key: cord-0016204-ctnjvux0 authors: Shim, JeongHyun; Kim, Kyoung‐Tae; Kim, Kwang Gi; Choi, Un‐Yong; Kyung, Jae Won; Sohn, Seil; Lim, Sang Heon; Choi, Hyemin; Ahn, Tae‐Keun; Choi, Hye Jeong; Shin, Dong‐Eun; Han, Inbo title: Safety and efficacy of Wharton's jelly‐derived mesenchymal stem cells with teriparatide for osteoporotic vertebral fractures: A phase I/IIa study date: 2020-12-16 journal: Stem Cells Transl Med DOI: 10.1002/sctm.20-0308 sha: 10d64b19d9c93653709ebc3456377146c695a072 doc_id: 16204 cord_uid: ctnjvux0 Osteoporotic vertebral compression fractures (OVCFs) are serious health problems. We conducted a randomized, open‐label, phase I/IIa study to determine the feasibility, safety, and effectiveness of Wharton's jelly‐derived mesenchymal stem cells (WJ‐MSCs) and teriparatide (parathyroid hormone 1‐34) in OVCFs. Twenty subjects with recent OVCFs were randomized to teriparatide (20 μg/day, daily subcutaneous injection for 6 months) treatment alone or combined treatment of WJ‐MSCs (intramedullary [4 × 10(7) cells] injection and intravenous [2 × 10(8) cells] injection after 1 week) and teriparatide (20 μg/day, daily subcutaneous injection for 6 months). Fourteen subjects (teriparatide alone, n = 7; combined treatment, n = 7) completed follow‐up assessment (visual analog scale [VAS], Oswestry Disability Index [ODI], Short Form‐36 [SF‐36], bone mineral density [BMD], bone turnover measured by osteocalcin and C‐terminal telopeptide of type 1 collagen, dual‐energy x‐ray absorptiometry [DXA], computed tomography [CT]). Our results show that (a) combined treatment with WJ‐MSCs and teriparatide is feasible and tolerable for the patients with OVCFs; (b) the mean VAS, ODI, and SF‐36 scores significantly improved in the combined treatment group; (c) the level of bone turnover markers were not significantly different between the two groups; (d) BMD T‐scores of spine and hip by DXA increased in both control and experimental groups without a statistical difference; and (e) baseline spine CT images and follow‐up CT images at 6 and 12 months showed better microarchitecture in the combined treatment group. Our results indicate that combined treatment of WJ‐MSCs and teriparatide is feasible and tolerable and has a clinical benefit for fracture healing by promoting bone architecture. Clinical trial registration: https://nedrug.mfds.go.kr/, MFDS: 201600282‐30937. Osteoporosis and osteoporotic fractures lead to decreased life quality and high health care costs. An osteoporotic fracture can be defined as a fracture resulting from a fall from a standing height or less, without major trauma such as a motor vehicle accident. Osteoporotic vertebral compression fractures (OVCFs) are the most common single osteoporotic fractures worldwide. [1] [2] [3] The incidence of OVCFs increases with age, with a higher rate in women than in men, and OVCFs carry an increased risk of mortality. 2 Osteoporosis is thought to be caused in part by a decreased number of mesenchymal stem cells (MSCs) and their preferential differentiation into adipocytes rather than osteoblasts in the aging skeleton. 4, 5 This could lead to decreased osteoblast number and function and increased bone marrow fat in the aging bone. 4,6 Therefore, age-related decreases in MSC number and function may result in diminished bone formation and compromised bone microarchitecture, leading to further vertebral fractures and reduced fracture healing. 4, 7 The goals of treatment for OVCFs include back pain relief, restoration of function, and prevention of future fractures. Conservative treatments, including bed rest, pain medication, and a thoracolumbar hyperextension brace, are usually recommended to alleviate back pain. However, the failure of fracture healing after conservative treatment can lead to intractable back pain associated with nonunion and increased morbidity and mortality rates. 8 The incidence of nonunion after conservative treatments is approximately 13.5%. 8, 9 Once nonunion advances to osteonecrosis and delayed vertebral collapse (Kummell's disease), the collapsed vertebrae causes a progressive kyphotic deformity and severe neural tissue compression with neurological deficits. 10, 11 According to previous randomized controlled trials, vertebral augmentation procedures (percutaneous vertebroplasty or balloon kyphoplasty) cause serious complications, including bone cement leakage, infection, and pulmonary embolism, 8, 12 and are not superior to sham procedures for pain relief. 8 In addition, vertebral augmentation procedures do not affect bone metabolism and cannot prevent new fractures, but they can increase the risk of adjacent vertebral compression fractures. 12 Therefore, vertebral augmentation procedures may be applied to control back pain for those in whom conservative treatment fails or, accompanying immobilization, carries serious risks. 12 In terms of fracture healing after osteoporotic fractures, both MSCs and teriparatide (recombinant human parathyroid hormone [PTH] 1-34, an osteogenic osteoporosis agent) have been studied. Transplantation of MSCs has gained considerable attention to treat osteoporosis and OVCFs because implanted healthy MSCs could be differentiated into osteoblasts and reduce the susceptibility of fractures by facilitating new bone formation, as has been shown in animal studies. 4, 7, 13, 14 Teriparatide is a form of PTH consisting of the first (Nterminal) 34 amino acids and is an effective anabolic agent. Compared with bisphosphonate (antiresorptive drugs), teriparatide has been proven to induce bone formation through stimulation of osteoblast proliferation, prevention of osteoblast apoptosis, and increased osteoblast activity. 11 In addition, teriparatide could be effective in preventing secondary OVCFs, increasing spine bone mineral density (BMD), and accelerating fracture healing and union rate in • Compared with bisphosphonate (antiresorptive drugs), teriparatide has been proven to induce bone formation through stimulation of osteoblast proliferation, prevention of osteoblast apoptosis, and increased osteoblast activity. • The mechanism involved in bone formation of teriparatide is the activation of resident mesenchymal stem OVCFs. 11, 15, 16 Actually, the mechanism involved in bone formation of teriparatide is the activation of resident MSCs. 4, 17, 18 (e) participants in other clinical trials within 30 days of the start of the trial; and (f) severe comorbidities that could affect or interfere with therapeutic outcomes, including tumor, infection, uncontrolled hypertension and diabetes, renal disease, or liver disease. After informed consent was obtained, eligible subjects were randomized to teriparatide treatment alone or in combination with WJ-MSCs. A target sample size of 20 subjects randomized 1:1 to combination therapy or treatment with teriparatide alone was selected to be sufficient to provide an initial assessment of the safety and efficacy of combination therapy in patients with OVCFs. After random allocation, clinical, rheological, and radiological evaluations were performed at baseline and at 1, 3, 6, 9, and 12 months from the start of treatment. The primary endpoints were assessments of the safety and tolerability Safety and tolerability were assessed in all subjects who received teriparatide alone or combined treatment of teriparatide and WJ-MSCs at each visit. Study physicians assessed vital signs and laboratory examination of blood samples, adverse events (AEs), and serious AEs (SAEs) and determined whether each AE or SAE was related to the study treatment. The impact of study treatment on pain, function, and health-related quality of life was assessed by a VAS, ODI, and SF-36 questionnaire from the baseline and at each visit (1, 3, 6, 9, and 12 months after treatment initiation). Differences in mean VAS, ODI, and SF-36 values between control and experimental treatment groups were evaluated. BMD was measured at the posteroanterior lumbar spine, total hip, and femoral neck by DXA using a Hologic QDR 4500A densitometer (Hologic, Waltham, Massachusetts). All scans of individual subjects were performed on the same densitometer. Lumbar spine BMD was measured from lumbar 2 to lumbar 4 vertebral bodies. Differences in mean BMD T-scores between control and experimental treatment groups were evaluated at baseline and at the 6-and 12-month time points after treatment initiation. Fasting morning blood samples were obtained at each visit (baseline, 3, 6, 9, and 12 months after treatment initiation). Serum osteocalcin, a marker for bone formation, was measured via electrochemiluminescent BMD can also be assessed with quantitative computed tomography (CT), but quantitative CT was not available for BMD measurements at our hospital. Spine CT was performed at baseline and 6-month and 12-month follow-ups to assess the occurrence of new fractures and fracture progression and to determine changes in BMD of the index level of the OVCF using radiomic feature extraction. [20] [21] [22] Via Picture Archiving and Communication System, the average Hounsfield unit (HU) was obtained. We collected axial images of the index level of the OVCFs at baseline and 6 and 12 months after treatment initiation ( Figure 2 ). Because the HU of the CT images used in this study had a value of −1024~1024, it was normalized to a value of 0~255 pixels for the purpose of the extraction of radiomic features. 22 To analyze bone architecture from the normalized CT image, the region of interest (ROI) was defined as the rectangular box in an axial plane of the fractured vertebra, and the ROI was extracted using rectangle manual segmentation ( Figure 2 ). Radiomic feature extraction was performed using the PyRadiomics 2.2.0 library to analyze the texture in the ROI and included first-order statistics and gray-level cooccurrence matrix (GLCM). 20 to adjust for multiple testing using the sequentially rejective Bonferroni method. For analyzing the significant difference BMD of fractured bone between the control and experimental groups, a twoway repeated measures analysis of variance (ANOVA) with two factors (month and feature value) was used. 24 Using the two-way repeated measures ANOVA, the P value was less than .05. It was interpreted that the control and experimental groups showed a significant difference, and the specific features were selected. Twenty-eight subjects with OVCFs were assessed for eligibility, and 20 subjects were enrolled through the randomization: 10 in the control Table S1 . There were no significant differences in the baseline characteristics between control and experimental groups. AEs occurred in four (40%) subjects in the experiment group and three (30%) subjects in the control group (Table S2 ). According to the Common Terminology Criteria for Adverse Events (CTCAE), version 5.0, there were no grade 4 or 5 AEs. In the control group, three subjects complained of similar AEs such as nausea, vomiting, or dizziness after subcutaneous teriparatide injection, and they dropped out of the study. AEs or SAEs were found in four subjects in the experimental group. One subject was diagnosed with urinary tract infection due to Candida albicans 2 weeks after stem cell injection and fully recovered after antifungal therapy. However, the subject dropped out because of follow-up loss thereafter. Another subject complained of redness, itching, pain, and swelling at the site of injection after intravenous infusion of stem cells. The injection site reaction was mild and disappeared within 2 weeks. In another subject in the experimental group, pulmonary emboli were found on chest CT during the study. Five years before the start of the clinical trial, a lung examination was performed because of blood-tinged sputum and had shown no significant radiological findings. At day 30 after intravenous infusion of stem cells, a chest CT scan was performed because of blood-tinged sputum appearing after taking pain relief medications The mean ± SD VAS was 6.4 ± 1.1 in the experimental group and 7.1 ± 1.1 in the control group, and there was no statistical difference (P = .368) at baseline. From the first month of the treatment, the experimental group improved significantly compared with the control group. Although subjects in the control group improved during the study period, 0.42 ± 0.14, respectively, at baseline (P = .537). After treatment, the two groups showed an increase in CTX up to 6 months, which then decreased and showed no statistical difference. The experimental and control groups were, respectively, 0.70 ± 0.07 and 0.72 ± 0.08 at baseline and 0.70 ± 0.13 and 0.64 ± 0.19 at the last follow-up, with no statistical difference between the two groups. We found no occurrence of new fracture and fracture progression along a sagittal plane. The results of feature analysis using a two-way repeated measures ANOVA are shown in Table S3 , and the feature values using PyRadiomics are shown by the variance charts of 10th percentile, mean, and energy ( Figure 6 ). For the repeated measures ANOVA, a significant difference between the control and experimental groups was shown with a P value of less than .05 for 10th percentile, mean, and energy. In the experi- The most common type of osteoporotic fracture is OVCF, affecting nearly 25% of the elderly population who are older than 50 years. 1, 25 OVCF raises the risk for new vertebral fractures 5fold in the first year, and the presence of two or more OVCFs increases the risk up to 12-fold. 26 The number of subsequent fractures is associated with increased mortality risk. 15 Back pain that persists after an OVCF is partly associated with vertebral instability (nonunion or slow-forming union) at the fractured site, which may require surgical intervention such as vertebroplasty or spinal reconstruction. In addition, once nonunion of OVCF progresses to vertebral osteonecrosis, the collapsed vertebrae cause neurological deficits resulting from a progressive kyphotic deformity and severe neural tissue compression. 10, 11 Therefore, the top priority in the treatment of OVCF is to ensure fracture healing and preventing secondary OVCFs. 8, 11 In this study, we compared the efficacy of the combined injection of WJ-MSCs and teriparatide to that of teriparatide treatment alone. The primary findings of this study were as follows: The appropriate route of stem cell administration is an essential step for a successful treatment. Osteoporosis is a systemic disease, characterized by a decrease in bone mass and a deterioration in bone microstructure leading to an increased risk of bone fractures. 32 In elderly patients with osteoporosis, the number of osteoblast progenitor MSCs is decreased, and the capacity of MSCs to differentiate into osteoblasts was found to be lower than that from healthy people. 32 It has been shown that intravenous transplantation of MSCs significantly increased BMD in osteoporotic animals. 32 18 In addition, many researchers emphasize early fracture healing after OVCFs to prevent subsequent recurrent fractures. 34 PTH was shown to induce fracture repair in animals by activating MSCs, and MSCs can also enhance bone repair by modulating the process of inflammation. 18,34 Therefore, we injected PTH and WJ-MSCs and used two routes of administration of MSCs (intramedullary and intravenous). Osteoporosis is characterized by low bone strength, which is determined by not only bone mass but also bone quality. Bone mass is mainly expressed by BMD, and bone quality is composed of microarchitecture, bone turnover rate, mineralization, and microdamage accumulation. 35 Bone turnover markers have been used for monitoring early responses to antiosteoporosis therapy because of a delayed response of BMD to clinical treatment. Because both BMD and bone turnover markers are independent and essential, many researchers have tried to study the relationship between the changes in bone turnover markers and BMD in treated and untreated patients with osteoporosis. However, the results of these studies are sometimes controversial and generally exhibit significant disparity across studies. 36 It has been reported that there is a rapid rise in bone formation marker during the first month of teriparatide treatment and a subsequent increase in bone resorption markers during the entire 24-month treatment course, and the increase in bone formation markers exceeds increases in bone resorption markers during the entire 24-month treatment course. 37 In this study, the levels of bone formation (osteocalcin) and resorption (CTX) markers were not significantly different between the control and experimental groups. In the control group, the levels of osteocalcin and CTX increased at 1 month after treatment initiation, maintained similar levels up to 6 months, and gradually decreased. However, there seemed to be a more rapid rise in osteocalcin compared with CTX. These findings are consistent with previous reports demonstrating that increases in bone formation markers exceeded increases in CTX. 37 In the experimental group, osteocalcin increased rapidly at 1 month after stem cell injection, gradually increased up to 6 months, and then gradually decreased, whereas CTX gradually increased up to 6 months and gradually decreased, showing that the increase in osteocalcin exceeded the increase in CTX during the study. Both control and experimental groups showed that early significant increases in osteocalcin (bone formation marker) were followed by a subsequent increase in CTX (bone resorption marker), showing that the increase in the bone formation marker exceeded the increase in the bone resorption marker during the study. BMD T-scores of spine and hip have been reported to increase significantly after 24 months of treatment with teriparatide. [37] [38] [39] In terms of the effect of stem cell implantation on BMD, preclinical studies of the therapeutic role of stem cell therapy in animal models of osteoporosis have shown inconsistent results, but a meta-analysis showed that stem cell implantation was associated with significantly improved BMD as compared with that observed in controls in animal models of osteoporosis. 13 In this study, BMD T-scores of the spine and hip by DXA increased in both the control and experimental groups, but there was no significant difference between them. The risk factors for vertebral nonunion have been reported to be associated with thoracolumbar fracture, decreased BMD, and posterior wall fracture. 9 Especially, BMD was negatively correlated with the osteonecrosis (intervertebral vacuum) occurrence rate. 9, 39 Thus, sensitive detection of changes in BMD is a key issue in monitoring and evaluating the individual bone health status as well as bone metabolism and bone mineral status. 40 Additionally, bone microarchitecture is very important for the treatment of osteoporosis and OVCF and assessment of bone microarchitecture in complement to a BMD exam could improve the prediction of OVCFs. 41 Texture analysis has been reported to be independent and complementary with BMD for determining the odds ratio of fractures. 41, 42 Therefore, we assessed bone microarchitecture in complement to BMD using radiomics-based feature extraction after collecting baseline CT images and follow-up CT images at 6 months and 12 months. 41, 43, 44 MSCs have been reported to be a new therapeutic strategy to treat osteoporosis and OVCF, mainly because of their ability to secrete factors that are directly or indirectly involved in bone repair, as well as their ability to graft into tissues and differentiate into functional osteoblasts. Emerging evidence suggests that inflammation exerts a significant influence on bone turnover, thereby in osteoporosis. 5 Once the bone repair process has started, the inflammatory response must be stopped to avoid more damage. 5 In this study, intravenous injection of WJ-MSCs (2 × 10 8 cells) occurred 1 week after intramedullary injection (4 × 10 7 cells) because osteoporosis is a systemic skeletal disease. The exact mechanism by which the combined injection of WJ-MSCs and teriparatide led to improvement of back pain and bone microarchitecture of the fractured vertebra in the present study remains unclear. Although we found no significant difference in BMD by DXA between control and experimental groups in this study, we assume that teriparatide enhances WJ-MSCs migration into the fractured vertebra and differentiation of WJ-MSCs into osteoblasts and intramedullary and intravenous injection of WJ-MSCs improve fracture healing by inhibiting inflammatory response. 5, 18, 19 In terms of AEs, stem cell injection-related AEs were reported in two subjects in the experimental group. After intravenous injection, one subject complained of an injection site reaction, which was mild and disappeared within 2 weeks. The other subject was diagnosed with pulmonary embolism at day 30 after intravenous infusion of stem cells. Actually, intravenous injection of MSCs leads to the accumulation of fewer than 10% of administered MSCs, with many cells captured in the lung. 33 In this study, all subjects in the experimental group did not undergo a chest CT scan. One subject had a medical history of blood-tinged sputum 5 years before the start of the study and Our study is a randomized, open-label, phase I/IIa study to determine the feasibility and efficacy of combined treatment of WJ-MSCs and PTH in OVCFs, and thus caution should be applied when drawing any conclusions regarding long-term safety and efficacy. In addition, our study has limitations including a limited experimental group with a small number of participants, an absence of animal study, and insufficient examination of bone turnover markers. In this study, we did not include subjects who received WJ-MSCs monotherapy and included only subjects with PTH monotherapy and subjects with combined treatments of WJ-MSCs and PTH. Based on the literature 18 showing that MSC monotherapy was less effective for repairing osteoporotic vertebral bone defect compared with combined treatments of WJ-MSCs and PTH, we did not include a WJ-MSC monotherapy group. Second, we did not complete an animal study to explore possible mechanisms of combined treatments of WJ-MSCs and PTH, including paracrine action of transplanted WJ-MSCs and bone formation resulting from engraftment of cells and differentiation into osteoblasts. Third, we checked only two bone turnover markers (osteocalcin and CTX) in our study. Bone turnover markers have been used as short-term tools for monitoring the adherence and response to treatment with antiosteoporotic agents. Unfortunately, the levels of bone turnover markers are highly variable according to multiple contributors such as patients and time for measurements. [47] [48] [49] Factors that can be adjusted and minimized, termed controllable factors, include circadian rhythm variations, food intake, exercise level, alcohol intake, seasonal variation, and medications such as oral glucocorticoids and aromatase inhibitors. 48, 49 Factors contributing to preanalytical variability that cannot be controlled, known as uncontrollable factors, include age, degree of mobility/immobility, ethnicity, presence of fracture, and menopausal state. Additionally, the aminoterminal propeptide of type 1 procollagen (P1NP), the preferred marker for bone formation, is more stable compared with osteocalcin, 47 but we did not measure the P1NP level because our hospital did not provide services for measuring P1NP. Therefore, large-scale clinical trials assessing the optimal cell dose, optimal cell administration routes, optimal biomaterials loaded with stem cells, and relevant clinical endpoints are needed to define the long-term safety and efficacy of cell therapy for OVCFs. However, we propose that combined treatments of WJ-MSCs and PTH may provide a feasible and tolerable treatment for OVCFs. The authors declared no potential conflicts of interest. 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