Table of Contents
Welcome
Table Of Contents
Title Page
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PREFACE
List of Contributors
The Emerging Role of Mesenchymal Stem Cell Secretome in Cartilage Regeneration
Abstract
Introduction
Mesenchymal Stem Cells
Mesenchymal Stem Cell-based Therapies for Cartilage Repair
MSC Secretome
Cell Recruitment
Cell Survival
Cell Proliferation
Cell Differentiation and Matrix Synthesis
Immunomodulation
MSC Extracellular Vesicles
Cell-free Therapies for Cartilage Repair
Growth Factors, Cytokines and Chemokines
Exosomes
Conclusion
CONFLICT OF INTEREST
ACKNOWLEDGEMENTS
References
The Potential Clinical Application of Mesenchymal Stem Cells from the Dental Pulp (DPSCs) for Bone Regeneration
Abstract
INTRODUCTION
DENTAL STEM CELLS
Tooth Anatomy and the DPSCs Niche
Stem Cells from Human Permanent Teeth Dental Pulp
Phenotypic Features
Differentiation Capacities
Stem Cells from Human Deciduous Teeth Dental Pulp
Stem Cells from Other Dental-Related Tissues
Stem Cells from Human Permanent Teeth Apical Dental Pulp (APDCs/SCAPs)
Stem Cells from Human Dental Follicle (DF-MSCs)
Stem Cells from Human Periodontal Ligament (PDLSCs)
DPSCs from Other Species
Isolation and Culture Methods
Immunomodulatory Properties
Pro-Angiogenic Properties
Cryopreservation of Pulp Tissues and Cells
DPSCs Potential in Tissue Regeneration Applications
IN VITRO INDUCTION OF MINERALIZED TISSUE FORMATION
Association to Biomaterials for Mineralized Tissue Regeneration
Bone Tissue Regeneration
DPSCs for Dental Tissue Regeneration
Other Tissue Regeneration Applications
Other Neural Damage Models have also been Explored
FINAL REMARKS
CONFLICT OF INTEREST
ACKNOWLEDGEMENTS
Abbreviations
REFERENCES
Mesenchymal Stem Cells in Regenerative Medicine: The Challenges and the Opportunities
Abstract
INTRODUCTION
PERSPECTIVE OF MSCs IN REGENERATIVE MEDICINE
THE PHENOTYPICAL CHARACTERISTICS OF MSCs AND THE RELATED CHALLENGES
Typical In Vitro Morphological Features of MSCs
Candidate Markers of MSC
Main Challenges
Other Essential Phenotypical Characteristics of MSCs
Stemness: Self-renewal and Differentiation Capacity of MSCs
Main Challenges
Immunoprivileged Status of MSCs
Main Challenges
Immunomodulatory Effects of MSCs
Main Challenges
Trophic Effects of MSCs
Main Challenges
Anti-apoptotic Effects of MSCs
Main Challenges
The Safety Profile
Main Challenges
Easy Accessibility
Main Challenges
THE CLINICAL OPPORTUNITIES AND THE CHALLENGES
The Common Theoretical Challenges
The Common Practical Challenges
MSCs in Tissue Repair
The Challenges
MSCs in Bone and Cartilage Diseases
The Challenges
CONCLUSION AND FUTURE DIRECTIONS
CONFLICT OF INTEREST
ACKNOWLEDGEMENTS
REFERENCES
Liver Regeneration: An update on the Role of Non-Parenchymal Cells
Abstract
introduction
Non Parenchymal Cells within the Liver Microenvironment
Non Parenchymal Cells in Liver Regeneration
Liver Sinusoidal Endothelial Cells
Liver Macrophages
Hepatic Stellate Cells
NPCs Role in the Development of Bioartificial Liver Systems
Concluding Remarks
CONFLICT OF INTEREST
ACKNOWLEDGEMENTS
references
Cardiogenesis and Repair: Insights from Development and Clinical Trials
Abstract
INTRODUCTION
MODEL ORGANISMS IN THE STUDY OF CARDIOGENESIS
Introduction to Cardiac Development
Fruit Fly Cardiac Development
Zebrafish Cardiac Development
Toad Cardiac Development
Chicken Cardiac Development
Mouse Cardiac Development
Core Developmental Pathways
INSIGHTS FROM STEM CELL BIOLOGY
PRE-CLINICAL APPLICATIONS
Hematopoietic Stem and Progenitor Cells
Mesenchymal Stem Cells
Skeletal Myoblasts
Cardiac and Endothelial Stem and Progenitor Cells
Embryonic and Induced Pluripotent Stem Cells
Exosomes and Secretory Factors
CLINICAL APPLICATIONS
CONCLUSION
CONFLICT OF INTEREST
ACKNOWLEDGEMENTS
REFERENCES
The Role of Ca2+ Signalling in the Differentiation of Embryonic Stem Cells (ESCs) into Cardiomyocytes
Abstract
Introduction
Methods TO INDUCE in vitro cardiomyocyte differentiation
THE ROLE OF Ca2+ SIGNALLING IN THE DIFFERENTIATION OF ESCs INTO CARDIOMYOCYTES
Inositol 1,4,5-Trisphosphate Receptors (IP3Rs)
L-Type Ca2+ Channels (LTCCs) and Ryanodine Receptors (RyRs)
Cluster of Differentiation 38 (CD38) and Cyclic Adenosine Diphosphate Ribose (cADPR)
Store-Operated Ca2+ Entry (SOCE)
Transient Receptor Potential (TRP) Channels: TRPV1 and TRPC3
Ca2+ Clearance Mechanisms (SERCA and NCX)
Calreticulin
Conclusion and Future Perspectives
CONFLICT OF INTEREST
ACKNOWLEDGEMENTS
REFERENCES
Regenerative Cell-Based Therapies to Combat Neurodegenerative Disorders
Abstract
INTRODUCTION
Neural Stem/Precursor Cells (NSPCs)
Embryonic Stem Cells (ESCs)
Induced Pluripotent Stem Cells (iPSCs)
Induced Neural Progenitor Cells (iNPCs)
Limitations of Cell-based Therapies
CONCLUSION
CONFLICT OF INTEREST
ACKNOWLEDGEMENTS
REFERENCES
Stem Cell Transplantation: Is this the Future Solution for Parkinson's Disease?
Abstract
INTRODUCTION
Parkinson's Disease
Parkinson's Disease Treatment
Understanding the Graft in Basal Ganglia Circuitry
Some Controversial Facts about Basal Ganglia Models
Basal Ganglia Grafts
The Graft in PD Patients: The Past and the Current
Adrenal Medulla
Ventral Mesencephalon Tissue
Stem Cells
Other Graft Sources
Limitations
The Neurogenic Niche: Permissive or Not
The Central Nervous System Grafts: Are We Still Facing the Same Problems?
CONCLUSION
CONFLICT OF INTEREST
ACKNOWLEDGEMENTS
ABBREVIATIONS
Frontiers in Stem Cell and Regenerative
Medicine Research
(Volume 6)
Edited By
Atta-ur-Rahman, FRS
Honorary Life Fellow,Kings College,University of Cambridge
Cambridge,UK
&
Shazia Anjum
Department of Chemistry,Cholistan Institute of Desert Studies
The Islamia University of Bahawalpur,Pakistan
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PREFACE
Research in the rapidly emerging field of stem cells is playing a pivotal role in regenerative medicine. The first 3 chapters of this volume deal with mesenchymal stem cell research. In chapter 1, Wei Seong Toh presents the emerging role of mesenchymal stem cell (MSC) secretome as a new paradigm in treating cartilage regeneration. The study of MSC secretome allows a better mechanistic understanding of the role of MSCs in tissue repair and disease treatment.
Mauricio et al. describe a promising strategy for the optimization of hybrid systems through the association of biomaterials to dental pulp stem cells, in chapter 2. Dental pulp stem cells can be easily isolated from deciduous and definitive teeth. In chapter 3, Kan et al. describe the recent progress and the opportunities as well as challenges in MSC research.
Arteta et al., in chapter 4, discuss current studies that underline the importance of liver progenitor cells (LPCs) for constructing bioartificial livers and as the source of cells for transplantation.
In the next chapter, Ward et al. present new exciting developments in cardiogenesis from bench-to-bedside. They review the heart development in different organisms, supplemented with insights from stem cell biology and clinical studies, which will throw light on the development of effective stem cell treatments for myocardial infarction and other cardiac diseases. Yue et al., in their chapter 6, present an overview of different Ca2+ signalling events in the differentiation of embryonic stem cells into cardiomyocytes.
The last two chapters deal with neurodegenerative diseases. Zareen Amtul, in Chapter 7, highlights the regenerative cell-based therapies that can be used to combat neurodegenerative disorders. In the last chapter, García-Montes and Drucker-Colín discuss in detail about the central role of stem cell transplantation to cure Parkinson´s Disease. They discuss the current challenges in optimizing stem cell therapy for the treatment of Parkinson’s disease.
We hope that the readers will enjoy the comprehensive reviews on new developments in stem cell and regenerative medicine research. We wish to thank the editorial staff of Bentham Science Publishers, particularly Dr. Faryal Sami, Mr. Shehzad Naqvi and Mr. Mahmood Alam for their constant help and support.
Prof. Atta-ur-Rahman, FRS
Honorary Life Fellow
Kings College
University of Cambridge
Cambridge
UK
&Dr. Shazia Anjum
Department of Chemistry
Cholistan Institute of Desert Studies
The Islamia University of Bahawalpur
List of Contributors
Aitor BenedictoDepartment Cell Biology and Histology, School of Medicine and Enfermery, University of Basque Country, E-48940 Leioa, SpainAlister C. WardSchool of Medicine, Deakin University, Geelong, Australia
Centre for Molecular and Medical Research, Deakin University, Geelong, AustraliaAna Colette MaurícioCentro de Estudos de Ciência Animal (CECA), Instituto de Ciências, Tecnologias e Agroambiente (ICETA) da Universidade do Porto, Praça Gomes Teixeira, Apartado 55142, 4051-401, Porto, Portugal
Departamento de Clínicas Veterinárias, Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), Universidade do Porto (UP), Rua de Jorge Viterbo Ferreira, nº 228, 4050-313 Porto, PortugalAna Rita CaseiroCentro de Estudos de Ciência Animal (CECA), Instituto de Ciências, Tecnologias e Agroambiente (ICETA) da Universidade do Porto, Praça Gomes Teixeira, Apartado 55142, 4051-401, Porto, Portugal
Departamento de Clínicas Veterinárias, Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), Universidade do Porto (UP), Rua de Jorge Viterbo Ferreira, nº 228, 4050-313 Porto, Portugal
CEMUC, Departamento de Engenharia Metalúrgica e Materiais, Faculdade de Engenharia, Universidade do Porto, Rua Dr. Roberto Frias s/n, 4200-465 Porto, PortugalAndrew L. MillerDivision of Life Science and State Key Laboratory of Molecular Neuroscience, HKUST, Clear Water Bay, Hong Kong, ChinaAntonia AlvarezDepartment Cell Biology and Histology, School of Medicine and Enfermery, University of Basque Country, E-48940 Leioa, SpainBaixia HaoDivision of Life Science and State Key Laboratory of Molecular Neuroscience, HKUST, Clear Water Bay, Hong Kong, ChinaBeatriz ArtetaDepartment Cell Biology and Histology, School of Medicine and Enfermery, University of Basque Country, E-48940 Leioa, SpainCarla MendonçaCentro de Estudos de Ciência Animal (CECA), Instituto de Ciências, Tecnologias e Agroambiente (ICETA) da Universidade do Porto, Praça Gomes Teixeira, Apartado 55142, 4051-401, Porto, Portugal
Departamento de Clínicas Veterinárias, Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), Universidade do Porto (UP), Rua de Jorge Viterbo Ferreira, nº 228, 4050-313 Porto, PortugalChen KanDepartment of Pathophysiology, School of Basic Medical Sciences, Anhui Medical University, Hefei, Anhui, ChinaDaniel McCullochSchool of Medicine, Deakin University, Geelong, Australia
Centre for Molecular and Medical Research, Deakin University, Geelong, AustraliaEnrique HilarioDepartment Cell Biology and Histology, School of Medicine and Enfermery, University of Basque Country, E-48940 Leioa, SpainHaimei LuDepartment of Pathophysiology, School of Basic Medical Sciences, Anhui Medical University, Hefei, Anhui, ChinaInês Leal ReisCentro de Estudos de Ciência Animal (CECA), Instituto de Ciências, Tecnologias e Agroambiente (ICETA) da Universidade do Porto, Praça Gomes Teixeira, Apartado 55142, 4051-401, Porto, Portugal
Departamento de Clínicas Veterinárias, Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), Universidade do Porto (UP), Rua de Jorge Viterbo Ferreira, nº 228, 4050-313 Porto, PortugalJianbo YueDepartment of Biomedical Sciences, City University of Hong Kong, Hong Kong, ChinaJoana MarquezDepartment Cell Biology and Histology, School of Medicine and Enfermery, University of Basque Country, E-48940 Leioa, SpainJosé Domingos SantosCEMUC, Departamento de Engenharia Metalúrgica e Materiais, Faculdade de Engenharia, Universidade do Porto, Rua Dr. Roberto Frias s/n, 4200-465 Porto, PortugalJosé Rubén García- MontesDepartamento de Neurobiología funcional y de sistemas, Instituto Cajal. Madrid, España,José Miguel CamposCentro de Estudos de Ciência Animal (CECA), Instituto de Ciências, Tecnologias e Agroambiente (ICETA) da Universidade do Porto, Praça Gomes Teixeira, Apartado 55142, 4051-401, Porto, Portugal
Departamento de Clínicas Veterinárias, Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), Universidade do Porto (UP), Rua de Jorge Viterbo Ferreira, nº 228, 4050-313 Porto, Portugal
Escola Universitária Vasco da Gama (EUVG), Hospital Veterinário Universitário de Coimbra (HVUC), Av. José R. Sousa Fernandes, Campus Universitário – Bloco B, Lordemão, 3020-210 Coimbra, PortugalLijun ChenDepartment of Pathophysiology, School of Basic Medical Sciences, Anhui Medical University, Hefei, Anhui, ChinaLixin KanDepartment of Pathophysiology, School of Basic Medical Sciences, Anhui Medical University, Hefei, Anhui, China
Department of Medical Laboratory Science, Bengbu Medical College, Bengbu, Street no., Bengbu, China
Department of Neurology, Northwestern University, Chicago, USALuís Miguel AtaydeCentro de Estudos de Ciência Animal (CECA), Instituto de Ciências, Tecnologias e Agroambiente (ICETA) da Universidade do Porto, Praça Gomes Teixeira, Apartado 55142, 4051-401, Porto, Portugal
Departamento de Clínicas Veterinárias, Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), Universidade do Porto (UP), Rua de Jorge Viterbo Ferreira, nº 228, 4050-313 Porto, PortugalRené Drucker- ColínDepartamento de Neuropatología Molecular, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Ciudad de México, MéxicoRui AlvitesCentro de Estudos de Ciência Animal (CECA), Instituto de Ciências, Tecnologias e Agroambiente (ICETA) da Universidade do Porto, Praça Gomes Teixeira, Apartado 55142, 4051-401, Porto, Portugal
Departamento de Clínicas Veterinárias, Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), Universidade do Porto (UP), Rua de Jorge Viterbo Ferreira, nº 228, 4050-313 Porto, PortugalSarah E. WebbDivision of Life Science and State Key Laboratory of Molecular Neuroscience, HKUST, Clear Water Bay, Hong Kong, ChinaSheena RajuRegenerative Medicine, Reliance Life Sciences Pvt Ltd, Mumbai, India
School of Medicine, Deakin University, Geelong, AustraliaSílvia Santos PedrosaCentro de Estudos de Ciência Animal (CECA), Instituto de Ciências, Tecnologias e Agroambiente (ICETA) da Universidade do Porto, Praça Gomes Teixeira, Apartado 55142, 4051-401, Porto, Portugal
Departamento de Clínicas Veterinárias, Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), Universidade do Porto (UP), Rua de Jorge Viterbo Ferreira, nº 228, 4050-313 Porto, PortugalWei Seong TohFaculty of Dentistry, National University of Singapore, Singapore
Tissue Engineering Program, Life Sciences Institute, National University of Singapore, SingaporeWenjie WeiDepartment of Biomedical Sciences, City University of Hong Kong, Hong Kong, ChinaYangyang HuDepartment of Pathophysiology, School of Basic Medical Sciences, Anhui Medical University, Hefei, Anhui, ChinaZareen AmtulDr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan
The Emerging Role of Mesenchymal Stem Cell Secretome in Cartilage Regeneration
Wei Seong Toh*
Faculty of Dentistry, National University of Singapore, Singapore
Tissue Engineering Program, Life Sciences Institute, National University of Singapore, Singapore
Abstract
Articular cartilage has a limited capacity to repair following injury. As a result, cartilage injuries often progress to serious joint disorders such as osteoarthritis. Mesenchymal stem cells (MSCs) are currently being evaluated in clinical trials as the therapeutic cell source for treatment of cartilage lesions and osteoarthritis. In addition to their differentiation potential, it is widely accepted that the beneficial actions of MSCs can also be mediated by their secretome. Of note, it has been demonstrated that MSCs are able to secrete a broad range of trophic factors and matrix molecules in their secretome to modulate the injured tissue environment and direct regenerative processes including cell migration, proliferation and differentiation to mediate overall tissue regeneration. The study of MSC secretome not only allows a better mechanistic understanding of the role of MSCs in tissue repair and disease treatment, but also enables the potential development of the next-generation, ready-to-use, highly-amenable and ‘cell-free’ therapeutics for clinical application. In this chapter, we present the latest understanding of MSC secretome and its components as a new paradigm for the treatment of cartilage lesions and osteoarthritis.
Keywords: Cartilage, Exosomes, Extracellular vesicles, Immunomodulation, Mesenchymal stem cells, Osteoarthritis, Secretome, Tissue regeneration.
*Corresponding author Wei Seong Toh: Faculty of Dentistry, National University of Singapore, 11 Lower Kent Ridge Road, Singapore 119083; Tel: +65 6779 5555 ext. 1619; Fax: +65 6778 5742; Email:
[email protected]Introduction
Articular cartilage is a unique hypocellular, avascularized and aneural load-bearing tissue, supported by the underlying subchondral bone [1]. Due to the lack of vascularization, articular cartilage has a limited capacity for regeneration upon injury. Articular cartilage injuries have a high incidence and therefore a high socio-economic and healthcare impact that cannot be underestimated. In knee joint alone, ~60% of patients who underwent arthroscopy displayed cartilage lesions [2]. When left untreated, these lesions can lead to osteoarthritis (OA), an
inflammatory and degenerative joint disease characterized by the degradation of the articular cartilage, subchondral bone, meniscus, ligaments, and the formation of painful osteophytes. OA is the most common form of arthritis affecting numerous joints including the knee joint, hip joint, and the temporomandibular joint (TMJ), and is the leading cause of disability worldwide [3, 4].
Current treatment options for articular cartilage injuries include arthroscopic lavage and debridement, microfracture, osteochondral grafting, and autologous chondrocyte implantation (ACI) [2]. While there are tissue repair with symptomatic relief, most cartilage repair techniques lead to fibrocartilaginous tissue repair that lacks the structural organization and matrix composition of the native articular cartilage.
Stem cells represent a promising cell source for cartilage repair [5, 6]. Currently, stem cells are classified into embryonic or ‘pluripotent’ stem cells, and non-embryonic ‘somatic’, ‘adult’ or ‘tissue’ stem / progenitor cells [6]. Embryonic stem cells (ESCs) are pluripotent stem cells derived from the inner cell mass of a blastocyst, an early-stage preimplantation embryo, and are defined by two distinct properties: pluripotency and unlimited self-renewal. They are able to differentiate into cell derivatives of the three primary germ layers including ectoderm, endoderm and mesoderm [7]. With advances in stem cell biology, personalized pluripotent stem cells, also known as induced pluripotent stem cells (iPSCs) can be derived from somatic cells through reprogramming using defined gene and protein factors [8]. Of note, several groups have reported differentiation of human ESCs and iPSCs to chondrocytes [9-12], and demonstrated the functional efficacy of these cells for cartilage repair in animal studies [13-16].
Adult stem / progenitor cells are undifferentiated multipotent cells present in various adult tissues as they contribute to the physiological cell turnover as well as to tissue repair. Among these adult stem cells, mesenchymal stromal/stem cells (MSCs) are the most extensively studied and used cell type in clinical trials and have been heralded as the next major development for treatment of tissue injuries and diseases (http://www.clinicaltrials.gov). Of note, MSCs are currently being evaluated in clinical trials for treatment of cartilage injuries and osteoarthritis (OA) [17, 18]. While it is clear that MSCs are able to differentiate in vitro into a variety of cell types including chondrocytes, osteoblasts and adipocytes, MSCs are increasingly being investigated and harnessed for their trophic functional abilities [6, 19]. This book chapter aims to discuss the role of MSCs in cartilage regeneration and to present the latest development of MSC secretome and its components as a new paradigm for treatment of cartilage injuries and osteoarthritis.
Mesenchymal Stem Cells
Mesenchymal stromal/stem cells (MSCs) are multipotent adult stem cells capable of self-renewal and multi-lineage differentiation into osteoblasts, chondrocytes and adipocytes [20]. They are easily isolated from a wide variety of tissues including bone marrow, muscle, adipose tissue, blood, and synovium [21-23]. MSCs are isolated as a heterogeneous cell population and characterized by their ability to adhere to plastic, form colonies in colony-forming unit-fibroblast (CFU-F) assay, and differentiate into osteoblasts, chondrocytes and adipocytes [20, 24]. According to the minimal criteria defined by the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy (ISCT), MSCs are positive for cell surface markers CD73, CD90 and CD105, and negative for CD34, CD45, CD11b, CD14, CD19, CD79a and human leukocyte antigen (HLA)-DR surface molecules [25].
Mesenchymal Stem Cell-based Therapies for Cartilage Repair
Several MSC-based strategies for cartilage repair have been reported in animal [26] and clinical studies [17]. MSCs can be used in direct cell transplantation, and/or in combination with growth factors and scaffolds [26]. Direct transplantation of MSCs occur commonly in the form of fresh marrow or monolayer expanded and selected cells [27]. The use of fresh marrow or freshly isolated mononuclear cells is gaining interest due to their rapid availability without the need for cell expansion [28]. Furthermore, fresh marrow comprises not only MSCs but also accessory cells and growth factors.
However, in all above described cell-based strategies for cartilage repair, the culture conditions remains an issue, and there is currently poor standardization for the culture conditions and the number of cells needed for transplantation with respect to various sizes and types of cartilage lesions [29]. As with all cell-based therapies, there exist significant logistical and operational challenges associated with proper handling and cell storage to maintain the vitality and viability of the cells for transplantation [30]. With advances in proteomics, it is becoming clear that MSCs not only exhibits ability to differentiate into multiple lineages, but also secrete a broad spectrum of trophic factors in the secretome that are mediating various aspects and processes of tissue repair and regeneration [19] (Fig. 1). In the past decade, the investigation of MSC secretome has therefore gained much attention, with the interest to decipher the factor (s) mediating the biological activity of MSCs in tissue repair.
Fig. (1))
Therapeutic strategies utilizing cell-based and cell-free MSC therapies for cartilage repair and regeneration. In cell-based strategy, MSCs are directly transplanted for treatment of cartilage defects. Alternatively, secretome factors can be isolated from the MSCs and utilized as a cell-free therapy for treatment of cartilage defects.
MSC Secretome
Besides their differentiation potential, MSCs secrete a broad spectrum of trophic factors, including growth factors, cytokines and chemokines, which mediate the paracrine activity of these cells in tissue repair [19]. The paracrine effects of MSCs on chondrocytes were first discovered in co-culture studies that showed ability of MSC conditioned medium to enhance proliferation and matrix synthesis of cultured chondrocytes. In those studies, MSCs from different sources including bone marrow, adipose tissue and synovial membrane were observed to exert similar trophic effects, irrespective of the tissue origin and culture conditions [31-33]. Further analysis of MSC conditioned medium showed that several of the secreted factors are implicated in cell recruitment, survival, proliferation, differentiation, and immunomodulation during cartilage repair. Some of the trophic factors including growth factors, cytokines and ECM molecules are described below, based on their potential roles in cartilage regeneration, and summarized in Table 1.
Table 1Summary of key trophic factors and their functions in cartilage repair.Cellular FunctionSecreted FactorsReferencesCell recruitment / ChemoattractionSDF-1α,
IL-8
MIP-3α[40, 56]
[35, 40]
[40]Cell survival / anti-apoptoticTGF-β
HGF
IGF-1
FGF-2
Collagen VI[45]
[45]
[46]
[45]
[62]Cell proliferationFGF-1
FGF-2
SIP
Collagen VI[54]
[50]
[55]
[56, 61]Cell differentiation / matrix synthesis / anabolic functionsTGF-β
FGF-2
IGF-1
HGF
TSP-2
Laminins[47, 66]
[50, 53]
[47, 66]
[67, 68]
[56, 69]
[56, 57]Immunomodulation / anti-inflammationHGF
IL-10
TGF-β
PGE-2[72, 73]
[74, 77]
[72, 74]
[76]
Cell Recruitment
During articular cartilage injury, there is upregulated expression of pro-inflammatory cytokines including interleukin (IL)-1β and tumor necrosis factor (TNF)-α [34], that in turn induce the release of chemokines to recruit cells into the defect site. Apart from immune cells, MSCs respond to inflammation by secreting a wide variety of cytokines and chemokines [35]. Among these chemoattractant molecules secreted by MSCs, CXC chemokine ligand 12a (CXCL12a), also known as stromal cell-derived factor-1α (SDF-1α), has been shown to target endogenous MSCs, chondrogenic progenitors and chondrocytes to induce cartilage repair and regeneration [36, 37]. Indeed, expression of SDF-1α and its receptor CXCR4 are upregulated in many cartilaginous tissues including temporomandibular joint (TMJ) [38] and intervertebral disc (IVD) [39] during early stages of osteoarthritic and/or degenerative changes, supporting their role in recruitment of cells for tissue repair.
Besides SDF-1α, IL-8 and macrophage inflammatory protein (MIP)-3α are also secreted by MSCs and have been recently shown to recruit endogenous MSCs and immune cells (monocytes, macrophages and lymphocytes) to induce repair of osteochondral defects in beagles [40]. Other chemokines such as RANTES has been shown in cartilaginous tissues to be a potent chemoattractant of MSCs [38]. However, many of these chemokines also attract the migration of other cell types including endothelial [41] and neural progenitor cells [42] that could result in risk of angiogenesis and innervation, and lead to an undesirable response [43]. Notably, SDF-1α is recently shown to promote hypertrophic maturation of primary chondrocytes and endochondral ossification, and thus implicated in OA development [44].
Cell Survival
During cartilage injury, there is deleterious inflammation that results in cell death and matrix degradation, compromising the overall tissue homeostasis. To restore tissue homeostasis, there is a need to enhance cell survival and proliferation to increase the live cells to replace the dead cells. MSCs secrete several anti-apoptotic factors that could mediate enhanced cell survival following tissue injury. These factors include transforming growth factor (TGF)-β, hepatocyte growth factor (HGF), insulin growth factor (IGF)-1 and fibroblast growth factor (FGF)-2 [45, 46]. It has been shown that TGF-β and IGF-1 are capable of attenuating IL-1β-induced caspase activation in human chondrocytes, and thus enhancing the cell survival [47].
Cell Proliferation
Previous studies have active cellular proliferation of mesenchymal cells at the defect site which is required to initiate a chondrogenic reparative response that is followed by matrix synthesis and tissue formation [48, 49]. It has been further reported that MSCs express FGF-2, and autocrine FGF-2/FGF receptor 1 signalling is required for the proliferation of both bone marrow and adipose tissue-derived MSCs [50]. This explains for the common use of FGF-2 during MSC expansion to maintain the MSC proliferation ability and multi-lineage differentiation potency [51-53]. More recently, FGF-1 was identified as a potent factor secreted by MSCs that is capable of stimulating proliferation of OA chondrocytes in co-culture [54].
Apart from growth factors, bioactive lipids such as sphingosine 1-phosphate (SIP) are secreted by MSCs and have been shown to stimulate proliferation and counteract IL-1β-induced nitric oxide production and glycosaminoglycan (GAG) depletion from chondrocytes and cartilage explants [55]. MSCs also secrete ECM molecules include collagen I, collagen VI and laminins that exert profound effects on chondrocyte proliferation and differentiation [56]. Many of these matrix molecules are present in the cartilage ECM with distinct functions during cartilage development, health and disease [57-60]. Among these ECM molecules, collagen VI has been recently shown to stimulate proliferation [61], counteract IL-1β-induced expression of matrix metalloproteinases (MMPs), and protect against monoiodoacetate-induced apoptosis of chondrocytes [62].
Cell Differentiation and Matrix Synthesis
MSCs secrete several factors including TGF-β, FGF-2, IGF-1, HGF and thrombospondin-2 (TSP-2) that are capable of enhancing chondrogenic differentiation and expression of cartilage matrix molecules [19]. Among these factors, TGF-βs are commonly used in chondrogenic differentiation of MSCs and chondrocytes in both pellet and cell-seeded scaffold cultures [63-65]. IGF-1 is a well-established anabolic factor to promote biosynthetic activity of chondrocytes and has been shown to act in synergy with TGF-β1 to promote matrix synthesis of chondrocytes [66], and counteracts IL-1β-induced expression of cyclooxygenase (COX)-2 and MMP-13 [47]. Similarly, HGF has been shown to promote matrix synthesis in chondrocytes [67] and reduced hypertrophy and dedifferentiation in OA chondrocytes [68]. Recently, Jeong and colleagues showed that human umbilical cord blood-derived MSCs promoted differentiation and matrix synthesis of chondroprogenitor cells by paracrine action of secretome factors, of which TSP-2 was identified as the factor mediating the chondrogenic effects [69].
Among the ECM molecules secreted by MSCs, collagen VI and laminins are implicated in MSC chondrogenesis [70] and chondrocyte matrix biosynthesis [57, 61]. Collagen VI and laminin were found to maintain cartilage-specific collagen II expression by cultured chondrocytes [57], and knockdown of collagen VI has been found to negatively impact the biomechanical integrity during chondrogenesis [71].
Immunomodulation
Upon cartilage injury, there is rapid upregulation in expression of pro-inflammatory cytokines including IL-1β, IL-6 and IL-8, and MMPs that mediate inflammatory responses, matrix degradation, and contribute to the onset and development of OA. MSCs have the capacity to produce immunomodulatory factors to modulate the inflammatory responses.
MSCs secrete several immunomodulatory factors including HGF, IL-10, TGF-β and prostaglandin E2 (PGE-2) [19, 72-74].
In a study by van Buul and team, it was found that conditioned medium prepared by stimulation of human bone marrow MSCs with inflammatory cytokines (TNF-α and IFN-γ) contained immunomodulatory factors that downregulated IL-1β, MMP-1 and MMP-13, but upregulated IL-1 receptor antagonist (IL-1RA) expression to reduce matrix degradation and NO production by synovium and cartilage explants [75]. Manferdini and colleagues subsequently showed that adipose tissue MSCs are able to respond to inflammatory factors produced by OA chondrocytes and synoviocytes to produce PGE-2 that mediate the anti-inflammatory effects of MSCs through COX-2/PGE-2 pathway [76].
Macrophage polarization (M1 versus M2) and the associated cytokine production have been found to impact chondrogenesis and cartilage repair [77, 78]. Notably, M1 polarized macrophages present in OA synovium tissues have been found to suppress in vitro chondrogenic differentiation of MSCs, at least in part by effects of IL-6 [78]. MSCs have the capability to influence chondrogenesis by effects on macrophage polarization and associated cytokine production. It was found that bone marrow MSCs induced macrophage polarization to regenerative M2 phenotype to support survival of the cartilage graft by production of anti-inflammatory IL-10 to suppress adverse inflammation [77].
MSC Extracellular Vesicles
As described above, much of the initial efforts to identify the active therapeutic factor in MSC secretome focused on growth factors, cytokines and chemokines [19]. However, in recent years, the underlying biological activity of MSCs is increasingly attributed to exosomes that are released by the cells into the surrounding [79, 80]. These exosomes serve as intercellular communication vehicle and function to transfer lipids, nucleic acids (mRNAs and microRNAs) and proteins to elicit biological responses from recipient cells.
Exosomes are one class of EVs that are produced by many cell types, including MSCs in our body system. Other classes of EVs include ectosomes, membrane particles, exosome-like vesicles or apoptotic bodies [81]. Exosomes are 40-100nm in size, endosomal in origin, and possess exosome-associated marker proteins such as ALIX, TSG101, and tetraspanins (CD9, CD63 and CD81). Additionally, exosomes bear numerous membrane proteins and lipids that have binding affinity to ligands on cell membrane, including growth factor receptors, integrins and tetraspanins.
In a seminal study, Lai and colleagues identified MSC exosomes as the principal mediator underlying the therapeutic ability of MSCs to reduce the infarct size in the mouse model of myocardial ischemia/reperfusion (I/R) injury with comparable efficacy as that of unfractionated conditioned medium [79]. Subsequently, more studies showed that exosomes are the principal component present in the MSC secretome that mediates the underlying biological effects of MSCs in treatment of various diseases [82-85].
A detailed analysis revealed that MSC exosomes carry a complex cargo of nucleic acids, proteins and lipids, with 857 unique gene products [86] (www.exocarta.org) and >150 microRNAs [87]. Although the function of many of these proteins, mRNAs, miRNAs, lipids remains unclear, this functional complexity suggest the potential of MSC exosomes to elicit diverse cellular responses and to interact with numerous cell types [80]. To date, MSC exosomes have been reported to protect against myocardial I/R injury [79], attenuate limb ischemia [85], promote wound healing [83], ameliorate graft-versus-host-disease [88], reduce renal injury [84], and more recently improve bone and cartilage regeneration [89, 90].
Cell-free Therapies for Cartilage Repair
The identification of factors from the MSC secretome opens new avenues and opportunities for development of cell-free strategies in place of cell-based MSC therapies for cartilage injuries and osteoarthritis. Cell-free strategies could be administered by ways of direct intra-articular administration of the therapeutic factor [48], or loading into scaffolds [91] for sustained release of the factor at the defect site to facilitate cartilage repair over time.
There are several advantages of cell-free strategies over cell transplantation for cartilage repair. Cell-free strategies orchestrate endogenous tissue repair, and thus overcome some of the key logistical, operational, commercialization and regulatory issues associated with cell transplantation, including proper handling and cell storage, excessive cost, and risks of immune rejection and pathogen transmission [92]. Cell-free strategies utilizing growth factors, cytokines, or even exosomes, can be packaged as off-the-shelf products and administered in single-step procedure to patients in possibly an outpatient setting, thus providing better accessibility and convenience. Of note, there are already prior instances of regulatory approval for growth factor and cytokine delivery. Here, we describe the latest developments in cell-free therapies for cartilage repair.
Growth Factors, Cytokines and Chemokines
As described earlier, several of the factors secreted by MSCs are involved in various aspects and processes (cell recruitment, survival, proliferation, differentiation and matrix synthesis, and immunomodulation) during the course of cartilage repair and regeneration. These factors offer opportunities for development of a defined, standardized and cell-free therapeutic strategy for cartilage repair [27].
Several groups have investigated the use of TGF-β for cartilage repair in animal studies [91, 93]. Lee and colleagues showed that the rabbit humeral heads were resurfaced using three-dimensional (3-D) printed hydroxyapatite/poly-ε- caprolactone scaffold impregnated with TGF-β3 containing collagen I gel. Notably, the articular surface was repaired within 4 months through mechanisms of cell recruitment and differentiation [91]. Similarly, FGF-2 has been reported to be a potent factor to induce migration and proliferation of mesenchymal cells at the injured site to facilitate subsequent cartilage repair [48, 49]. Other studies have employed SDF-1α to attract migration of endogenous MSCs to the defect site to facilitate cartilage repair [36, 94]. Sukegawa and co-workers demonstrated the use of ultra-purified alginate (UPAL) gel loaded with SDF-1α to promote repair of full-thickness osteochondral defects in rabbits. In that study, the local administration of SDF-1α in UPAL gel promoted hyaline-like cartilage regeneration over a period of 16 weeks. Conversely, administration of AMD3100, an antagonist of CXCR4, greatly impaired the repair of osteochondral defects. These findings highlight the important role of SDF-1α/CXCR4 pathway in cartilage repair, especially in the initial phase of the repair process to attract host cells to the injured site [94].
However, in these approaches, there are still questions regarding the long-term phenotypic stability of the repaired tissue. For instance, it has been reported that TGF-β induced hypertrophy maturation of bone marrow MSCs with upregulated expression of collagen X and MMP-13 [95]. Similarly, SDF-1α/CXCR4 signalling is implicated in chondrocyte hypertrophy and OA development [44, 96-98]. In view of these concerns, there is still a need to better understand the growth factors and signalling pathways involved in chondrogenesis and OA development. Of note, a combinatorial approach utilizing multiple factors may be needed to induce a stable chondrocyte phenotype of the regenerated tissue [99-101].
Exosomes
Zhang and co-workers first reported that exosomes isolated from human MSCs could promote regeneration of full-thickness cartilage defects in an immuno-competent rat model [89]. It was found that MSC exosomes promoted early cellular proliferation to mediate accelerated neotissue formation and enhanced matrix synthesis. Concurrently, there was macrophage polarization to regenerative M2 macrophage phenotype induced by exosome treatment [102]. By the end of 12 weeks, exosome-treated defects displayed complete restoration of cartilage and subchondral bone with characteristic features including hyaline cartilage, good surface regularity and integration with adjacent cartilage. Conversely, saline-treated defects displayed only fibrous tissue repair [89]. The underlying mechanism of exosomes in mediating cartilage repair and regeneration remains to be determined. However, one would postulate that MSC exosomes being secreted by MSCs mediate cartilage regeneration through MSC associated mechanisms of cell recruitment, survival, proliferation, differentiation and matrix synthesis, and immunomodulation, as already mentioned [103].
In the above mentioned studies [89, 102, 104], exosomes were stored at -20oC until use, which highlights the potential of human MSC exosomes as an off-the- shelf and cell-free alternative to cell-based MSC therapy for cartilage repair. Apart from being ready-to-use and cell-free, MSC exosomes have several unique advantages over the growth factor approach for cartilage repair. The use of MSC exosomes overcome issues of high cost associated with the use of growth factors, and risk of adverse tissue reactions from high dose of growth factors [105, 106].
Similar to the growth factor/cytokine approach for cartilage repair, exosomes could be loaded into a liposomal delivery system and/or scaffold for sustained and localized release at the defect site to facilitate cartilage repair over time [107-109]. Additionally, the surface antigen profile and cargo content of MSC exosomes could be modified to enhance the target specificity, uptake efficiency and therapeutic efficacy required for cartilage regeneration [110].
Conclusion
Undoubtedly, there is a paradigm shift from cell-based MSC therapies to cell-free strategies for cartilage repair. Notably, the MSC secretome provides a discovery platform for identification and isolation of secretome factors that mediate the biological functions of MSCs in cartilage repair. These secretome factors that range from growth factors, cytokines and chemokines to extracellular matrix molecules and vesicles including exosomes hold significant potential for cartilage tissue engineering and regeneration. However, their therapeutic potential will only be realized through further research to better understand the role(s) of these secretome factors in chondrogenesis and cartilage regeneration, and their associated mechanisms. With advances in material science [111-113], tissue engineering strategies that combined the use of scaffolds and/or delivery systems for sustained and localized release of select therapeutic factor(s) hold strong promise to provide an off-the-shelf and cell-free solution for cartilage repair.
CONFLICT OF INTEREST
The author confirms that author has no conflict of interest to declare for this publication.
ACKNOWLEDGEMENTS
This work was supported by grants from the National University of Singapore (R221000090112) and National Medical Research Council Singapore (R221000080511).
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