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Engineering scaffolds to augment the repair of hard-to-soft multitissue musculoskeletal tissue units, such as bone-tendon, to simultaneously support tissue healing and functional movement has had limited success. Overcoming this challenge will require not only precise spatial control of bone- and tendon-like biomechanical properties, but also consideration of the resultant biomechanical cues, as well as the embedded biochemical cues imparted by these scaffolds. Here, we report on the effects of a spatially engineered combination of stiffness and growth factor (GF) cues to control bone-tendon-like differentiation in vitro and tissue formation in vivo. This was achieved using mechanically graded, bone- and tendon-like QHM polyurethane (QHM: Q: Quadrol; H: hexamethylene diisocyanate; M: methacrylic anhydride) scaffolds selectively biopatterned with osteogenic bone morphogenetic protein-2 (BMP-2) and tenogenic fibroblast growth factor-2 (FGF-2). First, material characterization, including porosity, surface roughness, contact angle, and microindentation measurements, was performed. Second, in vitro studies demonstrated that increased material stiffness promoted GF-mediated osteoblast differentiation and reduced tenocyte differentiation. Sustained GF exposure masked this stiffness effect. Third, in vivo studies involving subcutaneous implantation of mechanically graded and biochemically patterned QHM scaffolds (composed of these bone- and tendon-promoting GFs biopatterned on biphasic bone and tendon biomechanically mimicking regions) in mice demonstrated spatial control of bone- and tendon-like tissue formation. Altogether, these data provide new insights for future engineering of scaffolds to augment hard-to-soft multitissue repair.Schematic illustration showing our overall approach, studies performed, and envisioned application. Our strategy combines biomechanical and biochemical cues from a mechanically graded biomaterial and GF biopatterning, respectively, to spatially control bone- and tendon-like differentiation. Here, experiments (i) characterized our biomaterial, (ii) investigated the interplay between biomechanical and biochemical cues in vitro, and (iii) assessed the ability of our GF-biopatterned and biphasic biomaterial to spatially control bone- and tendon-like tissue formation in vivo. The envisioned goal of this study is to develop a biomaterial for treating large-to-massive tendon injuries.

This protocol details a procedure, known as the modified preplate technique, which is currently used in our laboratory to isolate muscle cells on the basis of selective adhesion to collagen-coated tissue culture plates. By employing this technique to murine skeletal muscle, we have been able to isolate a rapidly adhering cell (RAC) fraction within the earlier stages of the process, whereas a slowly adhering cell (SAC) fraction containing muscle-derived stem cells is obtained from the later stages of the process. This protocol outlines the methods and materials needed to isolate RAC and SAC populations from murine skeletal muscle. The procedure involves mechanical and enzymatic digestion of skeletal muscle tissue with collagenase XI, dispase and trypsin followed by plating the resultant muscle slurry on collagen type I-coated flasks where the cells adhere at different rates. The entire preplate technique requires 5 d to obtain the final preplate SAC population. Two to three additional days are usually required before this population is properly established. We also detail additional methodologies designed to further enrich the resultant cell population by continuing the modified preplating process on the SAC population. This process is known as replating and requires further time.

We document anatomic, molecular and developmental relationships between endothelial and myogenic cells within human skeletal muscle. Cells coexpressing myogenic and endothelial cell markers (CD56, CD34, CD144) were identified by immunohistochemistry and flow cytometry. These myoendothelial cells regenerate myofibers in the injured skeletal muscle of severe combined immunodeficiency mice more effectively than CD56 + myogenic progenitors. They proliferate long term, retain a normal karyotype, are not tumorigenic and survive better under oxidative stress than CD56 + myogenic cells. Clonally derived myoendothelial cells differentiate into myogenic, osteogenic and chondrogenic cells in culture. Myoendothelial cells are amenable to biotechnological handling, including purification by flow cytometry and long-term expansion in vitro , and may have potential for the treatment of human muscle disease.

Direct intracardiac cell injection for heart repair is hindered by numerous limitations including: cell death, poor spreading of the injected cells, arrhythmia, needle injury, etc. Tissue-engineered cell sheet implantation has the potential to overcome some of these limitations. We evaluated whether the transplantation of a muscle-derived stem cell (MDSC) sheet could improve the regenerative capacity of MDSCs in a chronic model of myocardial infarction. MDSC sheet-implanted mice displayed a reduction in left ventricle (LV) dilation and sustained LV contraction compared with the other groups. The MDSC sheet formed aligned myotubes and produced a significant increase in capillary density and a reduction of myocardial fibrosis compared with the other groups. Hearts transplanted with the MDSC sheets did not display any significant arrhythmias and the donor MDSC survival rate was higher than the direct myocardial MDSC injection group. MDSC sheet implantation yielded better functional recovery of chronic infarcted myocardium without any significant arrhythmic events compared with direct MDSC injection, suggesting this cell sheet delivery system could significantly improve the myocardial regenerative potential of the MDSCs.

Based on growing evidence that some adult multipotent cells necessary for tissue regeneration reside in the walls of blood vessels and the clinical success of vein wrapping for functional repair of nerve damage, we hypothesized that the repair of nerves via vein wrapping is mediated by cells migrating from the implanted venous grafts into the nerve bundle. To test the hypothesis, severed femoral nerves of rats were grafted with venous grafts from animals of the opposite sex. Nerve regeneration was impaired when decellularized or irradiated venous grafts were used in comparison to untreated grafts, supporting the involvement of venous graft-derived cells in peripheral nerve repair. Donor cells bearing Y chromosomes integrated into the area of the host injured nerve and participated in remyelination and nerve regeneration. The regenerated nerve exhibited proper axonal myelination, and expressed neuronal and glial cell markers. These novel findings identify the mechanism by which vein wrapping promotes nerve regeneration.

Oral squamous cell carcinomas are characterized by complex, often near-triploid karyotypes with structural and numerical variations superimposed on the initial clonal chromosomal alterations. We used immunohistochemistry combined with classical cytogenetic analysis and spectral karyotyping to investigate the chromosomal segregation defects in cultured oral squamous cell carcinoma cells. During division, these cells frequently exhibit lagging chromosomes at both metaphase and anaphase, suggesting defects in the mitotic apparatus or kinetochore. Dicentric anaphase chromatin bridges and structurally altered chromosomes with consistent long arms and variable short arms, as well as the presence of gene amplification, suggested the occurrence of breakage-fusion-bridge cycles. Some anaphase bridges were observed to persist into telophase, resulting in chromosomal exclusion from the reforming nucleus and micronucleus formation. Multipolar spindles were found to various degrees in the oral squamous cell carcinoma lines. In the multipolar spindles, the poles demonstrated different levels of chromosomal capture and alignment, indicating functional differences between the poles. Some spindle poles showed premature splitting of centrosomal material, a precursor to full separation of the microtubule organizing centers. These results indicate that some of the chromosomal instability observed within these cancer cells might be the result of cytoskeletal defects and breakage-fusion-bridge cycles.

Advances in stem cell research and media publicity of stem cell potential have raised the hopes of patients with severe disabilities and conditions which lack a cure. While stem-cell-based therapies are the clinical standard of care for a few hematological conditions, stem cell tourism continues to rise worldwide. This rise is driven in part by patients’ need for alternative treatment for difficult conditions and increased online access to health information. Unfortunately, clinics around the world are exploiting patients’ hopes by offering supposed stem cell therapies, without credible scientific rationale, oversight, or patient protections. Occurring particularly in Asia and South America, treatments which are illegal in most countries are being offered for what are often considered incurable conditions. A survey of health organization websites showed that while 71% of these websites provided information about stem cell treatment/research, only 18% of those sites included a direct warning or caution about stem cell treatments abroad. A number of professional organizations have published guidance documents to shine a light on the dangers of stem cell tourism. Comprehensive government regulations exist in the US, Europe, and several other countries, so that approvals for stem cell treatments occur only after extensive safety testing has occurred. In this review, we discuss issues related to stem cell treatments, including the patients’ needs for educational resources, and we describe the process of regulatory approval for cell therapies using the US system as an example.

Understanding stem cell (SC) population dynamics is essential for developing models that can be used in basic science and medicine, to aid in predicting cells fate. These models can be used as tools e.g. in studying patho-physiological events at the cellular and tissue level, predicting (mal)functions along the developmental course and personalized regenerative medicine. Using time-lapsed imaging and statistical tools, we show that the dynamics of SC populations involve a heterogeneous structure consisting of multiple sub-population behaviors. Using non-Gaussian statistical approaches, we identify the co-existence of fast and slow dividing subpopulations and quiescent cells, in stem cells from three species. The mathematical analysis also shows that, instead of developing independently, SCs exhibit a time-dependent fractal behavior as they interact with each other through molecular and tactile signals. These findings suggest that more sophisticated models of SC dynamics should view SC populations as a collective and avoid the simplifying homogeneity assumption by accounting for the presence of more than one dividing sub-population and their multi-fractal characteristics.

Limited autologous vascular graft availability and poor patency rates of synthetic grafts for bypass or replacement of small-diameter arteries remain a concern in the surgical community. These limitations could potentially be improved by a tissue engineering approach. We report here our progress in the development and in vivo testing of a stem-cell-based tissue-engineered vascular graft for arterial applications. Poly(ester urethane)urea scaffolds (length = 10 mm; inner diameter = 1.2 mm) were created by thermally induced phase separation (TIPS). Compound scaffolds were generated by reinforcing TIPS scaffolds with an outer electrospun layer of the same biomaterial (ES-TIPS). Both TIPS and ES-TIPS scaffolds were bulk-seeded with 10 × 10 6 allogeneic, LacZ-transfected, muscle-derived stem cells (MDSCs), and then placed in spinner flask culture for 48 h. Constructs were implanted as interposition grafts in the abdominal aorta of rats for 8 weeks. Angiograms and histological assessment were performed at the time of explant. Cell-seeded constructs showed a higher patency rate than the unseeded controls: 65% (ES-TIPS) and 53% (TIPS) versus 10% (acellular TIPS). TIPS scaffolds had a 50% mechanical failure rate with aneurysmal formation, whereas no dilation was observed in the hybrid scaffolds. A smooth-muscle-like layer of cells was observed near the luminal surface of the constructs that stained positive for smooth muscle α-actin and calponin. LacZ+ cells were shown to be engrafted in the remodeled construct. A confluent layer of von Willebrand Factor–positive cells was observed in the lumen of MDSC-seeded constructs, whereas acellular controls showed platelet and fibrin deposition. This is the first evidence that MDSCs improve patency and contribute to the remodeling of a tissue-engineered vascular graft for arterial applications.

Skeletal muscle injuries are among the most common and frequently disabling injuries sustained by athletes. Repair of injured skeletal muscle is an area that continues to present a challenge for sports medicine clinicians and researchers due, in part, to complete muscle recovery being compromised by development of fibrosis leading to loss of function and susceptibility to re-injury. Injured skeletal muscle goes through a series of coordinated and interrelated phases of healing including degeneration, inflammation, regeneration and fibrosis. Muscle regeneration initiated shortly after injury can be limited by fibrosis which affects the degree of recovery and predisposes the muscle to reinjury. It has been demonstrated in animal studies that antifibrotic agents that inactivate transforming growth factor (TGF)-β1 have been effective at decreasing scar tissue formation. Several studies have also shown that vascular endothelial growth factor (VEGF) can increase the efficiency of skeletal muscle repair by increasing angiogenesis and, at the same time, reducing the accumulation of fibrosis. We have isolated and thoroughly characterised a population of skeletal muscle-derived stem cells (MDSCs) that enhance repair of damaged skeletal muscle fibres by directly differentiating into myofibres and secreting paracrine factors that promote tissue repair. Indeed, we have found that MDSCs transplanted into skeletal and cardiac muscles have been successful at repair probably because of their ability to secrete VEGF that works in a paracrine fashion. The application of these techniques to the study of sport-related muscle injuries awaits investigation. Other useful strategies to enhance skeletal muscle repair through increased vascularisation may include gene therapy, exercise, neuromuscular electrical stimulation and, potentially, massage therapy. Based on recent studies showing an accelerated recovery of muscle function from intense eccentric exercise through massage-based therapies, we believe that this treatment modality offers a practical and non-invasive form of therapy for skeletal muscle injuries. However, the biological mechanism(s) behind the beneficial effect of massage are still unclear and require further investigation using animal models and potentially randomised, human clinical studies.