Bone regeneration and stem cells

This invited review covers research areas of central importance for orthopaedic and maxillofacial bone tissue repair, including normal fracture healing and healing problems, biomaterial scaffolds for tissue engineering, mesenchymal and foetal stem cells, effects of sex steroids on mesenchymal stem cells, use of platelet-rich plasma for tissue repair, osteogenesis and its molecular markers. A variety of cells in addition to stem cells, as well as advances in materials science to meet specific requirements for bone and soft tissue regeneration by addition of bioactive molecules, are discussed.     

Stem cell-derived exosomes in bone healing: focusing on their role in angiogenesis

Fractures in bone, a tissue critical in protecting other organs, affect patients’ quality of life and have a heavy economic burden on societies. Based on regenerative medicine and bone tissue engineering approaches, stem cells have become a promising and attractive strategy for repairing bone fractures via differentiation into bone-forming cells and production of favorable mediators. Recent evidence suggests that stem cell-derived exosomes could mediate the therapeutic effects of their counterpart cells and provide a cell-free therapeutic strategy in bone repair. Since bone is a highly vascularized tissue, coupling angiogenesis and osteogenesis is critical in bone fracture healing; thus, developing therapeutic strategies to promote angiogenesis will facilitate bone regeneration and healing. To this end, stem cell-derived exosomes with angiogenic potency have been developed to improve fracture healing. This review summarizes the effects of stem cell-derived exosomes on the repair of bone tissue, focusing on the angiogenesis process.     

Dysfunctional stem and progenitor cells impair fracture healing with age

Successful fracture healing requires the simultaneous regeneration of both the bone and vasculature; mesenchymal stem cells(MSCs) are directed to replace the bone tissue, while endothelial progenitor cells(EPCs) form the new vasculature that supplies blood to the fracture site. In the elderly, the healing process is slowed, partly due to decreased regenerative function of these stem and progenitor cells. MSCs from older individuals are impaired with regard to cell number, proliferative capacity, ability to migrate, and osteochondrogenic differentiation potential. The proliferation, migration and function of EPCs are also compromised with advanced age. Although the reasons for cellular dysfunction with age are complex and multidimensional, reduced expression of growth factors, accumulation of oxidative damage from reactive oxygen species,and altered signaling of the Sirtuin-1 pathway are contributing factors to aging at the cellular level of both MSCs and EPCs. Because of these geriatric-specific issues, effective treatment for fracture repair may require new therapeutic techniques to restore cellular function. Some suggested directions for potential treatments include cellular therapies, pharmacological agents, treatments targeting age-related molecular mechanisms, and physical therapeutics.Advanced age is the primary risk factor for a fracture, due to the low bone mass and inferior bone quality associated with aging; a better understanding of the dysfunctional behavior of the aging cell will provide a foundation for new treatments to decrease healing time and reduce the development of complications during the extended recovery from fracture healing in the elderly.     

New insights on the reparative cells in bone regeneration and repair

Bone possesses a remarkable repair capacity to regenerate completely without scar tissue formation. This unique characteristic, expressed during bone development, maintenance and injury (fracture) healing, is performed by the reparative cells including skeletal stem cells (SSCs) and their descendants. However, the identity and functional roles of SSCs remain controversial due to technological difficulties and the heterogeneity and plasticity of SSCs. Moreover, for many years, there has been a biased view that bone marrow is the main cell source for bone repair. Together, these limitations have greatly hampered our understanding of these important cell populations and their potential applications in the treatment of fractures and skeletal diseases. Here, we reanalyse and summarize current understanding of the reparative cells in bone regeneration and repair and outline recent progress in this area, with a particular emphasis on the temporal and spatial process of fracture healing, the sources of reparative cells, an updated definition of SSCs, and markers of skeletal stem/progenitor cells contributing to the repair of craniofacial and long bones, as well as the debate between SSCs and pericytes. Finally, we also discuss the existing problems, emerging novel technologies and future research directions in this field.     

Adipose tissue in bone regeneration - stem cell source and beyond

Adipose tissue (AT) is recognized as a complex organ involved in major home-ostatic body functions, such as food intake, energy balance, immunomodulation, development and growth, and functioning of the reproductive organs. The role of AT in tissue and organ homeostasis, repair and regeneration is increasingly recognized. Different AT compartments (white AT, brown AT and bone marrow AT) and their interrelation with bone metabolism will be presented. AT-derived stem cell populations - adipose-derived mesenchymal stem cells and pluripotent-like stem cells. Multilineage differentiating stress-enduring and dedifferentiated fat cells can be obtained in relatively high quantities compared to other sources. Their role in different strategies of bone and fracture healing tissue engineering and cell therapy will be described. The current use of AT- or AT-derived stem cell populations for fracture healing and bone regenerative strategies will be presented, as well as major challenges in furthering bone regenerative strategies to clinical settings.     

Mobilization of endogenous stem cell populations enhances fracture healing in a murine femoral fracture model

Background aimsDelivery of bone marrow–derived stem and progenitor cells to the site of injury is an effective strategy to enhance bone healing. An alternate approach is to mobilize endogenous, heterogeneous stem cells that will home to the site of injury. AMD3100 is an antagonist of the chemokine receptor 4 (CXCR4) that rapidly mobilizes stem cell populations into peripheral blood. Our hypothesis was that increasing circulating numbers of stem and progenitor cells using AMD3100 will improve bone fracture healing.MethodsA transverse femoral fracture was induced in C57BL/6 mice, after which they were subcutaneously injected for 3 d with AMD3100 or saline control. Mesenchymal stromal cells, hematopoietic stem and progenitor cells and endothelial progenitor cells in the peripheral blood and bone marrow were evaluated by means of flow cytometry, automated hematology analysis and cell culture 24 h after injection and/or fracture. Healing was assessed up to 84 d after fracture by histomorphometry and micro–computed tomography.ResultsAMD3100 injection resulted in higher numbers of circulating mesenchymal stromal cells, hematopoietic stem cells and endothelial progenitor cells. Micro-computed tomography data demonstrated that the fracture callus was significantly larger compared with the saline controls at day 21 and significantly smaller (remodeled) at day 84. AMD3100-treated mice have a significantly higher bone mineral density than do saline-treated counterparts at day 84.ConclusionsOur data demonstrate that early cell mobilization had significant positive effects on healing throughout the regenerative process. Rapid mobilization of endogenous stem cells could provide an effective alternative strategy to cell transplantation for enhancing tissue regeneration.     

Current evidence on potential of adipose derived stem cells to enhance bone regeneration and future projection

Injuries to the postnatal skeleton are naturally repaired through successive steps involving specific cell types in a process collectively termed “bone regeneration”. Although complex, bone regeneration occurs through a series of well-orchestrated stages wherein endogenous bone stem cells play a central role. In most situations, bone regeneration is successful; however, there are instances when it fails and creates non-healing injuries or fracture nonunion requiring surgical or therapeutic interventions. Transplantation of adult or mesenchymal stem cells (MSCs) defined by the International Society for Cell and Gene Therapy (ISCT) as CD105+CD90+CD73+CD45-CD34-CD14orCD11b-CD79αorCD19-HLA-DR- is being investigated as an attractive therapy for bone regeneration throughout the world. MSCs isolated from adipose tissue, adipose-derived stem cells (ADSCs), are gaining increasing attention since this is the most abundant source of adult stem cells and the isolation process for ADSCs is straightforward. Currently, there is not a single Food and Drug Administration (FDA) approved ADSCs product for bone regeneration. Although the safety of ADSCs is established from their usage in numerous clinical trials, the bone-forming potential of ADSCs and MSCs, in general, is highly controversial. Growing evidence suggests that the ISCT defined phenotype may not represent bona fide osteoprogenitors. Transplantation of both ADSCs and the CD105^- sub-population of ADSCs has been reported to induce bone regeneration. Most notably, cells expressing other markers such as CD146, AlphaV, CD200, PDPN, CD164, CXCR4, and PDGFRα have been shown to represent osteogenic sub-population within ADSCs. Amongst other strategies to improve the bone-forming ability of ADSCs, modulation of VEGF, TGF-β1 and BMP signaling pathways of ADSCs has shown promising results. The U.S. FDA reveals that 73% of Investigational New Drug applications for stem cell-based products rely on CD105 expression as the “positive” marker for adult stem cells. A concerted effort involving the scientific community, clinicians, industries, and regulatory bodies to redefine ADSCs using powerful selection markers and strategies to modulate signaling pathways of ADSCs will speed up the therapeutic use of ADSCs for bone regeneration.     

A mathematical framework to study the effects of growth factor influences on fracture healing

During fracture healing, multipotential stem cells differentiate into specialized cells responsible for producing the different tissues involved in the bone regeneration process. This cell differentiation has been shown to be regulated by locally expressed growth factors. The details of their regulatory mechanisms need to be understood. In this work, we present a two-dimensional mathematical model of the bone healing process for moderate fracture gap sizes and fracture stability. The inflammatory and tissue regeneration stages of healing are simulated by modeling mesenchymal cell migration; mesenchymal cell, chondrocyte and osteoblast proliferation and differentiation, and extracellular matrix synthesis and degradation over time. The effects of two generic growth factors on cell differentiation are based on the experimentally studied chondrogenic and osteogenic effects of bone morphogenetic proteins-2 and 4 and transforming growth factor-beta-1, respectively. The model successfully simulates the progression of healing and predicts that the rate of osteogenic growth factor production by osteoblasts and the duration of the initial release of growth factors upon injury are particularly important parameters for complete ossification and successful healing. This temporo-spatial model of fracture healing is the first model to consider the effects of growth factors. It will help us understand the regulatory mechanisms involved in bone regeneration and provides a mathematical framework with which to design experiments and understand pathological conditions.     

Informing tendon tissue engineering with embryonic development

Tendon is a strong connective tissue that transduces muscle-generated forces into skeletal motion. In fulfilling this role, tendons are subjected to repeated mechanical loading and high stress, which may result in injury. Tissue engineering with stem cells offers the potential to replace injured/damaged tissue with healthy, new living tissue. Critical to tendon tissue engineering is the induction and guidance of stem cells towards the tendon phenotype. Typical strategies have relied on adult tissue homeostatic and healing factors to influence stem cell differentiation, but have yet to achieve tissue regeneration. A novel paradigm is to use embryonic developmental factors as cues to promote tendon regeneration. Embryonic tendon progenitor cell differentiation in vivo is regulated by a combination of mechanical and chemical factors. We propose that these cues will guide stem cells to recapitulate critical aspects of tenogenesis and effectively direct the cells to differentiate and regenerate new tendon. Here, we review recent efforts to identify mechanical and chemical factors of embryonic tendon development to guide stem/progenitor cell differentiation toward new tendon formation, and discuss the role this work may have in the future of tendon tissue engineering.     

Why Adult Stem Cell Functionality Declines with Age? Studies from the Fruit Fly Drosophila Melanogaster Model Organism

Highly regenerative adult tissues are supported by rare populations of stem cells that continuously divide to self-renew and generate differentiated progeny. This process is tightly regulated by signals emanating from surrounding cells to fulfill the dynamic demands of the tissue. One of the hallmarks of aging is slow and aberrant tissue regeneration due to deteriorated function of stem and supporting cells. Several Drosophila regenerative tissues are unique in that they provide exact identification of stem and neighboring cells in whole-tissue anatomy. This allows for precise tracking of age-related changes as well as their targeted manipulation within the tissue. In this review we present the stem cell niche of Drosophila testis, ovary and intestine and describe the major changes and phenotypes that occur in the course of aging. Specifically we discuss changes in both intrinsic properties of stem cells and their microenvironment that contribute to the decline in tissue functionality. Understanding these mechanisms in adult Drosophila tissues will likely provide new paradigms in the field of aging.     

Perinatal stem cells: A promising cell resource for tissue engineering of craniofacial bone

In facing the mounting clinical challenge and suboptimal techniques of craniofacial bone defects resulting from various conditions, such as congenital malformations, osteomyelitis, trauma and tumor resection, the ongoing research of regenerative medicine using stem cells and concurrent advancement in biotechnology have shifted the focus from surgical reconstruction to a novel stem cell-based tissue engineering strategy for customized and functional craniofacial bone regeneration. Given the unique ontogenetical and cell biological properties of perinatal stem cells, emerging evidence has suggested these extraembryonic tissue-derived stem cells to be a promising cell source for extensive use in regenerative medicine and tissue engineering. In this review, we summarize the current achievements and obstacles in stem cell-based craniofacial bone regeneration and subsequently we address the characteristics of various types of perinatal stem cells and their novel application in tissue engineering of craniofacial bone. We propose the promising feasibility and scope of perinatal stem cell-based craniofacial bone tissue engineering for future clinical application.     

Biological Characteristics and Effect of Human Umbilical Cord Mesenchymal Stem Cells (hUC-MSCs) Grafting with Blood Plasma on Bone Regeneration in Rats

We evaluated the biological characteristics/effect of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) grafting with blood plasma on bone regeneration in rat tibia nonunion. SD rats (142) were randomly divided into four groups: fracture group (positive control); nonunion group (negative control); hUC-MSCs grafting with blood plasma group; and hUC-MSCs grafting with saline group. Rats were administered tetracycline (30 mg/kg) and calcein blue (5 mg/kg) 8 days before killing. The animals were killed under deep anesthesia at 4 and 8 weeks post fracture for radiological evaluation and histological/immunohistological studies. The hUC-MSCs grafting with blood plasma group was similar to fracture group: the fracture line blurred in 4 weeks and disappeared in 8 weeks postoperatively. Histological/immunohistological studies showed that hUC-MSCs were of low immunogenicity which merged in rat bone tissue, differentiated into osteogenic lineages, and completed the healing of nonunion. After stem cell transplantation, regardless of whether plasma or saline was used, new multi-center bone formation was observed; fracture site density was better in stem cell grafting with blood plasma group. We, therefore, concluded that the biological characteristics of hUC-MSCs-treated nonunion were different from the standard fracture healing process, and the proliferative and localization capacity of hUC-MSCs might benefit from the use of blood plasma.     

Mathematical Modeling in Wound Healing,Bone Regeneration and Tissue Engineering

The processes of wound healing and bone regeneration and problems in tissue engineering have been an active area for mathematical modeling in the last decade. Here we review a selection of recent models which aim at deriving strategies for improved healing. In wound healing, the models have particularly focused on the inflammatory response in order to improve the healing of chronic wound. For bone regeneration, the mathematical models have been applied to design optimal and new treatment strategies for normal and specific cases of impaired fracture healing. For the field of tissue engineering, we focus on mathematical models that analyze the interplay between cells and their biochemical cues within the scaffold to ensure optimal nutrient transport and maximal tissue production. Finally, we briefly comment on numerical issues arising from simulations of these mathematical models.     

Intestinal stem cell proliferation and epithelial homeostasis in the adult Drosophila midgut

Adult tissue homeostasis requires a tight balance between the removal of old or damaged cells and the production of new ones. Such processes are usually driven by dedicated stem cells that reside within specific tissue locations or niches.The intestinal epithelium has a remarkable regenerative capacity, which has made it a prime paradigm for the study of stem cell-driven tissue self-renewal. The discovery of the presence of stem cells in the adult midgut of the fruit fly Drosophila melanogaster has significantly impacted our understanding of the role of stem cells in intestinal homeostasis. Here we will review the current knowledge of the main mechanisms involved in the regulation of tissue homeostasis in the adult Drosophila midgut, with a focus on the role of stem cells in this process. We will also discuss processes involving acute or chronic disruption of normal intestinal homeostasis such as damage-induced regeneration and ageing.     

Dedifferentiated fat cells: A cell source for regenerative medicine

The identification of an ideal cell source for tissue regeneration remains a challenge in the stem cell field. The ability of progeny cells to differentiate into other cell types is important for the processes of tissue reconstruction and tissue engineering and has clinical, biochemical or molecular implications. The adaptation of stem cells from adipose tissue for use in regenerative medicine has created a new role for adipocytes. Mature adipocytes can easily be isolated from adipose cell suspensions and allowed to dedifferentiate into lipid-free multipotent cells, referred to as dedifferentiated fat (DFAT) cells. Compared to other adult stem cells, the DFAT cells have unique advantages in their abundance, ease of isolation and homogeneity. Under proper condition in vitro and in vivo, the DFAT cells have exhibited adipogenic, osteogenic, chondrogenic, cardiomyogenc, angiogenic, myogenic, and neurogenic potentials. In this review, we first discuss the phenomena of dedifferentiation and transdifferentiation of cells, and then dedifferentiation of adipocytes in particular. Understanding the dedifferentiation process itself may contribute to our knowledge of normal growth processes, as well as mechanisms of disease. Second, we highlight new developments in DFAT cell culture and summarize the current understanding of DFAT cell properties. The unique features of DFAT cells are promising for clinical applications such as tissue regeneration.     

Human adult pluripotency: Facts and questions

Cellular reprogramming and induced pluripotent stem cell(IPSC) technology demonstrated the plasticity of adult cell fate, opening a new era of cellular modelling and introducing a versatile therapeutic tool for regenerative medicine.While IPSCs are already involved in clinical trials for various regenerative purposes, critical questions concerning their medium-and long-term genetic and epigenetic stability still need to be answered. Pluripotent stem cells have been described in the last decades in various mammalian and human tissues(such as bone marrow, blood and adipose tissue). We briefly describe the characteristics of human-derived adult stem cells displaying in vitro and/or in vivo pluripotency while highlighting that the common denominators of their isolation or occurrence within tissue are represented by extreme cellular stress. Spontaneous cellular reprogramming as a survival mechanism favoured by senescence and cellular scarcity could represent an adaptative mechanism. Reprogrammed cells could initiate tissue regeneration or tumour formation dependent on the microenvironment characteristics. Systems biology approaches and lineage tracing within living tissues can be used to clarify the origin of adult pluripotent stem cells and their significance for regeneration and disease.