Thoracic giant cell tumor after two total en bloc spondylectomies including one emergency surgery: A case report.

BACKGROUND: For patients with acute paraplegia caused by spinal giant cell tumor (GCT) who require emergency decompressive surgery, there is still a lack of relevant reports on surgical options. This study is the first to present the case of an acute paraplegic patient with a thoracic spinal GCT who underwent an emergency total en bloc spondylectomy (TES). Despite tumor recurrence, three-level TES was repeated after denosumab therapy. CASE SUMMARY: A 27-year-old female patient who underwent single-level TES in an emergency presented with sudden severe back pain and acute paraplegia due to a thoracic spinal tumor. After emergency TES, the patient's spinal cord function recovered, and permanent paralysis was avoided. The postoperative histopathological examination revealed that the excised neoplasm was a rare GCT. Unfortunately, the tumor recurred 9 months after the first surgery. After 12 months of denosumab therapy, the tumor size was reduced, and tumor calcification. To prevent recurrent tumor progression and provide a possible cure, a three-level TES was performed again. The patient returned to an active lifestyle 1 month after the second surgery, and no recurrence of GCT was found at the last follow-up. CONCLUSION: This patient with acute paraplegia underwent TES twice, including once in an emergency, and achieved good therapeutic results. TES in emergency surgery is feasible and safe when conditions permit; however, it may increase the risk of tumor recurrence.

Spine Surgery with Electronic Conductivity Device: A Prospectively Multicenter Randomized Clinical Trial and Literature Review.

OBJECTIVE: Improving accuracy and safety of pedicle screw placement is of great clinical importance. Electronic conductivity device (ECD) can be a promising technique with features of affordability, portability, and real-time detection capabilities. This study aimed to validate the safety and effectiveness of a modified ECD. METHODS: The ECD underwent a modification where six lamps of various colors, and it was utilized in a prospectively multicenter randomized controlled clinical trial involving 96 patients across three hospitals from June 2018 to December 2018. The trial incorporated a self-control randomization with an equal distribution of left or right side of vertebral pedicle among two groups: the free-hand group and the ECD group. A total of 496 pedicle screws were inserted, with 248 inserted in each group. The primary outcomes focused on the accuracy of pedicle screw placement and the frequency of intraoperative X-rays. Meanwhile, the secondary indicator measured the time required for pedicle screw placement. Results were presented as means ± SD. Paired samples t-test and χ2 -test were used for comparison. Furthermore, an updated review was conducted, which included studies published from 2006 onwards. RESULTS: Baseline patient characteristics were recorded. The primary accuracy outcome revealed a 96.77% accuracy rate in the ECD group, compared to a 95.16% accuracy rate in the free-hand group, with no significant differences noted. In contrast, ECD demonstrated a significant reduction in radiation exposure frequency when compared to the free-hand group (1.11 ± 0.32 vs. 1.30 ± 0.53; p < 0.001), resulting in a 14.6% reduction. Moreover, ECD displayed a decrease of 30.38% in insertion time (70.88 ± 30.51 vs. 101.82 ± 54.00 s; p < 0.001). According to the results of the 21 studies, ECD has been utilized in various areas of the spine such as the atlas, thoracic and lumbar spine, as well as sacral 2-alar-iliac. The accuracy of ECD ranged from 85% to 100%. CONCLUSION: The prospectively randomized trial and the review indicate that the use of ECD presents a secure and precise approach to the placement of pedicle screws, with the added benefit of reducing both procedure time and radiation exposure.

Glycolytic enzyme PKM2 regulates cell senescence but not inflammation in the process of osteoarthritis.

Chondrocyte senescence is an important mechanism underlying osteoarthritis in the senile population and is characterized by reduced expressions of the extracellular matrix proteins. The involvement of glycolysis and the tricarboxylic acid cycle in the development of osteoarthritis is inclusive. The present study aims to investigate the role of the glycolytic enzyme M2 isoform of pyruvate kinase (PKM2) in chondrocytes in senescence and inflammation. Primary chondrocytes are isolated from the knee joints of neonatal mice. Small interfering RNAs (siRNAs) against PKM2 are transfected using lipofectamine. RNA sequencing is conducted in primary chondrocytes with the PKM2 gene deleted. Cell apoptosis, autophagy, reactive oxygen species measurement, and senescent conditions are examined. The glycolytic rate in cells is measured by Seahorse examination. Interleukin 1-ß (IL-1ß) increases the protein expressions of matrix metallopeptidases (MMP)13 and PKM2 and reduces the protein expression of collagen type II (COL2A1) in primary chondrocytes. Silencing of PKM2 alters the protein expressions of MMP13, PKM2, and COL2A1 in the same pattern in quiescent and stimulated chondrocytes. RNA sequencing analysis reveals that PKM2 silencing reduces senescent biomarker p16 INK4a expression. Compared with low-passage chondrocytes, high-passage chondrocytes exhibit increased expression of p16 INK4a and reduced expression of COL2A1. Silencing of PKM2 reduces SA-ß-Gal signals and increases COL2A1 expression in high-passage chondrocytes. Seahorse assay reveals that PKM2 deletion favors the tricarboxylic acid cycle in mitochondria in low- but not in high-passage chondrocytes. In summary, the glycolytic enzyme PMK2 modulates chondrocyte senescence but does not participate in the regulation of inflammation.

Contribution of immune cells to intervertebral disc degeneration and the potential of immunotherapy.

Substantial evidence supports that chronic low back pain is associated with intervertebral disc degeneration (IDD), which is accompanied by decreased cell activity and matrix degradation. The role of immune cells, especially macrophages, in a variety of diseases has been extensively studied; therefore, their role in IDD has naturally attracted widespread scholarly interest. The IVD is considered to be an immunologically-privileged site given the presence of physical and biological barriers that include an avascular microenvironment, a high proteoglycan concentration, high physical pressure, the presence of apoptosis inducers such as Fas ligand, and the presence of notochordal cells. However, during IDD, immune cells with distinct characteristics appear in the IVD. Some of these immune cells release factors that promote the inflammatory response and angiogenesis in the disc and are, therefore, important drivers of IDD. Although some studies have elucidated the role of immune cells, no specific strategies related to systemic immunotherapy have been proposed. Herein, we summarize current knowledge of the presence and role of immune cells in IDD and consider that immunotherapy targeting immune cells may be a novel strategy for alleviating IDD symptoms.

Effect of different structures fabricated by additive manufacturing on bone ingrowth.

OBJECTIVE: To study the effects of different structures (solid/hollow) and pore diameters (300/600 µm) on bone ingrowth. METHODS: Porous titanium alloy scaffolds (3.2 * 10.5 mm) were printed using electron beam melting. The implants were divided into either Hollow or Solid Group. The upper half of each implant was printed with a pore diameter of 600 µm while the bottom half was printed with a pore diameter of 300 µm. Visualization of the structural morphology was done using Scanning Electron Microscope (SEM). Cell proliferation was evaluated with the cell counting kit-8 assay and live/dead staining assay. The different lateral femoral condyles of 15 New Zealand rabbits were implanted with different groups of scaffolds. The rabbits were randomly sacrificed at the 4th, 8th, and 12th week postoperatively. Bone mineral density (BMD) and bone volume fraction (BV/TV) evaluation was completed by quantitative Micro-Computed Tomography (Micro-CT). Tissue histology were stained with toluidine blue to observe bone ingrowth under an optical microscope, and the percentage of new bone area were calculated using Image Pro-Plus 6.0. RESULTS: SEM images showed a significant decrease in residual powder in the hollow implant and cell studies showed no obvious cytotoxicity for the Ti6Al4V scaffolds. Micro-CT reconstruction revealed high levels of new bone formation around the scaffolds. The trabeculae around the implants showed a gradual increase with each week, and new bone filled the scaffold pores gradually. BMD, BV/TV, and tissue histology revealed the 300 µm pore diameter is more conducive to bone ingrowth than the 600 µm (p < .05). CONCLUSION: Our study revealed that Ti6Al4V implants with hollow structure could reduce the residual metal powder and implants with 300 µm pore diameter were more effective on bone formation than a 600 µm.

Effect of silicon-doped calcium phosphate cement on angiogenesis based on controlled macrophage polarization.

Vascularization is an important early indicator of osteogenesis involving biomaterials. Bone repair and new bone formation are associated with extensive neovascularization. Silicon-based biomaterials have attracted widespread attention due to their rapid vascularization. Although calcium phosphate cement (CPC) is a mature substitute for bone, the application of CPC is limited by its slow degradation and insufficient promotion of neovascularization. Calcium silicate (CS) has been shown to stimulate vascular endothelial proliferation. Thus, CS may be added to CPC (CPC-CS) to improve the biocompatibility and neovascularization of CPC. In the early phase of bone repair (the inflammatory phase), macrophages accumulate around the biomaterial and exert both anti- and pro-inflammatory effects. However, the effect of CPC-CS on macrophage polarization is not known, and it is not clear whether the effect on neovascularization is mediated through macrophage polarization. In the present study, we explored whether silicon-mediated macrophage polarization contributes to vascularization by evaluating the CPC-CS-mediated changes in the immuno-environment under different silicate ion contents both in vivo and in vitro. We found that the silicon released from CPC-CS can promote macrophage polarization into the M2 phenotype and rapid endothelial neovascularization during bone repair. Dramatic neovascularization and osteogenesis were observed in mouse calvarial bone defects implanted with CPC-CS containing 60% CS. These findings suggest that CPC-CS is a novel biomaterial that can modulate immune response, promote endothelial proliferation, and facilitate neovascularization and osteogenesis. Thus, CPC-CS shows potential as a bone substitute material.

Clinical outcomes of lumbar spinal surgery in patients 80 years or older with lumbar stenosis or spondylolisthesis: a systematic review and meta-analysis.

PURPOSE: This systematic review and meta-analysis of all available evidence was performed to assess the safety and efficacy of surgery for lumbar stenosis and spondylolisthesis in patients 80 years or older versus those younger than 80 years. METHODS: A search of the literature was conducted in PubMed/MEDLINE, EMBASE and the Cochrane Collaboration Library. Relevant studies comparing the clinical outcomes of lumbar surgery in octogenarians and younger patients were selected according to the eligibility criteria. The predefined endpoints were extracted and meta-analysed from the identified studies. RESULTS: Data from 16 observational studies including 374,197 patients were included in the final analysis. The pooled data revealed that patients 80 years or older had a significantly higher incidence of overall complication, mortality, readmission and longer length of hospital stay than younger patients. There was a similar improvement in the clinical symptoms (Oswestry Disability Index and pain) of patients in the two groups. No significant differences in overall wound complication, reoperation rate, operative time and intraoperative blood loss were found between the groups. CONCLUSIONS: Our results revealed that the clinical improvement in pain and disability did not significantly differ according to age, although the patients aged 80 years or older had increased incidences of mortality and complication than younger patients. Age alone is not a contraindication for lumbar surgery in very old patients. A careful preoperative evaluation, proper patient selection and appropriate surgical approach are important to achieve successful surgical outcomes. These slides can be retrieved under Electronic Supplementary Material.

Epidemiology of ossification of the spinal ligaments and associated factors in the Chinese population: a cross-sectional study of 2000 consecutive individuals.

BACKGROUND: The epidemiology and cause of ossification of the spinal ligaments (OSL) remains obscure. To date, there is no study that comprehensively evaluates the prevalence, distribution, and concomitance of each type of OSL by CT among general Chinese population. We therefore aimed to comprehensively investigate epidemiological characteristics of OSL using whole spine CT in the Chinese population and examine the factors that correlate with the presence of OSL. METHODS: Ossification of the posterior longitudinal ligament (OPLL), ligamentum flavum (OLF), anterior longitudinal ligament (OALL), nuchal ligament (ONL), and diffuse idiopathic skeletal hyperostosis (DISH) were evaluated from the subjects who underwent PET/CT for the purpose of cancer screening in our hospital. Prevalence, distribution, and concomitance of OSL were reviewed. Logistic regression analysis was performed to identify the risk factors of OSL. RESULTS: A total of 2000 subjects (1335 men and 665 women) were included. The prevalence rate of cervical OPLL (C-OPLL) was 4.1%, thoracic OPLL (T-OPLL) 2.25%, lumbar OPLL (L-OPLL) 0.8%, thoracic OLF (T-OLF) 37.65%, lumbar OLF (L-OLF) 1.45%, ONL 31.5%, DISH 3.85%. The most commonly involved level was C5 for C-OPLL, T1 for T-OPLL, T10 for T-OLF, and T8/9 for OALL. 21% of subjects with C-OPLL had T-OPLL, 44% of C-OPLL had T-OLF, 38% of T-OPLL had C-OPLL, 53% of T-OPLL had T-OLF, 44% of L-OPLL had T-OPLL, and 56% of L-OPLL had T-OLF. The average age of OSL-positive subjects was significantly higher than that of OSL-negative subjects. The results of the multiple regression analysis revealed that males had a strong association with DISH (odds ratio, 3.15; 95% confidence interval, 1.27-7.78; P = 0.013). CONCLUSION: The prevalence of OSL in the Chinese was revealed. Tandem ossification is not uncommon in people with OSL. There is a high incidence of multiple-regional OPLL in the whole spine. Approximately half of the subjects with OPLL coexist with T-OLF. For patients with clinical symptoms induced by OPLL, thorough evaluation of whole spine using CT is recommended.

Decompression plus fusion versus decompression alone for degenerative lumbar spondylolisthesis: a systematic review and meta-analysis.

PURPOSE: To compare the clinical effectiveness of decompression plus fusion and decompression alone for patients with degenerative lumbar spondylolisthesis, a systematic review and meta-analysis of all available evidence was performed. METHODS: A search of the literature was conducted on PubMed/MEDLINE, EMBASE, and the Cochrane Collaboration Library. Relevant studies comparing decompression plus fusion and decompression alone were selected according to eligibility criteria. Predefined endpoints were extracted and meta-analyzed from the identified studies. RESULTS: Four randomized controlled trials and 13 observational studies were eligible. The pooled data revealed that fusion was associated with significantly higher rates of satisfaction and lower leg pain scores when compared with decompression alone. However, fusion significantly increased the intraoperative blood loss, operative time and hospital stay. Both techniques had similar ODI, back pain scores, complication rate, and reoperation rate. CONCLUSIONS: Based on the available evidence, decompression plus fusion maybe be better than decompression alone in the treatment of degenerative spondylolisthesis. Fusion had advantages of improvement of clinical satisfaction, as well as reduction of postoperative leg pain, with similar complication rate to decompression alone.

Isolation and identification of stem cells from degenerated human intervertebral discs and their migration characteristics.

Mesenchymal stem cells (MSCs) have been isolated and identified separately from the three components of intervertebral disc, i.e. annulus fibrosus (AF), nucleus pulposus (NP), and cartilage endplate (CEP). However, few studies have been carried out to compare the properties of these three kinds of stem cells, especially their migration ability which is essential for their potential clinical application. In this study, MSCs were isolated from AF, NP, and CEP, respectively, of human degenerated discs and identified by surface markers and multilineage differentiation assay at passage 3. These three types of stem cells were named as AF-MSCs, NP-MSCs, and CEP-MSCs. Then, their biological characteristics were compared in terms of proliferation, passage, colony formation, migration, and invasion capacity. Results showed that all the three types of cells were identified as MSCs and had similar characteristics in proliferation, passage, and colony formation capacity. CEP-MSCs showed the highest migration and invasion potency, while NP-MSCs showed the lowest migration ability and almost no invasion potency, suggesting that CEP-MSCs had the most powerful properties of migration and invasion when compared with AF-MSCs and NP-MSCs. It was also found that the expression of CXCR4 was higher in CEP-MSCs than in the other two, suggesting that SDF-1/CXCR4 axis may play significant roles in the migration of these cells.

A biomechanical comparison of 3 different posterior fixation techniques for 2-level lumbar spinal disorders.

OBJECT: This study sought to make a biomechanical comparison of 3 different posterior fixation techniques for 2-level lumbar spinal disorders. METHODS: Eight fresh-frozen human cadaver lumbar spines (4 from L-1 to L-5, 4 from L-1 to S-1) were tested by applying pure moments of ± 8 Nm. Each specimen was first tested intact, and then the left facetectomies of L3-4 and L4-5 were performed to establish an unstable condition without removal of discs. Three instrumentation systems were then tested randomly: unilateral pedicle screw (UPS), UPS with contralateral translaminar facet screw (UPSFS), and bilateral pedicle screw (BPS). The range of motion (ROM) and the neutral zone (NZ) of L3-5 were measured. RESULTS: All fixation types could reduce the ROM of L3-5 significantly in flexion, extension, and lateral bending, compared with the intact state. In axial torsion, only BPS reduced the ROM significantly, compared with the intact state. The UPSFS technique provided intermediate stability, which was superior to the UPS in flexion-extension and lateral bending, and inferior to the BPS in lateral bending. Compared with the intact state, the NZs decreased significantly for UPS, UPSFS, and BPS in flexion-extension, while not significantly in lateral bending and axial torsion. CONCLUSIONS: In this study, among the 3 fixation techniques, BPS offered the highest stability, UPSFS provided intermediate stability, and UPS was the least stable for 2-level lumbar spinal disorders. UPSFS appeared to be able to offer a less invasive choice than BPS in well-selected patients with 2-level lumbar spinal disorders.

Changes of number of cells expressing proliferation and progenitor cell markers with age in rabbit intervertebral discs.

Basic knowledge about the normal regeneration process within the intervertebral disc (IVD) is important to the understanding of the underlying biology. The presence of progenitor and stem cells in IVD has been verified. However, changes of number of progenitor and stem cells with age are still unknown. In this study, changes of cell proliferation and progenitor cell markers with age in IVD cells from rabbits of two different ages were investigated using flow cytometry, immunohistochemistry, real-time polymerase chain reaction, and western blot analysis. Proliferating cell nuclear antigen (PCNA) was chosen as a marker for proliferation, and Notch1, Jagged1, C-KIT, CD166 were chosen as stem/progenitor cell markers. Cell cycle analysis showed that cell number in the G2/M phase of the young rabbits was significantly higher than that of mature rabbits. Immunohistochemical staining demonstrated the expression of PCNA, C-KIT, CD166, Notch1, and Jagged1 in both young and mature annulus fibrosus (AF). Protein expressions of these cell markers in the young rabbits were all significantly higher than those in the mature rabbits. The expression levels of PCNA, CD166, C-KIT, Jagged1 were significantly higher in the AF, and PCNA, C-KIT in the nucleus pulposus from young rabbits than those from the mature rabbits. These findings demonstrated that both proliferation and progenitor cells exist in rabbit IVDs and the number of cells expressing proliferation and progenitor cell markers decreases with age in the rabbit IVD cells. Methods that are designed to maintain the endogenous progenitor cells and stimulate their proliferation could be successful in preventing or inhibiting degenerative disc disease.

Modified total en bloc spondylectomy in thoracic vertebra tumour.

Total en bloc spondylectomy (TES) for vertebral tumour was previously reported by Tomita through a single posterior approach using a T-saw. A modified total en bloc spondylectomy (MTES) technique is reported in the present study. The disc puncture needle with a sleeve was used to obliquely puncture from the posterior to the anterior direction. A T-saw was inserted through the sleeve and led out to the operator's side by the leading clamp. The disc was partially cut with the saw from its medial to lateral aspect. After a spinal fixation rod was applied on the operator's side, the residual discs on the opposite side were cut as described above. Six patients with thoracic vertebral tumours were operated on using the MTES technique. Five patients showed improvement in their neurological deficits postoperatively. There was no evidence of tumour recurrence at the final follow-up. The MTES is technically feasible with improved practicality and safety.

Osteogenic growth peptide enhances the proliferation of bone marrow mesenchymal stem cells from osteoprotegerin-deficient mice by CDK2/cyclin A.

To promote bone formation is one of the fundamental strategies in osteoporosis treatment and fractures repair. As one of the stimulators on bone formation, osteogenic growth peptide (OGP) increases both proliferation and differentiation of the osteoblasts in vitro and in vivo, in which osteoprotegerin (OPG) has been suggested being involved. In this study, we evaluated the effects of OGP on bone marrow mesenchymal stem cells (MSCs) from OPG-deficient mice in vitro by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, alkaline phosphatase (ALP) activity assay, real-time polymerase chain reaction, and western blot analysis. Results showed that OGP stimulated MSC proliferation and increased the expression of CDK2 and cyclin A in MSCs both at mRNA and protein levels. However, no differentiative effect of OGP was shown as ALP activity and the expression levels of Runx2 and Osterix were not increased significantly by OGP. Our study suggested that OGP may increase the bone formation in OPG-deficient mice by stimulating MSC proliferation rather than differentiation, and probably by triggering CDK2/cyclin A pathway.

[Bone inductive effects of rhBMP-2 loaded porous CPC implants on spine fusion in rabbits].

OBJECTIVE: To investigate the effect of rhBMP-2 combined with porous CPC on spine fusion in rabbits. METHODS: rhBMP-2 (1 mg) was loaded with 1 g CPC and 6.0 cm x 2.0 cm x 0.5 cm absorbable gelatin sponge (AGS), respectively, and thereafter frozen to prepare the biomaterial of rhBMP-2/CPC and rhBMP-2/AGS. Forty-five 24-week-old New Zealand rabbits (weight 2.5-3.5 kg) were randomly divided into 3 groups: group A (n=17), group B (n=11) and group C (n=17). With the exposure and removal of L5,6 transverse process's posterior bone cortex in all the rabbits, the corresponding cancellous bones were exposed and the posterior bilateral intertransverse bone grafting of L5,6 were performed on the three groups, then the rhBMP-2/CPC, rhBMP-2/AGS and CPC was implanted into the rabbits of group A, B and C, respectively. Gross observation, histology assay and image examination were conducted 4, 8 and 24 weeks after operation. RESULTS: Decalcified hard tissue section demonstrated obvious callus connections in group A, small pieces of callus in group B, and fibrous connection and few cartilage in group C at 4 and 8 weeks after operation. By Kacena measurement standard, the score of group A, B and C at 4 weeks after operation was (7.30 +/- 0.76), (3.68 +/- 1.60) and (1.75 +/- 0.54) points, respectively, and their score at 8 weeks after operation were (8.32 +/- 1.11), (3.75 +/- 1.23) and (1.47 +/- 0.23) points, respectively, indicating there were significant differences between group A and group B as well as between group A and group C at different time points (P < 0.05). Undecalcified hard tissue section demonstrated that there was cancellous bone-like tissue regeneration in group A, and fiber connection around the implants and little ossification in group C at 4 and 8 weeks after operation. By three dimensions reconstructed CT, group A, B and C scored (2.50 +/- 0.57), (1.00 +/- 0.00) and (1.00 +/- 0.00) points respectively, indicating there was a significant difference between group C and groups A and B as well as between group A and group B (P < 0.05). CONCLUSION: As a carrier of rhBMP-2, the CPC is capable of promoting spine bone fusion in rabbits and is a new type of artificial bone repair material.

Pedicle screw fixation strength: a biomechanical comparison between 4.5-mm and 5.5-mm diameter screws in osteoporotic upper thoracic vertebrae.

The purpose of this study was to evaluate the difference between 4.5-mm and 5.5-mm diameter pedicle screws inserted into the pedicles of upper thoracic vertebrae (T2 to T5). Seven fresh human spines were obtained. The bone mineral density was measured by dual-energy radiograph absorptiometry. The 4.5-mm and 5.5-mm diameter screws were inserted alternately in the right or left pedicle of each vertebra. The insertion torque and force (applied in the cephalic direction) to produce loosening of the screw were measured. The average bone mineral density for the seven thoracic spines was 0.710 g/cm(2). All of the vertebrae were classified as osteoporotic. The torque of insertion for the 5.5-mm diameter screws was significantly greater (59% greater on average) than that for the 4.5-mm diameter screws (p =. 001). Although the average force to loosening for the 5.5-mm diameter screws was higher than the average force to loosening for the 4.5-mm diameter screws (14%), the difference was not significant (p =. 33).

A comparison of three cell types as potential candidates for intervertebral disc therapy: annulus fibrosus cells, chondrocytes, and bone marrow derived cells.

OBJECTIVES: Candidate cell types for disc cell transplantation therapy include anulus fibrosus (AF) cells, chondrocytes, and bone marrow derived cells (BMDCs). We compared the disc matrix production in these three types of cells, before and after stimulation with rhBMP-2. There is no study extant that compares these three cell types to determine the best candidate for the disc cell therapy. METHODS: AF cells, chondrocytes, and BMDCs (iliac crest and femur) were isolated and grown in monolayer. They were treated for 3 days with rhBMP-2. After 3 days, proteoglycan (sGAG) content in the media was quantified. The results were normalized by cell numbers. The mRNA expression of aggrecan, type I collagen, and type II collagen was measured using real-time PCR. Each cell type was also cultured in chamber slides and immunostained for aggrecan, type I collagen, and type II collagen after 3 days of treatment with rhBMP-2. RESULTS: (1) Without rhBMP-2 the chondrocytes produced more proteoglycan (sGAG) as compared to the other two cell types (AF cells and BMDCs). After stimulation with rhBMP-2 the chondrocytes produce even more proteoglycan than the other two cell types. (2) As compared to the other two cell types, in terms of mRNA expression, the chondrocytes expressed more aggrecan, type I collagen, and type II collagen before stimulation with rhBMP-2. After rhBMP-2 stimulation, the chondrocytes expressed even more aggrecan, type I collagen, and type II collagen in proportion to the concentration of rhBMP-2. For the BMDCs there were no changes in type I and II collagen. (3) rhBMP-2 stimulation produced increases in the protein levels of aggrecan, type I and II collagen in all three types of cells. CONCLUSIONS: On balance, according to these results, it would seem that chondrocytes are the best candidate for the disc cell therapy.

Can TGF-beta1 and rhBMP-2 act in synergy to transform bone marrow stem cells to discogenic-type cells?

INTRODUCTION: The recombinant human bone morphogenic protein-2 (rhBMP-2) is known to increase the proteoglycan production and chondrogenic gene expression in the disc cells. The transforming growth factor-beta 1 (TGF-beta(1)) can transform the bone marrow stem cells (BMDCs) into the disc-like cells. MATERIALS AND METHODS: We carried out an experiment to determine if TGF-beta(1) and rhBMP-2 can act in synergy on BMDCs by increasing the production of sulfated-glycosaminoglycan (sGAG) and affecting the mRNA expression of aggrecan, type I collagen, and type II collagen. The BMDCs were isolated from the iliac crest and femur of a New Zealand white rabbit (1 year). The BMDCs were culured in monolayer and treated for 6 days with TGF-beta(1) 10 ng/ml (group 1), rhBMP-2 200 ng/ml (group 2), and both TGF-beta(1) 10 ng/ml and rhBMP-2 200 ng/ml (group 3: the combined group) in Dulbecco's modified Eagle medium/F-12 with 1% fetal bovine serum. After 6 days, the sGAG content in the media was quantified using 1,9-dimethylmethylene blue staining and the mRNA expression of aggrecan, type I collagen, type II collagen, Sox-9, BMP-2, and BMP-7 were measured with the real-time PCR. The same BMDCs were also cultured in the chamber slide at 3 x 10(4) cells/chamber. After 6 days treatment, the treated cells were immunofluorescence stained with aggrecan, type I collagen, type II collagen, anti-BMP-2, anti-BMP-7 antibodies. After that, we compared the number of positive immunofluorescence stained cells with fluorescence microscope. The sGAG production and mRNA expression for each group were normalized against the same parameters for a non-treatment group. RESULTS AND DISCUSSION: The sGAG production was increased 1.15*, 1.34*, and 1.45* times in the TGF-beta(1) 10 ng/ml group, the rhBMP-2 200 ng/ml group, and the combined group respectively. The mRNA expression of aggrecan was increased 1.28, 3.42*, and 5.34* times, the mRNA expression of type I collagen was increased 0.86, 1.09, 1.17 times, the mRNA expression of type II collagen was increased 3.58*, 3.77*, and 10.78* times, the mRNA expression of Sox-9 was increased 1.29, 2.45, 2.75* times, the mRNA expression of BMP-2 was increased 1.14, 2.07, 4.43* times, and the mRNA expression of BMP-7 was increased 1.16, 1.49, 1.97* times, respectively for each group (* indicates p < 0.05). On the immunofluorescence staining of antibodies, the average positively immunofluorescence stained cells number for aggrecan were 4.2, 15.8*, 10*, and 22* according to the non-treatment group, TGF-beta(1) 10 ng/ml group, rhBMP-2 200 ng/ml group, and the combined group respectively. The average positively immunofluorescence stained cells number for type I collagen were 7, 14.2*, 9.2*, 17.4* and the average positively immunofluorescence stained cells number for type II collagen were 8.5, 28.25*, 20.25*, 42.25* and the average positively immunofluorescence stained cells number for anti-BMP-2 were 5, 16.75*, 8.75*, 27.25* and the average positively immunofluorescence stained cells number for anti-BMP-7 were 3.25, 7.5*, 8.75*, 15.25* (* indicates p < 0.05). CONCLUSIONS: Both TGF-beta(1) and rhBMP-2 alone, can increase proteoglycan production in the BMDCs. However, if they were used in combination, there is a synergistic effect. Similarly, the mRNA expressions of both aggrecan, type II collagen, Sox-9, BMP-2, and BMP-7 except for type I collagen were increased significantly when TGF-beta(1) and rhBMP-2 were combined. The positive immunofluorescence stained cell numbers for aggrecan, type I, II collagen, BMP-2 and BMP-7 were also increased after each TGF-beta(1) and rhBMP-2 treatment, and also more increased significantly in the aggrecan, type I, II collagen, BMP-2, and 7 when they were used jointly.

Truncated human LMP-1 triggers differentiation of C2C12 cells to an osteoblastic phenotype in vitro.

LIM mineralization protein-1 (LMP-1) is a novel intracellular osteoinductive protein that has been shown to induce bone formation both in vitro and in vivo. LMP-1 contains an N-terminal PDZ domain and three C-terminal LIM domains. In this study, we investigated whether a truncated form of human LMP-1 (hLMP-1[t]), lacking the three C-terminal LIM domains, triggers the differentiation of pluripotent myoblastic C(2)C(12) cells to the osteoblast lineage. C(2)C(12) cells were transiently transduced with Ad5-hLMP-1(t)-green fluorescent protein or viral vector control. The expression of hLMP-1(t) RNA and the truncated protein were examined. The results showed that hLMP-1(t) blocked myotube formation in C(2)C(12) cultures and significantly enhanced the alkaline phosphatase (ALP) activity. In addition, the expressions of ALP, osteocalcin, and bone morphogenetic protein (BMP)-2 and BMP-7 genes were also increased. The induction of these key osteogenic markers suggests that hLMP-1(t) can trigger the pluripotent myoblastic C2C12 cells to differentiate into osteoblastic lineage, thus extending our previous observation that LMP-1 and LMP-1(t) enhances the osteoblastic phenotype in cultures of cells already committed to the osteoblastic lineage. Therefore, C(2)C(12) cells are an appropriate model system for the examination of LMP-1 induction of the osteoblastic phenotype and the study of mechanisms of LMP-1 action.

rhBMP-2 enhancement of posterolateral spinal fusion in a rabbit model in the presence of concurrently administered doxorubicin.

BACKGROUND CONTEXT: Spinal fusions can be necessary in patients undergoing chemotherapy with doxorubicin. In a previous study, doxorubicin was shown to decrease spinal fusion rates in a rabbit model of lumbar intertransverse process spinal fusion with autograft iliac crest bone. In the current study, we determine whether spinal fusion with recombinant human bone morphogenetic protein-2 (rhBMP-2) can overcome the inhibitory effect of doxorubicin in spinal fusion. PURPOSE: To determine if rhBMP-2 can overcome the inhibitory effects of doxorubicin (adriamycin) in an animal model of posterolateral spinal fusion. STUDY DESIGN/SETTING: Prospective, controlled, rabbit model of posterolateral lumbar fusion. OUTCOME MEASURES: Spine fusion was assessed by manual palpation (by observers blinded to the treatment group) at the level of arthrodesis. Fusion was graded according to a five-tiered classification (0-4). Posteroanterior radiographs of the excised spines were also graded in a blinded fashion using a six-point scoring system (0-5) devised to describe the amount of bone observed between the L5-L6 transverse processes. METHODS: Thirty-two New Zealand White rabbits underwent posterolateral fusion at L5-L6 with either autograft (iliac crest autograft bone) or rhBMP-2 (rhBMP-2/absorbable collagen sponge (0.86 mg/level). All animals received a dose of doxorubicin (2.5 mg/kg) known to inhibit spine fusion via the central vein of the ear immediately postoperatively. Five weeks postoperatively the rabbits were euthanized. Spine fusion was assessed by manual palpation, and graft quality was assessed with posteroanterior radiographs. RESULTS: Four of the 16 spines (25%) in the autograft group and 16 of the 16 spines (100%) in the rhBMP-2 group fused in the presence of doxorubicin administration (p<.05). There was significantly increased bone formation in the rhBMP-2 group (p<.05). One unilateral, subclinical wound infection was observed in each group at the time of euthanization (autograft [n=1, 6%] and rhBMP-2 [n=1, 6%]). CONCLUSIONS: We confirm that when autograft is used, doxorubicin decreases spinal fusion rate (25%) compared with historical controls (60-75%). More importantly, using rhBMP-2 overcomes the inhibitory effect of doxorubicin, resulting in 100% fusion in our animal model. This study suggests that rhBMP-2 has the potential to improve fusion rates in human patients undergoing chemotherapy with doxorubicin.