Subcutaneous toxicity of a dual ionically cross-linked atelocollagen and sodium hyaluronate gel: Rat in vivo study for biological safety evaluation of the injectable hydrogel.

Hydrogels are commonly used in wound dressing, as they retain moisture, accelerate healing, and break down necrotic tissue. This process enhances patient comfort levels while simultaneously reducing pain caused by dead tissue. The purpose of this study was to investigate the in vivo toxicity of a dual hydrogel consisting of type I atelocollagen cross-linked with sodium hyaluronate hydrogel used for wound dressing. Porcine type I atelocollagen was cross-linked with sodium hyaluronate to form the hydrogel. For subcutaneous implantation, 0.5 ml of dual hydrogel was injected into two different sites of twenty rats per group. High density polyethylene rods were implanted subcutaneously to serve as a control material. Hematological assessment, blood biochemistry, histopathological, and histological evaluations were scored and graded after 4 weeks. A bioreactivity rating was used for evaluation of subacute toxicity. Differences observed in blood chemical analysis and hematological analysis between control and test groups were within normal variations and considered unrelated to the test article implantation. No significant implantation-related lesions were observed in any of the major organs of all test animals. The overall histopathological index of the test article implantation sites was evaluated as 0. The bioreactivity rating was evaluated as non-irritant after 4-week subcutaneous implantation. Overall, these results indicate that the dual hydrogel of type I atelocollagen and sodium hyaluronate is biologically and chemically safe for clinical application as a wound dressing.

Tendon-bone interface healing using an injectable rhBMP-2-containing collagen gel in a rabbit extra-articular bone tunnel model.

This study examines the hypothesis that injectable collagen gel can be an effective carrier for recombinant human bone morphogenetic protein-2 (rhBMP-2)'s localization to the healing tendon-bone interface. In 36 mature New Zealand White rabbits, the upper long digital extensor tendon was cut and inserted into the proximal tibial bone tunnel. Then a rhBMP-2-containing collagen gel was injected into the tendon-bone tunnel interface, using a syringe. Histological and biomechanical assessments of the tendon-bone interface were conducted at 3 and 6 weeks after implantation. In vitro testing showed that the semi-viscous collagen gel at room temperature was transformed into a firm gel state at 37°C. The rhBMP-2 release profile showed that rhBMP-2 was released from the collagen gel for more than 28 days. In vivo testing showed that fibrocartilage and new bone are formed at the interface at 6 weeks after injection of rhBMP-2. On radiography, spotty calcification appeared and enthesis-like tissue was produced successfully in the tendon at 6 weeks after injection of rhBMP-2. Use of the viscous collagen gel and rhBMP-2 mixture increased the fusion rate between the bone tunnel and tissue graft. This study demonstrates that viscous collagen gel can be an effective carrier for rhBMP-2 delivery into surgical sites, and that the injectable rhBMP-2-containing collagen gel may be applied for the enhancement of tendon-bone interface healing in the future. Copyright © 2015 John Wiley & Sons, Ltd.

Vibrotactile perception assessment for a haptic interface on an antigravity suit.

Haptic technology is used in various fields to transmit information to the user with or without visual and auditory cues. This study aimed to provide preliminary data for use in developing a haptic interface for an antigravity (anti-G) suit. With the structural characteristics of the anti-G suit in mind, we determined five areas on the body (lower back, outer thighs, inner thighs, outer calves, and inner calves) on which to install ten bar-type eccentric rotating mass (ERM) motors as vibration actuators. To determine the design factors of the haptic anti-G suit, we conducted three experiments to find the absolute threshold, moderate intensity, and subjective assessments of vibrotactile stimuli. Twenty-six fighter pilots participated in the experiments, which were conducted in a fixed-based flight simulator. From the results of our study, we recommend 1) absolute thresholds of ∼11.98-15.84 Hz and 102.01-104.06 dB, 2) moderate intensities of 74.36 Hz and 126.98 dB for the lower back and 58.65 Hz and 122.37 dB for either side of the thighs and calves, and 3) subjective assessments of vibrotactile stimuli (displeasure, easy to perceive, and level of comfort). The results of this study will be useful for the design of a haptic anti-G suit.

Effective healing of chronic rotator cuff injury using recombinant bone morphogenetic protein-2 coated dermal patch in vivo.

Biologic augmentation for rotator cuff repair is a challenging treatment in patients with chronic large, massive, and irreparable rotator cuff injuries. Particularly, the use of an extracellular matrix (ECM) patch such as dermal tissue offered improved biomechanical properties in previous studies. Cytokines induce cell chemotaxis, proliferation, matrix synthesis, and cell differentiation. Moreover, osteoinductive growth factors such as bone morphogenetic protein-2 (BMP-2) affect the formation of new bone and fibrocartilage in lesions. However, the effects of using a dermal patch in combination with BMP-2 have not been evaluated to date, although many researchers have recognized the importance thereof. In this study, rhBMP-2-coated dermal patch (1 cm × 2 cm) isolated from human cadaveric donor was inserted in a rabbit model of chronic rotator cuff injury for in vivo evaluation. Bone mineral density and biomechanical strength were tested and histological and histomorphometric analyses were performed. The results showed that insertion of an rhBMP-2-coated acellular dermal patch not only significantly ameliorated new bone formation, it also improved biomechanical properties such as ultimate tensile strength. Thus, the use of this combination may improve the chronic rotator cuff injury-healing rate and clinical outcomes after rotator cuff repair. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1840-1846, 2017.

Mechanical-chemical analyses and sub-chronic systemic toxicity of chemical treated organic bovine bone.

Sequentially chemical-treated bovine bone was not only evaluated by mechanical and chemical analyses but also implanted into the gluteal muscles of rats for 12 weeks to investigate potential local pathological effects and systemic toxicities. The test (chemical treated bone) and control (heat treated bone) materials were compared using scanning electron microscope (SEM), x-ray diffraction pattern, inductively coupled plasma analysis, and bending strength test. In the SEM images, the micro-porous structure of heat-treated bone was changed to sintered ceramic-like structure. The structure of bone mineral from test and control materials was analyzed as100% hydroxyapatite. The ratio of calcium (Ca) to potassium (P), the main inorganic elements, was same even though the Ca and P percentages of the control material was relatively higher than the test material. No death or critical symptoms arose from implantation of the test (chemical treated bone) and control (physiological saline) materials during 12 weeks. The implanted sites were macroscopically examined, with all the groups showing non-irritant results. Our results indicate that chemical processed bovine bone has a better mechanical property than the heat treated bone and the implantation of this material does not produce systemic or pathological toxicity.

Herniated intervertebral disk induces hypertrophy and ossification of ligamentum flavum.

STUDY DESIGN: In vitro experiment using degenerated human ligamentum flavum (LF) and herniated intervertebral disk (IVD). OBJECTIVES: To investigate the role and effect of degenerated and herniated IVDs on LF hypertrophy and ossification. SUMMARY OF BACKGROUND DATA: Spinal stenosis is caused, in part, by hypertrophy and ossification of the LF, which are induced by aging and degenerative process. It is well known that degenerated IVDs spontaneously produce inflammatory cytokines. Therefore, we hypothesized that degenerated IVD may affect adjacent LF through secreted inflammatory cytokines. METHODS: LF and herniated lumbar IVD tissues were obtained during surgical spinal procedures. LF fibroblasts were isolated by enzymatic digestion of LF tissue. LF cell cultures were treated with disk supernatant from herniated IVDs. Secreted cytokines from IVD tissue culture were detected by enzyme-linked immunosorbent assay. After analysis of cytotoxicity, DNA synthesis was measured. Reverse transcription-polymerase chain reaction for mRNA expressions of types I, II, III, V, and XI collagen and osteocalcin, and histochemical stains were performed. RESULTS: Supernatant from tissue culture of herniated IVD showed increased production of interleukin-1α, interleukin-6, tumor necrosis factor-α, prostaglandin E2, and nitric oxide compared with disk tissue culture from traumatic condition. There was no cytotoxicity in LF cells treated with disk supernatant from herniated IVDs. There was significant increase in DNA synthesis, upregulation in mRNA expression of types III, XI collagen and osteocalcin, whereas variable expression pattern of type I and V, and strong positive stains for Von Kossa and alkaline phosphatase in LF cultures with disk supernatant. CONCLUSIONS: Degenerated and herniated IVDs provide an important pathomechanism in hypertrophy and ossification of the LF through inflammatory cytokines.

Mechanical properties of decellularized tendon cultured by cyclic straining bioreactor.

Decellularized tissues have been successfully used in tissue engineering and regenerative medicine for the purpose of removing antigens present in the cellular components. However, this decellularization technique uses ionic solutions or chemical treatments such as enzyme treatments that might damage the biophysical properties or reduce the physical strength of tissue. This study aimed to improve the strength of decellularized tissues. We designed a tissue bioreactor that can repeatedly deliver physical stimulation, such as tensile and torsional deformation, to the upper and lower parts of a tissue. To decellularized porcine Tibialis tendons, we used an enzymatic solution to remove the primary cells, and then applied ultrasonic cleansing using a combination of ionic solution and distilled water to destroy residual cells by differing from the osmotic pressure between the inside and outside of the cell membrane. The total DNA content of decellularized tissue was decreased by 77% compared with that of the original tissue and the ultimate tensile strength of the decellularized tissue was 20% lower than that of the normal tissue. Decellularized tissues were then cultivated in the tissue bioreactor with repeated physical stimulation of 110% tension, 90° torsion, and frequency of once per a second, and the ultimate tensile strength was found to be greater than that of the normal ligament at 7 day culture. This study showed that decellularization using enzyme and mechanical treatment is safe and use of a tissue bioreactor can increase the physical strength of tendons, making this a potential mechanism to reconstruct human ligaments.

Inflammatory cytokines induce fibrosis and ossification of human ligamentum flavum cells.

STUDY DESIGN: In vitro experiment using degenerated human ligamentum flavum (LF) and various inflammatory cytokines. OBJECTIVES: To examine the effect of inflammatory cytokines on LF cells and to identify their roles in the pathogenesis of LF hypertrophy and ossification. SUMMARY OF BACKGROUND DATA: Spinal stenosis is caused, in part, by hypertrophy and ossification of the LF, which are induced by the degenerative processes (ie, increased collagen synthesis and chondroid metaplasia) of ligament fibroblasts. Degenerated intervertebral disk spontaneously produces inflammatory cytokines, which might affect the adjacent LF through local milieu of the spinal canal. METHODS: The interlaminar portion of the LF was collected during surgical spinal procedures in 15 patients (age range, 49-78 y) with lumbar spinal stenosis. LF fibroblasts were isolated by enzymatic digestion of LF tissue. LF cell cultures were treated with various inflammatory cytokines: interleukin (IL)-1α, IL-6, tumor necrosis factor-α (TNF-α), prostaglandin E2 (PGE2), and nitric oxide (NO). Cytotoxicity was analyzed by MTT assays. DNA synthesis was measured with H-thymidine incorporation, and mRNA expression of types I, III, V, and XI collagen and osteocalcin were performed by reverse transcription-polymerase chain reaction. Histochemical stains such as Von Kossa were also performed to detect bone nodule formation. RESULTS: There was no cytotoxicity in the LF cells treated with each cytokine. There were significant increases in DNA synthesis and upregulated mRNA expression of types I, V, XI collagen and osteocalcin in LF cultures treated with various cytokines. LF cultures treated with IL-6, TNF-α, PGE2, and NO showed positive Von Kossa staining, indicating bone nodule formation from LF cells. CONCLUSIONS: Inflammatory cytokines (IL-6, TNF-α, PGE2, and NO) seem to play a crucial role in hypertrophy and ossification of LF. Degenerated, herniated intervertebral disks, and facet arthrosis may influence LF through inflammatory cytokines and cause hypertrophy and ossification of LF.

Inactivation of enveloped and non-enveloped viruses in the process of chemical treatment and gamma irradiation of bovine-derived grafting materials.

BACKGROUND: Xenografts, unlike other grafting products, cannot be commercialized unless they conform to stringent safety regulations. Particularly with bovine-derived materials, it is essential to remove viruses and inactivate infectious factors because of the possibility that raw materials are imbrued with infectious viruses. The removal of the characteristics of infectious viruses from the bovine bone grafting materials need to be proved and inactivation process should satisfy the management provision of the Food and Drug Administration (FDA). To date, while most virus inactivation studies were performed in human allograft tissues, there have been almost no studies on bovine bone. METHODS: To evaluate the efficacy of virus inactivation after treatment of bovine bone with 70% ethanol, 4% sodium hydroxide, and gamma irradiation, we selected a variety of experimental model viruses that are known to be associated with bone pathogenesis, including bovine parvovirus (BPV), bovine herpes virus (BHV), bovine viral diarrhea virus (BVDV), and bovine parainfluenza-3 virus (BPIV-3). The cumulative virus log clearance factor or cumulative virus log reduction factor for the manufacturing process was obtained by calculating the sum of the individual virus log clearance factors or log reduction factors determined for individual process steps with different physicochemical methods. RESULTS: The cumulative log clearance factors achieved by three different virus inactivation processes were as follows: BPV ≥ 17.73, BHV ≥ 20.53, BVDV ≥ 19.00, and BPIV-3 ≥ 16.27. On the other hand, the cumulative log reduction factors achieved were as follows: BPV ≥ 16.95, BHV ≥ 20.22, BVDV ≥ 19.27, and BPIV-3 ≥ 15.58. CONCLUSIONS: Treatment with 70% ethanol, 4% sodium hydroxide, or gamma irradiation was found to be very effective in virus inactivation, since all viruses were at undetectable levels during each process. We have no doubt that application of this established process to bovine bone graft manufacture will be effective and essential.

Clinical significance of right hepatectomy along the main portal fissure on donors in living donor liver transplantation.

There might be discordance between inter-lobar borders of the main portal fissure (MPF) using the middle hepatic vein (MHV) and of the portal segmentation. Forty-five living donors who underwent right hepatectomy for the adult recipients from 2007 to 2011 in a tertiary hospital were retrospectively analyzed. The donors were classified into conventional right hepatectomy along the MPF (cRL group, n = 26) and modified right hepatectomy along right-side shifted transection plane from the MPF (mRL group, n = 19). The cRL donors had higher postoperative peak level of INR (1.84 vs. 1.62; P = 0.022), and bilirubin (3.37 mg/dl vs. 2.74 mg/dl; P = 0.065) than the mRL donors. cRL donors experienced greater depression of platelet count (144 per nL vs. 168 per nL; P = 0.042) and enlargement of splenic volume (52% vs. 37%; P = 0.025) than mRL donors for 7 days after hepatectomy. The regeneration of the left lateral sector was more accelerated in the cRL donors than the mRL donors for postoperative 3 months (148% vs. 84%; P = 0.015). There were no differences in the post-transplant graft function, incidence of complications, and graft survival rates between the two groups of recipients (P > 0.05). This study suggests that the conventional right hepatectomy along the MHV might increase donor risk by reducing parenchymal liver volume of the segment IV.

Osteoinductive activity of biphasic calcium phosphate with different rhBMP-2 doses in rats.

OBJECTIVE: The aim of the current study was to determine whether a hydroxyapatite (HA)/beta-tricalcium phosphate (ß-TCP) ratio of 20/80 impregnated with recombinant human bone morphogenetic protein (rhBMP-2) enhances new bone formation and to evaluate the dose-dependent response of rhBMP-2. STUDY DESIGN: Critical-sized calvarial defects were made in rats, and biphasic calcium phosphate (BCP) with different rhBMP-2 doses was loaded into rat calvarial defects. The animals were allowed to heal for either 2 or 8 weeks. RESULTS: The percentages of new bone after 2 and 8 weeks of healing were significantly greater in the rhBMP-2-treated groups (at all doses) than in the control groups. The percentage of remaining BCP was significantly lower at 8 weeks than at 2 weeks in all groups that included BCP. CONCLUSIONS: rhBMP-2 administered using a BCP carrier significantly induces new bone formation. A dose-dependent response was not shown in the present study.

Volumetric bone regenerative efficacy of biphasic calcium phosphate-collagen composite block loaded with rhBMP-2 in vertical bone augmentation model of a rabbit calvarium.

Block-type biphasic calcium phosphate (BCP) carriers are more effective at delivering recombinant human bone morphogenetic protein-2 (rhBMP-2) in various clinical situations than are particle-type carriers, due to their potential for highly successful three-dimensional bone regeneration. The aim of this study was to confirm the bone-regenerative capabilities of three-dimensional BCP blocks with a low hydroxyapatite/ß-tricalcium phosphate ratio (20/80) combined with collagen (10% wt) as an rhBMP-2 delivery system in a craniofacial vertical bone augmentation model. BCP blocks and BCP-collagen blocks (with average macropore sizes of 296 and 390 µm, respectively) with or without rhBMP-2 were fixed with osteosynthesis screws to the calvarial surface of rabbits. After 8 weeks, histologic and histomorphometric analyses were performed to evaluate the resulting new bone area, augmented area, bone density, and degree of integration. The area of new bone was significantly greater in specimens containing rhBMP-2 than in the control group (p < 0.05). Moreover, the area fractions of newly formed bone within the augmented area and a degree of integration between the regenerative bone and the calvarium were both significantly greater in the BCP-collagen/rhBMP-2 group than in the BCP/rhBMP-2 group (p < 0.05), whereas the two carrier systems exhibited similar rhBMP-2 release profiles, with sustained and linear release. The BCP and BCP/rhBMP-2 blocks exhibited excellent structural integrity, with large fragments of residual ceramic. In conclusion, the BCP-collagen composite block exhibited enhanced osteoinductive potential and could be a good candidate as a carrier of rhBMP-2 due to its characteristics of favorable volumetric stability, ease of handling, and excellent remodeling properties.

Developmental pattern of Ginkgo biloba levopimaradiene synthase (GbLPS) as probed by promoter analysis in Arabidopsis thaliana.

Levopimaradiene synthase (GbLPS) of Ginkgo biloba catalyzes the first committed step in ginkgolide biosynthesis by converting geranylgeranyl diphosphate into levopimaradiene, which subsequently undergoes complex oxidation step and rearrangement of carbon skeleton, leading to formation of ginkgolides. To assess the organ-specificity and developmental characteristics of GbLPS expression, the GbLPS promoter-driven GUS expression in transgenic Arabidopsis was studied. Histological analysis of the transgenic Arabidopsis plant showed that the GUS accumulation was mainly localized in the epidermis of leaves, phloem of the shoots, ovaries and stamens of flowers, and vasculature of roots. These observations correlate with the occurrence of LPS transcripts in roots and male strobili of G. biloba. Treatment of methyl jasmonate on the transformant exhibited significant upregulation of the reporter gene in the roots with little change in leaves and flowers. The present findings support biosynthesis of ginkgolide in the roots of Ginkgo plant and suggest translocation occurs through the phloem.

Tissue engineering of the intervertebral disc with cultured nucleus pulposus cells using atelocollagen scaffold and growth factors.

STUDY DESIGN: In vitro experiment using rabbit nucleus pulposus (NP) cells seeded in atelocollagen scaffolds under the stimulation of growth factors. OBJECTIVE: To demonstrate the effect of anabolic growth factors in rabbit NP cells cultured in atelocollagen type I and type II. SUMMARY OF BACKGROUND DATA: Atelocollagen provides intervertebral disc (IVD) cells for a biocompatible environment to produce extracellular matrix. IVD cells with exogenous transforming growth factor-beta 1 (TGF-ß1) and bone morphogenetic protein-2 (BMP-2) also render an increase in matrix synthesis. However, the effect of anabolic growth factors in NP cells cultured in atelocollagens was not elucidated before. METHODS: Rabbit NP cell was harvested, enzymatically digested, and cultured. The NP cells were seeded to atelocollagen type I and type II scaffolds, and then cultures were exposed to TGF-ß1 (10 ng/mL) and/or BMP-2 (100 ng/mL). DNA synthesis was measured using [4H]-thymidine incorporation. Newly synthesized proteoglycan was measured using [35S]-sulfate incorporation. Reverse transcription-polymerase chain reactions (RT-PCRs) for mRNA expression of aggrecan, collagen type I, collagen type II, and osteocalcin were performed. RESULTS: Rabbit NP cells cultured in atelocollagen type I scaffold showed an increase (1.7 to 2.4-fold) in DNA synthesis in response to TGF-ß1 and/or BMP-2 (P < 0.05), whereas NP cultures in atelocollagen type II demonstrated a 30% increase in DNA synthesis only with combination of both growth factors compared with control (P < 0.05). Rabbit NP cells in atelocollagen type II scaffold with TGF-ß1 and combination of both growth factors exhibited robust 5.3- and 5.4-fold increases in proteoglycan synthesis (P < 0.05), whereas any cultures in atelocollagen type I failed to show any significant increase compared with control. Rabbit NP cells in atelocollagen type I and type II scaffolds with TGF-ß1 and/or BMP-2 demonstrated the upregulation of aggrecan, collagen type I, and collagen type II mRNA expression compared with saline control (P < 0.05). The response in transcriptional level was more robust in atelocollagen type II than in type I. In any event, there is no recognizable expression of osteocalcin (P < 0.05). CONCLUSION: NP cells in atelocollagens under the stimulation of TGF-ß1 and BMP-2 exhibited anabolic responses in transcriptional and translational levels. Hence, such an approach can provide a suitable engineered tissue for IVD regeneration with potential for robust refurbishment of matrix.

Volumetry-based selection of right posterior sector grafts for adult living donor liver transplantation.

To determine the feasibility of volumetric criteria without anatomic exclusion for the selection of right posterior sector (RPS) grafts for adult-to-adult living donor liver transplantation (LDLT), we reviewed and compared our transplant data for RPS grafts and right lobe (RL) grafts. Between January 2008 and September 2010, adult-to-adult LDLT was performed 65 times at our institute; 13 of the procedures (20%) were performed with RPS grafts [the posterior sector (PS) group], and 39 (60%) were performed with RL grafts (the RL group). The volumetry of the 13 RPS donor livers showed that the RPS volume was 39.8% ± 7.6% of the total liver volume. Ten of the 13 donors had to donate RPS grafts because the left liver volume was inadequate. All donor procedures were performed successfully, and all donors recovered from hepatectomy. However, longer operative times were required for the procurement of RPS grafts versus RL grafts (418 ± 40 versus 345 ± 48 minutes, P < 0.001). The postoperative recovery of liver function was smoother for the donors of the PS group versus the donors of the RL group. The RPS grafts had significantly smaller hepatic artery and bile duct openings than the RL grafts. All recipients with RPS grafts survived LDLT. No recipients experienced vascular graft complications or small-for-size graft dysfunction. There were no significant differences in the incidence of posttransplant complications between the donors and recipients of the PS and RL groups. The 3-year graft survival rates were favorable in both groups (100% in the PS group versus 91% in the RL group). In conclusion, the selection of RPS grafts by volume criteria is a feasible strategy for an adult-to-adult LDLT program.

The risk assessment of a fall in patients with lumbar spinal stenosis.

STUDY DESIGN: A prospective case control study. OBJECTIVES: To investigate the risk of a fall by using functional mobility tests in patients with lumbar spinal stenosis (LSS) via a comparison with patients with knee osteoarthritis (KOA). SUMMARY OF BACKGROUND DATA: LSS is a degenerative arthritic disease in the spine that results in decreasing function, impaired balance, and gait deficit, with increased levels of leg and back pain. This physical impairment may result in an increased risk of fall later in the disease process, as shown in KOA. However, there has been no study regarding the association between the risk of a fall and LSS. METHODS: The study was an age- and weight-matched case control study consisting of two groups: one group consisting of 40 patients with LSS who were scheduled to undergo spine surgery (LSS group) and the other group consisting of 40 patients with advanced osteoarthritis in both knees, scheduled to undergo TKA on both knees (KOA group). For both groups, four functional mobility tests, such as a Six-Meter-Walk Test (SMT), Sit-to-Stand test (STS), Alternative-Step Test (AST), and Timed Up and Go Test (TUGT), were performed. RESULTS: There was no difference in demographic data between both groups except for body mass index. For the SMT and STS, the patients in the LSS group spent significantly more time performing these tests than the patients in the KOA. For the AST, however, patients in the KOA group presented a statistically worse performance in functional mobility, compared with the LSS group. The mean TUGT time was not statistically different between the two groups. CONCLUSIONS: The current study highlights that patients with symptomatic LSS have a risk of a fall comparable with the patients who had degenerative KOA based on the results of functional mobility tests (SMT, STS, AST, and TUGT).

De novo osteogenesis from human ligamentum flavum by adenovirus-mediated bone morphogenetic protein-2 gene transfer.

STUDY DESIGN: In vitro and in vivo experiment using degenerated human ligamentum flavum (LF) and Type 5 adenovirus construct with bone morphogenetic protein-2 (BMP-2) cDNA. OBJECTIVES: To demonstrate in vitro and in vivo osteogenic effect of BMP-2 gene transfer to human LF and to propose genetically modified LF as a substitute for autogenous bone graft in spinal fusion. SUMMARY OF BACKGROUND DATA: Spinal fusion is still considered to be an important option for treating various spinal disorders. To induce solid spinal fusion, osteoinductive and/or osteoconductive agents have been widely adopted. Autogenous LF, however, has never been seriously considered as a carrier for ex vivo osteoinductive gene therapy for spinal fusion. METHODS: In vitro experiment: Degenerated human LF was harvested and cultured. Type 5 adenovirus lacZ (Ad/lacZ) and BMP-2 construct (Ad/BMP-2) were produced. LF cell cultures were then exposed to Ad/BMP-2. Expressions of osteocalcin and BMP-2 mRNA were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). Western blot analysis was performed to detect osteocalcin protein. Alkaline phosphatase and von Kossa stains were used to detect osteogenic markers and bone nodule formation, respectively. In vivo experiment: Human LF tissues treated with Ad/lacZ, Ad/BMP-2, and saline were implanted into the subcutaneous tissue of nude mice. After 4 weeks, nude mice were radiographed and killed. Implanted LF tissues were harvested and histologically stained. RESULTS: LF cell cultures with Ad/BMP-2 revealed strong expression of BMP-2 and osteocalcin mRNA in RT-PCR and osteocalcin protein in western blot analysis. LF cell culture with saline showed baseline expression of BMP-2, osteocalcin mRNA, and osteocalcin protein, respectively. Furthermore, LF cell culture with Ad/BMP-2 demonstrated the expression of alkaline phosphatase and bone nodule formation in the aforementioned histochemical stain. LF tissues with Ad/BMP-2 revealed de novo osteogenesis in nude mice, whereas LF with Ad/lacZ or saline showed only remaining LF tissue without sign of bone formation. CONCLUSION: Human LF cells transduced with Ad/BMP-2 exhibited the expression of osteogenic phenotype and bone nodule formation. Additionally, genetically modified human LF with BMP-2 cDNA clearly demonstrated de novo osteogenesis, which supports the concept that biologically modified LF can be a substitute for autogenous bone graft in spinal fusion surgery.

Comparison of the predictive value of myelography, computed tomography and MRI on the treadmill test in lumbar spinal stenosis.

To date, there have been no prospective, objective studies comparing the accuracy of the MRI, myelo-CT and myelography. The purpose of this study is to compare the diagnostic and predictive values of MRIs, myelo-CTs, and myelographies. Myelographies with dynamic motion views, myelo-CTs, MRIs and exercise treadmill tests were performed in 35 cases. The narrowest AP diameter of the dural sac was measured by myelography. At the pathologic level, dural cross-sectional area (D-CSA) was calculated in the MRI and Myelo-CT. The time to the first symptoms (TAF) and the total ambulation time (TAT) were measured during the exercise treadmill test and used as the standard in the comparison of correlation between radiographic parameters and walking capacity. The mean D-CSA by CT was 58.3 mm(2) and 47.6 mm(2) by MRI. All radiographic parameters such as AP diameters and D-CSA have no correlation to TAF or TAT (p > 0.05). Our data showed no statistically significant differences in the correlation of the patients' walking capacity to the severity of stenosis as assessed by myelography, myelo-CT and MRI.

Radiographic and functional outcome after surgical management of severe scoliosis in skeletally immature patients with muscular dystrophy.

OBJECTIVE: Most patients with muscular dystrophy (MD) develop progressive scoliosis after losing ambulatory status, but some cases develop severe scoliosis at a skeletally immature age before losing ambulatory status. Only a few studies have been conducted in skeletally immature patients with severe scoliosis. The purpose of this study was to assess the functional and cosmetic outcome in skeletally immature patients with severe scoliosis. METHODS: Preoperative, immediate postoperative, and final follow-up radiographs were analyzed in 10 consecutive skeletally immature patients with respect to the Cobb angle degree and the pelvic obliquity angle correction, how long the correction was maintained, and the development of the crankshaft phenomenon. In the functional assessment, the ability to sit balanced, according to the Mulcahy method, and the ability to use hands, according to the Rhyu method, were evaluated. Furthermore, the degree of subjective satisfaction was evaluated in these patients. RESULTS: The average age of the patients was 10.4 years, and the average follow-up period was 33 months with minimum 2 years' follow-up. All 10 patients survived and were available at the follow-up. The mean Cobb and pelvic obliquity angles were 80 degrees and 17 degrees at the time of the surgery, 31 degrees and 3.7 degrees immediately after the surgery, and 35 degrees and 4.7 degrees at the time of the final follow-up, respectively. The initial mean Cobb angle correction averaged 61%, with 78% of pelvic obliquity corrected. These corrections were maintained over time in most cases. At the time of the surgery, the mean volume of blood loss was 1111 mL, with an average operation time of 411 minutes. There were no major complications. At the time of the last follow-up, no patient showed development of the crankshaft phenomenon. The average score for the ability to sit balanced improved from 4.4 to 6.6 according to the Mulcahy evaluation method. The scores for hand use were 2.2-2.7. However, the forced vital capacity of the lungs decreased from a preoperative 48% to 46.1%. CONCLUSIONS: These results indicate that even in very young MD patients with severe scoliosis, acceptable curve correction can be achieved and maintained with surgery. The improved pelvic obliquity and scoliosis angle stabilized the spine, freeing the upper extremities and allowing productive activities characteristic of childhood.