Transplantation of mesenchymal stem cells in a canine disc degeneration model

Transplantation of mesenchymal stem cells (MSCs) is effective in decelerating disc degeneration in small animals; much remains unknown about this new therapy in larger animals or humans. Fas‐ligand (FasL), which is only found in tissues with isolated immune privilege, is expressed in IVDs, particularly in the nucleus pulposus (NP). Maintaining the FasL level is important for IVD function. This study evaluated whether MSC transplantation has an effect on the suppression of disc degeneration and preservation of immune privilege in a canine model of disc degeneration. Mature beagles were separated into a normal control group (NC), a MSC group, and the disc degeneration (nucleotomy‐only) group. In the MSC group, 4 weeks after nucleotomy, MSCs were transplanted into the degeneration‐induced discs. The animals were followed for 12 weeks after the initial operation. Subsequently, radiological, histological, biochemical, immunohistochemical, and RT‐PCR analyses were performed. MSC transplantation effectively led to the regeneration of degenerated discs. FACS and RT‐PCR analyses of MSCs before transplantation demonstrated that the MSCs expressed FasL at the genetic level, not at the protein level. GFP‐positive MSCs detected in the NP region 8 weeks after transplantation expressed FasL protein. The results of this study suggest that MSC transplantation may contribute to the maintenance of IVD immune privilege by the differentiation of transplanted MSCs into cells expressing FasL. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:589–600, 2008     

Effect of cell number on mesenchymal stem cell transplantation in a canine disc degeneration model

Transplantation of mesenchymal stem cells (MSCs) inhibits the progression of disc degeneration in animal models. We know of no study to determine the optimal number of cells to transplant into the degenerated intervertebral disc (IVD). To determine the optimal donor cell number for maximum benefit, we conducted an in vivo study using a canine disc degeneration model. Autologous MSCs were transplanted into degenerative discs at 10⁵, 10⁶, or 10⁷ cells per disc. The MSC‐transplanted discs were evaluated for 12 weeks using plain radiography, magnetic resonance imaging, and gross and microscopic evaluation. Preservation of the disc height, annular structure was seen in MSC‐transplantation groups compared to the operated control group with no MSC transplantation. Result of the number of remaining transplanted MSCs, the survival rate of NP cells, and apoptosis of NP cells in transplanted discs showed both structural microenvironment and abundant extracellular matrix maintained in 10⁶ MSCs transplanted disc, while less viable cells were detected in 10⁵ MSCs transplanted and more apoptotic cells in 10⁷ MSCs transplanted discs. The results of this study demonstrate that the number of cells transplanted affects the regenerative capability of MSC transplants in experimentally induced degenerating canine discs. It is suggested that maintenance of extracellular matrix by its production from transplanted cells and/or resident cells is important for checking the progression of structural disruption that leads to disc degeneration. Published by Wiley Periodicals, Inc. J Orthop Res 28:1267–1275, 2010     

Scale‐up of MSC under hypoxic conditions for allogeneic transplantation and enhancing bony regeneration in a rabbit calvarial defect model

To realize the therapeutic potential of mesenchymal stem cells (MSCs), we aimed to develop a method for isolating and expanding New Zealand rabbit MSCs in a great scale. Rabbit MSCs expanded under hypoxic and normoxic conditions were compared in terms of replication capacity, differentiation potential, and the capacity for allogeneic transplantation in a calvarial defect model. The cells from all tested rabbits were expanded more rapidly when plated at low‐density under hypoxic conditions compared to under normoxic conditions. Moreover, cells expanded under hypoxic conditions increased in the potential of osteoblastic, adipocytic, and chondrocytic differentiation. More importantly, radiographic analysis and micro‐CT measurement of bone volume revealed the hypoxic cells when transplanted in the calvarial defects of another rabbit increased in the ability to repair bone defect compared to the normoxic cells. Six weeks after allogeneic transplantation of hypoxic MSCs, histological analysis revealed a callus spanned the length of the defect, and several bone tissues spotted in the implant. At 12 weeks, new bone had formed throughout the implant. Using BrdU labeling to track the transplanted cells, the hypoxic cells were more detected in the newly formed bone compared to the normoxic cells. For defects treated with allogeneic MSCs, no adverse host response could be detected at any time‐point. In conclusion, we have developed a robust method for isolation and expansion of rabbit MSCs by combining low‐density with hypoxic culture, which can be applied for the design of clinical trials in allogeneic transplantation of MSCs for bone healing. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1213–1220, 2012     

Effects of Intradiscal Injection of Local Anesthetics on Intervertebral Disc Degeneration in Rabbit Degenerated Intervertebral Disc

Analgesic discoblock is widely used for the diagnosis or treatment of discogenic low back pain by injecting local anesthetics. The purpose of this study was to investigate the deleterious effects of local anesthetics on degenerated rabbit intervertebral disks (IVDs) using an organotypic culture model and in vivo long‐term follow‐up model. To induce IVD degeneration, a rabbit annular puncture model was used. For the organotypic culture model, degenerated IVDs were harvested 1 month after the initial annular puncture and cultured for 3 or 7 days after intradiscal injection of local anesthetics (1% lidocaine and 0.5% bupivacaine). To perform in vivo analysis, local anesthetics were injected into degenerated IVDs, and IVDs were prepared for histological analysis after 6 or 12 months. In the organotypic model, terminal deoxynucleotidyl transferase dUTP nick end labeling‐positive nucleus pulposus (NP) cells were significantly increased in the bupivacaine group compared with the other groups. In the in vivo study, the number of NP cells was significantly decreased in the saline and local anesthetics groups compared with the untreated control and puncture‐only groups. However, there was no significant difference among the saline, lidocaine, and bupivacaine groups. In addition, histological analysis showed no significant difference of IVD degeneration among the puncture‐only, saline, lidocaine, and bupivacaine groups. Although bupivacaine induced apoptotic NP cell death in the organotypic culture model, in vivo observations did not show any definitive proof to suggest that local anesthetics were capable of promoting degeneration in the degenerated IVD, except for pressurized injection‐induced damage. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1963–1971, 2019     

Optimization of puncture injury to rat caudal disc for mimicking early degeneration of intervertebral disc

The caudal discs of rats have been proposed as a puncture model in which intervertebral disc (IVD) degeneration can be induced and novel therapies can be tested. For biological repair, treatments for ongoing IVD degeneration are ideally administered during the earlier stages. The purpose of this study was to elucidate the optimal puncture needle size for creating a model that mimicked the earlier stages of IVD degeneration. According to the disc height index, histologic score, and MRI grading, a puncture needle sized 21G or larger induced rapid degenerative processes in rat caudal discs during the initial 2–4 weeks. The degenerative changes were severe and continued deteriorating after 4 weeks. Conversely, puncture injury induced by needles sized 25G or smaller also produced degenerative changes in rat caudal discs during initial 2–4 weeks; however, the changes were less severe. Furthermore, the degenerative process became stabilized and showed no further deterioration or spontaneous recovery after 4 weeks. In the discs punctured by 25G needles, the expression of collagen I was increased at 2–4 weeks with a gradually fibrotic transformation thereafter. The expressions of collagen II and SOX9 were enhanced initially but returned to pre‐injury levels at 4–8 weeks. The above‐mentioned findings were more compatible with earlier degeneration in discs punctured by needles sized 25G or smaller than by needles sized 21G or larger, and the appropriate timing for intradiscal administration of proposed therapeutic agents would be 4 weeks or longer after puncture. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:202–211, 2018.     

Differential phenotypic behaviors of human degenerative nucleus pulposus cells under normoxic and hypoxic conditions: influence of oxygen concentration during isolation,expansion, and cultivation

Background contextIntervertebral discs (IVDs) are the largest avascular structures in the body; therefore, cells within these discs might be adapted to low-oxygen conditions. Although it has been demonstrated that a low oxygen concentration could promote synthesis of the extracellular matrix by IVD cells in the in vitro culture, isolation, expansion, and cultivation of IVD cells under classical tissue culture O₂ saturation could still be detrimental.PurposeTo investigate the phenotypic differences between human degenerative nucleus pulposus (NP) cells during isolation and expansion under normoxic (Nx: 21% O₂) or hypoxic (Hx: 3.5% O₂) conditions.Study designWe investigated in vitro isolation, expansion, and cultivation of human NP cells.MethodsHuman NP tissue samples were obtained from patients who underwent lumbar disc surgeries. Nucleus pulposus cells were then isolated, expanded, and cultivated under normoxic or hypoxic conditions. To determine whether the effects of normoxic expansion are reversible, another group of cells was isolated and expanded in normoxic conditions and then cultivated under hypoxic conditions (Nx→Hx group). Cellular proliferation, RNA expression of selected genes, and immunohistochemical staining were performed to evaluate the phenotypic behaviors of human NP cells under different conditions.ResultsExpressions of Type II collagen and aggrecan in the Nx→Hx group were significantly higher than those in the normoxic group but were significantly lower than those in the hypoxic group. The normoxic group showed higher expression of matrix metalloproteinase (MMP)-2 and MMP-13 than did the other groups. Expression levels of hypoxia-inducible factors (HIFs) were significantly higher in the normoxic groups; however, a greater degree of HIF-1α staining was found in the hypoxic group, whereas a greater degree of HIF-2α staining was found in the normoxic group.ConclusionsHuman degenerative NP cells isolated, expanded, and cultivated in hypoxic conditions could better preserve the cells' regenerative potential. Compromised properties that were observed during isolation and expansion under normoxic conditions could only be partially rescued by later hypoxic cultivation. The superior phenotypic behaviors of human NP cells under hypoxia may be related to higher HIF-1α production and lower HIF-2α production. Cells that are isolated, expanded, and cultivated under hypoxic conditions may show better regenerative results when transplanted; therefore, the isolation and expansion processes of human degenerative NP cells should be managed in a hypoxic environment.     

移植骨髓间充质干细胞对兔退变椎间盘髓核细胞凋亡的影响

目的 观察骨髓间充质干细胞(MSCs)移植对兔退变椎间盘髓核细胞凋亡的影响.方法 以各兔L2/3、L3/4、L4/5、L5/6节段分为正常组、退变组、成纤维细胞(SFs)移植对照组、MSCs移植治疗组.MSCs和SFs分别经绿色荧光蛋白(GFP)转染后,注射植入退变椎间盘的髓核.通过透射电镜观察退变椎间盘凋亡髓核细胞形态;用实时定量聚合酶链反应(PCR)检测退变组织中髓核细胞凋亡相关基因bcl-2和box mRNA的表达;免疫荧光法标记髓核细胞凋亡相关蛋白Caspase-3,并通过TUNEL法标记凋亡髓核细胞,激光共聚焦显微镜检测髓核细胞凋亡蛋白表达率和细胞凋亡比率.结果 透射电镜下,退变椎间盘中凋亡髓核细胞呈现出核染色质边集,空泡形成,核膜断裂,凋亡小体形成等变化.MSCs移植治疗组bcl-2 mRNA的表达量高于退变组和SFs移植对照组(P<0.05),bax mRNA的表达量与退变组差异无统计学意义(P>0.05).MSCs移植治疗组细胞凋亡率和Caspase-3表达率均高于正常组[细胞凋亡率分别为(16.75±2.14)%和(6.86±1.08)%;Caspase-3表达率分别为[(20.34±1.03)%和(6.09±0.77)%](P<0.05),低于退变组和SFs移植对照组[细胞凋亡率分别为(31.87±4.16)%和(29.02±2.16)%;Caspase-3表达率分别为(31.50±3.78)%和(30.20±4.93)%](P<0.05).结论 髓核细胞凋亡在椎间盘退变过程中起重要作用.MSCs移植能有效抑制椎间盘髓核细胞凋亡,延缓椎间盘退变过程.     

Human bone morphogenetic protein 7 transfected nucleus pulposus cells delay the degeneration of intervertebral disc in dogs

The main reason for intervertebral disc (IVD) degeneration is the decrease in the quantity and activity of IVD cells with subsequent reduction of the extracellular matrix (ECM). In this study, we investigated a cell‐based repair strategy by injecting nucleus pulposus cells (NPCs) transduced with human bone morphogenetic protein (hBMP7) by adeno‐associated virus‐2 into the canine degenerative IVD to determine whether NPCs expressing hBMP7 could delay the degeneration of the IVD. Fourteen canines received annular punctures to induce disc degeneration. Eight weeks later, saline (group A), allogeneic NPCs (group B), or allogeneic NPCs transduced with hBMP7 (group C) were injected into the degenerative discs. Twelve weeks after the injection, MRI scan showed that the degeneration process of groups C was slower and less severe compared with that of groups B and C. The IVD stability in group C was superior to that in groups A and B in left‐right bending and rotation. HE, safranin‐O staining, and ELISA indicated that the degenerative degree of the IVD in group C was significantly milder than that in groups A and B. The study demonstrated that the implantation of NPCs‐hBMP7 could effectively maintained the structural integrity, ECM, and biomechanical properties of the canine degenerated discs. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1311–1322, 2017.

     

Intradiscal injections of osteogenic protein-1 restore the viscoelastic properties of degenerated intervertebral discs

BACKGROUND CONTEXT: Using biochemical, histological, and radiological parameters in a rabbit model of intervertebral disc (IVD) degeneration, the intradiscal injection of a growth factor, such as osteogenic protein-1 (OP-1), has been shown to regenerate the IVD. However, very little is known about how such a biological therapeutic approach affects the biomechanical properties of the degenerated IVD. PURPOSE: To investigate the effects of an intradiscal injection of OP-1 on the biomechanical properties of IVDs in the rabbit annular-puncture disc degeneration model and to determine their relationship to biochemical properties. STUDY DESIGN/SETTING: In vivo study on the effects of intradiscally administered OP-1 on the biomechanical and biochemical properties of IVDs in the rabbit annular-puncture disc degeneration model. METHODS: New Zealand White rabbits (n=16) underwent annulus fibrosus (AF) puncture, using an 18-gauge needle, at L2-L3 and L4-L5 (L3-L4: nonpunctured control). Four weeks later, the punctured discs received an injection of either 5% lactose (10 microL) or OP-1 (100 microg/10 microL of 5% lactose) into the nucleus pulposus (NP). The disc height was radiographically monitored biweekly. After sacrifice and removal of bone-disc-bone complexes 8 weeks postinjection, the dynamic viscoelastic properties of the IVDs were tested by applying a cycle of sinusoidal strain in uniaxial compression at six loading frequencies (0.05 to 2 Hz). The biochemical properties of the dissected IVDs were then analyzed and correlated with the biomechanical properties. RESULTS: A single injection of OP-1 significantly restored disc height when compared with the lactose-injected discs (OP-1 vs. lactose, p<.001). The elastic modulus of the IVDs in the OP-1-injected discs was significantly higher than that in the lactose-injected discs at all frequencies (mean: +43%, p<.001). The viscous modulus in the OP-1-injected discs was significantly higher at 0.05, 0.2, 0.5, and 1 Hz (mean: +55%, p<.001) and showed higher tendencies at other frequencies (p=.08-.09). For both moduli, no significant differences were observed between the OP-1-injected and the nonpunctured control discs. The OP-1 injection significantly increased the proteoglycan (PG) content in the NP and AF, and the collagen content in the NP (p<.001-.05). Both elastic and viscous moduli showed significant positive correlations with PG content in the NP and collagen content in the NP and AF (Rho=.357-.466, p=.010-.047). CONCLUSIONS: We have shown for the first time that an injection of the growth factor, OP-1, restored the biomechanical properties of IVDs in a rabbit model of IVD degeneration. Comparing biomechanical with biochemical data suggests that the OP-1-induced biomechanical restoration was a consequence of increased activities of anabolic pathways that resulted in biochemical changes in the IVD.     

The effects of microenvironment in mesenchymal stem cell–based regeneration of intervertebral disc

Background contextRecent studies have demonstrated new therapeutic strategy using transplantation of mesenchymal stem cells (MSCs), especially bone marrow–derived MSCs (BM-MSCs), to preserve intervertebral disc (IVD) structure and functions. It is important to understand whether and how the MSCs survive and thrive in the hostile microenvironment of the degenerated IVD. Therefore, this review majorly examines how resident disc cells, hypoxia, low nutrition, acidic pH, mechanical loading, endogenous proteinases, and cytokines regulate the behavior of the exogenous MSCs.PurposeTo review and summarize the effect of the microenvironment in biological characteristics of BM-MSCs for IVD regeneration; the presence of endogenous stem cells and the state of the art in the use of BM-MSCs to regenerate the IVD in vivo were also discussed.Study designLiterature review.MethodsMEDLINE electronic database was used to search for articles concerning stem/progenitor cell isolation from the IVD, regulation of the components of microenvironment for MSCs, and MSC-based therapy for IVD degeneration. The search was limited to English language.ResultsStem cells are probably resident in the disc, but exogenous stem cells, especially BM-MSCs, are currently the most popular graft cells for IVD regeneration. The endogenous disc cells and the biochemical and biophysical components in the degenerating disc present a complicated microenvironment to regulate the transplanted BM-MSCs. Although MSCs regenerate the mildly degenerative disc effectively in the experimental and clinical trials, many underlying questions are in need of further investigation.ConclusionsThere has been a dramatic improvement in the understanding of potential MSC-based therapy for IVD regeneration. The use of MSCs for IVD degeneration is still at the stage of preclinical and Phase 1 studies. The effects of the disc microenvironment in MSCs survival and function should be closely studied for transferring MSC transplantation from bench to bedside successfully.     

Joint analysis of IVD herniation and degeneration by rat caudal needle puncture model

Intervertebral disc (IVD) degeneration is responsible for various spine pathologies and present clinical treatments are insufficient. Concurrently, the mechanisms behind IVD degeneration are still not completely understood, so as to allow development of efficient tissue engineering approaches. A model of rat IVD degeneration directly coupled to herniation is here proposed in a pilot study. Disc injury is induced by needle puncture, using two different needles gauges: a low caliber 25‐G needle and a high caliber 21‐G needle. Histological, biochemical, and radiographic degeneration was evaluated at 2 and 6 weeks post‐injury. We show that the larger caliber needle results in a more extended histological and radiographic degeneration within the IVD, compared to the smaller one. TUNEL quantification indicates also increased cell death in the 21‐G group. Analyses of collagen type I (Picrosirius red staining), collagen type II (immunofluorescence), and GAG content (Blyscan assay) indicate that degeneration features spontaneously recover from 2 to 6 weeks, for both needle types. Moreover, we show the occurrence of hernia proportional to the needle gauge. The number of CD68+ macrophages present, as well as cell apoptosis within the herniated tissue are both proportional to hernia volume. Moreover, hernias formed after lesion tend to spontaneously diminish in volume after 6 weeks. Finally, MMP3 is increased in the hernia in the 21‐G group at 2 weeks. This model, by uniquely combining IVD degeneration and IVD herniation in the same animal, may help to understand mechanisms behind IVD pathophysiology, such as hernia formation and spontaneous regression. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:258–268, 2017.

     

Combination therapy with intra‐articular injection of mesenchymal stem cells and articulated joint distraction for repair of a chronic osteochondral defect in the rabbit

The present study investigated intra‐articular injection of bone‐marrow‐derived mesenchymal stem cells (MSCs) combined with articulated joint distraction as treatment for osteochondral defects. Large osteochondral defects were created in the weight‐bearing area of the medial femoral condyle in rabbit knees. Four weeks after defect creation, rabbits were divided into six groups: control group, MSC group, distraction group, distraction + MSC group, temporary distraction group, and temporary distraction + MSC group. Groups with MSC received intra‐articular injection of MSCs. Groups with distraction underwent articulated distraction arthroplasty. Groups with temporary distraction discontinued the distraction after 4 weeks. The rabbits were euthanized at 4, 8, and 12 weeks after treatment except temporary distraction groups which were euthanized at only 12 weeks. Histological scores in the distraction + MSC group were significantly better than in the control, MSC group or distraction group at 4 and 8 weeks, but showed no further improvement. At 12 weeks, the temporary distraction + MSC group showed the best results, demonstrating hyaline cartilage repair with regeneration of the osteochondral junction. In conclusion, joint distraction with intra‐articular injection of MSCs promotes early cartilage repair, and compressive loading of the repair tissue after temporary distraction stimulates articular cartilage regeneration. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1466–1473, 2015.     

应用微创技术建立恒河猴腰椎间盘早期退变模型

目的 应用CT定位,经皮穿刺纤维环诱导恒河猴腰椎间盘退变,建立灵长类动物腰椎间盘早期退变模型.方法 恒河猴13只,随机分为三组:(1)造模组:在CT定位下,用20G穿刺针从左侧后方入路经皮穿刺L1,2:(n=12),L2,3、L3,4、L4,5、L5,6(n=13)椎间盘的纤维环全层至椎间盘髓核正中,共64个椎间盘.(2)穿刺对照组:15G穿刺针穿刺1只猴的L1,2椎间盘.(3)正常对照组:L6,7,L7-S1,共26个椎间盘.造模前及造模后4、8、12周对各组椎间盘行MRI检查,并行HE、Masson、番红O、免疫组织化学染色组织学观察.结果 (1)MRI:20G穿刺针穿刺的造模组椎间盘造模前及造模后4、8、12周,椎间盘信号强度按Pfirmann分级均为Ⅰ级.15G穿刺针穿刺椎间盘4周时信号降低(Pfirrmann Ⅲ级),8周时为黑色椎间盘(Pfirmann Ⅳ级).正常对照组椎间盘为Pfirmann Ⅰ级.(2)组织学:造模组椎间盘造模后4周未见改变,8周时HE染色示髓核内细胞数减少,12周时较为明显.Masson染色4周未见改变,8周时各层纤维间出现裂隙,12周时裂隙增宽.番红O染色见8、12周髓核内蛋白聚糖进行性减少.免疫组织化学结果显示4周和8周时同正常椎间盘比较差异无统计学意义(P＞0.05),12周时,Ⅱ型胶原合成减少(P＜0.05).15G穿刺对照组在8周时HE染色见髓核内细胞减少明显,Masson染色见纤维环各层间裂隙明显,呈波浪状.番红O染色示髓核内蛋白聚糖数量明显减少.免疫组织化学染色示Ⅱ型胶原合成减少.正常对照组在各时间点未见到形态学改变.结论 20G穿刺针可以诱发椎间盘缓慢进展的轻度退变.MRI平均信号强度观察椎间盘轻度退变时,不是敏感的指标,需要依靠组织学证实.     

Intradiscal injection of simvastatin results in radiologic,histologic, and genetic evidence of disc regeneration in a rat model of degenerative disc disease

Background contextA large percentage of back pain can be attributed to degeneration of the intervertebral disc (IVD). Bone morphogenetic protein 2 (BMP-2) is known to play an important role in chondrogenesis of the IVD. Simvastatin is known to upregulate expression of BMP-2. Thus, we hypothesized that intradiscal injection of simvastatin in a rat model of degenerative disc disease (DDD) would result in retardation of DDD.PurposeThe purpose of the present study was to develop a novel conservative treatment for DDD and related discogenic back pain.Study design/settingThe setting of this study is the laboratory investigation.MethodsDisc injury was induced in 272 rats via 21-ga needle puncture. After 6 weeks, injured discs were treated with simvastatin in a saline or hydrogel carrier. Rats were sacrificed at predetermined time points. Outcome measures assessed were radiologic, histologic, and genetic. Radiologically, the magnetic resonance imaging (MRI) index (number of pixels multiplied by the corresponding image densities) was determined. Histologically, disc spaces were read by three blinded scorers using a previously described histologic grading scale. Genetically, nuclei pulposi were harvested, and polymerase chain reaction was run to determine relative levels of aggrecan, collagen type II, and BMP-2 gene expression.ResultsRadiologically, discs treated with 5 mg/mL of simvastatin in hydrogel or saline demonstrated MRI indices that were normal through 8 weeks after treatment, although this was more sustained when delivered in hydrogel. Histologically, discs treated with 5 mg/mL of simvastatin in hydrogel demonstrated improved grades compared with discs treated at higher doses. Genetically, discs treated with 5 mg/mL of simvastatin in hydrogel demonstrated higher gene expression of aggrecan and collagen type II than control.ConclusionsDegenerate discs treated with 5 mg/mL of simvastatin in a hydrogel carrier demonstrated radiographic and histologic features resembling normal noninjured IVDs. In addition, the gene expression of aggrecan and collagen type II (important constituents of the IVD extracellular matrix) was upregulated in treated discs. Injection of simvastatin into degenerate IVDs may result in retardation of disc degeneration and represents a promising investigational therapy for conservative treatment of DDD.     

Therapeutic effects of adenovirus-mediated growth and differentiation factor-5 in a mice disc degeneration model induced by annulus needle puncture

Background contextThe therapeutic strategies that have thus far been used for the treatment of intervertebral disc degeneration (IDD) have focused on relieving the symptoms, although reversal of the degeneration remains an important challenge for the effective treatment of IDD. Growth and differentiation factor-5 (GDF5), of which deficiency leads to early disc degeneration changes, has the potential to increase proliferation of disc cells and expression of extracellular matrix proteins.PurposeThe purpose of the study was to develop a lumbar disc degeneration model in mice and determine the effect of adenoviral GDF5 gene therapy.Study designThe study design was to compare the degeneration changes of discs punctured by different-size needles to develop a mice lumbar disc degeneration model and to evaluate the effects of in vivo gene therapy for the mice disc degeneration model by an adenoviral vector carrying GDF5 gene.MethodsA lumbar disc degeneration model was developed by needle punctures to the discs in Balb/c mice. Afterward, a gene therapy treatment to disc degeneration was evaluated. Two of the mice lumbar discs were randomly chosen to be punctured by a 30-gauge needle and then injected with adenovirus that had been engineered to express either the luciferase gene (Ad-Luc) or the GDF5 gene (Ad-GDF5). Animals were analyzed by bioluminescent imaging, radiographic, and magnetic resonance imaging (MRI) scanning, then sacrificed at 1, 2, 4, or 8 weeks after operation, and subjected to histological and biochemical assays.ResultsBy the detection of T2-weighted MRI scanning and histological study, the degeneration was found in all of the discs punctured by different-size needles. But the development of the degeneration in the discs injured by the 30-gauge needle was more reliable and moderate compared with that in other groups. The detection of luciferase activity by bioluminescent imaging revealed that adenovirus survived and the introduced genes were expressed over 6 weeks after injection. There were no T2-weighted MRI signals in the mice injected with either Ad-Luc or Ad-GDF5 up to 4 weeks after operation. At 6 and 8 weeks, T2-weighted signals were detected in the Ad-GDF5 group but none in the Ad-Luc control group. The percent disc height index (%DHI) was significantly decreased (～20%) by 1 week after injury in both groups, indicating the development of disc degeneration. At 2 weeks, the %DHI in the mice injected with Ad-GDF5 increased significantly compared with that of the mice injected with Ad-Luc; the increase was sustained for the rest of the experiment period. The disc histology treated with Ad-GDF5 was improved compared with that in the control group. Glycosaminoglycan (GAG) levels were significantly decreased in the Ad-Luc injection group since 2 weeks after injury, and the DNA content had diminished by 4 weeks after the operation. In contrast, in the discs injected with Ad-GDF5, there was no decrease in the GAG and DNA levels after injury throughout the 8-week treatment period.ConclusionsDisc degeneration animal model can be developed by using needle puncture to the discs in mice. The adenovirus is an effective vehicle for gene delivery with rapid and prolonged expression of target protein and resulting improvement in markers of disc degeneration. Ad-GDF5 gene therapy could restore the functions of injured discs and has the potential to be an effective treatment.     

Papain-induced in vitro disc degeneration model for the study of injectable nucleus pulposus therapy

Background contextProteolytic enzyme digestion of the intervertebral disc (IVD) offers a method to simulate a condition of disc degeneration for the study of cell-scaffold constructs in the degenerated disc.PurposeTo characterize an in vitro disc degeneration model (DDM) of different severities of glycosaminoglycans (GAG) and water loss by using papain, and to determine the initial response of the human mesenchymal stem cells (MSCs) introduced into this DDM.Study designDisc degeneration model of a bovine disc explant with an end plate was induced by the injection of papain at various concentrations. Labeled MSCs were later introduced in this model.MethodsPhosphate-buffered saline (PBS control) or papain in various concentrations (3, 15, 30, 60, and 150 U/mL) were injected into the bovine caudal IVD explants. Ten days after the injection, GAG content of the discs was evaluated by dimethylmethylene blue assay and cell viability was determined by live/dead staining together with confocal microscopy. Overall matrix composition was evaluated by histology, and water content was visualized by magnetic resonance imaging. Compressive and torsional stiffness of the DDM were also recorded. In the second part, MSCs were labeled with a fluorescence cell membrane tracker and injected into the nucleus of the DDM or a PBS control. Mesenchymal stem cell viability and distribution were evaluated by confocal microscopy.ResultsA large drop of GAG and water content of the bovine disc were obtained by injecting >30 U/mL papain. Magnetic resonance imaging showed Grade II, III, and IV disc degeneration by injecting 30, 60, and 150 U/mL papain. A cavity in the center of the disc could facilitate later injection of the nucleus pulposus tissue engineering construct while retaining an intact annulus fibrosus. The remaining disc cell viability was not affected. Mesenchymal stem cells injected into the protease-treated DDM disc showed significantly higher cell viability than when injected into the PBS-injected control disc.ConclusionsBy varying the concentration of papain for injection, an increasing amount of GAG and water loss could be induced to simulate the different severities of disc degeneration. MSC suspension introduced into the disc has a very low short-term survival. However, it should be clear that this bovine IVD DDM does not reflect a clinical situation but offers exciting possibilities to test novel tissue engineering protocols.     

Therapeutic effects of cell therapy with neonatal human dermal fibroblasts and rabbit dermal fibroblasts on disc degeneration and inflammation

BACKGROUND CONTEXT

Increasing evidence suggests transplanting viable cells into the degenerating intervertebral disc (IVD) may be effective in treating disc degeneration and back pain. Clinical studies utilizing autologous or allogeneic mesenchymal stem cells to treat patients with back pain have reported some encouraging results. Animal studies have shown that cells injected into the disc can survive for months and have regenerative effects. Studies to determine the advantages and disadvantages of cell types and sources for therapy are needed.

Needle puncture injury of the rat intervertebral disc affects torsional and compressive biomechanics differently

Needle puncture is a common method of inducing intervertebral disc (IVD) degeneration in small animal models and may have some similarities to IVD injury conditions such as herniation. Yet, the influence of puncture injuries on IVD biomechanics is not well understood. This study quantified the acute effects of anular injury on the biomechanics of rat caudal IVDs in compression and torsion following puncture with 30, 25 and 21 G needles. In compression, puncture injury reduced elastic stiffness by 20% for all needle sizes, but differences between control and punctured discs did not remain after compressive overload. In contrast, torsional parameters associated with anular fiber tension were affected proportionally with needle size. We conclude that IVD injuries that penetrate through the thickness of the annulus affect IVD biomechanics through different mechanisms for compression and torsion. Anular injuries affect torsional properties in a manner directly related to the amount of fiber disruption and compressive properties in a manner that affects pressurization.