Lih-Huei Chen

Enhancement of posterolateral lumbar spine fusion using low-dose rhBMP-2 and cultured marrow stromal cells.

We tested the hypothesis that the dose of recombinant human bone morphogenetic protein‐2 (rhBMP‐2) required to induce spine fusion can be reduced by combination with mesenchymal stem cells (MSCs). Twenty‐four adult rabbits underwent posterolateral intertransverse fusion at the L4–L5 level. The animals were divided into four groups based on the implant material: autologous iliac graft, Alginate‐MSCs composite, Alginate‐BMP‐2‐MSCs composite, and Alginate‐BMP‐2 composite. After 16 weeks, the rabbits were euthanized for radiographic examination, manual palpation, biomechanical testing, and histology. Radiographic union of 12 intertransverse fusion areas for the autogenous iliac graft, Alginate‐MSCs, Alginate‐BMP‐2‐MSCs, and Alginate‐BMP‐2 groups was 11, 8, 11, and 0, respectively. Moreover, manual palpation of six fusion segments in each subgroup found solid union to be 6, 1, 5, and 0, respectively. The average torques at failure of the first three groups were 2278 ± 135, 1943 ± 140, and 2334 ± 187 N‐mm, respectively. The failure torque did not differ significantly between the autograft and Alginate‐BMP‐2‐MSCs groups; both groups were significantly higher than the Alginate‐MSCs group. The results indicate that MSCs delivered with in vitro cellular doses of rhBMP‐2 are more osteoinductive than MSCs without rhBMP‐2. In combination with MSCs, a low dose (2.5 µg) of rh‐BMP‐2 could enhance bone formation and posterolateral spine fusion success in the rabbit model.

New Vertebral Fracture After Vertebroplasty

BACKGROUND Because the complications of vertebroplasty are serious and can include new fracture, we retrospectively evaluated potential risk factors for new fracture after vertebroplasty. Our hypothesis is that thoracolumbar vertebrae adjacent to a vertebroplasty site have a higher incidence of new compression fracture than do other vertebrae. METHODS Between March 2001 and December 2002, a total of 271 patients underwent vertebroplasty and a retrospective review of charts was performed. Patients reached 24 months of follow-up were included for analyses. Age, gender, bone mineral density, the numbers of prior vertebroplasty procedures, cement volume, postoperative kyphotic angle, the vertebral level, and kyphotic changes were assessed in relation to surgical outcome. RESULTS The 220 patients had a mean age of 72.7 years (range, 53-97 years) and a mean follow-up interval of 25.6 months (range, 24-36 months). A total of 15 patients had 18 new fractures, and 11 new fractures were at the thoracolumbar junction (T12-L1) (p = 0.61). New fractures were in vertebrae adjacent to a treated vertebra in 55.6% (10 of 18 cases) of cases. Analysis of potential predictors for new vertebral fracture failed to identify statistically significant risk factors, despite a large sample size. CONCLUSIONS Overall, 6.16% (18 of 292) of all treated vertebra developed associated new vertebral fractures. Cranial vertebrae were most likely to fracture at the adjacent level, whereas thoracic vertebrae were least likely to fracture at the adjacent level. Additional risk factors for new vertebral fracture are poorly understood.

Back pain after thoracolumbar fracture treated with long instrumentation and short fusion

The purposes of this retrospective study are to evaluate the degeneration of facets in unfused segments immobilized by Harrington rods and to see how often back pain developed after treatment with this method. Sixty patients with thoracolumbar fracture were treated with the Harrington rod long-fuse short technique. Harrington rods are removed an average of 14 months after initial surgery. Every case showed solid fusion during implant removal exploration. Thereafter, all cases were followed for a minimum of 5 years. Roentgenographic studies and a bone scan were performed to evaluate the degeneration of facet joints. Back pain complaint was found in 11 patients (18.3%). The degeneration of unfused facets was visualized in six patients by roentgenograms. Increased activity in the instrumented spine by bone scan examination also was noted in six patients: three cases were at unfused area, one at a fused area, and two at fused and unfused areas. In conclusion, degeneration in the immobilized and unfused segments after rod long and fused short was rarely seen clinically after long-term follow-up. Back pain was experienced in only 18.3% of the patients and seldom required medication.

Use of fluorescence labeled mesenchymal stem cells in pluronic F127 and porous hydroxyapatite as a bone substitute for posterolateral spinal fusion

Posterolateral spinal fusion is used to treat patients with degenerative spinal disorders. We investigated the effectiveness of a mesenchymal stem cell (MSC)/Pluronic F127/Interpore hybrid graft for spinal fusion in rabbits. Spinal fusion was examined using radiography, manual palpation, computed tomography (CT), torsional loading tests, and histological analysis. Using a PKH fluorescence labeling system, we also examined whether the newly formed bone was derived from the transplanted MSCs. We found that the MSCs adhered to the Interpore surface and within its pores, and differentiated into osteoblasts. Radiographs and CT images showed a continuous bone bridge and a satisfactory fusion mass incorporated into the transverse processes. The results of manual palpation and biomechanical data did not differ significantly from an autograft group. Histology from both groups revealed the presence of fibrous tissue, cartilage, and endochondral ossification in the gaps between the grafted fragments. In both groups, the degree of mature bone formation was greater at 12 weeks than at 6 weeks after grafting. Quantitative histomorphometry revealed no significant differences between the two groups at either time point. In situ tracing of the PKH 67‐labeled MSCs indicated that the transplanted MSCs were partly responsible for the new bone formation in both the repaired transverse processes and the grafted fragments. Thus, the MSC/Pluronic F127/Interpore hybrid graft could be used effectively to achieve posterolateral spinal fusion.

Beneficial effects of hyperbaric oxygen on human degenerated intervertebral disk cells via suppression of IL-1β and p38 MAPK signal.

Nucleus pulposus cells (NPCs) from degenerating disks produce catabolic and inflammatory factors, including interleukin (IL)‐1, nitric oxide (NO), prostaglandin E2 (PGE‐2), and matrix metalloproteinaes (MMPs). An imbalance between MMPs and tissue inhibitors of matrix metalloproteinases (TIMPs) has been proposed to exist in the degenerating disk. This study evaluates the effects of hyperbaric oxygen (HBO) on the human degenerated NPCs. NPCs were maintained in alginate bead culture. All hyperoxic cells were exposed to 100% O2 at 2.5 atmospheres absolute (ATA) in a hyperbaric chamber. p38 MAPK phosphorylation of the NPCs was detected using the phosphor‐kinase array kit. RNA was isolated for real‐time quantitative polymerase chain reaction (Q‐PCR) analysis of aggrecan and type II collagen gene expression. The amounts of IL‐1β, NO, PGE‐2, MMP‐3, and TIMP‐1 in the conditioned media were quantified by enzyme‐linked immunosorbent assay (ELISA). Our data showed that HBO treatment decreased expression of IL‐1β, increased the gene expression of aggrecan and type II collagen, suppressed the phosphorylation of p38 MAPK, decreased NO, PGE‐2, and MMP‐3, and increased TIMP‐1 expression in NPCs as compared with the atmospheric treatment. These results support the hypothesis that IL‐1β and the p38 MAPK signal may be responsible for many of the inflammatory and catabolic changes seen in the human disk degeneration, and support our proposal that HBO treatment‐induced increase of the anabolic factor (TIMP‐1)/catabolic factor (MMP‐3) ratio may provide a therapeutic approach to slow the course of intervertebral disk degeneration.

The use of fluorescence-labeled mesenchymal stem cells in poly(lactide-co-glycolide)/hydroxyapatite/collagen hybrid graft as a bone substitute for posterolateral spinal fusion.

BACKGROUND Posterolateral spinal fusion is used to treat patients with degenerative spinal disorders. In this study, we investigated the effectiveness of a mesenchymal stem cell (MSC)/hydroxyapatite/type I collagen hybrid graft for posterolateral spinal fusion in a rabbit model. METHODS In vitro study, the hybrid graft was cultured in complete or osteogenic medium for 7 days and 14 days and examined by scanning electron microscopy. The alkaline phosphatase activity of the MSCs was assessed and the expression of osteogenic gene was determined by reverse transcription polymerase chain reaction. In vivo investigation, spinal fusion was examined using radiography, manual palpation, computed tomography, torsional loading tests, and histologic analysis. Furthermore, using a PKH fluorescence labeling system, we examined whether the newly formed bone was derived from the transplanted MSCs. RESULTS Our data suggested that the MSCs differentiated into osteoblasts and produced extracellular matrix in the hybrid graft. Increased alkaline phosphatase activity was noted and mRNA of Cbfa-1 and osteopontin were detected. Radiographs and computed tomography images showed a continuous bone bridge and a satisfactory fusion mass incorporated into the transverse processes. The results of manual palpation and biomechanical data did not significantly differ between the two groups. Histologic examination of both groups revealed the presence of cartilage and endochondral ossification in the gaps between the grafted fragments. In situ tracing of the PKH 67-labeled MSCs indicated that the transplanted MSCs were partly responsible for the new bone formation. CONCLUSION The hybrid graft could be effectively used to achieve posterolateral spinal fusion.

Advanced glycation end products in degenerative nucleus pulposus with diabetes

Diabetes mellitus (DM) has been clinically proved as a risk factor of disc degeneration, and the accumulation of advanced glycation end products (AGEs) is known to be potentially involved in diabetes. The purpose of this study is to investigate the effect of AGEs in the degeneration process of diabetic nucleus pulposus (NP) in rats and humans. Diabetic NP cells from rat coccygeal discs were treated with different concentrations of AGEs (0, 50, and 100 µg/ml) for 3 days, and mRNA expressions of MMP‐2 and RAGE were measured by real‐time RT‐PCR. In addition, conditioned medium from NP cells was used to analyze protein expression of MMP‐2 activity and ERK by gelatin zymography and Western blot. These experiments were repeated using human intervertebral disc samples. The immunohistochemical expression of AGEs was significantly increased in diabetic discs. In response to AGEs, an increase of MMP‐2, RAGE, and ERK at both mRNA and protein expression levels was observed in diabetic NP cells. The findings suggest that AGEs and DM are associated with disc degeneration in both species. Hyperglycemia in diabetes enhances the accumulation of AGEs in the NP and triggers disc degeneration.

Hyperbaric oxygen treatment suppresses MAPK signaling and mitochondrial apoptotic pathway in degenerated human intervertebral disc cells

Nucleus pulposus cells (NPCs) from degenerating discs produce catabolic and inflammatory factors, including interleukin (IL)‐1 and nitric oxide (NO). Enhanced production of NO has been implicated in the apoptosis of degenerating disc cells. This study evaluates the effects of hyperbaric oxygen (HBO) on degenerated human NPCs. All hyperoxic cells were exposed to 100% O2 at 2.5 atmospheres absolute (ATA). Phosphorylation of extracellular signal‐regulated kinase 1/2 (ERK1/2), c‐Jun N‐terminal kinase (JNK), and p38 mitogen‐activated protein kinase (MAPK) in NPCs was detected using the phosphor‐kinase array kit. RNA was isolated for real‐time polymerase chain reaction (PCR) analysis of aggrecan and type II collagen gene expression. The levels of IL‐1β and NO were quantified by enzyme‐linked immunosorbent assay (ELISA). To identify the HBO‐induced anti‐apoptotic pathways, expression of Bcl‐2 and Bax proteins as well as activation of cysteine‐containing aspartate‐specific proteases (caspases) 3, 8, and 9 was evaluated using Western blotting after HBO treatment. Our data showed that HBO treatment decreased the expression of IL‐1β, suppressed phosphorylation of ERK1/2, JNK, and p38 MAPK, decreased synthesis of NO, and increased the gene expression of aggrecan and type II collagen in NPCs as compared with the atmospheric treatment. HBO up‐regulated the ratio of Bcl‐2 to Bax expression and reduced the activity of caspases 9 and 3 but not of caspase 8, indicating a selective effect over the mitochondrial apoptosis pathway in degenerated NPCs. These results support our hypothesis that HBO treatment suppresses MAPK signaling and mitochondrial apoptotic pathway in degenerated human intervertebral disc cells.

An Unusual Complication of Anterior Spinal Instrumentation: Hemothorax Contralateral to the Side of the Incision. A Case Report*

Anterior procedures on the spine, initially popularized for the treatment of spinal tuberculosis8, are now frequently used for the treatment of a variety of complex spinal disorders. Several authors have discussed the perioperative complications of such procedures5,10,12,17,18,19,22, however, to our knowledge, development of a hemothorax contralateral to the side of the incision has not been reported. We report such a complication in a patient who had a burst fracture of the twelfth thoracic vertebra, which was osteoporotic. The patient was managed with a decompression and arthrodesis from the eleventh thoracic to the first lumbar vertebra. The procedure was performed through a left thoracoabdominal approach and consisted of corpectomy of the twelfth thoracic vertebra, placement of a strut graft, and internal fixation with screws and a plate. Postoperatively, a hemothorax developed on the right side and necessitated a second procedure. This unexpected, serious complication was iatrogenic; it developed because a protruding screw tip lacerated the diaphragm on the contralateral side. A seventy-four-year-old woman was first seen by us because of a two-month history of back pain following a motor-vehicle accident. The patient reported that the pain had been increasing during the previous two weeks and that there was progressive motor weakness of the lower extremities. Neurological examination revealed a symmetrical decrease in the strength of the muscles of the lower extremities, with the patient having a full range of motion against gravity but not against resistance. There was a sensory loss distal to the inguinal level. The neurological deficit was recorded as grade C on the scale of Frankel et al.6. A magnetic resonance imaging scan revealed a burst fracture of the twelfth thoracic vertebra, which was osteoporotic, and an associated loss of …

Increased periostin gene expression in degenerative intervertebral disc cells.

BACKGROUND CONTEXT Disc degeneration is a multifactorial disease that may cause clinical symptoms such as chronic back pain or radiculopathy in the extremities. Periostin, an extracellular matrix protein involved in the process of fibrosis, expressed in tissues subjected to mechanical stress such as intervertebral disc. However, the expression of periostin during disc degeneration has not yet been studied. PURPOSE The aim of this study is to elucidate the difference in gene expression profiles between degenerative and nondegenerative intervertebral discs for a better understanding of disc degeneration. STUDY DESIGN Degenerative and nondegenerative nucleus pulposus cells were isolated from elderly patients with degenerative disc disease and younger patients with adolescent idiopathic scoliosis, respectively. METHODS Affymetrix GeneChip Human arrays were used to derive gene expression profiles for disc degeneration, and gene expressions of periostin and other degeneration-related markers were confirmed by reverse transcription-polymerase chain reaction (RT-PCR), real-time RT-PCR, and western blot analysis. Immunohistochemical analysis of periostin and Gomori trichrome stain was performed to show the relationship of periostin, fibrosis, and disc degeneration. The mechanical stress experiment was designed to demonstrate the relationship of periostin, stress, and disc degeneration. RESULTS Fourteen genes were identified to express at significantly different levels between degenerative and nondegenerative groups. An increase of periostin gene expression was observed in human degenerative nucleus pulposus cells for the messenger RNA and protein levels. Histological examination demonstrated an increased positive staining of periostin in degenerative discs from human tissues and rat needle-punctured tails and more fibrosis with architectural disorder and fragmentation in human degenerative disc as compared with nondegenerative discs. The expression of periostin was significantly induced by stress in human degenerative nucleus pulposus cells but not in nondegenerative cells. CONCLUSIONS This study demonstrates for the first time an upregulation of periostin in addition to the expression levels of Type I collagen and matrix metalloproteinase-2 in human disc degeneration. It suggests that periostin may be a candidate gene that shows promise as a new prognostic marker and a therapeutic target that is worth further study to expand our knowledge of its role in disc degeneration.