Manman Gao

Shock absorbing function study on denucleated intervertebral disc with or without hydrogel injection through static and dynamic biomechanical tests in vitro.

Hydrogel injection has been recently proposed as a novel therapy for disc degenerative diseases, with the potential to restore the spine motion and the intervertebral disc height. However, it remains unknown whether the new technique could also maintain the shock absorbing property of the treated intervertebral disc. In this study, 18 porcine lumbar bone-disc-bone specimens were collected and randomly divided into three groups: the normal with intact intervertebral discs, the mimic for the injection of disulfide cross-linked hyaluronan hydrogels following discectomy, and the control disc with discectomy only. In the static compression test, specimens in the mimic group exhibited displacements similar to those in the normal discs, whereas the control group showed a significantly larger displacement range in the first two steps (P < 0.05). With the frequency increasing, all specimens generally displayed an increasing storage modulus, decreasing loss modulus, and tanδ. At any frequency point, the control group exhibited the largest value in all the three parameters among three groups while the normal group was the lowest, with the mimic group being mostly close to the normal group. Therefore, the hydrogel injection into the intervertebral discs greatly restored their shock absorbing function, suggesting that the technique could serve as an effective approach to maintaining biomechanical properties of the degenerative intervertebral disc.

Pingyangmycin-Induced In Vivo Lumbar Disc Degeneration Model of Rhesus Monkeys

Study Design. Animal experimental study. Objective. To establish a slowly progressive and reproducible intervertebral disc degeneration model and determine the performance of T1&rgr; magnetic resonance imaging in the evaluation of disc degeneration. Summary of Background Data. Recently, one of the hotspots of research efforts was related to management of early stage of disc degeneration. To our knowledge, a functional animal model that mimics ischemic and slowly progressive disc degeneration of humans does not exist. Methods. The subchondral bone adjacent to the lumbar intervertebral discs (from L3–L4 to L6–L7) of 8 rhesus monkeys was randomly injected with 4 mL of Pingyangmycin (PYM) solution (1.5 mg/mL, PYM), or 4 mL of phosphate buffered saline (Vehicle control), or exteriorized but not injected anything (Sham), respectively. The degenerative process was investigated by using radiography and T1&rgr; magnetic resonance imaging at 1, 3, 6, 9, 12, and 15 months postoperatively. Histological scoring, immunohistochemistry, and real-time polymerase chain reaction were performed at 15 months. Results. The mean T1&rgr; values of nucleus pulposus and annulus fibrosus in the PYM group significantly decreased after 3 and 6 months, respectively, followed by slow decrease, and the histological score was significantly higher at 15 months, compared with the control groups. The results of molecular analysis revealed a significant increase matrix metalloprotease-3, A disintegrin and metalloproteinase with thrombospondin motifs -5, tumor necrosis factor &agr;, interleukin-1&bgr;, interleukin-6 expressions, and marked reduction in aggrecan, type II collagen, von Willebrand factor expressions at the messenger RNA levels in the PYM group. Spearman correlation analysis of Pfirrmann grades showed significantly inverse correlation with T1&rgr; values of nucleus pulposus and annulus fibrosus (r = −0.634, −0.617, respectively, P < 0.01). Conclusion. Injection of PYM into the subchondral bone adjacent to the lumbar intervertebral discs of rhesus monkeys can results in mild, slowly progressive disc degeneration, which mimics the onset of human disc degeneration, and the T1&rgr; magnetic resonance imaging is suited for evaluating intervertebral disc degeneration. Level of Evidence: N/A

Induction of chondrogenic differentiation of mouse embryonic mesenchymal stem cells through an in vitro pellet model

This study employed transforming growth factor beta 3 (TGF‐β3) induction of the C3H10T1/2 mesenchymal stem cell (MSC) line to construct an in vitro chondrogenic differentiation model for MSCs. A C3H10T1/2 MSC cell line was cultured, amplified, and the seventh generation of cells was centrifuged to construct pellets, which were divided into a non‐induced group and an induced group (treated with TGF‐β3, vitamin C, dexamethasone, and ITS). Specimens were taken after 7, 14, 21, and 28 days under non‐induced and induced culture to compare these two groups by Alcian Blue staining, collagen type II immunohistochemical staining, and transmission election microscopy (TEM) on days 21 and 42. Cell pellets in the non‐induced group were smaller than those in the induced group on days 7, 14, 21, and 28 with the pellet morphology of the induced group being more regular and nearly spherical. Alcian blue staining in the induced group was consistently stronger than that in non‐induced group across all time points, and type II collagen immunohistochemical IOD values were significantly higher in the induced group over the non‐induced group across all time points. On days 21 and 42, TEM revealed that the induced group displayed greater karyokinesis and a higher euchromatin ratio compared to the non‐induced group. This specially constructed pellet model treated with TGF‐β3‐containing chondrogenic medium can effectively promote chondrogenic differentiation of C3H10T1/2 MSC cells in vitro. This in vitro pellet model should be of value in providing a preliminary cell model reference for further studies of the mechanism of chondroblast differentiation of stem cells.

FOXO1-suppressed miR-424 regulates the proliferation and osteogenic differentiation of MSCs by targeting FGF2 under oxidative stress

Recently, microRNAs (miRNAs) have been identified as key regulators of the proliferation and differentiation of mesenchymal stem cells (MSCs). Our previous in vivo study and other in vitro studies using miRNA microarrays suggest that miR-424 is involved in the regulation of bone formation. However, the role and mechanism of miR-424 in bone formation still remain unknown. Here, we identified that the downregulation of miR-424 mediates bone formation under oxidative stress, and we explored its underlying mechanism. Our results showed that miR-424 was significantly downregulated in an anterior lumbar interbody fusion model of pigs and in a cell model of oxidative stress induced by H2O2. The overexpression of miR-424 inhibited proliferation and osteogenic differentiation shown by a decrease in alkaline phosphatase (ALP) activity, mineralization and osteogenic markers, including RUNX2 and ALP, whereas the knockdown of miR-424 led to the opposite results. Moreover, miR-424 exerts its effects by targeting FGF2. Furthermore, we found that FOXO1 suppressed miR-424 expression and bound to its promoter region. FOXO1 enhanced proliferation and osteogenic differentiation in part through the miR-424/FGF2 pathway. These results indicated that FOXO1-suppressed miR-424 regulates both the proliferation and osteogenic differentiation of MSCs via targeting FGF2, suggesting that miR-424 might be a potential novel therapeutic strategy for promoting bone formation.

In Vivo Magnetic Resonance Imaging Evaluation of Porous Tantalum Interbody Fusion Devices in a Porcine Spinal Arthrodesis Model.

Study Design. Animal experimental study. Objective. To investigate the use of magnetic resonance (MR) imaging in the assessment of lumbar interbody fusion using porous tantalum implants in a porcine spinal fusion model. Summary of Background Data. Porous tantalum has been used successfully as a spinal interbody fusion device. However, to our knowledge, there has been no consensus on the optimal technique for evaluating spinal fusion when using porous tantalum implants. Methods. Twelve 12-week-old female Danish Landrace pigs underwent 3 levels of anterior lumbar interbody fusion at L2–3, L4–5 and L6–7. One level was fused using a solid porous tantalum cage with pedicle screw fixation. The other 2 levels were fused using a hollow porous tantalum ring packed with autograft and stabilized anteriorly with staples. Six months postoperatively, T1-weighted, T2-weighted, and gadolinium-DTPA contrast-enhanced MRI were obtained on a GE 1.5-T unit. After sacrifice, conventional radiograph and histological examination were carried out. Results. Eleven pigs went through the experiment without complications. On all the MR sequences, there were significant higher signal intensity bands at the vertebrae-implant interface of nonfused segments compared with that of fused segments and the vertebral bone and implants themselves (P < 0.001). There was an agreement between histological findings of fibrous tissues surrounding the implants and the high signal intensity band on T1- and T2-weighted MR images respectively (sensitivity 69.6% and 56.5%, specificity 90.7% and 95.3%), both of which were superior to conventional radiograph images (sensitivity 52.2%, specificity 97.7%), especially the T1-weighted MR images. Conclusion. MR imaging could be an effective and noninvasive way to determine the fusion status of tantalum metal implants. Compared with T2-weighted MR imaging and conventional radiograph, T1-weighted spin-echo MR imaging is more sensitive and specific in detecting nonunion via the lucency between the vertebral body and tantalum metal device. Level of Evidence: N/A

Time-sequential changes of differentially expressed miRNAs during the process of anterior lumbar interbody fusion using equine bone protein extract, rhBMP-2 and autograft

The precise mechanism of bone regeneration in different bone graft substitutes has been well studied in recent researches. However, miRNAs regulation of the bone formation has been always mysterious. We developed the anterior lumbar interbody fusion (ALIF) model in pigs using equine bone protein extract (BPE), recombinant human bone morphogenetic protein-2 (rhBMP-2) on an absorbable collagen sponge (ACS), and autograft as bone graft substitute, respectively. The miRNA and gene expression profiles of different bone graft materials were examined using microarray technology and data analysis, including self-organizing maps, KEGG pathway and Biological process GO analyses. We then jointly analyzed miRNA and mRNA profiles of the bone fusion tissue at different time points respectively. Results showed that miRNAs, including let-7, miR-129, miR-21, miR-133, miR-140, miR-146, miR-184, and miR-224, were involved in the regulation of the immune and inflammation response, which provided suitable inflammatory microenvironment for bone formation. At late stage, several miRNAs directly regulate SMAD4, Estrogen receptor 1 and 5-hydroxytryptamine (serotonin) receptor 2C for bone formation. It can be concluded that miRNAs play important roles in balancing the inflammation and bone formation.

Biomimetic matrix fabricated by LMP-1 gene-transduced MC3T3-E1 cells for bone regeneration

Bone healing is regulated by multiple microenvironmental signals provided by the extracellular matrix (ECM). This study aimed to mimic the native osteoinductive microenvironment by developing an ECM using gene-transduced cells. The LIM mineralization protein-1 (LMP-1) gene was transferred to murine pre-osteoblast cells (MC3T3-E1) using lentiviral vectors. Western blotting assay indicated that the MC3T3-E1 cells expressed an increased level of bone morphologic protein-2, -4 and -7 (BMP-2, -4 and -7) after LMP-1 gene transduction. The transduced cells were then seeded into calcined bovine bone scaffolds and cultured for 7, 14, and 21 days to construct ECMs on the scaffolds. The ECM-scaffold composites were then decellularized using the freeze-drying method. Scaffolds without ECM deposition were used as controls. The composites and controls were implanted into critical-sized bone defects created in the distal femurs of New Zealand rabbits. Twelve weeks after the surgery, both microcomputed tomography and histologic results indicated that the 7-day-cell-modified ECM-scaffold composites induced bone regeneration with significantly larger volume, trabecular thickness and connectivity than the controls. However, the 14- and 21-day-cell-modified ECM-scaffold composites triggered sustained inflammation response even at 12 weeks after the surgery and showed less bone ingrowth and integration than their 7-day-cell-modified counterparts. In conclusion, these results highlight the viable gene transfer techniques for manipulating cells in a constructed microenvironment of ECM for bone regeneration. However, the unresolved inflammation relating to the duration of ECM modification needs to be considered.