Abstract: Some treatments for full thickness defects of the articular cartilage, such as the transplantation of cultured chondrocytes have already been performed. However, in order to overcome osteoarthritis, we must further study the partial thickness defects of articular cartilage. It is much more difficult to repair a partial thickness defect because few repair cells can address such injured sites. We herein show that bioengineered and layered chondrocyte sheets using temperature-responsive culture dishes may be a potentially useful treatment for the repair of partial thickness defects. We also show that a chondrocyte-plate using a rotational culture system without the use of a scaffold may also be useful as a core cartilage of an articular cartilageous defect. We evaluated the properties of these sheets and plates using histological findings, scanning electrical microscopy, and photoacoustic measurement methods, which we developed to evaluate the biomechanical properties of tissue-engineered cartilage. In conclusion, the layered chondrocyte sheets and chondrocyte-plates were able to maintain the cartilageous phenotype, thus suggesting that they could be a new and potentially effective therapeutic product when attached to the sites of cartilage defects.

Abstract: We investigated whether articular chondrocytes could form three-dimensional tissue-engineered cartilage in a rotational culture system without a scaffold. A suspension of chondrocytes derived from Japanese white rabbits was inoculated into a mold. Eight hours later, the cell suspension in the mold showed cell aggregation, forming a chondrocyte plate. The mold was removed, and the plate was cultured under static conditions. After 7 days of primary static culture, the plate was cultured under dynamic conditions, using rotational culture. After 2-3 weeks of rotational culture, the chondrocyte plate maintained a constant form and was considered stable enough to be handled with surgical pincers. Conversely, after 3 weeks of static culture, the plate gradually changed into an arch over that time. Histological and immunohistochemical evaluations indicated that the plate had cartilaginous qualities in terms of cell distribution and organization and the production of glycosaminoglycans and type II collagen in rotational cultures. Chondron units were detected with scanning electron microscopy. In contrast, a plate cultivated in static culture for 3 weeks was irregular in shape, and histological analysis indicated irregularly accumulated glycosaminoglycans. TUNEL-positive cells had increased significantly in the central region in 3-week static cultures, compared with those in 3-week rotational cultures. In this study, cartilaginous tissue in a scaffold-free environment has been produced. Significantly rotational cultures produce a construct, which is stable enough to be handled with surgical forceps after only 2 weeks of rotational culture. This system should be useful for implantation in the future.

Abstract: If a tissue-engineered cartilage transplant is to succeed, it needs to integrate with the host tissue, to endure physiological loading, and to acquire the phenotype of the articular cartilage. Although there are many reported treatments for osteochondral defects of articular cartilage, problems remain with the use of artificial matrices (scaffolds) and the stage of implantation. We constructed scaffold-free three-dimensional tissue-engineered cartilage allografts using a rotational culture system and investigated the optimal stage of implantation and repair of the remodeling site. We evaluated the amounts of extracellular matrix and gene expression levels in scaffold-free constructs and transplanted the constructs for osteochondral defects using a rabbit model. Allografted 2-week constructs expressed high levels of proteoglycan and collagen per DNA content, integrated with the host cartilage successfully, and were able to counter physiological loads, and the chondrocyte plate contributed reparative mesenchymal stem cells to the final phenotype of the articular cartilage.

Abstract: The specific aim of our investigation is to study the potential use of a collagen/heparin-carrying polystyrene (HCPS) composite extracellular matrix for articular cartilage tissue engineering. Here, we created a high-performance extracellular matrix (HpECM) scaffold to build an optimal extracellular environment using an HCPS we originally developed, and an atelocollagen honeycomb-shaped-scaffold (ACHMS-scaffold) with a membrane seal. This scaffold was coated with HCPS to enable aggregation of heparin-binding growth factors such as FGF-2 and TGF-beta1 within the scaffold. Three-dimensional culture of rabbit articular chondrocytes within the HpECM-scaffold and subsequent preparation of a tissue-engineered cartilage were investigated. The results showed remarkably higher cell proliferative activity within the HpECM-pretreated-FGF-2 scaffold and the sustenance of phenotype within the HpECM-pretreated-TGF-beta1 scaffold. It was thought that both FGF-2 and TGF-beta1 were stably immobilized in the HpEMC-scaffold since HCPS generated an extracellular environment similar to that of heparan sulfate proteoglycan within the scaffold. These results suggest that an ACHMS-scaffold immobilized with HCPS can be a HpECM for cartilage regeneration to retain the heparin-binding growth factors within the scaffolds.

Abstract: The extracellular matrix (ECM) of articular cartilage has several functions that are unique to joints. Although a technique for transplanting cultured chondrocytes has already been introduced, it is difficult to collect intact ECM when using enzymes to harvest samples. Temperature-responsive culture dishes have already been clinically applied in the fields of myocardial and corneal transplantation. Earlier studies have shown that a sheet of cultured cells with intact ECM and adhesive factors can be harvested using such culture dishes, which allow the surface properties of the dish to be reversibly altered by changing the temperature. Human chondrocytes were subjected to enzymatic digestion and then were seeded in temperature-responsive culture dishes. A sheet of chondrocytes was harvested by only reducing the temperature after the cultured cells reached confluency. A real-time PCR analysis of the chondrocyte sheets confirmed that type II collagen, aggrecan, and fibronectin were present. These results suggested that, although chondrocytes undergo dedifferentiation in a monolayer culture, multilayer chondrocyte sheets grown in a similar environment to that of three-dimensional culture may be able to maintain a normal phenotype. A histological examination suggested that multilayer chondrocyte sheets could thus prevent the loss of proteoglycans because the area covered by the sheets was well stained by safranin-O. The present experiments suggested that temperature-responsive culture dishes are useful for obtaining cultured chondrocytes, which may then be clinically employed as a substitute for periosteal patches because such sheets can be applied without a scaffold.

Abstract: Adipose tissue derived stromal cells (ATSCs), which were isolated from adipose tissue of rabbit, have shown to possess multipotential, that is, they differentiate into osteoblasts and adipocytes in plate-culturing and into chondrocytes in an established aggregate culture using defined differentiation-inductive medium. The aim of this study was to evaluate the utility of ATSCs in tissue engineering procedures for repair of articular cartilage-defects using the atelocollagen honeycomb-shaped scaffold with a membrane sealing (ACHMS-scaffold). We intended to repair full-thickness articular cartilage defects in rabbit knees using autologously cultured ATSCs embedded in the ACHMS-scaffold. ATSCs were incubated within the ACHMS-scaffold to allow a high density and three-dimensional culture with control medium. An articular cartilage defect was created on the patellar groove of the femur, and the defect was filled with the ATSCs-containing ACHMS-scaffold, ACHMS-scaffold alone, or empty (control). Twelve weeks after the operation, the histological analyses showed that only the defects treated with the ATSCs-containing ACHMS-scaffold were filled with reparative hyaline cartilage, highly expressed Type II collagen. These results indicate that transplantation of autologous ATSCs-containing ACHMS-scaffold is effective in repairing articular cartilage defects.

Abstract: Human adipose tissue, obtained by liposuction, was processed to obtain a fibroblast-like population of cells or adipose tissue-derived stromal cells (ATSCs). The ATSCs, as well as bone marrow-derived mesenchymal stem cells (BMSCs), have the capacity for renewal and the potential to differentiate into multiple lineages of mesenchymal tissues. These cells are capable of forming bone when implanted ectopically in an appropriate scaffold. The aim of this study was to evaluate a beta-tricalcium phosphate (beta-TCP) as a scaffold and to compare the potential of osteogenic differentiation of ATSCs with BMSCs. Both cell types were loaded into beta-TCP disk and cultured in an osteogenic induction medium. Optimal osteogenic differentiation in ATSCs in vitro, as determined by secretion of osteocalcin, scanning electron microscope, and histology, were obtained in the culturing with the beta-TCP disk. Furthermore, bone formation in vivo was examined by using the ATSC- or BMSC-loaded scaffolds in nude mice. The present results show that ATSCs have a similar ability to differentiate into osteoblasts and to synthesize bone in beta-TCP disk as have BMSCs.

Abstract: Some treatments for full thickness defects of articular cartilage, such as cultured chondrocyte transplantation, have already been done. However, to overcome osteoarthritis, we must further study the partial thickness defect of articular cartilage. It is much more difficult to repair a partial thickness defect because few repairing cells can address such injured sites. We herein show that bioengineered layered chondrocyte sheets using temperature-responsive culture dishes may be a potentially useful treatment for partial thickness defects. We evaluated the property of these sheets using real-time PCR and histological findings, and allografted these sheets to evaluate the effect of treatment using a rabbit partial model. In conclusion, layered chondrocyte sheets were able to maintain the cartilageous phenotype, and could be attached to the sites of cartilage damage which acted as a barrier to prevent a loss of proteoglycan from these sites and to protect them from catabolic factors in the joint.

Abstract: OBJECTIVES: Intramedullary spinal cord metastasis (ISCM) is a rare complication of cancer. This complication has been studied by magnetic resonance imaging (MRI) more frequently in the clinical context to improve the survival time of cancer patients. However, the number of the reports of its clinical management or radiographic findings is small. To investigate the clinical and imaging characteristics of ISCM, seven cases of ISCM of a nonneurogenic origin were examined retrospectively. METHOD: Data regarding the site of the primary neoplasm, location of other metastases, initial symptoms, interval from the original diagnosis of cancer to the onset of neurologic symptoms, time course from initial symptoms to urinary retention, MRI data, and outcome after radiologic or surgical intervention were investigated. RESULTS: Five patients had lung cancer metastases, and one each had breast or uterine cancer metastases. The progression of neurologic deficit was rapid, and the average period between the occurrence of initial symptoms and urinary retention was 25 days. The averaged survival period from the diagnosis of ISCM was 5 months in three patients. (One patient is alive, and the data could not be obtained in another three patients.) MRI characteristics were useful for diagnosis, including large high signal intensity areas on T2-weighted images and strong ring or inhomogeneous enhancement on gadopentetate dimeglumine-enhanced T1-weighted images. Surgery was performed on two patients, but total removal could not be achieved. Radiotherapy was effective in treating neurologic deficit in five patients. CONCLUSION: Surgical treatment is seldom indicated for ISCM, and radiotherapy is recommended as soon as possible after a diagnosis is made from MRI.

Abstract: BACKGROUND AND OBJECTIVES: We demonstrated that photoacoustic measurement enables viscoelastic characterization of biological tissue. The purpose of this study was to develop a practical photoacoustic measurement system for diagnosis of osteoarthritis (OA) by viscoelastic characterization of articular cartilage. STUDY DESIGN/MATERIALS AND METHODS: The portable system consists of a commercially available 3rd harmonic Q-switched Nd:YAG laser as a light source and a transducer, which is arranged coaxially with an optical fiber. Cell proliferation tests were performed to study the effect of laser irradiation on chondrocytes. Photoacoustic measurements were performed using enzymatically treated cartilage as a model of OA. RESULTS: There was no significant damage of chondrocytes caused by laser irradiation (100 microJ/mm2, 5 Hz, 30 shots). The change in relaxation times measured by the photoacoustic measurement had a positive correlation with time of enzymatic treatment, that is, the degree of cartilage degeneration. CONCLUSIONS: We have developed a noninvasive photoacoustic measurement system designed for arthroscopic use and have demonstrated the applicability of this system to the diagnosis of OA-like cartilage degeneration.

Abstract: Monostotic fibrous dysplasia of the spine is extremely rare. We present a 57-year-old man who complained of persistent low back pain with monostotic fibrous dysplasia of the lumbar spine. Computed tomography revealed a lytic expansile lesion and marginal sclerosis in the L2 posterior element, although a bone scan did not reveal increased uptake in the lesion. The patient underwent total excision of the tumor via a posterior approach. Two years later, he is asymptomatic with no recurrence of the lesion, as confirmed by imaging.

Abstract: The aim of this study was to investigate with tissue engineering procedures the possibility of using atelocollagen honeycomb-shaped scaffolds sealed with a membrane (ACHMS scaffold) for the culturing of chondrocytes to repair articular cartilage defects. Chondrocytes from the articular cartilage of Japanese white rabbits were cultured in ACHMS scaffolds to allow a high-density, three-dimensional culturing for up to 21 days. Although the DNA content in the scaffold increased at a lower rate than monolayer culturing, scanning electron microscopy data showed that the scaffold was filled with grown chondrocytes and their produced extracellular matrix after 21 days. In addition, glycosaminoglycan (GAG) accumulation in the scaffold culture was at a higher level than the monolayer culture. Cultured cartilage in vitro for 14 days showed enough elasticity and stiffness to be handled in vivo. An articular cartilage defect was initiated in the patellar groove of the femur of rabbits and was subsequently filled with the chondrocyte-cultured ACHMS scaffold, ACHMS scaffold alone, or non-filled (control). Three months after the operations, histological analysis showed that only defects inserted with chondrocytes being cultured in ACHMS scaffolds were filled with reparative hyaline cartilage, and thereby highly expressing type II collagen. These results indicate that implantation of allogenic chondrocytes cultured in ACHMS scaffolds may be effective in repairing articular cartilage defects.

Abstract: OBJECTIVE: Most patients with traumatic spondylolisthesis of the axis are treated nonsurgically. Some patients do not develop symptoms, but others experience strong and persistent neck pain. To clarify the factor that plays a major role in residual neck pain after this fracture, we reviewed nine patients who underwent conservative treatment. METHODS: Patients were assessed via telephone interviews an average of 62 months after injury and were divided into two groups: those with and those without neck pain. Angulation and anterior translation between the second and third cervical vertebrae were measured on lateral radiograms at the time of injury, removal of brace, and final follow-up. Fracture lines were also investigated by computed tomography. RESULTS: At the time of final follow-up, the average angulation was 6.3 degrees and the average translation was 5.3 mm in the neck-pain group and 0.6 degrees and 0.2 mm, respectively, in the no-symptom group. In patients with fracture lines present on the articular surface of the inferior face joints, angulation persisted or increased, even though the fractures were hemilateral. This was compatible with severe neck pain. In contrast, angulation and translation were improved in patients with an intact inferior articular surface of the axis, and pain was not reported. CONCLUSIONS: Injury on the inferior articular surface of the axis may disturb spontaneous healing of C2-C3 subluxation and cause residual neck pain. It is necessary to assess the presence of injury to the inferior facet bilaterally with plain or computed tomograms.

Abstract: There is a demand in the field of regenerative medicine for measurement technology that enables functions of engineered tissue to be determined. For meeting this demand, we previously proposed a noninvasive method for determination of the viscoelasticity of a tissue phantom based on photoacoustic measurements. The purpose of this study was to verify the usefulness of the photoacoustic measurement method for evaluation of the viscoelastic properties of actual engineered tissue and to determine the correlation between biochemical characteristics and photoacoustic signals. The relaxation times measured by the photoacoustic method agreed well with the intrinsic viscoelastic parameters with a correlation coefficient of 0.98 when tissue-engineered cartilage tissues cultured for various periods (up to 12 weeks) were used as samples. By comparison of the results of biochemical analyses and biomechanical studies, we proved that the photoacoustic signal is a good indicator for evaluating extracellular matrix formation in order to determine the characteristics of tissue-engineered cartilage. To our knowledge, this is the first report on noninvasive and time-dependent viscoelastic evaluation of engineered tissue for determining functions of engineered tissues.

Abstract: The intervertebral disc is classified as fibrocartilage, and has distinct functional and mechanical properties. The disc, composed of the nucleus pulposus and annulus fibrosus, varies in density, anatomic features, and in the morphology of their constituent cells. This heterogeneity has hampered detailed investigations of disc cellular metabolism. Focusing on the heterogeneity, we cultured disc cells under various conditions to examine the cellular metabolism, specifically glycosaminoglycan accumulation and pericellular microenvironment, and succeeded in mechanically isolating disc chondrons from annulus fibrosus. We believe that disc chondron also plays an important role in maintaining cartilageous phenotype in terms of cell-matrix interaction of the intervertebral disc.

Abstract: We immunohistochemically studied expressions of inducible heat shock protein 70 (HSP 72) and apoptosis of corneas ablated with an ArF excimer laser. The temperature of corneal surfaces and laser-induced optical emission spectra were measured in real time as direct physical parameters related to the ablation mechanism. To the best of the authors' knowledge, there have been no experimental studies regarding the influence of physical parameters directly related to the ablation mechanism on corneal reactions at the cell level after laser ablation. The expression of HSP 72 was mainly localized in the regenerative epithelium, which was confirmed to be caused by laser ablation. The HSP 72 positive cell ratios had a correlation with thermal dose, which was derived from the measured time courses of temperature. Expressions of both HSP 72 and apoptosis depended on the thermal dose and elapsed time after ablation. HSP 72 and apoptosis could be seen up to a few hundred micrometers into the stroma, only at a fluence with an optical breakdown emission. This could have been caused by shock waves induced by the optical breakdown.

Abstract: Adult bone marrow contains mesenchymal stem cells (bone marrow-derived mesenchymal stem cells; BMSCs) which contribute to the generation of mesenchymal tissue such as bone, cartilage, muscle and adipose. However, using bone marrow as a source of stem cells has the limitation of a low cell number. An alternate source of adult stem cells that could be obtained in large quantities, under local anesthesia, with minimal discomfort would be advantageous. Human adipose tissue obtained by liposuction was processed to obtain a fibroblast-like population of cells or adipose tissue-derived stromal cells (ATSCs). In this study, we compared the osteogenic differentiation of ATSCs with that of BMSCs. Both cell types were cultured in atelocollagen honeycomb-shaped scaffolds with a membrane seal (ACHMS scaffold) for three-dimensional culturing in a specific osteogenic induction medium. Optimal osteogenic differentiation in both cell types, as determined by alkaline phosphatase cytochemistry, secretion of osteocalcin, mineral (calcium phosphate) deposition and scanning electron microscopy, was obtained with the same three-dimensional culture. Furthermore, osteoblastic lining in vivo was examined using ATSC-seeded or BMSC-seeded scaffolds in nude mice. The present results show that ATSCs have a similar ability to differentiate into osteoblasts to that of BMSCs.

Abstract: Although many transpedicular screw systems have been developed and have undergone wide clinical use, experience with semirigid transpedicular systems has rarely been reported. We evaluated the efficacy and safety of the Crock-Yamagishi (C-Y) system for posterior spinal fusion in lumbar degenerative diseases. The outcomes for 26 patients (14 men, 12 women) with lumbar degenerative diseases who underwent posterior spinal fusion using the C-Y system were analyzed (posterior lumbar interbody fusion (PLIF), 11 patients; posterolateral fusion (PLF), 14; and facet fusion (FF), 1. Symptoms were evaluated using the Japanese Orthopaedic Association Assessment of Treatment for Low Back Pain (JOA score). Preoperative scores ranged from -1 to 23 points (mean, 12.8), while postoperative scores ranged from 19 to 29 points (mean, 26.4). Degree of recovery ranged from 23.1% to 100% (mean, 83.2%). Overall fusion rate was 96.2% (25/26). Neither breakage nor loosening of implants was observed radiographically. Intra- and postoperative complications included one case of transient L5 nerve root palsy attributable to surgical technique, and one deep postoperative infection. The C-Y system, categorized as semirigid, is effective when used with one- or two-level PLIF or PLF for lumbar degenerative disorders, grade I to II spondylolisthesis, and failed back syndrome.

Abstract: Application of ultraviolet (UV) irradiation to a photocrosslinkable chitosan (Az-CH-LA) aqueous solution resulted within 10 s in an insoluble, flexible hydrogel. A low molecular weight acidic molecule like trypan blue and various high molecular weight molecules such as bovine serum albumin (BSA), heparin and protamine were all retained within the hydrogel, while a low molecular weight basic molecule like toluidine blue was rapidly released from the hydrogel. In the present work, we examined the retaining capability of the chitosan hydrogel for growth factors and controlled release of growth factors from the chitosan hydrogel in vitro and in vivo. Fibroblast growth factor-1 (FGF-1), fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor(165) (VEGF(165)), heparin-binding epidermal growth factor (HB-EGF) in phosphate buffered saline (PBS) were mixed with Az-CH-LA aqueous solution to form growth factor-incorporated chitosan hydrogels. About 10-25% of the growth factor was released from a growth factor-incorporated chitosan hydrogel into PBS within the first day, after which no further substantial release took place. The growth factors interacted with Az-CH-LA molecules poly-ion complexation, and probably were unable to be released after the first day under the in vitro nondegradation conditions of the hydrogel. Although the FGF-1, FGF-2, and VEGF(165)-incorporated chitosan hydrogels on a culture plate significantly stimulated HUVEC growth, the stimulating activity of the growth factor-incorporated chitosan hydrogel was completely cancelled out by washing the hydrogel with PBS solution for 3 days or more. The stimulating activity on the HUVEC growth were however highly recovered by treating the washed growth factor-incorporated chitosan hydrogel during 7 days with chitinase and chitosanase to partly degrade the hydrogel, strongly suggesting that the growth factors within the hydrogel retained their biologically active forms. The chitosan hydrogel (100 microl) when implanted into the back of a mouse was biodegraded in about 10-14 days. When FGF-1- and FGF-2-incorporated chitosan hydrogels were subcutaneously implanted into the back of a mouse, significant neovascularization was induced near the implanted site of the FGF-1- and FGF-2-incorporated chitosan hydrogels. Furthermore, addition of heparin with either FGF-1 or FGF-2 into the hydrogel resulted in a significantly enhanced and prolonged vascularization effect. These results indicate that the controlled release of biologically active FGF-1 and FGF-2 with heparin is caused by biodegradation of the chitosan hydrogel, and subsequent induction of vascularization.

Abstract: The objective of the study was to investigate the regeneration of intervertebral discs after laser discectomy using tissue engineering procedures. Annulus fibrosus (AF) cells from the intervertebral discs of Japanese white rabbits were cultured in an atelocollagen honeycomb-shaped scaffold with a membrane seal (ACHMS scaffold), to produce a high-density, three-dimensional culture for up to 3 weeks. Although the DNA content in the scaffold increased at a lower rate than that in the monolayer culture, expression of type II collagen and glycosaminoglycan accumulation in the scaffold were at higher levels than in the monolayer. The AF cells that had been cultured in the scaffold for 7 days were allografted into the lacunae of intervertebral discs of recipients (40 rabbits, 14-16 weeks old; average weight, 3.2 kg), whose nucleus pulposus (NP) had been vaporised with an ICG dye-enhanced laser. The allografted cultured AF cells survived and produced hyaline-like cartilage. Furthermore, the narrowing of the intervertebral disc space of the cell-containing scaffold insertion groups was significantly inhibited after 12 post-operative weeks.

Abstract: BACKGROUND: Separation of the posterior ring apophysis of an adjacent vertebral body can sometimes accompany lumbar intervertebral disc herniation. The condition can be both difficult to detect in conventional radiographs and is somewhat controversial to treat. Although there is general agreement on the frequent need for surgery, there is no consensus on the choice of operation. One procedure, posterior lumbar interbody fusion (PLIF), has never been examined for effectiveness. METHOD: Computed tomography (CT) either or with discography (CTD) was compared to plane radiographs and magnetic resonance imaging (MRI) for its ability to reveal the fragmentation. Five cases were identified and the severity of the condition evaluated using the Japanese Orthopaedic Association's Assessment of Treatment for Low Back Pain (JOA score). The fragments were removed by impaction with a shoe shaped device and posterior lumbar interbody fusion (PLIF) was performed using autogenous iliac crest bone. A second evaluation using the JOA score was performed after a 4 to 5 year follow-up. FINDINGS: CT identified the separation in all 5 cases while plain films showed the defect in two and MRI in none. JOA scores prior to surgery were between 5 and 19 (mean, 13) and between 25 and 29 (mean, 27.4) at follow-up. The scores represented a recovery of 80% to 100%t (mean, 89.4). INTERPRETATION: CT and CT discography (CTD) are the diagnostic tools of choice for detecting this condition. Posterior lumbar interbody fusion (PLIF) is an effective procedure for patients who have low back pain due to lumbar disc herniation accompanied by a separation of the posterior ring apophysis.

Abstract: BACKGROUND: A variety of procedures for reconstructing the spine following the removal of spinal cord and cauda equina tumours have been developed to prevent postoperative spinal deformities and nerve entrapment. The purpose of this paper is to introduce a new reconstructive procedure based on rotational laminoplasty and to report preliminary results in a small series. METHOD: The trough is drilled at the border of the laminae and articular processes and the ligamentum flavum is resected on its cephalocaudal aspect, so the vertebral arch can be separated as a single mass. After tumour resection, the vertebral arch is removed en bloc with the laminae, and is rotated 90 degrees and placed on the articular facets and fixed using suture passing through holes drilled in the bone. FINDINGS: One man and six women underwent rotational laminoplasty following resection of spinal or cauda equina tumours. Operative exposure was good and permitted complete resection. Patients did well postoperatively from both spine-surgical and neurosurgical points of view. Computed tomography documented a bony union with preservation of widely patent spinal canal. INTERPRETATION: Rotational laminoplasty affords a satisfactory operative exposure for the resection of large, complex lesions. It creates a widely patent, stable spinal canal easily, without the need for special tools.

Abstract: The aim of this study was to investigate the possibility of using the atelocollagen honeycomb-shaped scaffold with a membrane seal (ACHMS-scaffold) for the culture of annulus fibrosus (AF) cells in tissue engineering procedures of intervertebral disc repair. AF cells from the intervertebral discs of Japanese white rabbits were cultured for up to 3 weeks in the ACHMS-scaffold to allow a high density, three-dimensional culture. Although the DNA content in the scaffold increased at a lower rate than in the monolayer culture, scanning electron microscopy data showed that the scaffold was filled with the grown AF cells and produced extracellular matrix on day 21. The amount of type II collagen and its mRNA expression by the scaffold cultured cells were determined using Western blotting and Northern blotting analyses, respectively, and remained at a higher level than in the monolayer cultured cells. Furthermore, glycosaminoglycan (GAG) accumulation in the scaffold culture was at a higher level than in the monolayer culture. Western blot analysis for extracted proteoglycans from the scaffold culture also exhibited a much higher proteoglycan accumulation than the monolayer culture. These results indicate that the AF cells are able to grow and remain phenotypically stable in the scaffold.

Abstract: STUDY DESIGN: Cultured annulus fibrosus cells within an atelocollagen honeycomb-shaped scaffold with a membrane seal were allografted into the lacunas of intervertebral discs of which the nucleus pulposus had been vaporized using an indocyanine green dye-enhanced laser. Regeneration of the intervertebral disc was assessed based on the viability and histologic status of the allografted annulus fibrosus cells, as well as the prevention of narrowing disc space. OBJECTIVES: To study the regeneration of intervertebral disc after laser discectomy using tissue-engineering methods. SUMMARY OF BACKGROUND DATA: Intervertebral disc is the most avascular tissue in the human body, and its ability to regenerate is as low as that of articular cartilage. When nucleotomy is carried out, little regeneration of the annulus fibrosus is observed; consequently, intervertebral disc degeneration is inevitable. METHODS: Annulus fibrosus cells isolated from 20 Japanese white rabbits were labeled with a PKH-26 fluorescent dye and seeded within an atelocollagen honeycomb-shaped scaffold with a membrane seal. Annulus fibrosus cells cultured in atelocollagen honeycomb-shaped scaffold with a membrane seal for 1 week were allografted into the lacunas of intervertebral discs of recipient rabbit of which the nucleus pulposus had been vaporized using an ICG dye-enhanced laser. Soft radiograph photographs of the lumbar spine of these anesthetized rabbits were taken, the disc space measured, and the lumbar spine extracted 2, 4, 8, and 12 weeks after the operation. The proliferation of allografted annulus fibrosus cells with 5-bromo-2'-deoxyuridine/PKH-26 fluorescent labels was assessed using consecutive frozen sections, and safranin-O staining carried out for histologic evaluation. RESULTS: The allografted annulus fibrosus cells were viable and showed proliferation activity with a hyaline-like cartilage being produced. The narrowing of the intervertebral disc space of the cell translation group was significantly prevented, as shown, up to 12 postoperative weeks. CONCLUSION: The annulus fibrosus cells cultured in an ACHMS-scaffold were allografted into the lacunae of nucleus pulposus (obtained using laser vaporization), as well as the hole of annulus fibrosus (obtained by laser fiber insertion) of rabbit intervertebral discs. These cells were viable and showed cell proliferation in the disc tissues of recipients.

Abstract: Application of ultraviolet light (UV-) irradiation to a photocrosslinkable chitosan (Az-CH-LA) aqueous solution resulted in an insoluble, flexible hydrogel like soft rubber within 60 s. The chitosan hydrogel could completely stop bleeding from a cut mouse tail within 30 s of UV-irradiation and could firmly adhere two pieces of sliced skins of mouse to each other. In order to evaluate its accelerating effect on wound healing, full thickness-skin incisions were made on the back of mice and subsequently an Az-CH-LA aqueous solution was added into the wound and irradiated with UV light for 90 s. Application of the chitosan hydrogel significantly induced wound contraction and accelerated wound closure and healing. Histological examinations also have demonstrated an advanced granulation tissue formation and epithelialization in the chitosan hydrogel treated wounds. The chitosan hydrogel due to its accelerating healing ability is considered to become an excellent dressing for wound occlusion and tissue adhesive in urgent hemostasis situations.

Abstract: In most reported cases, a posterior approach to the cervical spine was used to remove an endodermal cyst or to place a shunt for cyst drainage. The authors performed partial excision, shunt insertion, and anterior spinal fusion in a patient with endodermal cyst of the cervical spine. Symptoms resolved after surgery and magnetic resonance images indicated morphologic restoration of the spinal cord.

Abstract: Heparin-carrying polystyrene (HCPS), consisting of low molecular weight heparin chains linked to a synthetic polystyrene core, is able to attach to polymeric surfaces. In this study, HCPS has efficiently bound to collagen-coated micro-plates and collagen membranes thereby retaining the binding of heparin-binding growth factors, such as vascular endothelial growth factor (VEGF)(165) or fibroblast growth factor (FGF)-2. Both human skin fibroblast cells and human umbilical vein endothelial cells have shown a good adherence to both collagen- and HCPS-bound collagen substrata. The growth rate of the fibroblast cells on the HCPS-bound collagen substratum in the presence of low concentrations of FGF-2 is higher than on a collagen surface. The fibroblast cells grow at a significantly higher rate on the HCPS-bound collagen substratum retained with FGF-2. Similarly, the growth rate of the endothelial cells on the HCPS-bound collagen substrata in the presence of low concentrations of either FGF-2 or VEGF(165) is higher than on collagen. The endothelial cells also grow at a significantly higher rate on the HCPS-bound collagen substratum retained with either FGF-2 or VEGF(165). These results indicate that HCPS-bound collagen substrata with various bioactive heparin-binding molecules may provide novel biomaterials controlling cellular activities such as growth and differentiation.

Abstract: BACKGROUND: In various surgical cases, effective tissue adhesives are required for both hemostasis (eg, intraoperative bleeding) and air sealing (eg, thoracic surgery). We have designed a chitosan molecule (Az-CH-LA) that can be photocrosslinked by ultraviolet (UV) light irradiation, thereby forming a hydrogel. The purpose of this work was to evaluate the effectiveness and safety of the photocrosslinkable chitosan hydrogel as an adhesive with surgical applications. METHODS: The sealing ability of the chitosan hydrogel, determined as a bursting pressure, was assessed with removed thoracic aorta, trachea, and lung of farm pigs and in a rabbit model. The carotid artery and lung of rabbits were punctured with a needle, and the chitosan hydrogel was applied to, respectively, stop the bleeding and the air leakage. In vivo chitosan degradability and biologic responses were histologically assessed in animal models. RESULTS: The bursting pressure of chitosan hydrogel (30 mg/mL) and fibrin glue, respectively, was 225 +/- 25 mm Hg (mean +/- SD) and 80 +/- 20 mm Hg in the thoracic aorta; 77 +/- 29 mm Hg and 48 +/- 21 mm Hg in the trachea; and in the lung, 51 +/- 11 mm Hg (chitosan hydrogel), 62 +/- 4 mm Hg (fibrin glue, rubbing method), and 12 +/- 2 mm Hg (fibrin glue, layer method). The sealing ability of the chitosan hydrogel was stronger than that of fibrin glue. All rabbits with a carotid artery (n = 8) or lung (n = 8) that was punctured with a needle and then sealed with chitosan hydrogel survived the 1-month observation period without any bleeding or air leakage from the puncture sites. Histologic examinations demonstrated that 30 days after application, a fraction of the chitosan hydrogel was phagocytosed by macrophages, had partially degraded, and had induced the formation of fibrous tissues around the hydrogel. CONCLUSIONS: A newly developed photocrosslinkable chitosan has demonstrated strong sealing ability and a great potential for use as an adhesive in surgical operations.

Abstract: Application of ultraviolet light irradiation to a photocrosslinkable chitosan aqueous solution resulted in an insoluble, flexible hydrogel like soft rubber within 60 seconds. In order to evaluate its accelerating effect on wound healing, full-thickness skin incisions were made on the backs of mice and subsequently a photocrosslinkable chitosan aqueous solution was added into the wound and irradiated with UV light for 90 seconds. Application of the chitosan hydrogel significantly induced wound contraction and accelerated wound closure and healing compared with the untreated controls. Histological examination also showed an advanced contraction rate on the first 2 days and tissue fill rate on days 2 to 4 in the chitosan hydrogel-treated wounds. Furthermore, in cell culture studies, chitosan hydrogel culture medium supplemented with 5% fetal-bovine serum was found to be chemoattractant for human dermal fibroblasts in an invasion chamber assay using filters coated with Matrigel and in a cell migration assay. Due to its ability to accelerate wound contraction and healing, chitosan hydrogel may become accepted as an occlusive dressing for wound management.

Abstract: BACKGROUND AND OBJECTIVE: The lasers used today for Percutaneous Laser Disc Decompression (PLDD) can not selectively ablate the nucleus pulposus (NP). We hypothesized that if indocyanine green dye were injected into the NP, 805 nm diode laser irradiation would result in selective and safe removal of NP tissue without damaging nearby tissues. STUDY DESIGN/MATERIALS AND METHODS: Twelve beagle dogs were used for three experiments, i.e., determination of attenuation coefficients of NP and AF, determination of weight of NP before and after laser irradiation, and histopathological study. RESULTS: The attenuation coefficient at 805 nm of NP which had been homogenized in the presence of ICG was 2521.3/cm. Upon application of the diode laser at a power of 1, 3, or 5 W (the intensity of laser irradiation: 0.353, 1.061, or 1.768 x 10(3) W/cm(2)) to NPs into which ICG had been injected, the weight of the NPs decreased by a mean 20, 45, and 65%, respectively. Macroscopic and microscopic examination of the discs after PLDD showed that only the NP where the tissues were stained by ICG were removed. CONCLUSIONS: These results indicated that the combined use of ICG and diode laser irradiation effectively and selectively ablates the NP with low laser power.

Abstract: STUDY DESIGN: With the heterogeneity of the intervertebral disc as the focus, intervertebral discs from normal young rabbits were separated into nucleus pulposus (NP), inner anulus fibrosus (IAF), and outer anulus fibrosus (OAF) zones. Disc cells from each zone were isolated and propagated under monolayer and within agarose gel culture. The metabolism of these cultured disc cells was examined in terms of glycosaminoglycan (GAG) accumulation. OBJECTIVES: The object was to study the metabolism of disc cells derived from each zone and characterize them on the basis of GAG accumulation and composition. SUMMARY OF BACKGROUND DATA: It has been shown that three-dimensional culture systems, such as within-agarose gels or in alginate beads, permit long-term maintenance of the articular chondrocyte phenotype in vitro. However, little has been reported on how the metabolism of intervertebral disc cells, especially GAG accumulation, is affected by different culture conditions. METHODS: Cells from each zone were subjected to monolayer or three-dimensional culture for up to 12 days. GAG accumulation in the different culture systems was analyzed using chemical, histologic, and immunohistologic methods. Differences of GAG and DNA content among NP, IAF, and OAF cells were statistically evaluated by analysis of variance. The data of keratin sulfate content in three-dimensional culture were compared with that in monolayer culture using nonparametric Mann-Whitney U test. RESULTS: Monolayer culture revealed that increases in GAG content were significantly higher in IAF cells than in OAF cells. However, in three-dimensional culture GAG content was similar in the two groups. AF cells in three-dimensional cultures showed immunohistochemical localization of chondroitin sulfate and keratan sulfate, suggesting the existence of pericellular matrix. High performance liquid chromatography confirmed the expression of keratan sulfate in cultured cells. CONCLUSIONS: GAG accumulation in cultures of cells from different zones of the intervertebral disc varied according to the culture conditions used. The importance of choosing the appropriate culture systems to meet the objectives of a study should be emphasized.