Kai-Jow Tsai

The expression of anabolic cytokines in intervertebral discs in age-related degeneration.

Study Design. Quantitative analysis of endogenous messenger ribonucleic acid (mRNA) expression of anabolic cytokines in the anulus fibrosus and nucleus pulposus tissue from the intervertebral discs of young and old rabbits was performed. Objectives. To measure the expression of anabolic cytokines bone morphogenetic protein-2 (BMP-2), BMP-7, transforming growth factor-β (TGF-β), and insulin-like growth factor-I (IGF-I) in the anulus fibrosus and nucleus pulposus tissue from young and old rabbits to determine if there are differences with age. Summary of Background Data. Disc degeneration increases with age and is associated with compromised disc chondrocytic function. Molecules such as BMP-2, BMP-7, TGF-β, and IGF-I are known to up-regulate disc cell synthesis of key chondrocytic matrix molecules in vitro, and have been proposed as therapeutic agents to prevent disc degeneration. Previous studies have shown that exogenous anabolic cytokines can up-regulate disc-cell function both in vitro and in vivo, however, the endogenous expression of anabolic cytokines in the disc is still unknown. Methods. New Zealand white rabbits aged 3 years (old) and 6 months (young) were used. Quantitative real-time polymerase chain reaction was performed to measure the mRNA levels of BMP-2, BMP-7, TGF-β1, and IGF-I from anulus fibrosus and nucleus pulposus tissue from young and old rabbits. The discs form the young rabbits represent nondegenerated discs, and the discs from the old rabbits represent discs at the onset of degeneration. Results. In the nucleus pulposus, the mRNA levels, given as a ratio of old to young, were 3.6 for BMP-2 (P = 0.004), 61 for BMP-7 (P = 0.02), 4.0 for TGF-β1 (P = 0.3), and 0.6 for IGF-I (P = 0.2). In the anulus fibrosus, the mRNA levels, given as a ratio of old to young, were 1.6 for BMP-2 (P = 0.07), 4.6 for BMP-7 (P = 0.004), 2.9 for TGF- β1 (P = 0.01), and 2.0 for IGF-I (P = 0.1). Conclusion. The disc tissue from the old rabbits as compared to the young rabbits showed, in general, significantly higher mRNA levels of endogenous BMP-2, BMP-7, and TGF-β in both the anulus fibrosus and nucleus pulposus. The similar patterns of up-regulation in gene expression with age shown by these 3 anabolic cytokines suggest a common pathway in terms of regulation and transcription in the early stage of disc degeneration. The knowledge of the age-related pattern in endogenous gene expression of these anabolic cytokines could provide important information for clinical interventional therapy.

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.

A biomechanical study of 3 different types of sublaminar wire used for constructs in the thoracic spine.

A biomechanical study was carried out on 3 different types of sublaminar wire used in constructs to secure the thoracic spine: stainless steel monofilament wire (steel wire), titanium cable (cable), and ultra-high molecular weight polyethylene tape (tape). Two experiments were carried out. Experiment 1: Thirty-one fresh human thoracic vertebrae classified as osteoporotic (bone mineral density of <0.8 g/cm2) were used. The steel wire, cable, or tape was placed sublaminarly and a tensile force was applied until the steel wire, cable, or tape cut 5 mm through the lamina, and the force at this point was noted. Experiment 2: Seven fresh human thoracic spines (T7–T10) were biomechanically tested as follows: axial compression (250 N), flexion (7.5 Nm), extension (7.5 Nm), left lateral bending (7.5 Nm), right lateral bending (7.5 Nm), left axial torsion (10 Nm), and right axial torsion (10 Nm). This sequence was applied to the intact spine. The spine was then de-stabilized and then restabilized using one or other of the 3 different types of sublaminar wires. The biomechanical testing was then repeated on the restabilized spine and stiffness curves were generated. In the laminar cut-through test, the cut-through force for tape was higher than that for either steel wire or cable. In the biomechanical stiffness testing, there was no significant difference between the 3 different sublaminar wiring constructs in any of the loading modes tested. The results of both experiments suggest that tape is as good, if not better, than steel wire or cable as a sublaminar wiring construct material.

A biomechanical assessment of infra-laminar hooks as an alternative to supra-laminar hooks in thoracolumbar fixation

Study Design. Biomechanical study, using human cadaveric spines, of two types of posterior fixation in the thoracolumbar spine. Objectives. To compare fixation in the thoracolumbar spine using pedicle screws combined with infra-laminar hooks (at both the cranial and caudal ends of the same vertebra) with fixation using pedicle screws alone. Summary of Background Data. The standard method of using laminar hooks is to place supra-laminar hooks at the level above the vertebra being fixed by the cranial pedicle screws, and infra-laminar hooks at the level of the vertebra receiving the caudal pedicle screws. However, using this method, a single normal motion segment is killed. In the proposed technique, infra-laminar hooks will be used at the same level as the cranial pedicle screws, thus sparing the superior adjacent segment. Methods. Pedicle screw loosening test. Nine vertebrae (T9–L2) had pedicle screws inserted into both pedicles. On one side, the pedicle screw was connected to a fixation rod, while on the other side the pedicle screw was combined with an infra-laminar hook and connected to a rod. The rod on one side was then pushed with increasing force until loosening of the screw. The same test was repeated on the other rod until loosening of the screw. The reverse test (i.e., pulling until loosening) was done in a similar manner on another nine vertebrae. Biomechanical stiffness test. Eight spines (T12–L2) were biomechanically tested in: axial compression, flexion, extension, left and right lateral bending, and left and right axial torsion. This sequence was applied to: 1) the intact spine; 2) after applying pedicle screws and infra-laminar hooks with rods to the destabilized spine; and 3) after removal of the hooks (with the pedicle screw and rods still inserted). From the load-deformation curves obtained, stiffness values were calculated. Results. Pedicle screw loosening test. The pedicle screw loosening tests showed that a pedicle screw combined with an infra-laminar hook offers significantly greater fixation strength as compared to a pedicle screw alone. Biomechanical stiffness test. Fixation by pedicle screws combined with infra-laminar hooks, placed on the vertebra fixed by pedicle screws at both the cranial and caudal ends, offered a stiffer construct as compared to fixation by pedicle screws alone. Conclusions. This method offers a firm construct without sacrificing a normal motion segment.

The AdLMP-1 transfection in two different cells; AF cells, chondrocytes as potential cell therapy candidates for disc degeneration

SummaryBackground. LMP-1 is known to increase proteoglycan production through the upregulating the BMPs and it is also known that BMP-2 acts on anulus fibrosus cells and chondrocytes to increase proteoglycan production. Method. We carried out an experiment, the effect of AdLMP-1 transfection on AF cells and chondrocytes in the production of sulfated-glycosaminoglycans, mRNA expression (aggrecan, type I, II collagen, LMP-1, BMP-2, and BMP-7), and immunofluorescence staining.AF cells and chondrocytes were grown in monolayer and treated for 6 days with AdLMP1-green fluorescence protein (GFP) (10, 20, and 30 multiplicity of infection [MOI]). After 6 days, the sGAG content in the media was quantified using 1,9-dimethylmethylene blue staining. The mRNA expression was measured with real-time PCR after 20 MOI infection of AdLMP1-GFP. The each cells treated with 20 MOI infection of AdGFP was used as a control group for the mRNA expression.The each cell group was immunofluorescence stained with each antibodies in the chamber slide at 3 × 104 cells/chamber. Findings. 1) The sGAG production was maximum in 20 MOI AdLMP1-GFP infection on the AdLMP-1 treatment for both of AF cells and chondrocytes. 2) The mRNA expression of aggrecan, type I collagen, type II collagen, LMP-1, BMP-2, and BMP-7 is increased in both AF cells and chondrocytes in 20 MOI AdLMP1-GFP infection. 3) On the immunofluorescence staining results, the positive immunofluorescence stained cell numbers are increased after 20 MOI AdLMP1-GFP infection concordant with upregulation of mRNA expression. Conclusions. The AdLMP-1 treatments in AF cells and chondrocytes may be useful for cell transplantation therapy in disc degeneration.

A sonography assisted technique for the removal of a femoral interlocking nail – a technical note

BackgroundOpen methods for removal of femoral interlocking nails involve an incision (up to 10 cm) over the trochanter to find the tip of the nail. The distal locking screws are some times difficult to palpate and an incision (up to about 5 cm) is often needed for exposure. Intra-operative fluoroscopy is often used as an adjunct technique to minimize the surgical wound. However, patients and surgeons are exposed to a radiation hazard. Sonography can provide a real-time and efficient alternative to fluoroscopy.MethodsSonography of soft tissue has been established to identify a foreign body. A metallic implant has a hyperechoic image; therefore, we can identify the correct position of the screws preoperatively and intraoperatively.ResultsWe have developed a technique using sonography and minimal incisions for the removal of a femoral interlocking nail. The proximal wound is 2.5 cm in length and the wound is 0.5 cm in length for each distal locking screw.ConclusionThe sonography can be used to minimize the length of incision and prevent radiation exposure in the removal of intramedullary femoral nails.