Advances in mesenchymal stem cell transplantation for the treatment of osteoporosis

Abstract Osteoporosis is a systemic metabolic bone disease with characteristics of bone loss and microstructural degeneration. The personal and societal costs of osteoporosis are increasing year by year as the ageing of population, posing challenges to public health care. Homing disorders, impaired capability of osteogenic differentiation, senescence of mesenchymal stem cells (MSCs), an imbalanced microenvironment, and disordered immunoregulation play important roles during the pathogenesis of osteoporosis. The MSC transplantation promises to increase osteoblast differentiation and block osteoclast activation, and to rebalance bone formation and resorption. Preclinical investigations on MSC transplantation in the osteoporosis treatment provide evidences of enhancing osteogenic differentiation, increasing bone mineral density, and halting the deterioration of osteoporosis. Meanwhile, the latest techniques, such as gene modification, targeted modification and co‐transplantation, are promising approaches to enhance the therapeutic effect and efficacy of MSCs. In addition, clinical trials of MSC therapy to treat osteoporosis are underway, which will fill the gap of clinical data. Although MSCs tend to be effective to treat osteoporosis, the urgent issues of safety, transplant efficiency and standardization of the manufacturing process have to be settled. Moreover, a comprehensive evaluation of clinical trials, including safety and efficacy, is still needed as an important basis for clinical translation.


| INTRODUC TI ON
Osteoporosis is characterized as a quantitative and qualitative deterioration of bone tissues causing increased risks of fracture. 1 It is classified as primary (with unknown cause) and secondary (with traceable aetiology) osteoporosis. Primary osteoporosis is further classified as Type-I post-menopausal (between 50 and 70 years old) and Type-II age related (more than 70 years old affecting both trabecular and cortical bone), while secondary causes of osteoporosis include hypercortisolism, hyperthyroidism, hyperparathyroidism, alcohol abuse and immobilization. 2 Diagnosis of osteoporosis is mainly on the basis of T-score, which reflects the bone mineral density (BMD) of lumbar vertebrae and the femoral necks. Under the unified definition of WHO, patients with a T-score < −2.5 standard deviation (SD) of the young female adult mean are diagnosed as having osteoporosis, while those with a T-score between −1 SD and −2.5 SD of the young female adult mean are categorized as having osteopenia. 3 Moreover, the WHO Fracture Risk Assessment Tool (FRAX) is considered to be efficient in estimating the longterm risk of fracture. 4 Currently, the prevalence of osteoporosis among people over 50 years old in Europe and the United States is 4%-6%, 5,6 while in Asia it is above 15%. 7,8 With the increasing prevalence resulting from the ageing population, osteoporosis has been recognized as a major public health concern.
The mainstream treatment of osteoporosis is to stimulate osteogenesis or inhibit bone resorption through drug-based agents. 9 Bisphosphonates, the predominant first-line drugs to treat osteoporosis, decrease bone resorption by promoting osteoclast apoptosis. 10 Alternative anti-resorption drugs include denosumab and calcitonin. 11,12 Oestrogen and raloxifene have been applied in hormone therapy to retard the process of bone breakdown and reduce fracture risk in post-menopausal women. 13 Chinese medicines, such as rhizoma drynariae 14 and icariin, 15 have been shown to maintain BMD in osteoporosis. In addition, non-pharmacological treatments such as vitamin D and calcium intake have also been used. 16 However, drug-based treatments have two obvious drawbacks: First, they cannot reverse the existing bone loss, and second, they always lead to serious side effects, including osteonecrosis of the jaw, cancer, risk of thromboembolic events, and strokes. 17 Therefore, there is an urgent need for alternative therapeutic methods for osteoporosis.
Mesenchymal stem cells (MSCs) are a breed of undifferentiated cells with self-proliferation and multi-linage differentiation capabilities, which have been proven to be closely related to the progression of osteoporosis. 18 During recent decades, MSCs are high-profile, not only because their widespread application in basic research, but also because their potential capabilities to develop therapeutic strategies for a wide range of pathophysiological disorders in regenerative medicine. 19 MSCs also have promising application in the treatment of osteoporosis.
In this review, we summarize the effects, mechanisms, and potential clinical applications of MSCs in the field of primary osteoporotic therapy. Meanwhile, reported progress in preclinical studies as well as several strategies aiming to enhance the therapeutic effects of MSCs is discussed. Furthermore, we introduce recent completed or ongoing clinical trials. Finally, the major obstacles to the development of MSC transplantation and future trends are discussed. • Only original research articles, but not reviews, were included.

| ME THODS
• Studies based on MSC transplantation in osteoporotic models, including the treatment of systematic osteoporosis, osteoporotic fractures, and bone defects under osteoporotic conditions, were included.
A total of 1723 articles were retrieved after the initial search of the databases and then 230 reviews were excluded. After screening the titles and abstracts, 1410 articles were excluded mainly because they were not considered to be of relevance to the current analysis, or they were letters, editorials, or duplicate reports. Among the 83 potentially relevant studies, 42 were further excluded after reviewing the full texts because 29 studies were unrelated to the treatment of osteoporosis, 12 studies were unrelated to stem cell therapy and one paper represented repetition of the same studies. Reference tracking was performed on the full texts of the resulting articles to find missing articles that met the inclusion criteria. Two articles fulfilled the inclusion criteria. The final number of included articles was 43 ( Figure 1A). During the last decade, the number of publications in this field has been increasing year by year, which indicates the research value and practical significance of cell therapy ( Figure 1B).
Among the included studies, bone marrow mesenchymal stem cells (BMMSCs), and adipose-derived mesenchymal stem cells (ASCs) were the most common MSCs used to treat osteoporosis, accounting for more than three quarters of the total. Dental related MSCs and MSCs from other tissue sources have also received attention in recent years ( Figure 1C).

| MSC s IN THE PATHOG ENE S IS OF OS TEOP OROS IS
The pathogenesis of primary osteoporosis is generally recognized as the imbalance between bone formation and resorption during bone reconstruction, in which the speed of bone absorption is greater than that of bone formation, leading to increased bone turnover.
Homing disorders, impaired capability of osteogenic differentiation, and senescence of MSCs are important pathogeneses of primary osteoporosis. An imbalanced microenvironment and disordered immunoregulation also have key impacts on the occurrence and development of osteoporosis ( Figure 2).

| Homing disorders
Homing is the first step of bone repair, in which MSCs migrate to bone marrow to exert a local functional and restorative role. Common knowledge is that MSCs follow similar steps to leukocyte homing. 20 F I G U R E 1 Overview of the included articles for mesenchymal stem cell (MSC) transplantation in the treatment of osteoporosis. A, Flow diagram illustrating the study screening and inclusion process. B, Statistics for the numbers of publications in different years. C, Types of cells of the included articles. ASCs, adipose-derived mesenchymal stem cells; BMMSCs, bone marrow mesenchymal stem cells; DPSCs, dental pulp stem cells; SHEDs, stem cells from human exfoliated deciduous teeth, PDSCs, placenta-derived mesenchymal stem cells; TMSCs, tonsil-derived mesenchymal stem cells; UCMSCs, umbilical cord blood mesenchymal stem cells The first step is the cells contact with the endothelium by tethering and rolling, bringing about the cells decelerating in bloodstream. The second step is the activation of cells by G-protein coupled receptors, and integrin-mediated, activation-dependent arrest come next in the third step. The last step is the cells migrate through endothelial cells and underlying basement membrane.
In the case of reduced homing ability, it is difficult to ensure that enough MSCs can reach the damaged tissue, which hinders bone repair. 21 Sanghani et al 22 showed that both ageing and osteoporosis impaired MSC migration, and this might be referable to a significant reduction in bone formation in patients with osteoporosis.
More importantly, their study emphasized the positive effect of C-X-C motif receptor 4 (CXCR4) overexpression on MSC migration.
Haasters et al 23 found MSCs from patients with osteoporosis showed a surge in the migration upon bone morphogenetic protein 2 (BMP-2) stimulation, as well as their invasion increased significantly upon BMP-2 or BMP-7 stimulation. Nevertheless, the invasion and migration capacity decreased significantly compared with those of the healthy controls. Therefore, increasing the total number of MSCs through cell transplantation or enhancing the homing of MSCs through gene modification or targeted peptides would be helpful to solve this problem.

| Impaired capability of osteogenic differentiation
Common mesenchymal progenitor cells differentiate into various types of skeleton-related cells is determined by multiple transcription factors and signalling pathways. The initial step in osteoblastic differentiation is the determination of a MSC to become an osteoprogenitor, in which mesenchymal progenitor cells are directed to preosteoblasts by runt-related transcription factor 2 (RUNX2), while chondrocyte and adipocyte differentiation are inhibited. 24 Next, RUNX2 and Osterix (OSX) guide preosteoblasts to immature osteoblasts expressing bone matrix protein genes, completely eliminating the potential for chondrocytic differentiation. 25 Furthermore, the BMP signalling pathway is generally acknowledged to play important roles in regulating the adipogenic and osteogenic differentiation of MSCs. 26 BMP-2 accelerates the osteogenic differentiation of stem cells. 27 However, BMP-2 can act as a potent adipogenic agent if presented together with activators of peroxisome proliferator-activated receptor γ (PPARγ). 28 The reduction of osteogenic differentiation is the core of os-

| Senescence
Osteoporosis is also associated with the senescence of MSCs. Zhou et al 32 discovered that the number of MSCs in elderly patients with osteoporosis was much lower than that in young people; the doubling time in MSCs from the older was 1.7-fold longer than those from the younger subjects, and the content of β-galactosidase re-

| Imbalanced microenvironment
Bone remodelling is a complex coordinated event requiring various cell types to activate synchronously in the microenvironment to ensure that both bone formation and bone resorption occurs successively to sustain bone mass. 34 This process starts at the initiation stage by activating osteoclasts under the regulation of osteoclastogenic factors, including receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF), 35 followed by osteoblast-mediated bone formation. In this process, exosomes are regarded as paracrine regulators. The number of mature phenotypes differentiate from osteoclasts stimulated by osteoclast precursor-derived exosomes is significantly larger than that in the absence of exosomes. 36 Nevertheless, osteoblast-derived exosomes which contain RANKL can arouse osteoclast formation by activating RANK signalling in osteoclast precursors through the RANKL-RANK interaction. 37 Xu et al 38 reported the existence of microRNAs (miRNAs) in exosomes during BMMSC osteogenic differentiation, which have been proven to repress adipogenesis and activate osteogenesis by enhancing key osteoblast signalling molecules. Moreover, this cycle is in the charge of bone lining cells and osteocytes. 39 Several coupling factors, including BMP, transforming growth factor β (TGF-β), fibroblast growth factor (FGF), insulin-like growth factor (IGF) and platelet-derived growth factor (PDGF), are also involved in the process. 40,41 Dalle et al 42 found a lower OPG (osteoprotegerin)/RANKL ratio in the supernatants of osteoblastic culture from patients with osteoporosis than that from normal donors, which caused an alteration of osteoblastic F I G U R E 2 Mesenchymal stem cells (MSCs) in the pathogenesis of primary osteoporosis. Homing disorder results in a decreased number of MSCs in bone tissue under osteoporotic conditions. Impaired osteogenic ability and enhanced adipogenic ability of MSCs leads to less mature osteoblasts and more adipocytes. Senescence of MSCs further aggravates the imbalance of osteoblasts and adipocytes. In addition, abnormal activation of immune cells and impaired immunoregulatory ability of MSCs causes immune disorders in the bone niche, with altered cellular interactions and imbalanced paracrine secretion of many key signalling factors, such as RANK-RANKL-OPG axis. ↑indicates an increase in the number of cells/factors.↓indicates a decrease in the number of cells/factors. (+) represents the enhancement of the process. (−) represents the inhibition of the process. BMP2, bone morphogenetic protein 2; FGF, fibroblast growth factor; HSCs, hematopoietic stem cells; IGF, insulin-like growth factor; OPG, osteoprotegerin; PDGF, platelet-derived growth factor; PPARγ, peroxisome proliferator-activated receptor γ; RANK, receptor activator of NF-κB; RANKL, receptor activator of NF-κB ligand differentiation and might contribute to the pathogenesis of osteoporosis. Abnormal miRNA levels are also involved in the occurrence of primary osteoporosis through regulating osteoclast and osteoblast differentiation. 43,44 Therefore, disorders of important factors and signalling pathways regulating MSC differentiation in the microenvironment may cause an imbalance of bone metabolism, eventually leading to osteoporosis. Exogenous MSC transplantation is expected to redress the imbalance of microenvironment by regulating related factors and signalling pathways through paracrine.

| Disordered immunoregulation
Recently, the close relationship between bones and the immune system has been recognized, particularly when both systems are activated under pathological conditions. 45  On the other hand, MSC-mediated osteoimmunology was also altered under osteoporotic conditions. Available evidence suggests that MSCs may stimulate the differentiation of Treg cells, and induce the apoptosis of the pro-inflammatory Th1 and Th17 cells. 52,53 Meanwhile, Corcione et al 54  In addition, MSCs can also affect monocytes, DCs and NKs by secreting chemoattractant molecules. 45 Therefore, the interaction between immune cells and MSCs is paramount to bone metabolism, and the abnormal levels of inflammatory factors lead to the excessive activation of osteoclasts, leading to pathologic bone destruction and bone loss.

| PRECLINIC AL INVE S TIG ATIONS
MSCs can be insulated from amount of tissues (eg bone marrow, dental pulp, adipose tissue, umbilical cord, placenta and tonsil) and selective cultured prior to clinical use. According to their capacity to differentiate towards multiple mesenchymal lineages, MSCs have shown promises for wide applications in regenerative medicine and tissue engineering. Intra-bone marrow and intra-tail venous injections are common methods for MSC transplantation to treat osteoporosis ( Figure 3).

| Direct MSC transplantation
Direct MSC transplantation has long been the focus of researchers, and the results based on osteoporotic animal models are relatively mature (Table 1) ASCs have the advantages of easy accessibility, less donor site morbidity, satisfactory proliferative capacity and the ability to differentiate into multilineage cells, including osteoblasts and adipocytes. 77 In the cell therapy of osteoporosis, ASCs have been reported as effective autologous cells. The mechanism of improving OVX-induced osteoporosis is similar to that of BMMSCs, which is mainly reflected in three aspects: (a) Significant increases in cortical thickness, bone volume density and bone load 63 , (b) improved trabecular microstructure 64 , and (c) increased serum calcium and OCN levels. 65 Ye et al 66  Dental mesenchymal stem cells have aroused research interests since their early discovery. Studies focusing on stem cells from the dental pulp of permanent and deciduous teeth to regenerate or repair non-dental tissues have proved their effectiveness in bone, skin, nervous tissue, and vascular tissue regeneration. 79 Dental pulp stem cells (DPSCs) were reported to have a higher osteogenic capability compared with that of BMMSCs, and their adipogenic potential was found to be weaker than that of BMMSCs. 80 Kong et al 70

| Gene-modified MSC transplantation
To achieve improved osteogenic and angiogenic capabilities of in osteochondral progenitors inhibits chondrogenic differentiation to enhance osteoblastic differentiation. 25 Conversely, inhibition of RUNX2 prevents MSCs from differentiating into osteoblasts. 109 OSX, a member of specificity protein 1 family (Sp1) of transcription factors with three zinc finger motifs, acts as a downstream factor of RUNX2. 110 The expression of RUNX2 plays a role at the initial  115 and stabilize newly formed vessels. 116 Chen et al 100

| Targeted modification of MSCs
To improve the bone-targeted efficacy of transplanted MSCs, tar-  During bone formation, angiogenesis and osteogenesis are mutually interdependent. 126 The application of endothelial progenitor cells (EPCs) has been shown to initiate and facilitate neovascularization. 127 He et al 128

| CLINI C AL TRIAL S
To date, clinical trials of MSC transplantation for osteoporosis have mainly focused on the application of autologous cells; however, no results have been reported (Table 3).

| Autologous ASCs
In addition to bone marrow-derived cells, ASCs have also been stud- However, the trial was terminated and no results were reported (NCT01532076).

| CHALLENG E S IN CLINI C AL TR ANS FORMATION
Although preclinical experiments using MSCs to treat osteoporosis have been established for years with positive effects and recognized mechanisms, there are still many challenges and hurdles to be faced in the process of clinical transformation, including safety issues, transplant efficiency and standardization of proliferation and the manufacturing processes.

| Transplant efficiency
A majority of MSCs were trapped inside the lungs following intravenous infusion, which was termed the pulmonary first-pass effect. 141 After the MSCs were concentrated in the lungs after intravenous injection, 1-2.7% of the MSCs migrated to each organ, of which less than 1/8 of the MSCs homed to the bone marrow. 142 Huang et al 143 used an in vivo imaging system (IVIS) to observe the number of MSCs homing at different time slots after transplantation. They found that after intravenous injection, MSCs were initially retained in lungs for around 8-9 days and then gradually remigrated to the fracture site. It was also reported that intra-bone marrow injected MSCs could rapidly home to damaged bone tissue; however, the apoptosis rate was high, and less than 3% remained at the fracture site after 5 weeks. 143  the standardization of MSC growth and their functional amplification, which is a mandatory objective of cell therapies. However, no unified standardized process has been proposed so far. 146 Next, there is no unified standard for the MSC injection volume at present and 1-5 × 10 6 /kg is commonly used in animal experiments. 147 The last issue is that different administrations might lead to different therapeutic effects. Agata et al 148  The results indicated that intra-bone marrow administration of pure MSCs might be a safer and more effective method to treat osteoporosis.

| PER S PEC TIVE S
Recently, growing attention has been focused on extracellular vesicles (EVs), which are secreted by MSCs 149 and play a critical role in cell-cell communication. 150 Unlike MSCs, implanted EVs interact with their targets via signal transduction by docking at the plasma membrane of the target cell and/or via releasing the bioactive cargo upon fusion or endocytosis followed by fusing with the delimiting membrane of the endosomal compartment in bone-remodelling microenvironment. 149,151 In addition to inhibiting the inflammatory response 152 and promoting vascularization, 153 which are similar to the effects of MSC transplantation, EVs have been found to promote bone formation by repairing the function of impaired MSCs 150 and improving the activity of osteoblasts, 154 suggesting that EV is a prospective therapeutic target for osteoporosis. 155 Li et al 156

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

AUTH O R CO NTR I B UTI O N S
JY searched the literatures and wrote the paper. PZ and XZ searched the literatures and revised the manuscript. LL and ZY conceived the review, revised the manuscript, final approval of the manuscript and financial support. All authors read and approved the final version of the manuscript.

CO N S E NT FO R PU B LI C ATI O N
All authors agree to submit the manuscript for consideration for publication in the journal.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article as no new data were created or analysed in this study.