SPINE Volume 39, Number 26, pp E1553-E1559 ©2014, Lippincott Williams & Wilkins
BASIC SCIENCE
Does Extracorporeal Shock Wave Introduce Alteration of Microenvironment in Cell Therapy for Chronic Spinal Cord Injury? Ju-Yup Lee, MD, Kee-Yong Ha, MD, Jang-Woon Kim, MS, Jun-Yeong Seo, MD,* and Young-Hoon Kim, MD, PhD
Study Design. Animal experimental study. Objective. To present experimental evidence for cell therapy for spinal cord injury (SCI). Summary of Background Data. In chronic SCI, the efficacy of cell engraftment has been known to be low due to its distinct pathology. Alteration of microenvironment was tried using extracorporeal shock waves (ESW) for chronic SCI, and the efficacy of cell therapy was investigated. Methods. A chronic contusive SCI model was made in 36 SpragueDawley rats. The rats were allocated into (1) control group (SCI only), (2) ESW control group (SCI + ESW), (3) IV group (SCI + intravenous transplantation of mesenchymal stem cells; MSCs), and (4) ESW + IV group (SCI + MSCs IV transplantation after ESW). ESW were applied at the energy determined by our preliminary trials. Engraftment of the cells and expressions of growth factors (brain-derived neurotrophic factor, neuronal growth factor) and cytokines (SDF-1, CXCR4, VEGF) at the epicenter were assessed. The Basso, Beattie, and Bresnahan locomotor scale was used for the clinical assessment. Results. The mean numbers of engrafted cells were higher in the ESW+ IV than that in the IV with a statistical significance. The expression of SDF-1 was higher in the ESW groups than that in the control or IV group. CXCR4 was highly expressed in the transplanted groups. The expressions of growth factors in the treated group were higher in the treated group than those in the control group. However, various statistical significances were noted. The improvement of locomotor was higher in the transplanted groups than that in the control and ESW only group. From the Department of Orthopedic Surgery, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea. *Department of Orthopedic Surgery, Jeju National University Hospital, Jeju, Korea. Acknowledgment date: April 30, 2014. First revision date: July 27, 2014. Second revision date: August 10, 2014. Acceptance date: September 9, 2014. The manuscript submitted does not contain information about medical device(s)/drug(s). No funds were received in support of this work. Seoul St. Mary’s Clinical Medicine Research Program year of 2012 through the Catholic University of Korea funds was received to support this work No relevant financial activities outside the submitted work. Address correspondence and reprint requests to Young-Hoon Kim, MD, PhD, Department of Orthopedic Surgery, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, # 222 banpodae-ro, seocho-gu, Seoul, 137-701, Korea; E-mail: [email protected] DOI: 10.1097/BRS.0000000000000626 Spine
Conclusion. At a given energy level, ESW presented more engraftment of the transplanted MSCs without any clinical deterioration in a chronic SCI. Based on this promising result and possible explanations, ESW may cause an alteration of the microenvironment for the cell therapy in chronic SCI. Key words: spinal cord injuries, mesenchymal stem cells, shock waves. Level of Evidence: N/A Spine 2014;39:E1553–E1559
P
romising results from the basic research of cell therapy for spinal cord injury (SCI) have induced human clinical trials.1–4 However, lack of knowledge about the fate of the transplanted cells and few reliable clinical results limit the availability of those clinical trials.5–7 Therefore, more accumulative evidences regarding these issues should be provided prior to safe and useful clinical trials. Timing and route of transplantation are ones of these issues that should be addressed. Transplantation in the acute phase of SCI results in a low rate of engraftment by severe inflammatory environment, and transplantation in the chronic phase results in a low rate of engraftment and functional restoration due to a glial scar in contrast.4 Although our previous study presented a successful engraftment of the transplanted cells by intravenous routes in the chronic SCI, the efficacy was lower than that of the intralesional transplantation.8 With this background, a modulation of the environment of the chronic SCI is required to enhance the efficacy of the transplantation. Extracorporeal shock waves (ESW) have been used for various orthopedic diseases over the last decades. The therapeutic mechanisms are thought to be the induction of neovascularization and production of growth factors by the focused shock waves in a theoretical manner. Clinical trials for nonunions or delayed unions of fracture and chronic tendinitis have presented favorable results.9 Not only these orthopedic diseases but also myocardial ischemia was also experimentally tried with these theoretical backgrounds.10 With this background, we hypothesized that an application of ESW in the chronic phase of SCI could alter the microenvironment of the injured tissue and promote the engraftment of the transplanted stem cells. We studied possibilities of ESW as an enhancing method for cell therapy by investigating the engraftment of the stem www.spinejournal.com
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Alteration of Microenvironment in Cell Therapy for Chronic Spinal Cord Injury• Lee et al
cells and expression of the related growth factors and cytokines as well as the clinical results.
MATERIALS AND METHODS SCI Model and Group Allocation A total of 36 adult male Sprague-Dawely rats (body weights ranging between 290 to 340 g) were used. All animals were kept under standardized conditions (2 rats per cage, 20–24°C, 45%–65% humidity and a 12-hr of daily light) and given free access to standard food and drinking water. The rats were anesthetized with intraperitoneal injection of ketamine (50 mg/kg) and Rompun (2 mg/kg) after body weight measurement. Briefly, backs of the rat were shaved and sterilized with antiseptic Betadine. Preoperatively, 5-mg gentamicin was intramuscularly administrated. The spinal cords of the rats were exposed by performing laminectomy at T9 after exposure of the paravertebral muscles from T8 to T10. An SCI was induced by a 25-g·cm contusion using the Multicenter Animal Spinal Cord Injury Study impactor (a rod weighing 10 g and dropped from a height of 2.5 cm). Postoperatively, 5-mg ketoprofen was administrated for 3 days, and the bladder was manually emptied during the experiment. At 4 weeks postinjury, the rats were randomly assigned to one of the following four groups: (1) the control group (n = 9, SCI only), (2) the ESW control group (n = 9, SCI + ESW only), (3) the IV group (n = 9, SCI + intravenous mesenchymal stem cell [MSC] transplantation), and (4) the ESW + IV group (n = 9, SCI and IV MSCs transplantation after ESW). All surgical and physical therapies and the presurgical and postsurgical animal care were provided in accordance with the Laboratory Animal Welfare Act and the Guidelines and Policies for Rodent Survival Surgery, as provided by the Animal Studies Committee of the Catholic University of Korea (IACUC approval NO.2013-0070-02).
Preparation of MSCs Bone marrow was obtained from the femoral bone of rats. The femoral bone was harvested and both ends were cut. Bone marrow was aspirated with an 18-gauge needle and then diluted to 45 mL with Dulbecco’ Eagles medium (SigmaAldrich, Milwaukee, WI) supplemented with 20% heatinactivated fetal bovine serum (GibcoBRL, Grand island, NY), 2 mM L-glutamate (Sigma-Aldrich), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (Sigma-Aldrich). The bone marrow aspirates were plated and then incubated in a humidified atmosphere of 5% CO2 at 37ºC. For selecting the MSCs, the nonadherent cells were eliminated by replacing the medium 4 days after cell seeding. For each passage, the cells were plated at about 8000 cells/cm2 and were grown to confluence. Flow cytometric analysis of the cultured MSC was performed. In brief, the cells were detached with 0.05% trypsin-EDTA solution (Sigma-Aldrich) for 3 minutes and washed twice with PBS containing 0.1% bovine serum albumin. For direct assays, aliquots of the cells at a concentration of 1 × 106 cells per mL were immunolabeled at room temperature for 30 minutes with the following antibodies: FITC-conjugated CD 45, 29, and 90. MSCs are known to have the immunophenotype E1554
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of CD 29, and 90, and they lack the CD 45 hematopoietic immunophenotype. All the monoclonal antibodies were purchased from Pharmingen/Becton Dickinson (Franklin Lakes, NJ). Mouse immunoglobulin G1-FITC or mouse immunoglobulin G1-PE was used as an isotype-matched control. The labeled cells were analyzed by a FACSCalibur flow cytometer (Becton Dickinson) with the use of CellQuest software.
Determination of ESW Energy Level and Application A pilot study was conducted to determine the energy level of ESW. The Swiss Dolorcast (EMS, Lyon, Switzerland) with 10-mm focus applicator was used for ESW devices. This device was clinically applied for therapeutic purposes in various orthopedic diseases such as nonunion or delayed union of fracture, tendinitis, and avascular necrosis of femoral head. Six rats were used after laminectomy without any SCI. Three levels of energy (level 1, 0.01 mJ/mm2; level 2, 0.04 mJ/mm2; and level 3, 0.11 mJ/mm2) were applied at 1000 impulses. Neurological impairment was not noted in all subjects after ESW at all levels of energy. However, histological examination revealed cystic changes at the level 3 of ESW application region. There were no significant histological changes at level 1 or 2 (Figure 1). Based on this preliminary study, the energy level 2 (0.04 mJ/mm2) at 1000 impulses was chosen.
Transplantation of MSCs
In the IV and ESW + IV groups, the fourth passage 1 × 106 MSCs with a 0.1 mL total volume was injected through the tail vein after labeling with PKH26 (red fluorescence, 10−3 M, Sigma-Aldrich) at 4 weeks postinjury. In the ESW + IV group, ESW were applied with a determined level 24 hours before transplantation.
Histological Analysis Three rats in each group were assigned to immune staining for histological analysis. Each injured spinal cord was obtained after transcardiac perfusion at 6 weeks post-transplantation. The tissue was cut to 5-μm thickness. Immunofluorescence staining was performed for histological analysis to trace the transplanted cells and expression of chemokines (SDF-1, VEGF, and CXCR-4). The primary antibodies to the SDF-1 (stromal cell–derived factor-1 a, 1: 20, Abcam, Cambridge, England), VEGF (vascular endothelial growth factor, 1:50, Santa Cruz, Dallas, TX), and CXCR-4 (CXC chemokine receptor 4, 1:20, Abcam) were used. The details followed the previously described procedures.6 To address the efficacy of engraftment of the transplanted cells, the positive cells with colocalization by expressing DAPI, PKH26 (red fluorescence) in highpowered (×400) magnification were counted using a confocal microscope. The means of the counted numbers at 6 separate fields in each rat were recorded and used for statistical analysis.
Expression of Growth Factors and Chemokines Western blot was used for the evaluation of neurotrophic factors and chemokines in the injured spinal cord. Spinal cord tissue blocks (∼1.5-cm length) were harvested encompassing the epicenter and stored at −80°C. The samples were December 2014
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Alteration of Microenvironment in Cell Therapy for Chronic Spinal Cord Injury• Lee et al
Figure 1. Histological examination of the spinal cord under each extracorporeal shock waves (ESW) energy level (H-E stain). Three different energy levels of ESW were tried after laminectomy. Although no neurological impairment was noted at all energy levels, cystic changes were noted at the energy level 3. Representative images on (A, B) energy level 1 (0·01 mJ/mm2) and (C, D) energy level 2 (0·04 mJ/mm2) did not show any gross histological changes. E and F, At energy level 3 (0·11 mJ/mm2), cystic changes were noted on posterior part in the epicenter of the subjected spinal cord. Ant. indicates anterior; Post, posterior.
homogenized on tissue protein extraction reagent (Thermo scientific, Rockford, IL) with 1 mM PMSF, 10 mg/mL aprotinin, and 5 mg/mL leupeptin. The lysate was centrifuged at 16,000 rpm for 10 minutes at 4°C. The proteins were separated by SDS-polyacrylamide gel electrophoresis and they were transferred to a polyvinylidene difluoride membrane (Hybond-P, Amersham Pharmacia Biotech, Buckinghamshire, England). The membrane was blocked with 3% bovine serum albumin for 1 hour in Tris-buffered saline (0.1% Tween-20, 20 mM Tris-HCl, 137 mM NaCl, pH 7.4) and then incubated with the primary antibodies overnight at 4°C. The antibodies used for immunoblotting were as follows: brain-derived neurotrophic factor (BDNF; 1:700, Santa Cruz), neuronal growth factor (NGF; 1:500; Santa Cruz), SDF-1 (1:500, Abcam), VEGF (1:500, Santa Cruz), and CXCR-4 (1:500, Abcam). After the membranes were washed, they were incubated with secondary peroxidase-conjugated antimouse antibodies (Amersham Pharmacia Biotech) that were diluted 1:2000 in Tris-buffered saline with 0.01% Tween 20. An antibody detection system (ECL, Thermo scientific) was used, and the membranes were exposed to LAS 3000 (Biorad, Hercules, CA). The protein band intensities were quantified with a multi gauge V 3.0 software (Fuji photo film, Tokyo, Japan).
BBB Locomotor Test Basso, Beattie, and Bresnahan (BBB) locomotor rating scale values were recorded every week. Two researchers independently recorded BBB scale after 2 minutes of observation. BBB scales were checked and compared for 10 weeks postinjury.
Statistical Analysis
All results in the figures and text were expressed as means ± SEMs. The results were analyzed by Kruskal-Wallis analysis, Spine
followed by a Mann-Whitney analysis for an intergroup comparison. A P value of less than 0.05 was considered to be statistically significant.
RESULTS Engraftment of the Transplanted Cells At 4 weeks post-transplantation, an engraftment of the transplanted MSCs was found in the IV and ESW + IV groups (Figure 2) (3-dimensional reconstruction images were supplied as supplemental videos). The MSCs costained with PKH 26 and DAPI were counted in 6 fields under high-power magnification (×400). Three nonconsecutive sections at the epicenter were used for cell counts in each group (n = 3). The mean numbers of PKH 26 positive cells in the IV group and ESW + IV group were 23 ± 3.6 and 30·8 ± 5.7, respectively. The number of engrafted MSCs was higher in the ESW + IV group than that in the IV group, with statistical significance (P < 0.05).
Expression of SDF-1 and CXCR4
The expressions for SDF-1 of the ESW control group (0.1 ± 0.03) and ESW + IV (0.1 ± 0.02) group were significantly higher than those of the control group (0.06 ± 0.01) and the IV group (0.08 ± 0.03) (n = 6, P < 0.05). On histological examination using immunofluorescence staining, higher SDF-1 expressions were noted at blood vessels and ependyma around the posterior part of the spinal cord (Figure 3) (3-dimensional reconstruction images were supplied as Supplemental Digital Content videos, available at http://links .lww.com/BRS/A911). On higher power magnification, a highly stromal expression was noted in the ESW control and ESW + IV groups than in the control and IV groups. Expressions of CXCR4 demonstrated significantly higher in the IV www.spinejournal.com
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Alteration of Microenvironment in Cell Therapy for Chronic Spinal Cord Injury• Lee et al
Figure 2. Transplantation after extracorporeal shock waves (ESW) enhanced engraftment of the intravenously transplanted mesenchymal stem cells (MSCs). Engraftment of the transplanted MSCs was found in the epicenter of the spinal cord. Compared with the IV group (A–C), significantly higher number of engrafted MSCs (red fluorescence, PKH 26 tagged) was noted in the ESW+ IV group (D–F). Engraftment was quantitatively analyzed by counting the colocalized cells in high power magnification at 6 consecutive fields (×400, n = 3) (G). The boxed areas are magnified one. *P < 0.05. ESWT indicates extracorporeal shock waves therapy; IV, intravenous transplantation.
(0.22 ± 0.04) and MSC + IV (0.29 ± 0.03) groups than those of the control (0.16 ± 0.06) and ESW control (0.19 ± 0.05) groups (P < 0.05). On histological examination, a higher expression of CXCR4 was noted in the transplanted group, and a colocalization of the transplanted cells with CXCR4 was also noted in the transplanted cells (Figure 4).
Expression of VEGF and Neurotrophic Factors
The ESW + IV group (0.18 ± 0.01) presented only a significantly higher level of VEGF expression than the control group (0.1 ± 0.04) (n = 6, P < 0.05). The ESW control (0.15 ± 0.03) and IV groups (0.15 ± 0.04) did not show
significant differences compared with the control group. And there was no significant difference between the treated groups (Figure 5). The BDNF expressions in the ESW control (0.18 ± 0.03), IV (0.18 ± 0.01), and ESW + IV groups (0.25 ± 0.1) were significantly higher than those in the control group (0.15 ± 0.02) (n = 6, P < 0.05). However, there was no significant difference between the treated groups. Only the NGF expression in the ESW + IV group (0.24 ± 0.07) presented a significantly higher level than that in the control group (0.12 ± 0.03) (n = 6, P < 0.05). The ESW control (0.15 ± 0.05) and IV groups (0.17 ± 0.07) did not show significant differences compared with the control group (Figure 6).
Figure 3. Higher expressions of the SDF-1 in the injured spinal cord were noted in the ESW groups. Expressions of SDF-1 were noted at blood vessels and ependyma around the posterior part of the spinal cord on immunofluorescence stain. In the ESW control (C, D) and ESW+ IV (G, H) groups, higher expression was noted compared with the control (A, B) and IV (E, F) groups. Western blotting analysis revealed higher expressions for SDF-1 in the ESW control (0.1 ± 0.03) and ESW+ IV (0.1 ± 0.02) groups than those in the control (0.06 ± 0.01) and the IV (0.08 ± 0.03) groups (I). The boxed area indicates the magnified one. *P < 0.05. SDF-1 indicates stromal derived factor-1; ESW, extracorporeal shock waves; IV, intravenous transplantation.
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December 2014
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Alteration of Microenvironment in Cell Therapy for Chronic Spinal Cord Injury• Lee et al
Figure 4. Expression of CXCR4 in the injured spinal cord. CXCR4-expressing cells were noted in all groups (A, B: control, C, D: extracorporeal shock waves [ESW] control). Colocalization of the transplanted cells with CXCR4 was also noted in the IV (E, F) and ESW + IV (G, H) groups. Quantitative analysis using western blotting showed higher expression of CXCR4 in the IV (0.22 ± 0.04) and mesenchymal stem cell (MSC) + IV (0.29 ± 0.03) groups than that in the control (0.16 ± 0.06) and ESW control (0.19 ± 0.05) groups (I). The boxed area indicates the magnified one. *P < 0.05. CXCR4 indicates c-x-c chemokine receptor type 4; ESWT, extracorporeal shock waves therapy; IV, intravenous transplantation.
Behavioral Improvement There was no significant statistical difference in the pretreatment BBB scores between each group. However, the mean BBB scores in the control, ESW control, IV, and ESW + IV groups were 2·5 ± 0·8, 3·7 ± 0·8, 6 ± 0·8, and 6·2 ± 1·2 at 4 weeks post-treatment, respectively. The treatment groups presented significant clinical improvement compared with the control group (P < 0.05) (Figure 7).
DISCUSSIONS Stem cell therapy for SCI could be a new hope considering the extensive promising results of the experimental studies. However, prior to safe and efficient clinical trials, we should accumulate more basic evidences regarding the fate of transplanted cells and interaction of the cells with the microenvironment.
In this study, we tried to overcome the limitations of the chronic phase of SCI such as the low engraftment of the transplanted cells. ESW have been tried in various diseases such as nonunion or ischemic heart disease with many experimental and clinical evidences.9–11 The proposed mechanisms are an alteration of the microenvironment of the pathologic conditions by enhancing neovascularization and an expression of various growth factors. In this study, a higher engraftment of the transplanted cells in the chronic phase of SCI by intravenous transplantation after ESW was presented compared with the IV transplantation. The expression of SDF-1 in the western blot analysis and also the histological examination support this phenomenon considering the role of SDF-1.2,12,13 Moreover, a higher expression of CXCR4 in the ESW + IV group and the IV group may also support those results. And
Figure 5. Expression of VEGF in the injured spinal cord. VEGF expressions were profoundly noted at blood vessels and the injured site in all groups (A, B: control; C, D: ESW control; E, F: IV; G, H: ESW + IV). On quantitative analysis, only the ESW + IV (0.18 ± 0.01) group was significantly higher than the control (0.1 ± 0.04) group (I). Boxed area indicates the magnified one. *P < 0.05. VEGF indicates vascular endothelial growth factor; ESWT, extracorporeal shock waves therapy; IV, intravenous transplantation. Spine
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Alteration of Microenvironment in Cell Therapy for Chronic Spinal Cord Injury• Lee et al
Figure 6. Expression of BDNF and NGF in the injured spinal cord. The expression of neuronal growth factors showed divergent results. In the BDNF expression (A), the experimental groups showed significant higher expression than the control group. However, in the NGF expression (B), the ESW + IV group presented only statistical significance. *P < 0.05. BDNF indicates brain-derived neurotrophic factor; ESW, extracorporeal shock waves; IV, intravenous transplantation; NGF, neuronal growth factor.
a colocalization of CXCR4 with the transplanted cells was also noted in the transplanted groups. There was a higher expression of BDNF, NGF, and VEGF in the treated groups than in the control group. However, we could not find any significant differences among the ESW control, ESW + IV, and the IV groups. By analyzing the expression of growth factors, this study could not confirm whether ESW may introduce more favorable conditions by induction of these growth factors. Several factors such as the type of transplanted cells (MSCs) and time of evaluation (6 wk post-transplantation) may influence these inconsistent results. Further studies might
Figure 7. Behavioral improvement assessed by BBB scale 10 weeks postinjury. At 4 weeks postinjury, transplantation and application of ESW was done. There was no significant difference in the neurological status at this intervention time. A gradual recovery of the hindlimb was observed and a significant improvement was noted in all experimental groups compared with that of the control group at 10 weeks postinjury. BBB indicates Basso, Beattie, and Bresnahan; ESW, extracorporeal shock waves; IV, intravenous transplantation.
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be required to support ESW effects on these growth factors for enhanced engraftment of the transplanted cells. A peculiar pathologic condition represented by a glial scar is one of the obstacles in the chronic phase of SCI and decreases the efficient engraftment of the transplanted cells. Moreover, although intralesional administration has been investigated in many experimental and clinical trials, this transplantation has inherent risks by secondary injury due to direct damage and injection pressure. In contrast, intravenous transplantation has also been tried to reduce the secondary damage under the experimental evidence of stem cells homing effects. However, this transplantation route has also limitations such as an entrapment of the transplanted cells by the first pass effect. Therefore, it is necessary to determine less invasive transplantation routes and to enhance the efficient cell engraftment. For these purposes, manipulations of the transplanted cells have been tried, and trials using various types of cells have been conducted.7,14 Also, trials to overcome the pathologic condition of the chronic SCI have been reported.15 In terms of the fate of the transplanted cells, there are contradictory results along the type of the used cells,6,7 the timing, as well as the type of transplantation. And these results preclude convincing results. However, most authors are in agreement that the effects of MSCs transplantation on SCI result from the modulation of microenvironment rather than from direct differentiation into functional cells. In this study, we did not investigate the fate of the transplanted cells in each experimental condition. Investigation on this issue would be one of topics following studies. Moreover, considering recruitment of endogenous stem cells including neural/progenitor cells of the subventricular zone and ependyma after SCI, studies for the effects of ESW on these endogenous stem cells would be another issues to be investigated. Because there have been no studies regarding ESW trials for cell therapy of SCI, determination of the energy level and frequency would be one of the limitations of this study. Although skin eruptions at the applied site were noted in a December 2014
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Alteration of Microenvironment in Cell Therapy for Chronic Spinal Cord Injury• Lee et al
few animals at the energy level 2 and 3 through our preliminary trials, there was no gross neurological deficit during the observation period. However, a cystic change at the posterior part of the spinal cord was noted on histological examination at energy level 3. As an explorative study, we focused on the detection of clinical changes (neurological deficit) and gross histological changes after ESW in this study. However, as few studies have demonstrated the vulnerability of the spinal cord by ESW and its dose-dependent tendency,16,17 further studies on different energy level and frequency in conjunction with multifaceted evaluation such as inflammation and apoptosis in the subjected spinal cord are prerequisites to address safety issue of ESW trial. In addition, the effects of ESW on the spinal cord without laminectomy have to be addressed on following studies. Another limitation of this study is timing of transplantation. Although there are many debates on defining the chronicity of SCI, most experimental studies on pathophysiology presented that most acute secondary injury persisted till 2 weeks postinjury.18,19 Phagocytic response after injury is maximal during this period. Moreover, more efficacy of cell transplantation after the subacute phase has been reported than that after the other phase.20 This is a reason why authors adopted 4 weeks postinjury as a time point of transplantation. However, rodent models could not fully represent the pathophysiology of human SCI. Spontaneous remyelination in conjunction with less gliosis formation was frequently observed in the rodent model compared with human SCI. Although this study has its limitations as mentioned previously, ESW at a given condition (energy level and frequency) introduce more engraftment of the transplanted MSCs without any deterioration of clinical improvements in a chronic SCI. Based on this promising result and possible explanation, ESW may be one of the possible measures for an alteration of microenvironment for the stem cell therapy in chronic SCI.
➢ Key Points ESW were tried for cell therapy to alter the microenvironment of chronic SCI. ESW introduced more engraftment of the intravenously transplanted cells into the lesion without any clinical deterioration. This finding was also confirmed by the expression of factors which is related to stem cell migration. Based on this promising result and as a possible explanation, ESW may be one of the possible measures for an alteration of the microenvironment for the stem cell therapy in chronic SCI.
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References
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BASIC SCIENCE
Does Extracorporeal Shock Wave Introduce Alteration of Microenvironment in Cell Therapy for Chronic Spinal Cord Injury? Ju-Yup Lee, MD, Kee-Yong Ha, MD, Jang-Woon Kim, MS, Jun-Yeong Seo, MD,* and Young-Hoon Kim, MD, PhD
Study Design. Animal experimental study. Objective. To present experimental evidence for cell therapy for spinal cord injury (SCI). Summary of Background Data. In chronic SCI, the efficacy of cell engraftment has been known to be low due to its distinct pathology. Alteration of microenvironment was tried using extracorporeal shock waves (ESW) for chronic SCI, and the efficacy of cell therapy was investigated. Methods. A chronic contusive SCI model was made in 36 SpragueDawley rats. The rats were allocated into (1) control group (SCI only), (2) ESW control group (SCI + ESW), (3) IV group (SCI + intravenous transplantation of mesenchymal stem cells; MSCs), and (4) ESW + IV group (SCI + MSCs IV transplantation after ESW). ESW were applied at the energy determined by our preliminary trials. Engraftment of the cells and expressions of growth factors (brain-derived neurotrophic factor, neuronal growth factor) and cytokines (SDF-1, CXCR4, VEGF) at the epicenter were assessed. The Basso, Beattie, and Bresnahan locomotor scale was used for the clinical assessment. Results. The mean numbers of engrafted cells were higher in the ESW+ IV than that in the IV with a statistical significance. The expression of SDF-1 was higher in the ESW groups than that in the control or IV group. CXCR4 was highly expressed in the transplanted groups. The expressions of growth factors in the treated group were higher in the treated group than those in the control group. However, various statistical significances were noted. The improvement of locomotor was higher in the transplanted groups than that in the control and ESW only group. From the Department of Orthopedic Surgery, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea. *Department of Orthopedic Surgery, Jeju National University Hospital, Jeju, Korea. Acknowledgment date: April 30, 2014. First revision date: July 27, 2014. Second revision date: August 10, 2014. Acceptance date: September 9, 2014. The manuscript submitted does not contain information about medical device(s)/drug(s). No funds were received in support of this work. Seoul St. Mary’s Clinical Medicine Research Program year of 2012 through the Catholic University of Korea funds was received to support this work No relevant financial activities outside the submitted work. Address correspondence and reprint requests to Young-Hoon Kim, MD, PhD, Department of Orthopedic Surgery, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, # 222 banpodae-ro, seocho-gu, Seoul, 137-701, Korea; E-mail: [email protected] DOI: 10.1097/BRS.0000000000000626 Spine
Conclusion. At a given energy level, ESW presented more engraftment of the transplanted MSCs without any clinical deterioration in a chronic SCI. Based on this promising result and possible explanations, ESW may cause an alteration of the microenvironment for the cell therapy in chronic SCI. Key words: spinal cord injuries, mesenchymal stem cells, shock waves. Level of Evidence: N/A Spine 2014;39:E1553–E1559
P
romising results from the basic research of cell therapy for spinal cord injury (SCI) have induced human clinical trials.1–4 However, lack of knowledge about the fate of the transplanted cells and few reliable clinical results limit the availability of those clinical trials.5–7 Therefore, more accumulative evidences regarding these issues should be provided prior to safe and useful clinical trials. Timing and route of transplantation are ones of these issues that should be addressed. Transplantation in the acute phase of SCI results in a low rate of engraftment by severe inflammatory environment, and transplantation in the chronic phase results in a low rate of engraftment and functional restoration due to a glial scar in contrast.4 Although our previous study presented a successful engraftment of the transplanted cells by intravenous routes in the chronic SCI, the efficacy was lower than that of the intralesional transplantation.8 With this background, a modulation of the environment of the chronic SCI is required to enhance the efficacy of the transplantation. Extracorporeal shock waves (ESW) have been used for various orthopedic diseases over the last decades. The therapeutic mechanisms are thought to be the induction of neovascularization and production of growth factors by the focused shock waves in a theoretical manner. Clinical trials for nonunions or delayed unions of fracture and chronic tendinitis have presented favorable results.9 Not only these orthopedic diseases but also myocardial ischemia was also experimentally tried with these theoretical backgrounds.10 With this background, we hypothesized that an application of ESW in the chronic phase of SCI could alter the microenvironment of the injured tissue and promote the engraftment of the transplanted stem cells. We studied possibilities of ESW as an enhancing method for cell therapy by investigating the engraftment of the stem www.spinejournal.com
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Alteration of Microenvironment in Cell Therapy for Chronic Spinal Cord Injury• Lee et al
cells and expression of the related growth factors and cytokines as well as the clinical results.
MATERIALS AND METHODS SCI Model and Group Allocation A total of 36 adult male Sprague-Dawely rats (body weights ranging between 290 to 340 g) were used. All animals were kept under standardized conditions (2 rats per cage, 20–24°C, 45%–65% humidity and a 12-hr of daily light) and given free access to standard food and drinking water. The rats were anesthetized with intraperitoneal injection of ketamine (50 mg/kg) and Rompun (2 mg/kg) after body weight measurement. Briefly, backs of the rat were shaved and sterilized with antiseptic Betadine. Preoperatively, 5-mg gentamicin was intramuscularly administrated. The spinal cords of the rats were exposed by performing laminectomy at T9 after exposure of the paravertebral muscles from T8 to T10. An SCI was induced by a 25-g·cm contusion using the Multicenter Animal Spinal Cord Injury Study impactor (a rod weighing 10 g and dropped from a height of 2.5 cm). Postoperatively, 5-mg ketoprofen was administrated for 3 days, and the bladder was manually emptied during the experiment. At 4 weeks postinjury, the rats were randomly assigned to one of the following four groups: (1) the control group (n = 9, SCI only), (2) the ESW control group (n = 9, SCI + ESW only), (3) the IV group (n = 9, SCI + intravenous mesenchymal stem cell [MSC] transplantation), and (4) the ESW + IV group (n = 9, SCI and IV MSCs transplantation after ESW). All surgical and physical therapies and the presurgical and postsurgical animal care were provided in accordance with the Laboratory Animal Welfare Act and the Guidelines and Policies for Rodent Survival Surgery, as provided by the Animal Studies Committee of the Catholic University of Korea (IACUC approval NO.2013-0070-02).
Preparation of MSCs Bone marrow was obtained from the femoral bone of rats. The femoral bone was harvested and both ends were cut. Bone marrow was aspirated with an 18-gauge needle and then diluted to 45 mL with Dulbecco’ Eagles medium (SigmaAldrich, Milwaukee, WI) supplemented with 20% heatinactivated fetal bovine serum (GibcoBRL, Grand island, NY), 2 mM L-glutamate (Sigma-Aldrich), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (Sigma-Aldrich). The bone marrow aspirates were plated and then incubated in a humidified atmosphere of 5% CO2 at 37ºC. For selecting the MSCs, the nonadherent cells were eliminated by replacing the medium 4 days after cell seeding. For each passage, the cells were plated at about 8000 cells/cm2 and were grown to confluence. Flow cytometric analysis of the cultured MSC was performed. In brief, the cells were detached with 0.05% trypsin-EDTA solution (Sigma-Aldrich) for 3 minutes and washed twice with PBS containing 0.1% bovine serum albumin. For direct assays, aliquots of the cells at a concentration of 1 × 106 cells per mL were immunolabeled at room temperature for 30 minutes with the following antibodies: FITC-conjugated CD 45, 29, and 90. MSCs are known to have the immunophenotype E1554
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of CD 29, and 90, and they lack the CD 45 hematopoietic immunophenotype. All the monoclonal antibodies were purchased from Pharmingen/Becton Dickinson (Franklin Lakes, NJ). Mouse immunoglobulin G1-FITC or mouse immunoglobulin G1-PE was used as an isotype-matched control. The labeled cells were analyzed by a FACSCalibur flow cytometer (Becton Dickinson) with the use of CellQuest software.
Determination of ESW Energy Level and Application A pilot study was conducted to determine the energy level of ESW. The Swiss Dolorcast (EMS, Lyon, Switzerland) with 10-mm focus applicator was used for ESW devices. This device was clinically applied for therapeutic purposes in various orthopedic diseases such as nonunion or delayed union of fracture, tendinitis, and avascular necrosis of femoral head. Six rats were used after laminectomy without any SCI. Three levels of energy (level 1, 0.01 mJ/mm2; level 2, 0.04 mJ/mm2; and level 3, 0.11 mJ/mm2) were applied at 1000 impulses. Neurological impairment was not noted in all subjects after ESW at all levels of energy. However, histological examination revealed cystic changes at the level 3 of ESW application region. There were no significant histological changes at level 1 or 2 (Figure 1). Based on this preliminary study, the energy level 2 (0.04 mJ/mm2) at 1000 impulses was chosen.
Transplantation of MSCs
In the IV and ESW + IV groups, the fourth passage 1 × 106 MSCs with a 0.1 mL total volume was injected through the tail vein after labeling with PKH26 (red fluorescence, 10−3 M, Sigma-Aldrich) at 4 weeks postinjury. In the ESW + IV group, ESW were applied with a determined level 24 hours before transplantation.
Histological Analysis Three rats in each group were assigned to immune staining for histological analysis. Each injured spinal cord was obtained after transcardiac perfusion at 6 weeks post-transplantation. The tissue was cut to 5-μm thickness. Immunofluorescence staining was performed for histological analysis to trace the transplanted cells and expression of chemokines (SDF-1, VEGF, and CXCR-4). The primary antibodies to the SDF-1 (stromal cell–derived factor-1 a, 1: 20, Abcam, Cambridge, England), VEGF (vascular endothelial growth factor, 1:50, Santa Cruz, Dallas, TX), and CXCR-4 (CXC chemokine receptor 4, 1:20, Abcam) were used. The details followed the previously described procedures.6 To address the efficacy of engraftment of the transplanted cells, the positive cells with colocalization by expressing DAPI, PKH26 (red fluorescence) in highpowered (×400) magnification were counted using a confocal microscope. The means of the counted numbers at 6 separate fields in each rat were recorded and used for statistical analysis.
Expression of Growth Factors and Chemokines Western blot was used for the evaluation of neurotrophic factors and chemokines in the injured spinal cord. Spinal cord tissue blocks (∼1.5-cm length) were harvested encompassing the epicenter and stored at −80°C. The samples were December 2014
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Alteration of Microenvironment in Cell Therapy for Chronic Spinal Cord Injury• Lee et al
Figure 1. Histological examination of the spinal cord under each extracorporeal shock waves (ESW) energy level (H-E stain). Three different energy levels of ESW were tried after laminectomy. Although no neurological impairment was noted at all energy levels, cystic changes were noted at the energy level 3. Representative images on (A, B) energy level 1 (0·01 mJ/mm2) and (C, D) energy level 2 (0·04 mJ/mm2) did not show any gross histological changes. E and F, At energy level 3 (0·11 mJ/mm2), cystic changes were noted on posterior part in the epicenter of the subjected spinal cord. Ant. indicates anterior; Post, posterior.
homogenized on tissue protein extraction reagent (Thermo scientific, Rockford, IL) with 1 mM PMSF, 10 mg/mL aprotinin, and 5 mg/mL leupeptin. The lysate was centrifuged at 16,000 rpm for 10 minutes at 4°C. The proteins were separated by SDS-polyacrylamide gel electrophoresis and they were transferred to a polyvinylidene difluoride membrane (Hybond-P, Amersham Pharmacia Biotech, Buckinghamshire, England). The membrane was blocked with 3% bovine serum albumin for 1 hour in Tris-buffered saline (0.1% Tween-20, 20 mM Tris-HCl, 137 mM NaCl, pH 7.4) and then incubated with the primary antibodies overnight at 4°C. The antibodies used for immunoblotting were as follows: brain-derived neurotrophic factor (BDNF; 1:700, Santa Cruz), neuronal growth factor (NGF; 1:500; Santa Cruz), SDF-1 (1:500, Abcam), VEGF (1:500, Santa Cruz), and CXCR-4 (1:500, Abcam). After the membranes were washed, they were incubated with secondary peroxidase-conjugated antimouse antibodies (Amersham Pharmacia Biotech) that were diluted 1:2000 in Tris-buffered saline with 0.01% Tween 20. An antibody detection system (ECL, Thermo scientific) was used, and the membranes were exposed to LAS 3000 (Biorad, Hercules, CA). The protein band intensities were quantified with a multi gauge V 3.0 software (Fuji photo film, Tokyo, Japan).
BBB Locomotor Test Basso, Beattie, and Bresnahan (BBB) locomotor rating scale values were recorded every week. Two researchers independently recorded BBB scale after 2 minutes of observation. BBB scales were checked and compared for 10 weeks postinjury.
Statistical Analysis
All results in the figures and text were expressed as means ± SEMs. The results were analyzed by Kruskal-Wallis analysis, Spine
followed by a Mann-Whitney analysis for an intergroup comparison. A P value of less than 0.05 was considered to be statistically significant.
RESULTS Engraftment of the Transplanted Cells At 4 weeks post-transplantation, an engraftment of the transplanted MSCs was found in the IV and ESW + IV groups (Figure 2) (3-dimensional reconstruction images were supplied as supplemental videos). The MSCs costained with PKH 26 and DAPI were counted in 6 fields under high-power magnification (×400). Three nonconsecutive sections at the epicenter were used for cell counts in each group (n = 3). The mean numbers of PKH 26 positive cells in the IV group and ESW + IV group were 23 ± 3.6 and 30·8 ± 5.7, respectively. The number of engrafted MSCs was higher in the ESW + IV group than that in the IV group, with statistical significance (P < 0.05).
Expression of SDF-1 and CXCR4
The expressions for SDF-1 of the ESW control group (0.1 ± 0.03) and ESW + IV (0.1 ± 0.02) group were significantly higher than those of the control group (0.06 ± 0.01) and the IV group (0.08 ± 0.03) (n = 6, P < 0.05). On histological examination using immunofluorescence staining, higher SDF-1 expressions were noted at blood vessels and ependyma around the posterior part of the spinal cord (Figure 3) (3-dimensional reconstruction images were supplied as Supplemental Digital Content videos, available at http://links .lww.com/BRS/A911). On higher power magnification, a highly stromal expression was noted in the ESW control and ESW + IV groups than in the control and IV groups. Expressions of CXCR4 demonstrated significantly higher in the IV www.spinejournal.com
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Alteration of Microenvironment in Cell Therapy for Chronic Spinal Cord Injury• Lee et al
Figure 2. Transplantation after extracorporeal shock waves (ESW) enhanced engraftment of the intravenously transplanted mesenchymal stem cells (MSCs). Engraftment of the transplanted MSCs was found in the epicenter of the spinal cord. Compared with the IV group (A–C), significantly higher number of engrafted MSCs (red fluorescence, PKH 26 tagged) was noted in the ESW+ IV group (D–F). Engraftment was quantitatively analyzed by counting the colocalized cells in high power magnification at 6 consecutive fields (×400, n = 3) (G). The boxed areas are magnified one. *P < 0.05. ESWT indicates extracorporeal shock waves therapy; IV, intravenous transplantation.
(0.22 ± 0.04) and MSC + IV (0.29 ± 0.03) groups than those of the control (0.16 ± 0.06) and ESW control (0.19 ± 0.05) groups (P < 0.05). On histological examination, a higher expression of CXCR4 was noted in the transplanted group, and a colocalization of the transplanted cells with CXCR4 was also noted in the transplanted cells (Figure 4).
Expression of VEGF and Neurotrophic Factors
The ESW + IV group (0.18 ± 0.01) presented only a significantly higher level of VEGF expression than the control group (0.1 ± 0.04) (n = 6, P < 0.05). The ESW control (0.15 ± 0.03) and IV groups (0.15 ± 0.04) did not show
significant differences compared with the control group. And there was no significant difference between the treated groups (Figure 5). The BDNF expressions in the ESW control (0.18 ± 0.03), IV (0.18 ± 0.01), and ESW + IV groups (0.25 ± 0.1) were significantly higher than those in the control group (0.15 ± 0.02) (n = 6, P < 0.05). However, there was no significant difference between the treated groups. Only the NGF expression in the ESW + IV group (0.24 ± 0.07) presented a significantly higher level than that in the control group (0.12 ± 0.03) (n = 6, P < 0.05). The ESW control (0.15 ± 0.05) and IV groups (0.17 ± 0.07) did not show significant differences compared with the control group (Figure 6).
Figure 3. Higher expressions of the SDF-1 in the injured spinal cord were noted in the ESW groups. Expressions of SDF-1 were noted at blood vessels and ependyma around the posterior part of the spinal cord on immunofluorescence stain. In the ESW control (C, D) and ESW+ IV (G, H) groups, higher expression was noted compared with the control (A, B) and IV (E, F) groups. Western blotting analysis revealed higher expressions for SDF-1 in the ESW control (0.1 ± 0.03) and ESW+ IV (0.1 ± 0.02) groups than those in the control (0.06 ± 0.01) and the IV (0.08 ± 0.03) groups (I). The boxed area indicates the magnified one. *P < 0.05. SDF-1 indicates stromal derived factor-1; ESW, extracorporeal shock waves; IV, intravenous transplantation.
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December 2014
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Alteration of Microenvironment in Cell Therapy for Chronic Spinal Cord Injury• Lee et al
Figure 4. Expression of CXCR4 in the injured spinal cord. CXCR4-expressing cells were noted in all groups (A, B: control, C, D: extracorporeal shock waves [ESW] control). Colocalization of the transplanted cells with CXCR4 was also noted in the IV (E, F) and ESW + IV (G, H) groups. Quantitative analysis using western blotting showed higher expression of CXCR4 in the IV (0.22 ± 0.04) and mesenchymal stem cell (MSC) + IV (0.29 ± 0.03) groups than that in the control (0.16 ± 0.06) and ESW control (0.19 ± 0.05) groups (I). The boxed area indicates the magnified one. *P < 0.05. CXCR4 indicates c-x-c chemokine receptor type 4; ESWT, extracorporeal shock waves therapy; IV, intravenous transplantation.
Behavioral Improvement There was no significant statistical difference in the pretreatment BBB scores between each group. However, the mean BBB scores in the control, ESW control, IV, and ESW + IV groups were 2·5 ± 0·8, 3·7 ± 0·8, 6 ± 0·8, and 6·2 ± 1·2 at 4 weeks post-treatment, respectively. The treatment groups presented significant clinical improvement compared with the control group (P < 0.05) (Figure 7).
DISCUSSIONS Stem cell therapy for SCI could be a new hope considering the extensive promising results of the experimental studies. However, prior to safe and efficient clinical trials, we should accumulate more basic evidences regarding the fate of transplanted cells and interaction of the cells with the microenvironment.
In this study, we tried to overcome the limitations of the chronic phase of SCI such as the low engraftment of the transplanted cells. ESW have been tried in various diseases such as nonunion or ischemic heart disease with many experimental and clinical evidences.9–11 The proposed mechanisms are an alteration of the microenvironment of the pathologic conditions by enhancing neovascularization and an expression of various growth factors. In this study, a higher engraftment of the transplanted cells in the chronic phase of SCI by intravenous transplantation after ESW was presented compared with the IV transplantation. The expression of SDF-1 in the western blot analysis and also the histological examination support this phenomenon considering the role of SDF-1.2,12,13 Moreover, a higher expression of CXCR4 in the ESW + IV group and the IV group may also support those results. And
Figure 5. Expression of VEGF in the injured spinal cord. VEGF expressions were profoundly noted at blood vessels and the injured site in all groups (A, B: control; C, D: ESW control; E, F: IV; G, H: ESW + IV). On quantitative analysis, only the ESW + IV (0.18 ± 0.01) group was significantly higher than the control (0.1 ± 0.04) group (I). Boxed area indicates the magnified one. *P < 0.05. VEGF indicates vascular endothelial growth factor; ESWT, extracorporeal shock waves therapy; IV, intravenous transplantation. Spine
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Alteration of Microenvironment in Cell Therapy for Chronic Spinal Cord Injury• Lee et al
Figure 6. Expression of BDNF and NGF in the injured spinal cord. The expression of neuronal growth factors showed divergent results. In the BDNF expression (A), the experimental groups showed significant higher expression than the control group. However, in the NGF expression (B), the ESW + IV group presented only statistical significance. *P < 0.05. BDNF indicates brain-derived neurotrophic factor; ESW, extracorporeal shock waves; IV, intravenous transplantation; NGF, neuronal growth factor.
a colocalization of CXCR4 with the transplanted cells was also noted in the transplanted groups. There was a higher expression of BDNF, NGF, and VEGF in the treated groups than in the control group. However, we could not find any significant differences among the ESW control, ESW + IV, and the IV groups. By analyzing the expression of growth factors, this study could not confirm whether ESW may introduce more favorable conditions by induction of these growth factors. Several factors such as the type of transplanted cells (MSCs) and time of evaluation (6 wk post-transplantation) may influence these inconsistent results. Further studies might
Figure 7. Behavioral improvement assessed by BBB scale 10 weeks postinjury. At 4 weeks postinjury, transplantation and application of ESW was done. There was no significant difference in the neurological status at this intervention time. A gradual recovery of the hindlimb was observed and a significant improvement was noted in all experimental groups compared with that of the control group at 10 weeks postinjury. BBB indicates Basso, Beattie, and Bresnahan; ESW, extracorporeal shock waves; IV, intravenous transplantation.
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be required to support ESW effects on these growth factors for enhanced engraftment of the transplanted cells. A peculiar pathologic condition represented by a glial scar is one of the obstacles in the chronic phase of SCI and decreases the efficient engraftment of the transplanted cells. Moreover, although intralesional administration has been investigated in many experimental and clinical trials, this transplantation has inherent risks by secondary injury due to direct damage and injection pressure. In contrast, intravenous transplantation has also been tried to reduce the secondary damage under the experimental evidence of stem cells homing effects. However, this transplantation route has also limitations such as an entrapment of the transplanted cells by the first pass effect. Therefore, it is necessary to determine less invasive transplantation routes and to enhance the efficient cell engraftment. For these purposes, manipulations of the transplanted cells have been tried, and trials using various types of cells have been conducted.7,14 Also, trials to overcome the pathologic condition of the chronic SCI have been reported.15 In terms of the fate of the transplanted cells, there are contradictory results along the type of the used cells,6,7 the timing, as well as the type of transplantation. And these results preclude convincing results. However, most authors are in agreement that the effects of MSCs transplantation on SCI result from the modulation of microenvironment rather than from direct differentiation into functional cells. In this study, we did not investigate the fate of the transplanted cells in each experimental condition. Investigation on this issue would be one of topics following studies. Moreover, considering recruitment of endogenous stem cells including neural/progenitor cells of the subventricular zone and ependyma after SCI, studies for the effects of ESW on these endogenous stem cells would be another issues to be investigated. Because there have been no studies regarding ESW trials for cell therapy of SCI, determination of the energy level and frequency would be one of the limitations of this study. Although skin eruptions at the applied site were noted in a December 2014
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Alteration of Microenvironment in Cell Therapy for Chronic Spinal Cord Injury• Lee et al
few animals at the energy level 2 and 3 through our preliminary trials, there was no gross neurological deficit during the observation period. However, a cystic change at the posterior part of the spinal cord was noted on histological examination at energy level 3. As an explorative study, we focused on the detection of clinical changes (neurological deficit) and gross histological changes after ESW in this study. However, as few studies have demonstrated the vulnerability of the spinal cord by ESW and its dose-dependent tendency,16,17 further studies on different energy level and frequency in conjunction with multifaceted evaluation such as inflammation and apoptosis in the subjected spinal cord are prerequisites to address safety issue of ESW trial. In addition, the effects of ESW on the spinal cord without laminectomy have to be addressed on following studies. Another limitation of this study is timing of transplantation. Although there are many debates on defining the chronicity of SCI, most experimental studies on pathophysiology presented that most acute secondary injury persisted till 2 weeks postinjury.18,19 Phagocytic response after injury is maximal during this period. Moreover, more efficacy of cell transplantation after the subacute phase has been reported than that after the other phase.20 This is a reason why authors adopted 4 weeks postinjury as a time point of transplantation. However, rodent models could not fully represent the pathophysiology of human SCI. Spontaneous remyelination in conjunction with less gliosis formation was frequently observed in the rodent model compared with human SCI. Although this study has its limitations as mentioned previously, ESW at a given condition (energy level and frequency) introduce more engraftment of the transplanted MSCs without any deterioration of clinical improvements in a chronic SCI. Based on this promising result and possible explanation, ESW may be one of the possible measures for an alteration of microenvironment for the stem cell therapy in chronic SCI.
➢ Key Points ESW were tried for cell therapy to alter the microenvironment of chronic SCI. ESW introduced more engraftment of the intravenously transplanted cells into the lesion without any clinical deterioration. This finding was also confirmed by the expression of factors which is related to stem cell migration. Based on this promising result and as a possible explanation, ESW may be one of the possible measures for an alteration of the microenvironment for the stem cell therapy in chronic SCI.
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References
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