In vitro lifespan and senescence mechanisms of human nucleus pulposus chondrocytes

Background contextOur previous in vivo study demonstrated that human nucleus pulposus chondrocytes (NPCs) in aging discs exhibited characteristic senescent features such as an increased senescence-associated β-galactosidase (SA-β-gal) expression, shortened telomere, and decreased telomerase activity. The replicative p53-p21-pRB pathway, rather than the stress-induced p16-pRB pathway, played a more important role in the senescence of NPCs in an in vivo condition, although there is a situation in which both the pathways can be activated simultaneously. However, the in vitro lifespan and senescence mechanisms of human NPCs remain unclear.PurposeTo evaluate the underlying mechanisms of in vitro lifespan and in vitro senescence of the human NPCs and to verify whether the in vitro senescence mechanisms of the human NPC can represent the in vivo aging mechanisms of the cell.Study design/settingAn in vitro study.MethodsWe serially cultivated human NPCs from patients of different ages (35, 42, 55, 66, and 76 years) until the cells reached the end of their in vitro lifespan. During each subcultivation, we calculated NPCs cumulative population doubling level (PDL) and examined senescence markers (SA-β-gal, telomere length, telomerase activity, and p53, p21, pRB, and p16 expressions).ResultsThe cumulative PDLs of the NPCs from patients aged 35, 42 55, 66, and 76 years were 32, 29, 11, 9, and 11, respectively. The younger patients (35 and 42 years) had a higher mean of cumulative PDLs than the elderly patients did (55, 66, and 76 years; 30.5 vs. 10.3; p=.001). In addition, there was a significant, negative correlation between the cumulative PDLs and patient's age (r=?0.89; p=.04). With advancing culture passages, the NPCs irrespective of patient's age exhibited characteristic senescent features, such as an increase in SA-β-gal expression, shortening of telomeres, decrease in telomerase activity, and activation of both replicative p53-p21-pRB and stress-induced p16-pRB pathways. However, all the senescent features occurred at the earlier passages in elderly compared with younger patients.ConclusionsThe present study demonstrated that the human NPCs had a finite in vitro lifespan, which declined with host aging. The in vitro lifespan was determined by both replicative and stress-induced senescence mechanisms. The similarity in the in vitro senescent features with those apparent in the previous in vivo study suggests a possibility of the in vitro senescence mechanisms of the human NPC as a model of the in vivo aging mechanisms of the cell for future studies.     

The effects of simulated microgravity on intervertebral disc degeneration

Background contextAstronauts experience back pain, particularly low back pain, during and after spaceflight. Recent studies have described histologic and biochemical changes in rat intervertebral discs after space travel, but there is still no in vitro model to investigate the effects of microgravity on disc metabolism.PurposeTo study the effects of microgravity on disc degeneration and establish an in vitro simulated microgravity study model.Study designDiscs were cultured in static and rotating conditions in bioreactor, and the characteristics of disc degeneration were evaluated.MethodsThe mice discs were cultured in a rotating wall vessel bioreactor where the microgravity condition was simulated. Intervertebral discs were cultured in static and microgravity condition. Histology, biochemistry, and immunohistochemical assays were performed to evaluate the characteristics of the discs in microgravity condition.ResultsIntervertebral discs cultured in rotating bioreactors were found to develop changes of disc degeneration manifested by reduced red Safranin-O staining within the annulus fibrosus, downregulated glycosaminoglycan (GAG) content and GAG/hydroxyproline ratio, increased matrix metalloproteinase 3 expression, and upregulated apoptosis.ConclusionsWe conclude that simulated microgravity induces the molecular changes of disc degeneration. The rotating bioreactor model will provide a foundation to investigate the effects of microgravity on disc metabolism.     

Intradiscal pressure after intradiscal injection of hypertonic saline: an experimental study

Although chemonucleolysis with chymopapain is a long-established treatment for lumbar intervertebral disc herniation, serious complications have been reported. Accordingly, alternative substances for chemonucleolysis have been sought. The main beneficial effect of chemonucleolysis derives from the decrease in intradiscal pressure. Several previous studies have investigated the relationship between physiological saline injection and disc mechanics in cadaveric specimens [2, 5, 16]. However, no previous study has assessed the intradiscal pressure after intradiscal injection of “hypertonic saline” in living animals. The present study compared the changes in intradiscal pressure after intradiscal injection of hypertonic saline with those after chymopapain injection. The lumbar intervertebral discs of 26 living rabbits were examined: 10% hypertonic saline was injected in ten rabbits, and chymopapain (10 pikokatal units) was injected intradiscally in another ten, with the remaining six being used as controls. The intradiscal pressure was measured at 1, 4, and 12 weeks after injection. The intradiscal pressure of the hypertonic saline-injected group at 4 weeks was significantly lower than that of the control group, but by 12 weeks it had recovered. On the other hand, that of the chymopapain-injected group remained significantly lower than that of the control group at 12 weeks. The results of this study found that hypertonic saline injected into the intervertebral discs temporarily decreased the intradiscal pressure. Received: 26 July 1999 Revised: 26 November 1999 Accepted: 22 December     

Experimental studies on the effects of recombinant human matrix metalloproteinases on herniated disc tissues--how to facilitate the natural resorption process of herniated discs.

PURPOSE: Recently, MMP-7 and MMP-3 have been found to play a crucial role in the natural resorption process of herniated discs. We therefore examined the role of these recombinant human matrix metalloproteinases (rh MMPs) in the treatment of herniated discs. METHODS: (a) Surgical samples of herniated disc were cultured in the presence or absence of rh MMPs, and wet weight was measured 24h later. (b) The rh MMPs were administered into normal rabbit intervertebral discs, and after 1 week spine samples were stained with Safranin O. (c) The rh MMPs were administered into canine herniated discs in vivo. Myelography and MRI were performed prior to and 1 week after administration. Spine samples were examined histologically. Whole disc tissue was collected, total protein was extracted, and Western blot analysis was performed. RESULTS: (a) Proteoglycan degradation was found in MMP-7, MMP-3, and chymopapain-treated samples. MMP-7 and chymopapain-treated samples displayed a significant loss in wet weight (p<0.01). (b) Normal disc tissues after administration of rh MMP-7, MMP-3, and chymopapain showed an extensive loss of Safranin O staining. (c) The rh MMP-7-treated discs had a marked decrease in protruded herniation by MRI. Herniated discs after administration of MMP-7 and chymopapain showed a significant decrease in protruded mass 7 days after administration compared with saline-treated discs when evaluated by myelography (p<0.01). The rh MMP-7-treated discs displayed a clear loss of Safranin O staining in the nucleus pulposus. Proteoglycan expression was barely detectable in disc tissues after MMP-7 administration, whereas obvious expression was obtained in saline-treated or untreated disc tissues. CONCLUSIONS: Exposure to rh MMP-7 resulted in promising proteoglycan loss in human surgical samples, normal rabbit intervertebral discs, and natural canine herniated discs. Administration of rh MMP-7 may facilitate the resorption process of herniated discs.     

Thermoreversible hyaluronan-based hydrogel supports in vitro and ex vivo disc-like differentiation of human mesenchymal stem cells

Background contextThe fate of human mesenchymal stem cells (hMSCs) supplied to the degenerating intervertebral disc (IVD) is still not fully understood and can be negatively affected by low oxygen, pH, and glucose concentration of the IVD environment. The hMSC survival and yield upon injection of compromised IVD could be improved by the use of an appropriate carrier and/or by predifferentiation of hMSCs before injection.PurposeTo optimize hMSC culture conditions in thermoreversible hyaluronan-based hydrogel, hyaluronan-poly(N-isopropylacrylamide) (HA-pNIPAM), to achieve differentiation toward the disc phenotype in vitro, and evaluate whether preconditioning contributes to a better hMSC response ex vivo.Study designIn vitro and ex vivo whole-organ culture of hMSCs.MethodsIn vitro cultures of hMSCs were conducted in HA-pNIPAM and alginate for 1 week under hypoxia in chondropermissive medium alone and with the supplementation of transforming growth factor β1 or growth and differentiation factor 5 (GDF-5). Ex vivo, hMSCs were either suspended in HA-pNIPAM and directly supplied to the IVDs or predifferentiated with GDF-5 for 1 week in HA-pNIPAM and then supplied to the IVDs. Cell viability was evaluated by Live-Dead assay, and DNA, glycosaminoglycan (GAG), and gene expression profiles were used to assess hMSC differentiation toward the disc phenotype.ResultsThe HA-pNIPAM induced hMSC differentiation toward the disc phenotype more effectively than alginate: in vitro, higher GAG/DNA ratio and higher collagen type II, SOX9, cytokeratin-19, cluster of differentiation 24, and forkhead box protein F1 expressions were found for hMSCs cultured in HA-pNIPAM compared with those cultured in alginate, regardless of the addition of growth factors. Ex vivo, direct combination of HA-pNIPAM with the disc environment induced a stronger disc-like differentiation of hMSCs than predifferentiation of hMSCs followed by their delivery to the discs.ConclusionsHyaluronan-based thermoreversible hydrogel supports hMSC differentiation toward the disc phenotype without the need for growth factor supplementation in vitro and ex vivo. Further in vivo studies are required to confirm the suitability of this hydrogel as an effective stem cell carrier for the treatment of IVD degeneration.     

Intradiscal pressure after repeat intradiscal injection of hypertonic saline: an experimental study

Chemonucleolysis with chymopapain is an effective alternative to an operation for the treatment of some patients who have a lumbar intervertebral disc herniation. However, chymopapain is associated with rare but serious complications. Accordingly, alternative substances for chemonucleolysis have been sought. The main beneficial effect of chemonucleolysis derives from the decrease in the intradiscal pressure. We have previously reported that hypertonic saline injected into the intervertebral discs decreased the intradiscal pressure, but only temporarily. The present experimental study investigated changes in the intradiscal pressure after a repeat intradiscal injection of hypertonic saline. The lumbar intervertebral discs of 18 living rabbits were examined: 10% hypertonic saline was injected intradiscally just once in 12 rabbits, and the same dosage was injected again, 4 weeks later, in the same animals. The intradiscal pressure was measured at 1, 4, 8, and 12 weeks after the second injection. The remaining six rabbits were used as controls, without puncture and without injection. The intradiscal pressure of the group with repeat hypertonic saline injection at 4 weeks was significantly lower than that of the control group. The decreased pressure showed a tendency to increase at 8 weeks, and it had recovered at 12 weeks. The results of this study suggest that repeat hypertonic saline injections may be clinically useful.     

Effects of chondroitinase ABC on intradiscal pressure in sheep: an in vivo study

STUDY DESIGN: In vivo intradiscal measurements of pressure in lumbar discs treated with chondroitinase ABC were performed. OBJECTIVE: To determine the decrease in lumbar intradiscal pressure after chemonucleolysis by chondroitinase ABC in sheep. SUMMARY OF BACKGROUND DATA: No previous study has assessed in vivo intradiscal pressure after chemonucleolysis. This study investigated the effect of chondroitinase ABC on intradiscal pressure in terms of a dose and time relation. It also included roentgenographic observations and evaluation of the correlation between disc space narrowing and decrease in intradiscal pressure. METHODS: Chondroitinase ABC was injected in the lumbar intervertebral discs of sheep at doses of 1, 5, and 50 U. Phosphate buffered saline also was injected as a negative control measure. One week before injection, then 1 and 4 weeks afterward, intradiscal pressure was measured using a catheter microtip pressure transducer. Simultaneously, standard lateral roentgenographs were taken, and the disc height index was calculated. RESULTS: The intradiscal pressure clearly was decreased 1 week after chondroitinase ABC injection. A further decrease was observed up to 4 weeks. This pressure decrease was dose dependent. The disc height indexes also decreased with time, but the state of the change was different from that of the changes in intradiscal pressure. No clear quantitative correlation was found between intradiscal pressure and disc height index. CONCLUSIONS: Chondroitinase ABC can induce the reduction of intradiscal pressure in the lumbar spine.     

腰部椎间盘造影及椎间盘内加压注射疗法

目的:探讨椎间盘内加压注射疗法对脱出型腰椎间盘突出症的治疗效果.方法:突出的椎间盘内加压注射3～20ml生理盐水,要避免用力过大,加压注射的压力低于3kg/cm^2.结果:脱出的椎间盘组织进入硬膜外腔后接触血液系统,逐渐被血液系统中的T细胞及单核细胞清除吸收.加压注射疗法能够促进这一吸收过程.结论:L1～5椎间盘和L5～S1椎间盘的穿刺体位和穿刺方向虽然有一定的差异,但是椎间盘内加压注射疗法是一种有效的治疗腰椎椎间盘突出症的微创疗法.     

Anterior or posterior approach of thoracic disc herniation? A comparative cohort of mini-transthoracic versus transpedicular discectomies

Background contextThe optimal surgical treatment of thoracic disc herniations remains controversial and depends on the consistency of the herniation and its location related to the spinal cord.PurposeTo compare the outcomes of patients with symptomatic thoracic disc herniations treated with anterolateral mini-transthoracic approach (TTA) versus posterior transpedicular discectomy.Study designThis is a prospective comparative cohort study.Patient sampleOne hundred consecutive patients with symptomatic thoracic herniated discs were operated by mini-TTA (56 patients) or transpedicular discectomy (44 patients).Outcome measuresNeurologic assessment by American Spinal Injury Association (ASIA) Impairment Scale and patients' self reported perceived recovery and complications.MethodsThe consistency and location of the herniated disc in relation to the spinal cord was evaluated by preoperative computed tomography and magnetic resonance imaging. Patients were assessed neurologically before surgery and at regular outpatient controls at 2 months or later. Long-term follow-up was achieved by questionnaires sent by mail.ResultsIn both groups, most patients had symptoms of myelopathy and radicular pain; patients who underwent mini-TTA, more frequently suffered from spasticity. Fifty-eight percent of the herniated discs were calcified and 77% were larger than one-third of the spinal canal. All patients presented with ASIA Grade C or D (64%) or ASIA Grade E (36%). Postoperatively, 50% of the patients treated with mini-TTA and 37% of the transpedicular group improved at least one grade on the ASIA scale (p=.19). The duration of surgery, blood loss, hospital stay, and complication rate were significantly higher in patients treated with mini-TTA and were mainly related to the magnitude and consistency of the herniated disc. At long-term follow-up, 72% of the mini-TTA patients reported good outcome versus 76% of the transpedicular discectomy group (p=.80).ConclusionsSurgical treatment of a symptomatic herniated disc contributed to a clinical improvement in most cases. The approach is dependent on the location, the magnitude, and the consistency of the herniated thoracic disc. Medially located large calcified discs should be operated through an anterolateral approach, whereas noncalcified or lateral herniated discs can be treated from a posterior approach as well. For optimal treatment of this rare entity, the treatment should be performed in selected centers.     

Radiographic and histologic effects of chondroitinase ABC on normal canine lumbar intervertebral disc

Twelve dogs were divided into two equal groups and given lumbar intradiscal injections of 10, 50, or 100 U/ml of chondroitinase ABC reconstituted in sodium acetate buffer. Radiographs of the lumbar spine were made before and after surgery in both groups. Additional films were made at 5 days after surgery in Group I and at 7, 14, and 21 days after surgery in Group II. All spaces injected with 50 or 100 U/ml chondroitinase ABC demonstrated significant radiographic narrowing in both groups compared with uninjected control and buffer injected discs (P less than 0.001). Discs injected with 10 U/ml of chondroitinase ABC showed increased narrowing over time from 7 to 21 days (P less than 0.05). A zone of safranin O depletion was present in the ventral anulus fibrosus adjacent to the nucleus pulposus in all treated discs, indicating proteoglycan loss. All histologic effects of chondroitinase ABC were confined to intervertebral disc tissues. Chondroitinase ABC appears to be effective for chemonucleolysis in dogs.     

The influence of vertebral body fracture, intradiscal injection, and partial discectomy on the radial bulge and height of human lumbar discs

The effects of vertebral body fracture, intradiscal injection, and partial discectomy on the radial extension and height of human lumbar discs have been investigated in vitro. Fracture and discectomy result in an increase of the radial disc bulge and a decrease of the disc height; intradiscal injection results in a decrease of the radial disc bulge and in an increase of the disc height. The significance of these changes is assessed by comparing them with those changes induced by load decrease or increase within the physiologic range. The results may explain clinical observations with regard to the persistence of or relief from low-back pain after fractures or after application of the therapeutic measures simulated in this experiment.     

Discitis following chemonucleolysis. An experimental study

Although infection following intradiscal injections has been recognized as a distinct entity, discitis following chemonucleolysis has been often attributed to a chemical reaction from chymopapain. In the first part of this study the effect of chymopapain and Conray 280 on a wide range of bacteria was measured in vitro. Chymopapain was found to have a bactericidal effect on all bacteria tested, which was more pronounced with gram positive organisms, whereas Conray 280 showed very little if any antibacterial effect after 48 hours. The aim of the second part of the study was to test the hypothesis that discitis following intradiscal chymopapain injection is due to infection and not to a chemical reaction. Multiple level lumbar intradiscal injections were carried out in eight mature sheep. Sixteen discs in four sheep were injected with a mixture of reconstituted chymopapain and a Staphylococcus epidermidis suspension. Sixteen discs in another four sheep were injected with reconstituted chymopapain only. All sheep were sacrificed at 6 weeks and the discs and end-plates were examined radiologically, and by histopathology and nuclear material was cultured for bacteria. None of the controls showed any evidence of discitis, whereas all sheep injected with bacteria had typical radiologic and histopathologic changes of discitis. However, in most cases in which end-plate lesions were well established there was no evidence of bacteria at sacrifice. These findings support the opinion that discitis following intradiscal injection is always due to infection introduced by the needle tip.     

Anaphylactoid reaction to chymopapain

Chymopapain is a proteolytic enzyme used in the chemonucleolysis of the herniated nucleus pulposus of lumbar intervertebral discs. It causes rapid hydrolysis of the noncollagenous polypeptides that maintain the tertiary structure of the chondromucoprotein of the nucleus pulposus. We report here an anaphylactoid reaction after the intervertebral injection of chymopapain.     

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.     

Matrix replenishment by intervertebral disc cells after chemonucleolysis in vitro with chondroitinase ABC and chymopapain

BACKGROUND CONTEXT: One of the advantages of chemonucleolysis for the treatment of a herniated intervertebral disc is the potential for the disc to self-repair. It has been suggested that the enzymes used for chemonucleolysis differentially affect the potential of the disc cells to promote repair. PURPOSE: To test the ability of nucleus pulposus and anulus fibrosus cells to repair the extracellular matrix degraded in vitro by either chondroitinase ABC or chymopapain. STUDY DESIGN: An alginate cell culture system was used to monitor the progress of matrix repair after chemonucleolysis in vitro. METHODS: Rabbit nucleus pulposus or anulus fibrosus cells precultured for 10 days in alginate gel were briefly exposed to low concentrations of chondroitinase ABC or chymopapain and then returned to normal culture conditions for up to 4 weeks. At each time point, the contents of DNA and matrix macromolecules and proteoglycan synthesis were measured. RESULTS: The DNA content of enzyme-treated alginate beads during the following 4 weeks of culture was higher in the chondroitinase ABC group than in the chymopapain group (NP, p<.01, and AF, p<.05). The content of proteoglycan in beads containing nucleus pulposus and anulus fibrosus cells in the chondroitinase ABC group was higher than that in the chymopapain group (NP and AF, p<.001). The rate of proteoglycan synthesis and the content of collagen did not, however, differ between those two groups. CONCLUSIONS: Intervertebral disc cells exposed to chondroitinase ABC reestablish a matrix richer in proteoglycan than cells exposed to chymopapain. This may be because of differences in the substrate spectrum of each enzyme. Although these results cannot be translated directly to the in vivo situation, they suggest the possibility that cells in discs subjected to chondroitinase ABC-induced chemonucleolysis retain a greater ability to replenish their extracellular matrix with proteoglycans than cells in discs exposed to chymopapain.     

Detrimental effects of discectomy on intervertebral disc biology can be decelerated by growth factor treatment during surgery: a large animal organ culture model

Background contextLumbar discectomies are common surgical interventions that treat radiculopathy by removing herniated and loose intervertebral disc (IVD) tissues. However, remaining IVD tissue can continue to degenerate resulting in long-term clinical problems. Little information is available on the effects of discectomy on IVD biology. Currently, no treatments exist that can suspend or reverse the degeneration of the remaining IVD.PurposeTo improve the knowledge on how discectomy procedures influence IVD physiology and to assess the potential of growth factor treatment as an augmentation during surgery.Study designTo determine effects of discectomy on IVDs with and without transforming growth factor beta 3 (TGFβ3) augmentation using bovine IVD organ culture.MethodsThis study determined effects of discectomy with and without TGFβ3 injection using 1-, 6-, and 19-day organ culture experiments. Treated IVDs were injected with 0.2 μg TGFβ3 in 20 μL phosphate-buffered saline+bovine serum albumin into several locations of the discectomy site. Cell viability, gene expression, nitric oxide (NO) release, IVD height, aggrecan degradation, and proteoglycan content were determined.ResultsDiscectomy significantly increased cell death, aggrecan degradation, and NO release in healthy IVDs. Transforming growth factor beta 3 injection treatment prevented or mitigated these effects for the 19-day culture period.ConclusionsDiscectomy procedures induced cell death, catabolism, and NO production in healthy IVDs, and we conclude that post-discectomy degeneration is likely to be associated with cell death and matrix degradation. Transforming growth factor beta 3 injection augmented discectomy procedures by acting to protect IVD tissues by maintaining cell viability, limiting matrix degradation, and suppressing NO. We conclude that discectomy procedures can be improved with injectable therapies at the time of surgery although further in vivo and human studies are required.     

Translational research of herniated discs: current status of diagnosis and treatment

Lumbar herniated discs commonly occur in patients 20–40 years of age, and result in acute symptoms of shooting and intractable pain in the low back and/or lower extremities. However, the prognosis of these patients is considered to be very good. Moreover, 70 % of these patients have been reported to be free from sciatica at approximately 6 months after the first onset. Magnetic resonance imaging (MRI) studies have described the spontaneous resorption process of herniated discs, which is a major cause of the reduction of symptoms in patients. New advancements in MRI have recently been developed that have facilitated the examination of nerve tract fibers and identification of symptomatic nerve tissue. Furthermore, the mechanism underlying the resorption process of a herniated disc has been determined. Inflammatory cytokines such as TNF (tumor necrosis factor)-α, angiogenic factors such as vascular endothelial growth factor, and enzymes such as matrix metalloproteinases are intricately related to each other. In our previous studies, matrix metalloproteinase-7 (MMP-7) has been shown to play a crucial role in the initiation of herniated disc resorption. Therefore, we developed recombinant human MMP-7 for intradiscal therapy through an industry–university joint research program. We have already performed in vitro and in vivo experiments to confirm its efficacy; this therapy avoids the side effects associated with surgery, such as nerve tissue damage. Moreover, the phase 1/2 studies of recombinant human (rh) MMP-7 are currently ongoing in the United States, and careful monitoring is required for these clinical trials. In conclusion, patients with lumbar herniated discs may benefit from the development of a less invasive treatment for disc herniation, which can be applied even immediately after the onset of disease symptoms.     

Small molecule antagonist of C-C chemokine receptor 1 (CCR1) reduces disc inflammation in the rabbit model

BACKGROUND CONTEXTTargeting chemokines or chemokine receptors is a promising treatment strategy for diseases with chronic inflammation such as rheumatoid arthritis and discogenic pain. Identifying specific molecules and determining their effectiveness in animal models are the first steps in developing these treatments. Macrophage markers have been detected in the intervertebral disc tissues of patients with disc degenerative disease and discogenic pain and in different animal models. Macrophage recruitment into the disc may play a role in initiation of inflammation and if unresolved may lead to chronic inflammation and subsequent back pain.PURPOSEThe objectives of these studies are to (1) identify chemokine receptor antagonists that can block macrophage migration induced by disc cells in vitro and (2) determine if intradiscal treatment with these antagonists can reduce disc inflammation and degeneration in vivo.STUDY DESIGNIn vitro migration assays were used to test effectiveness of chemokine receptor antagonists to block macrophage migration induced by disc cells. The rabbit annular puncture model was used to test for anti-inflammatory and regenerative effects of chemokine receptor antagonist treatment in vivo.METHODSIn vitro – THP-1 human monocytic cell line and freshly isolated rabbit primary splenocytes were assayed for migration using 3 µm Corning Transwell inserts with conditioned media of interleukin (IL)-1β treated human or rabbit disc cells. Inhibition of macrophage migration was evaluated using different concentrations of small molecule antagonists of C-C chemokine receptor (CCR)1 and CCR2. In vivo – New Zealand White rabbits (n=40) underwent disc puncture and intradiscal treatment with saline, CCR1 or CCR2 antagonists within the same procedure. X-ray and magnetic resonance (MR) images and serum samples were taken for disc height, MRI grade and IL-8 serum level analyses. Intervertebral discs were isolated for RNA analysis of inflammatory and disc phenotypic markers and for immunohistochemical analysis of macrophage marker, RAM11. The outcome measures were compared between the three treatment groups. These studies were funded by a research grant from AO Foundation, Switzerland (Project no S-14-86A; 120000 CHF). CCR1 and CCR2 antagonists were kindly provided by ChemoCentryx (Mountain View, CA).RESULTSIn vitro migration assays showed that THP-1 migration induced by disc cells was blocked by CCR2 antagonist more effectively than CCR1 antagonist, while rabbit splenocyte migration was inhibited by CCR1 antagonist and not the other. In the rabbit annular puncture model, rabbit discs treated with CCR1 antagonist had significantly better MRI grades than those treated with CCR2 antagonist at 6 weeks post-treatment. Gene expression studies demonstrate that discs treated with CCR1 or CCR2 antagonists expressed less inflammatory markers than saline-treated discs at 3 weeks post-treatment. Although CCR2 antagonist treatment did not reduce inflammatory marker expression at 6 weeks, discs treated with CCR1 antagonist expressed less inflammatory markers and also a higher ratio of collagen type 2 to collagen type 1 genes indicating favorable disc matrix production. There were no significant differences between all three treatment groups in regards to disc height indexes, IL-8 serum levels or macrophage marker detection.CONCLUSIONSThese studies have identified that small molecule antagonists against CCR2 and CCR1 were respectively effective in blocking THP-1 and rabbit splenocyte migration induced by disc cells in vitro. Further, both CCR2 and CCR1 antagonist intradiscal treatments were effective in reducing disc inflammation at an early time point of 3 weeks. Lastly, only CCR1 antagonist demonstrated anti-inflammatory effects and better MRI grades at 6 weeks.CLINICAL SIGNIFICANCEOur preclinical studies demonstrate that CCR1 and CCR2 antagonist delivery through intradiscal injection is sufficient to reduce disc inflammation at early time points, whereas CCR1 antagonists had longer term anti-inflammatory effects. Clinical studies have found that CCR1 antagonist was safe, tolerable and clinically active in reducing inflammation in rheumatoid arthritis patients. These studies suggest that CCR1 antagonist may be a promising biological treatment to reduce disc inflammation that translates to back pain relief.     

Double-blind evaluation of chemonucleolysis for herniated lumbar discs. Late results.

Sixty-six patients with symptomatic herniated lumbar discs refractory to the usual conservative management were allocated at random into one of two treatment groups according to a double-blind protocol: 31 received chymopapain intradiscally (chemonucleolysis) and 35 received a placebo intradiscally. Symptoms remained significantly improved 1 year or more after injection for 55% of those treated with chymopapain and for 46% of those treated with placebo. The difference is not statistically significant. However, to discard chemonucleolysis on the basis of this one small clinical trial may be premature. Since continuing controversy has re-established a climate in which another double-blind study of chemonucleolysis is ethically feasible and scientifically desirable, we favor additional clinical trials under a tightly controlled protocol to help resolve the issue.     

Lumbar facet joint and intervertebral disc loading during simulated pelvic obliquity

Background contextIntervertebral disc and facet joints are the two primary load-bearing structures of the lumbar spine, and altered loading to these structures may be associated with frontal plane spinal deviations.PurposeTo determine the load on the lumbar facet joint and intervertebral disc under simulated frontal plane pelvic obliquity combined loading, an in vitro biomechanical study was conducted.Study design/settingAn in vitro biomechanical study using a repeated-measures design was used to compare L4–L5 facet joint and intervertebral disc loading across pure moment and combined loading conditions.MethodsEight fresh-frozen lumbosacral specimens were tested under five loading conditions: flexion/extension, lateral bending, axial rotation using pure moment bending (±10 Nm), and two additional tests investigating frontal plane pelvic obliquity and axial rotation (sacrum tilted left 5° and at 10° followed by a ±10-Nm rotation moment). Three-dimensional kinematics, facet load, and intradiscal pressures were recorded from the L4–L5 functional spinal unit.ResultsSagittal and frontal plane loading resulted in significantly smaller facet joint forces compared with conditions implementing a rotation moment (p<.05). The facet joint had the highest peak load during the 10° combined loading condition (124.0±30.2 N) and the lowest peak load in flexion (26.8±16.1 N). Intradiscal pressure was high in lateral flexion (495.6±280.9 kPa) and flexion (429.0±212.9 kPa), whereas intradiscal pressures measured in rotation (253.2±135.0 kPa) and 5° and 10° combined loading conditions were low (255.5±132.7 and 267.1±127.1 kPa, respectively).ConclusionsFacet loading increased during simulated pelvic obliquity in frontal and transverse planes, whereas intradiscal pressures were decreased compared with sagittal and frontal plane motions alone. Altered spinopelvic alignment may increase the loads experienced by spinal tissue, especially the facet joints.