Bone regeneration: stem cell therapies and clinical studies in orthopaedics and traumatology

Regenerative medicine seeks to repair or replace damaged tissues or organs, with the goal to fully restore structure and function without the formation of scar tissue. Cell based therapies are promising new therapeutic approaches in regenerative medicine. By using mesenchymal stem cells, good results have been reported for bone engineering in a number of clinical studies, most of them investigator initiated trials with limited scope with respect to controls and outcome. With the implementation of a new regulatory framework for advanced therapeutic medicinal products, the stage is set to improve both the characterization of the cells and combination products, and pave the way for improved controlled and well-designed clinical trials. The incorporation of more personalized medicine approaches, including the use of biomarkers to identify the proper patients and the responders to treatment, will be contributing to progress in the field. Both translational and clinical research will move the boundaries in the field of regenerative medicine, and a coordinated effort will provide the clinical breakthroughs, particularly in the many applications of bone engineering.     

Extracellular matrix considerations for scar-free repair and regeneration: Insights from regenerative diversity among vertebrates

The extracellular matrix (ECM) is an essential feature of development, tissue homeostasis and recovery from injury. How the ECM responds dynamically to cellular and soluble components to support the faithful repair of damaged tissues in some animals but leads to the formation of acellular fibrotic scar tissue in others has important clinical implications. Studies in highly regenerative organisms such as the zebrafish and the salamander have revealed a specialist formulation of ECM components that support repair and regeneration, while avoiding scar tissue formation. By comparing a range of different contexts that feature scar-less healing and full regeneration vs. scarring through fibrotic repair, regenerative therapies that incorporate ECM components could be significantly enhanced to improve both regenerative potential and functional outcomes. This article is part of a directed issue entitled: Regenerative Medicine: the challenge of translation.     

The extracellular matrix as a scaffold for tissue reconstruction

The extracellular matrix (ECM) consists of a complex mixture of structural and functional proteins and serves an important role in tissue and organ morphogenesis, maintenance of cell and tissue structure and function, and in the host response to injury. Xenogeneic and allogeneic ECM has been used as a bioscaffold for the reconstruction of many different tissue types in both pre-clinical and human clinical studies. Common features of ECM-associated tissue remodeling include extensive angiogenesis, recruitment of circulating progenitor cells, rapid scaffold degradation and constructive remodeling of damaged or missing tissues. The ECM-induced remodeling response is a distinctly different phenomenon from that of scar tissue formation.     

Nanotechnology in regenerative medicine: the materials side

Regenerative medicine is an emerging multidisciplinary field that aims to restore, maintain or enhance tissues and hence organ functions. Regeneration of tissues can be achieved by the combination of living cells, which will provide biological functionality, and materials, which act as scaffolds to support cell proliferation. Mammalian cells behave in vivo in response to the biological signals they receive from the surrounding environment, which is structured by nanometre-scaled components. Therefore, materials used in repairing the human body have to reproduce the correct signals that guide the cells towards a desirable behaviour. Nanotechnology is not only an excellent tool to produce material structures that mimic the biological ones but also holds the promise of providing efficient delivery systems. The application of nanotechnology to regenerative medicine is a wide issue and this short review will only focus on aspects of nanotechnology relevant to biomaterials science. Specifically, the fabrication of materials, such as nanoparticles and scaffolds for tissue engineering, and the nanopatterning of surfaces aimed at eliciting specific biological responses from the host tissue will be addressed.     

Imaging stem cell distribution,growth, migration,and differentiation in 3‐D scaffolds for bone tissue engineering using mesoscopic fluorescence tomography

Regenerative medicine has emerged as an important discipline that aims to repair injury or replace damaged tissues or organs by introducing living cells or functioning tissues. Successful regenerative medicine strategies will likely depend upon a simultaneous optimization strategy for the design of biomaterials, cell‐seeding methods, cell‐biomaterial interactions, and molecular signaling within the engineered tissues. It remains a challenge to image three‐dimensional (3‐D) structures and functions of the cell‐seeded scaffold in mesoscopic scale (>2 ～ 3 mm). In this study, we utilized angled fluorescence laminar optical tomography (aFLOT), which allows depth‐resolved molecular characterization of engineered tissues in 3‐D to investigate cell viability, migration, and bone mineralization within bone tissue engineering scaffolds in situ.     

Neurotrophic regulation of fibroblast dedifferentiation during limb skeletal regeneration in the axolotl (Ambystoma mexicanum)

The ability of animals to repair tissue damage is widespread and impressive. Among tissues, the repair and remodeling of bone occurs during growth and in response to injury; however, loss of bone above a threshold amount is not regenerated, resulting in a “critical-size defect” (CSD). The development of therapies to replace or regenerate a CSD is a major focus of research in regenerative medicine and tissue engineering. Adult urodeles (salamanders) are unique in their ability to regenerate complex tissues perfectly, yet like mammals do not regenerate a CSD. We report on an experimental model for the regeneration of a CSD in the axolotl (the Excisional Regeneration Model) that allows for the identification of signals to induce fibroblast dedifferentiation and skeletal regeneration. This regenerative response is mediated in part by BMP signaling, as is the case in mammals; however, a complete regenerative response requires the induction of a population of undifferentiated, regeneration-competent cells. These cells can be induced by signaling from limb amputation to generate blastema cells that can be grafted to the wound, as well as by signaling from a nerve and a wound epithelium to induce blastema cells from fibroblasts within the wound environment.     

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic diseases¹. However, most organs are not easily accessible, necessitating the need to develop surgical methods to gain access to these structures. In this video article, we describe a method for transplanting cells directly into the kidney of adult zebrafish, a popular model to study regeneration and disease². Recipient fish are pre-conditioned by irradiation to suppress the immune rejection of the injected cells³. We demonstrate how the head kidney can be exposed by a lateral incision in the flank of the fish, followed by the injection of cells directly in to the organ. Using fluorescently labeled whole kidney marrow cells comprising a mixed population of renal and hematopoietic precursors, we show that nephron progenitors can engraft and differentiate into new renal tissue - the gold standard of any cell-based regenerative therapy. This technique can be adapted to deliver purified stem or progenitor cells and/or small molecules to the kidney as well as other internal organs and further enhances the zebrafish as a versatile model to study regenerative medicine.     

Exploring the role of microRNAs in axolotl regeneration

The axolotl, Ambystoma mexicanum, is used extensively for research in developmental biology, particularly for its ability to regenerate and restore lost organs, including in the nervous system, to full functionality. Regeneration in mammals typically depends on the healing process and scar formation with limited replacement of lost tissue. Other organisms, such as spiny mice (Acomys cahirinus), salamanders, and zebrafish, are able to regenerate some damaged body components. Blastema is a tissue that is formed after tissue injury in such organisms and is composed of progenitor cells or dedifferentiated cells that differentiate into various cell types during regeneration. Thus, identifying the molecules responsible for initiation of blastema formation is an important aspect for understanding regeneration. Introns, a major source of noncoding RNAs (ncRNAs), have characteristic sizes in the axolotl, particularly in genes associated with development. These ncRNAs, particularly microRNAs (miRNAs), exhibit dynamic regulation during regeneration. These miRNAs play an essential role in timing and control of gene expression to order and organize processes necessary for blastema creation. Master keys or molecules that underlie the remarkable regenerative abilities of the axolotl remain to be fully explored and exploited. Further and ongoing research on regeneration promises new knowledge that may allow improved repair and renewal of human tissues.     

脂肪干细胞在再生医学中的应用

再生医学是一门研究如何促进创伤与组织再生及功能重建的新兴学科,主要通过研究干细胞分化、机体等正常组织创伤修复与再生等机制来维持、修复、再生或改善损伤组织和器官功能。脂肪干细胞（adipose-derived stem cells,ASCs）是近年来从脂肪组织中分离得到的一种具有多向分化潜能的干细胞,是一种足量的、可用于实际的、有一定吸引力的自体细胞代替的供体资源,并能够广泛的用于组织修复、再生、发育的可塑性及细胞治疗等研究中。阐述了脂肪干细胞在旁分泌、软组织重建及损伤修复、骨骼肌重建、心血管重建、神经系统重建及癌症转移与入侵方面的作用模式,概括总结了目前利用脂肪干细胞参与的临床治疗方法,以期对脂肪干细胞在再生医学中应用研究提供参考。     

Regenerative biology: the emerging field of tissue repair and restoration

Regenerative biology has now been recognized as a new field with certain aims and goals. One direction of this new field is to understand the basic mechanisms by which tissues can be repaired and restored. The other direction examines the possibility of using this basic knowledge to apply it to medicine with the goal to clinically repair damaged tissues. Regeneration of tissues can occur by the differentiation of stem cells (local or non-local) or by the transdifferentiation of local terminally differentiated cells. While the transdifferentiation aspects are old, during the past few years many data have accumulated regarding the existence of stem cells and their participation in tissue renewal. This review will present an overview of the potential of all vertebrate organs to regenerate and of the basic mechanisms involved.     

Multi-Tissue Microarray Analysis Identifies a Molecular Signature of Regeneration

The inability to functionally repair tissues that are lost as a consequence of disease or injury remains a significant challenge for regenerative medicine. The molecular and cellular processes involved in complete restoration of tissue architecture and function are expected to be complex and remain largely unknown. Unlike humans, certain salamanders can completely regenerate injured tissues and lost appendages without scar formation. A parsimonious hypothesis would predict that all of these regenerative activities are regulated, at least in part, by a common set of genes. To test this hypothesis and identify genes that might control conserved regenerative processes, we performed a comprehensive microarray analysis of the early regenerative response in five regeneration-competent tissues from the newt Notophthalmus viridescens. Consistent with this hypothesis, we established a molecular signature for regeneration that consists of common genes or gene family members that exhibit dynamic differential regulation during regeneration in multiple tissue types. These genes include members of the matrix metalloproteinase family and its regulators, extracellular matrix components, genes involved in controlling cytoskeleton dynamics, and a variety of immune response factors. Gene Ontology term enrichment analysis validated and supported their functional activities in conserved regenerative processes. Surprisingly, dendrogram clustering and RadViz classification also revealed that each regenerative tissue had its own unique temporal expression profile, pointing to an inherent tissue-specific regenerative gene program. These new findings demand a reconsideration of how we conceptualize regenerative processes and how we devise new strategies for regenerative medicine.     

Stem cells for osteoarticular and vascular tissue engineering

Tissue damages or loss of organs often result in structural and metabolic changes that can cause serious complications. The therapeutic objective of tissue engineering (TE) is to recreate, regenerate or restore function of damaged tissue. TE is based on the coalescence of three components: a scaffold or matrix from natural or synthetic origin biodegradable or not, reparative cells and signals (hypoxia, mechanical stress, morphogens…). Articular cartilage, bone and blood vessels are tissues for which TE has progressed significantly, from basic research to clinical trials. If biomaterials must exhibit different properties depending on the tissue to regenerate, the cellular component of TE is mostly represented by stem cells notably adult mesenchymal stem cells harvested from bone marrow or adipose tissue. In recent years, progress has been made in our understanding of the biological mechanisms that govern stem cell differentiation and in the development of materials with controlled physicochemical and biological properties. However, many technological barriers and regulations concerns have to be overcome before tissue engineering enters into the therapeutic arsenal of regenerative medicine. This review aims at highlighting the progress in the use of stem cells for engineering osteoarticular and vascular tissues.     

Multiplexed targeting of microRNA in stem cell-derived extracellular vesicles for regenerative medicine

Regenerative medicine is a research field that develops methods to restore damaged cell or tissue function by regeneration, repair or replacement. Stem cells are the raw material of the body that is ultimately used from the point of view of regenerative medicine, and stem cell therapy uses cells themselves or their derivatives to promote responses to diseases and dysfunctions, the ultimate goal of regenerative medicine. Stem cell-derived extracellular vesicles (EVs) are recognized as an attractive source because they can enrich exogenous microRNAs (miRNAs) by targeting pathological recipient cells for disease therapy and can overcome the obstacles faced by current cell therapy agents. However, there are some limitations that need to be addressed before using miRNA-enriched EVs derived from stem cells for multiplexed therapeutic targeting in many diseases. Here, we review various roles on miRNA-based stem cell EVs that can induce effective and stable functional improvement of stem cell-derived EVs. In addition, we introduce and review the implications of several miRNA-enriched EV therapies improved by multiplexed targeting in diseases involving the circulatory system and nervous system. This systemic review may offer potential roles for stem cell-derived therapeutics with multiplexed targeting.     

Extracellular matrix-derived products modulate endothelial and progenitor cell migration and proliferation in vitro and stimulate regenerative healing in vivo

Most adult mammals heal without restorative replacement of lost tissue and instead form scar tissue at an injury site. One exception is the adult MRL/MpJ mouse that can regenerate ear and cardiac tissue after wounding with little evidence of scar tissue formation. Following production of a MRL mouse ear hole, 2 mm in diameter, a structure rapidly forms at the injury site that resembles the amphibian blastema at a limb amputation site during limb regeneration. We have isolated MRL blastemal cells (MRL-B) from this structure and adapted them to culture. We demonstrate by RT-PCR that even after continuous culturing of these cells they maintain expression of several progenitor cell markers, including DLK (Pref-1), and Msx-1. We have isolated the underlying extracellular matrix (ECM) produced by these MRL-B cells using a new non-proteolytic method and studied the biological activities of this cell-free ECM. Multiplex microELISA analysis of MRL-B cell-free ECM vs. cells revealed selective enrichment of growth factors such as bFGF, HGF and KGF in the matrix compartment. The cell-free ECM, degraded by mild enzyme treatment, was active in promoting migration and proliferation of progenitor cells in vitro and accelerating wound closure in a mouse full thickness cutaneous wound assay in vivo. In vivo, a single application of MRL-B cell matrix-derived products to full thickness cutaneous wounds in non-regenerative mice, B6, induced re-growth of pigmented hair, dermis and epidermis at the wound site whereas scar tissue replaced these tissues at wound sites in mice treated with vehicle alone. These studies suggest that matrix-derived products can stimulate regenerative healing and avert scar tissue formation in adult mammals.     

Recellularization of decellularized human adipose-tissue-derived extracellular matrix sheets with other human cell types

Decellularized human extracellular matrices (ECMs) are an extremely appealing biomaterial for tissue engineering and regenerative medicine. In this study, we decellularized human adipose tissue, fabricated a thin ECM sheet and explored the potential of this human adipose-derived ECM sheet as a substrate to support the formation of tissues other than adipose tissue. Acellular ECM sheets were fabricated from human adipose tissue through successive physical and chemical treatments: homogenization, centrifugation, casting, freeze-drying and sodium dodecyl sulfate treatment. The ECM sheets exhibited good mechanical properties, despite their porous structure. They degraded quickly in the presence of collagenase and the degradation rate increased with the collagenase concentration in phosphate-buffered saline. Five different human cell types, covering a broad range of cells and applications (normal human dermal fibroblasts, human aortic smooth muscle cells, human chondrocytes, human umbilical vein endothelial cells and human adipose-derived stem cells), were seeded onto the ECM sheets. All the human cell types spread well, proliferated and were successfully integrated into the decellularized ECM sheet. Overall, the results suggest that recellularized ECM sheets are a promising substitute for defective or damaged human tissues.     

Known data on applied regenerative medicine in tendon healing

Tendons and ligaments are important structures in the musculoskeletal system. Ligaments connect various bones and provide stability in complex movements of joints in the knee. Tendon is made of dense connective tissue and transmits the force of contraction from muscle to bone. They are injured due to direct trauma in sports or roadside accidents. Tendon healing after repair is often poor due to the formation of fibro vascular scar tissues with low mechanical property. Regenerative techniques such as PRP (platelet-rich plasma), stem cells, scaffolds, gene therapy, cell sheets, and scaffolds help augment repair and regenerate tissue in this context. Therefore, it is of interest to document known data (repair process, tissue regeneration, mechanical strength, and clinical outcome) on applied regenerative medicine in tendon healing.     

Advances in using MRI probes and sensors for in vivo cell tracking as applied to regenerative medicine

The field of molecular and cellular imaging allows molecules and cells to be visualized in vivo non-invasively. It has uses not only as a research tool but in clinical settings as well, for example in monitoring cell-based regenerative therapies, in which cells are transplanted to replace degenerating or damaged tissues, or to restore a physiological function. The success of such cell-based therapies depends on several critical issues, including the route and accuracy of cell transplantation, the fate of cells after transplantation, and the interaction of engrafted cells with the host microenvironment. To assess these issues, it is necessary to monitor transplanted cells non-invasively in real-time. Magnetic resonance imaging (MRI) is a tool uniquely suited to this task, given its ability to image deep inside tissue with high temporal resolution and sensitivity. Extraordinary efforts have recently been made to improve cellular MRI as applied to regenerative medicine, by developing more advanced contrast agents for use as probes and sensors. These advances enable the non-invasive monitoring of cell fate and, more recently, that of the different cellular functions of living cells, such as their enzymatic activity and gene expression, as well as their time point of cell death. We present here a review of recent advancements in the development of these probes and sensors, and of their functioning, applications and limitations.KEY WORDS: Regenerative medicine, Stem cells, Magnetic resonance imaging, Paramagnetic contrast agents, CEST, Perfluorocarbon particles, Biosensor, Cell labeling, Cellular function     

Adult stem cell therapy for the heart

The purpose of this review is to summarize current data leading to and arising from recent clinical application of cellular therapy for acute myocardial infarct (heart attack) and congestive heart failure. We specifically focus on use of adult stem cells and compare and contrast bone marrow and adipose tissue; two different sources from which stem cells can be harvested in substantial numbers with limited morbidity. Cellular therapy is the latest in a series of strategies applied in an effort to prevent or mitigate the progressive and otherwise irreversible loss of cardiac function that frequently follows a heart attack. Unlike surgical, pharmacologic, and gene transfer approaches, cellular therapy has the potential to restore cardiac function by providing cells capable of regenerating damaged myocardium and/or myocardial function. Skeletal muscle myoblast expansion and transfer allows delivery of cells with contractile function, albeit without any evidence of cardiomyogenesis or electrical coupling to remaining healthy myocardium. Delivery of endothelial progenitor cells (EPCs) which drive reperfusion of infarct zone tissues is also promising, although this mechanism is directed at halting ongoing degeneration rather than initiating a regenerative process. By contrast, demonstration of the ability of adult stem cells to undergo cardiomyocyte differentiation both in vitro and in vivo suggests a potential for regenerative medicine. This potential is being examined in early clinical studies.