depended on cell type, culture condition, and ATP dose. In addition, gene expression of P2X4 purinergic receptor was found in both cell types. Inhibition of the ATP-induced response by pyridoxalphosphate-6-azophenyl-2', 4'-disulfonate (PPADS), a non-competitive inhibitor of P2 receptors, suggests that ATP may modulate the biological activities of IVD cells via P2 purinergic receptors.

Changes in dynamic plantar pressure during loaded gait.

BACKGROUND: Lower extremity overuse injuries are detrimental to military readiness. Extremes of arch height and heavy loads carried by military personnel are associated with increased risk for overuse injury. Little is known regarding the impact of load carriage on plantar pressure distributions during gait. OBJECTIVE: The objective of this study was to determine how load carriage affects plantar pressure distributions during gait in individuals with varying arch types. DESIGN: A cross-sectional, repeated-measures design was used for the study. METHODS: The study was performed at a research laboratory at Fort Sam Houston, Texas. Service members who were healthy and weighing ≥70 kg were enrolled in the study. The participants (97 men, 18 women; mean age=31.3 years, SD=5.6; mean weight=86.0 kg, SD=11.0) were categorized as having high-, normal-, or low-arched feet on the basis of published cutoff values for the arch height index. Plantar pressure measurements were obtained with the use of an in-shoe pressure measurement system while participants wore combat boots and walked on a treadmill under 3 loaded conditions (uniform, 20-kg load, and 40-kg load). Maximal force (MaxF) and force-time integral (FTI) were assessed with the use of a 9-sector mask to represent regions of the foot. A 3 × 3 repeated-measures analysis of variance was used for analysis across the levels of load and arch type. RESULTS: A significant interaction existed between arch type and load for MaxF and FTI in the medial midfoot, with greater force in low-arched feet. In the medial forefoot, MaxF and FTI were greatest in high-arched feet across all load conditions. In the great toe region, low-arched and normally arched feet had greater MaxF and FTI. The relative distribution of FTI increased proportionately in all regions of the foot regardless of arch type for all load conditions. LIMITATIONS: The influence of fatigue, greater loads, and different walking speeds was not assessed. CONCLUSIONS: Regardless of arch type, increases in load did not alter the relative distribution of force over the plantar foot during gait. Participants with high-arched feet had greater force in the medial forefoot region, whereas those with normally arched or low-arched feet had greater force in the great toe region, regardless of load. These differences in force distribution may demonstrate different strategies to generate a rigid lever during toe-off.