Current status of drug therapies for osteoporosis and the search for stem cells adapted for bone regenerative medicine
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- Mikami, Y., Matsumoto, T., Kano, K. et al. Anat Sci Int (2014) 89: 1. doi:10.1007/s12565-013-0208-8
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A number of factors can lead to bone disorders such as osteoporosis, in which the balance of bone resorption vs. bone formation is upset (i.e., more bone is resorbed than is formed). The result is a loss of bone mass, with a concomitant decrease in bone density. Drugs for osteoporosis can be broadly classified as “bone resorption inhibitors”, which impede bone resorption by osteoclasts, and “bone formation accelerators”, which augment bone formation by osteoblasts. Here, we describe representative drugs in each class, i.e., the bisphosphonates and the parathyroid hormone. In addition, we introduce two novel bone formation accelerators, SST-VEDI and SSH-BMI, which are currently under investigation by our research group. On the other hand, regenerative therapy, characterized by (ideally) the use of a patient’s own cells to regenerate lost tissue, is now a matter of global interest. At present, candidate cell sources for regenerative therapy include embryonic stem cells (created from embryos based on the fertilization of oocytes), induced pluripotent stem cells (created artificially by using somatic cells as the starting material), and somatic stem cells (found in the tissues of the adult body). This review summarizes the identifying features and the therapeutic potential of each of these stem cell types for bone regenerative medicine. Although a number of different kinds of somatic stem cells have been reported, we turn our attention toward two that are of particular interest for prospective applications in bone repair: the dedifferentiated fat cell, and the deciduous dental pulp-derived stem cell.
Due to the increase in the size of the elderly population, osteoporosis, osteoarthritic bone, joint disease, and other disorders characterized by tissue loss are currently on the rise. Thus, the development of new methods for preventing and treating osteoporosis and related conditions is of paramount importance. Generally, when bone and other bodily tissues and organs are lost or become dysfunctional, drug therapy or a transplant must be undertaken to induce their regeneration. Drug therapy is the first choice and the primary form of treatment for regenerating lost tissue or organs. However, pharmacological agents have a limited ability to promote the repair of lesioned tissue. Indeed, it is difficult to regenerate a large tissue mass or an organ with drug therapy alone. Furthermore, tissue or organ transplantation, while highly effective, poses problems with respect to the extent and source of the replacement tissue in autologous transplantation (i.e., relocation of the patient’s own tissue to the affected site). Osteoporosis, for example, often involves a systemic decrease in bone density, rendering it impossible to collect enough healthy bone from the afflicted individual for transplantation. While allogeneic transplantation (transplantation of tissue or organs from another person) is also an option, this procedure has disadvantages of its own, including chronic donor shortages and immune rejection responses.
To overcome these problems, regenerative therapy with autologous cells is now an area of intensive exploration. This strategy involves the use of the patient’s own cells to replace damaged tissue, and poses less of a burden on the patient because a limited number of cells are collected and expanded ex vivo prior to their clinical employment. Moreover, because the cells are autologous, rejection by the immune system is avoided. At present, it is possible to culture skin, cartilage, and corneal cells, among others, in sufficient quantities for utilization in regenerative medicine applications.
This review introduces the latest findings from other investigators, together with our own breakthroughs, in regard to drug therapies and cell sources for the management of tissue loss associated with bone disease.
Bone remodeling refers to the repetitive process whereby bone is replaced. Osteoclasts first resorb bone from the cancellous bone surface and the perivascular cortical bone, followed by the generation of new bone through the actions of osteoblasts (Schaffler and Kennedy 2012; Kular et al. 2012). Bone remodeling occurs throughout life and is required for the normal growth and repair of the skeletal structure. However, a number of factors can lead to osteoporosis, in which the balance of bone resorption vs. bone formation is upset in favor of the former. The result is a loss of bone mass and a decrease in bone density (Lippuner 2012).
Drugs for osteoporosis are broadly classified as “bone resorption inhibitors”, which impede bone resorption by osteoclasts, and “bone formation accelerators”, which augment bone formation by osteoblasts. Here, we describe representative drugs in each class: the bisphosphonates and the parathyroid hormone (PTH). In addition, we discuss two novel drugs that are currently under investigation by our research group for the bone regenerative medicine.
Bisphosphonate preparations are currently the most common treatment for osteoporosis. Bisphosphonates function to improve bone mass by inhibiting resorption by osteoclasts (McClung et al. 2013). Although bone mass increases accordingly, bone remodeling is halted, and aged tissue begins to accumulate. As a result, the bone is changed into a brittle and glass-like structure, which is dangerous to the patient (Rizzoli and Reginster 2011; McClung et al. 2013). If a strong force is applied to the bone, complex bone crushing can ensue.
Osteonecrosis of the jaw is another adverse event attendant to bisphosphonate use (Ruggiero and Mehrotra 2009). Bisphosphonate-related osteonecrosis of the jaw is defined as osteonecrosis affecting patients who have continuously taken a bisphosphonate for 8 or more weeks. Several aspects of osteonecrosis are poorly understood, such as the mechanism of onset and the most appropriate tactic to prevent and treat the disorder. However, “oncological conditions” reportedly predominate in the majority (94 %) of cases associated with osteonecrosis of the jaw (Bagan et al. 2009). Because cancer patients often take multiple immunosuppressant drugs (e.g., steroids, chemotherapeutics, and other agents), this implies that osteonecrosis of the jaw is an early indication of immunosuppression. Recent work using mice suggests that the onset of osteonecrosis stems from an imbalance between regulatory T-cells and T-helper 17 cells, the latter representing a subset of T-helper cells that produce interleukin-17 (Kikuiri et al. 2010).
Although bisphosphonates have strong side effects, they are presently, as noted above, the drug of choice to treat osteoporosis. Many clinical trials have demonstrated the efficacy of bisphosphonates for the treatment of osteoporosis, but their utility in managing other bone disorders, such as metastatic bone disease, is more controversial. Therefore, careful attention must be paid to the risks of employing this class of drug, while concurrently attempting to take full advantage of their benefits.
Human PTH is an 84-amino acid, single-chain polypeptide with a molecular weight of 9,425 Da. The section of human PTH between the N terminus and amino acid 34 (PTH1–34) retains the biological activity of full-length PTH (Morley et al. 2001) and is marketed as a drug. PTH aggressively stimulates the creation of new bone, and improves bone density as well as bone quality. Therefore, PTH generates strong new bone that is resistant to fracture (Esbrit and Alcaraz 2013). When PTH1–34 was first approved for the treatment of bone disorders, it seemed likely that the hormone together with a bisphosphonate would be more effective than either drug alone for increasing bone mass. Concomitant therapy combining the two drug types was thus highly anticipated. However, subsequent research indicated that the acceleration of bone turnover by PTH was canceled by the inhibition of bone resorption by the bisphosphonate, making it impossible to achieve significant additive effects of the joint pharmacological therapy (Black et al. 2003; Finkelstein et al. 2003).
PTH is normally secreted from the parathyroid gland and is imperative for regulating calcium levels in the blood. Any decrease in blood calcium content will promote PTH synthesis and secretion. The PTH that is generated stimulates bone metabolism, which in turn releases calcium from the bone into the blood. In this manner, blood calcium levels are again restored. Consequently, the administration of exogenous PTH1–34 as a drug can lead to nausea, vomiting, feelings of weakness, and other adverse events due to hypercalcemia, or excess calcium in the blood (Rizzoli and Reginster 2011).
Intermittent administration of once-daily PTH1–34 promotes osteoblast differentiation and inhibits osteoblast apoptosis. Hence, bone formation is rapidly accelerated, bone mass is increased, and bone microarchitecture is improved (Lombardi et al. 2011; Drake et al. 2011). However, in severe hyperparathyroidism, bone mass is paradoxically reduced (Lowe et al. 2007). This occurs because a persistent state of excess PTH promotes bone resorption rather than bone formation, reducing bone mass and leading to secondary osteoporosis. Experiments in which rats were continuously injected with human PTH1–34 augmented the expression of the osteoclast differentiation-promoting factor RANKL and decreased the expression of the osteoclastogenesis inhibitory factor, osteoprotegerin. PTH also decreased the expression levels of the osteoblast-specific transcription factor, osteocalcin, and other biomolecules related to bone formation (Komrakova et al. 2010). Accordingly, as with the bisphosphonates, the use of PTH1–34 necessitates extreme caution, given that the timing or the dosage of the drug might induce adverse events. Rat testing also indicated the occurrence of osteosarcoma with prolonged PTH administration (Subbiah et al. 2010). Although it remains to be confirmed that exogenous PTH similarly causes osteosarcoma in human patients, utmost care is required for long-term dosing.
Interestingly, SSH-BM-type compounds not only stimulate osteoblast activity, but also inhibit osteoclast activity in the cultured scales of goldfish (Suzuki et al. 2008). The teleost scale is a calcified tissue that contains both osteoclasts and osteoblasts (Yamada 1971; Bereither-Hahn and Zylberberg 1993). Bone matrix components, including type I collagen, osteocalcin, and osteonectin, are present in these scales, as they are in mammalian bone. Hydroxyapatite is also found in teleost scales, as in bone. Teleost scales contain as much as 20 % of the organism’s total calcium and function as internal calcium reservoirs throughout periods of increased calcium demand, such as during sexual maturation or starvation (Takagi et al. 1989). Thus, many similarities exist between the teleost scale and the mammalian membrane bone. Although the mechanism of action of SST-VEDI and SSH-BMI remains to be fully elucidated, we anticipate that these compounds will find utility as therapeutic and preventative anti-osteoporosis agents. The fact that SST-VEDI and SSH-BMI are artificially created, small synthetic chemical compounds adds to their arsenal of beneficial properties. For example, they are more readily synthesized than protein or peptide drugs, with enhanced potential for mass production. Their small size also lends the compounds to increased oral bioavailability. Therefore, it seems likely that SST-VEDI and SSH-BMI will be considerably more cost-effective than PTH and other peptide drugs as agents for the management of osteoporosis.
Adaptation of stem cells for bone regenerative therapy
At present, the candidate cell sources for use in regenerative bone therapy include embryonic stem (ES) cells, which are derived from embryos following the fertilization of oocytes; induced pluripotent stem (iPS) cells, which are created artificially from somatic cells; and somatic stem cells (tissue stem cells or adult stem cells), which are endogenous to the tissues of the body. A summary of each of these stem cell types is given below.
ES and iPS cells
ES cells are created from the inner cell mass of the blastocyst, an early-stage embryo. These stem cells have a high proliferative capacity in addition to pluripotency, indicating that they can differentiate into any of the tissues or organs of the body (Evans and Kaufman 1981; Thomson et al. 1998). The high differentiation potential of ES cells makes them a central focus of regenerative therapy, and accordingly, ES cells have been the subject of exhaustive research. A recent report finally confirmed the efficacious use of ES cells for the replacement of lost tissue (Schwartz et al. 2012). In this report, Advanced Cell Technology Inc., a Santa Monica, California-based biotechnology company, conducted a clinical trial in which 50,000 retinal pigment epithelial cells were differentiated from ES cells. The differentiated retinal pigment epithelial cells were then transplanted into one eye of a 70-year-old woman who presented with age-related macular degeneration and reduced visual acuity, and into one eye of a 50-year-old woman who presented with Stargardt macular degenerative disease. Both women regained significant visual acuity in the transplanted eye. As of 4 months after the surgery, the transplanted cells showed no observable changes and were not associated with any obvious adverse events.
The demonstration of clinical efficacy clearly raises expectations for the expansion of regenerative ES cell therapies. Nonetheless, the creation of ES cells from fertilized eggs poses ethical considerations regarding their use. Transplantation of “foreign” ES cells also runs the risk of a rejection response in the recipient. Therefore, somatic cell nuclear transplantation (SCNT) is occasionally used to create ES cells for allogenic transplantation that are derived from an enucleated embryo and the patient’s own DNA. While SCNT can resolve the problem of immune system rejection, the SCNT-generated embryo can still potentially be utilized for the creation of a cloned organism as in the case of Dolly the sheep, the first mammal to be cloned (Wilmut et al. 1997). For this reason, iPS cells are gaining interest as alternatives to ES cells for tissue and organ replacement.
iPS cells are formed when somatic cells are provided with a known transcription factor that initiates a return to the immature (or ES cell-like) state, followed by their differentiation into the component cells of somatic tissues or organs. This process is termed “reprogramming”. iPS cells are pluripotent and can differentiate into any cell type within the body. They were first successfully established from the mouse in 2006 and from the human in 2007 (Takahashi and Yamanaka 2006; Yu et al. 2007). Unlike ES cells, somatic cells from a patient’s own body can be used to create iPS cells in the absence of an enucleated embryo, indicating that iPS cells are largely free from ethical issues or the possibility of rejection by the immune system. However, the establishment of iPS cells involves genetic transfer, with a related risk of carcinogenesis. In addition, it is still unclear whether the tissues or organs generated from iPS cells are capable of long-term in vivo behavior that is similar to that of tissues and organs generated via normal developmental pathways.
Despite these concerns, we anticipate that ES and iPS cells will be of great use in applications designed to increase our understanding of disease etiology and drug actions, in addition to the regeneration of lost tissue. Nevertheless, many ethical and technological issues have yet to be overcome (Vitale et al. 2011; Zhang et al. 2011).
Somatic stem cells
Somatic stem cells are found in various tissues of the adult body. Unlike ES and iPS cells, somatic stem cells are not pluripotent but are instead multipotent. That is, somatic stem cells can differentiate into several different cell lineages, but not into all the cells, tissues, and organs of the body. Somatic stem cells, like iPS cells, are collected from the affected individual, and therefore, these cells are also largely free from ethical and immune system rejection concerns. Furthermore, there is virtually no risk of carcinogenesis with somatic stem cells, and somatic stem cells can be stored as a raw material for the production of conventional iPS cells. Because of the aforementioned advantages, somatic stem cells represent an extremely promising cell source for regenerative medicine.
Many different types of somatic stem cells are discussed in the literature; however, this review describes only the two that form the primary focus of our current research for bone repair: the dedifferentiated fat (DFAT) cell and the deciduous dental pulp-derived stem cell (DDPSC).
The “ceiling culture” of mature adipocytes isolated from the adipose tissue of mammals, including humans, yields groups of cells with a fibroblast-like morphology. These fibroblast-like cells acquire a high proliferative ability and multipotency (Matsumoto et al. 2008). The multipotent cells so-derived from adult adipocytes are called DFAT cells. DFAT cells are artificially generated cells; thus, they are fundamentally different from somatic stem cells. However, they also share essential similarities with somatic stem cells. For example, DFAT cells, like somatic stem cells, are multipotent rather than pluripotent.
DFAT cells completely lack protein expression of leptin, glucose transporter type 4, and all other adipocyte markers. On the other hand, these cells exhibit an expression profile of surface antigens that is substantially consistent with that of bone marrow mesenchymal stem cells (BMMSCs) and ASCs (Matsumoto et al. 2008), although, as noted above, DFAT cells are not ASCs. Under appropriate conditions, DFAT cells can differentiate into adipocytes, osteoblasts, chondrocytes, and other cells derived from the paraxial mesoderm, as well as into vascular endothelial cells, vascular smooth muscle cells, and myocardial cells derived from the visceral mesoderm (Yagi et al. 2004; Oki et al. 2008; Kazama et al. 2008; Sakuma et al. 2009; Jumabay et al. 2009). Notably, the transplantation of DFAT cells at an affected site improves blood flow in a lower limb ischemia model, cardiac function in a myocardial infarction model, motor function in a spinal cord injury model, and renal function in a chronic renal dysfunction model. DFAT cells also exhibit a broad range of other tissue repair abilities (Ohta et al. 2008; Nur et al. 2008).
DFAT cells can be prepared from a limited amount of adipose tissue, irrespective of the maturation state of the organism. This indicates that DFAT cells have great potential as a new cell source for regenerative medicine in patients for whom autologous stem cell transplantation was previously thought to be difficult because of conditions such as severe heart failure or aging. In addition, DFAT cells can be mass-produced in short amounts of time by relatively simple methods that do not employ genetic engineering or viral vectors. Therefore, these cells benefit from fewer complicating ethical or technical issues compared with other types of stem cells, and accordingly, clinical application of DFAT cells for tissue replacement is eagerly foreseen.
Dental pulp-derived stem cells
We next compared the differentiation potential between CD271-positive and CD271-negative DDPSCs. We originally presumed that the CD271-positive cells would more readily and rapidly differentiate into osteoblasts than the CD271-negative cells. However, contrary to our expectations, CD271-positive DDPSCs exhibited an impaired ability to differentiate into osteoblasts (as well as adipocytes) during the early stages of culture. By contrast, continuous in vitro induction for longer periods of time (12–24 days) resulted in the initiation of rapid differentiation (Fig. 6c) and a reduction in CD271 mRNA expression levels (Fig. 6d). Conversely, forced expression of CD271 inhibited the in vitro differentiation of murine mesenchymal stem cell-like C3H10T1/2 cells into osteogenic, adipogenic, chondrogenic, and myogenic lineages. These observations indicate that CD271-positive DDPSCs are limited in their capacity for differentiation and are instead upheld in an immature state.
For applications in regenerative medicine, collected DDPSCs must be expanded in vitro to ensure a sufficient number of cells for transplantation. In the steps preceding induced differentiation and transplantation into the recipient, the DDPSCs must be stored and maintained in an undifferentiated state. Nonetheless, an in-depth understanding of the intracellular mechanisms behind the perpetuation of the undifferentiated state of mesenchymal stem cells remains elusive. A more detailed investigation of CD271-positive DDPSCs may provide important clues regarding the requisite mechanisms. We propose that the provision of such clues may very well be of equal or greater significance to the field of regenerative medicine than the mere identification of an easily differentiable stem cell type.
The primary drugs approved for use in clinical practice as anti-osteoporosis agents are those that inhibit bone resorption, exemplified by the bisphosphonates. To date, the only drug known to promote bone formation is PTH1–34. Bisphosphonates and PTH1–34 are both associated with numerous adverse effects, and their administration demands caution. A safer drug that promotes osteoblast differentiation and bone formation would be extremely advantageous in bone regenerative therapy in conjunction with stem cells, because the identified drug would presumably promote bone formation by the transplanted stem cells, as well as by endogenous cells. Thus, the development of new bone formation regulators for the treatment of osteoporosis and related disorders represents a matter of great need.
The complete removal of old bone by osteoclasts during the initial stages of bone remodeling necessitates the suppression of bone formation while bone resorption is accomplished. However, the mechanism by which osteoclasts block bone formation is unknown. Recently, Negishi-Koga et al. (2011) identified semaphorin-4D as a bone formation-inhibiting protein produced by osteoclasts. Semaphorin-4D is broadly categorized as a member of the semaphorin family based on its amino acid sequence. The authors succeeded in employing primary antibodies against semaphorin-4D to treat osteoporosis in mice by promoting bone formation (Negishi-Koga et al. 2011). Semaphorin-4D is also expressed in activated T-cells, and inhibits the activation of B-cells and dendritic cells. Binding of the protein to the plexin B1 receptor on osteoblasts in turn activates the low-molecular weight G protein RhoA, thereby preventing osteoblasts from forming new bone. On the other hand, SST-VEDI-1 and SSH-BMI may inhibit RhoA activation in cultured osteoblasts (unpublished data), whereas CD271 contains a binding domain for Rho guanine nucleotide inhibitor (Rho GDI). Hence, CD271 acts as a displacement factor to release active Rho from Rho GDI (Yamashita et al. 1999). Given that the expression of CD271 in DDPSCs blocks their differentiation into osteoblasts, Rho (A) activation may somehow be involved in maintaining DDPSCs in a dedifferentiated state. Taken together, these observations indicate that semaphorin-4D/Rho signaling is a powerful new molecular target in the future development of therapies for bone formation.
The question of the most effective cell source for the replacement of bone is of critical significance. There is absolutely no doubt regarding the therapeutic utility of pluripotent cells (e.g., ES and iPS cells). However, the use of these cells has major disadvantages that have yet to be addressed, including ethical concerns surrounding the attainment of human ES cells from embryos, and the danger of tumor formation by iPS cells. When considering bone regeneration, it seems probable that pluripotency, as with ES and iPS cells, is not entirely necessary. Fortunately, somatic stem cells are largely devoid of the complications associated with ES and iPS cells. BMMSCs and umbilical cord blood cells have already been used in clinical applications, with the establishment of bone marrow and umbilical cord blood banks. On the other hand, bone marrow collection is a burdensome and painful operation for the donor, while umbilical cord blood can only be collected at the time of birth. We propose that DFAT and DDPSCs cells are among the most viable candidates as cell sources for regenerative medicine, for the reasons discussed above.
Stem cell research has the potential to make major and diverse contributions not only to the fields of embryology and regenerative medicine, but also to our understanding of normal aging and metabolic processes, as well as the canceration of normal cells. Moreover, such research is anticipated to be extremely useful for the development of new therapeutic strategies, where previous strategies have failed. Our fervent hope is that our present research yields quantifiable results that contribute toward the same.
This work was supported in part by grants from A-STEP; Adaptable and Seamless Technology Transfer Program through target-driven R&D (Exploratory Research).
Conflict of interest