Increased migratory activity and cartilage regeneration by superficial-zone chondrocytes in enzymatically treated cartilage explants.

BACKGROUND: Limited chondrocyte migration and impaired cartilage-to-cartilage healing is a barrier in cartilage regenerative therapy. Collagenase treatment and delivery of a chemotactic agent may play a positive role in chondrocyte repopulation at the site of cartilage damage. This study evaluated chondrocyte migratory activity after enzymatic treatment in cultured cartilage explant. Differential effects of platelet-derived growth factor (PDGF) dimeric isoforms on the migratory activity were investigated to define major chemotactic factors for cartilage. METHODS: Full-thickness cartilage (4-mm3 blocks) were harvested from porcine femoral condyles and subjected to explant culture. After 15 min or 60 min of actinase and collagenase treatments, chondrocyte migration and infiltration into a 0.5-mm cartilage gap was investigated. Cell morphology and lubricin, keratan sulfate, and chondroitin 4 sulfate expression in superficial- and deep-zone chondrocytes were assessed. The chemotactic activities of PDGF-AA, -AB, and -BB were measured in each zone of chondrocytes, using a modified Boyden chamber assay. The protein and mRNA expression and histological localization of PDGF-ß were analyzed by western blot analysis, real-time reverse transcription polymerase chain reaction (RT-PCR), and immunohistochemistry, and results in each cartilage zone were compared. RESULTS: Superficial-zone chondrocytes had higher migratory activity than deep-zone chondrocytes and actively bridged the cartilage gap, while metachromatic staining by toluidine blue and immunoreactivities of keratan sulfate and chondroitin 4 sulfate were detected around the cells migrating from the superficial zone. These superficial-zone cells with weak immunoreactivity for lubricin tended to enter the cartilage gap and possessed higher migratory activity, while the deep-zone chondrocytes remained in the lacuna and exhibited less migratory activity. Among PDGF isoforms, PDGF-AB maximized the degree of chemotactic activity of superficial zone chondrocytes. Increased expression of PDGF receptor-ß was associated with higher migratory activity of the superficial-zone chondrocytes. CONCLUSIONS: In enzymatically treated cartilage explant culture, chondrocyte migration and infiltration into the cartilage gap was higher in the superficial zone than in the deep zone. Preferential expression of PDGF receptor-ß combined with the PDGF-AB dimeric isoform may explain the increased migratory activity of the superficial-zone chondrocytes. Cells migrating from superficial zone may contribute to cartilage regeneration.

Compressive mechanical stress enhances susceptibility to interleukin-1 by increasing interleukin-1 receptor expression in 3D-cultured ATDC5 cells.

BACKGROUND: Mechanical overload applied on the articular cartilage may play an important role in the pathogenesis of osteoarthritis. However, the mechanism of chondrocyte mechanotransduction is not fully understood. The purpose of this study was to assess the effects of compressive mechanical stress on interleukin-1 receptor (IL-1R) and matrix-degrading enzyme expression by three-dimensional (3D) cultured ATDC5 cells. In addition, the implications of transient receptor potential vanilloid 4 (TRPV4) channel regulation in promoting effects of compressive mechanical loading were elucidated. METHODS: ATDC5 cells were cultured in alginate beads with the growth medium containing insulin-transferrin-selenium and BMP-2 for 6 days. The cultured cell pellet was seeded in collagen scaffolds to produce 3D-cultured constructs. Cyclic compressive loading was applied on the 3D-cultured constructs at 0.5 Hz for 3 h. The mRNA expressions of a disintegrin and metalloproteinases with thrombospondin motifs 4 (ADAMTS4) and IL-1R were determined with or without compressive loading, and effects of TRPV4 agonist/antagonist on mRNA expressions were examined. Immunoreactivities of reactive oxygen species (ROS), TRPV4 and IL-1R were assessed in 3D-cultured ATDC5 cells. RESULTS: In 3D-cultured ATDC5 cells, ROS was induced by cyclic compressive loading stress. The mRNA expression levels of ADAMTS4 and IL-1R were increased by cyclic compressive loading, which was mostly prevented by pyrollidine dithiocarbamate. Small amounts of IL-1ß upregulated ADAMTS4 and IL-1R mRNA expressions only when combined with compressive loading. TRPV4 agonist suppressed ADAMTS4 and IL-1R mRNA levels induced by the compressive loading, whereas TRPV4 antagonist enhanced these levels. Immunoreactivities to TRPV4 and IL-1R significantly increased in constructs with cyclic compressive loading. CONCLUSION: Cyclic compressive loading induced mRNA expressions of ADAMTS4 and IL-1R through reactive oxygen species. TRPV4 regulated these mRNA expressions, but excessive compressive loading may impair TRPV4 regulation. These findings suggested that TRPV4 regulates the expression level of IL-1R and subsequent IL-1 signaling induced by cyclic compressive loading and participates in cartilage homeostasis.

High-molecular-weight Hyaluronan Administration Inhibits Bone Resorption and Promotes Bone Formation in Young-age Osteoporosis Rats.

Osteoporosis poses a significant global health concern, affecting both the elderly and young individuals, including athletes. Despite the development of numerous antiosteoporotic drugs, addressing the unique needs of young osteoporosis patients remains challenging. This study focuses on young rats subjected to ovariectomy (OVX) to explore the impact of high-molecular-weight hyaluronan (HA) on preventing OVX-induced osteoporosis. Twenty-four rats underwent OVX, while 12 underwent sham procedures (sham control group). Among the OVX rats, half received subcutaneous injections of HA (MW: 2700 kDa) at 10 mg/kg/week into their backs (OVX-HA group), whereas the other half received saline injections (0.5 ml/week) at the same site (OVX-saline group). OVX-HA group exhibited significantly higher percentages of osteoclast surface (Oc. S/BS), osteoblast surface per bone surface (Ob. S/BS), and bone volume/tissue volume (BV/TV) compared with OVX-saline group at the same age. The proportions of Ob. S/BS and BV/TV in the OVX-HA group closely resembled those of the sham control group, whereas the proportion of Oc. S/BS in the OVX-HA group was notably higher than that in the sham control group. In summary, the administration of HA significantly mitigated bone resorption and enhanced bone formation, suggesting a crucial role for HA in the treatment of young adult osteoporosis.

Overexpression of Interleukin-1α Suppresses Liver Metastasis of Lymphoma: Implications for Antitumor Effects of CD8+ T-cells.

Interleukin (IL)-1 plays a key role in carcinogenesis, tumor progression, and metastasis. Although IL-1 may enhance the expansion of CD8+ T-cells, the pathological contribution of IL-1-activated CD8+ T-cells to tumor metastasis remains unclear. This study used a liver metastasis model of the EL4 T-cell lymphoma cells transplanted into human IL (hIL)-1α conditional transgenic (hIL-1α cTg) mice. Overproduction of hIL-1α suppressed both macroscopic and histological liver metastasis of EL4 T-cell lymphoma. The hIL-1α-induced inflammatory state increased the number of CD8+ T-cells both within and around metastatic tumors. Moreover, larger numbers of CD8+ T-cells showed greater infiltration of liver blood vessels in hIL-1α cTg mice than in control wild-type mice. Terminal deoxynucleotidyl transferase dUTP nick-end labeling staining of liver tissue from hIL-1α cTg mice indicated increased apoptosis of cells in the tumor. Localization of apoptosis cells resembled that of CD8+ T-cells. In addition, cytotoxicity assay showed that CD8+ T-cell counts from tumor-bearing hIL-1α cTg mice correlated with cytotoxicity against EL4. In summary, IL-1α suppresses lymphoma metastasis, and IL-1α-activated CD8+ T-cells may play important roles in inhibiting both tumor metastasis and metastatic tumor growth.

Adipose-Derived Stem Cell Sheets Improve Early Biomechanical Graft Strength in Rabbits After Anterior Cruciate Ligament Reconstruction.

BACKGROUND: Although various reconstruction techniques are available for anterior cruciate ligament (ACL) injuries, a long recovery time is required before patients return to sports activities, as the reconstructed ACL requires time to regain strength. To date, several studies have reported use of mesenchymal stem cells in orthopaedic surgery; however, no studies have used adipose-derived stem cell (ADSC) sheets in ACL reconstruction (ACLR). HYPOTHESIS: ADSC sheet transplantation can improve biomechanical strength of the autograft used in ACLR. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 68 healthy Japanese white rabbits underwent unilateral ACLR with a semitendinosus tendon autograft after random enrollment into a control group (no sheet; n = 34) and a sheet group (ADSC sheet; n = 34). At 2, 4, 8, 16, and 24 weeks after surgery, rabbits in each group were sacrificed to evaluate tendon-bone healing using histological staining, micro-computed tomography, and biomechanical testing. At 24 weeks, scanning transmission electron microscopy of the graft midsubstance was performed. RESULTS: The ultimate failure load for the control and sheet groups, respectively, was as follows: 17.2 ± 5.5 versus 37.3 ± 10.3 (P = .01) at 2 weeks, 28.6 ± 1.9 versus 47.4 ± 10.4 (P = .003) at 4 weeks, 53.0 ± 14.3 versus 48.1 ± 9.3 (P = .59) at 8 weeks, 66.2 ± 9.3 versus 95.2 ± 43.1 (P = .24) at 16 weeks, and 66.7 ± 27.3 versus 85.3 ± 29.5 (P = .39) at 24 weeks. The histological score was also significantly higher in the sheet group compared with the control group at early stages up to 8 weeks. On micro-computed tomography, relative to the control group, the bone tunnel area was significantly narrower in the sheet group at 4 weeks, and the bone volume/tissue volume of the tendon-bone interface was significantly greater at 24 weeks. Scanning transmission electron microscopy at 24 weeks indicated that the mean collagen fiber diameter in the midsubstance was significantly greater, as was the occupation ratio of collagen fibers per field of view, in the sheet group. CONCLUSION: ADSC sheets improved biomechanical strength, prevented bone tunnel enlargement, and promoted tendon-bone interface healing and graft midsubstance healing in an in vivo rabbit model. CLINICAL RELEVANCE: ADSC sheets may be useful for early tendon-bone healing and graft maturation in ACLR.

Temporal effects of cyclic stretching on distribution and gene expression of integrin and cytoskeleton by ligament fibroblasts in vitro.

Cyclic stretching is pivotal to maintenance of the ligaments. However, it is still not clear when ligament fibroblasts switch on expression of genes related to the mechanotransduction pathway in response to cyclic stretching. This in vitro study investigated, using ligament fibroblasts, the time-dependent changes in distribution and gene expression of beta1 integrin, the cytoskeleton, and collagens after the application of 6% cyclic stretching at a frequency of 0.1 Hz for 3 hr on silicon membranes. We carried out confocal laser scanning microscopy to demonstrate changes in distribution of these components as well as quantitative real-time RT-PCR to quantify levels of these gene expression both during application of cyclic stretching and at 0, 2, 6, 12, and 18 hr after the termination of stretching. Control (unstretched) cells were used at each time point. Within 1 hr of the application of stretching, the fibroblasts and their actin stress fibers became aligned in a direction perpendicular to the major axis of stretch, whereas control (unstretched) cells were randomly distributed. In response to cyclic stretching, upregulation of actin at the mRNA level was first observed within 1 hr after the onset of stretching, while upregulation of beta1 integrin and type I and type III collagens was observed between 2 and 12 hr after the termination of stretching. These results indicate that the fibroblasts quickly modify their morphology in response to cyclic stretching, and subsequently they upregulate the expression of genes related to the mechanotransduction pathway mainly during the resting period after the termination of stretching.

Rapid 3-dimensional imaging of embryonic craniofacial morphology using microscopic computed tomography.

OBJECTIVE: Microscopic computed tomography (microCT) has been recently applied to morphological evaluation of mouse embryos with or without congenital malformations, and 3-dimensional (3D) digital images of the whole embryo can be obtained. In the present study, the authors report a modified, rapid technique of 3D embryonic microCT without processing with osmium tetroxide. METHODS: Normal embryonic days 10.5 to 11 mouse embryos, as well as those with craniofacial anomalies treated with teratogens, were examined. After fixation, we processed the embryo samples with hexamethyldisilazane, instead of highly toxic osmium tetroxide in the original method. RESULTS: Our protocol enabled clear 3D craniofacial imaging of the normal and anomalous mouse embryos within a short period of 20 minutes or 1 hour. In addition, some anatomical landmarks were clearly detected in the reconstituted craniofacial section images. CONCLUSION: Our present data suggest a possible role of microCT for high-throughput morphological screening of the mouse embryos with craniofacial anomalies.

Upregulated expression of inducible nitric oxide synthase plays a key role in early apoptosis after anterior cruciate ligament injury.

The reason that the anterior cruciate ligament (ACL) has a very poor healing potential after injury is not well understood. In this study, we investigated the role of nitric oxide (NO) in the apoptotic cell death of ACL cells using a rabbit model and in vitro cell culture. The apoptosis of ACL cells in vivo was analyzed by TUNEL assay and electron microscopy. NO synthase (NOS) expression was observed by immunohistochemical analysis. ACL cells were cultured and the susceptibility to NO-induced apoptosis was tested. Inducible NOS (iNOS) expression after treatment with cytokines was examined by immunohistochemical and RT-PCR analyses. Mitogen-activated protein kinase (MAPK) inhibitors were used for the analysis of downstream signals. A significant number of apoptotic cells were observed on days 1 to 3 after injury; the apoptotic rate returned to the control level by day 7. Upregulation of iNOS in the ACL remnant was observed at day 1. Intraarticular injection of NOS inhibitor suppressed the apoptotic rate. Isolated ACL cells showed much higher susceptibility to NO-induced apoptosis than did medial collateral ligament cells. IL-1beta stimulated ACL cells to upregulate iNOS mRNA and increase NO production. p38 MAPK inhibitor decreased NO-induced apoptosis. Rapid iNOS induction after injury contributes to the high apoptotic rate of ACL cells, and this may partly account for the poor healing capacity of this ligament. iNOS and NO production is suggested to be stimulated by IL-1beta, and NO activates the p38 MAPK pathway and triggers an apoptotic signal in ACL cells.

Anterior cruciate reconstruction with bioactive Leeds-Keio ligament (LKII): preliminary report.

The scaffold type Leeds-Keio artificial ligament (LK) for ligament reconstruction of the knee provides sufficient strength to require minimal sacrifice of autogenous tissue. This implant works not only as prosthesis but also as scaffold onto which natural tissue grows from the synovium. LK has been in clinical use for anterior cruciate ligament (ACL) reconstruction since 1982, although the operative procedure has been modified and has undergone significant development since that time. Recently, we developed radio frequency-generated glow discharge (RFGGD)-treated LK ligament (LKII, previously indicated as Bio-LK) to improve tissue induction and reported that cell proliferation and cell attachment to artificial fibers increased considerably with this hydrophilic treatment. In this study, we report the findings of reconstructed ACL using LKII, which has been in clinical use since 2003. At reconstruction, LKII was covered with the remnant of the original ACL as much as possible in order to preserve the nerve ending system. Thirteen cases with over 12 months' postoperative history were reviewed. Knee stability was regained after reconstruction without any complications such as joint effusion and chronic synovitis. In one case, postoperative arthroscopy showed that the reconstructed ACL was completely covered with newly formed tissue at 8 weeks postoperatively. Biopsy revealed abundant fibroblasts, collagenous fibers, and vessels around the artificial fibers without marked inflammatory findings. Transmission electron microscope study showed abundant thin collagen fibers, which demonstrated regular orientation to some extent. Fibroblasts were observed with extensive amount of rough endoplasmic reticulum. According to these results, we consider LKII to be superior to LK in tissue induction and maturation.

Usefulness of photoacoustic measurements for evaluation of biomechanical properties of tissue-engineered cartilage.

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.

An atelocollagen honeycomb-shaped scaffold with a membrane seal (ACHMS-scaffold) for the culture of annulus fibrosus cells from an intervertebral disc.

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.

Regional differences of type II collagen synthesis in the human temporomandibular joint disc: immunolocalization study of carboxy-terminal type II procollagen peptide (chondrocalcin).

The purpose of this study was to determine the regional differences of distribution of the carboxy-terminal type II procollagen peptide (pCOL-II-C; chondrocalcin) as markers of cartilaginous expression in the human temporomandibular joint (TMJ) disc. Twelve human TMJ discs without morphologic abnormalities were obtained from 12 fresh cadavers. All specimens were analysed for pCOL-II-C expression using polyclonal rabbit anti-human pCOL-II-C antibody in avidin-biotin-peroxidase complex staining. The results were demonstrated that the percentage of pCOL-II-C immunoreactive disc cells was significantly higher in the outer part (the articular surfaces) than in the inner part (the deep central areas) of the disc. These findings suggest that the tissue heterogeneity of cartilaginous expression reflects the functional demands of the remodelling process in the human TMJ disc.

Comparative study of the covered area of Leeds-Keio (LK) artificial ligament and radio frequency generated glow discharge treated Leeds-Keio (Bio-LK) ligament with synovial cells.

The Leeds-Keio artificial ligament (LK), which was developed not only as a ligament substitute but also as a scaffold for tissue induction in knee ligament reconstruction, has been in clinical use since 1982 in Europe and Japan. Recently, we have developed radio frequency generated glow discharge (RFGD)-treated LK ligament (Bio-LK) to expedite the process of tissue induction and its maturation. In this study of cell adhesion to the scaffold, we report the difference in the covered area with synovial cells when using scaffolds made from treated and untreated materials. Plasma clot methods were used in this study. The covered area on LK and Bio-LK by cells was stained by 0.1% toluidine blue and analyzed using NIH image. The covered area of Bio-LK was about three times higher than that of LK (untreated) at 3 weeks. In scanning electron microscopy, more cells were observed on fibers of Bio-LK, and these filled the space among the fibers more extensively. The spreading of covered area means that cell attachment, cell proliferation, and cell migration on the fibers are likely to be improved. Our experimental study indicates that Bio-LK will possibly speed up the process of induction of autogenous tissue from synovium.

Skeletal unloading induces a full-thickness patellar cartilage defect with increase of urinary collagen II CTx degradation marker in growing rats.

Mechanical stress plays an important role in tissue morphogenesis and extracellular matrix metabolism. However, little is known about the effects of reduced loading without restriction of joint motion on the patella. We investigated the effects of long-term skeletal unloading on patellar cartilage and subchondral bone and systemic collagen II metabolism. Nine-week-old male F344/N rats (n=128) were randomly divided into two groups: caged control (C) and tail suspended (TS). Hindlimbs of the TS rats were subjected to unloading for up to 12 weeks. Sequential changes in the patellar cartilage and subchondral bone were analyzed macroscopically, by pathological findings and histomorphologically. All animals received double tidemark fluorochrome labeling prior to sacrifice. Glycosaminoglycan (GAG) content in patellar cartilage, cross-linked C-telopeptide of type II collagen (CTx-II) in 24-h urine and type II procollagen-C-peptide (pCol-II-C) in sera were also measured by DMB assay, ELISA and EIA, respectively. In the TS group, GAG content was significantly reduced only during the first 3 weeks. No further significant decrease was found. Alkaline phosphatase (ALP) activity was increased, especially at the deep zone. Tidemark mineral apposition rate (MAR) was temporally increased, which resulted in an increase in the ratio of calcified cartilage to the entire cartilage. In the medial part, in addition, thickness of the entire cartilage was decreased by temporal acceleration of subchondral ossification advancement and, in the medial margin, a full-thickness cartilage defect was revealed in 88.6% of TS rats. However, the remaining articular surface was free from fibrillation. While urinary CTx-II was significantly increased during the experimental periods, serum pCol-II-C was significantly decreased at the early phase. There were significant correlations between the urinary CTx-II levels and tidemark MAR. Our results provided evidence that skeletal unloading increased ALP activity at the deep zone and temporally accelerated tidemark advancement associated with a decrease in proteoglycan content. In addition, skeletal unloading temporally accelerated subchondral ossification advancement in the medial part of the patella and finally induced a full-thickness patellar cartilage defect without any fibrillation at the remaining articular surface by additional subchondral bone modeling and possible retarded cartilage growth, which was through a different mechanism than overloading.

Differential sensitivity to NO-induced apoptosis between anterior cruciate and medial collateral ligament cells.

It is well known that the anterior cruciate ligament (ACL) of the knee joint has poorer healing responses than the medial collateral ligament (MCL). Nitric oxide (NO) induces free radicals and plays a key role in the induction of apoptosis in various wound-healing models. We hypothesized that the poor healing response of the ACL may be ascribed to high susceptibility to apoptosis, and we investigated the difference in susceptibility to apoptosis between ACL and MCL cells after treatment with sodium nitroprusside, a NO donor. Apoptosis was evaluated by phase contrast microscopy, electron microscopy, DNA gel electrophoresis, and flow cytometric analysis. Although morphological changes and DNA ladders were observed in both ACL and MCL cells after 2 mM sodium nitroprusside treatment, ACL cells were more prone to apoptosis at 1 mM. Based on flow cytometric analysis, DNA fragmentation at 1 mM sodium nitroprusside was significantly greater in ACL cells than in MCL cells (58.6% +/- 1.6% vs. 11.9% +/- 2.2%). Caspase-3 inhibitor (Ac-Asp-Glu-Val-Asp-CHO) and caspase-9 inhibitor (Ac-Leu-Glu-His-Asp-CHO) completely inhibited this DNA fragmentation. In conclusion, the ACL and MCL cells exhibit essential differences, and the differential sensitivity to NO-induced apoptosis between the ACL and MCL cells may be a reflection of these differences.

Type II collagen synthesis in the articular cartilage of a rabbit model of osteoarthritis: expression of type II collagen C-propeptide and mRNA especially during early-stage osteoarthritis.

BACKGROUND: The aim of this study was to observe time course changes in type II collagen synthesis in various regions of articular cartilage affected with osteoarthritis (OA) by examining the expression of type II collagen C-propeptide (pCOL II-C) and mRNA in a rabbit OA model. METHODS: Osteoarthritis was experimentally induced by partial lateral meniscectomy in the knees of Japanese white rabbits. The cartilage of the animals was then examined histologically over time. The degenerative area of articular cartilage was divided into three areas, according to the degree of degeneration. The ability to synthesize type II collagen was estimated by the immunohistological staining of pCOL II-C and the in situ hybridization of mRNA in type II collagen. RESULTS: The positive rate of pCOL II-C immunostaining in chondrocytes was highest in the central-degenerative region 1 week after surgery, and the highest rate in the para-degenerative region was observed 2 and 4 weeks after surgery. The percentage of pCOL II-C positive cells increased as the histological degeneration score increased to moderate degeneration and then decreased with further progression of the severity of cartilage degeneration. Examination by in situ hybridization revealed that the regions marked by strong pCOL II-C mRNA expression were similar to those indicated by the immunohistology results. CONCLUSIONS: These results suggest that the type II collagen-synthesizing potential of chondrocytes is highest in moderately degenerated areas of OA articular cartilage. Cartilage repair continues to be seen even as OA advances, although the reaction varies depending on the stage of OA.

Overexpression of the human interleukin 1a gene causes osteopenia in mice.

OBJECTIVE: Osteoporosis is a major complication of chronic inflammatory diseases such as rheumatoid arthritis (RA). We describe disordered bone metabolism in transgenic mice that overexpress human interleukin 1a (hIL-1a). METHODS: Bone mineral density (BMD), microcomputed tomography (microCT), histomorphometry, and blood biochemical data of hIL-1a transgenic mice and littermate wild-type mice were examined. RESULTS: The femoral BMD of transgenic mice was decreased by 27.7% compared with wild-type mice. microCT revealed a marked reduction in the trabecular bone, and cortical thinning with an enlarged cavity was observed in femora of transgenic mice. Histomorphometric analysis revealed inhibition of several measures of bone formation, while the serum alkaline phosphatase level was reduced in transgenic mice; however, their indices of bone resorption were not elevated. CONCLUSION: Overexpression of hIL-1a causes osteopenia in mice. It was suggested that the systemic osteopenia in these transgenic mice occurred primarily as a result of decreased bone formation, with a reduction of bone mineralization rather than increased osteoclastic bone resorption. This may be one aspect of bone metabolism in RA that results in disease complications.

An experimental study of the regeneration of the intervertebral disc with an allograft of cultured annulus fibrosus cells using a tissue-engineering method.

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.

Membrane-associated IL-1 contributes to chronic synovitis and cartilage destruction in human IL-1 alpha transgenic mice.

IL-1 molecules are encoded by two distinct genes, IL-1alpha and IL-1beta. Both isoforms possess essentially identical activities and potencies, whereas IL-1alpha, in contrast to IL-1beta, is known to act as a membrane-associated IL-1 (MA-IL-1) and plays an important role in a variety of inflammatory situations. The transgenic (Tg) mouse line (Tg1706), which was generated in our laboratory, overexpresses human IL-1alpha (hIL-1alpha) and exhibits a severe arthritic phenotype characterized by autonomous synovial proliferation with subsequent cartilage destruction. Because the transgene encoded Lys(64) to Ala(271) of the hIL-1alpha amino acid sequence, Tg mice may overproduce MA-IL-1 as well as soluble IL-1alpha. The present study investigated whether MA-IL-1 contributes to synovial proliferation and cartilage destruction in the development of arthritis. Flow cytometric analysis revealed that both macrophage-like and fibroblast-like synoviocytes constitutively produce MA-IL-1. D10 cell proliferation assay revealed MA-IL-1 bioactivity of paraformaldehyde-fixed synoviocytes and the further induction of endogenous mouse MA-IL-1 via autocrine mechanisms. MA-IL-1 expressed on synoviocytes triggered synoviocyte self-proliferation through cell-to-cell (i.e., juxtacrine) interactions and also promoted proteoglycan release from the cartilage matrix in chondrocyte monolayer culture. Interestingly, the severity of arthritis was significantly correlated with MA-IL-1 activity rather than with soluble IL-1alpha activity or concentration of serum hIL-1alpha. Moreover, when the Tg1706 line was compared with the Tg101 line, which selectively overexpresses the 17-kDa mature hIL-1alpha, the severity of arthritis was significantly higher in the Tg1706 line than in the Tg101 line. These results suggest that MA-IL-1 contributes to synoviocyte self-proliferation and subsequent cartilage destruction in inflammatory joint disease such as rheumatoid arthritis.

Distribution of type VI collagen in chondrocyte microenvironment: study of chondrons isolated from human normal and degenerative articular cartilage and cultured chondrocytes.

The chondron is the microanatomical unit composed of a chondrocyte and its pericellular microenvironment (PCME), including the pericellular matrix and capsule. In the present study, we extracted chondrons from human articular cartilages and investigated the relationship between the distribution of the matrix molecules, including type VI collagen, and the degeneration of articular cartilage. We also investigated the effects of interleukin-1beta (IL-1beta) and transforming growth factor beta-1 (TGF-beta1) on the distribution of type VI collagen in cultured chondrocytes. Chondrons were extracted by low-speed homogenization from cartilage pieces obtained from forensic autopsies and from patients with knee osteoarthritis (OA) undergoing total knee arthroplasty. Cartilage sections were classified into three groups (normal, slight degeneration, and moderate degeneration) based on the degree of degeneration according to Mankin's score. Extracted chondrons were immunostained, and the distribution of the matrix molecules, including type VI collagen, was investigated using a confocal laser scanning microscope (CLSM). The chondrocytes isolated by enzymic treatment were subjected to three-dimensional culture in agarose gel and then treated with IL-1beta or TGF-beta1. The distribution of newly synthesized type VI collagen in agarose gel was also investigated using the CLSM. Type VI collagen was localized specifically within the PCME of chondrons. The volume ratio of PCME to chondrocyte (P/C ratio) was significantly higher in the moderate degeneration group than in the other two groups. The accumulation of type VI collagen around a chondrocyte was obviously increased by the addition of TGF-beta1. The P/C ratio significantly increased as the severity of the OA progressed, suggesting that type VI collagen distributed specifically in the PCME was playing a protective role for chondrocytes by maintaining the pericellular microenvironment in OA.