Controlling scaffold conductivity and pore size to direct myogenic cell alignment and differentiation.

Skeletal muscle's combination of three-dimensional (3D) anisotropy and electrical excitability is critical for enabling normal movement. We previously developed a 3D aligned collagen scaffold incorporating conductive polypyrrole (PPy) particles to recapitulate these key muscle properties and showed that the scaffold facilitated enhanced myotube maturation compared with nonconductive controls. To further optimize this scaffold design, this work assessed the influence of conductive polymer incorporation and scaffold pore architecture on myogenic cell behavior. Conductive PPy and poly(3,4-ethylenedioxythiophene) (PEDOT) particles were synthesized and mixed into a suspension of type I collagen and chondroitin sulfate prior to directional freeze-drying to produce anisotropic scaffolds. Energy dispersive spectroscopy revealed homogenous distribution of conductive PEDOT particles throughout the scaffolds that resulted in a threefold increase in electrical conductivity while supporting similar myoblast metabolic activity compared to nonconductive scaffolds. Control of freezing temperature enabled fabrication of PEDOT-doped scaffolds with a range of pore diameters from 98 to 238 µm. Myoblasts conformed to the anisotropic contact guidance cues independent of pore size to display longitudinal cytoskeletal alignment. The increased specific surface area of the smaller pore scaffolds helped rescue the initial decrease in myoblast metabolic activity observed in larger pore conductive scaffolds while also promoting modestly increased expression levels of the myogenic marker myosin heavy chain (MHC) and gene expression of myoblast determination protein (MyoD). However, cell infiltration to the center of the scaffolds was marginally reduced compared with larger pore variants. Together these data underscore the potential of aligned and PEDOT-doped collagen scaffolds for promoting myogenic cell organization and differentiation.

Aligned and electrically conductive 3D collagen scaffolds for skeletal muscle tissue engineering.

Skeletal muscle is characterized by its three-dimensional (3D) anisotropic architecture composed of highly aligned and electrically-excitable muscle fibers that enable normal movement. Biomaterial-based tissue engineering approaches to repair skeletal muscle are limited due to difficulties combining 3D structural alignment (to guide cell/matrix organization) and electrical conductivity (to enable electrically-excitable myotube assembly and maturation). In this work we successfully produced aligned and electrically conductive 3D collagen scaffolds using a freeze-drying approach. Conductive polypyrrole (PPy) nanoparticles were synthesized and directly mixed into a suspension of type I collagen and chondroitin sulfate followed by directional lyophilization. Scanning electron microscopy (SEM), energy-dispersive spectroscopy (EDS), and confocal microscopy showed that directional solidification resulted in scaffolds with longitudinally aligned pores with homogeneously-distributed PPy content. Chronopotentiometry verified that PPy incorporation resulted in a five-fold increase in conductivity compared to non-PPy-containing collagen scaffolds without detrimentally affecting myoblast metabolic activity. Furthermore, the aligned scaffold microstructure provided contact guidance cues that directed myoblast growth and organization. Incorporation of PPy also promoted enhanced myotube formation and maturation as measured by myosin heavy chain (MHC) expression and number of nuclei per myotube. Together these data suggest that aligned and electrically conductive 3D collagen scaffolds could be useful for skeletal muscle tissue engineering.

Analysis and Modeling of Rat Gait Biomechanical Deficits in Response to Volumetric Muscle Loss Injury.

There is currently a substantial volume of research underway to develop more effective approaches for the regeneration of functional muscle tissue as treatment for volumetric muscle loss (VML) injury, but few studies have evaluated the relationship between injury and the biomechanics required for normal function. To address this knowledge gap, the goal of this study was to develop a novel method to quantify the changes in gait of rats with tibialis anterior (TA) VML injuries. This method should be sensitive enough to identify biomechanical and kinematic changes in response to injury as well as during recovery. Control rats and rats with surgically-created VML injuries were affixed with motion capture markers on the bony landmarks of the back and hindlimb and were recorded walking on a treadmill both prior to and post-surgery. Data collected from the motion capture system was exported for post-hoc analysis in OpenSim and Matlab. In vivo force testing indicated that the VML injury was associated with a significant deficit in force generation ability. Analysis of joint kinematics showed significant differences at all three post-surgical timepoints and gait cycle phase shifting, indicating augmented gait biomechanics in response to VML injury. In conclusion, this method identifies and quantifies key differences in the gait biomechanics and joint kinematics of rats with VML injuries and allows for analysis of the response to injury and recovery. The comprehensive nature of this method opens the door for future studies into dynamics and musculoskeletal control of injured gait that can inform the development of regenerative technologies focused on the functional metrics that are most relevant to recovery from VML injury.

Adipose-derived stem cells delay muscle atrophy after peripheral nerve injury in the rodent model.

INTRODUCTION: Injuries to peripheral nerves cause distal muscle atrophy. The effects of adipose-derived stem cell (ASC) injections into a muscle after injury were examined. METHODS: A 1.5 cm defect in the rat sciatic nerve was created, resulting in gastrocnemius muscle atrophy. The nerve defect was repaired with autograft; DiR-labeled ASCs were injected into the gastrocnemius immediately postoperatively. Quantitation of gross musculature and muscle fiber area, cell survival, fibrosis, lipid deposition, inflammation, and reconstructive responses were investigated. RESULTS: ASCs were identified in the muscle at 6 weeks, where injections showed increased muscle mass percentage retained, larger average fiber area, and less overall lipid content accumulated throughout the musculature. Muscles having received ASCs showed increased presence of interlukin-10 and Ki67, and decreased inducible nitric oxide synthase (iNOS). DISCUSSION: This investigation is suggestive that an ASC injection into denervated muscle post-operatively is able to delay the onset of atrophy. Muscle Nerve 59:603-603, 2019.

Characterization of a Murine Model of Bioequivalent Bladder Wound Healing and Repair Following Subtotal Cystectomy.

Previous work demonstrated restoration of a bioequivalent bladder within 8 weeks of removing the majority of the bladder (subtotal cystectomy or STC) in rats. The goal of the present study was to extend our investigations of bladder repair to the murine model, to harness the power of mouse genetics to delineate the cellular and molecular mechanisms responsible for the observed robust bladder regrowth. Female C57 black mice underwent STC, and at 4, 8, and 12 weeks post-STC, bladder repair and function were assessed via cystometry, ex vivo pharmacologic organ bath studies, and T2-weighted magnetic resonance imaging (MRI). Histology was also performed to measure bladder wall thickness. We observed a time-dependent increase in bladder capacity (BC) following STC, such that 8 and 12 weeks post-STC, BC and micturition volumes were indistinguishable from those of age-matched non-STC controls and significantly higher than observed at 4 weeks. MRI studies confirmed that bladder volume was indistinguishable within 3 months (11 weeks) post-STC. Additionally, bladders emptied completely at all time points studied (i.e., no increases in residual volume), consistent with functional bladder repair. At 8 and 12 weeks post-STC, there were no significant differences in bladder wall thickness or in the different components (urothelium, lamina propria, or smooth muscle layers) of the bladder wall compared with age-matched control animals. The maximal contractile response to pharmacological activation and electrical field stimulation increased over time in isolated tissue strips from repaired bladders but remained lower at all time points compared with controls. We have established and validated a murine model for the study of de novo organ repair that will allow for further mechanistic studies of this phenomenon after, for example, genetic manipulation.

Cell and Growth Factor-Loaded Keratin Hydrogels for Treatment of Volumetric Muscle Loss in a Mouse Model.

Wounds to the head, neck, and extremities have been estimated to account for ∼84% of reported combat injuries to military personnel. Volumetric muscle loss (VML), defined as skeletal muscle injuries in which tissue loss results in permanent functional impairment, is common among these injuries. The present standard of care entails the use of muscle flap transfers, which suffer from the need for additional surgery when using autografts or the risk of rejection when cadaveric grafts are used. Tissue engineering (TE) strategies for skeletal muscle repair have been investigated as a means to overcome current therapeutic limitations. In that regard, human hair-derived keratin (KN) biomaterials have been found to possess several favorable properties for use in TE applications and, as such, are a viable candidate for use in skeletal muscle repair. Herein, KN hydrogels with and without the addition of skeletal muscle progenitor cells (MPCs) and/or insulin-like growth factor 1 (IGF-1) and/or basic fibroblast growth factor (bFGF) were implanted in an established murine model of surgically induced VML injury to the latissimus dorsi (LD) muscle. Control treatments included surgery with no repair (NR) as well as implantation of bladder acellular matrix (BAM). In vitro muscle contraction force was evaluated at two months postsurgery through electrical stimulation of the explanted LD in an organ bath. Functional data indicated that implantation of KN+bFGF+IGF-1 (n = 8) enabled a greater recovery of contractile force than KN+bFGF (n = 8)***, KN+MPC (n = 8)**, KN+MPC+bFGF+IGF-1 (n = 8)**, BAM (n = 8)*, KN+IGF-1 (n = 8)*, KN+MPCs+bFGF (n = 9)*, or NR (n = 9)**, (*p < 0.05, **p < 0.01, ***p < 0.001). Consistent with the physiological findings, histological evaluation of retrieved tissue revealed much more extensive new muscle tissue formation in groups with greater functional recovery (e.g., KN+IGF-1+bFGF) when compared with observations in tissue from groups with lower functional recovery (i.e., BAM and NR). Taken together, these findings further indicate the general utility of KN biomaterials in TE and, moreover, specifically highlight their potential application in the treatment of VML injuries.

Keratin Hydrogel Enhances In Vivo Skeletal Muscle Function in a Rat Model of Volumetric Muscle Loss.

Volumetric muscle loss (VML) injuries exceed the considerable intrinsic regenerative capacity of skeletal muscle, resulting in permanent functional and cosmetic deficits. VML and VML-like injuries occur in military and civilian populations, due to trauma and surgery as well as due to a host of congenital and acquired diseases/syndromes. Current therapeutic options are limited, and new approaches are needed for a more complete functional regeneration of muscle. A potential solution is human hair-derived keratin (KN) biomaterials that may have significant potential for regenerative therapy. The goal of these studies was to evaluate the utility of keratin hydrogel formulations as a cell and/or growth factor delivery vehicle for functional muscle regeneration in a surgically created VML injury in the rat tibialis anterior (TA) muscle. VML injuries were treated with KN hydrogels in the absence and presence of skeletal muscle progenitor cells (MPCs), and/or insulin-like growth factor 1 (IGF-1), and/or basic fibroblast growth factor (bFGF). Controls included VML injuries with no repair (NR), and implantation of bladder acellular matrix (BAM, without cells). Initial studies conducted 8 weeks post-VML injury indicated that application of keratin hydrogels with growth factors (KN, KN+IGF-1, KN+bFGF, and KN+IGF-1+bFGF, n = 8 each) enabled a significantly greater functional recovery than NR (n = 7), BAM (n = 8), or the addition of MPCs to the keratin hydrogel (KN+MPC, KN+MPC+IGF-1, KN+MPC+bFGF, and KN+MPC+IGF-1+bFGF, n = 8 each) (p < 0.05). A second series of studies examined functional recovery for as many as 12 weeks post-VML injury after application of keratin hydrogels in the absence of cells. A significant time-dependent increase in functional recovery of the KN, KN+bFGF, and KN+IGF+bFGF groups was observed, relative to NR and BAM implantation, achieving as much as 90% of the maximum possible functional recovery. Histological findings from harvested tissue at 12 weeks post-VML injury documented significant increases in neo-muscle tissue formation in all keratin treatment groups as well as diminished fibrosis, in comparison to both BAM and NR. In conclusion, keratin hydrogel implantation promoted statistically significant and physiologically relevant improvements in functional outcomes post-VML injury to the rodent TA muscle.

In Silico and In Vivo Experiments Reveal M-CSF Injections Accelerate Regeneration Following Muscle Laceration.

Numerous studies have pharmacologically modulated the muscle milieu in the hopes of promoting muscle regeneration; however, the timing and duration of these interventions are difficult to determine. This study utilized a combination of in silico and in vivo experiments to investigate how inflammation manipulation improves muscle recovery following injury. First, we measured macrophage populations following laceration injury in the rat tibialis anterior (TA). Then we calibrated an agent-based model (ABM) of muscle injury to mimic the observed inflammation profiles. The calibrated ABM was used to simulate macrophage and satellite stem cell (SC) dynamics, and suggested that delivering macrophage colony stimulating factor (M-CSF) prior to injury would promote SC-mediated injury recovery. Next, we performed an experiment wherein 1 day prior to injury, we injected M-CSF into the rat TA muscle. M-CSF increased the number of macrophages during the first 4 days post-injury. Furthermore, treated muscles experienced a swifter increase in the appearance of PAX7+ SCs and regenerating muscle fibers. Our study suggests that computational models of muscle injury provide novel insights into cellular dynamics during regeneration, and further, that pharmacologically altering inflammation dynamics prior to injury can accelerate the muscle regeneration process.

Achieving Acetylcholine Receptor Clustering in Tissue-Engineered Skeletal Muscle Constructs In vitro through a Materials-Directed Agrin Delivery Approach.

Volumetric muscle loss (VML) can result from trauma, infection, congenital anomalies, or surgery, and produce permanent functional and cosmetic deficits. There are no effective treatment options for VML injuries, and recent advances toward development of muscle constructs lack the ability to achieve innervation necessary for long-term function. We sought to develop a proof-of-concept biomaterial construct that could achieve acetylcholine receptor (AChR) clustering on muscle-derived cells (MDCs) in vitro. The approach consisted of the presentation of neural (Z+) agrin from the surface of microspheres embedded with a fibrin hydrogel to muscle cells (C2C12 cell line or primary rat MDCs). AChR clustering was spatially restricted to areas of cell (C2C12)-microsphere contact when the microspheres were delivered in suspension or when they were incorporated into a thin (2D) fibrin hydrogel. AChR clusters were observed from 16 to 72 h after treatment when Z+ agrin was adsorbed to the microspheres, and for greater than 120 h when agrin was covalently coupled to the microspheres. Little to no AChR clustering was observed when agrin-coated microspheres were delivered from specially designed 3D fibrin constructs. However, cyclic stretch in combination with agrin-presenting microspheres led to dramatic enhancement of AChR clustering in cells cultured on these 3D fibrin constructs, suggesting a synergistic effect between mechanical strain and agrin stimulation of AChR clustering in vitro. These studies highlight a strategy for maintaining a physiological phenotype characterized by motor endplates of muscle cells used in tissue engineering strategies for muscle regeneration. As such, these observations may provide an important first step toward improving function of tissue-engineered constructs for treatment of VML injuries.

The effect of low-frequency electromagnetic field on human bone marrow stem/progenitor cell differentiation.

Human bone marrow stromal cells (hBMSCs, also known as bone marrow-derived mesenchymal stem cells) are a population of progenitor cells that contain a subset of skeletal stem cells (hSSCs), able to recreate cartilage, bone, stroma that supports hematopoiesis and marrow adipocytes. As such, they have become an important resource in developing strategies for regenerative medicine and tissue engineering due to their self-renewal and differentiation capabilities. The differentiation of SSCs/BMSCs is dependent on exposure to biophysical and biochemical stimuli that favor early and rapid activation of the in vivo tissue repair process. Exposure to exogenous stimuli such as an electromagnetic field (EMF) can promote differentiation of SSCs/BMSCs via ion dynamics and small signaling molecules. The plasma membrane is often considered to be the main target for EMF signals and most results point to an effect on the rate of ion or ligand binding due to a receptor site acting as a modulator of signaling cascades. Ion fluxes are closely involved in differentiation control as stem cells move and grow in specific directions to form tissues and organs. EMF affects numerous biological functions such as gene expression, cell fate, and cell differentiation, but will only induce these effects within a certain range of low frequencies as well as low amplitudes. EMF has been reported to be effective in the enhancement of osteogenesis and chondrogenesis of hSSCs/BMSCs with no documented negative effects. Studies show specific EMF frequencies enhance hSSC/BMSC adherence, proliferation, differentiation, and viability, all of which play a key role in the use of hSSCs/BMSCs for tissue engineering. While many EMF studies report significant enhancement of the differentiation process, results differ depending on the experimental and environmental conditions. Here we review how specific EMF parameters (frequency, intensity, and time of exposure) significantly regulate hSSC/BMSC differentiation in vitro. We discuss optimal conditions and parameters for effective hSSC/BMSC differentiation using EMF treatment in an in vivo setting, and how these can be translated to clinical trials.

Age-related alterations in regeneration of the urinary bladder after subtotal cystectomy.

Prior work documented that surgical removal of approximately 70% of the bladder (subtotal cystectomy) in 12-week-old female rats induced complete functional regeneration of the bladder within 8 weeks. To determine whether animal age affects bladder regeneration, female F344 rats aged 12 weeks (young) and 12 months (old) underwent subtotal cystectomy, and then were evaluated from 1 to 26 weeks after subtotal cystectomy. At 26 weeks after subtotal cystectomy, bladder capacity in young animals was indistinguishable from that in age-matched controls, but bladder capacity in old animals was only approximately 56% of that in age-matched controls. There was no detectable difference in residual volume among treatment groups, but the diminished regeneration in old animals was associated with a corresponding increase in the ratio of residual volume to micturition volume. The majority of old animals exhibited evidence of chronic kidney damage after subtotal cystectomy. Maximal contraction of bladder strips to electrical field stimulation, as well as activation with carbachol, phenylephrine, and KCl, were lower in old than in young animals at 26 weeks after subtotal cystectomy. Immunostaining with proliferating cell nuclear antigen and Von Willebrand factor revealed delayed and/or diminished proliferative and angiogenic responses, respectively, in old animals. These results confirm prior work and suggest that multiple mechanisms may contribute to an age-related decline in the regenerative capacity of the bladder.

The pharmacology of regenerative medicine.

Regenerative medicine is a rapidly evolving multidisciplinary, translational research enterprise whose explicit purpose is to advance technologies for the repair and replacement of damaged cells, tissues, and organs. Scientific progress in the field has been steady and expectations for its robust clinical application continue to rise. The major thesis of this review is that the pharmacological sciences will contribute critically to the accelerated translational progress and clinical utility of regenerative medicine technologies. In 2007, we coined the phrase "regenerative pharmacology" to describe the enormous possibilities that could occur at the interface between pharmacology, regenerative medicine, and tissue engineering. The operational definition of regenerative pharmacology is "the application of pharmacological sciences to accelerate, optimize, and characterize (either in vitro or in vivo) the development, maturation, and function of bioengineered and regenerating tissues." As such, regenerative pharmacology seeks to cure disease through restoration of tissue/organ function. This strategy is distinct from standard pharmacotherapy, which is often limited to the amelioration of symptoms. Our goal here is to get pharmacologists more involved in this field of research by exposing them to the tools, opportunities, challenges, and interdisciplinary expertise that will be required to ensure awareness and galvanize involvement. To this end, we illustrate ways in which the pharmacological sciences can drive future innovations in regenerative medicine and tissue engineering and thus help to revolutionize the discovery of curative therapeutics. Hopefully, the broad foundational knowledge provided herein will spark sustained conversations among experts in diverse fields of scientific research to the benefit of all.

The role of endothelial cells in myofiber differentiation and the vascularization and innervation of bioengineered muscle tissue in vivo.

Musculoskeletal disorders are a major cause of disability and effective treatments are currently lacking. Tissue engineering affords the possibility of new therapies utilizing cells and biomaterials for the recovery of muscle volume and function. A major consideration in skeletal muscle engineering is the integration of a functional vasculature within the regenerating tissue. In this study we employed fluorescent cell labels to track the location and differentiation of co-cultured cells in vivo and in vitro. We first utilized a co-culture of fluorescently labeled endothelial cells (ECs) and muscle progenitor cells (MPCs) to investigate the ability of ECs to enhance muscle tissue formation and vascularization in an in vivo model of bioengineered muscle. Scaffolds that had been seeded with both MPCs and ECs showed significantly greater vascularization, tissue formation and enhanced innervation as compared to scaffolds seeded with MPCs alone. Subsequently, we performed in vitro experiments using a 3-cell type system (ECs, MPCs, and pericytes (PCs)) to demonstrate the utility of fluorescent cell labeling for monitoring cell growth and differentiation. The growth and differentiation of individual cell types was determined using live cell fluorescent microscopy demonstrating the utility of fluorescent labels to monitor tissue organization in real time.

Bioengineered vascular access maintains structural integrity in response to arteriovenous flow and repeated needle puncture.

OBJECTIVE: Tissue-engineered blood vessels (TEBV) have been proposed as an alternative to prosthetic grafts for dialysis access. However, arteriovenous (AV) grafts must withstand extreme flow rates and frequent needle trauma. In a proof-of-concept study, we sought to determine whether scaffold-based TEBV could withstand the hemodynamic and mechanical challenges of chronic dialysis access. METHODS: TEBV were constructed using decellularized arterial scaffolds seeded with autologous ovine endothelial cells (EC) derived from circulating endothelial progenitor cells (EPC) using a novel high-affinity capture approach. Seeded scaffolds were preconditioned to arterial pressure and flow in a bioreactor for 2 weeks prior to implantation to create carotid artery to jugular vein AV grafts in each animal. TEBV were healed for 1 month before initiating percutaneous needle puncture 3 days/week. TEBV wall geometry and patency were monitored using duplex imaging and were either explanted for histologic analysis at 2 months (n = 5) or followed for up to 6 months until venous outflow stenosis threatened AV graft patency (n = 6). RESULTS: Despite high flow, TEBV maintained stable geometry with only modest wall dilation (under 6%) by 4 months after implantation. Needle access was well tolerated with a single puncture site complication, a small pseudoaneurysm, occurring in the late group. Time-to-hemostasis at puncture sites averaged 4 ± 2 minutes. Histologic analysis at 2 months demonstrated repopulation of the outer TEBV wall by host cells and healing of needle punctures by cellular ingrowth and new matrix deposition along the tract. TEBV followed beyond 2 months showed stable wall geometry but, consistent with the primary mode of clinical AV graft failure, all TEBV eventually developed venous anastomotic stenosis (mean, 4.4 ± 0.9 months; range, 3.3-5.6 months postimplantation; n = 6). CONCLUSIONS: This pilot study supports the concept of creating dialysis access from scaffold-based autologous TEBV. Engineered AV grafts were created within a clinically relevant time frame and demonstrated stable wall geometry despite high flow and repeated puncture. Cellular ingrowth and puncture site healing may improve wall durability, but venous outflow stenosis remains the primary mode of TEBV graft failure in the ovine model.

Further development of a tissue engineered muscle repair construct in vitro for enhanced functional recovery following implantation in vivo in a murine model of volumetric muscle loss injury.

Volumetric muscle loss (VML) can result from trauma and surgery in civilian and military populations, resulting in irrecoverable functional and cosmetic deficits that cannot be effectively treated with current therapies. Previous work evaluated a bioreactor-based tissue engineering approach in which muscle derived cells (MDCs) were seeded onto bladder acellular matrices (BAM) and mechanically preconditioned. This first generation tissue engineered muscle repair (TEMR) construct exhibited a largely differentiated cellular morphology consisting primarily of myotubes, and moreover, significantly improved functional recovery within 2 months of implantation in a murine latissimus dorsi (LD) muscle with a surgically created VML injury. The present report extends these initial observations to further document the importance of the cellular phenotype and composition of the TEMR construct in vitro to the functional recovery observed following implantation in vivo. To this end, three distinct TEMR constructs were created by seeding MDCs onto BAM as follows: (1) a short-term cellular proliferation of MDCs to generate primarily myoblasts without bioreactor preconditioning (TEMR-1SP), (2) a prolonged cellular differentiation and maturation period that included bioreactor preconditioning (TEMR-1SPD; identical to the first generation TEMR construct), and (3) similar treatment as TEMR-1SPD but with a second application of MDCs during bioreactor preconditioning (TEMR-2SPD); simulating aspects of "exercise" in vitro. Assessment of maximal tetanic force generation on retrieved LD muscles in vitro revealed that TEMR-1SP and TEMR-1SPD constructs promoted either an accelerated (i.e., 1 month) or a prolonged (i.e., 2 month postinjury) functional recovery, respectively, of similar magnitude. Meanwhile, TEMR-2SPD constructs promoted both an accelerated and prolonged functional recovery, resulting in twice the magnitude of functional recovery of either TEMR-1SP or TEMR-1SPD constructs. Histological and molecular analyses indicated that TEMR constructs mediated functional recovery via regeneration of functional muscle fibers either at the interface of the construct and the native tissue or within the BAM scaffolding independent of the native tissue. Taken together these findings are encouraging for the further development and clinical application of TEMR constructs as a VML injury treatment.

Impact of partial urethral obstruction on bladder function: time-dependent changes and functional correlates of altered expression of Ca²âº signaling regulators.

In animal models of partial urethral obstruction (PUO), altered smooth muscle function/contractility may be linked to changes in molecules that regulate calcium signaling/sensitization. PUO was created in male rats, and urodynamic studies were conducted 2 and 6 wk post-PUO. Cystometric recordings were analyzed for the presence or absence of nonvoiding contractions [i.e., detrusor overactivity (DO)]. RT-PCR and Western blots were performed on a subpopulation of rats to study the relationship between the expression of RhoA, L-type Ca(2+) channels, Rho kinase-1, Rho kinase-2, inositol 1,4,5-trisphosphate, ryanodine receptor, sarco(endo)plasmic reticulum Ca(2+)-ATPase 2 and protein kinase C (PKC)-potentiated phosphatase inhibitor of 17 kDa, and urodynamic findings in the same animal. Animals displayed DO at 2 (38%) and 6 wk (43%) post-PUO, increases were seen in in vivo pressures at 2 wk, and residual volume at 6 wk. Statistical analysis of RT-PCR and Western blot data at 2 wk, during the compensatory phase of detrusor hypertrophy, documented that expression of molecules that regulate calcium signaling and sensitization was consistently lower in obstructed rats without DO than those with DO or control rats. Among rats with DO at 2 wk, linear regression analysis revealed positive correlations between in vivo pressures and protein and mRNA expression of several regulatory molecules. At 6 wk, in the presence of overt signs of bladder decompensation, no clear or consistent alterations in expression of these same targets were observed at the protein level. These data extend prior work to suggest that molecular profiling of key regulatory molecules during the progression of PUO-mediated bladder dysfunction may shed new light on potential biomarkers and/or therapeutic targets.

A tissue-engineered muscle repair construct for functional restoration of an irrecoverable muscle injury in a murine model.

There are no effective clinical treatments for volumetric muscle loss (VML) resulting from traumatic injury, tumor excision, or other degenerative diseases of skeletal muscle. The goal of this study was to develop and characterize a more clinically relevant tissue-engineered muscle repair (TE-MR) construct for functional restoration of a VML injury in the mouse lattissimus dorsi (LD) muscle. To this end, TE-MR constructs developed by seeding rat myoblasts on porcine bladder acellular matrix were preconditioned in a bioreactor for 1 week and implanted in nude mice at the site of a VML injury created by excising 50% of the native LD. Two months postinjury and implantation of TE-MR, maximal tetanic force was ∼72% of that observed in native LD muscle. In contrast, injured LD muscles that were not repaired, or were repaired with scaffold alone, produced only ∼50% of native LD muscle force after 2 months. Histological analyses of LD tissue retrieved 2 months after implantation demonstrated remodeling of the TE-MR construct as well as the presence of desmin-positive myofibers, blood vessels, and neurovascular bundles within the TE-MR construct. Overall, these encouraging initial observations document significant functional recovery within 2 months of implantation of TE-MR constructs and provide clear proof of concept for the applicability of this technology in a murine VML injury model.

Oxidative stress status accompanying diabetic bladder cystopathy results in the activation of protein degradation pathways.

OBJECTIVE: • To investigate the role that oxidative stress plays in the development of diabetic cystopathy. MATERIALS AND METHODS: • Comparative gene expression in the bladder of non-diabetic and streptozotocin (STZ)-induced 2-month- old diabetic rats was carried out using microarray analysis. • Evidence of oxidative stress was investigated in the bladder by analyzing glutathione S-transferase activity, lipid peroxidation, and carbonylation and nitrosylation of proteins. • The activity of protein degradation pathways was assessed using Western blot analysis. RESULTS: • Analysis of global gene expression showed that detrusor smooth muscle tissue of STZ-induced diabetes undergoes significant enrichment in targets involved in the production or regulation of reactive oxygen species (P = 1.27 × 10(-10)). The microarray analysis was confirmed by showing that markers of oxidative stress were all significantly increased in the diabetic bladder. • It was hypothesized that the sequelae to oxidative stress would be increased protein damage and apoptosis. • This was confirmed by showing that two key proteins involved in protein degradation (Nedd4 and LC3B) were greatly up-regulated in diabetic bladders compared to controls by 12.2 ± 0.76 and 4.4 ± 1.0-fold, respectively, and the apoptosis inducing protein, BAX, was up-regulated by 6.76 ± 0.76-fold. CONCLUSION: • Overall, the findings obtained in the present study add to the growing body of evidence showing that diabetic cystopathy is associated with oxidative damage of smooth muscle cells, and results in protein damage and activation of apoptotic pathways that may contribute to a deterioration in bladder function.

Vascular smooth muscle enhances functionality of tissue-engineered blood vessels in vivo.

OBJECTIVES: There is significant room for improvement in the development of tissue-engineered blood vessels (TEBVs) for vascular reconstruction. Most commonly, TEBVs are seeded with endothelial cells (ECs) only. This provides an antithrombogenic surface but suboptimal physiologic characteristics compared with native arteries, due to lack of smooth muscle cells (SMCs) in the vessel media. Although SMCs are critical in vessel architecture and function throughout the vascular tree, few studies have incorporated SMCs in TEBVs implanted in vivo. As such, the goal of the present study was to evaluate the effect of SMC coseeding with ECs on TEBV maturation, structure, and function after prolonged in vivo maturation. METHODS: Dual-seeded TEBVs (dsTEBVs) were created by coseeding autologous ECs derived from circulating progenitor cells and SMCs from artery explants onto the lumen and outer surface of extracellular matrix scaffolds, respectively. Control vessels were seeded with ECs alone (ecTEBV). All vessels were preconditioned to pulsatile flow for 10 to 14 days in a bioreactor, implanted as arterial interposition grafts in sheep, and allowed to heal and adapt in vivo for 4 months before ex vivo physiologic testing and histologic analysis. RESULTS: All implants were patent at 4 months. There were no structural failures, aneurysms, or infectious complications. The dsTEBVs exhibited a greater degree of wall maturation, characterized by higher medial cellularity (P = .01) and greater percentage of α-actin (P = .005) and SMC-specific muscle myosin heavy chain (P = .005) staining compared with ecTEBVs. Contractile responses to phenylephrine and serotonin were significantly greater in isolated rings of dsTEBVs than those observed in ecTEBVs (P = .01). CONCLUSIONS: To our knowledge, this is the first study that demonstrates enhanced in vivo wall maturation and contractile function of TEBVs coseeded with autologous SMCs and ECs compared with EC seeding alone. These data suggest a coseeding strategy can be accomplished in a clinically relevant timeframe (typically 6 weeks) and may provide advantages for arterial reconstruction compared with vessels engineered only with endothelium.

Regenerative medicine: basic concepts, current status, and future applications.

A recent report demonstrated that a laboratory-grown neobladder tissue could be successfully used for cystoplasty in young patients with myelomeningocele who were otherwise healthy. This remarkable achievement portends well for the application of tissue engineering/regenerative medicine technologies to the treatment of end-organ failure due to a variety of causes (ie, congenital, acquired, age or disease related). Nonetheless, the broader clinical use of these groundbreaking technologies awaits improved understanding of endogenous regenerative mechanisms, more detailed knowledge of the boundary conditions that define the current limits for tissue repair and replacement in vivo, and the parallel development of critical enabling technologies (ie, improved cell source, biomaterials, bioreactors). This brief report will review a number of the most salient features and recent developments in this rapidly advancing area of medical research and detail some of our own experience with bladder and skeletal muscle regeneration and replacement as examples that highlight both the promise and challenges facing regenerative medicine/tissue engineering.