Abstract Background Context Disc degeneration is the leading cause of low back pain and is often characterized by a loss of disc height, resulting from cleavage of chondroitin sulfate proteoglycans ...(CSPGs) present in the nucleus pulposus. Intact CSPGs are critical to water retention and maintenance of the nucleus osmotic pressure. Decellularization of healthy nucleus pulposus tissue has the potential to serve as an ideal matrix for tissue engineering of the disc because of the presence of native disc proteins and CSPGs. Injectable in situ gelling matrices are the most viable therapeutic option to prevent damage to the anulus fibrosus and future disc degeneration. Purpose The purpose of this research was to create a gentle decellularization method for use on healthy nucleus pulposus tissue explants and to develop an injectable formulation of this matrix to enable therapeutic use without substantial tissue disruption. Study Design Porcine nuclei pulposi were isolated, decellularized, and solubilized. Samples were assessed to determine degree of cell removal, matrix maintenance, gelation ability, cytotoxic residuals, and native cell viability. Methods Nuclei pulposi were decellularized using serial detergent, buffer, and enzyme treatments. Decellularized nuclei pulposi were solubilized, neutralized, and buffered. Efficacy of decellularization was assessed by quantifying DNA removal and matrix preservation. An elution study was performed to confirm removal of cytotoxic residuals. Gelation kinetics and injectability were quantified. Long term in vitro experiments were performed with nucleus pulposus cells to ensure cell viability and native matrix production within the injectable decellularized nucleus pulposus matrices. Results This work resulted in the creation of a robust acellular matrix (>96% DNA removal) with highly preserved sulfated glycosaminoglycans (>47%), and collagen content and microstructure similar to native nucleus pulposus, indicating preservation of disc components. Furthermore, it was possible to create an injectable formulation that gelled in situ within 45 minutes and formed fibrillar collagen with similar diameters to native nucleus pulposus. The processing did not result in any remaining cytotoxic residuals. Solubilized decellularized nucleus pulposus samples seeded with nucleus pulposus cells maintained robust viability (>89%) up to 21 days of culture in vitro with morphology similar to native nucleus pulposus cells and exhibited significantly enhanced sulfated glycosaminoglycans production over 21 days. Conclusions A gentle decellularization of porcine nucleus pulposus, followed by solubilization enabled creation of an injectable tissue specific matrix that is well tolerated in vitro by nucleus pulposus cells. These matrices have the potential to be used as a minimally invasive nucleus pulposus therapeutic to restore disc height.
Hydrogel scaffolds hold promise for a myriad of tissue engineering applications, but often lack tissue-mimetic architecture. Therefore, in this work, we sought to develop a new technology for the ...incorporation of aligned tubular architecture within hydrogel scaffolds engineered from the bottom-up.
We report a platform fabrication technology-magnetic templating-distinct from other approaches in that it uses dissolvable magnetic alginate microparticles (MAMs) to form aligned columnar structures under an applied magnetic field. Removal of the MAMs yields scaffolds with aligned tubular microarchitecture that can promote cell remodeling for a variety of applications. This approach affords control of microstructure diameter and biological modification for advanced applications. Here, we sought to replicate the microarchitecture of the native nerve basal lamina using magnetic templating of hydrogels composed of glycidyl methacrylate hyaluronic acid and collagen I.
Magnetically templated hydrogels were characterized for particle alignment and micro-porosity. Overall MAM removal efficacy was verified by 96.8% removal of iron oxide nanoparticles. Compressive mechanical properties were well-matched to peripheral nerve tissue at 0.93 kPa and 1.29 kPa, respectively. In vitro, templated hydrogels exhibited approximately 36% faster degradation over 12 h, and were found to guide axon extension from dorsal root ganglia. Finally, in a pilot in vivo study utilizing a 10 mm rat sciatic nerve defect model, magnetically templated hydrogels demonstrated promising results with qualitatively increased remodeling and axon regeneration compared to non-templated controls.
This simple and scalable technology has the flexibility to control tubular microstructure over long length scales, and thus the potential to meet the need for engineered scaffolds for tissue regeneration, including nerve guidance scaffolds.
It has been hypothesized that obese and reduced-obese individuals have decreased oxidative capacity, which contributes to weight gain and regain. Recent data have challenged this concept.
To ...determine (1) whether total and dietary fat oxidation are decreased in obese and reduced-obese adults compared to lean but increase in response to an acute exercise bout and (2) whether regular physical activity attenuates these metabolic alterations.
We measured 24-hr total (whole-room calorimetry) and dietary fat (14C-oleate) oxidation in Sedentary Lean (BMI = 21.5±1.6; n = 10), Sedentary Obese (BMI = 33.6±2.5; n = 9), Sedentary Reduced-Obese (RED-SED; BMI = 26.9±3.7; n = 7) and in Physically Active Reduced-Obese (RED-EX; BMI = 27.3±2.8; n = 12) men and women with or without an acute exercise bout where energy expended during exercise was not replaced.
Although Red-SED and Red-EX had a similar level of fatness, aerobic capacity and metabolic profiles were better in Red-EX only compared to Obese subjects. No significant between-group differences were seen in 24-hr respiratory quotient (RQ, Lean: 0.831±0.044, Obese: 0.852±0.023, Red-SED: 0.864±0.037, Red-EX: 0.842±0.039), total and dietary fat oxidation. A single bout of exercise increased total (+27.8%, p<0.0001) and dietary (+6.6%, p = 0.048) fat oxidation across groups. Although exercise did not impact RQ during the day, it decreased RQ during sleep (p = 0.01) in all groups. Red-EX oxidized more fat overnight than Red-SED subjects under both resting (p = 0.036) and negative energy balance (p = 0.003) conditions, even after adjustment for fat-free mass.
Obese and reduced-obese individuals oxidize as much fat as lean both under eucaloric and negative energy balance conditions, which does not support the hypothesis of reduced oxidative capacity in these groups. Reduced-obese individuals who exercise regularly have markers of metabolic health similar to those seen in lean adults. Both the acute and chronic effects of exercise were primarily observed at night suggesting an important role of sleep in the regulation of lipid metabolism.
Peripheral nerve injury results in loss of motor and sensory function distal to the nerve injury and is often permanent in nerve gaps longer than 5 cm. Autologous nerve grafts (nerve autografts) ...utilize patients' own nerve tissue from another part of their body to repair the defect and are the gold standard in care. However, there is a limited autologous tissue supply, size mismatch between donor nerve and injured nerve, and morbidity at the site of nerve donation. Decellularized cadaveric nerve tissue alleviates some of these limitations and has demonstrated success clinically. We previously developed an alternative apoptosis-assisted decellularization process for nerve tissue. This new process may result in an ideal scaffold for peripheral nerve regeneration by gently removing cells and antigens while preserving delicate topographical cues. In addition, the apoptosis-assisted process requires less active processing time and is inexpensive. This study examines the utility of apoptosis-decellularized peripheral nerve scaffolds compared to detergent-decellularized peripheral nerve scaffolds and isograft controls in a rat nerve gap model. Results indicate that, at 8 weeks post-injury, apoptosis-decellularized peripheral nerve scaffolds perform similarly to detergent-decellularized and isograft controls in both functional (muscle weight recovery, gait analysis) and histological measures (neurofilament staining, macrophage infiltration). These new apoptosis-decellularized scaffolds hold great promise to provide a less expensive scaffold for nerve injury repair, with the potential to improve nerve regeneration and functional outcomes compared to current detergent-decellularized scaffolds.
Locomotive changes are often associated with disease or injury, and these changes can be quantified through gait analysis. Gait analysis has been applied to preclinical studies, providing ...quantitative behavioural assessment with a reasonable clinical analogue. However, available gait analysis technology for small animals is somewhat limited. Furthermore, technological and analytical challenges can limit the effectiveness of preclinical gait analysis. The Gait Analysis Instrumentation and Technology Optimized for Rodents (GAITOR) Suite is designed to increase the accessibility of preclinical gait analysis to researchers, facilitating hardware and software customization for broad applications. Here, the GAITOR Suite's utility is demonstrated in 4 models: a monoiodoacetate (MIA) injection model of joint pain, a sciatic nerve injury model, an elbow joint contracture model, and a spinal cord injury model. The GAITOR Suite identified unique compensatory gait patterns in each model, demonstrating the software's utility for detecting gait changes in rodent models of highly disparate injuries and diseases. Robust gait analysis may improve preclinical model selection, disease sequelae assessment, and evaluation of potential therapeutics. Our group has provided the GAITOR Suite as an open resource to the research community at www.GAITOR.org , aiming to promote and improve the implementation of gait analysis in preclinical rodent models.
Spinal cord injury (SCI) affects a quarter million individuals in the United States, and there is currently no clinical treatment. Both fresh and acellular peripheral nerve grafts can induce spinal ...axon regeneration and support functional recovery in experimental injury models. Nonetheless, a scaffold that can be injected into a spinal contusion would be far less invasive to apply. We aimed to develop the first injectable acellular nerve graft for promoting repair after contusion SCI.
We report a method to enzymatically solubilize optimized acellular (OA) nerve-a decellularized peripheral nerve graft developed in our laboratory and currently used clinically-to obtain an injectable solution that undergoes thermal gelation under physiological conditions. We quantified multiple physical and compositional properties of this novel material as well as tested its efficacy at acute and chronic time points following cervical contusion SCI.
This injectable optimized acellular (iOA) nerve graft retains native chemical cues such as collagens and glycosaminoglycans. By varying hydrogel concentration, the rheological properties and compressive modulus of iOA were similar to that previous reported for rat central nervous tissue. iOA solution was compatible with rat Schwann cells in culture, and hydrogel injection into a rat cervical contusion model significantly reduced the ratio of M1:M2 macrophages after one week, favoring regenerative phenotypes (p < 0.05). Furthermore, while iOA treatment did not affect locomotor or respiratory recovery over an eight week period, the percentage of axonal coverage increased at the distal tissue interface (p < 0.05), suggesting enhanced axonal extension within this region.
Our data indicate that this novel injectable form of acellular nerve grafts is amenable for use after contusion SCI and may bolster a simultaneous therapy by acutely modulating the inflammatory milieu and supporting axonal growth.
Data suggest women are more sensitive to the lipolytic action of epinephrine compared with men while maintaining similar glucoregulatory effects (Horton et al. J Appl Physiol 107: 200-210, 2009). ...This study aimed to determine the specific adrenergic receptor(s) that may mediate these sex differences. Lean women (n = 14) and men (n = 16) were studied on 4 nonconsecutive days during the following treatment infusions: saline (S: control), epinephrine E: mixed β-adrenergic (lipolytic) and α2-adrenergic (antilipolytic) stimulation, epinephrine + phentolamine (E + P: mixed β-adrenergic stimulation only), and terbutaline (T: selective β2-adrenergic stimulation). Tracer infusions of glycerol, palmitate, and glucose were administered to determine systemic lipolysis, free fatty acid (FFA) release, and glucose turnover, respectively. Following basal measurements, substrate and hormone concentrations were measured in all subjects over 90 min of treatment and tracer infusion. Women had greater increases in glycerol and FFA concentrations with all three hormone infusions compared with men (P < 0.01). Glycerol and palmitate rate of appearance (Ra) and rate of disappearance (Rd) per kilogram body weight were greater with E infusion in women compared with men (P < 0.05), whereas no sex differences were observed with other treatments. Glucose concentration and kinetics were not different between sexes with any infusion. In conclusion, these data support the hypothesis that the greater rate of lipolysis in women with infusion of E was likely due to lesser α2 antilipolytic activation. These findings may help explain why women have greater lipolysis and fat oxidation during exercise, a time when epinephrine concentration is elevated.
Decellularized tissues hold great potential for both regenerative medicine and disease modeling applications. The acellular extracellular matrix (ECM)-enriched scaffolds can be recellularized with ...patient-derived cells prior to transplantation, or digested to create thermally-gelling ECM hydrogels for 3D cell culture. Current methods of decellularization clear cellular components using detergents, which can result in loss of ECM proteins and tissue architectural integrity. Recently, an alternative approach utilizing apoptosis to decellularize excised murine sciatic nerves resulted in superior ECM preservation, cell removal, and immune tolerance in vivo. However, this apoptosis-assisted decellularization approach has not been optimized for other tissues with a more complex geometry, such as lungs. To this end, we developed an apoptosis-assisted lung tissue decellularization method using a combination of camptothecin and sulfobetaine-10 (SB-10) to induce apoptosis and facilitate gentle and effective removal of cell debris, respectively. Importantly, combination of the two agents resulted in superior cell removal and ECM preservation compared to either of the treatments alone, presumably because of pulmonary surfactants. In addition, our method was superior in cell removal compared to a previously established detergent-based decellularization protocol. Furthermore, thermally-gelling lung ECM hydrogels supported high viability of rat lung epithelial cells for up to 2 weeks in culture. This work demonstrates that apoptosis-based lung tissue decellularization is a superior technique that warrants further utilization for both regenerative medicine and disease modeling purposes.
Despite living in an environment that promotes weight gain in many individuals, some individuals maintain a thin phenotype while self‐reporting expending little or no effort to control their weight. ...When compared with obesity prone (OP) individuals, we wondered if obesity resistant (OR) individuals would have higher levels of spontaneous physical activity (SPA) or respond to short‐term overfeeding by increasing their level of SPA in a manner that could potentially limit future weight gain. SPA was measured in 55 subjects (23 OP and 32 OR) using a novel physical activity monitoring system (PAMS) that measured body position and movement while subjects were awake for 6 days, either in a controlled eucaloric condition or during 3 days of overfeeding (1.4× basal energy) and for the subsequent 3 days (ad libitum recovery period). Pedometers were also used before and during use of the PAMS to provide an independent measure of SPA. SPA was quantified by the PAMS as fraction of recording time spent lying, sitting, or in an upright posture. Accelerometry, measured while subjects were in an upright posture, was used to categorize time spent in different levels of movement (standing, walking slowly, quickly, etc.). There were no differences in SPA between groups when examined across all study periods (P > 0.05). However, 3 days following overfeeding, OP subjects significantly decreased the amount of time they spent walking (−2.0% of time, P = 0.03), whereas OR subjects maintained their walking (+0.2%, P > 0.05). The principle findings of this study are that increased levels of SPA either during eucaloric feeding or following short term overfeeding likely do not significantly contribute to obesity resistance although a decrease in SPA following overfeeding may contribute to future weight gain in individuals prone to obesity.
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