JPOR-270; No. of Pages 17 journal of prosthodontic research xxx (2015) xxx–xxx

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Cell-based bone regeneration for alveolar ridge augmentation – Cell source, endogenous cell recruitment and immunomodulatory function Masaru Kaku DDS, PhDa,*, Yosuke Akiba DDS, PhDa, Kentaro Akiyama DDS, PhDb, Daisuke Akita DDS, PhDc, Masahiro Nishimura DDS, PhDd a

Division of Bioprosthodontics, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan Department of Oral Rehabilitation and Regenerative Medicine, Okayama University Graduate School of Medicine Dentistry and Pharmaceutical Sciences, Okayama, Japan c Department of Partial Denture Prosthodontics, Nihon University School of Dentistry, Tokyo, Japan d Department of Oral Maxillofacial Prosthodontics, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan b

article info


Article history:

Alveolar ridge plays a pivotal role in supporting dental prosthesis particularly in edentulous

Received 5 January 2015

and semi-dentulous patients. However the alveolar ridge undergoes atrophic change after

Accepted 5 February 2015

tooth loss. The vertical and horizontal volume of the alveolar ridge restricts the design of

Available online xxx

dental prosthesis; thus, maintaining sufficient alveolar ridge volume is vital for successful


benefits in prosthetic dentistry, enabling regeneration of the atrophic alveolar ridge. In

oral rehabilitation. Recent progress in regenerative approaches has conferred marked Bone augmentation

order to achieve successful alveolar ridge augmentation, sufficient numbers of osteogenic

Bone regeneration

cells are necessary; therefore, autologous osteoprogenitor cells are isolated, expanded in

Alveolar ridge

vitro, and transplanted to the specific anatomical site where the bone is required. Recent

Mesenchymal stem cell

studies have gradually elucidated that transplanted osteoprogenitor cells are not only a

Cell transplantation

source of bone forming osteoblasts, they appear to play multiple roles, such as recruitment

Endogenous cell mobilization

of endogenous osteoprogenitor cells and immunomodulatory function, at the forefront of

Immunomodulatory function

bone regeneration. This review focuses on the current consensus of cell-based bone augmentation therapies with emphasis on cell sources, transplanted cell survival, endogenous stem cell recruitment and immunomodulatory function of transplanted osteoprogenitor cells. Furthermore, if we were able to control the mobilization of endogenous osteoprogenitor cells, large-scale surgery may no longer be necessary. Such treatment strategy may open a new era of safer and more effective alveolar ridge augmentation treatment options. # 2015 Japan Prosthodontic Society. Published by Elsevier Ireland. All rights reserved.

* Corresponding author at: 2-5274 Gakkochou-douri, Chuo-ku, Niigata 951-8514, Japan. Tel.: +81 25 227 2897; fax: +81 25 227 2898. E-mail address: [email protected] (M. Kaku). 1883-1958/# 2015 Japan Prosthodontic Society. Published by Elsevier Ireland. All rights reserved.

Please cite this article in press as: Kaku M, et al. Cell-based bone regeneration for alveolar ridge augmentation – Cell source, endogenous cell recruitment and immunomodulatory function. J Prosthodont Res (2015),

JPOR-270; No. of Pages 17


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Contents 1. 2.




Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell-based bone regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Cell sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1. Bone marrow mesenchymal stem cells . . . . . . . . . . . . . . 2.1.2. Periosteum-derived stem/progenitor cells . . . . . . . . . . . . 2.1.3. Mature adipocyte-derived dedifferentiated fat cells . . . . 2.2. Survival of transplanted cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Recruitment of endogenous osteoprogenitor cells . . . . . . . . . . . . 2.3.1. SDF-1 (CXCL12)/CXCR4 axis . . . . . . . . . . . . . . . . . . . . . . . 2.3.2. MCP-1 (CCL2)/CCR2 Axis . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.3. MSC conditioned medium and secretome . . . . . . . . . . . . Immunomodulatory properties of mesenchymal stem cells (MSCs) . . . 3.1. Systemic transplantation of MSCs for human immune diseases . 3.2. Local transplantation of MSCs and host immune system . . . . . . Future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


The alveolar ridge primarily supports natural dentition and environ the oral cavity proper together with teeth, palate and tongue. The alveolar ridge plays an important role in oral function, including mastication, bolus formation and speech. After the tooth loss, particularly in edentulous and semidentulous patients, the alveolar ridge plays a pivotal role in supporting dental prosthesis. The height and width of the alveolar ridge is crucial for the stability of removable dentures and installation of dental implants. In the case of dental implant restoration, the alveolar ridge directly supports the implant body via functional connections, termed osseointegration. The vertical and horizontal volume of the alveolar ridge restricts the size and angle of dental implant installation; thus, maintaining sufficient alveolar ridge volume is vital for successful implant-supported dental prostheses. As long recognized by prosthodontists, the alveolar ridge undergoes atrophic changes after tooth loss. It has been reported that reductions in the residual alveolar ridge are most prominent within 6 months of tooth extraction, but they continue at a slower rate throughout the lifetime [1]. The reduced alveolar ridge is not ideal for the stability and longterm prognosis of dental prosthesis; consequently, prosthodontists have attempted to cope with irreversible anatomical changes in the alveolar ridge by utilizing a variety of therapeutic approaches. Recent progress in regenerative approaches has conferred marked benefits in prosthetic dentistry, enabling regeneration of the atrophic alveolar ridge. The alveolar ridge can be regenerated by autograft (patient’s own tissue), allograft (human tissue from different individuals), xenograft (nonhuman tissue) or synthetic materials [2]. Among the surgical transplantation approaches, autograft has been the gold standard for alveolar ridge augmentation, as other types of grafting materials may lead to extra clinical complications such as pathogen transfer, immune rejection and suboptimal integration [2]. On the other hand, a major drawback of

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autograft is donor site morbidity [3]; therefore, to minimize host tissue morbidity, ‘‘bioengineering’’, which aims to develop biological substitutes to restore defective tissue, has become an area of emerging interest [4]. The key components of tissue engineering are cells, scaffolds and signaling molecules, the so-called ‘‘tissue engineering triad’’ [5]. Among these key factors, scaffold-based approaches have been attempted since the dawning of bioengineering-based bone augmentation (see recent review [6]). Guided bone regeneration (GBR) is a well-documented material-based bone regeneration approach [7,8]. Numerous studies have provided evidence that bone regeneration is significantly enhanced when osseous defects are mechanically protected with a synthetic membrane [9]. GBR membranes prevent the invasion of undesirable soft tissues into osseous defects, thereby allowing host tissue-derived osteogenic cells and their precursors to repopulate the osseous wound space [9–12]. Together with a membrane, three-dimensional porous scaffolds can maintain the space for bone regeneration, not only preventing the invasion of undesired cells, but also anchoring endogenous osteogenic cells and providing molecular cues for osteoblastic differentiation. The scaffold-based bone augmentation approach has now been combined with cell-based regeneration therapy. Pittenger et al. first reported that adherent cells isolated from human bone marrow aspirates maintain their undifferentiated state in long-term culture and differentiate toward osteogenic lineage when appropriate treatment is performed [13]. Based on this finding, numerous studies have aimed to regenerate bone tissue using bone marrow-derived mesenchymal stem cells (MSCs). However, if the cells are transplanted without a scaffold, the transplanted cells do not remain at the site of transplantation, and are washed out by extracellular fluids and/or the bloodstream. Therefore, cells should be transplanted with an appropriate scaffold, such as a porous scaffold, cell-sheet with secreted ECMs or cell encapsulated hydrogel [14]. Furthermore, the scaffold may provide bioactive cues for either proliferation or differentiation of transplanted cells.

Please cite this article in press as: Kaku M, et al. Cell-based bone regeneration for alveolar ridge augmentation – Cell source, endogenous cell recruitment and immunomodulatory function. J Prosthodont Res (2015),

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In order to achieve successful cell-based bone augmentation, sufficient numbers of osteogenic cells are necessary; thus autologous osteoprogenitor cells must be isolated, expanded in vitro, and transplanted to the specific anatomical site where the bone is required [15]. Previous studies have reported that transplantation of osteogenic cells results in significantly larger amounts of new bone formation [16–18]. Recent advances in molecular and cellular biology have shed light on the various functions of transplanted osteoprogenitor cells, including endogenous stem cell recruitment and immunomodulation [19]. As compared with tissue autografts, cellbased regenerative therapies could minimize donor site morbidity and associate complications. This review focuses on the current consensus of cell-based bone augmentation therapies with emphasis on cell sources, transplanted cell survival, endogenous stem cell recruitment and immunomodulatory function of transplanted osteoprogenitor cells.

collection, particularly in elderly patients. Therefore, reliable and safe cell expansion procedures are required for BMSCbased bone regeneration [19]. Several investigations have reported that greater bone formation is observed with human-derived BMSCs using bone morphogenetic protein2 (BMP-2), basic fibroblast growth factor (bFGF) or transforming growth factors-beta (TGF-b) as osteogenic factors, which promote cell proliferation and neovascularization [37–41]. BMP-2 and TGF-b are cytokines that belong to the TGF-b superfamily comprising approximately 40 members divided into several subgroups [42,43]. BMP and TGF-b are major signaling cascades responsible for osteogenesis and are involved in the vast majority of cellular processes that are responsible for bone formation [44–48]. Therefore, the use of combination BMSCs with osteogenic growth factor is recommended for bone tissue regeneration [40,41,49]. In other words, BMSCs are an important cell source for bone regeneration and tissue engineering strategies.




Cell-based bone regeneration Cell sources

MSCs are present in virtually all mesenchyme tissue and are able to differentiate into osteogenic cells in vitro. In this regard, osteoprogenitor cells can theoretically be obtained from all mesenchyme. On the other hand, the characteristics of MSCs vary from tissue to tissue [20]. In this article, 3 emerging types of tissue-derived cell, bone marrow-, periosteum-derived MSCs and mature adipocyte-derived dedifferentiated fat cells, and their osteogenic properties are discussed.


Bone marrow mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells (BMSCs) are most commonly applied to regenerative medicine owing to their high proliferative and multipotent properties [21,22]. BMSCs can differentiate into osteoblasts, chondrocytes, adipocytes, myocytes or neuronal cells in vitro and robustly form bone in vivo [13,23–26]. Therefore, BMSCs are the most applicable cell source because of their great osteogenic potential for bone regeneration [27,28]. BMSCs are easily isolated from not only the iliac crest, but may also be harvested from orofacial bone marrow aspirates during dental surgical procedures such as dental implant placement, wisdom tooth extraction, extirpation of cysts and orthodontic osteotomy. Several groups have indicated that grafted bone obtained from the craniofacial area for autologous bone grafting at craniofacial sites provides better results and significantly higher resultant bone volume than bone harvested from the iliac crest or rib [29–32]. In addition, the osteogenic differentiation ability of orofacial-derived BMSCs is reported to be higher than that of iliac-derived BMSCs [32,33]. These differences between orofacial- and iliac-derived BMSCs may be due to the embryological origin of cranial neural crest cells and mesoderm [33,34]. The properties of orofacialderived BMSCs may provide an advantage for bone regeneration. However, the collectable volume of orofacial bone marrow is less than that of the iliac crest [35,36]. Moreover, bone marrow aspiration is necessary to harvest BMSCs, and this is associated with drawbacks such as pain and difficulty in

Periosteum-derived stem/progenitor cells

Periosteum is a specialized connective tissue that covers surface of bone. This tissue is composed of two distinct layers; a thick outer fibrous layer, and an inner osteogenic cambium layer [50,51]. Although the osteogenic capacity of the periosteum was first reported in the 18th century [52], the osteogenic potential of periosteum was uncertain until recently, as stable results with free periosteum grafts were not obtained in experimental studies [53–55]. Because the periosteum membrane was found to form a mineralized extracellular matrix under appropriate in vitro conditions, several subsequent studies have addressed other aspects of periosteal osteogenesis, including long bone development and the periosteum [56], the relationship between the vasculature and periosteum [57], and the periosteal osteogenic capacity [58]. Clinically, maxillary periosteal was introduced for primary repair of alveolar clefts in the 1960s [59,60], and then tibia-derived free periosteal grafts were used for primary repair of the palate [61]. Many reports have indicated that the inner osteogenic layer of periosteum contains osteogenic progenitor cells, osteoblasts and fibroblasts, as well as micro vessels and sympathetic nerves [62,63]. In addition, periosteum-derived cells (PDCs) can differentiate into osteoblasts, adipocytes and chondrocytes, and express typical MSCs markers, such as Sca1, CD105, SSEA-4, CD29 and CD140 [64–67]. Therefore, PDCs could be useful for functional tissue engineering, particularly for bone regeneration, and have already been tested in clinical trials as well as BMSCs [19,68–70]. Clinically, harvesting periosteum is not only easier for dentists, but is also less invasive for patients when compared with bone marrow aspiration [71–75]. At Niigata University Medical and Dental Hospital (Niigata, Japan), cultured periosteal sheet transplantation, in which small pieces of periosteum are collected from the patient, expanded in vitro, and then applied to the periodontal defect with suitable scaffolds, has been used in clinical trials [72,73,75]. The results of these studies have demonstrated that autologous implantation of cultured PDCs combined with other elements (i.e., scaffold and/or plateletrich plasma), facilitates successive bone regeneration, and that PDC-containing cell sheets play an important role in bone healing and remodeling.

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In case of bone fracture healing, PDCs make major contributions to the initial phase of the bone-healing process. As early as 24–48 h after the injury, an acute inflammatory reaction can be seen at the periosteum [76,77]. Subsequently, periosteal cells start to proliferate, and a thickening of periosteum is observed. This process is defined as periosteal activation [78]. Cells derived from the endosteum and bone marrow do not participate in callus formation, but are primarily responsible for the regeneration of the new endosteum and bone marrow. These observations indicate that the contribution of periosteal cells is critical to callus formation. In fact, activation of PDCs induces robust chondrogenesis and osteogenesis, accompanied by marked induction of angiogenesis, which eventually leads to vascularization and remodeling of bone grafts [79,80]. Other sources of mesenchymal progenitor cells have also been proposed to participate in bonefracture healing, including local bone marrow, adjacent muscle tissue, endothelial cells and circulating mesenchymal stem cells [80–82]. However, the relative contribution to healing by each cellular phenotype remains unclear. Periosteum has been shown to vary among regions and to change with age. However, the precise characteristics and variations of the craniofacial periosteum are not well known. The craniofacial periosteum may have distinct biological characters when compared with periosteum from other locations. In fact, craniofacial PDCs appear to act differently when compared with long-bone PDCs [83]. Intramembranous ossification was observed when mandibular periosteal cells were transplanted into a tibial bony defect. In contrast, endochondral ossification was noted when tibial periosteal cells were transplanted into a mandibular defect. Therefore, the signaling pathways involved in periosteal-mediated bone healing may differ from those involved in craniofacial periosteal-mediated bone healing. However, regardless of the signaling pathways, given its richness in skeletal progenitors, preservation and harnessing of the periosteum may promote bone regeneration. The phenotypic profiles of human mandibular PDCs were comparable to those of maxillary tuberosityderived BMSCs, and both cell populations formed ectopic bone after subcutaneous implantation in mice [84]. In addition, PDCs showed higher proliferation than BMCs [64,85]. Therefore, the periosteum is a reliable source of stem/progenitor cells for bone regeneration, particularly for large defects. However, because of the limited number of cases reported and the lack of wellcontrolled clinical trials, further studies are necessary to evaluate the effectiveness of PDCs for bone regeneration.


Mature adipocyte-derived dedifferentiated fat cells

Adipocytes have long been regarded as simply heat insulators or stores of excess free fatty acids that could be released when needed. Now, they are considered to comprise a critical organ involved in energy balance regulation and the immune response through intricate signals [86,87]. Adipocytes include various cell types, such as endothelial cells, blood cells, macrophages and mesenchymal stem cells [88]. Many groups have demonstrated that the cultured stromal-vascular fraction, termed adipose-derived stem/stromal cells (ASCs), is able to differentiate into cells such as adipocytes, osteoblasts, chondrocytes, neuronal cells, myocytes, endothelial cells and hepatocytes [89], and are thus extensively used for bone tissue

engineering [90,91]. However, ASCs comprise a heterogeneous cell population and are often contaminated by endothelial and smooth muscle cells, and pericytes [92–95]. In contrast, mature adipocytes are functionally the cell type that shows typical and specific morphology characterized by the presence of a single, large cytoplasmic lipid droplet accounting for approximately 80% of its volume [96]. Until quite recently, mature adipocytes were considered to be a terminally differentiated lineage without the ability to proliferate, as the process of cellular differentiation in terminally differentiated mammalian cells is thought to be irreversible [97]. However, recent findings have suggested that mature adipocytes are easily isolated without postoperative pain procedures or donor site injury, and they can dedifferentiate into fibroblast-like cells with an in vitro dedifferentiation strategy, known as ceiling culture (Fig. 1a). This method exploits the buoyant properties of adipocytes, which allows them to adhere to the top inner surface of a culture flask filled completely with medium [98– 100]. These mature adipocyte-derived fibroblast-like cells lose expression of mature adipocytes marker genes, but retain or gain expression of mesenchymal lineage-committed marker genes and show proliferative activity [101,102]. These singlecell derived lipid-free fibroblast-like cells are known as dedifferentiated fat cells (DFAT cells; Fig. 1b). In addition, DFAT cells can differentiate into adipocytes [102–104], osteoblasts [102,104,105], chondrocytes [102], skeletal myocytes [106], smooth muscle cell lineage [107,108], cardiomyocytes [109] and endothelial cells [110,111] in vitro. Additional studies have also reported expression of neural and neuronal progenitor markers [112,113]. Several reports have revealed that DFAT cells are positive for CD13, CD29, CD44, CD90 and CD105 (stromal/stem cell-associated markers), but negative for CD31, CD34 and CD45 (platelet endothelial/vascular cellassociated markers) on flow cytometry [102,114–118]. Moreover, DFAT cells express embryonic stem cell markers such as Oct4, Sox2, c-Myc and Nanog [115]. Real-time RT-PCR revealed that DFAT cells do not express mature adipocyte markers, such as LPL, leptin and GLUT4, but they express osteogenesis and chondrogenesis transcription factors (Runx2/Cbfa1 and Sox9, respectively) [102]. These reports suggest that DFAT cells have lost the functional characteristics of mature adipocytes, but have retained multilineage potential in a more homogeneous cell population than ASCs. In addition, DFAT cells have higher telomerase activity [115,118]. Furthermore, some reports have indicated that osteogenic differentiation ability in human-derived DFAT cells is higher than that of ASCs [105,118]. Indeed, osteogenic DFAT cells with appropriate scaffolds showed newly formed bone in calvarial defects [119,120]. Previous and unpublished data of Akita et al. have shown that DFAT cells facilitate periodontal regeneration, including cementum and alveolar bone in inbred rat fenestration defects when compared with ASCs [121] (Fig. 1c). Collectively, DFAT cells have several properties that make them suited for regenerative dentistry. As DFAT cells are a nearly homogenous population, they are associated with better safety and efficacy for clinical cell-based therapies. Furthermore, DFAT cells can be obtained from patients regardless of their age and only a small portion of fat tissue is needed. DFAT cells can be obtained from buccal fat, the harvesting of which is a simple procedure that requires a

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Fig. 1 – Isolation and bone regenerative capacity of DFAT cells. (a) Dedifferentiation and proliferation of mature adipocytes by ceiling culture. Isolated unicolor mature adipocytes adhered to the top inner surface of a culture flask and generated fibroblast-like cells. (b) Microscopic view of primary cultured DFAT cells harvested from rat inguinal fat pads after 7 days of ceiling culture. Black arrows indicate lipid droplets in mature adipocytes. Scale bars = 250 mm. (c) DFAT cells transplantation showed alveolar bone regeneration (scale bar = 1000 mm; Hematoxylin and Eosin staining). Higher magnification of the framed area. Black arrows indicate newly formed alveolar bone. Black arrow heads indicate newly formed cementum (scale bar = 100 mm; Hematoxylin and Eosin staining).

minimal incision with local anesthesia and causes minimal donor site morbidity [102,105,122]. As reviewed above, each osteoprogenitor cells have advantages in different aspects. From the view point of osteogenic differentiation, many studies reported the predominance of BMSCs compared to other MSC-like cells, including PDCs and DFAT cells [64,123–125]. In contrast, Yoshimura et al. reported that PDCs have higher mineralization ability compared to BMSCs and ASCs [126]. In a study by Lee et al., BMSCs and ASCs have comparable differentiation ability, however; it was indicated that the proliferating ability and differentiation potential were variable according to the culture condition [127]. Interestingly, pretreatment with basicFGF made PDCs more sensitive to BMP-2 and more osteogenic in comparison with BMSCs [85]. These results suggested that the optimization of the culture condition and/or appropriate additives will maximally exert the differentiation ability of isolated osteoprogenitor cells. Besides, regarding the immunomodulatory function, ASCs and BMSCs are shown to have comparable ability [128,129]. At this moment, superiority of each osteoprogenitor cells for alveolar ridge augmentation is still controversial and there are still many fundamental objectives that need to be addressed.


Survival of transplanted cells

In order to improve cell-based bone regeneration, survival of transplanted cells until capillary angiogenesis; which supplies oxygen, nourishment and disposal of metabolic waste products, is crucial [130,131]. Numerous studies have analyzed

the fate of transplanted cells in bone defects (i.e., cell survival, osteoblastic differentiation and contributions in new bone formation); however, the outcomes of these studies remain controversial. Zimmermann et al. reported that when fluorescent-labeled BMSCs are seeded onto porous hydroxyapatite/b-tricalcium phosphate (HA/TCP) scaffold and subcutaneously transplanted to isogenic rats, transplanted BMSCs were no longer observed at 14 days after transplantation [132]. A similar study by Boukhechba et al. reported that transplanted BMSCs with HA/TCP scaffold did not survive for more than 3 weeks [133]. Quintavalla et al. also confirmed the shortterm survival of BMSCs by seeding fluorescently labeled autologous BMSCs on a gelatin carrier, followed by transplantation to osteochondral defects in goat [134]. At 1 and 2 days after transplantation, viable transplanted cells were clearly identified within the defects; but extensive loss of cells was evident at 7 days after transplantation. The authors presumed that the decreases in cell number were due to implant fragmentation, cell death and possibly cell migration toward the bone compartment. Numerous studies have reported the short-term survival of transplanted cells, but it is evident that cell transplantation results in faster bone defect healing in both animal studies [135–137] and human clinical trials [75,138,139]. The transplanted MSCs themselves differentiate, deposit bone matrix at the surface of biomaterials and contribute to bone regeneration [140,141]. However, if transplanted cells are a source of bone-forming osteoblasts, they need to survive for certain periods in order to exert osteogenic stimuli, which are indispensable for producing bone matrix and subsequent

Please cite this article in press as: Kaku M, et al. Cell-based bone regeneration for alveolar ridge augmentation – Cell source, endogenous cell recruitment and immunomodulatory function. J Prosthodont Res (2015),

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mineralization. Recent evidence indicates that transplanted cells are not only the source of bone-forming osteoblasts, they may have different roles, immediately after transplantation, such as endogenous osteogenic cell recruitment and immunomodulatory functions. Formation of vascular system plays an important role in bone regeneration process, not only by supply oxygen and nutrients, but also by delivering osteoprogenitor cells to repair site. For the strategy to stimulate angiogenesis in transplanted site, supplying angiogenic growth factors, activating hypoxia signaling pathway, or recruiting endothelial progenitor cells (EPCs) are important matters. Furthermore, recent study shows inhibition of miRNA (miR-92a) enhances promoting angiogenesis and leading to improve bone repair [142]. Whereas the numerous studies reported the relationship between angiogenesis and bone regeneration, it is unclear how to induce the vascular network effectively to cells/ scaffold complex transplanted site.


Recruitment of endogenous osteoprogenitor cells

The number of reports has shown that transplanted osteoprogenitor cells accelerate new bone formation [143–145], and it has been hypothesized that they differentiate into boneforming osteoblasts [125]. In contrast, the survival period of transplanted cells is thought to be around 2 weeks [132]; thus, there is a discrepancy between the lifespan of transplanted MSCs and the timing of bone regeneration [125,132]. Recent studies have found that recruitment of endogenous MSCs plays an important role in organ repair and regeneration of the heart [146], brain [147] and skin [148]. It has shown that 2 distinct molecular pathways, the SDF-1/CXCR4 and MCP-1/ CCR2 axis, are widely related to endogenous cell recruitment, migration and tissue regeneration.


SDF-1 (CXCL12)/CXCR4 axis

Stromal cell-derived factor 1 (SDF-1) also known as CXC chemokine ligand 12 (CXCL12), is a member of the CXC chemokine (chemotactic cytokine) family, which was originally identified as a pre-B-cell stimulatory factor and cloned from murine bone marrow cell supernatants [149]. SDF-1 binds to the G protein-coupled receptor CXC chemokine receptor 4 (CXCR4), which is also known as hematopoietic marker CD148. SDF-1 is the only known ligand of the receptor CXCR4 [150], but SDF-1 is able to bind and initiate signaling through its cognate receptors CXCR4 and CXCR7 [151]. SDF-1/ CXCR4 interaction plays a role in several physiological functions, including stem cell migration [152,153], tumor metastasis [154,155], maintenance of bone marrow niche [156], and anti-apoptotic cell survival [157]. SDF-1 is expressed by endothelial and stromal cells in bone marrow, cells with osteoblastic lineage and perivascular reticular, but not by hematopoietic cells [158]. On the other hand, CXCR4 is expressed hematopoietic cells, osteoblasts and MSCs [156,159]. CXCR4 is strongly expressed in BMSCs, but its expression is reduced during ex vivo culture when the cells tend to lose their migration and homing ability [160,161]. SDF-1 mediates cell homing and migration through binding with CXCR4. For instance, during bone marrow development, hematopoietic stem cells migrate from liver through the

blood circulation, homing to the bone marrow microenvironment using the SDF-1/CXCR4 axis [162]. SDF-1 is up-regulated at the injury site and provide as an endogenous chemoattractant to recruit circulating CXCR4-positive MSCs, which are required for repair or regeneration in many organs [163–165]. A previous study has shown that intravenously injected CXCR4-positive BMSCs migrated to ischemic brain regions where SDF-1 is strongly expressed [153]. Engrafted MSCs in ischemic myocardium release SDF-1, which cooperates with basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), contributing to homing of circulating endothelial progenitor cells that induces neovascularization [166]. A mouse ectopic bone formation model using BMP-2 containing pellets showed that circulating bone marrowderived osteoprogenitor cells gathered around the new bone formation area through SDF-1 expression on vascular endothelial cells [159]. It has also been reported that intravenously implanted BMSCs migrate to the bone fracture site in a CXCR4dependent manner [167], and that periosteum cells at the bone injury site secrete SDF-1 to recruit CXCR4-expressing BMSCs to the healing site [164]. In bone marrow stromal cells and osteoblast-like cell lines, SDF-1 synthesis was increased by interleukin-1beta (IL-1b), platelet-derived growth factor-BB (PDGF-BB), vascular endothelial growth factor (VEGF), tumor necrosis factor-alpha (TNF-a) and parathyroid hormone (PTH), but not by transforming growth factor-beta (TGF-b) [168].


MCP-1 (CCL2)/CCR2 Axis

Monocyte chemotactic protein-1 (MCP-1), also known as C–C motif chemokine ligand 2 (CCL2) is a small cytokine that belongs to the C–C chemokine ligand family. MCP-1 is produced by various cells, including endothelial cells, fibroblasts, epithelial cells, smooth muscle cells, meningeal cells, astrocytes, monocytes and microglial cells. Among these, monocytes and macrophages are the major source of MCP-1 [169], while MSCs also secrete MCP-1 [170]. C-C chemokine receptor type 2 (CCR2), also known as CD192 is the G proteincoupled receptor that binds multiple ligands, such as chemo attractant protein MCP-1 (CCL2), MCP-2 (CCL8), MCP-3 (CCL7) and MCP-4 (CCL13). CCR2 is reported to be expressed by monocytes, hematopoietic cells and MSCs [171]. Recombinant MCP-1 induces migration of MSCs and bone marrow cells and this induction is blocked by siRNA against MCP-1 [172]. In a mice distraction osteogenesis model, transplantation of MSC conditioned medium (MSC-CM) containing collagen hydrogel induced new callus formation, but this activity was blocked by neutralization antibody against MCP-1 [173]. These results indicate the importance of the MCP1/CCR2 axis to MSC recruitment and subsequent bone regeneration. On the other hand, another study cast doubt on the contribution of the MCP-1/CCR2 axis in the recruitment of MSCs [171]. Further research is needed in order to confirm the significance of the MCP-1/CCR2 axis in bone regeneration. In addition to the SDF1/CXCR4 and MCP-1/CCR2 axes, numerous chemokine/chemokine receptor axes are known to contribute MSC migration, including CX3CL1 (fractalkine)/ CX3CR1, CXCL16/CXCR6, CCL3/CCR1, MCP-3(CCL7)/CCR1 and CCL19/CCR7 [174]. Several growth factors are most likely involved in MSC migration, such as PDGF, hepatocyte growth

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Fig. 2 – Schematic diagram illustrating the concept of transplanted MSCs recruitment of endogenous osteogenic cells and contribution to bone formation. MSCs are transplanted with scaffold and growth factors. Transplanted MSCs differentiate and secrete a secretome containing chemokines and growth factors. Endogenous osteoprogenitor cells are recruited by chemokines and growth factors, and migrating to healing sites. Migrating cells and transplanted cells are stimulated by the secretome in an autocrine and paracrine manner, and contribute to bone regeneration.

factor (HGF), heparin binding epidermal growth factor like growth factor (HB-EGF), transforming growth factor alpha (TGF-a), insulin-like growth factor (IGF), epidermal growth factor (EGF) and angiopoietin-1 (Ang-1) [175,176].


MSC conditioned medium and secretome

With regard to clinical applications, usage of MSCs remains challenging because of the requirements for safety investigations, quality management of cell handling and handling costs. Recently, MSC conditioned medium (MSC-CM) and the MSC secretome have attracted much attention and have been studied as a substitute for MSC transplantation. Osugi et al. reported that hMSC-CM enhances the cell migration and gene expression of osteogenic markers, Osteocalcin and Runx2/ Cbfa1 [177]. In a rat calvarial critical size bone defect, hMSCCM containing agarose gel enhanced bone regeneration; hMSC-CM was positive for IGF-1 and VEGF, but not FGF-2, PDGF, BMP-2 or SDF-1, on ELISA assay. A similar study by Chen et al. showed that repeated injection of hMSC-CM into transplanted collagen gel matrix accelerated callus formation in a mouse distraction osteogenesis model. Furthermore, several soluble factors related to MSC recruitment, endothelial cell and endothelial precursor cell recruitment, osteogenesis, angiogenesis, cell proliferation and anti-inflammation, were detected in hMSC-CM, as confirmed by cytokine antibody array. In terms of cell recruitment and soluble osteogenesis factors, MCP-1, MCP-3, Regulated on Activation Normal T Cell Expressed and Secreted (RANTES), IL-6 and IL-3 were detected, but SDF-1 was not observed. In addition, treatment of neutralizing antibodies for MCP-1 and/or MCP-3 in hMSCCM resulted in significant reductions in the number of migrating bone marrow mononuclear cells, while SDF-1 neutralizing antibody showed no effect [173]. These data suggest that two distinct pathways, the SDF-1/CXCR4 and MCP-1/CCR2 axes, differentially regulate the MSC secretomemediated endogenous cell recruitment in the respective MSC micro-environment. At the site of transplantation, MSCs themselves sense the surrounding environment, stimulate their secretome, and differentiate into specific lineages. MSCs control their own characteristics and cell fate by changing

their secretome in an autocrine and paracrine manner. For example, it has reported that VEGF-A, TGF-2, TGF-3, IL-1, IL-6 and IL-8 were significantly elevated under hypoxic conditions, which are known to enhance tissue wound healing [178]. Numerous studies have indicated the limitations of single growth factors and the advantages of a combination of different growth factors for optimizing new bone formation [176,179]. The MSC secretome in MSC-CM is composed of several growth factors and cytokines, and the constitution changes with the surrounding environment. Transplanted MSCs probably monitor the surrounding environment based on factors such as physical stress, inflammation and hypoxia, and then differentiate into bone-forming osteoblasts. At the same time, the MSCs orchestrate the surrounding cells toward appropriate lineages and mobilize endogenous osteoprogenitor cells from remote locations by changing their secretome (Fig. 2). Further studies are needed in order to explain the functional roles of transplanted MSCs with regard to cell recruitment and bone regeneration. If it is able to control the mobilization of endogenous osteoprogenitor cells, this treatment strategy may be more effective therapy for alveolar ridge augmentation. On the other hand, recent study reported that CXCR4 expression is decreased with aging and this CXCR4 deficiency impairs osteogenic differentiation of MSCs [180]. Furthermore, it is also reported that angiogenesis and vasculogenesis are impaired with aging [181]. Thus, development of novel method to accelerate these factors might be important for alveolar bone augmentation especially in elderly patients.

3. Immunomodulatory properties of mesenchymal stem cells (MSCs) In the last 15 years, many studies have tried to explain the mechanisms of MSC immunomodulatory function by direct cell-to-cell contact or indirect interference by soluble factor secretion. More recently, most studies have described the inhibition mechanisms that prevent immune cell proliferation and inflammatory cytokine secretion. For instance, MSCs from

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Fig. 3 – MSC transplantation: systemic vs. local.

human bone marrow inhibit T lymphocyte proliferation by transforming growth factor beta 1 (TGFb1) and hepatocyte growth factor (HGF) in vitro [182]. MSCs induce arrest of T lymphocytes in the G1/G0 phase and down-regulate cyclin D2 expression in lymphocytes [183]. MSCs secrete indoleamine 2,3-dioxygenase (IDO), thus accelerating tryptophan degradation and kynureine synthesis, which results in inhibition of T lymphocyte proliferation [184]. Nitric oxide (NO) from MSCs suppresses phosphorylation of transcription factor, signal transducer and activator transcription-5 (STAT-5) [185]. Human leukocyte antigen-G5 (HLA-G5) from MSCs triggers inhibition of T lymphocyte function, followed by up-regulation of T helper type 2 (Th2) and regulatory T cells (Tregs) [186,187]. On the other hand, some studies have reported that MSCs are able to inhibit immune cells by direct cell-to-cell contact [188– 190]. Krampera et al. reported that MSCs physically hinder T lymphocytes from contact with antigen-presenting cells [191], while induction of immune cell apoptosis by MSCs has also been reported. IDO induces 3-hydroxyanthranilic acid (HAA) synthesis during tryptophan metabolism, thus inducing T cell apoptosis via inhibition of the NFkB pathway [192]. MSCs inhibit T lymphocytes by activation of the programmed death 1 pathway [193]. MSCs induce T lymphocyte apoptosis through the FAS ligand/FAS pathway and lead to immunotolerance via up-regulation of Tregs [194]. Although interactions between MSCs and B lymphocytes are gradually being elucidated, it remains controversial and the mechanisms are not fully understood. MSCs are able to inhibit CD40L antibody and IL-4 stimulated B cell proliferation in vitro [183,195]. MSCs inhibit the production of antibodies and the expression of chemokine receptors, including CXCR4, CXCR5 and CXCR7 for chemotaxis (14). In mechanistic studies, MSCs were found to produce IDO, leading to this inhibition [196]. However, some studies have shown that MSCs promote B lymphocyte proliferation and antibody production in vitro [197,198]. This discrepancy may depend on the maturation of B cells and microenviroments. MSCs also affect immunotolerance by inducing Tregs and suppressing Th17. Forkhead box p3 (Foxp3) is a critical transcriptional factor for Treg development and functional

maturation. Numerous studies have reported that MSCs promote Foxp3 expression in CD4+CD25+ T lymphocytes [187,190,199,200]. In the meantime, MSCs inhibit differentiation of Th17 from CD4+ T lymphocytes and suppress IL-17 secretion in vitro by PGE2. More interestingly, MSCs have the potential to convert Th17 into Tregs like anti-inflammatory T lymphocytes in vitro [201]. MSCs promote generation of Tregs, not only via soluble factors such as IDO, PGE2 and TGFb1 [202], but also by direct cell-to-cell contact. To obtain treatment effects, up-regulation of Tregs is thought to be critical for MSCbased immune therapy in autoimmune disease treatment [203,204]. However, the detailed mechanisms of how MSCs modulate immune tolerance through Tregs and Th17 remain under investigation.

3.1. Systemic transplantation of MSCs for human immune diseases Based on the in vivo and in vitro immunomodulatory evidence, various possibilities for MSC-based immunotherapy have been explored. Growing evidence of the therapeutic potential of MSCs transplantation in pre-clinical animal model has offered new hope for MSC-based immunotherapy for human disorders. Le Blank et al. firstly reported MSC therapy for graft versus host disease (GvHD) [205]. Systemic allogeneic MSC transplantation may ameliorate disease phenotypes in immunosuppressive drug refractory SLE patients [206]. In addition, autologous MSC transplantation failed to improve disease activity in SLE patients [208]. MSCs also showed efficacy and safety in several clinical trials for systemic sclerosis [194], Crohn’s disease (CD) [209], myocardial infarction [210] and diabetes [211]. Although systemic MSC transplantation is becoming a promising treatment tool, the detailed mechanisms remain uncertain.

3.2. Local transplantation of MSCs and host immune system In the case of local MSC transplantation, such as tissue regeneration, most animal studies use immune-compromised

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mice that lack components of the immune system in order to avoid ‘‘rejection’’. However, the immune system cannot be ignored in clinical settings. Therefore, regulation of the recipient immune system is considered to be a critical factor for MSC-mediated bone regeneration in vivo. Liu et al. reported that host immune cells and inflammatory cytokines regulate MSC-based bone regeneration [212]. Even when using autologous MSCs, ectopic bone formation failed when the cells were subcutaneously transplanted into wildtype mice with the HA/TCP carrier. On the other hand, abundant bone formation was observed when MSCs were transplanted into immune-compromised mice. Furthermore, when wild-type T cells were systemically injected into immune-compromised mice before MSCs transplantation, bone formation was significantly inhibited. These results strongly suggest that the host immune system, particularly T cells, affect tissue regeneration by transplanted MSCs. When regenerated tissue was analyzed, increased levels of IFN-g and TNF-a from host T cells were observed, and this led to transplanted MSC apoptosis. Moreover, in this study, calvarial bone regeneration in wild-type mice was enhanced by systemic infusion of Tregs or site-specific aspirin treatment, that resulting in suppression of IFN-g and TNF-a. On the other hand, Ren et al. reported that inflammatory niche including IFN-g, TNF-a, IL-1a and IL-1-b, activates the immunosuppressive properties of MSCs to secrete high levels of several chemokines and inducible nitric oxide synthase [213]. Chemokines induce T cell migration toward MSCs, where T cell responsiveness is suppressed by nitric oxide (NO). These reports indicate that the host environmental immune system at the grafting site affects not only MSC survival (or homing) but also MSC properties, resulting in MSC-mediated bone regeneration. Crosstalk between transplanted donor MSCs and recipient immune cells plays an essential role in determining the success of MSC-mediated tissue regeneration (Fig. 3). Moreover, these studies might imply the paradigm shift of bone regeneration therapy from transplantation of donor MSCs to emphasizing host MSCs.


Future directions

For successful bone regeneration, it is crucial to obtain sufficient numbers of osteogenic cells; therefore, isolation of autologous stem cells, expansion in vitro, and transplantation with a suitable scaffold is considered to be the most effective approach. However, as reviewed in this article, transplanted osteoprogenitor cells are not only a source of bone-forming osteoblasts, they play multiple roles at the forefront of bone regeneration (Fig. 4). Cell transplantation sequentially and synergistically accelerate the multiple factors which are contributing successful bone augmentation. Recent studies have gradually elucidated the molecular cues for recruiting endogenous osteoprogenitor cells both systemically and locally (see Section 2.3). It is known that each tissue harbors substantial amounts of endogenous stem cells, which contribute to tissue remodeling and regeneration. Furthermore, small numbers of bone marrow-derived stem cells are constantly circulating throughout the whole body via the bloodstream and some spontaneously reside in the


Fig. 4 – Multiple and synergistic roles of transplanted osteoprogenitor cells in bone augmentation.

respective mesenchymal tissues at the perivascular location (‘‘circulating MSCs’’) [214]. Interestingly, the number of circulating MSCs in peripheral blood significantly increases after injury or trauma such as skeletal muscle damage [215], acute burns [216] and bone fracture [217]. Otsuru et al. reported that bone marrow-derived osteoprogenitor cells are present in peripheral blood and that these cells have the ability to form ectopic bone formations when implanted with BMP-2-containing collagen pellets into the skeletal muscle beds of mice [218]. In a follow-up study, it was demonstrated that circulating bone marrow-derived osteoprogenitor cells were recruited to bone-forming sites via the SDF-1/CXCR4 axis [159]. There is an expanding focus on circulating MSCs due to their potential for bone regeneration, particularly from a safe and efficacious point of view. Taken together, by manipulating molecular cues that regulate the release and anchorage of circulating MSCs, it is theoretically possible to obtain sufficient numbers of osteoprogenitor cells without large-scale surgery, to the site where bone is required. Further investigations are required in order to realize this novel concept in clinical therapy. It is noteworthy that in patients with declining general status, congenital disorders or advanced age, increasing the number of osteogenic cells at specific anatomical site may not be sufficient. Because the activity of endogenous osteoprogenitor cells may be affected by host factors, general status of the patient and clinical history of the lost tissue need to be carefully assessed. From the viewpoint of prosthetic longevity, it is also necessary to consider postoperative tissue reduction. At present, the precise biological mechanisms of postoperative tissue reduction in regenerated bone are poorly understood; however, this is thought to be an atrophic tissue response. If this is the case, application of physiological occlusal force, after an appropriate healing period, may prevent further bone loss. In fact, it has been reported that placement of dental implants can prevent reductions in the alveolar ridge, which

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Fig. 5 – Cell-based alveolar ridge augmentation: current and future.

can be reconstructed by autografts or guided bone regeneration [219,220]. It has also been reported that the long-term wearing of unfit removable dentures can accelerate the reduction in alveolar ridge height [221,222]. For long-term prognosis of reconstructed tissue and dental prosthesis, it is of critical importance to elucidate the regulatory mechanisms of bone homeostasis, which is maintained by physiological occlusal loading. In conclusion, current evidences indicated that transplanted osteoprogenitor cells are not only a source of bone forming osteoblasts, they appear to play multiple roles in bone regeneration events. Furthermore, if we were able to control the mobilization of endogenous osteoprogenitor cells in systemic or local circulation, cell isolation procedures may no longer be necessary (Fig. 5). There might also be an advantage to intentionally using allogenic MSCs, particularly with regard to their immunomodulatory function, in some cases. Further clinical and basic research is necessary in order to understand the efficacy and underlying mechanisms of cell-based bone augmentation therapy. In addition, such fundamental research will open a new era of safer and more effective alveolar ridge augmentation treatment options.

Conflicts of interest statement The authors certify that there are no conflicts of interest with any financial organization regarding the material discussed in this manuscript.

Acknowledgements This review article was written as a project for the Journal of Prosthodontic Research (JPR) Editorial Committee with the support of the Japan Prosthodontic Society (JPS). Part of this review article was written based on a scientific session held at the 123rd General Meeting of the JPS. Some of the research discussed in this article was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS) (24792068 MK, 26462915 YA, 26293414 MN).


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Please cite this article in press as: Kaku M, et al. Cell-based bone regeneration for alveolar ridge augmentation – Cell source, endogenous cell recruitment and immunomodulatory function. J Prosthodont Res (2015),

Cell-based bone regeneration for alveolar ridge augmentation--cell source, endogenous cell recruitment and immunomodulatory function.

Alveolar ridge plays a pivotal role in supporting dental prosthesis particularly in edentulous and semi-dentulous patients. However the alveolar ridge...
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