Skeletal Regeneration Research Articles

Effect of low-energy laser irradiation and antioxidant supplementation on cell apoptosis during skeletal muscle post-injury regeneration in pigs.

The aim of this study was to evaluate the effect of low-energy laser irradiation, coenzyme Q10 and vitamin E supplementation on the apoptosis of macrophages and muscle precursor cells during skeletal muscle regeneration after bupivacaine-induced injury. The experiment was conducted on 75 gilts, divided into 5 experimental groups: I--control, II--low-energy laser irradiation, III--coenzyme Q10, IV--coenzyme Q10 and vitamin E, V--vitamin E. Muscle necrosis was induced by injection of 0.5% bupivacaine hydrochloride. The animals were euthanized on subsequent days after injury. Samples were formalin fixed and processed routinely for histopathology. Apoptosis was detected using the TUNEL method. The obtained results indicate that low-energy laser irradiation has a beneficial effect on macrophages and muscle precursor cell activity during muscle post-injury regeneration and protects these cells against apoptosis. Vitamin E has a slightly lower protective effect, limited mainly to the macrophages. Coenzyme Q10 co-supplemented with vitamin E increases the activity of macrophages and muscle precursor cells, myotube and young muscle formation. Importantly, muscle precursor cells seem to be more sensitive to apoptosis than macrophages in the environment of regenerating damaged muscle.

MSC signature associated with therapeutical effect in osteoarticular diseases

Adipose derived mesenchymal stromal cells (ASC) are adult stem cells exhibiting functional properties that have open the way for cell-based clinical therapies. Primarily, their capacity of multilineage differentiation has been explored in a number of strategies for skeletal tissue regeneration. More recently, MSCs have been reported to exhibit immunosuppressive as well as healing capacities, to improve angiogenesis and prevent apoptosis or fibrosis through the secretion of paracrine mediators including HGF, IL1RA, IL-6, PDL-1, TSG6, TGF-β1 and PGE2. In normal homeostasis, IL1Ra counterbalances the effect of IL1α and IL1β as a competitive receptor antagonist. MSC immunosuppressive effect is highly variable according to cell population heterogeneity. We compared gene expression of huMSC with high potential to inhibit T cells response with low efficient MSC through gene array. We identified a signature associated with high immunoregulatory function. High expression of ITPR1, NR4A3, ABCA1, LIPG ADAMTS4 as well as expression of CD109, ITGA10, IL6R was associated with increased T cell suppressive effect in vitro. We performed preclinical models of osteoarthritis, and showed that a local injection of ASC showed a reduction of synovitis, reduction of osteophytes, joint stabilization, reducing the score of cartilage lesions. We then investigated the role of iNOS, IL6 and IL1RA respectively using the inflammatory collagen-induced arthritis model. In contrast to wt MSCs, which reduced both the incidence and clinical signs of arthritis, IL1RA–/– MSCs were not able to prevent arthritis progression and even worsened the arthritic symptoms. μCT analysis showed a protection against bone erosion by wt MSCs but not in IL1RA–/–MSCs-treated mice. We found a decrease of the number of CD4 + IFN-γ+ Th1 and CD4 + IL-17+ Th17 lymphocytes in the spleens of wt MSCs-treated mice as compared to IL1RA–/– MSCs-treated mice. This work was completed by toxicology data showing the excellent tolerance of the local injection of ADSC and biodistribution showing the persistence of cells after 6 months in murine models. We conducted a open-label phase 1 trial including 18 patients with severe osteoarthritis of the knee in failure of conventional therapies (62.5% were KL IV) at two sites, Montpellier and Wurzburg. Mean age was 61 years, with a 10 years history of knee OA. The patient received a single injection of autologous ASC 15 days after lipoaspiration (2.106, 107 or 5.107) through intra-articular injection. The primary outcome measure of effectiveness was patient-reported WOMAC pain subscores by VAS in the affected knee at week 12. Secondary outcome measures included Outcome Measures in Rheumatology Clinical Trials and Osteoarthritis Research Society International (OMERACT OARSI) responses. We observed a decrease of the VAS Pain (73 ± 11 mm day 0 to 32 ± 23 month 3), and of WOMAC (50 ± 18 to 25 ± 7 month 3). This study confirms the feasibility and safety of local injection of autologous cells from adipose tissue and suggested that the most effective dose was 107 autologous cells. Moreover, a molecular signature associated with increase immunosuppressive potential is described.

Pluripotent Stem Cells and Skeletal Regeneration--Promise and Potential.

The bone is a regenerative tissue, capable of healing itself after fractures. However, some circumstances such as critical-size defects, malformations, and tumor destruction may exceed the skeleton's capacity for self-repair. In addition, bone mass and strength decline with age, leading to an increase in fragility fractures. Therefore, the ability to generate large numbers of patient-specific osteoblasts would have enormous clinical implications for the treatment of skeletal defects and diseases. This review will highlight recent advances in the derivation of pluripotent stem cells, and in their directed differentiation towards bone-forming osteoblasts.

THE ROLE OF GENDER FACTOR IN CHRONIC ALCOHOL MYOPATHY

Alcohol induced skeletal muscles lesion is one of the most frequent symptom of alcohol disease. Alcohol myopathy occurs in 40–60% of patients with chronic ethanol intoxication. The role of gender factor in manifest and course of chronic alcohol myopathy (CAM) is discussed in the article. The results of comprehensive examination of 42 patients (30 males and 12 females) with chronic ethanol intoxication are presented. Morphologic, morphometric and immunohystochemical examination of skeletal muscle biopsy specimens is a standard of diagnosis of CAM that gives the opportunity to access quantitively and qualitatively atrophic process. The basic pathogenic mechanisms of muscle fibers atrophy both in men and women with chronic alcohol intoxication are the damage of systemic protein synthesis and skeletal muscles regeneration. The severity of myopathic syndrome correlates with muscle atrophy both in men and women. But in women CAM was found to manifest earlier. The duration of alcohol intoxication in women was less but muscle lesion was more severe and widespread.

Dynamic remodeling of the extra cellular matrix during zebrafish fin regeneration

Extracellular matrix plays a dynamic role during the process of wound healing, embryogenesis and tissue regeneration. Caudal fin regeneration in zebrafish is an excellent model to study tissue and skeletal regeneration. We have analyzed the expression pattern of some of the well characterized ECM proteins during the process of caudal fin regeneration in zebrafish. Our results show that a transitional matrix analogous to the one formed during newt skeletal and heart muscle regeneration is synthesized during fin regeneration. Here we demonstrate that a provisional matrix rich in hyaluronic acid, tenascin C, and fibronectin is synthesized following amputation. Additionally, we observed that the link protein Hapln1a dependent ECM, consisting of Hapln1a, hyaluronan and proteoglycan aggrecan, is upregulated during fin regeneration. Laminin, the protein characteristic of differentiated tissues, showed only modest change in the expression pattern. Our findings on zebrafish fin regeneration implicates that changes in the extracellular milieu represent an evolutionarily conserved mechanism that proceeds during tissue regeneration, yet with distinct players depending on the type of tissue that is involved.

Self-renewal And Metabolism Of Muscle Stem Cells Are Regulated By Ampk

During skeletal muscle regeneration, muscle stem cells activate and recapitulate the myogenic program to repair the damaged myofibers. A subset of these cells will not enter into the myogenesis but will self-renew to return into quiescence. The control of this process is crucial to maintain skeletal muscle homeostasis and the molecular mechanism that control muscle stem cells self-renewal only start to be understood. Recent studies highlight the importance of the metabolism switch in the regulation of stem cell fate and particularly the exit from and the entry into the quiescent state. In this way, AMPK seems to be an excellent candidate in the control of stem cell fate choice. AMPK is a master metabolic regulator. Its activation mainly depends on the increase of the AMP/ATP ratio and it induces a regulation of the anabolic/catabolic balance. Although it is a well-studied molecule, its action on stem cell fate choice has never been investigated. In this project, we decipher the role of AMPKa1 in muscle stem cells self-renewal by ex vivo (isolated fibers) and in vitro (muscle stem cell primary cultures) experiments. These reliable procedures permit us to pointing out that in absence of AMPKa1 muscle stem cell differentiate in a lower extend and self-renew much more (+300%). We also performed in vivo experiments, using specific conditional mouse strains in which AMPKa1 is specifically deleted in muscle stem cells. The model of cardiotoxin injury in the Tibialis Anterior couple with the measurement of specific readouts of skeletal muscle regeneration allow us to analyze the different states of this process. Thus, a strong decrease in myofiber size 14 and 28 days post injury is observed. Moreover, a significant increase in the total number of fibers per muscle (+40%) associated with an important increase in the total number of satellite cell per muscle (+30%) 28 days post injury is noticed. Metabolism of muscle stem cells is explored in vitro by analyses of mitochondrial respiration and by measurement of the activity of key enzymes of glycolysis. In this way, our results support the hypothesis that in absence of AMPKa1 muscle stem cells are not able to use mitochondria to synthesize energy. To conclude, our work permits to establish a new and crucial role of AMPKa1 in muscle stem cell fate choice by switching the metabolism during skeletal regeneration, linking for the very first time self-renewal and metabolism in this context.

Role of muscle stem cells during skeletal regeneration.

Although the importance of muscle in skeletal regeneration is well recognized clinically, the mechanisms by which muscle supports bone repair have remained elusive. Muscle flaps are often used to cover the damaged bone after traumatic injury yet their contribution to bone healing is not known. Here, we show that direct bone-muscle interactions are required for periosteum activation and callus formation, and that muscle grafts provide a source of stem cells for skeletal regeneration. We investigated the role of satellite cells, the muscle stem cells. Satellite cells loss in Pax7(-/-) mice and satellite cell ablation in Pax7(Cre) (ERT) (2/) (+) ;DTA(f/f) mice impaired bone regeneration. Although satellite cells did not contribute as a large source of cells endogenously, they exhibited a potential to contribute to bone repair after transplantation. The fracture healing phenotype in Pax7(Cre) (ERT) (2/) (+) ;DTA(f/f) mice was associated with decreased bone morphogenetic proteins (BMPs), insulin-like growth factor 1, and fibroblast growth factor 2 expression that are normally upregulated in response to fracture in satellite cells. Exogenous rhBMP2 improved bone healing in Pax7(Cre) (ERT) (2/) (+) ;DTA(f/f) mice further supporting the role of satellite cells as a source of growth factors. These results provide the first functional evidence for a direct contribution of muscle to bone regeneration with important clinical implications as it may impact the use of muscle flaps, muscle stem cells, and growth factors in orthopedic applications.

Bioactive glass reinforced elastomer composites for skeletal regeneration: A review.

Biodegradable elastomers have clinical applicability due to their biocompatibility, tunable degradation and elasticity. The addition of bioactive glasses to these elastomers can impart mechanical properties sufficient for hard tissue replacement. Hence, a composite with a biodegradable polymer matrix and a bioglass filler can offer a method of augmenting existing tissue. This article reviews the applications of such composites for skeletal augmentation.

A distinct regulatory region of the Bmp5 locus activates gene expression following adult bone fracture or soft tissue injury

Bone morphogenetic proteins (BMPs) are key signaling molecules required for normal development of bones and other tissues. Previous studies have shown that null mutations in the mouse Bmp5 gene alter the size, shape and number of multiple bone and cartilage structures during development. Bmp5 mutations also delay healing of rib fractures in adult mutants, suggesting that the same signals used to pattern embryonic bone and cartilage are also reused during skeletal regeneration and repair. Despite intense interest in BMPs as agents for stimulating bone formation in clinical applications, little is known about the regulatory elements that control developmental or injury-induced BMP expression. To compare the DNA sequences that activate gene expression during embryonic bone formation and following acute injuries in adult animals, we assayed regions surrounding the Bmp5 gene for their ability to stimulate lacZ reporter gene expression in transgenic mice. Multiple genomic fragments, distributed across the Bmp5 locus, collectively coordinate expression in discrete anatomic domains during normal development, including in embryonic ribs. In contrast, a distinct regulatory region activated expression following rib fracture in adult animals. The same injury control region triggered gene expression in mesenchymal cells following tibia fracture, in migrating keratinocytes following dorsal skin wounding, and in regenerating epithelial cells following lung injury. The Bmp5 gene thus contains an “injury response” control region that is distinct from embryonic enhancers, and that is activated by multiple types of injury in adult animals.

MicroRNAs regulate bone development and regeneration.

MicroRNAs (miRNAs) are endogenous small noncoding ~22-nt RNAs, which have been reported to play a crucial role in maintaining bone development and metabolism. Osteogenesis originates from mesenchymal stem cells (MSCs) differentiating into mature osteoblasts and each period of bone formation is inseparable from the delicate regulation of various miRNAs. Of note, apprehending the sophisticated circuit between miRNAs and osteogenic homeostasis is of great value for artificial skeletal regeneration for severe bone defects. In this review, we highlight how different miRNAs interact with diverse osteo-related genes and endeavor to sketch the contours of potential manipulations of miRNA-modulated bone repair.

Influence of PCL molecular weight on mesenchymal stromal cell differentiation

The molecular weight of polycaprolactone was varied to investigate its effect on stem cell activity. Results showed that polymer molecular weight is an additional parameter to consider when designing scaffolds for skeletal regeneration.

NOTCH-Mediated Maintenance and Expansion of Human Bone Marrow Stromal/Stem Cells: A Technology Designed for Orthopedic Regenerative Medicine.

Human bone marrow-derived stromal/stem cells (BMSCs) have great therapeutic potential for treating skeletal disease and facilitating skeletal repair, although maintaining their multipotency and expanding these cells ex vivo have proven difficult. Because most stem cell-based applications to skeletal regeneration and repair in the clinic would require large numbers of functional BMSCs, recent research has focused on methods for the appropriate selection, expansion, and maintenance of BMSC populations during long-term culture. We describe here a novel biological method that entails selection of human BMSCs based on NOTCH2 expression and activation of the NOTCH signaling pathway in cultured BMSCs via a tissue culture plate coated with recombinant human JAGGED1 (JAG1) ligand. We demonstrate that transient JAG1-mediated NOTCH signaling promotes human BMSC maintenance and expansion while increasing their skeletogenic differentiation capacity, both ex vivo and in vivo. This study is the first of its kind to describe a NOTCH-mediated methodology for the maintenance and expansion of human BMSCs and will serve as a platform for future clinical or translational studies aimed at skeletal regeneration and repair.

Calluses Flex Their Muscles to Align Bone Fragments during Fracture Repair

Neonatal animals spontaneously reduce fractures, yet the mechanical forces influencing this process are poorly understood. In this issue of Developmental Cell, Rot etal. (2014) show that muscle and the fracture callus actively position fractured neonatal bone fragments to restore their alignment, highlighting the multifaceted roles of mechanical cues in skeletal regeneration.

Natural large-scale regeneration of rib cartilage in a mouse model.

The clinical need for methods to repair and regenerate large cartilage and bone lesions persists. One way to make new headway is to study skeletal regeneration when it occurs naturally. Cartilage repair is typically slow and incomplete. However, an exception to this observation can be found in the costal cartilages, where complete repair has been reported in humans but the cellular and molecular mechanisms have not yet been characterized. In this study, we establish a novel animal model for cartilage repair using the mouse rib costal cartilage. We then use this model to test the hypothesis that the perichondrium, the dense connective tissue that surrounds the cartilage, is a tissue essential for repair. Our results show that full replacement of the resected cartilage occurs quickly (within 1 to 2 months) and properly differentiates but that repair occurs only in the presence of the perichondrium. We then provide evidence that the rib perichondrium contains a special niche that houses chondrogenic progenitors that possess qualities particularly suited for mediating repair. Label-retaining cells can be found within the perichondrium that can give rise to new chondrocytes. Furthermore, the perichondrium proliferates and thickens during the healing period and when ectopically placed can generate new cartilage. In conclusion, we have successfully established a model for hyaline cartilage repair in the mouse rib, which should be useful for gaining a more detailed understanding of cartilage regeneration and ultimately for developing methods to improve cartilage and bone repair in other parts of the skeleton.

EPO promotes bone repair through enhanced cartilaginous callus formation and angiogenesis.

Erythropoietin (EPO)/erythropoietin receptor (EPOR) signaling is involved in the development and regeneration of several non-hematopoietic tissues including the skeleton. EPO is identified as a downstream target of the hypoxia inducible factor-α (HIF-α) pathway. It is shown that EPO exerts a positive role in bone repair, however, the underlying cellular and molecular mechanisms remain unclear. In the present study we show that EPO and EPOR are expressed in the proliferating, pre-hypertrophic and hypertrophic zone of the developing mouse growth plates as well as in the cartilaginous callus of the healing bone. The proliferation rate of chondrocytes is increased under EPO treatment, while this effect is decreased following siRNA mediated knockdown of EPOR in chondrocytes. EPO treatment increases biosynthesis of proteoglycan, accompanied by up-regulation of chondrogenic marker genes including SOX9, SOX5, SOX6, collagen type 2, and aggrecan. The effects are inhibited by knockdown of EPOR. Blockage of the endogenous EPO in chondrocytes also impaired the chondrogenic differentiation. In addition, EPO promotes metatarsal endothelial sprouting in vitro. This coincides with the in vivo data that local delivery of EPO increases vascularity at the mid-stage of bone healing (day 14). In a mouse femoral fracture model, EPO promotes cartilaginous callus formation at days 7 and 14, and enhances bone healing at day 28 indexed by improved X-ray score and micro-CT analysis of microstructure of new bone regenerates, which results in improved biomechanical properties. Our results indicate that EPO enhances chondrogenic and angiogenic responses during bone repair. EPO's function on chondrocyte proliferation and differentiation is at least partially mediated by its receptor EPOR. EPO may serve as a therapeutic agent to facilitate skeletal regeneration.

Skeletal tissue regeneration: where can hydrogels play a role?

The emerging field of tissue engineering reveals promising approaches for the repair and regeneration of skeletal tissues including the articular cartilage, bone, and the entire joint. Amongst the myriad of biomaterials available to support this strategy, hydrogels are highly tissue mimicking substitutes and thus of great potential for the regeneration of functional tissues. This review comprises an overview of the novel and most promising hydrogels for articular cartilage, osteochondral and bone defect repair. Chondro- and osteo-conductive and -instructive hydrogels are presented, highlighting successful combinations with inductive signals and cell sources. Moreover, advantages, drawbacks, and future perspectives of the role of hydrogels in skeletal regeneration are addressed, pointing out the current state of this rising approach.

Growth and differentiation of a long bone in limb development, repair and regeneration.

Repair from traumatic bone fracture is a complex process that includes mechanisms of bone development and bone homeostasis. Thus, elucidation of the cellular/molecular basis of bone formation in skeletal development would provide valuable information on fracture repair and would lead to successful skeletal regeneration after limb amputation, which never occurs in mammals. Elucidation of the basis of epimorphic limb regeneration in amphibians would also provide insights into skeletal regeneration in mammals, since the epimorphic regeneration enables an amputated limb to re-develop the three-dimensional structure of bones. In the processes of bone development, repair and regeneration, growth of the bone is achieved through several events including not only cell proliferation but also aggregation of mesenchymal cells, enlargement of cells, deposition and accumulation of extracellular matrix, and bone remodeling.

Stepwise Differentiation of Pluripotent Stem Cells into Osteoblasts Using Four Small Molecules under Serum-free and Feeder-free Conditions

SummaryPluripotent stem cells are a promising tool for mechanistic studies of tissue development, drug screening, and cell-based therapies. Here, we report an effective and mass-producing strategy for the stepwise differentiation of mouse embryonic stem cells (mESCs) and mouse and human induced pluripotent stem cells (miPSCs and hiPSCs, respectively) into osteoblasts using four small molecules (CHIR99021 [CHIR], cyclopamine [Cyc], smoothened agonist [SAG], and a helioxanthin-derivative 4-(4-methoxyphenyl)pyrido[4′,3′:4,5]thieno[2,3-b]pyridine-2-carboxamide [TH]) under serum-free and feeder-free conditions. The strategy, which consists of mesoderm induction, osteoblast induction, and osteoblast maturation phases, significantly induced expressions of osteoblast-related genes and proteins in mESCs, miPSCs, and hiPSCs. In addition, when mESCs defective in runt-related transcription factor 2 (Runx2), a master regulator of osteogenesis, were cultured by the strategy, they molecularly recapitulated osteoblast phenotypes of Runx2 null mice. The present strategy will be a platform for biological and pathological studies of osteoblast development, screening of bone-augmentation drugs, and skeletal regeneration.

Metallic Nanoparticles-Based Biochip with Multi-Channel for Immunoassay

Osteogenesis is the cellular and molecular foundation of skeletal development and bone regeneration related to fracture healing, revitalization of bone graft and therapies for osteoporosis. A hallmark of osteogenesis is the mineralization of extracellular matrix. Because of its potential impact on developmental biology and human health, understanding and regulation of osteogenesis are subjects of intensive study. Although significant advancements have been made over the past decades, there are still unsolved puzzles in regulations of osteogenesis. Angiogenesis generally refers to new blood vessels branching out from established vasculature. Besides of fundamental physiology, angiogenesis involves in pathology, such as growth of cancer, and is essential for the repair of virtually all types of tissues. It has long been recognized that osteogenesis and angiogenesis are coupling events during bone formation. The classic osteogenic and angiogenic pathways intertwine and cross-talk during bone formation. To better understand the signal pathways and coupling factors of osteogenesis and angiogenesis is critically important for enhancing bone regeneration and tissue engineering of bone. The conventional biological models, however, have very limited capacity of isolating angiogenic and osteogenic events from the cascade of bone regeneration, and precisely quantifying the effects of angiogenic and osteogenic factors on bone formation at a molecular level. Biochips and tissue chips provide a powerful tool to simulate and quantify angiogenic and osteogenic events on the chips and effectively untangle these biologically important and clinically relevant molecular events during bone formation.

The Molecular and Cellular Events That Take Place during Craniofacial Distraction Osteogenesis.

Summary:Gradual bone lengthening using distraction osteogenesis principles is the gold standard for the treatment of hypoplastic facial bones. However, the long treatment time is a major disadvantage of the lengthening procedures. The aim of this study is to review the current literature and summarize the cellular and molecular events occurring during membranous craniofacial distraction osteogenesis. Mechanical stimulation by distraction induces biological responses of skeletal regeneration that is accomplished by a cascade of biological processes that may include differentiation of pluripotential tissue, angiogenesis, osteogenesis, mineralization, and remodeling. There are complex interactions between bone-forming osteoblasts and other cells present within the bone microenvironment, particularly vascular endothelial cells that may be pivotal members of a complex interactive communication network in bone. Studies have implicated number of cytokines that are intimately involved in the regulation of bone synthesis and turnover. The gene regulation of numerous cytokines (transforming growth factor-β, bone morphogenetic proteins, insulin-like growth factor-1, and fibroblast growth factor-2) and extracellular matrix proteins (osteonectin, osteopontin) during distraction osteogenesis has been best characterized and discussed. Understanding the biomolecular mechanisms that mediate membranous distraction osteogenesis may guide the development of targeted strategies designed to improve distraction osteogenesis and accelerate bone regeneration that may lead to shorten the treatment duration.