Introduction
Recent advances in stem cell biology provides new promising approaches for understanding mechanisms and developing the treatment for a number of diseases including cardiovascular disease, neurodegenerative disease, diabetes, cancer and musculoskeletal disease. This review will highlight the recent advances on stem cells in bone biology and its potential translation into clinical applications, particularly related to treatment with osteoporosis and bone regeneration.
Bone is a specialized and dynamic organ that undergoes a continuous remodeling in an adult life, which includes bone resorption and bone formation in a coupled manner (
Martin and Seeman, 2008). The function of the unique process maintains the bone strength and bone mass by removing micro-damages. The two sequential steps are performed by osteoclasts for bone resorption and osteoblasts for bone formation, which are derived from different systems in the bone marrow. Osteoclast lineage shares a common progenitor cells with macrophage in hematopoietic system, while mensenchymal stem cells not only give rise to osteoblast lineages, but also others such as adipocytes, chondrocytes, muscles cells, endothelial cells and even neural cells. It has been well known that the lineage progression of the stem cells is tightly regulated by a number of local growth factors, systemic hormones and signaling pathways (
Martin et al., 2009). The deficiencies of these regulatory networks are believed to cause the development of metabolic bone diseases, for example, osteoporosis, a major public health disease (
Rowe and Lichtler, 2002). Osteoporosis is a disease characterized by low bone mass and structural deterioration of bone tissue, leading to bone fragility and an increased susceptibility to fractures, especially of the hip, spine and wrist, although any bone can be affected. Osteoporosis affects almost 70 million Chinese over the age of 50 and causes some 687 000 hip fractures in China each year [China Health Promotion Foundation (2008) White Paper China 2008, Osteoporosis a Summary Statement of China]. The incidence of hip fractures in Beijing increased by 34% in women and 33% in men from 1988 to 1992 (
Xu et al., 1996). There is a higher incidence of hip fractures in men than in women in China (
Xu et al., 1996;
Yan et al., 1999;
Huang et al., 2000). The average direct cost of a hip fracture in 2007 was $3603 and has been increasing at a rate of 6% per year. In 2006, China spent $1.5 billion for treating hip fractures. It is estimated that this will rise to $12.5 billion in 2020 and by 2050 to more than $264.7 billion (
Zhu et al., 2004;
Luo and Xu, 2005). A better understanding of bone cell biology will definitely provide a new avenue to treat the disease.
The deficiency of mesenchymal stem cells contributes to osteoporosis
Accumulated evidences point out that deficiency of stem cells in osteoblast lineage plays a pivotal role in the pathogenesis of osteoporosis (
Bonyadi et al., 2003;
Miura et al., 2005;
Raisz, 2005;
Buckbinder et al., 2007;
Holmes et al., 2007;
Jethva et al., 2009;
Mizoguchi et al., 2010) . Here, we take two examples to demonstrate the role of stem cells. It has been observed that Sca-1, a well known surface marker for its expression on hematopoietic stem cells, is present on a subset of bone marrow stromal cells, which potentially include mesenchymal stem cells. Sca-1(-/-) mice undergo normal bone development, but with age, exhibit dramatically decreased bone mass resulting in brittle bones (
Bonyadi et al., 2003). Both
in vivo and
in vitro analyses demonstrated that Sca-1 is required directly for the self-renewal of mesenchymal progenitors and indirectly for the regulation of osteoclast differentiation. Thus, defective mesenchymal stem or progenitor cell self-renewal may represent a mechanism of age-dependent osteoporosis in humans. The second example is the Zmpste24-null progeroid mice (Zmpste24
-/-), which exhibit nuclear lamina defects and accumulate unprocessed prelamin A (
Liu et al., 2005). Lamin A is a major component of the nuclear lamina and nuclear skeleton. Truncation in lamin A causes Hutchinson-Gilford progerial syndrome (HGPS), a severe form of early-onset premature aging. Unprocessed prelamin A and truncated lamin A act dominant negatively to perturb DNA damage response and repair, resulting in genomic instability which might contribute to laminopathy-based premature aging (
Liu et al., 2005). Defective prelamin A processing induced accelerated features of age-related bone loss, such as lower osteoblast and osteocyte numbers and higher levels of marrow adipogenesis (
Rivas et al., 2009;
Cunningham et al., 2010). Therefore, processing of prelamin A could be a new approach to regulate osteoblastogenesis and bone turnover for the prevention and treatment of senile osteoporosis.
As many adult tissues and adult organs preserve and maintain stem and progenitor cells that play a role in normal tissue homeostasis and regenerative processes in response to injuries, it is conceivable that a body’s own cells can be targeted
in vivo to augment regenerative potential (
Stevenson et al., 2009;
Daley, 2010). Understanding the stem cell biology in bone will certainly provide fundamental rationale for developing efficient therapeutic strategies for treatment of the disease. Therefore, a new concept of targeting the stem cells for enhancing new bone formation is attractive. A recent study by Muklerjee has demonstrated this feasibility with clinically available proteasome inhibitor, Bortezomib (Bzb) (
Mukherjee et al., 2008). In this work, Bzb induced mesenchymal stem cells (MSCs) to preferentially differentiate into osteoblasts through modulating a bone specific transcription factor, Runx2 in mice. When recipient mice received low doses of Bzb, implantation of normal mouse MSCs showed increased ectopic bone formation. Furthermore, treatment of osteoporotic mice with this drug also increased bone formation and importantly, rescue bone loss. The promising data shows a new way for treatment of osteoporosis by pharmacologic targeting a tissue-resident adult stem cells population to achieve the goal of promoting a regenerative function in adults.
Stem cells also play a vital role in regulation of hematopoietic stem cell (HSC). The HSC niche is currently defined as the specific microenvironment in the bone marrow (BM) which anatomically harbors HSCs and governs their fate by regulating the survival and self-renewal ability of HSCs, protecting them from exhaustion while preventing their excessive proliferation. Many different stromal cell types have been proposed as putative constituents of the niche, but their integrated function is still unrevealed. Mechanisms by which stem/progenitor cell behavior is regulated in the niche include cell-to-cell interaction and the production of growth factors, cytokines, and extracellular matrix proteins. The HSC niche is a dynamic entity reflecting and responding to the needs of the organism. An understanding of how the niche participates in the maintenance of tissue homeostasis and repair offers new opportunities for the development of novel therapeutic tools. Recent studies indicated that the dysfunction of bone progenitor cells induces myelodysplasia. Specific deletion of Dicer1 in mouse osteoprogenitor cells, rather than in mature osteoblasts, results in impaired osteoblastic differentiation and disruption of integrity of haematopoiesis by reduced expression of a gene mutated in Schwachman-Bodian-Diamond syndrome (Sbds) in mouse osteoprogenitor cells, which induced bone marrow dysfunction with myelodysplasia (
Raaijmakers et al., 2010). These results indicate a role of osteoprogenitor cells as a critical component in bone marrow niche, providing an evidence to support a concept of niche-induced oncogenesis. A most recent work indicated Nestin+ cells are mesenchymal stem cells with a self-renewal ability and multipotential to differentiate into osteoblasts, adipocyte and chondryocutes (
Méndez-Ferrer et al., 2010). Nestin+ cells maintain HSCs in the bone marrow and are required for HSC homing. These results may indicate a direct and promising approach for treatment of blood diseases in the future which should include both HSC and mesenchymal stem cells to be targeted.
A new promising strategy for treatment of osteoporosis
In the past decades, the treatment of osteoporosis is largely dependent on either inhibition of osteoclasts mediated bone resorption or stimulation of osteoblastic bone formation (
Ferrari, 2009). However, the outcome of the approach is largely beyond our expectation for preventing or curing the disease. It cannot restore loss of bone structure which is believed to be more important than increased bone mass. This inefficiency causes us to rethink a new therapeutic strategy with a way to improve it. In the natural recycle of bone remodeling, bone formation is always coupled by bone resorption in balance that preserves the physical structure of bone (
Martin et al., 2009). Targeting either side of bone remodeling only temporarily achieves new bone formation.
On the other hand, several lines of evidence from both bench to bedside suggest a new role of osteoclasts in the process of bone formation. In the high bone mass phenotype in Lrp5 mutation, the normal bone structure was observed indicating a balance of bone remodeling in which osteoclast function is also increased along with increased bone formation (
Johnson, 2004;
Yadav and Ducy, 2010). Secondly, long-term administration of bisphosphonates (BPs) causes a necrosis of mandibular bone, due to a long inhibition of osteoclast activity even although osteoblast activity remains unaffected (
Van den Wyngaert et al., 2006;
Khan et al., 2009;
Bedogni et al., 2010;
Vassiliou et al., 2010). In addition, combined use of BPs with parathyroid hormone (PTH) reduced the effect of PTH action on bone formation (
Delmas et al., 1995). Thirdly, several lines of data indicate that appropriate stimulation of osteoclast activity enhances new bone formation. Conditioned medium collected from human osteoclasts cultured on either bone or plastic was tested to check if osteoclast mediates the control of bone formation (
Karsdal et al., 2008). Interestingly, the conditioned medium induced bone nodule form in a dose-dependent manner, suggesting that osteoclasts secrete non-osteoblast derived factors that stimulate preosteoblast to differentiate into mature osteoblasts (
Karsdal et al., 2008). Osteoclasts are also found to produce cardiotrophin-1 (CT-1) which signals through gp130 and the leukaemia inhibitory factor (LIF) receptor (
Walker et al., 2008). It plays a major role in cardiac, neurological and liver biology. CT-1 is reported to have increased osteoblast activity and terminal differentiation both
in vitro and
in vivo; in addition, in CT-1 knockout mice, impaired bone resorption is associated with low bone mass and reduced osteoblast activity and many large osteoclasts (
Walker et al., 2008). With aging, the mutant mice developed an osteopetrotic phenotype. Using an ossicle model, cells of osteoclasts lineage at developmental stage of marrow localization and multinucleation before active bone resorption function as mediators of anabolic actions of parathyroid hormone in bone (
Koh et al., 2005). Osteoprotegerin blunt the anabolic action of PTH, while haematopoietic stem cells were recently observed to influence the fate of mesenchymal stem cells to enter into osteoblasts (
Jung et al., 2008). Isolated HSC with expression of signaling lymphocyte activation molecule (SLAM) family receptors (Sca-1+ cKit+CD105CD41-CD48-) from stressed animal significantly guided mesenchymal stem cell differentiation toward osteoblast lineage both
in vitro and
in vivo through HSC-derived bone morphogenetic protein 2 (BMP-2) and BMP-6. If transplantation of HSCs were able to alter the progression of mesenchymal stem cells, the clinical significance could be remarkable. Manipulation of HSC may be viable therapeutic regimen to treat metabolic bone diseases (
Horwitz et al., 2002). Further works remain to be performed in the future.
The collective results from both clinics and lab works lead us to propose a new promising strategy for curing osteoporosis in which a prominent bone formation can be achieved by not only stimulating stem cells/progenitor in osteoblast lineage, but also requiring a temporally increasing stem cell/progenitor in haematologic/osteoclast lineage. The role of osteoclasts in bone formation during bone remodeling demands further investigations. To appreciate the role of stem cell in bone lineages, bone biologists in the Chinese scientific community should be armed with more powerful research tools, such as green fluorescent protein (GFP) technology for mapping the stem cell lineage progression into osteoblasts and osteoclasts, fluorescent activated cell sorting (FACS) and functional genomics for discovering new genes in regulating the lineage processes with appropriate transgenic and tissue-specific knockout animal models.
Stem cells based bone regenerative medicine
The discovery of stem cells from different tissues and organ systems from embryonic and post-natal life opens a new avenue to offer novel therapeutic strategies for manipulating disease progression and regenerating damaged tissues and organs. Cell-based therapy for bone regeneration is one of the exciting investigational areas that is recognized as the most applicable to clinical applications, as a bone is a relatively simple organ which contains two major cell types—osteoblasts and osteoclasts, relatively to other tissues and organs. The therapy is remarkably demanded by patients suffering from limb amputation, damaged tissues and various bone-related diseases. Two challenges are required to be solved for the feasibility: (1) to properly define an optimal cell source that can be ex vivo expanded within a facility/a stem cell bioreactor from small numbers of stem cells/progenitor to a clinical scale usage; (2) to identify an ideal scaffold that should have natural bony properties for delivering the expanded stem/progenitor population into a required site in the body.
Attempts to isolate skeletal stem cells were dated back to the 1960s when Friedenstein first discovered human bone marrow stromal cells (hBMSCs) in adult bone marrow (1 in 10
4 to 10
6 marrow mononuclear cells)(
Friedenstein et al., 1966;
Friedenstein et al., 1968;
Friedenstein et al., 1970;). hBMSCs from bone marrow aspirates is able to form colony forming units, called CFU-Fs. These cells are also refereed to as osteogenic stem cells, mesenchymal stem cells or skeletal stem cells. Since then, a number of surface markers, CD 90, CD146, SH-10, SB-2, HOP-1 and STRO-1, have been identified as a marker for these cells. Recently, Nestin+ cells are also recognized as a fraction of hBMSCs that preserve ability of self-renewal and multipotential (
Méndez-Ferrer et al., 2010). Unlike HSC, until now, there are not clearly defined markers for isolating skeletal stem cells, as the population of CFU-F is highly heterogeneous in size, morphology and proliferation and potential for
in vivo bone formation. To address this issue, we should combine advanced technologies to map the lineage progression and isolate the different stages of relatively pure sub-population by FAC sorting based on stage-specific expression of certain transgenic genes for proteomic study of unique expression of surface markers.
To facilitate the clinical application of the stem cell based therapy, it is urgently required to develop a set of a closed/sterile bioreactor that enables to
ex vivo expand autologous bone marrow aspirate harvested from a patient. This system should specifically amplify the cells at early stages from stem cells to precursor cells for preserving the most potential of regeneration. Recently, we demonstrated a stronger bone formation potential in mixed population of stem cells and progenitor cells produced by a closed bioreactor preserve than that of hBMSCs cultured by a conventional culture procedure (
Yin et al., 2009). The mixed population not only contains cells that can differentiate into mesenchymal lineages (osteoblasts, adipocytes, chondrocytes and even endothelial cells), but also cells that belong to haematopoietic cells including precursor cells for osteoclasts as well as cells in endothelial lineage. Enhanced micro-vascular formation was observed in bone formation areas by the stem/progenitor cell product. Angiogenesis is believed to be a prerequisite for any tissue/organ regeneration. It remains largely unknown if osteoclasts play a role in the process of bone regeneration for creating local microenvironment for expansion and differentiation of implanted cells within a scaffold. With the combination of both and angiogenesis regeneration potential, the mixed stem/progenitor cells may be a suitable product for clinical application in bone regeneration. For designing an optimal cell-based therapy, we have to appreciate the stem/progenitor cells in the regenerative process. It has been assumed that the contribution to bone regeneration process should be made by both donor and recipient sides. However, we observed that only implanted stem/progenitor cells from a donor participate in the process visualized by bone specific promoter driven GFP transgene expression. Cells from recipient were hardly detected. This data indicated implanted stem/progenitor cells from donors contribute fundamentally to the regeneration process and it is crucial to supply stem/progenitor cells for facilitating the regeneration in critical-sized defect (
Yin et al., 2009)
Stem cells in dental field
Adult MSCs have also been recently isolated from teeth, including dental pulp stem cells (DPSCs) (
Gronthos et al., 2000), stem cells from human exfoliated deciduous teeth (SHED)(
Miura et al., 2003), periodontal ligament stem cells (PDLSCs) (
Seo et al., 2004), and stem cells from apical papilla (SCAP) (
Sonoyama et al., 2006). These stem cells also can be used to regenerate oral and maxillofacial tissues. Human PDLSCs could form substantial amounts of collagen fibers and improve facial wrinkles in mice. In contrast, bone marrow MSCs failed to survive at 8 weeks post-transplantation under the conditions used for the PDLSC transplantation (
Fang et al., 2007). Another dental MSCs, isolated from miniature pig deciduous teeth, an autologous and easily accessible stem cell source, were able to engraft and regenerate bone to repair critical-size mandibular defects (
Zheng et al., 2009).
Periodontitis is a periodontal tissue infectious disease and the most common cause for tooth loss in adults. It has been linked to many systemic disorders such as coronary artery disease, stroke, and diabetes. At the present, there is no ideal therapeutic approach to cure periodontitis and achieve optimal periodontal tissue regeneration. Liu et al. explored the potential of using autologous PDLSCs to treat periodontal defects in a porcine model of periodontitis. Autologous PDLSCs were obtained from extracted teeth of the miniature pigs and then expanded
ex vivo to enrich PDLSC numbers. When transplanted into the surgically created periodontal defect areas, PDLSCs were capable of regenerating periodontal tissues, leading to a favorable treatment for periodontitis (
Liu et al., 2008). Significant periodontal tissue regeneration was achieved in both the autologous and the allogeneic PDLSCs transplantation groups at 12 weeks post PDLSCs transplantation. There was no marked difference between the autologous and allogeneic PDLSCs transplantation groups, based on clinical assessments, CT scanning, and histological examination. Lack of immunological rejections in the animals that received the allogeneic PDLSCs transplantation was observed. Interestingly, human PDLSCs failed to express HLA-II DR and co-stimulatory molecules. PDLSCs were not able to elicit T cell proliferation and inhibit T cell proliferation when stimulated with mismatched major histocompatibility complex molecules. Furthermore, prostaglandin E2 (PGE2) was found to play a crucial role in PDLSCs-mediated immunomodulation and periodontal tissue regeneration
in vitro and
in vivo. PDLSCs possess low immunogenicity and marked immunosuppression via PGE2-induced T cell anergy. A standard technological procedure of using allogeneic PDLSCs has been developed to cure periodontitis in swine (unpublished data).
Regeneration of a functional and living tooth is one of the most promising therapeutic strategies for the replacement of a diseased or damaged tooth (
Thesleff, 2003;
Murray and Garcia-Godoy, 2004;
Chai et al., 2006). Recent advances in dental stem cell biotechnology and cell-mediated murine tooth regeneration have encouraged researchers to explore the potential for regenerating living teeth with appropriate functional properties (
Ohazama et al., 2004;
Shi et al., 2005). Murine teeth can be regenerated using many different stem cells to collaboratively form dental structures
in vivo (
Young et al., 2002;
Duailibi et al., 2004;
Young et al., 2005). Dentin/pulp tissue and cementum/periodontal complex have been regenerated by human DPSCs and PDLSCs, respectively, when transplanted into immunocompromised mice (
Gronthos et al., 2000;
Seo et al., 2004). However, owing to the complexity of human tooth growth and development, the regeneration of a whole tooth structure including enamel, dentin/pulp complex, and periodontal tissues as a functional entity in humans is not possible given available regenerative biotechnologies. Although dental implant therapies have achieved long-term success in the clinic for the recovery of tooth function, the dental implants require pre-existing high-quality bone structures for supporting the implants (
Heitz-Mayfield and Lang, 2004;
Park and Wang, 2005). Reconstruction of teeth in patients without adequate bone support would be a major advance. Stem cell-mediated root regeneration offers opportunities to regenerate a bio-root and its associated periodontal tissues, which are necessary for maintaining the physiological function of teeth. Using a minipig model, Wataru et al. transplanted human SCAP and PDLSCs to generate a root/periodontal complex capable of supporting a porcelain crown, resulting in normal tooth function. This work integrates a stem cell-mediated tissue regeneration strategy, engineered materials for structure, and current dental crown technologies. This hybridized tissue engineering approach led to recovery of tooth strength and appearance (
Wataru et al., 2005).
If dental MSCs could be used in allogeneic bodies, the source of seed cells for dental and maxillofacial tissue regeneration would be expanded; however, little information about the immunological properties of dental MSCs appears in the literature. The minipig dental system shares several anatomical and physiological characteristics with that of humans (
Wang et al., 2007). Thus, one dental MSCs-SCAP was investigating the immunogenicity and immunomodulatory effects in a swine dental model. They found that SCAP were weakly immunogenic and suppressed T cell proliferation
in vitro through an apoptosis-independent mechanism (
Ding et al., 2010).
Procedures to store and preserve MSCs for future clinical applications have not been explored. So human freshly isolated SCAP (fSCAP) was compared with cryopreserved SCAP (cSCAP) in terms of cell viability, colony-forming efficiency, cell proliferation rate, multilineage differentiation potential, profiles of MSC markers, karyotype analysis, and immunological assays. cSCAP showed a similar viable cell ratio, colony-forming efficiency, cell proliferation rate, multilineage differentiation potential, MSC surface markers, apoptotic rate, and G-banded karyotype when compared to fSCAP. There was no significant difference between fSCAP and cSCAP with regard to immune properties. In addition, cSCAP of miniature pig possessed the similar proliferation rate, differentiation potential, and immunomodulatory function as seen in fSCAP. This study demonstrates that cryopreservation does not affect the biological and immunological properties of SCAP, supporting the feasibility of SCAP cryopreservation in nitrogen (
Ding et al., 2010).
ES cells and iPS for understanding mechanisms of osteoblast differentiation, pathological mechanisms and bone regeneration
The breakthrough in development of embryonic stem cell and induced pluripotent stem cells (iPS cells) provide an alternative and exciting cell source for regenerative medicine. Because of ethical issue on the hESC cells for regeneration, there is scarcity of data on these cells for the potential, particularly in bone biology. The utilization of ES cells and iPS cells would be a significant advance in bone biology, because these cells can not only be genetically manipulated, but also provide an infinite resource of osteogenic precursors for bone formation, if the concern of histocompatibility and tumorigenesis could be conquered. In addition, the results from hESC models will certainly shed light on an individual’s own iPS cells. To translate these cell application to clinical scenarios, a large number of cells are required to meet the demand by developing a protocol that enables to generate a population of osteoblast lineage. It has been recently shown that hESCs can be induced into osteoblasts
in vitro with or without embryonic body formation by culturing them in medium supplemented with osteoblast differentiation medium (
Xu et al., 2004;
Tong et al., 2007;
Kim et al., 2008;
Brown et al., 2009;
Kärner et al., 2009;
Thyagarajan et al., 2009;
Woo et al., 2009;
Elçin et al., 2010;
Lee et al., 2010;
Mahmood et al., 2010). However, these studies have not demonstrated inconsistent bone formation
in vivo. Further works need to be performed to define osteoblast lineage progression derived from hESCs in both
in vitro and
in vivo conditions.
Recently, it has been demonstrated that somatic mouse and human cells can be reprogrammed into an ESC-like state-induced multipotent stem cells (iPS cells) by four transcriptional factors from different types of cells (
Takahashi and Yamanaka, 2006;
Takahashi et al., 2007;
Aoi et al., 2008;
Nakagawa et al., 2008;
Okita et al., 2008), and the iPS cells are able to generate all the types of cells in the body of a transgenic mice by an innovative work including cells in skeletal tissues (
Zhao et al., 2009;
Zhao et al., 2010a,
2010b), indicating the resulting iPS cells preserve a nature of germinal line cells with an ability of self-renewal and multiple differentiation potential. A few preliminary studies have been carried out for examining the induction of iPS cells into osteoblasts
in vitro. Using retroviral-delivered four factors (Oct4, Sox2, c-myc and Klf4), mouse tail fibroblasts were induced into iPS cells and treatment with transforming growth factor beta1 (TGF-β1) in the presence of retinoic acid enhanced generation of MSC-like cells that can further differentiate into osteoblasts (
Li et al., 2010). In addition, iPS cells have also been generated from human dental tissue origin (
Duan et al., 2010;
Tamaoki et al., 2010;
Yan et al., 2010), but there is no convincing histological data showing the potential of iPS cells in bone formation
in vivo in these preliminary works. Similar to hESCs, further work waits to be performed to demonstrate the important property of iPS cells for bone regeneration
in vivo with a suitable cell protocol that can expand the cells in a clinical scale.
Biodegradable scaffolds for bone regeneration
Regenerating new bone structure requires the design and fabrication of a porous 3D biomaterial scaffold to deliver implanted stem/progenitor cells. Several requirements have been identified to be crucial for the production of an ideal scaffold (
Bueno and Glowacki, 2009;
Zippel et al., 2010): (1) adequate mechanical property that matches the intended site of implantation; (2) hydrophilicity which can maintain nutrition for stem cell expansion and differentiation at an early stage of implantation; (3) controlled biodegradability that allows generated bone to eventually replace the scaffold itself; (4) appropriately orientated surface chemistry that favors cellular attachment, proliferation and differentiation; and (5) interconnected pores with appropriate scales to favor cells and tissue integration and vascularization. Scaffold materials include natural or synthetic polymers, ceramics, and composites. To create these kinds of scaffolds, scientists in the field of bone biology and stem cell biology should work closely with biomaterial scientists to design and evaluate the biological interactions between loaded stem/progenitor cells and scaffolds in both
in vitro and
in vivo states.
Osteogenic markers and phenotypes induced by
in vitro stimulation may have little relevance to the actual differentiation of these cells
in vivo (
Bennett et al., 1991; Derubeis et al., 2004;
Mendes et al., 2004;
De Kok et al., 2006;
Sacchetti et al., 2007;
Sudo et al., 2007). Therefore,
in vivo animal models are recognized as a proof of functionality of osteogenic capacity of the cells within the tested scaffolds. Scaffolds loaded with viable osteogenic committed cells have been implanted in immunocompromised (nude, SCID) mice and in athymic rats for a proof of function test. The available animal models currently include ectopic bone formation of subcutaneous/intramuscular implants, orthopedic bone formation using cranial defect implant and mouse/rat tibia long segmental defect.
Small molecules guide stem cell differentiation
For stem/progenitor cell-based bone regenerative medicine, utilization of bioactive factors is also an important issue for augmenting the cellular regenerative potential. These bioactive factors include systemic hormones like PTH and prostaglandins, numerous growth factors and factors involved in signaling transduction and synthetic small molecules. They are believed to play a role in guiding implanted stem cells entering the fate of osteoblast lineage, expansion and final differentiation. While PTH (1-34) has been used clinically to increase bone mass and reduce fracture risk in postmenopausal women with osteoporosis, there is increasing evidence from preclinical studies that PTH (1-34) may promote fracture healing (
Alkhiary et al., 2005;
Marsell et al., 2007;
Rozen et al., 2007;
Kaback et al., 2008;
Cipriano et al., 2009;
Warden et al., 2009). Pulsatile release of parathyroid hormone from an implantable scaffold is possible (
Wei et al., 2004;
Liu et al., 2007). Clinical studies are necessary to clarify the therapeutic utility of PTH in bone healing.
Recently it has been demonstrated that a number of small molecules can be used to selectively regulate stem cell fate and developmental signaling pathways. Such molecules will likely provide new insights into stem cell biology, and may ultimately contribute to effective medicine for tissue repair and regeneration. Those small molecules that can manipulate cells in osteoblast lineage progression will certainly provide an alternative way to increase bone formation. As many small molecule compounds have more or less side-effects when given systematically, local administration or release from a scaffold may avoid the disadvantage.
Prostaglandins (PGs) are multifunctional regulators of bone metabolism that stimulate both bone resorption and formation (
Raisz, 1999;
Fracon et al., 2008;
Blackwell et al., 2010). PGs have been implicated in bone resorption associated with inflammation and metastatic bone disease, and also in bone formation associated with fracture healing and heterotopic ossification (
Blackwell et al., 2010). However, systemic side effects have limited their clinical utility. The pharmacological activities of PGE2 are mediated through four G protein-coupled receptor subtypes, EP1-EP4. Recent studies have shown that EP2 and EP4 receptors play important roles in regulating bone formation and resorption (
Li et al., 2007). EP2 and EP4 receptor-selective agonists have been shown to stimulate local or systemic bone formation, augment bone mass and accelerate the healing of fractures or bone defects in animal models upon local or systemic administration (
Li et al., 2003;
Paralkar et al., 2003;
Tanaka et al., 2004;
Cameron et al., 2006), thus potentially offering new therapeutic options for enhancing bone formation and bone repair in humans. Purmorphamine, a 2,6,9-trisubstituted purine small molecule that was discovered through cell-based high-throughput screening from a heterocycle combinatorial library, induced differentiation of multipotent mesenchymal progenitor cells into an osteoblast lineage (
Wu et al., 2002;
Beloti et al., 2005a,
2005b). It will serve as a unique chemical tool to study the molecular mechanisms of osteogenesis of stem cells and bone development. It remains unknown for its role
in vivo for bone formation.
Wnt/Notch/BMPs signaling pathways are recognized as important mediators in regulation of osteoprogenitor proliferation and differentiation (
Klüppel and Wrana, 2005;
Sahlgren and Lendahl, 2006;
Bolós et al., 2007;
Katoh, 2007;
Bodine, 2008;
Canalis, 2008;
Chen and Alman, 2009;
Hoeppner et al., 2009;
Kubota et al., 2009;
Secreto et al., 2009;
Takada et al., 2009;
Itasaki and Hoppler, 2010;
Tamura et al., 2010;
Weber and Calvi, 2010;
Zanotti and Canalis, 2010). Discovery of small molecules that can mediate these signaling pathways will be a tool for understanding these pathways and osteoprogenitor cells. More importantly they may be translated into a therapeutic reagent for bone regenerative medicine. We used an innovated approach that combines structural and molecular biology, “
in silico” virtual screening, and biological assays to identify two small molecules as an antagonist for DKK1, an inhibitor of Wnt pathway. They stimulated osteoblast differentiation
in vitro and enhanced local bone formation when locally injected over mouse calvarias through disruption of the interaction between LRP5 and Wnt antagonist Dkk (data not published). It will be attractive to investigate the interaction of these small molecules with implanted stem/progenitor cells within a scaffold for achieving local bone regeneration (Fig.1 ).
Prospectives
Advances in developmental biology have guided the design of directed, stepwise differentiation of stem cells in ways that recapitulate the progression of embryonic development. The full comprehension of the biological processes driving the development of the engineered tissue could be pivotal to design new and more effective clinical strategies to treatment of osteoporosis and enhanced bone regeneration. We should invest more of our efforts into in vivo bone biology using new knowledge derived from stem cell biology to deeply investigate the roles of both stem cells in lineages of osteoclasts and osteoblasts during bone remodeling. Furthermore, we should translate the lessons from animal models to appreciate the complex regulation network for human stem cells in osteoporosis development. Multiple disciplinary collaborations are required to successfully develop stem cell therapy for bone regeneration.
Higher Education Press and Springer-Verlag Berlin Heidelberg
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