Promotion of bone formation by naringin in a titanium particle‐induced diabetic murine calvarial osteolysis model

Diabetic patients have an increased risk of prosthesis failure requiring revision surgery. Furthermore, skeletal defects are observed in conjunction with type 1 diabetes. Using a titanium particle‐induced calvarial osteolysis model in diabetic mice, we investigated the effect of diabetes on the osteolytic process and the role of naringin in its prevention. Three groups each of nondiabetic or diabetic mice were treated with vehicle only, with particles only, or with particles then naringin for 10 days. Alteration of bone indices near the midline suture were then analyzed by microcomputed tomography scanning and histology. Serum levels of osteocalcin (OCN) and cross‐linked N‐telopeptide of type I collagen (NTx) were measured by enzyme‐linked immunosorbent assay. The decreases in new bone formation (p < 0.05), calvaria thickness (p < 0.05), bone volume (p < 0.05), midline suture area (p < 0.05), and OCN concentration (p < 0.05) found in diabetic mice were normalized with naringin treatment. Diabetic state promoted particle‐induced osteolysis. Naringin, an osteoanabolic agent, improved bone indices apparently by stimulating bone formation. Therefore, naringin may be beneficial in preventing and treating debris‐mediated periprosthetic osteolysis after total joint replacement, especially in diabetics. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:451–456, 2010     

IKKβ activation is sufficient for RANK‐independent osteoclast differentiation and osteolysis

Monocytes differentiate into osteoclasts through stimulation of receptor activator of NF‐κB (RANK). Many downstream effectors of RANK play a positive role in osteoclastogenesis, but their relative importance in osteoclast differentiation is unclear. We report the discovery that activation of a single pathway downstream of RANK is sufficient for osteoclast differentiation. In this regard, introduction of constitutively activated IKKβ (IKKβ^SSEE) but not wild‐type IKKβ into monocytes stimulates differentiation of bona fide osteoclasts in the absence of RANK ligand (RANKL). This phenomenon is independent of upstream signals because IKKβ^SSEE induced the development of bone‐resorbing osteoclasts from RANK and IKKα knockout monocytes and in conditions in which NEMO‐IKKβ association was inhibited. NF‐κB p100 and p105, but not RelB, were critical mediators of this effect. Inflammatory autocrine signaling by tumor necrosis factor α (TNF‐α) and interleukin 1 (IL‐1) were dispensable for the spontaneous osteoclastogenesis driven by IKKβ^SSEE. More important, adenoviral gene transfer of IKKβ^SSEE induced osteoclasts and osteolysis in calvariae and knees of mice. Our data establish the sufficiency of IKKβ activation for osteolysis and suggest that IKKβ hyperactivation may play a role in conditions of pathologic bone destruction refractory to RANK/RANKL proximal therapeutic interventions. © 2010 American Society for Bone and Mineral Research     

Platelet‐rich plasma impairs osteoclast generation from human precursors of peripheral blood

Platelet‐rich plasma is used to accelerate bone repair for the release of osteogenic growth factors from activated platelets. To date, the effects on osteoclasts have been only scarcely investigated, even though these cells are crucial for bone remodeling. The aim of this research was the evaluation of the effects of thrombin‐activated platelets (PRP) on osteoclastogenesis from human blood precursors. We evaluated both the ability to influence osteoclast differentiation induced by the receptor activator of nuclear factor‐kappaB ligand (RANKL), and the ability to induce osteoclast differentiation without RANKL. In both assays, the incubation with PRP supernatant at 10% did not significantly affect the formation of tartrate‐resistant acid phosphatase (TRACP)‐positive multinucleated cells that were able to form the F‐actin ring. However, when PRP at 25 and 50% was added to the medium without RANKL, the generation of TRACP‐positive multinucleated cells was inhibited. PRP, even at 10%, reduced the osteoclast‐mediated bone collagen degradation, suggesting inhibition of osteoclast activation. Similarly, after incubation with PRP supernatant, calcitonin receptor mRNA was lower than the untreated samples. In conclusion, PRP at 10% interfered with the complete differentiation process of human osteoclast precursors. At higher concentration it impaired osteoclast formation also at an early stage of differentiation. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:792–797, 2010     

Local delivery of mutant CCL2 protein‐reduced orthopaedic implant wear particle‐induced osteolysis and inflammation in vivo

Total joint replacement (TJR) has been widely used as a standard treatment for late‐stage arthritis. One challenge for long‐term efficacy of TJR is the generation of ultra‐high molecular weight polyethylene wear particles from the implant surface that activates an inflammatory cascade which may lead to bone loss, prosthetic loosening and eventual failure of the procedure. Here, we investigate the efficacy of local administration of mutant CCL2 proteins, such as 7ND, on reducing wear particle‐induced inflammation and osteolysis in vivo using a mouse calvarial model. Mice were treated with local injection of 7ND or phosphate buffered saline (PBS) every other day for up to 14 days. Wear particle‐induced osteolysis and the effects of 7ND treatment were evaluated using micro‐CT, histology, and immunofluorescence staining. Compared with the PBS control, 7ND treatment significantly decreased wear particle‐induced osteolysis, which led to a higher bone volume fraction and bone mineral density. Furthermore, immunofluorescence staining showed 7ND treatment decreased the number of recruited inflammatory cells and osteoclasts. Together, our results support the feasibility of local delivery of 7ND for mitigating wear particle‐induced inflammation and osteolysis, which may offer a promising strategy for extending the life time of TJRs. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:58–64, 2016.     

Expression of XBP1s in fibroblasts is critical for TiAl6V4 particle‐induced RANKL expression and osteolysis

Wear particle‐induced osteolysis is a major cause of aseptic loosening, which is one of the most common reasons for total hip arthroplasty (THA) failure. Previous studies have shown that the expression of Receptor activation of nuclear factor (NF)‐kB (RANKL) by fibroblasts in periprosthetic membrane played a crucial role in wear particle‐induced osteolysis. However, the underlying mechanism of RANKL expression remains largely unknown. In the present study, we investigated the effect of TiAl₆V₄ particle (TiPs)‐induced XBP1s (spliced form of X‐box binding protein 1) on RANKL expression and osteoclastogenesis both in vitro and in vivo. The levels of XBP1s in peri‐implant membrane, animal models, and TiPs‐stimulated fibroblasts were determined by western blots. To assess the effect of XBP1s on RANKL expression, fibroblasts were treated with both a small interfering RNA (siRNA) and an inhibitor of XBP1 prior to exposure to TiPs. The effect of XBP1s on osteoclasts formation was determined by tartrate‐resistant acid phosphatase (TRAP) staining in vitro osteoclastogenesis assay and in animal models. The resorption of bone was assessed by micro‐computed tomography (micro‐CT) with three‐dimensional reconstruction. Our results demonstrated that XBP1s was activated in periprosthetic membrane, mouse calvaria models, and TiPs‐stimulated human synovial fibroblasts. Further, inhibition of XBP1s decreased the expression of RANKL and osteoclasts formation in vitro. In mouse calvaria models, both of the osteoclastogenesis and osteolysis were inhibited XBP1s inhibitor. Our results suggested that XBP1s mediated TiPs‐induced of RANKL expression in fibroblasts, and down regulating XBP1s may represent a potential therapy for wear particle‐induced osteolysis. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:752–759, 2017.

     

Understanding the tissue effects of tribo‐corrosion: Uptake,distribution, and speciation of cobalt and chromium in human bone cells

Cobalt and chromium species are released in the local tissues as a result of tribo‐corrosion, and affect bone cell survival and function. However we have little understanding of the mechanisms of cellular entry, intracellular distribution, and speciation of the metals that result in impaired bone health. Here we used synchrotron based X‐ray fluorescence (XRF), X‐ray absorption spectroscopy (XAS), and fluorescent‐probing approaches of candidate receptors P2X7R and divalent metal transporter‐1 (DMT‐1), to better understand the entry, intra‐cellular distribution and speciation of cobalt (Co) and chromium (Cr) in human osteoblasts and primary human osteoclasts. We found that both Co and Cr were most highly localized at nuclear and perinuclear sites in osteoblasts, suggesting uptake through cell membrane transporters, and supported by a finding that P2X7 receptor blockade reduced cellular entry of Co. In contrast, metal species were present at discrete sites corresponding to the basolateral membrane in osteoclasts, suggesting cell entry by endocytosis and trafficking through a functional secretory domain. An intracellular reduction of Cr⁶⁺ to Cr³⁺ was the only redox change observed in cells treated with Co²⁺, Cr³⁺, and Cr⁶⁺. Our data suggest that the cellular uptake and processing of Co and Cr differs between osteoblasts and osteoclasts. © 2014 The Authors. Journal of Orthopaedic Research published by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society. J Orthop Res 33:114–121, 2015.     

The effects of local and systemic alendronate delivery on wear debris‐induced osteolysis in vivo

We investigated the effects of locally and systemically administered alendronate on wear debris‐induced osteolysis in vivo. Endotoxin‐free titanium particles were injected into rabbit femurs, prior to insertion of a nonweight‐bearing polymethylmethacrylate plug into the distal femur canal. Then the particles were repeatedly injected into the knee 2, 4, and 6 weeks after the implantation. Alendronate was incorporated at three different concentrations (0.1, 0.5, and 1.0 wt %) into bone cement for local delivery. For systemic delivery, alendronate was subcutaneously injected (1.0 mg/kg/week) 1 week after the implantation and then once a week until sacrifice. Eight weeks postoperatively, there was significant evidence of osteolysis surrounding the plug in the control group compared with markedly blocked osteolysis in the 0.5 wt % and the 1.0 wt % groups, and the systemic group. There was a concentration‐dependent effect of alendronate‐loaded bone cement on the improvement of peri‐prosthetic bone stock. Notably, no significant differences were found between the 0.5 wt % and the systemic group in peri‐prosthetic bone stock and implant fixation. Collectively, although the biological efficacy after the systemic delivery of alendronate was slightly higher than that in the local treatment groups, alendronate‐loaded bone cement may be therapeutically effective in inhibiting titanium particle‐induced osteolysis in vivo. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:893–899, 2010     

Osteoprotegerin deficiency attenuates strontium‐mediated inhibition of osteoclastogenesis and bone resorption

Strontium (Sr) exerts an anabolic and antiresorptive effect on bone, but the mechanism remains unknown. Osteoprotegerin (OPG) expressed by osteoblasts plays an important role in regulating bone homeostasis by inhibiting osteoclastogenesis and bone resorption. This study aims at evaluating the role of OPG in Sr‐mediated inhibition of osteoclastogenesis and bone resorption. Six‐week‐old Opg knockout (KO) male mice and their wild‐type (WT) littermates were treated orally with vehicle (Veh) or Sr compound (4 mmol/kg) daily for 8 weeks. Bone mass and microstructure in the lumbar spine (L₄) and proximal tibia were analyzed with micro–computed tomography (µCT). Bone remodeling was evaluated with serum biochemical analysis and static and dynamic bone histomorphometry. Osteoclast differentiation potential and gene expression were analyzed in bone marrow cells. The findings demonstrate that Sr compound treatment results in greater bone volume and trabecular number than Veh treatment in WT mice. The anabolic response of trabecular bone to Sr treatment is attenuated in KO mice. Although Sr treatment significantly decreases in vitro osteoclastogenesis and bone resorption in WT mice, these effects are attenuated in KO mice. Furthermore, Sr treatment profoundly increases Opg gene expression in the tibias and OPG protein levels in the sera of WT mice. This study concludes that the inhibition of osteoclastogenesis and bone resorption is possibly associated with OPG upregulation by Sr treatment. © 2011 American Society for Bone and Mineral Research.     

Quantitative mouse model of implant‐associated osteomyelitis and the kinetics of microbial growth,osteolysis, and humoral immunity

Although osteomyelitis (OM) remains a serious problem in orthopedics, progress has been limited by the absence of an in vivo model that can quantify the bacterial load, metabolic activity of the bacteria over time, immunity, and osteolysis. To overcome these obstacles, we developed a murine model of implant‐associated OM in which a stainless steel pin is coated with Staphylococcus aureus and implanted transcortically through the tibial metaphysis. X‐ray and micro‐CT demonstrated concomitant osteolysis and reactive bone formation, which was evident by day 7. Histology confirmed all the hallmarks of implant‐associated OM, namely: osteolysis, sequestrum formation, and involucrum of Gram‐positive bacteria inside a biofilm within necrotic bone. Serology revealed that mice mount a protective humoral response that commences with an IgM response after 1 week, and converts to a specific IgG2b response against specific S. aureus proteins by day 11 postinfection. Real‐time quantitative PCR (RTQ‐PCR) for the S. aureus specific nuc gene determined that the peak bacterial load occurs 11 days postinfection. This coincidence of decreasing bacterial load with the generation of specific antibodies is suggestive of protective humoral immunity. Longitudinal in vivo bioluminescent imaging (BLI) of luxA‐E transformed S. aureus (Xen29) combined with nuc RTQ‐PCR demonstrated the exponential growth phase of the bacteria immediately following infection that peaks on day 4, and is followed by the biofilm growth phase at a significantly lower metabolic rate (p < 0.05). Collectively, these studies demonstrate the first quantitative model of implant‐associated OM that defines the kinetics of microbial growth, osteolysis, and humoral immunity following infection. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J. Orthop Res 26:96–105, 2008     

Effects of sequential treatment with intermittent parathyroid hormone and zoledronic acid on particle‐induced implant loosening: Evidence from a rat model

Particle‐induced implant loosening is a major challenge to long‐term survival of joint prostheses. Administration of intermittent parathyroid hormone (PTH) has shown potential in the treatment of cases of early‐stage periprosthetic osteolysis, while sequential administration of intermittent PTH (iPTH) and bisphosphonates (Bps) has achieved significant effects on treatment of postmenopausal osteoporosis. The objective of this study was to determine whether sequential treatment could preserve bone mass and implant fixation during a pathological course of peri‐implant osteolysis in a rat model. Ninety male Sprague Dawley rats were randomly divided into nine groups, four of which were used for confirmation of establishment of the peri‐implant osteolysis model at two time points, while the other five were used to determine the efficiency of the sequential treatment on peri‐implant osteolysis. Implant fixation and peri‐implant bone mass were evaluated using biomechanical testing, micro‐CT analysis, and histology at 6 and 12 weeks postoperative. The biomechanical test demonstrated that the maximum loading force during a push‐out test was significantly elevated in the sequential treatment group compared to the osteolysis group and iPTH withdrawal group at 12 weeks. Peri‐implant bone morphology also indicated a robust increase in bone volume in the sequential treatment group. Sequential administration of iPTH and Bps was effective in preventing experimental peri‐implant osteolysis, resulting in improved implant fixation and increased peri‐implant bone volume. Clinical significance: The innovative application of sequential treatment in peri‐implant osteolysis could be used clinically to improve the prognosis of patients with early‐stage periprosthetic osteolysis. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1489–1497, 2019.     

The roles of TNFR1 in lipopolysaccharide‐induced bone loss: Dual effects of TNFR1 on bone metabolism via osteoclastogenesis and osteoblast survival

LPS (lipopolysaccharide), a major constituent of Gram‐negative bacteria, regulates proliferation and differentiation of osteoclasts directly or indirectly. This study sought to investigate the functions of the RANK/RANKL pathway in LPS‐induced bone loss in vivo. Wild‐type mice or TNFR1?/? mice were injected LPS with or without osteoprotegerin (OPG) and analyzed histologically. Bone volume was reduced by LPS injection in all groups, and OPG administration prevented the LPS‐induced bone loss regardless of genotypes. LPS‐induced enhancement of osteoclastogenesis in wild‐type mice was blocked by OPG administration. LPS or OPG did not affect osteoclastogenesis in TNFR1?/? mice. Interestingly, osteoblast surface was remarkably reduced in LPS‐treated TNFR1?/? mice as a result of enhanced osteoblast apoptosis. TRAIL, induced by TNF‐α in BMC, triggered apoptosis of primary osteoblast only when TNFR1 signal was ablated in vitro. In conclusion, RANK signaling plays a prominent role in osteoclastogenesis downstream of LPS. Furthermore, TNFR1 regulates bone metabolism through not only the regulation of osteoclast differentiation but also osteoblast survival. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:657–663, 2010     

CA‐074Me compound inhibits osteoclastogenesis via suppression of the NFATc1 and c‐FOS signaling pathways

The osteoclast is an integral cell of bone resorption. Since osteolytic disorders hinge on the function and dysfunction of the osteoclast, understanding osteoclast biology is fundamental to designing new therapies that curb osteolytic disorders. The identification and study of lysosomal proteases, such as cathepsins, have shed light on mechanisms of bone resorption. For example, Cathepsin K has already been identified as a collagen degradation protease produced by mature osteoclasts with high activity in the acidic osteoclast resorption pits. Delving into the mechanisms of cathepsins and other osteoclast related compounds provides new targets to explore in osteoclast biology. Through our anti‐osteoclastogenic compound screening experiments we encountered a modified version of the Cathepsin B inhibitor CA‐074: the cell membrane‐permeable CA‐074Me (L‐3‐trans‐(Propylcarbamoyl) oxirane‐2‐carbonyl]‐L‐isoleucyl‐L‐proline Methyl Ester). Here we confirm that CA‐074Me inhibits osteoclastogenesis in vivo and in vitro in a dose‐dependent manner. However, Cathepsin B knockout mice exhibited unaltered osteoclastogenesis, suggesting a more complicated mechanism of action than Cathepsin B inhibition. We found that CA‐074Me exerts its osteoclastogenic effect within 24 h of osteoclastogenesis stimulation by suppression of c‐FOS and NFATc1 pathways. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1474–1486, 2015.     

Role of myeloid early endothelial progenitor cells in bone formation and osteoclast differentiation in tissue construct based on hydroxyapatite poly(ester‐urethane) scaffolds

Engineering of a vascularized bone construct is a highly challenging task which needs to take into account the impact of different components on the bone regeneration process. Bone repair influencing factors in such constructs range from the material properties and scaffold design, to the interaction of different cell types contributing to bone formation and remodeling or neovascularization, respectively. In this context, early endothelial progenitor cells (EPC), mononuclear cells isolated from the peripheral blood, express the endothelial marker CD31 but also a series of myeloid markers and have been shown to support the formation of vessel‐like structures. These cells are also characterized by a highly adaptable phenotype influenced by other cells creating an instructive niche. The present study was designed to investigate the impact of EPC on bone formation or remodeling using a co‐culture system of outgrowth endothelial cells, mature endothelial cells isolated from the peripheral blood cell cultures, and mesenchymal stem cells grown on hydroxyapatite poly(ester‐urethane) scaffolds. The formation of vessel‐like structures in these constructs was shown by CLSM and immunohistochemistry and further evaluated by real time RT‐PCR. Osteogenic differentiation in these constructs was investigated by von Kossa, Alizarin Red, and real time PCR. Data indicated that osteogenic differentiation occurred within the constructs after 14 days of culture but without a direct influence by EPC in this process. Finally, although we observed a series of osteoclast related makers in the constructs when EPC were included, no indications for an increased osteoclast‐like activity, which might lead to increased bone resorption, were observed. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1922–1932, 2016.     

Hypoxia suppresses serum deprivation‐induced degradation of the nucleus pulposus cell extracellular matrix through the JNK and NF‐κB pathways

Intervertebral disc (IVD) degeneration is associated with the imbalance between anabolism and catabolism of the nucleus pulposus (NP) extracellular matrix (ECM). Serum deprivation (SD) has been reported to exacerbate IVD degeneration; however, the effect of SD on ECM metabolism is not fully understood. Hypoxia plays important roles in maintaining the physiological functions of IVD cells; however, whether hypoxia has any effect on NP ECM production under conditions of SD is still unclear. In the current study, we established an in vitro SD model by exposing NP cells to serum‐free medium. SD decreased the expression of aggrecan and collagen II, as well as the production of sulfated glycosaminoglycan (sGAG) in a time‐dependent manner. However, hypoxia abolished SD‐mediated down‐regulation of aggrecan and collagen II expression via JNK1/2 activation. Moreover, hypoxia abolished SD‐induced MMP‐3 and MMP‐13 expression by inhibiting NF‐κB activation, p65 translocation, and MMP‐3 and MMP‐13 promoter activity. These results indicated that, hypoxia maintained ECM production under conditions of SD. This effect was elicited in part through JNK1/2‐mediated up‐regulation of matrix gene expression and down‐regulation of MMP expression, through the inhibition of NF‐κB. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2059–2066, 2017.

     

Selective tyrosine kinase inhibition of insulin‐like growth factor‐1 receptor inhibits human and mouse breast cancer–induced bone cell activity,bone remodeling,and osteolysis

Insulin‐like growth factor 1 (IGF‐1) plays an important role in both bone metabolism and breast cancer. In this study, we investigated the effects of the novel IGF‐1 receptor tyrosine kinase inhibitor cis‐3‐[3‐(4‐methyl‐piperazin‐l‐yl)‐cyclobutyl]‐1‐(2‐phenyl‐quinolin‐7‐yl)‐imidazo[1,5‐a]pyrazin‐8‐ylamine (PQIP) on osteolytic bone disease associated with breast cancer. Human MDA‐MB‐231 and mouse 4T1 breast cancer cells enhanced osteoclast formation in receptor activator of NF‐κB ligand (RANKL) and macrophage colony‐stimulating factor (M‐CSF) stimulated bone marrow cultures, and these effects were significantly inhibited by PQIP. Functional studies in osteoclasts showed that PQIP inhibited both IGF‐1 and conditioned medium–induced osteoclast formation by preventing phosphatidylinositol 3‐kinase (PI3K)/protein kinase B (Akt) activation without interfering with RANKL or M‐CSF signaling. Treatment of osteoblasts with PQIP significantly inhibited the increase in RANKL/osteoprotegerin (OPG) ratio by IGF‐1 and conditioned medium and totally prevented conditioned medium–induced osteoclast formation in osteoblast–bone marrow (BM) cell cocultures, thereby suggesting an inhibitory effect on osteoblast–osteoclast coupling. PQIP also inhibited IGF‐1–induced osteoblast differentiation, spreading, migration, and bone nodule formation. Treatment with PQIP significantly reduced MDA‐MB‐231 conditioned medium–induced osteolytic bone loss in a mouse calvarial organ culture system ex vivo and in adult mice in vivo. Moreover, once daily oral administration of PQIP significantly decreased trabecular bone loss and reduced the size of osteolytic bone lesions following 4T1 intratibial injection in mice. Quantitative histomorphometry showed a significant reduction in bone resorption and formation indices, indicative of a reduced rate of cancer‐associated bone turnover. We conclude that inhibition of IGF‐1 receptor tyrosine kinase activity by PQIP suppresses breast cancer–induced bone turnover and osteolysis. Therefore, PQIP, and its novel derivatives that are currently in advanced clinical development for the treatment of a number of solid tumors, may be of value in the treatment of osteolytic bone disease associated with breast cancer. © 2013 American Society for Bone and Mineral Research.     

Apoptosis of human intervertebral discs after trauma compares to degenerated discs involving both receptor‐mediated and mitochondrial‐dependent pathways

Post‐traumatic disc degeneration with consecutive loss of reduction and kyphosis remains a debatable issue within both the operative and nonoperative treatment regimen of thoracolumbar spine fractures. Intervertebral disc (IVD) cell apoptosis has been suggested to play a vital role in promoting the degeneration process. To evaluate and compare apoptosis‐regulating signaling mechanisms, IVDs were obtained from patients with thoracolumbar spine fractures (n = 21), patients suffering from symptomatic IVD degeneration (n = 6), and from patients undergoing surgical resection of a primary vertebral tumor (n = 3 used as control samples). All tissues were prospectively analyzed in regards to caspase‐3/7, ‐8, and ‐9 activity, apoptosis‐receptor expression levels, and gene expression of the mitochondria‐bound apoptosis‐regulating proteins Bax and Bcl‐2. Morphologic changes characteristic for apoptotic cell death were confirmed by H&E staining. Statistical significance was designated at p < 0.05 using the Student's t‐test. Both traumatic and degenerative IVD demonstrated a significant increase of caspase‐3/7 activity with evident apoptosis. Although caspase‐3/7 activation was significantly greater in degenerated discs, both showed equally significant activation of the initiator caspases 8 and 9. Traumatic IVD alone demonstrated a significant increase of the Fas receptor (FasR), whereas the TNF receptor I (TNFR I) was equally up‐regulated in both morbid IVD groups. Only traumatic IVD showed distinct changes in up‐regulated TNF expression, in addition to significantly down‐regulated antiapoptotic Bcl‐2 protein. Our results suggest that post‐traumatic disc changes may be promoted and amplified by both the intrinsic mitochondria‐mediated and extrinsic receptor‐mediated apoptosis signaling pathways, which could be, in part, one possible explanation for developing subsequent disc degeneration. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:999–1006, 2008