The Effect of Bone Morphogenetic Protein-2 on the Irradiated Allogenic Cartilage of Rats.

ABSTRACT: Autogenous cartilage graft is associated with the problem of chondrocyte dedifferentiation. Bone morphogenetic protein-2 (BMP-2) plays an important role in the differentiation and matrix maturation of chondrocytes, and preventing their dedifferentiation.This study was performed on 48 rats, divided equally into 3 groups. In group I, the xiphoid process cartilage was harvested and irradiated, and broken into 2 pieces. Each piece was implanted on the back. The same process was performed in Groups II and III, but further treated with BMP-2, Group II with 25 µg, and Group III with 50 µg. The implanted cartilage pieces were reharvested at postoperative weeks 2 and 4. The weight change was measured and histological evaluation was performed.The extent of the weight change was higher in Groups II and III than in Group I. The extracellular matrix between the chondrocytes showed increased in Groups II and III. The fibrous tissue on the surface of the cartilage increased in Groups II and III. Ossification of the chondrocytes was observed in Groups II and III.The use of BMP-2 increased the matrix between chondrocytes and the fibrous tissue of the cartilage and facilitated the ossification of chondrocytes. The effect of BMP-2 increased with its increasing concentration, and maintenance of its effectiveness over time was confirmed.

Biophysical stimulation of bone and cartilage: state of the art and future perspectives.

INTRODUCTION: Biophysical stimulation is a non-invasive therapy used in orthopaedic practice to increase and enhance reparative and anabolic activities of tissue. METHODS: A sistematic web-based search for papers was conducted using the following titles: (1) pulsed electromagnetic field (PEMF), capacitively coupled electrical field (CCEF), low intensity pulsed ultrasound system (LIPUS) and biophysical stimulation; (2) bone cells, bone tissue, fracture, non-union, prosthesis and vertebral fracture; and (3) chondrocyte, synoviocytes, joint chondroprotection, arthroscopy and knee arthroplasty. RESULTS: Pre-clinical studies have shown that the site of interaction of biophysical stimuli is the cell membrane. Its effect on bone tissue is to increase proliferation, synthesis and release of growth factors. On articular cells, it creates a strong A2A and A3 adenosine-agonist effect inducing an anti-inflammatory and chondroprotective result. In treated animals, it has been shown that the mineralisation rate of newly formed bone is almost doubled, the progression of the osteoarthritic cartilage degeneration is inhibited and quality of cartilage is preserved. Biophysical stimulation has been used in the clinical setting to promote the healing of fractures and non-unions. It has been successfully used on joint pathologies for its beneficial effect on improving function in early OA and after knee surgery to limit the inflammation of periarticular tissues. DISCUSSION: The pooled result of the studies in this review revealed the efficacy of biophysical stimulation for bone healing and joint chondroprotection based on proven methodological quality. CONCLUSION: The orthopaedic community has played a central role in the development and understanding of the importance of the physical stimuli. Biophysical stimulation requires care and precision in use if it is to ensure the success expected of it by physicians and patients.

Laser surface modification of decellularized extracellular cartilage matrix for cartilage tissue engineering.

The implantation of autologous cartilage as the gold standard operative procedure for the reconstruction of cartilage defects in the head and neck region unfortunately implicates a variety of negative effects at the donor site. Tissue-engineered cartilage appears to be a promising alternative. However, due to the complex requirements, the optimal material is yet to be determined. As demonstrated previously, decellularized porcine cartilage (DECM) might be a good option to engineer vital cartilage. As the dense structure of DECM limits cellular infiltration, we investigated surface modifications of the scaffolds by carbon dioxide (CO2) and Er:YAG laser application to facilitate the migration of chondrocytes inside the scaffold. After laser treatment, the scaffolds were seeded with human nasal septal chondrocytes and analyzed with respect to cell migration and formation of new extracellular matrix proteins. Histology, immunohistochemistry, SEM, and TEM examination revealed an increase of the scaffolds' surface area with proliferation of cell numbers on the scaffolds for both laser types. The lack of cytotoxic effects was demonstrated by standard cytotoxicity testing. However, a thermal denaturation area seemed to hinder the migration of the chondrocytes inside the scaffolds, even more so after CO2 laser treatment. Therefore, the Er:YAG laser seemed to be better suitable. Further modifications of the laser adjustments or the use of alternative laser systems might be advantageous for surface enlargement and to facilitate migration of chondrocytes into the scaffold in one step.

Wave velocities in articular cartilage measured by micro-Brillouin scattering technique.

Micro-Brillouin scattering was used to measure gigahertz ultrasonic wave velocities in the articular cartilage of a bovine femur. Velocities propagating parallel to the surface of the subchondral bone were 3.36-3.83 × 103 m/s in a dry cartilage sample. Anisotropy measurements were also performed in a 10-µm-diameter local area of the cartilage matrix. A weak velocity anisotropy reflected characteristics of the layers. The velocity also depended on the water content. In the middle layer, the velocity in the dry sample was 3.58 × 103 m/s, whereas that for a fully wet sample was 2.04 × 103 m/s.

The Potential Application of Pulsed Ultrasound on Bone Defect Repair via Developmental Engineering: An In Vitro Study.

Repairing bone defect by recapitulation of endochondral bone formation, known as developmental engineering, has been a promising approach in bone tissue engineering. The critical issue in this area is how to effectively construct the hypertrophic cartilaginous template in vitro and enhance in vivo endochondral ossification process after implantation. Pulsed ultrasound stimulation has been widely used in the clinic for accelerating bone healing in fractures and nonunions. The aim of this study was to investigate whether ultrasound (US) could accelerate in vitro chondrogenesis and the hypertrophic process in certain microenvironments. Rat bone marrow mesenchymal stem cells were chondrogenic or hypertrophic differentiated in a three-dimensional pellet culture system with different media, and treated with different intensities of US. US exposure promoted chondrogenic differentiation of stem cells and inhibited their transition into the hypertrophic stage in a chondrogenic-friendly microenvironment. US significantly advanced hypertrophic differentiation of bone marrow stem cell pellets in hypertrophic medium after chondrogenesis. Our data indicated that pulsed US promoted in vitro chondrogenic and hypertrophic differentiation of stem cell pellets in specific culture conditions. The present study proves the potential application of US in the in vitro stage of "developmental engineering" for bone development and repair.

Dependence of optical attenuation coefficient and mechanical tension of irradiated human cartilage measured by optical coherence tomography.

As banked human tissues are not widely available, the development of new non-destructive and contactless techniques to evaluate the quality of allografts before distribution for transplantation is very important. Also, tissues will be processed accordingly to standard procedures and to minimize disease transmission most tissue banks will include a decontamination or sterilization step such as ionizing radiation. In this work, we present a new method to evaluate the internal structure of frozen or glycerol-processed human cartilages, submitted to various dosis of irradiation, using the total optical attenuation coefficient retrieved from optical coherence tomography (OCT) images. Our results show a close relationship between tensile properties and the total optical attenuation coefficient of cartilages. Therefore, OCT associated with the total optical attenuation coefficient open a new window to evaluate quantitatively biological changes in processed tissues.

Prenatal Exposure to Continuous Constant Light Alters Endochondral Ossification of the Tibiae of Rat Pups.

UNLABELLED: Clinical and experimental studies suggest that prenatal exposure to stress can impact the growth and development of offspring. The effect of prenatal exposure to constant light, applied as a chronic stressor, on endochondral ossification of the tibiae of 3-day-old and 15-day-old pups was histomorphometrically evaluated. Pregnant rats were divided into 2 groups: mothers chronically exposed to a 12:12-hour light/light cycle (LL) and control mothers maintained on a 12:12-hour light/dark cycle on days 10-20 of pregnancy. On postnatal days 3 and 15, the pups were weighed and euthanized. The tibiae were resected and histologically processed to obtain sections for hematoxylin and eosin staining (HE) and tartrate-resistant acid phosphatase (TRAP) histochemistry, in order to perform histomorphometric determinations. The data were statistically analyzed. A significant decrease in hypertrophic cartilage thickness was observed in the tibiae of the 3-day-old (LL: 0.134 ± 0.02 vs. CONTROLS: 0.209 ± 0.023 mm; p < 0.01) and 15-day-old (LL: 23.32 ± 3.98 vs. CONTROLS: 22.96 ± 1.93 mm; p < 0.05) prenatally stressed pups. The subchondral bone volume was significantly lower in the tibiae of the 3-day-old LL pups (38.83 ± 6.14%) than in the controls (62.83 ± 10.67%; p < 0.01). The decrease in subchondral bone volume and hypertrophic cartilage thickness shows that the normal growth process of the tibia is impaired in prenatally stressed pups.

Effects of low-level laser therapy on joint pain, synovitis, anabolic, and catabolic factors in a progressive osteoarthritis rabbit model.

The aim of this study was to investigate the effect of low-level laser therapy (LLLT) on short-term and long-term joint pain, synovitis, anabolic, and catabolic factors in the cartilage of a rabbit model with progressive osteoarthritis (OA) induced by anterior cruciate ligament transection (ACLT). A total of 160 New Zealand white rabbits were randomly assigned into two groups (ACLT group and LLLT group). All rabbits received ACLT surgery, and 2-, 4-, 6-, and 8-week treatment after the surgery, with 20 rabbits being tested biweekly over every study period. The LLLT group received LLLT with a helium-neon (He-Ne) laser (830 nm) of 1.5 J/cm(2) three times per week, and the ACLT group received placebo LLLT with the equipment switched off. Long-term and short-term pain was tested via weight-bearing asymmetry; synovitis was assessed histologically; and knee joint cartilage was evaluated by gross morphology, histology, and gene expression analysis of anabolic and catabolic factors. The histological assessment of pain and synovitis showed that at least 6-week intermittent irradiation of LLLT could relief knee pain and control synovium inflammation. Gross morphologic inspection and histological evaluation showed that 6 weeks of LLLT could decrease cartilage damage of medical femoral condyle and 8 weeks of LLLT could decrease cartilage damage of medical and lateral femoral condyles and medical tibial plateau. Gene expression analysis revealed two results: At least 6 weeks of LLLT could decrease production of catabolic factors, for example, interleukin 1ß (IL-1ß), inducible nitric oxide synthase (iNOS), and MMP-3, and slow down the loss of anabolic factors, mainly TIMP-1. Eight weeks of LLLT treatment could slow down the loss of collagen II, aggrecan, and anabolic factors, mainly transforming growth factor beta (TGF-ß). The study suggests that LLLT plays a protective role against cartilage degradation and synovitis in rabbits with progressive OA by virtue of the regulation of catabolic and anabolic factors in the cartilage.

A 2-year follow-up of irradiated homologous costal cartilage used as a septal extension graft for the correction of contracted nose in Asians.

BACKGROUND: Septal extension graft is a useful method for the correction of contracted nose. When septal or costal cartilage is not available, irradiated homologous costal cartilage (IHCC) may be an alternative choice. This preliminary study is focused on noninfective absorption and other complications and postoperative changes of nasal length after septal extension graft using IHCC. METHODS: Thirty patients who could be observed for more than 2 years postoperatively were assessed. All patients had contracted nose deformities with a history of an inflammatory contracture after previous aesthetic surgical procedures. The IHCC was used only as a septal extension graft in each patient. Nasal length was analyzed by comparing preoperative and 2-year postoperative photographs. RESULTS: There was no noticeable graft warping or infective IHCC absorption. However, 3 patients underwent revision: 2 cases of graft avulsion fracture and 1 case of minimal nasal obstruction. Nasal length, which had increased an average of 8.5% after the procedure, was maintained at follow-up 2 years postoperatively. CONCLUSIONS: The IHCC has the advantages of avoiding donor-site scars, and also provides the same benefits as autogenous costal cartilage. It can therefore be a reliable alternative material for rhinoplasty. A longer follow-up may be necessary to confirm the structural stability of the IHCC graft in the lengthened caudal septum.

Effects of ionizing radiation and preservation on biomechanical properties of human costal cartilage.

Tissue banks around the world store human cartilage obtained from cadaveric donors for use in diverse reconstructive surgical procedures. To ensure this tissue is sterile at the time of distribution, tissues may be sterilized by ionizing radiation. In this work, we evaluate the physical changes in deep frozen costal cartilage (-70 °C) or costal cartilage preserved in high concentrations of glycerol (>98 %) followed by a terminal sterilization process using ionizing radiation, at 3 different doses (15, 25 and 50 kGy). Tension and compression tests were carried out to determine the mechanical changes related both to the different preservation methods and irradiation doses. For both methods of preservation, tension strength was increased by about 24 %, when cartilage tissue was irradiated with 15 kGy. Deep frozen samples, when irradiated with 25 or 50 kGy, had a decrease in their mechanical performance, albeit to a lesser extent than when tissues were preserved in high concentration of glycerol and equally irradiated. In conclusion, processing in high concentration of glycerol did not increase tissue protection against radiation damage; while cartilage preserved in high concentrations of glycerol withstands radiation up to 25 kGy, deep frozen human costal cartilage may be sterilized with a doses up to 50 kGy without significant mechanical impact.

[Forensic medical implications of histomorphological changes in the bone and cartilage tissues under effect of radiation].

The objective of the present work was to study roentgenological, microscopic, and histomorphological changes in the bone and cartilage tissues under effect of different doses of gamma-ray radiation from Gammatron-2 (GUT Co 400) and betatron bremsstrahlung radiation (25 MeV). The total radiation dose varied from 9.6 Gy to 120 Gy per unit area during 5-8 weeks. The study included 210 patients at the age from 7 to 82 years (97 men and 113 women). Histomorphological studies were carried out using samples of bone and cartilage tissues taken from different body regions immediately after irradiation and throughout the follow-up period of up to 4 years 6 months. Control samples were the unexposed bone and cartilage tissues from the same subjects (n = 14). The tissues were stained either with eosin and hematoxylin or by Van Gieson's and Mallory's methods. Gomori's nonspecific staining was used to detect acid and alkaline phosphatase activities. Moreover, argyrophilic substance was identified in the cartilaginous tissue. Best's carmine was used for glycogen staining and Weigert's stain for elastic fibers. Metachromasia was revealed by toluidine blue staining and fat by the sudan III staining technique. In addition, the ultrastructure of cartilaginous tissue was investigated. Taken together, these methods made it possible to identify the signs of radiation-induced damage to the bone and cartilage tissues in conjunction with complications that are likely to develop at different periods after irradiation including such ones as spontaneous fractures, deforming arthrosis and radiation-induced tumours.

Chondroprotective effects of pulsed electromagnetic fields on human cartilage explants.

This study investigated the effects of pulsed electromagnetic fields (PEMFs) on proteoglycan (PG) metabolism of human articular cartilage explants from patients with osteoarthritis (OA). Human cartilage explants, recovered from lateral and medial femoral condyles, were classified according to the International Cartilage Repair Society (ICRS) and graded based on Outerbridge scores. Explants cultured in the absence and presence of IL-1ß were treated with PEMF (1.5 mT, 75 Hz) or IGF-I alone or in combination for 1 and 7 days. PG synthesis and release were determined. Results showed that explants derived from lateral and medial condyles scored OA grades I and III, respectively. In OA grade I explants, after 7 days exposure, PEMF and IGF-I significantly increased (35) S-sulfate incorporation 49% and 53%, respectively, compared to control, and counteracted the inhibitory effect of IL 1ß (0.01 ng/ml). The combined exposure to PEMF and IGF-I was additive in all conditions. Similar results were obtained in OA grade III cartilage explants. In conclusion, PEMF and IGF-I augment cartilage explant anabolic activities, increase PG synthesis, and counteract the catabolic activity of IL-1ß in OA grades I and III. We hypothesize that both IGF-I and PEMF have chondroprotective effects on human articular cartilage, particularly in early stages of OA.

Chondrogenesis of Adipose-Derived Stem Cells on Irradiated Cartilage.

BACKGROUND: Irradiated allogeneic costal cartilage is an alternative option of cartilage graft in patients with insufficient autologous cartilage. However, complications can occur during long-term follow-up. This study investigated whether Tutoplast-processed cartilage, one of the irradiated allogeneic costal cartilages, acts as a scaffold for adipose-derived stem cells and chondrogenesis. METHODS: In vitro setting, human adipose-derived stem cells seeded onto Tutoplast-processed cartilage were cultured in chondrogenic medium and observed using a scanning electron microscope. Next, 3 types of irradiated cartilage-including Tutoplast-processed cartilage, undifferentiated stem cells on Tutoplast-processed cartilage (undifferentiated group), and chondrogenic differentiated stem cells on Tutoplast-processed cartilage (chondrogenic group)-were implanted subcutaneously into nude mice. Gross, histologic, and gene expression analyses of Tutoplast-processed cartilages were performed at postoperative weeks 2 and 4. RESULTS: Human adipose-derived stem cells subjected to in vitro three-dimensional culture differentiated into chondrocytes and expressed cartilage-specificgenes. Adipose-derived stem cells seeded onto Tutoplast-processed cartilage were differentiated into chondrocytes in chondrogenic medium. In the chondrogenic group, the chondrogenic-differentiated cells attached to the surface of the Tutoplast-processed cartilage were maintained during the follow-up and were distinct from the existing Tutoplast-processed cartilage. Moreover, the chondrogenic group had higher expression of cartilage-specific genes compared with the undifferentiated group. CONCLUSIONS: Adipose-derived stem cells seeded onto Tutoplast-processed cartilage underwent chondrogenic differentiation, generating new cartilage, which was maintained after implantation without critical complications. The findings are clinically valuable in terms of overcoming the limitations of irradiated allogeneic costal cartilage, and broaden the surgical options for treatments requiring cartilage.

Bone and cartilage turnover markers, bone mineral density, and radiographic damage in men with ankylosing spondylitis.

PURPOSE: To determine the levels of bone and cartilage turnover markers in men with ankylosing spondylitis (AS) and to investigate their associations with disease activity, bone mineral density, and radiographic damage of the spine. PATIENTS AND METHODS: This cross-sectional study enrolled 35 men with newly diagnosed AS. The bone mineral densities (BMD) of their lumbar spines and proximal femurs, Bath AS Disease Activity Index (BASDAI), and Bath AS Radiographic Index (BASRI) were evaluated. Urinary C-terminal telopeptide fragments of type I collagen (CTX-I) and type II collagen (CTX-II) levels were determined by enzyme-linked immunosorbent assay, and serum levels of bone-specific alkaline phosphatase (BALP) and osteocalcin were determined by an enzyme immunoassay. Levels of biochemical markers were compared with those of 70 age-matched healthy men. RESULTS: Patients with AS had significantly higher mean urinary CTX-I and CTX-II levels than control subjects (p<0.05). Elevated urinary CTX-I levels correlated well with BASDAI, femoral BMD, and femoral T score (p<0.05), and elevated urinary CTX-II levels correlated well with spinal BASRI (p<0.05) in patients with AS. Mean serum BALP and osteocalcin levels did not differ between patients and controls and did not show any significant correlations with BMD, BASDAI, or BASRI in men with AS. CONCLUSIONS: Elevated CTX-I reflects disease activity and loss of femoral BMD while elevated CTX-II levels correlate well with radiographic damage of the spine, suggesting the usefulness of these markers for monitoring disease activity, loss of BMD, and radiographic damage in men with AS.

Radial shockwave treatment promotes human mesenchymal stem cell self-renewal and enhances cartilage healing.

BACKGROUND: Shockwaves and mesenchymal stem cells (MSCs) have been widely accepted as useful tools for many orthopedic applications. However, the modulatory effects of shockwaves on MSCs remain controversial. In this study, we explored the influence of radial shockwaves on human bone marrow MSCs using a floating model in vitro and evaluated the healing effects of these cells on cartilage defects in vivo using a rabbit model. METHODS: MSCs were cultured in vitro, harvested, resuspended, and treated with various doses of radial shockwaves in a floating system. Cell proliferation was evaluated by growth kinetics and Cell Counting Kit-8 (CCK-8) assay. In addition, the cell cycle and apoptotic activity were analyzed by fluorescence activated cell sorting. To explore the "stemness" of MSCs, cell colony-forming tests and multidifferentiation assays were performed. We also examined the MSC subcellular structure using transmission electron microscopy and examined the healing effects of these cells on cartilage defects by pathological analyses. RESULTS: The results of growth kinetics and CCK-8 assays showed that radial shockwave treatment significantly promoted MSC proliferation. Enhanced cell growth was also reflected by an increase in the numbers of cells in the S phase and a decrease in the numbers of cells arrested in the G0/G1 phase in shockwave-treated MSCs. Unexpectedly, shockwaves caused a slight increase in MSC apoptosis rates. Furthermore, radial shockwaves promoted self-replicating activity of MSCs. Transmission electron microscopy revealed that MSCs were metabolically activated by shockwave treatment. In addition, radial shockwaves favored MSC osteogenic differentiation but inhibited adipogenic activity. Most importantly, MSCs pretreated by radial shockwaves exhibited an enhanced healing effect on cartilage defects in vivo. Compared with control groups, shockwave-treated MSCs combined with bio-scaffolds significantly improved histological scores of injured rabbit knees. CONCLUSIONS: In the present study, we found that radial shockwaves significantly promoted the proliferation and self-renewal of MSCs in vitro and safely accelerated the cartilage repair process in vivo, indicating favorable clinical outcomes.

Preconditioning of mesenchymal stem cells with low-intensity ultrasound for cartilage formation in vivo.

The purpose of this study was to evaluate the benefits of in vitro preconditioning of mesenchymal stem cells (MSCs) using low-intensity ultrasound (US) in the induction of chondrogenic differentiation of MSCs in vivo. After rabbit bone marrow-derived MSCs were seeded onto a polyglycolic acid (PGA) scaffold, the PGA-MSCs constructs were divided into 4 subgroups: untreated control, low-intensity US group, transforming growth factor-beta [TGF]-treated group and low-intensity US/TGF group. The chondrocyte-seeded PGA construct served as a positive control. For 1 week before implantation, the low-intensity US groups were subjected to ultrasound treatment for 20 min daily at an intensity of 200 mW/cm(2). The TGF groups were treated with 10 ng/mL TGF-beta1. The cells were then implanted into the nude mouse subcutaneously. Retrieved 1, 2, 4, and 6 weeks after implantation, each construct underwent gross examination, histology, biochemical assays, mechanical testing, and reverse transcriptase polymerase chain reaction (RT-PCR). Substantial size reduction and blood invasion were found much earlier in the groups that did not undergo low-intensity US than in those that did. Safranin O/Fast green staining revealed that the chondrogenic differentiation of MSCs was more widespread throughout the constructs in the low-intensity US groups. In the biochemical and mechanical analyses, the low-intensity US and low-intensity US/TGF groups were significantly better in forming hyaline cartilage-like tissue by 4 weeks than the non-low-intensity US groups. Presented by von Kossa staining, the development of osteogenic phenotypes was highly suppressed until 4 weeks in the low-intensity US groups, along with compressive strength comparable to the positive control. In the RT-PCR analysis before implantation, the messenger RNA levels of Sox-9, aggrecan, and tissue inhibitors of metalloproteinase-2 were higher in the low-intensity US groups, while those of type I and type X collagens and matrix metalloproteinase-13 were higher in the non-low-intensity US groups. Blood invasion into the constructs was also considerably hindered in the low-intensity US groups. These results strongly indicate that low-intensity US preconditioning in vitro could be an effective cue to upregulate chondrogenic differentiation of MSCs in vivo.

The biologic effects and the therapeutic mechanism of action of electric and electromagnetic field stimulation on bone and cartilage: new findings and a review of earlier work.

BACKGROUND: Muscle, ligament, bone, cartilage, blood, and adult stem-cell production all respond to electric and electromagnetic fields, and these biophysical field agents can be applied in therapeutic contexts. Postulated mechanisms at the cellular, subcellular, and molecular level are discussed. Electric and electromagnetic field stimulation enhance the repair of bone through the mediation of three areas at the cellular level: (1) the complex interplay of the physical environment; (2) growth factors; and (3) the signal transduction cascade. Studies of electric and electromagnetic fields suggest that an intermediary mechanism of action may be an increase in morphogenetic bone proteins, transforming growth factor-beta, and the insulin-like growth factor II, which results in an increase of the extracellular matrix of cartilage and bone. Investigations have begun to clarify how cells respond to biophysical stimuli by means of transmembrane signaling and gene expression for structural and signaling proteins. METHODS: Review of meta-analysis trials of electrical stimulation of all types. CONCLUSIONS: Further research in the form of methodologically sound, randomized, controlled studies are needed. Inter alia, resolutions are needed for the significant disparities between clinical targets, types of electrical stimulation, and clinical outcomes.

Irradiated homograft cartilage in laryngotracheal reconstruction--a preliminary experience.

OBJECTIVE: To determine the suitability of irradiated homograft cartilage in pediatric laryngotracheal reconstruction (LTR). METHODS: Retrospective case series at a tertiary care children's hospital. Pediatric patients with subglottic stenosis who had LTRs performed using irradiated homograft costal cartilage. Data was collected on postoperative complications, findings at subsequent endoscopic examinations and final outcome. RESULTS: Minor degrees of graft resorption were observed that did not affect eventual decannulation. CONCLUSION: Irradiated homograft costal cartilage can serve as a successful alternative to the traditional autologous costal cartilage in lower grades of laryngotracheal stenosis.

[The study of apoptosis factors released from laser injured cartilage inducing apoptosis of chondrocyte].

OBJECTIVE: To explore the role of pro-apoptotic signals following tissue injury and how these may promote a progression of further cell death. METHODS: Laser treated porcine articular cartilage disks were maintained in culture media. The collected media at various time periods (3, 6, 9, 12, 24 and 48 h), was called treated conditioned media (TCM). Non-laser treated cartilage disks were used to create control conditioned media (CCM). Each disk was subsequently maintained for 28 days and used in confocal microscopic assessment to document the progression of the damaged area. Isolated porcine chondrocytes were cultured in monolayer, and were exposed to TCM, CCM or normal culture medium (NM). As a positive inducer of apoptosis, the monolayer cells were exposed to UV radiation for 10 min and cultured in NM. Following 24 h exposure, the cells were harvested and stained with the appropriate combination of fluorescent dyes and processed via flow cytometry. RESULTS: All cultured cells exposed to TCM displayed a caspase-3 positive subpopulation, a loss of CMXRos, and with a reduced or lost NO signal. CCM exposure signals were comparable to the NM treatments with all having retained CMXRos, NO and without evidence of caspase-3 activity. UV treatment also induced a reduction in NO, but both CMXRos and caspase-3 positive, representing an earlier stage of apoptosis and suggesting that the mode of cell death via UV and TCM exposure are via different processes. The investigation of a dose (100%, 50%, 25% and 12.5%) and time (0.5, 1, 3, 9, 12 h) response to TCM exhibited that all treatments observed an increase in caspase-3 positive cells and a reduction in NO and CMXRos. CONCLUSION: The usefulness of FCM can be used in the study of cell viability and apoptosis. Such a system may be useful in the study of mechanisms of disease such as osteoarthritis, thus may be of practical use for the pharmaceutical industry for screening associated drugs.

Expression of MMP-13 in osteoblast cells and rat tibia after exposure to gamma rays or accelerated carbon ions.

In past research, we found that carbon ion irradiation increased bone volume in rats, and a significant amount of cartilage remained inside the carbon ion-irradiated trabeculae. The amounts of matrix metalloproteinase 13 (MMP-13) mRNA in osteoblast-like MC3T3-E1 cells tended to decrease after carbon ion irradiation. The level of MMP-13 mRNA in non-irradiated cells was stable during the experimental period, but in gamma ray-irradiated cells it tended to increase. When localization of MMP-13 in locally irradiated experimental rats was investigated, it was found in the marginal trabeculae in both non-irradiated and gamma ray-irradiated animals. MMP-13 was detected in osteoid and neogenetic bone in the trabeculae surface. The trabeculae in carbon ion-irradiated bone remained cartilaginous. Carbon ion-irradiated rats exhibited weak expression of MMP-13 around the cartilage inside the trabeculae. We conclude that carbon ion irradiation reduced expression of MMP-13, thus suppressing both chondrocyte maturation and cartilage resorption. Increases in hyperplasia of the bone trabeculae and of bone volume were caused by ongoing bone addition and calcification in the absence of sufficient cartilage resorption.