Functionally Graded, Bone- and Tendon-Like Polyurethane for Rotator Cuff Repair.

Critical considerations in engineering biomaterials for rotator cuff repair include bone-tendon-like mechanical properties to support physiological loading and biophysicochemical attributes that stabilize the repair site over the long-term. In this study, UV-crosslinkable polyurethane based on quadrol (Q), hexamethylene diisocyante (H), and methacrylic anhydride (M; QHM polymers), which are free of solvent, catalyst, and photoinitiator, is developed. Mechanical characterization studies demonstrate that QHM polymers possesses phototunable bone- and tendon-like tensile and compressive properties (12-74 MPa tensile strength, 0.6-2.7 GPa tensile modulus, 58-121 MPa compressive strength, and 1.5-3.0 GPa compressive modulus), including the capability to withstand 10 000 cycles of physiological tensile loading and reduce stress concentrations via stiffness gradients. Biophysicochemical studies demonstrate that QHM polymers have clinically favorable attributes vital to rotator cuff repair stability, including slow degradation profiles (5-30% mass loss after 8 weeks) with little-to-no cytotoxicity in vitro, exceptional suture retention ex vivo (2.79-3.56-fold less suture migration relative to a clinically available graft), and competent tensile properties (similar ultimate load but higher normalized tensile stiffness relative to a clinically available graft) as well as good biocompatibility for augmenting rat supraspinatus tendon repair in vivo. This work demonstrates functionally graded, bone-tendon-like biomaterials for interfacial tissue engineering.

Characterization of a compartment syndrome-like injury model.

INTRODUCTION: Acute compartment syndrome (CS) is caused by an elevation of pressure within a muscular compartment that can be caused by numerous factors, including blunt trauma. In this study, we characterized a rodent model of CS-like injury. METHODS: Forty male athymic rats received a standardized injury of ischemia and compression to their hindlimbs, while the intracompartmental pressure (ICP) was measured using an implantable transmitter. Tetanic muscle function was evaluated, and histology was performed on the tibialis anterior (TA) muscle. RESULTS: ICPs were held at 260.70 ± 2.70 mm Hg during injury. Injured muscles recovered 59% of their total function 4 weeks after injury, and histology showed high levels of edema, inflammation (CD68(+) ), angiogenesis (CD31(+) ), and fibrosis within 72 hours after injury. CONCLUSIONS: We describe a novel CS-like injury model and a novel method to measure ICP, which could potentially be used to develop innovative therapies to manage CS injury in patients.

Muscle-derived stem cell sheets support pump function and prevent cardiac arrhythmias in a model of chronic myocardial infarction.

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.

Stem cells, angiogenesis and muscle healing: a potential role in massage therapies?

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)-ß1 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.

Stem cells, angiogenesis and muscle healing: a potential role in massage therapies?

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)-ß1 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.

Biological approaches to improve skeletal muscle healing after injury and disease.

Skeletal muscle injury and repair are complex processes, including well-coordinated steps of degeneration, inflammation, regeneration, and fibrosis. We have reviewed the recent literature including studies by our group that describe how to modulate the processes of skeletal muscle repair and regeneration. Antiinflammatory drugs that target cyclooxygenase-2 were found to hamper the skeletal muscle repair process. Muscle regeneration phase can be aided by growth factors, including insulin-like growth factor-1 and nerve growth factor, but these factors are typically short-lived, and thus more effective methods of delivery are needed. Skeletal muscle damage caused by traumatic injury or genetic diseases can benefit from cell therapy; however, the majority of transplanted muscle cells (myoblasts) are unable to survive the immune response and hypoxic conditions. Our group has isolated neonatal skeletal muscle derived stem cells (MDSCs) that appear to repair muscle tissue in a more effective manner than myoblasts, most likely due to their better resistance to oxidative stress. Enhancing antioxidant levels of MDSCs led to improved regenerative potential. It is becoming increasingly clear that stem cells tissue repair by direct differentiation and paracrine effects leading to neovascularization of injured site and chemoattraction of host cells. The factors invoked in paracrine action are still under investigation. Our group has found that angiotensin II receptor blocker (losartan) significantly reduces fibrotic tissue formation and improves repair of murine injured muscle. Based on these data, we have conducted a case study on two hamstring injury patients and found that losartan treatment was well tolerated and possibly improved recovery time. We believe this medication holds great promise to optimize muscle repair in humans.

Follistatin improves skeletal muscle healing after injury and disease through an interaction with muscle regeneration, angiogenesis, and fibrosis.

Recovery from skeletal muscle injury is often incomplete because of the formation of fibrosis and inadequate myofiber regeneration; therefore, injured muscle could benefit significantly from therapies that both stimulate muscle regeneration and inhibit fibrosis. To this end, we focused on blocking myostatin, a member of the transforming growth factor-ß superfamily and a negative regulator of muscle regeneration, with the myostatin antagonist follistatin. In vivo, follistatin-overexpressing transgenic mice underwent significantly greater myofiber regeneration and had less fibrosis formation compared with wild-type mice after skeletal muscle injury. Follistatin's mode of action is likely due to its ability to block myostatin and enhance neovacularization. Furthermore, muscle progenitor cells isolated from follistatin-overexpressing mice were significantly superior to muscle progenitors isolated from wild-type mice at regenerating dystrophin-positive myofibers when transplanted into the skeletal muscle of dystrophic mdx/severe combined immunodeficiency mice. In vitro, follistatin stimulated myoblasts to express MyoD, Myf5, and myogenin, which are myogenic transcription factors that promote myogenic differentiation. Moreover, follistatin's ability to enhance muscle differentiation is at least partially due to its ability to block myostatin, activin A, and transforming growth factor-ß1, all of which are negative regulators of muscle cell differentiation. The findings of this study suggest that follistatin is a promising agent for improving skeletal muscle healing after injury and muscle diseases, such as the muscular dystrophies.

A role for cell sex in stem cell-mediated skeletal muscle regeneration: female cells have higher muscle regeneration efficiency.

We have shown that muscle-derived stem cells (MDSCs) transplanted into dystrophic (mdx) mice efficiently regenerate skeletal muscle. However, MDSC populations exhibit heterogeneity in marker profiles and variability in regeneration abilities. We show here that cell sex is a variable that considerably influences MDSCs' regeneration abilities. We found that the female MDSCs (F-MDSCs) regenerated skeletal muscle more efficiently. Despite using additional isolation techniques and cell cloning, we could not obtain a male subfraction with a regeneration capacity similar to that of their female counterparts. Rather than being directly hormonal or caused by host immune response, this difference in MDSCs' regeneration potential may arise from innate sex-related differences in the cells' stress responses. In comparison with F-MDSCs, male MDSCs have increased differentiation after exposure to oxidative stress induced by hydrogen peroxide, which may lead to in vivo donor cell depletion, and a proliferative advantage for F-MDSCs that eventually increases muscle regeneration. These findings should persuade researchers to report cell sex, which is a largely unexplored variable, and consider the implications of relying on cells of one sex.

Timing of the administration of suramin treatment after muscle injury.

INTRODUCTION: It has been reported that suramin treatment can improve muscle healing; however, details about optimizing the dosing requirements remain unclear. The purpose of this study was to determine the optimal timing of suramin administration and investigate the effects it had on the expression of myostatin, follistatin, and muscle vascularity after muscle injury. METHODS: Contusion injured muscles of mice were treated with suramin at 1, 2, or 3 weeks post-injury and evaluated histologically and physiologically at 1, 2, and 10 days after injection. RESULTS: Suramin treatment initiated at 2 weeks post-injury was observed to promote muscle regeneration and muscle strength, and to decrease fibrosis. Suramin reduced myostatin expression and increased follistatin expression and vascularity in injured skeletal muscle. CONCLUSIONS: Suramin's positive effect on muscle regeneration is thought to be due to its enhancement of follistatin expression which increases neoangiogenesis and inhibits myostatin's promotion of fibrosis.

Lentivirus-mediated Wnt11 gene transfer enhances Cardiomyogenic differentiation of skeletal muscle-derived stem cells.

Wnt signaling plays a crucial role in regulating cell proliferation, differentiation and inducing cardiomyogenesis. Skeletal muscle-derived stem cells (MDSCs) have been shown to be multipotent; however, their potential to aid in the healing of the heart after myocardial infarction appears to be due to the paracrine effects they impart on the host environment. The goal of this study was to investigate whether Wnt11 could promote the differentiation of MDSCs into cardiomyocytes and enhance the repair of infarcted myocardium. MDSCs transduced with a lentivirus encoding for Wnt11 increased mRNA and protein expression of the early cardiac markers NK2 transcription factor related 5 (NKx2.5) and Connexin43 (Cx43) and also led to an increased expression of late-stage cardiac markers including: α, ß-myosin heavy chain (MHC) and brain natriuretic protein (BNP) at the mRNA level, and MHC and Troponin I (TnI) at the protein level. We also observed that Wnt11 expression significantly enhanced c-jun N-terminal kinase activity in transduced MDSCs, and that some of the cells beat spontaneously but are not fully differentiated cardiomyocytes. Finally, lentivirus-Wnt11-transduced MDSCs showed greater survival and cardiac differentiation after being transplanted into acutely infarct-injured myocardium. These findings could one day lead to strategies that could be utilized in cardiomyoplasty treatments of myocardial infarction.

Biologic approaches to enhance rotator cuff healing after injury.

BACKGROUND: Despite the advances in surgical procedures to repair the rotator cuff, there is a high incidence of failure. Biologic approaches, such as growth factor delivery and stem cell and gene therapy, are potential targets for optimization to improve the outcome of rotator cuff therapies and reduce rates of reinjury. This article outlines the current evidence for growth factor and stem cell therapy in tendon healing and the augmentation of rotator cuff repair. METHODS: Literature on the PubMed-National Center for Biotechnology Information database was searched using the keywords growth factor, factor, gene therapy, stem cell, mesenchymal, or bone marrow in combination with rotator cuff, supraspinatus, or infraspinatus. Articles that studied growth factors or stem cells alone in rotator cuff repair were selected. Only 3 records showed use of stem cells in rotator cuff repair; thus, we expanded our search to include selected studies on stem cells and Achilles or patellar tendon repairs. Bibliographies and proceedings of meetings were searched to include additional applicable studies. We also included hitherto unpublished data by our group on the use of stem cell transplantation for rotator cuff therapy. RESULTS: More than 70 articles are summarized, with focus on recent original research papers and significant reviews that summarized earlier records. CONCLUSIONS: Use of growth factors, stem cell therapy, and other tissue-engineering means serve to augment classical surgical rotator cuff repair procedures. The combination of stem cells and growth factors resulted in enhanced repair that emulated uninjured tissue, but the literature search reflected paucity of research in this field. Preclinical evidence from gene therapy and stem cell studies can be used as a start to move therapy from the experimental phase to clinical translation in patients.

Growth and differentiation factor-7 immobilized, mechanically strong quadrol-hexamethylene diisocyanate-methacrylic anhydride polyurethane polymer for tendon repair and regeneration.

Biological and mechanical cues are both vital for biomaterial aided tendon repair and regeneration. Here, we fabricated mechanically tendon-like (0 s UV) QHM polyurethane scaffolds (Q: Quadrol, H: Hexamethylene diisocyanate; M: Methacrylic anhydride) and immobilized them with Growth and differentiation factor-7 (GDF-7) to produce mechanically strong and tenogenic scaffolds. In this study, we assessed QHM polymer cytocompatibility, amenability to fibrin-coating, immobilization and persistence of GDF-7, and capability to support GDF-7-mediated tendon differentiation in vitro as well as in vivo in mouse subcutaneous and acute rat rotator cuff tendon resection models. Cytocompatibility studies showed that QHM facilitated cell attachment, proliferation, and viability. Fibrin-coating and GDF-7 retention studies showed that mechanically tendon-like 0 s UV QHM polymer could be immobilized with GDF-7 and retained the growth factor (GF) for at least 1-week ex vivo. In vitro differentiation studies showed that GDF-7 mediated bone marrow-derived human mesenchymal stem cell (hMSC) tendon-like differentiation on 0 s UV QHM. Subcutaneous implantation of GDF-7-immobilized, fibrin-coated, QHM polymer in mice for 2 weeks demonstrated de novo formation of tendon-like tissue while implantation of GDF-7-immobilized, fibrin-coated, QHM polymer in a rat acute rotator cuff resection injury model indicated tendon-like tissue formation in situ and the absence of heterotopic ossification. Together, our work demonstrates a promising synthetic scaffold with human tendon-like biomechanical attributes as well as immobilized tenogenic GDF-7 for tendon repair and regeneration. STATEMENT OF SIGNIFICANCE: Biological activity and mechanical robustness are key features required for tendon-promoting biomaterials. While synthetic biomaterials can be mechanically robust, they often lack bioactivity. To biologically augment synthetic biomaterials, numerous drug and GF delivery strategies exist but the large tissue space within the shoulder is constantly flushed with saline during arthroscopic surgery, hindering efficacious controlled release of therapeutic molecules. Here, we coated QHM polymer (which exhibits human tendon-to-bone-like biomechanical attributes) with fibrin for GF binding. Unlike conventional drug delivery strategies, our approach utilizes immobilized GFs as opposed to released GFs for sustained, localized tissue regeneration. Our data demonstrated that GF immobilization can be broadly applied to synthetic biomaterials for enhancing bioactivity, and GDF-7-immobilized QHM exhibit high clinical translational potential for tendon repair.

Prospective identification of myogenic endothelial cells in human skeletal muscle.

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.

Malignant transformation of multipotent muscle-derived cells by concurrent differentiation signals.

Recent studies have shown that germ-line determination occurs early in development and that extracellular signaling can alter this fate. This denial of a cell's fate by counteracting its intrinsic signaling pathways through extrinsic stimulation is believed to be associated with oncogenesis. Using specific populations of multipotent skeletal muscle-derived stem cells (MDSCs), we have been able to generate tumors by subjecting cells with specific lineage predilections to concomitant differentiation signals. More specifically, when a stem cell that had a predilection toward osteogenesis was implanted into a skeletal muscle, tumors formed in 25% of implanted mice. When cells predilected to undergo myogenesis were pretreated with bone morphogenetic protein 4 (BMP4) for 4 days prior to implantation, they formed tumors in 25% of mice. These same myogenic predilected cells, when transduced to express BMP4 and implanted into either a long-bone or cranial defect, formed bone, but they formed tumors in 100% of mice when implanted into the skeletal muscle. The tumors generated in this latter study were serially transplantable as long as they retained BMP4 expression. Furthermore, when we impeded the ability of the cells to undergo myogenic differentiation using small interfering RNA to the myogenic regulator MyoD1, we stopped transformation. Based on our findings, we postulate that specific MDSC populations can undergo concomitant signal-induced transformation and that the initial stages of transformation may be due to changes in the balance between the inherent nature of the cell and extrinsic signaling pathways. This theory represents a potential link between somatic stem cells and cancer and suggests an involvement of the niche/environment in transformation.

Skeletal muscle derived stem cells microintegrated into a biodegradable elastomer for reconstruction of the abdominal wall.

A variety of techniques have been applied to generate tissue engineered constructs, where cells are combined with degradable scaffolds followed by a period of in vitro culture or direct implantation. In the current study, a cellularized scaffold was generated by concurrent deposition of electrospun biodegradable elastomer (poly(ester urethane)urea, PEUU) and electrosprayed culture medium + skeletal muscle-derived stem cells (MDSCs) or electrosprayed culture medium alone as a control. MDSCs were obtained from green fluorescent protein (GFP) transgenic rats. The created scaffolds were implanted into allogenic strain-matched rats to replace a full thickness abdominal wall defect. Both control and MDSC-integrated scaffolds showed extensive cellular infiltration at 4 and 8 wk. The number of blood vessels was higher, the area of residual scaffold was lower, number of multinucleated giant cells was lower and area of connective tissue was lower in MDSC-integrated scaffolds (p < 0.05). GFP + cells co-stained positive for VEGF. Bi-axial mechanical properties of the MDSC-microintegrated constructs better approximated the anisotropic behavior of the native abdominal wall. GFP + cells were observed throughout the scaffold at ∼5% of the cell population at 4 and 8 wk. RNA expression at 4 wk showed higher expression of early myogenic marker Pax7, and b-FGF in the MDSC group. Also, higher expression of myogenin and VEGF were seen in the MDSC group at both 4 and 8 wk time points. The paracrine effect of donor cells on host cells likely contributed to the differences found in vivo between the groups. This approach for the rapid creation of highly-cellularized constructs with soft tissue like mechanics offers an attractive methodology to impart cell-derived bioactivity into scaffolds providing mechanical support during the healing process and might find application in a variety of settings.

Heterogeneous structure of stem cells dynamics: statistical models and quantitative predictions.

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.

The use of blood vessel-derived stem cells for meniscal regeneration and repair.

PURPOSE: Surgical repairs of tears in the vascular region of the meniscus usually heal better than repairs performed in the avascular region; thus, we hypothesized that this region might possess a richer supply of vascular-derived stem cells than the avascular region. METHODS: In this study, we analyzed 6 menisci extracted from aborted human fetuses and 12 human lateral menisci extracted from adult human subjects undergoing total knee arthroplasty. Menisci were immunostained for CD34 (a stem cell marker) and CD146 (a pericyte marker) in situ, whereas other menisci were dissected into two regions (peripheral and inner) and used to isolate meniscus-derived cells by flow cytometry. Cell populations expressing CD34 and CD146 were tested for their multilineage differentiation potentials, including chondrogenic, osteogenic, and adipogenic lineages. Fetal peripheral meniscus cells were transplanted by intracapsular injection into the knee joints of an athymic rat meniscal tear model. Rat menisci were extracted and histologically evaluated after 4 wk posttransplantation. RESULTS: Immunohistochemistry and flow cytometric analyses demonstrated that a higher number of CD34- and CD146-positive cells were found in the peripheral region compared with the inner region. The CD34- and CD146-positive cells isolated from the vascular region of both fetal and adult menisci demonstrated multilineage differentiation capacities and were more potent than cells isolated from the inner (avascular) region. Fetal CD34- and CD146-positive cells transplanted into the athymic rat knee joint were recruited into the meniscal tear sites and contributed to meniscus repair. CONCLUSIONS: The vascularized region of the meniscus contains more stem cells than the avascular region. These meniscal-derived stem cells were multipotent and contributed to meniscal regeneration.

Human myogenic endothelial cells exhibit chondrogenic and osteogenic potentials at the clonal level.

We have previously reported the high regenerative potential of murine muscle-derived stem cells (mMDSCs) that are capable of differentiating into multiple mesodermal cell lineages, including myogenic, endothelial, chondrocytic, and osteoblastic cells. Recently, we described a putative human counterpart of mMDSCs, the myogenic endothelial cells (MECs), in adult human skeletal muscle, which efficiently repair/regenerate the injured and dystrophic skeletal muscle as well as the ischemic heart in animal disease models. Nevertheless it remained unclear whether human MECs, at the clonal level, preserve mMDSC-like chondrogenic and osteogenic potentials and classic stem cell characteristics including high proliferation and resistance to stress. Herein, we demonstrated that MECs, sorted from fresh postnatal human skeletal muscle biopsies, can be grown clonally and exhibit robust resistance to oxidative stress with no tumorigeneity. MEC clones were capable of differentiating into chondrocytes and osteoblasts under inductive conditions in vitro and participated in cartilage and bone formation in vivo. Additionally, adipogenic and angiogenic potentials of clonal MECs (cMECs) were observed. Overall, our study showed that cMECs not only display typical properties of adult stem cells but also exhibit chondrogenic and osteogenic capacities in vitro and in vivo, suggesting their potential applications in articular cartilage and bone repair/regeneration.

Terminal differentiation is not a major determinant for the success of stem cell therapy - cross-talk between muscle-derived stem cells and host cells.

We have found that when muscle-derived stem cells (MDSCs) are implanted into a variety of tissues only a small fraction of the donor cells can be found within the regenerated tissues and the vast majority of cells are host derived. This observation has also been documented by other investigators using a variety of different stem cell types. It is speculated that the transplanted stem cells release factors that modulate repair indirectly by mobilizing the host's cells and attracting them to the injury site in a paracrine manner. This process is loosely called a 'paracrine mechanism', but its effects are not necessarily restricted to the injury site. In support of this speculation, it has been reported that increasing angiogenesis leads to an improvement of cardiac function, while inhibiting angiogenesis reduces the regeneration capacity of the stem cells in the injured vascularized tissues. This observation supports the finding that most of the cells that contribute to the repair process are indeed chemo-attracted to the injury site, potentially through host neo-angiogenesis. Since it has recently been observed that cells residing within the walls of blood vessels (endothelial cells and pericytes) appear to represent an origin for post-natal stem cells, it is tempting to hypothesize that the promotion of tissue repair, via neo-angiogenesis, involves these blood vessel-derived stem cells. For non-vascularized tissues, such as articular cartilage, the regenerative property of the injected stem cells still promotes a paracrine, or bystander, effect, which involves the resident cells found within the injured microenvironment, albeit not through the promotion of angiogenesis. In this paper, we review the current knowledge of post-natal stem cell therapy and demonstrate the influence that implanted stem cells have on the tissue regeneration and repair process. We argue that the terminal differentiation capacity of implanted stem cells is not the major determinant of the cells regenerative potential and that the paracrine effect imparted by the transplanted cells plays a greater role in the regeneration process.

Engineering spatial control of multiple differentiation fates within a stem cell population.

The capability to engineer microenvironmental cues to direct a stem cell population toward multiple fates, simultaneously, in spatially defined regions is important for understanding the maintenance and repair of multi-tissue units. We have previously developed an inkjet-based bioprinter to create patterns of solid-phase growth factors (GFs) immobilized to an extracellular matrix (ECM) substrate, and applied this approach to drive muscle-derived stem cells toward osteoblasts 'on-pattern' and myocytes 'off-pattern' simultaneously. Here this technology is extended to spatially control osteoblast, tenocyte and myocyte differentiation simultaneously. Utilizing immunofluorescence staining to identify tendon-promoting GFs, fibroblast growth factor-2 (FGF-2) was shown to upregulate the tendon marker Scleraxis (Scx) in C3H10T1/2 mesenchymal fibroblasts, C2C12 myoblasts and primary muscle-derived stem cells, while downregulating the myofibroblast marker α-smooth muscle actin (α-SMA). Quantitative PCR studies indicated that FGF-2 may direct stem cells toward a tendon fate via the Ets family members of transcription factors such as pea3 and erm. Neighboring patterns of FGF-2 and bone morphogenetic protein-2 (BMP-2) printed onto a single fibrin-coated coverslip upregulated Scx and the osteoblast marker ALP, respectively, while non-printed regions showed spontaneous myotube differentiation. This work illustrates spatial control of multi-phenotype differentiation and may have potential in the regeneration of multi-tissue units.