SPINE Volume 39, Number 11, pp E669-E675 ©2014, Lippincott Williams & Wilkins
BASIC SCIENCE
Effects of Transplantation of hTIMP-1–Expressing Bone Marrow Mesenchymal Stem Cells on the Extracellular Matrix of Degenerative Intervertebral Discs in an In Vivo Rabbit Model Zhou Yi, MD,*† Tu Guanjun, MD, PhD,* Cong Lin, MD,* and Pei Zifeng, MD†
Study Design. Prospective, randomized, and controlled animal study. Objective. To observe extracellular matrix (ECM) changes in degenerative intervertebral disc (IVD) after transplantation of bone marrow mesenchymal stem cells (BMSCs) virally transfected with a construct expressing “human tissue inhibitor of metalloproteinase 1” (hTIMP-1), and to discuss the feasibility of using this approach to treat IVD degeneration. Summary of Background Data. Intervertebral disc (IVD) degeneration is characterized by decreased cell numbers, bioactivity of the nucleus pulposus, and remodeled ECM. Exogenous genes can be targeted into cells to produce inhibition of ECM degradation and increase ECM content in IVDs, and thereby potentially stop or reverse degenerative processes and modify disc structure. Methods. BMSCs were isolated from a pure New Zealand white rabbit and identified by flow cytometry. Transgenic BMSCs were acquired by transfection with a recombinant adenovirus vector carrying the hTIMP-1 gene. Animal models of IVD degeneration were established by annulus puncture and then given intra-nucleus pulposus injections according to their random assignment into 3 groups: (1) a transgenic BMSC transplantation (TgBT) group that received BMSCs transfected with an hTIMP-1–expressing adenovirus vector; (2) a BMSC transplantation (BT) group that received unaltered BMSCs; and (3) a control group that received cell-free phosphate-buffered saline. Degree of degeneration was From the *Department of Orthopedics, The First Affiliated Hospital, China Medical University, Shenyang, China; and †Department of Orthopedics, The Second Affiliated Hospital, Shenyang Medical College, Shenyang, China. Acknowledgment date: September 18, 2013. Revision date: November 21, 2013. Acceptance date: January 7, 2014. The manuscript submitted does not contain information about medical device(s)/drug(s). National Natural Science Foundation of China (grant no. 81070971) and Shenyang Medical College Technology Foundation (grant no. 20121014) funds were received in support of this work. No relevant financial activities outside the submitted work. Address correspondence and reprint requests to Tu Guanjun, MD, PhD, Department of Orthopedics, The First Affiliated Hospital, China Medical University, Shenyang 110001, China; E-mail: [email protected] DOI: 10.1097/BRS.0000000000000316 Spine
evaluated 12 weeks after modeling. ECM content was quantified using immunohistochemistry and spectrophotography. Expression of hTIMP-1 was observed via quantitative polymerase chain reaction, western blot, and immunohistochemistry. Results. Significantly fewer degenerative changes and increased ECM content were observed in the TBT and BT groups than the control group animals (P < 0.05). The TBT group had greater ECM content than did the BT group (P < 0.05), as well as higher levels of hTIMP-1 mRNA and protein. Conclusion. Transplantation of BMSCs transfected with hTIMP-1 can increase ECM content by inhibiting ECM degradation and promoting ECM synthesis. Key words: intervertebral disc degeneration, gene therapy, cell transplantation, BMSCs, TIMP. Level of Evidence: N/A Spine 2014;39:E669–E675
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ower back pain is one of the main symptoms of degenerative disc disease, which has a measurable impact on human health.1 The generation and development of degenerative disc disease are both relevant to intervertebral disc degeneration (IDD). Low levels of cellular components and abundant extracellular matrix (ECM) are characteristic of the intervertebral disc (IVD), with most biomechanical functions of the IVD being accomplished by the high ECM content, especially collagen type II and proteoglycans (PGs). Although the etiological factors and pathophysiological process of IDD are not yet well understood, degeneration is known to involve decreased cell numbers, bioactivity of the nucleus pulposus (NP), and reduced ECM content. The main cause of IDD is an imbalance between anabolism and catabolism.2–5 Previous studies have suggested that decreased collagen type II and PGs ultimately lead to morphological and biomechanical changes and decreased biofunction of the IVD. Gene therapy research has been devoted to selecting reasonable target molecules to interfere with the IDD process. Matrix metalloproteinases (MMPs) are important proteinases that can destroy the ECM in the IVD. MMPs participate throughout the process of IDD, and elevated www.spinejournal.com
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hTIMP-1–Expressing BMSC Transplantation on ECM • Yi et al
MMP expression levels are found with increasing degree of degeneration.6–8 The component variation and content diminution of ECM from excessive levels of activated MMPs not only leads to changes in the morphology and biochemistry of the IVD, but also diminishes the biomechanical function of the IVD.9 Tissue inhibitor of metalloproteinases (TIMPs) are a group of specific endogenous inhibitors of MMPs that can lessen ECM degradation via interaction with the zinc-binding site of MMPs. Some studies have indicated that an imbalance between MMPs and TIMPs is the primary reason for excessive degradation of the ECM.10 The strategy of increasing ECM content through inhibition of catabolism makes TIMP a popular candidate gene for IDD gene therapy. Bone marrow mesenchymal stem cells (BMSCs) are important participants in tissue repair in vivo. BMSCs can not only differentiate as NP-like cells within the IVD or in coculture with NP cells,11–12 but can also increase the PG content of the ECM in vivo after transplantation into a rabbit degenerative disc model.13 Adenoviral-mediated transfection can be used to deliver genes to BMSCs efficiently and has negligible effects on the phenotype and differentiation of these cells.14 Hence, adenoviral-mediated gene transfer as an approach has potential for enabling high-performance expression of a product in BMSCs without altering BMSC growth and differentiation. We constructed a recombinant adenoviral vector carrying hTIMP-1 (Ad-hTIMP-1) as part of a previous study.15 In this study, we aimed to transfect rabbit BMSCs via an adenovirus carrying this vector, and transplant these TIMP-1 expressing cells into the degenerative IVD of IDD model rabbits. To our knowledge, transplantation of BMSCs expressing exogenous TIMP-1 has not been performed in an in vivo disc degeneration model. This study investigates the effect of transplanted hTIMP-1–expressing BMSCs on the ECM content in a rabbit IDD model, and explored the feasibility of reversing or delaying IDD via gene therapy and BMSC transplantation (BT). We hypothesized that transgenic BT (TgBT) could postpone or reverse the process of IDD more significantly than BT alone.
Abcam, ab119335; CD90: Abcam, ab226) at a concentration of 0.1–10 μg/mL and incubated with the cells for at least 30 minutes at room temperature (RT) in the dark. Fluorochrome-labeled secondary antibody (115-095-062; Jackson Immunoresearch Laboratories Inc., Baltimore Pike, PA) was diluted in phosphate-buffered saline and incubated with the cells for at least 30 minutes at RT in the dark. For immunohistochemistry (IHC), BMSCs were harvested, washed, and adjusted to a concentration of 2 × 105 cells/mL in a 24-well plate. The viral vector pYr-adshuttle-4 (Yingrun Biotechnologies Inc., Changsha, China; 1 × 1010 pfu/mL virus activity and expressing green fluorescent protein [GFP]) was added at 125, 250, 500, or 1000 multiplicity of infection (MOI) values, respectively. GFP expression was observed via an inverted fluorescence microscope and the transfection efficiency was calculated. For transplantation, BMSCs were harvested and the cell suspension was adjusted to a concentration of 1-5 × 106 cells/ mL. The virus Ad-TIMP-1 (1 × 1010 IU/mL) was added at the optimal MOI and incubated for 45 minutes. The cells were then washed and the BMSC suspension was adjusted to a concentration of 1 × 106 cells/μL for transplantation.
MATERIALS AND METHODS
Radiographs were obtained 12 weeks postpuncture. The scanning parameters of the radiographs were 60 kV, 100 mA, and 50 ms. A previously validated method was used to evaluate the relative height of the intervertebral space.18 The value of the relative intervertebral space height (RISH) of each segment was calculated: RISH =X/Y, where X is the value of each segment ISH, and Y is the value of the nondegenerative L2–L3 segment ISH. The degree of IVD was evaluated by comparing the RISH of each segment.
Sample Preparation The hTIMP-1 cDNA was polymerase chain reaction (PCR) amplified from the pMD-hTIMP-1 vector (Sino Biological Inc., Beijing, China), which contains the human TIMP-1 ORF sequence. As a part of our previous study,15 the recombinant adenoviral vector carrying hTIMP-1 (Ad-hTIMP-1) with 1 × 1010 IU/mL virus activity was stored in the preclinical medicine laboratory at China Medical University. BMSCs were harvested from tibia bone marrow of a New Zealand white rabbit (Experimental Animal Center of Shenyang Pharmaceutical University, Shenyang, China; weight, 1.5 kg; age, 3 mo) using lymphocyte separation in vitro with gradient centrifugation. For fluorescence-activated cell sorting, BMSCs were harvested and washed and the cell suspension was adjusted to a concentration of 1-5 × 106 cells/mL in cold phosphatebuffered saline. Primary antibody was added (CD45: SANTA, SC-70690; CD34: Abcam, ab131589; CD44: E670
Animals and Surgery IDD was induced via a validated rabbit puncture model in 15 healthy New Zealand white rabbits (age, 3 mo; weight, 1.5 kg).16–17 The L3–L4, L4–L5, and L5–L6 discs were exposed and punctured with a 16-gauge needle at the middle point between the endplates to a depth of 5 mm for 5 seconds. Then the rabbits were divided randomly into 3 groups (n = 15) and 30 μL of transgenic BMSC suspension containing about 30 × 106 cells (TgBT group), 30 μL of BMSC suspension containing about 30 × 106 cells (BT group), or 30 μL of phosphate-buffered saline of 0.01 mol/L (PCon group) were injected with a microinjector in the same manner as the puncture model procedure.
Radiographs
Killing of Rabbits and Their Anatomical Observations All rabbits were killed 12 weeks after the initial puncture surgery. The spinal column was dissected out immediately en bloc (L2–L6) and the L3–L4, L4–L5, and L5–L6 discs were exposed and observed.
Pathological and IHC Analysis For pathological analysis, the sections were dewaxed, hydrated, and stained with hematoxylin and eosin using a
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BASIC SCIENCE standard histology protocol, and photographed via a light microscope. For IHC of collagen type II and hTIMP-1 expression, the sections were dewaxed, hydrated, repaired with citrate, and blocked using 3% hydrogen peroxide– formalin. Anticollagen type II or hTIMP-1 primary antibody and diluted horseradish peroxidase–labeled secondary antibody were applied as instructed by the Elivision plus kit (Maixin Biotechnology, Inc., Fuzhou, China). The sections were stained with freshly prepared diaminobenzidine, counterstained with hematoxylin, dehydrated, and mounted with neutral gum. The yellow-stained ECM or stained cytoplasm were positive for collagen type II or hTIMP-1 expression in the NP, and the average optical value was measured for quantification of collagen type II expression with Image-Pro Plus 6.0 software (Media Cybernetics, Bethesda, MD ).
Glycosaminoglycan Assay by Spectrophotography On the basis of changes in the absorption spectrum of 1,9-dimethylmethylene blue (DMMB) dye when bound to glycosaminoglycans (GAGs), we used a spectrophotometric method to study the total GAG content in NP tissue extracts.19 Preparation of working DMMB solution was derived from that reported by Isabelle Barbosa et al20 chondroitin sulfate (CS) standard solution (0.4 mL) was diluted with distilled water (0.1–1.0 mg/mL). A 3-mL working DMMB solution was then added to 1 mL of diluted CS standard solution and the absorbance of the mixture was measured at 580 nm. A calibration curve of CS solutions was described according to the results obtained for absorbance at concentrations with a range of 0.1 to 1 mg/mL. The 10-mg NP tissue extracts were digested in a solution of 200 μL/mL of papain at 56 °C overnight. A 225 μL of aliquot of working DMMB solution was added to NP tissue treated with 25 μL of papain (200 μL/mL) and the mixture was measured at 580 nm. The GAG content in the NP tissue was determined by comparison with a calibration curve of standard CS solutions.
Fluorescent Q-PCR Analysis of hTIMP-1 mRNA Expression RNA extracted from NP tissue was harvested according to the RNA extraction kit protocol. The sequence of the primers used was as follows: hTIMP-1 forward: 5′-CAG AAC CGC AGC GAG GAG T-3′; reverse: 5′-CAG CGT AGG TCT TGG TGA AGC-3′; GAPDH forward: 5′-GGT GAA GGT CGG TGT GAA CG-3′; and reverse: 5′-CTC GCT CCT GGA AGA TGG TG-3′. The resulting products from the BioEasy (Bioer, Hangzhou, China) SYBR Green I Q-PCR kit were analyzed by the line-gene fluorescent quantified PCR system. The amplification efficiency and solubility curve were evaluated and quantitative analysis of the product was done using the 2−△△ct method. The reaction components used for real-time PCR were (50-μL total reaction): 2XSYBR Mix, 25 μL; sense primer, 1 μL; antisense primer, 1 μL; Taq DNA polymerase, 0.3 μL; cDNA, 2 μL; and ddH2O, 20.7 μL. PCR reaction conditions were as follows: 95 °C for 120 seconds, then 45 cycles of 95 °C for 20 seconds, annealing at 60 °C for 25 seconds, and Spine
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elongation at 72 °C for 30 seconds, with a final ramp from 65 °C to 95 °C at 0.5 °C/s.
Western Blot Analysis of hTIMP-1 Protein Expression Protein was harvested from NP tissue extracts routinely; 20 μg of each sample underwent SDS-polyacrylamide gel electrophoresis, and were electrically transferred to polyvinylidene fluoride (PVDF) membranes. Nonspecific binding was blocked with Tris-buffered saline and tween20 containing 5% skim milk for 4 hours at RT, and then stained with anti-hTIMP-1 (1:100) and anti-βactin (1:2000) separately overnight at RT, followed by incubation with an horseradish peroxidase– tagged goat antirabbit secondary antibody (1:10,000) for 4 hours. The blot was incubated in 3,3′-diaminobenzidine and exposed to photographic film. Grayscale analysis was completed using LabWorks software (UVP, Upland, CA) .
Statistical Analysis
The data are presented as means ± standard deviations and SPSS 17.0 (SPSS Inc., Chicago, IL) was used for statistical analysis by simple factor analysis of variance and independent sample t test. A difference of P < 0.05 was considered statistically significant.
RESULTS Cultivation and Identification of BMSCs With time in culture, adherent cells reached 70% confluence and proliferated rapidly within 3 to 7 days. With continued culture, these cells could reach 90% confluence and assume a radial and swirled arrangement. Fluorescence-activated cell sorting analysis indicated that the cultured BMSCs were 95.61% and 96.53% positive for the surface antigen markers CD44 and CD90, respectively, and were 0.76% and 12.77% positive for CD34 and CD45, respectively. These results indicated that the cells were BMSCs
Identification of MOI Assessment of BMSC GFP expression was performed using an inverted fluorescent microscope 24 hours after transfection. At an MOI of 1000, 90% of the BMSCs expressed GFP, but the cells remained morphologically normal.
Evaluation of IVD Digital Radiography Results According to the digital radiographical analysis of the spinal column, the PCon and BT groups demonstrated more severe sclerosis of the IVD endplate than the TgBT group (Figure 1). Lower RISH values were obtained for the BT group (0.842 ± 0.0428) than in the TgBT group (0.896 ± 0.039), and this difference was significant (P < 0.05). RISH values were the lowest in the PCon group (0.791 ± 0.036), which was significantly different from the experimental groups (P < 0.05).
Anatomical and Pathological Observation of IVD Anatomical observation of morphological alterations indicated a range of degenerative changes, including loss of tight linkage between the NP and the endplate, partial or complete www.spinejournal.com
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Figure 1. Digital radiographical analysis of the spinal columns revealed more severe sclerosis of the endplate of the intervertebral disc and shorter RISH in the PCon group and BT group than the hTIMP-1 TgBT group (*P < 0.05). hTIMP indicates human tissue inhibitor of metalloproteinase; TgBT, transgenic BMSC transplantation 1; BMSC, bone marrow mesenchymal stem cell; PCon, control group; RISH, relative intervertebral space height.
loss of NP, undefined boundary between the NP and annulus fibrosis, derangement or partial disruption of the annulus fibrosis, loss of the characteristic oval appearance, and lackluster color of the NP (Figure 2A). Analysis of hematoxylin and eosin–stained sections also revealed degenerative changes, including decreased numbers and irregularly shaped NP cells, karyopyknosis in some NP cells, looseness in the ECM alignment, disappearance of the mucoid cartilage matrix and substitution by fibrous tissue (Figure 2B). The degenerative IVDrelated changes were more pronounced in the PCon group than in the BT or TgBT groups.
Spectrophotographic Analysis of GAGs The results of the assessment of PG contents for each group are described in Figure 3A. The PG contents were significantly reduced in the BT group (6.160 ± 0.569) compared with the TgBT group (7.910 ± 0.452, P < 0.05). PG content was lowest in the PCon group (4.263 ± 0.570, P < 0.05).
Type II Collagen Assays and hTIMP-1 Expression Quantitative analysis of the collagen type II contents for each group is described in Figure 3B. Collagen type II content was significantly lower in the BT group (0.272 ± 0.014) than in the TgBT group (0.354 ± 0.022, P < 0.05). Collagen type II content was lowest in the PCon group (0.218 ± 0.013, P < 0.05). Our IHC experiment (Figure 4A) demonstrated type II collagen expression in all 3 groups, but the TgBT group exhibited
the highest levels of staining, followed by the BT group, and the PCon group. IHC analysis of hTIMP-1 protein expression (Figure 4B) revealed hTIMP-1 protein expression in the TgBT group only, which exhibited buffy color positive staining localized to the cytoplasm of NP cells. There was negligible, if any, hTIMP-1 expression in the BT and PCon groups.
Q-PCR and Western Blot Analysis of hTIMP-1 Expression The fluorescent detection of our PCR results indicated an amplification efficiency of 98.74% with a unimodal solubility curve (Figure 5A). Quantitative analysis results using the 2−△△ct method are described in Figure 5B. We observed greater expression of hTIMP-1 mRNA in the TgBT group (136.531 ± 15.231) than in the BT (5.818 ± 0.989) and PCon (1.119 ± 0.375) groups (Ps < 0.05). As shown in Figure 5C, D, western blot analysis of hTIMP-1 protein expression showed that the TgBT group (0.781 ± 0.079) exhibited higher levels of hTIMP-1 protein expression (P < 0.05) than the BT (0.181 ± 0.035) and PCon (0.120 ± 0.038) groups.
DISCUSSION In this study, we transfected BMSCs with the hTIMP-1 gene by recombinant adenovirus vector in vitro and transplanted these transgenic BMSCs into the NP of an injuryinduced model of degenerative disc. Compared with the BT group, the TgBT group showed less degeneration and
Figure 2. Comparison of tissues across groups. A, Example discs obtained from rabbits in each group showing anatomical and morphological differences between the groups. B, Hematoxylin and eosin– stained sections (400×) demonstrating fewer and more irregularly shaped NP cells in animals from the PCon group relative to the hTIMP-1 TgBT group. NP indicates nucleus pulposus; human tissue inhibitor of metalloproteinase 1; TgBT, transgenic BMSC transplantation 1; BMSC, bone marrow mesenchymal stem cell; PCon, control group.
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Figure 3. Quantification of PG and collagen type II content. A, Spectrographic analysis revealed that PG content was significantly lower in the PCon group and BT group than in the hTIMP-1 TgBT group (*P < 0.05). B, Likewise, spectrographic analysis revealed that collagen type II content was significantly lower in the PCon group and BT group than in the hTIMP-1 TgBT group (*P < 0.05). hTIMP indicates human tissue inhibitor of metalloproteinase 1; TgBT, transgenic BMSC transplantation 1; BMSC, bone marrow mesenchymal stem cell; PCon, control group; PBS, phosphate-buffered saline; PG, proteoglycan.
Figure 4. Protein expression in NP tissue specimens from each group after 12 weeks, with buffy color indicating positive staining. A, Type II collagen– labeled sections (400×). B, hTIMP-1–labeled sections. (400×). hTIMP indicates human tissue inhibitor of metalloproteinase; NP, nucleus pulposus.
greater ECM content, as well as higher levels of hTIMP-1 expression. Therefore, the slowed progression of IDD we observed likely resulted from hTIMP-1 activity, which can inhibit the MMP activity and decrease the excess degradation of the ECM. A previous study demonstrated that transforming growth factor β1 can promote PG synthesis in NP cells,21 whereas another study showed that adenoviral-mediated transfection of NP cells with hTGF-β1 increased the content of PGs in
the disc.22 These represent the first attempts to repair and treat IDD with growth factor transplantation and transgenic technology. However, other studies have shown that transfection with latent membrane protein-1, bone morphogenetic protein-2,7), insulin-like growth factor, and Sox9 can also increase ECM content.23–26 Wallach et al27 introduced the technique of adenoviral-mediated transfection of tissue inhibitor of metalloproteinase-1 (TIMP-1) into human degenerative NP cells in 2003, and showed that the PG content was
Figure 5. Summary of data demonstrating successful elevation of hTIMP-1 expression in hTIMP-1 TgBT group. A, hTIMP-1 solubility curve obtained after fluorescent Q-PCR. Note the unimodal curve, indicating that one PCR product was obtained. B, Comparison of hTIMP-1 mRNA expression between groups. There was elevated expression of hTIMP-1 mRNA in the hTIMP-1 TgBT group (*P < 0.05 vs. BT group). C, Western blot for hTIMP-1 protein expression. We observed higher expression of hTIMP-1 protein in the hTIMP-1 TgBT group. D, Quantitative analysis of hTIMP-1 protein expression. There was elevated expression of hTIMP-1 protein in the hTIMP-1 TgBT group (*P < 0.05 vs. BT group). hTIMP indicates human tissue inhibitor of metalloproteinase 1; TgBT, transgenic BMSC; BMSC, bone marrow mesenchymal stem cell; PCR, polymerase chain reaction; PBS, phosphate-buffered saline. Spine
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BASIC SCIENCE increased by up to 5-fold, representing the first attempt to intervene in the process of IDD via inhibition of catabolism. The composition of the ECM depends on the balance between tissue formation and breakdown, which is regulated by 2 functionally antagonistic proteinases, the TIMPs and MMPs. Therefore, the balance between TIMPs and MMPs is key to maintaining ECM homeostasis. TIMP is an endogenous inhibitor that can combine with the zinc site of the MMP enzyme, block its activity and play an important role in remodeling and maintaining the balance of the ECM. As a broad-spectrum inhibitor of MMPs, TIMP-1 is a secreted glycoprotein with a relative molecular weight of 23.5 kD. Previous studies have demonstrated the effectiveness of TIMP-1 in treating IDD. For instance, Steven et al28 showed that injection of AAV2 (adeno-associated virus serotype 2)-bone morphogenetic protein-2 and AAV2-TIMP1 into the NP can slow the course of IDD in an in vivo rabbit model. Wei Jian-wei et al29 also indicated that successful delivery of anticatabolic TIMP-1 could result in the increase of PG in cultured degenerated NP cells. NP cells are typically present in low numbers and are difficult to amplify and cultivate in vitro, which limits their application in gene therapy. With the development of stem cell transplantation techniques, BT has been widely applied in IDD treatment because pluripotent BMSCs are conveniently acquired and easily cultivated in vitro. Some studies have shown that BMSCs can promote proliferation of NP cells and augment ECM synthesis after coculture with NP cells.12 Other studies have shown that BMSCs can recognize changes in the microenvironment in degenerated discs and differentiate into NP cells.11–12 Furthermore, previous work has shown that transplantation of BMSCs into degenerated intervertebral discs can increase the height of the intervertebral disc and PG content.30 Exogenous genes could be introduced to express proteins in BMSCs, and adenoviral-mediated gene vectors could be used for efficient transfection with almost no effect on the phenotype and differentiation status of the BMSCs.14 Therefore, adenoviral-mediated gene transfer has potential as a high-performance expression product in BMSCs.31 We demonstrated that BMSCs could be transfected by hTIMP-1 recombinant adenovirus effectively in vitro and hTIMP-1 mRNA and protein was expressed efficiently and stably at a high level in BMSCs. The main advantage of BMSCs as target cells is that they can be induced to differentiate as NP cells after transplantation by integrating into the changed microenvironment of the degenerated disc and assuming NP cell physiological function. But transgenic BMSCs can also provide a source of TIMP-1, thereby inhibiting excess ECM degradation. Our results suggest that TgBT was more effective at inhibiting disc degeneration and enhancing ECM content than BT alone. However, the BT group also showed improvement over controls in terms of PG and collagen type II content. Reduced degenerative change is closely related to ECM content, especially PG and collagen type II. Therefore, the synergistic effects of increasing ECM, stimulating NP cell production E674
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from transplanted BMSCs, and the expression of hTIMP-1 combine effectively to inhibit degradation of the ECM.
CONCLUSION This study is novel in that we have shown for the first time that the combination of transgenic hTIMP-1 expression, which can inhibit the catabolic pathway, and transplantation of BMSCs can ameliorate the course of IDD in vivo. The injury-induced degenerative disc rabbit model was demonstrated to be effective via radiograph, pathology analysis, and IHC. Adenoviral-mediated transfection resulted in high levels of hTIMP-1 expression in the TgBT group, as demonstrated through realtime PCR, western blotting, and IHC. Finally, compared with simple BT, TgBT resulted in better augmentation of PG and collagen type II content. Consequently, hTIMP-1 transfected BMSCs were able to reverse or delay IDD through the double action of exogenous hTIMP-1 expression (which inhibited ECM degradation) and injection of BMSCs to promote the synthesis of additional ECM.
➢ Key Points This study shows for the first time that a combination of transgenic hTIMP-1 expression, which can inhibit the catabolic pathway, and transplantation of BMSCs can ameliorate the course of IDD in vivo. The mechanism by which hTIMP-1 transfected BMSCs reverse or delay IDD may involve exogenous hTIMP-1 expression inhibiting ECM degradation combined with promotion of ECM synthesis by transplanted BMSCs. The slowed progression of IDD that we observed likely resulted from hTIMP-1 activity, which can inhibit MMP activity and decrease excess ECM degradation.
Acknowledgments The authors thank Medjaden Bioscience Limited, for assisting in the preparation of this manuscript.
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30. 31.
in biological extracts and its use in skin and muscle tissue studies. Glycobiology 2003;13:647–53. Thompson JP, Oegema TR, Bradford DS. Stimulation of mature canine intervertebral disc by growth factors. Spine 1991;16: 253–60. Nishida K, Kang JD, Gilbertson LG, et al. Modulation of the biologic activity of the rabbit intervertebral disc by gene therapy: an in vivo study of adenovirus-mediated transfer of the human transforming growth factor β1 encoding gene. Spine 1999;24: 2419–25. Yoon ST, Park JS, Kim KS, et al. LMP-1 upregulates intervertebral disc cell production of proteoglycans and BMPs in vitro and in vivo. Spine 2004;29:2603–11. Zhao Xin, Huang Shi, Zhang Hai-long, et al. The effects of bone mesenchymal stem cell transferred by adenovirus vector–mediated hBMP-7 gene on proteoglycan of degenerative intervertebral disc of rabbits. Chin J Clin Anat 2008;26:413–5 Moon S, Nishida K, Gilbertson JG, et al. Biologic response of human intervertebral disc cell to gene therapy cocktail. Spine 2008;33:1850–5. Paul R, Haydon RC, Cheng H, et al. Potential use of Sox9 gene therapy for intervertebral degenerative disc disease. Spine 2003;28:755–63. Wallach CJ, Sobajima S, Watanabe Y, et al. Gene transfer of the catabolic inhibitor TIMP-1 increases measured proteoglycans in cells from degenerated human intervertebral discs. Spine 2003;28: 2331–7. Steven K, Bernard P, Robert A, et al. Injection of AAV2-BMP2 and AAV2-TIMP1 into the nucleus pulposus slows the course of intervertebral disc degeneration in an in vivo rabbit model. Spine J 2012;12:7–20. Wei Jian-wei, Wang De-chun, Hu You-gu. An in vitro study of nucleus pulposus cells of human degenerated lumbar intervertebral discs: the biological effects of tissue inhibitor of metalloproteinase 1–mediated by adeno-associated virus vector. Chin J Spine Spinal Cord 2008;18:680–3. Sakai D, Mochida J, Iwashina T, et al. Regenerative effects of transplanting mesenchymal stem cells embedded in a telocollagen to the degenerated intervertebral disc. Biomaterials 2006;27:335–45. Sobajima S, Vadala G, Shimer A, et al. Feasibility of stem cell therapy for intervertebral disc degeneration. Spine J 2008;8: 888–96
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Effects of Transplantation of hTIMP-1–Expressing Bone Marrow Mesenchymal Stem Cells on the Extracellular Matrix of Degenerative Intervertebral Discs in an In Vivo Rabbit Model Zhou Yi, MD,*† Tu Guanjun, MD, PhD,* Cong Lin, MD,* and Pei Zifeng, MD†
Study Design. Prospective, randomized, and controlled animal study. Objective. To observe extracellular matrix (ECM) changes in degenerative intervertebral disc (IVD) after transplantation of bone marrow mesenchymal stem cells (BMSCs) virally transfected with a construct expressing “human tissue inhibitor of metalloproteinase 1” (hTIMP-1), and to discuss the feasibility of using this approach to treat IVD degeneration. Summary of Background Data. Intervertebral disc (IVD) degeneration is characterized by decreased cell numbers, bioactivity of the nucleus pulposus, and remodeled ECM. Exogenous genes can be targeted into cells to produce inhibition of ECM degradation and increase ECM content in IVDs, and thereby potentially stop or reverse degenerative processes and modify disc structure. Methods. BMSCs were isolated from a pure New Zealand white rabbit and identified by flow cytometry. Transgenic BMSCs were acquired by transfection with a recombinant adenovirus vector carrying the hTIMP-1 gene. Animal models of IVD degeneration were established by annulus puncture and then given intra-nucleus pulposus injections according to their random assignment into 3 groups: (1) a transgenic BMSC transplantation (TgBT) group that received BMSCs transfected with an hTIMP-1–expressing adenovirus vector; (2) a BMSC transplantation (BT) group that received unaltered BMSCs; and (3) a control group that received cell-free phosphate-buffered saline. Degree of degeneration was From the *Department of Orthopedics, The First Affiliated Hospital, China Medical University, Shenyang, China; and †Department of Orthopedics, The Second Affiliated Hospital, Shenyang Medical College, Shenyang, China. Acknowledgment date: September 18, 2013. Revision date: November 21, 2013. Acceptance date: January 7, 2014. The manuscript submitted does not contain information about medical device(s)/drug(s). National Natural Science Foundation of China (grant no. 81070971) and Shenyang Medical College Technology Foundation (grant no. 20121014) funds were received in support of this work. No relevant financial activities outside the submitted work. Address correspondence and reprint requests to Tu Guanjun, MD, PhD, Department of Orthopedics, The First Affiliated Hospital, China Medical University, Shenyang 110001, China; E-mail: [email protected] DOI: 10.1097/BRS.0000000000000316 Spine
evaluated 12 weeks after modeling. ECM content was quantified using immunohistochemistry and spectrophotography. Expression of hTIMP-1 was observed via quantitative polymerase chain reaction, western blot, and immunohistochemistry. Results. Significantly fewer degenerative changes and increased ECM content were observed in the TBT and BT groups than the control group animals (P < 0.05). The TBT group had greater ECM content than did the BT group (P < 0.05), as well as higher levels of hTIMP-1 mRNA and protein. Conclusion. Transplantation of BMSCs transfected with hTIMP-1 can increase ECM content by inhibiting ECM degradation and promoting ECM synthesis. Key words: intervertebral disc degeneration, gene therapy, cell transplantation, BMSCs, TIMP. Level of Evidence: N/A Spine 2014;39:E669–E675
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ower back pain is one of the main symptoms of degenerative disc disease, which has a measurable impact on human health.1 The generation and development of degenerative disc disease are both relevant to intervertebral disc degeneration (IDD). Low levels of cellular components and abundant extracellular matrix (ECM) are characteristic of the intervertebral disc (IVD), with most biomechanical functions of the IVD being accomplished by the high ECM content, especially collagen type II and proteoglycans (PGs). Although the etiological factors and pathophysiological process of IDD are not yet well understood, degeneration is known to involve decreased cell numbers, bioactivity of the nucleus pulposus (NP), and reduced ECM content. The main cause of IDD is an imbalance between anabolism and catabolism.2–5 Previous studies have suggested that decreased collagen type II and PGs ultimately lead to morphological and biomechanical changes and decreased biofunction of the IVD. Gene therapy research has been devoted to selecting reasonable target molecules to interfere with the IDD process. Matrix metalloproteinases (MMPs) are important proteinases that can destroy the ECM in the IVD. MMPs participate throughout the process of IDD, and elevated www.spinejournal.com
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hTIMP-1–Expressing BMSC Transplantation on ECM • Yi et al
MMP expression levels are found with increasing degree of degeneration.6–8 The component variation and content diminution of ECM from excessive levels of activated MMPs not only leads to changes in the morphology and biochemistry of the IVD, but also diminishes the biomechanical function of the IVD.9 Tissue inhibitor of metalloproteinases (TIMPs) are a group of specific endogenous inhibitors of MMPs that can lessen ECM degradation via interaction with the zinc-binding site of MMPs. Some studies have indicated that an imbalance between MMPs and TIMPs is the primary reason for excessive degradation of the ECM.10 The strategy of increasing ECM content through inhibition of catabolism makes TIMP a popular candidate gene for IDD gene therapy. Bone marrow mesenchymal stem cells (BMSCs) are important participants in tissue repair in vivo. BMSCs can not only differentiate as NP-like cells within the IVD or in coculture with NP cells,11–12 but can also increase the PG content of the ECM in vivo after transplantation into a rabbit degenerative disc model.13 Adenoviral-mediated transfection can be used to deliver genes to BMSCs efficiently and has negligible effects on the phenotype and differentiation of these cells.14 Hence, adenoviral-mediated gene transfer as an approach has potential for enabling high-performance expression of a product in BMSCs without altering BMSC growth and differentiation. We constructed a recombinant adenoviral vector carrying hTIMP-1 (Ad-hTIMP-1) as part of a previous study.15 In this study, we aimed to transfect rabbit BMSCs via an adenovirus carrying this vector, and transplant these TIMP-1 expressing cells into the degenerative IVD of IDD model rabbits. To our knowledge, transplantation of BMSCs expressing exogenous TIMP-1 has not been performed in an in vivo disc degeneration model. This study investigates the effect of transplanted hTIMP-1–expressing BMSCs on the ECM content in a rabbit IDD model, and explored the feasibility of reversing or delaying IDD via gene therapy and BMSC transplantation (BT). We hypothesized that transgenic BT (TgBT) could postpone or reverse the process of IDD more significantly than BT alone.
Abcam, ab119335; CD90: Abcam, ab226) at a concentration of 0.1–10 μg/mL and incubated with the cells for at least 30 minutes at room temperature (RT) in the dark. Fluorochrome-labeled secondary antibody (115-095-062; Jackson Immunoresearch Laboratories Inc., Baltimore Pike, PA) was diluted in phosphate-buffered saline and incubated with the cells for at least 30 minutes at RT in the dark. For immunohistochemistry (IHC), BMSCs were harvested, washed, and adjusted to a concentration of 2 × 105 cells/mL in a 24-well plate. The viral vector pYr-adshuttle-4 (Yingrun Biotechnologies Inc., Changsha, China; 1 × 1010 pfu/mL virus activity and expressing green fluorescent protein [GFP]) was added at 125, 250, 500, or 1000 multiplicity of infection (MOI) values, respectively. GFP expression was observed via an inverted fluorescence microscope and the transfection efficiency was calculated. For transplantation, BMSCs were harvested and the cell suspension was adjusted to a concentration of 1-5 × 106 cells/ mL. The virus Ad-TIMP-1 (1 × 1010 IU/mL) was added at the optimal MOI and incubated for 45 minutes. The cells were then washed and the BMSC suspension was adjusted to a concentration of 1 × 106 cells/μL for transplantation.
MATERIALS AND METHODS
Radiographs were obtained 12 weeks postpuncture. The scanning parameters of the radiographs were 60 kV, 100 mA, and 50 ms. A previously validated method was used to evaluate the relative height of the intervertebral space.18 The value of the relative intervertebral space height (RISH) of each segment was calculated: RISH =X/Y, where X is the value of each segment ISH, and Y is the value of the nondegenerative L2–L3 segment ISH. The degree of IVD was evaluated by comparing the RISH of each segment.
Sample Preparation The hTIMP-1 cDNA was polymerase chain reaction (PCR) amplified from the pMD-hTIMP-1 vector (Sino Biological Inc., Beijing, China), which contains the human TIMP-1 ORF sequence. As a part of our previous study,15 the recombinant adenoviral vector carrying hTIMP-1 (Ad-hTIMP-1) with 1 × 1010 IU/mL virus activity was stored in the preclinical medicine laboratory at China Medical University. BMSCs were harvested from tibia bone marrow of a New Zealand white rabbit (Experimental Animal Center of Shenyang Pharmaceutical University, Shenyang, China; weight, 1.5 kg; age, 3 mo) using lymphocyte separation in vitro with gradient centrifugation. For fluorescence-activated cell sorting, BMSCs were harvested and washed and the cell suspension was adjusted to a concentration of 1-5 × 106 cells/mL in cold phosphatebuffered saline. Primary antibody was added (CD45: SANTA, SC-70690; CD34: Abcam, ab131589; CD44: E670
Animals and Surgery IDD was induced via a validated rabbit puncture model in 15 healthy New Zealand white rabbits (age, 3 mo; weight, 1.5 kg).16–17 The L3–L4, L4–L5, and L5–L6 discs were exposed and punctured with a 16-gauge needle at the middle point between the endplates to a depth of 5 mm for 5 seconds. Then the rabbits were divided randomly into 3 groups (n = 15) and 30 μL of transgenic BMSC suspension containing about 30 × 106 cells (TgBT group), 30 μL of BMSC suspension containing about 30 × 106 cells (BT group), or 30 μL of phosphate-buffered saline of 0.01 mol/L (PCon group) were injected with a microinjector in the same manner as the puncture model procedure.
Radiographs
Killing of Rabbits and Their Anatomical Observations All rabbits were killed 12 weeks after the initial puncture surgery. The spinal column was dissected out immediately en bloc (L2–L6) and the L3–L4, L4–L5, and L5–L6 discs were exposed and observed.
Pathological and IHC Analysis For pathological analysis, the sections were dewaxed, hydrated, and stained with hematoxylin and eosin using a
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BASIC SCIENCE standard histology protocol, and photographed via a light microscope. For IHC of collagen type II and hTIMP-1 expression, the sections were dewaxed, hydrated, repaired with citrate, and blocked using 3% hydrogen peroxide– formalin. Anticollagen type II or hTIMP-1 primary antibody and diluted horseradish peroxidase–labeled secondary antibody were applied as instructed by the Elivision plus kit (Maixin Biotechnology, Inc., Fuzhou, China). The sections were stained with freshly prepared diaminobenzidine, counterstained with hematoxylin, dehydrated, and mounted with neutral gum. The yellow-stained ECM or stained cytoplasm were positive for collagen type II or hTIMP-1 expression in the NP, and the average optical value was measured for quantification of collagen type II expression with Image-Pro Plus 6.0 software (Media Cybernetics, Bethesda, MD ).
Glycosaminoglycan Assay by Spectrophotography On the basis of changes in the absorption spectrum of 1,9-dimethylmethylene blue (DMMB) dye when bound to glycosaminoglycans (GAGs), we used a spectrophotometric method to study the total GAG content in NP tissue extracts.19 Preparation of working DMMB solution was derived from that reported by Isabelle Barbosa et al20 chondroitin sulfate (CS) standard solution (0.4 mL) was diluted with distilled water (0.1–1.0 mg/mL). A 3-mL working DMMB solution was then added to 1 mL of diluted CS standard solution and the absorbance of the mixture was measured at 580 nm. A calibration curve of CS solutions was described according to the results obtained for absorbance at concentrations with a range of 0.1 to 1 mg/mL. The 10-mg NP tissue extracts were digested in a solution of 200 μL/mL of papain at 56 °C overnight. A 225 μL of aliquot of working DMMB solution was added to NP tissue treated with 25 μL of papain (200 μL/mL) and the mixture was measured at 580 nm. The GAG content in the NP tissue was determined by comparison with a calibration curve of standard CS solutions.
Fluorescent Q-PCR Analysis of hTIMP-1 mRNA Expression RNA extracted from NP tissue was harvested according to the RNA extraction kit protocol. The sequence of the primers used was as follows: hTIMP-1 forward: 5′-CAG AAC CGC AGC GAG GAG T-3′; reverse: 5′-CAG CGT AGG TCT TGG TGA AGC-3′; GAPDH forward: 5′-GGT GAA GGT CGG TGT GAA CG-3′; and reverse: 5′-CTC GCT CCT GGA AGA TGG TG-3′. The resulting products from the BioEasy (Bioer, Hangzhou, China) SYBR Green I Q-PCR kit were analyzed by the line-gene fluorescent quantified PCR system. The amplification efficiency and solubility curve were evaluated and quantitative analysis of the product was done using the 2−△△ct method. The reaction components used for real-time PCR were (50-μL total reaction): 2XSYBR Mix, 25 μL; sense primer, 1 μL; antisense primer, 1 μL; Taq DNA polymerase, 0.3 μL; cDNA, 2 μL; and ddH2O, 20.7 μL. PCR reaction conditions were as follows: 95 °C for 120 seconds, then 45 cycles of 95 °C for 20 seconds, annealing at 60 °C for 25 seconds, and Spine
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elongation at 72 °C for 30 seconds, with a final ramp from 65 °C to 95 °C at 0.5 °C/s.
Western Blot Analysis of hTIMP-1 Protein Expression Protein was harvested from NP tissue extracts routinely; 20 μg of each sample underwent SDS-polyacrylamide gel electrophoresis, and were electrically transferred to polyvinylidene fluoride (PVDF) membranes. Nonspecific binding was blocked with Tris-buffered saline and tween20 containing 5% skim milk for 4 hours at RT, and then stained with anti-hTIMP-1 (1:100) and anti-βactin (1:2000) separately overnight at RT, followed by incubation with an horseradish peroxidase– tagged goat antirabbit secondary antibody (1:10,000) for 4 hours. The blot was incubated in 3,3′-diaminobenzidine and exposed to photographic film. Grayscale analysis was completed using LabWorks software (UVP, Upland, CA) .
Statistical Analysis
The data are presented as means ± standard deviations and SPSS 17.0 (SPSS Inc., Chicago, IL) was used for statistical analysis by simple factor analysis of variance and independent sample t test. A difference of P < 0.05 was considered statistically significant.
RESULTS Cultivation and Identification of BMSCs With time in culture, adherent cells reached 70% confluence and proliferated rapidly within 3 to 7 days. With continued culture, these cells could reach 90% confluence and assume a radial and swirled arrangement. Fluorescence-activated cell sorting analysis indicated that the cultured BMSCs were 95.61% and 96.53% positive for the surface antigen markers CD44 and CD90, respectively, and were 0.76% and 12.77% positive for CD34 and CD45, respectively. These results indicated that the cells were BMSCs
Identification of MOI Assessment of BMSC GFP expression was performed using an inverted fluorescent microscope 24 hours after transfection. At an MOI of 1000, 90% of the BMSCs expressed GFP, but the cells remained morphologically normal.
Evaluation of IVD Digital Radiography Results According to the digital radiographical analysis of the spinal column, the PCon and BT groups demonstrated more severe sclerosis of the IVD endplate than the TgBT group (Figure 1). Lower RISH values were obtained for the BT group (0.842 ± 0.0428) than in the TgBT group (0.896 ± 0.039), and this difference was significant (P < 0.05). RISH values were the lowest in the PCon group (0.791 ± 0.036), which was significantly different from the experimental groups (P < 0.05).
Anatomical and Pathological Observation of IVD Anatomical observation of morphological alterations indicated a range of degenerative changes, including loss of tight linkage between the NP and the endplate, partial or complete www.spinejournal.com
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Figure 1. Digital radiographical analysis of the spinal columns revealed more severe sclerosis of the endplate of the intervertebral disc and shorter RISH in the PCon group and BT group than the hTIMP-1 TgBT group (*P < 0.05). hTIMP indicates human tissue inhibitor of metalloproteinase; TgBT, transgenic BMSC transplantation 1; BMSC, bone marrow mesenchymal stem cell; PCon, control group; RISH, relative intervertebral space height.
loss of NP, undefined boundary between the NP and annulus fibrosis, derangement or partial disruption of the annulus fibrosis, loss of the characteristic oval appearance, and lackluster color of the NP (Figure 2A). Analysis of hematoxylin and eosin–stained sections also revealed degenerative changes, including decreased numbers and irregularly shaped NP cells, karyopyknosis in some NP cells, looseness in the ECM alignment, disappearance of the mucoid cartilage matrix and substitution by fibrous tissue (Figure 2B). The degenerative IVDrelated changes were more pronounced in the PCon group than in the BT or TgBT groups.
Spectrophotographic Analysis of GAGs The results of the assessment of PG contents for each group are described in Figure 3A. The PG contents were significantly reduced in the BT group (6.160 ± 0.569) compared with the TgBT group (7.910 ± 0.452, P < 0.05). PG content was lowest in the PCon group (4.263 ± 0.570, P < 0.05).
Type II Collagen Assays and hTIMP-1 Expression Quantitative analysis of the collagen type II contents for each group is described in Figure 3B. Collagen type II content was significantly lower in the BT group (0.272 ± 0.014) than in the TgBT group (0.354 ± 0.022, P < 0.05). Collagen type II content was lowest in the PCon group (0.218 ± 0.013, P < 0.05). Our IHC experiment (Figure 4A) demonstrated type II collagen expression in all 3 groups, but the TgBT group exhibited
the highest levels of staining, followed by the BT group, and the PCon group. IHC analysis of hTIMP-1 protein expression (Figure 4B) revealed hTIMP-1 protein expression in the TgBT group only, which exhibited buffy color positive staining localized to the cytoplasm of NP cells. There was negligible, if any, hTIMP-1 expression in the BT and PCon groups.
Q-PCR and Western Blot Analysis of hTIMP-1 Expression The fluorescent detection of our PCR results indicated an amplification efficiency of 98.74% with a unimodal solubility curve (Figure 5A). Quantitative analysis results using the 2−△△ct method are described in Figure 5B. We observed greater expression of hTIMP-1 mRNA in the TgBT group (136.531 ± 15.231) than in the BT (5.818 ± 0.989) and PCon (1.119 ± 0.375) groups (Ps < 0.05). As shown in Figure 5C, D, western blot analysis of hTIMP-1 protein expression showed that the TgBT group (0.781 ± 0.079) exhibited higher levels of hTIMP-1 protein expression (P < 0.05) than the BT (0.181 ± 0.035) and PCon (0.120 ± 0.038) groups.
DISCUSSION In this study, we transfected BMSCs with the hTIMP-1 gene by recombinant adenovirus vector in vitro and transplanted these transgenic BMSCs into the NP of an injuryinduced model of degenerative disc. Compared with the BT group, the TgBT group showed less degeneration and
Figure 2. Comparison of tissues across groups. A, Example discs obtained from rabbits in each group showing anatomical and morphological differences between the groups. B, Hematoxylin and eosin– stained sections (400×) demonstrating fewer and more irregularly shaped NP cells in animals from the PCon group relative to the hTIMP-1 TgBT group. NP indicates nucleus pulposus; human tissue inhibitor of metalloproteinase 1; TgBT, transgenic BMSC transplantation 1; BMSC, bone marrow mesenchymal stem cell; PCon, control group.
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hTIMP-1–Expressing BMSC Transplantation on ECM • Yi et al
Figure 3. Quantification of PG and collagen type II content. A, Spectrographic analysis revealed that PG content was significantly lower in the PCon group and BT group than in the hTIMP-1 TgBT group (*P < 0.05). B, Likewise, spectrographic analysis revealed that collagen type II content was significantly lower in the PCon group and BT group than in the hTIMP-1 TgBT group (*P < 0.05). hTIMP indicates human tissue inhibitor of metalloproteinase 1; TgBT, transgenic BMSC transplantation 1; BMSC, bone marrow mesenchymal stem cell; PCon, control group; PBS, phosphate-buffered saline; PG, proteoglycan.
Figure 4. Protein expression in NP tissue specimens from each group after 12 weeks, with buffy color indicating positive staining. A, Type II collagen– labeled sections (400×). B, hTIMP-1–labeled sections. (400×). hTIMP indicates human tissue inhibitor of metalloproteinase; NP, nucleus pulposus.
greater ECM content, as well as higher levels of hTIMP-1 expression. Therefore, the slowed progression of IDD we observed likely resulted from hTIMP-1 activity, which can inhibit the MMP activity and decrease the excess degradation of the ECM. A previous study demonstrated that transforming growth factor β1 can promote PG synthesis in NP cells,21 whereas another study showed that adenoviral-mediated transfection of NP cells with hTGF-β1 increased the content of PGs in
the disc.22 These represent the first attempts to repair and treat IDD with growth factor transplantation and transgenic technology. However, other studies have shown that transfection with latent membrane protein-1, bone morphogenetic protein-2,7), insulin-like growth factor, and Sox9 can also increase ECM content.23–26 Wallach et al27 introduced the technique of adenoviral-mediated transfection of tissue inhibitor of metalloproteinase-1 (TIMP-1) into human degenerative NP cells in 2003, and showed that the PG content was
Figure 5. Summary of data demonstrating successful elevation of hTIMP-1 expression in hTIMP-1 TgBT group. A, hTIMP-1 solubility curve obtained after fluorescent Q-PCR. Note the unimodal curve, indicating that one PCR product was obtained. B, Comparison of hTIMP-1 mRNA expression between groups. There was elevated expression of hTIMP-1 mRNA in the hTIMP-1 TgBT group (*P < 0.05 vs. BT group). C, Western blot for hTIMP-1 protein expression. We observed higher expression of hTIMP-1 protein in the hTIMP-1 TgBT group. D, Quantitative analysis of hTIMP-1 protein expression. There was elevated expression of hTIMP-1 protein in the hTIMP-1 TgBT group (*P < 0.05 vs. BT group). hTIMP indicates human tissue inhibitor of metalloproteinase 1; TgBT, transgenic BMSC; BMSC, bone marrow mesenchymal stem cell; PCR, polymerase chain reaction; PBS, phosphate-buffered saline. Spine
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BASIC SCIENCE increased by up to 5-fold, representing the first attempt to intervene in the process of IDD via inhibition of catabolism. The composition of the ECM depends on the balance between tissue formation and breakdown, which is regulated by 2 functionally antagonistic proteinases, the TIMPs and MMPs. Therefore, the balance between TIMPs and MMPs is key to maintaining ECM homeostasis. TIMP is an endogenous inhibitor that can combine with the zinc site of the MMP enzyme, block its activity and play an important role in remodeling and maintaining the balance of the ECM. As a broad-spectrum inhibitor of MMPs, TIMP-1 is a secreted glycoprotein with a relative molecular weight of 23.5 kD. Previous studies have demonstrated the effectiveness of TIMP-1 in treating IDD. For instance, Steven et al28 showed that injection of AAV2 (adeno-associated virus serotype 2)-bone morphogenetic protein-2 and AAV2-TIMP1 into the NP can slow the course of IDD in an in vivo rabbit model. Wei Jian-wei et al29 also indicated that successful delivery of anticatabolic TIMP-1 could result in the increase of PG in cultured degenerated NP cells. NP cells are typically present in low numbers and are difficult to amplify and cultivate in vitro, which limits their application in gene therapy. With the development of stem cell transplantation techniques, BT has been widely applied in IDD treatment because pluripotent BMSCs are conveniently acquired and easily cultivated in vitro. Some studies have shown that BMSCs can promote proliferation of NP cells and augment ECM synthesis after coculture with NP cells.12 Other studies have shown that BMSCs can recognize changes in the microenvironment in degenerated discs and differentiate into NP cells.11–12 Furthermore, previous work has shown that transplantation of BMSCs into degenerated intervertebral discs can increase the height of the intervertebral disc and PG content.30 Exogenous genes could be introduced to express proteins in BMSCs, and adenoviral-mediated gene vectors could be used for efficient transfection with almost no effect on the phenotype and differentiation status of the BMSCs.14 Therefore, adenoviral-mediated gene transfer has potential as a high-performance expression product in BMSCs.31 We demonstrated that BMSCs could be transfected by hTIMP-1 recombinant adenovirus effectively in vitro and hTIMP-1 mRNA and protein was expressed efficiently and stably at a high level in BMSCs. The main advantage of BMSCs as target cells is that they can be induced to differentiate as NP cells after transplantation by integrating into the changed microenvironment of the degenerated disc and assuming NP cell physiological function. But transgenic BMSCs can also provide a source of TIMP-1, thereby inhibiting excess ECM degradation. Our results suggest that TgBT was more effective at inhibiting disc degeneration and enhancing ECM content than BT alone. However, the BT group also showed improvement over controls in terms of PG and collagen type II content. Reduced degenerative change is closely related to ECM content, especially PG and collagen type II. Therefore, the synergistic effects of increasing ECM, stimulating NP cell production E674
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from transplanted BMSCs, and the expression of hTIMP-1 combine effectively to inhibit degradation of the ECM.
CONCLUSION This study is novel in that we have shown for the first time that the combination of transgenic hTIMP-1 expression, which can inhibit the catabolic pathway, and transplantation of BMSCs can ameliorate the course of IDD in vivo. The injury-induced degenerative disc rabbit model was demonstrated to be effective via radiograph, pathology analysis, and IHC. Adenoviral-mediated transfection resulted in high levels of hTIMP-1 expression in the TgBT group, as demonstrated through realtime PCR, western blotting, and IHC. Finally, compared with simple BT, TgBT resulted in better augmentation of PG and collagen type II content. Consequently, hTIMP-1 transfected BMSCs were able to reverse or delay IDD through the double action of exogenous hTIMP-1 expression (which inhibited ECM degradation) and injection of BMSCs to promote the synthesis of additional ECM.
➢ Key Points This study shows for the first time that a combination of transgenic hTIMP-1 expression, which can inhibit the catabolic pathway, and transplantation of BMSCs can ameliorate the course of IDD in vivo. The mechanism by which hTIMP-1 transfected BMSCs reverse or delay IDD may involve exogenous hTIMP-1 expression inhibiting ECM degradation combined with promotion of ECM synthesis by transplanted BMSCs. The slowed progression of IDD that we observed likely resulted from hTIMP-1 activity, which can inhibit MMP activity and decrease excess ECM degradation.
Acknowledgments The authors thank Medjaden Bioscience Limited, for assisting in the preparation of this manuscript.
References
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