Humanoid Robotic Loading Enhances Mechanotransduction in Tendon Tissue Engineering

Tendon injury is the most common disease in the musculoskeletal system. The current treatment methods have many limitations, such as poor therapeutic effects, functional loss of donor site, and immune rejection. Tendon tissue engineering provides a new treatment strategy for tendon repair and regeneration. In this review, we made a retrospective analysis of applying mechanical stimulation in tendon tissue engineering, and its potential as a direction of development for future clinical treatment strategies. For this purpose, the following topics are discussed; (1) the context of tendon tissue engineering and mechanical stimulation; (2) the applications of various mechanical stimulations in tendon tissue engineering, as well as their inherent mechanisms; (3) the application of magnetic force and the synergy of mechanical and biochemical stimulation. With this, we aim at clarifying some of the main questions that currently exist in the field of tendon tissue engineering and consequently gain new knowledge that may help in the development of future clinical application of tissue engineering in tendon injury.

To the Editor: The article by Xue et al1 on human embryonic stem cell-derived pacemakers illustrates that embryonic stem cells differentiated into spontaneously beating cardiocytes may function as biological pacemakers and mentions a potential limitation: The intrinsic pacemaker rate was slower than desirable. They suggest that incorporating HCN pacemaker channel genes might achieve more desirable rates, an idea consistent with our published results using HCN2 in gene- and adult human mesenchymal stem cell (hMSC)-based therapies.2–4 However, certain of the comments by Xue et al misinterpret our own work on HCN-loaded hMSCs. They state that “…these modified, undifferentiated, human mesenchymal stem cells are incapable of pacing quiescent cells because the former are neither electrically active nor genuine cardiac cells” (p 19). This statement suggests a misunderstanding of the rationale and underlying biophysics of the hMSC experiments. In fact, generation of pacemaker activity does not require delivery of an “excitable differentiated cardiac cell,” but only that the delivered cell (1) carry sufficient pacemaker current and (2) make gap junctions; thus, the hMSC-myocyte pair should behave as a pacemaker unit entirely equivalent to a single heart cell with substantial if. We clearly demonstrated both …

In quantitative proteomics, the false discovery rate (FDR) can be defined as the number of false positives within statistically significant changes in expression. False positives accumulate during the simultaneous testing of expression changes across hundreds or thousands of protein or peptide species when univariate tests such as the Student's t test are used. Currently most researchers rely solely on the estimation of p values and a significance threshold, but this approach may result in false positives because it does not account for the multiple testing effect. For each species, a measure of significance in terms of the FDR can be calculated, producing individual q values. The q value maintains power by allowing the investigator to achieve an acceptable level of true or false positives within the calls of significance. The q value approach relies on the use of the correct statistical test for the experimental design. In this situation, a uniform p value frequency distribution when there are no differences in expression between two samples should be obtained. Here we report a bias in p value distribution in the case of a three-dye DIGE experiment where no changes in expression are occurring. The bias was shown to arise from correlation in the data from the use of a common internal standard. With a two-dye schema, where each sample has its own internal standard, such bias was removed, enabling the application of the q value to two different proteomics studies. In the case of the first study, we demonstrate that 80% of calls of significance by the more traditional method are false positives. In the second, we show that calculating the q value gives the user control over the FDR. These studies demonstrate the power and ease of use of the q value in correcting for multiple testing. This work also highlights the need for robust experimental design that includes the appropriate application of statistical procedures.

ABSTRACTTendons are frequently damaged and fail to regenerate, leading to pain, loss of function, and reduced quality of life. Mesenchymal stem cells (MSCs) possess clinically useful tissue-regenerative properties and have been exploited for use in tendon tissue engineering and cell therapy. However, MSCs exhibit phenotypic heterogeneity based on the donor tissue used, and the efficacy of cell-based treatment modalities may be improved by optimizing cell source based on relative differentiation capacity. Equine MSCs were isolated from bone marrow (BM), adipose (AD), and tendon (TN), expanded in monolayer prior to seeding on decellularized tendon scaffolds (DTS), and cell-laden constructs were placed in a bioreactor designed to mimic the biophysical environment of the tendon. It was hypothesized that TN MSCs would differentiate toward a tendon cell phenotype better than BM and AD MSCs in response to a conditioning period involving cyclic mechanical stimulation for 1 hour per day at 3% strain and 0.33 Hz. All cell types integrated into DTS adopted an elongated morphology similar to tenocytes, expressed tendon marker genes, and improved tissue mechanical properties after 11 days. TN MSCs expressed the greatest levels of scleraxis, collagen type-I, and cartilage oligomeric matrix protein. Major histocompatibility class-II protein mRNA expression was not detected in any of the MSC types, suggesting low immunogenicity for allogeneic transplantation. The results suggest that TN MSCs are the ideal cell type for regenerative medicine therapies for tendinopathies, exhibiting the most mature tendon-like phenotype in vitro. When TN MSCs are unavailable, BM or AD MSCs may serve as robust alternatives.

There is significant clinical demand for functional tendon grafts in human and veterinary medicine. Tissue engineering techniques combining cells, scaffolds, and environmental stimuli may circumvent the shortcomings of traditional transplantation processes. In this study, the influence of cyclic mechanical stimulation on graft maturation and cellular phenotype was assessed in an equine model. Decellularized tendon scaffolds from four equine sources were seeded with syngeneic bone marrow-derived mesenchymal stem cells and subjected to 0%, 3%, or 5% strain at 0.33 Hz for up to 1 h daily for 11 days. Cells cultured at 3% strain integrated deep into their scaffolds, altered extracellular matrix composition, adopted tendon-like gene expression profiles, and increased construct elastic modulus and ultimate tensile strength to native levels. This bioreactor protocol is therefore suitable for cultivating replacement tendon material or as an in vitro model for studying differentiation of stem cells toward tendon.

It has been proposed that mitochondrial dysfunction is involved in the pathogenesis of type 2 diabetes (T2D). To dissect the underlying mechanisms, we performed a multiplexed proteomics study on liver mitochondria isolated from a spontaneous diabetic rat model before/after they were rendered diabetic. Altogether, we identified 1091 mitochondrial proteins, 228 phosphoproteins, and 355 hydroxyproteins. Mitochondrial proteins were found to undergo expression changes in a highly correlated fashion during T2D development. For example, proteins involved in beta-oxidation, the tricarboxylic acid cycle, oxidative phosphorylation, and other bioenergetic processes were coordinately up-regulated, indicating that liver cells confronted T2D by increasing energy expenditure and activating pathways that rid themselves of the constitutively increased flux of glucose and lipid. Notably, activation of oxidative phosphorylation was immediately related to the overproduction of reactive oxygen species, which caused oxidative stress within the cells. Increased oxidative stress was also evidenced by our post-translational modification profiles such that mitochondrial proteins were more heavily hydroxylated during T2D development. Moreover, we observed a distinct depression of antiapoptosis and antioxidative stress proteins that might reflect a higher apoptotic index under the diabetic stage. We suggest that such changes in systematic metabolism were causally linked to the development of T2D. Comparing proteomics data against microarray data, we demonstrated that many T2D-related alterations were unidentifiable by either proteomics or genomics approaches alone, underscoring the importance of integrating different approaches. Our compendium could help to unveil pathogenic events in mitochondria leading to T2D and be useful for the discovery of diagnosis biomarker and therapeutic targets of T2D.

Historical Perspectives and Advances in Mesenchymal Stem Cell Research for the Treatment of Liver Diseases

A clinically-feasible protocol for using human platelet lysate and mesenchymal stem cells in regenerative therapies

Tendons bridge muscle and bone, translating forces to the skeleton and increasing the safety and efficiency of locomotion. When tendons fail or degenerate, there are no effective pharmacological interventions. The lack of available options to treat damaged tendons has created a need to better understand and improve the repair process, particularly when suitable autologous donor tissue is unavailable for transplantation. Cells within tendon dynamically react to loading conditions and undergo phenotypic changes in response to mechanobiological stimuli. Tenocytes respond to ultrastructural topography and mechanical deformation via a complex set of behaviors involving force-sensitive membrane receptor activity, changes in cytoskeletal contractility, and transcriptional regulation. Effective ex vivo model systems are needed to emulate the native environment of a tissue and to translate cell-matrix forces with high fidelity. While early bioreactor designs have greatly expanded our knowledge of mechanotransduction, traditional scaffolds do not fully model the topography, composition, and mechanical properties of native tendon. Decellularized tendon is an ideal scaffold for cultivating replacement tissue and modeling tendon regeneration. Decellularized tendon scaffolds (DTS) possess high clinical relevance, faithfully translate forces to the cellular scale, and have bulk material properties that match natural tissue. This review summarizes progress in tendon tissue engineering, with a focus on DTS and bioreactor systems.

Inhaled anesthetics bind specifically to many proteins in the mammalian brain. Within the subgroup of proteins whose activity is substantially modulated by anesthetic binding, it is reasonable to expect anesthetic-induced alterations in host expression level. Thus, in an attempt to define the group of functional targets for these commonly used drugs, we examined changes in protein expression after anesthetic exposure in both intact rodent brains and in neuronal cell culture. Differential in-gel electrophoresis was used to minimize variance, in order to detect small changes. Quantitative analysis shows that 5 h exposures to 1 minimum alveolar concentration (1 MAC) halothane caused changes in the expression of approximately 2% of detectable proteins, but only at 2-24 h after awakening, and only in the cortex. An equipotent concentration of isoflurane altered the expression of only approximately 1% of detectable proteins, and only in the hippocampus. Primary cortical neurons were exposed to three-fold higher concentrations of anesthetics with no evidence of cytotoxicity. Small changes in protein expression were elicited by both drugs. Despite the fact that anesthetics produce profound changes in neurobiology and behavior, we found only minor changes in brain protein expression. A pronounced degree of regional selectivity was noted, indicating an under appreciated degree of specificity for these promiscuous drugs.

Urological reconstructive surgery is sometimes hampered by a lack of tissue. In some cases, autologous urothelial cells (UCs) are not available for cell expansion and ordinary tissue engineering. In these cases, we wanted to explore whether autologous mesenchymal stem cells (MSCs) from bone marrow could be used to create urological transplants. MSCs from human bone marrow were cultured in vitro with medium conditioned by normal human UCs or by indirect co-culturing in culture well inserts. Changes in gene expression, protein expression and cell morphology were studied after two weeks using western blot, RT-PCR and immune staining. Cells cultured in standard epithelial growth medium served as controls. Bone marrow MSCs changed their phenotype with respect to growth characteristics and cell morphology, as well as gene and protein expression, to a UC lineage in both culture methods, but not in controls. Urothelial differentiation was also accomplished in human bone marrow MSCs seeded on a three-dimensional poly(ε-caprolactone) (PCL)-collagen construct. Human MSCs could easily be harvested by bone marrow aspiration and expanded and differentiated into urothelium. Differentiation could take place on a three-dimensional hybrid PCL-reinforced collagen-based scaffold for creation of a tissue-engineered autologous transplant for urological reconstructive surgery.

Fiber-based approaches hold great promise for tendon tissue engineering enabling the possibility of manufacturing aligned hydrogel filaments that can guide collagen fiber orientation, thereby providing a biomimetic micro-environment for cell attachment, orientation, migration, and proliferation. In this study, a 3D system composed of cell-laden, highly aligned hydrogel yarns is designed and obtained via wet spinning in order to reproduce the morphology and structure of tendon fascicles. A bioink composed of alginate and gelatin methacryloyl (GelMA) is optimized for spinning and loaded with human bone morrow mesenchymal stem cells (hBM-MSCs). The produced scaffolds are subjected to mechanical stretching to recapitulate the strains occurring in native tendon tissue. Stem cell differentiation is promoted by addition of bone morphogenetic protein 12 (BMP-12) in the culture medium. The aligned orientation of the fibers combined with mechanical stimulation results in highly preferential longitudinal cell orientation and demonstrates enhanced collagen type I and III expression. Additionally, the combination of biochemical and mechanical stimulations promotes the expression of specific tenogenic markers, signatures of efficient cell differentiation towards tendon. The obtained results suggest that the proposed 3D cell-laden aligned system can be used for engineering of scaffolds for tendon regeneration.

Proteomic analysis of cisplatin-induced cochlear damage: Methods and early changes in protein expression

The original complex structure and mechanical properties are not fully restored after ligament and tendon injuries. Due to their high proliferation rate and differentiation potential, Bone Marrow Stromal Cells (BMSC) are considered to be an ideal cell source for tissue engineering to optimize the healing process. Ideal matrices for tissue engineering of ligaments and tendons should allow for homogenous cell seeding and offer sufficient stability. A mixture of human BMSC and liquid fibrin glue was injected into a standardized full-thickness window defect of the patellar tendon of immunodeficient rats (BMSC group). The histology of the tissue was analysed 10 and 20 days postoperatively and compared to four control groups. These groups consisted of a cohort with a mixture of human fibroblasts and fibrin glue, fibrin glue without cells, a defect group without treatment, and a group with uninjured patellar tendon tissue. Tendon defects in the BMSC group revealed dense collagen fibres and spindle-shaped cells, which were mainly orientated along the loading axis. Histologic sections of the control groups, especially of untreated defects and of defects filled with fibrin glue only, showed irregular patterns of cell distribution, irregular formed cell nucleoli and less tissue maturation. Compared to healthy tendon tissue, higher numbers of cells and less intense matrix staining was observed in the BMSC group. No ectopic bone or cartilage formation was observed in any specimen. Injection of human BMSC in a fibrin glue matrix appears to lead to more mature tissue formation with more regular patterns of cell distribution. Advantages of this "in-vivo" tissue engineering approach are a homogenous cell-matrix mixture in a well-known and approved biological matrix, and simple, minimally-invasive application by injection.

Magnetic Systems for Regenerative Medicine