The therapeutic effect of low-level laser on repair of osteochondral defects in rabbit knee

INTRODUCTION: Low level laser therapy (LLLT) has been shown to enhance collagen production and wound healing but its effect on cartilage repair from biomechanical point of view is not known yet. The aim of present study was to evaluate the biomechanical behaviour of repairing osteochondral defect in rabbits which received a pulsed low-level gallium-arsenide (Ga-As) laser irradiation. MATERIALS AND METHODS: Osteochondral defects with 5mm diameter and 4mm in depth induced by drilling in right femoral patellar grooves of 41 adolescent male rabbits. They were divided into experimental and control groups. Experimental group received pulsed Ga-As (890nm) laser irradiation with energy density of 4.8J/cm(2). The rabbits in control group received placebo LLLT with shut-down equipment. The control defects were allowed to heal spontaneously. Each group were divided into three subgroups: A, B and C. Subgroups A, B and C were sacrificed on 4, 8, and 16 weeks after surgery. The knee joint were removed, and the defects were examined biomechanically by in situ-indentation method. The thickness, instantaneous and equilibrium indentation stiffness was measured during the test. Data were analysed using ANOVA and independent sample t-test. RESULT: While no difference was observed in the repaired cartilage biomechanical properties among 4th, 8th, 16th weeks in study groups. The equilibrium indentation stiffness of experimental group was significantly higher in 8th week in comparison with control group. CONCLUSION: LLLT significantly enhances the stiffness of repairing tissue in the 8th week post injury in osteochondral defects in rabbits.     

Effect of low-level laser therapy on the healing of second-degree burns in rats: a histological and microbiological study

This paper presents the results of a study on the effects of two different doses of low-level laser therapy on healing of deep second-degree burns. Sixty rats were randomly allocated to one of four groups. A deep second-degree burn was inflicted in each rat. In the control group burns remained untreated; in two laser treated groups the burns were irradiated daily with low-level helium-neon laser with energy densities of 1.2 and 2.4 J/cm2, respectively. In the fourth group the burns were treated topically with 0.2% nitrofurazone cream every day. The response to treatments was assessed histologically at 7, 16 and 30 days after burning, and microbiologically at Day 15. The number of macrophages at day 16, and the depth of new epidermis at day 30, was significantly less in the laser treated groups in comparison with control and nitrofurazone treated groups (P=0.000). Staphylococcus epidermidis was found in the 70% of rat wounds in the laser treated groups in comparison with 100% of rats in the control group. S. aureus was found in the 40% rat wounds in the nitrofurazone treated group, but there was not found in the wounds of laser treated, and control groups. It is concluded that low-level laser therapy of deep second-degree burn caused significant decrease in the number of macrophage and depth of new epidermis. In addition, it decreased incidence of S. epidermidis and S. aureus.     

Effect of low-level helium-neon laser therapy on the healing of third-degree burns in rats

This paper presents the results of a study on the effects of low-level helium-neon laser therapy (LL He-Ne LT) on the healing of burns. Seventy-eight adult male rats, having been subjected to third-degree burns, were randomly divided into four groups: two laser treated groups (n=20, each), one control group (n=19) and one nitrofurazone treated group (n=19). In the two laser treated groups, the burns were treated on a daily basis with LL He-Ne LT with an energy density of 1.2 and 2.4 J/cm(2), respectively. The response to treatment was assessed histologically at 7, 16 and 30 days after burning, and microbiologically at day 15. Analysis of variance showed that the mean of blood vessel sections in the 1.2J/cm(2) laser group was significantly higher than those in the other groups and the mean of the depth of new epidermis in the 2.4 J/cm(2) laser group on day 16 was significantly lower than in the nitrofurazone treated group (P=0.025, P=0.047, respectively). When Staphylococcus aureus and Pseudomonas aeruginosa grew in more than 50% of samples obtained from control group, there were no S. aureus and P. aeruginosa in the samples of 2.4 J/cm(2) laser group. It is concluded that LL He-Ne LT induced the destruction of S. aureus and P. aeruginosa in third-degree burns of rats, yet at the same time our histological findings showed that LL He-Ne LT caused a significant increase in the mean of blood vessel sections on day 7 after third degree burns and a decrease in the mean of the depth of new epidermis on day 16 after the same burns in rats.     

Therapeutic Effect of Irradiation of Magnetic Infrared Laser on Osteoarthritis Rat Model

Osteoarthritis (OA) is a degenerative joint disease caused by articular cartilage loss. Many complementary and alternative medicines for OA have been reported so far, but the effectiveness is controversial. Previously, we have shown anti‐inflammatory effects of low level laser therapy with static magnetic field, magnetic infrared laser (MIL), in various animal models. Therefore, the beneficial effects were examined in OA rat model. Rats were divided by six groups; no treatment controls of sham and OA model, three MIL treatment groups of OA model at 6.65, 2.66 and 1.33 J cm^?2, and Diclofenac group of OA model with 2 mg kg^?1 diclofenac sodium. The OA control exhibited typical symptoms of OA, but 4‐week MIL treatment improved the functional movement of knee joint with reduced edematous changes. In addition, cartilage GAGs were detected more in all MIL treatment groups than OA control. It suggests that 4‐week MIL irradiation has dose‐dependent anti‐inflammatory and chondroprotective effects on OA. Histopathological analyses revealed that MIL treatment inhibits the cartilage degradation and enhances chondrocyte proliferation. The fact that MIL has an additional potential for the cartilage formation and no adverse effects can be regarded as great advantages for OA treatment. These suggest that MIL can be useful for OA treatment.     

Evaluation of mitochondrial respiratory chain activity in wound healing by low-level laser therapy

Laser therapy is used in many biomedical sciences to promote tissue regeneration. Many studies involving low-level laser therapy have shown that the healing process is enhanced by such therapy. In this work, we evaluated mitochondrial respiratory chain complexes II and IV and succinate dehydrogenase activities in wounds after irradiation with low-level laser. The animals were divided into two groups: group 1, the animals had no local nor systemic treatment and were considered as control wounds; group 2, the wounds were treated immediately after they were made and every day after with a low-level laser (AsGa, wavelength of 904 nm) for 10 days. The results showed that low-level laser therapy improved wound healing. Besides, our results showed that low-level laser therapy significantly increased the activities of complexes II and IV but did not affect succinate dehydrogenase activity. These findings are in accordance to other works, where cytochrome c oxidase (complex IV) seems to be activated by low-level laser therapy. Besides, we showed, for the first time, that complex II activity was also activated. More studies are being carried out in order to evaluate other mitochondrial enzymes activities after different doses and irradiation time of low-level laser.     

Boswellic acid captivated hydroxyapatite carboxymethyl cellulose composites for the enhancement of chondrocytes in cartilage repair

Recently tissue repairing bone grafted materials have been greater properties than the recapitulating intramembranous regeneration of natural bone especially cartilage damage regeneration. In this present work was designed and developed for the enhancement of chondrocyte generation for cartilage repair. Boswellic acid (BA) is a traditional compound used for the treatment of osteoarthritis. Boswellic acid subjected to involve the preparation of hydroxyapatite (HAP) and HAP-BA compounds were functionalized with carboxymethyl cellulose (CMC) to promote the extra-cellular matrix. HAP, HAP-BA and HAP-BA-CMC composites were characterized via their physicochemical properties through FTIR, XRD, SEM and TEM techniques. The antibacterial activity and chondrocyte cell variability were tested. At 14 days, HAP-BA-CMC composite was observed 67% of cell viability. The chondrocyte cell adhesion on the HAP-BA-CMC composite was investigated and it observed polygonal filopodia. From the results suggest that HAP-BA-CMC composite can improve the chondrocyte production for repair of damaged cartilage.     

Immobilization of individual cells by local photo-polymerization on a chip

A novel separation method for random screening of target cells from a large heterogeneous population by using a local photo-polymerization is developed. A photo-crosslinkable resin solution is mixed with the sample liquid and we controlled the state from sol to gel by irradiating the near ultraviolet (UV) light with the mercury lamp and He-Cd laser near the target cell. We applied three types of immobilization methods such as direct immobilization method, caging method, and direct immobilization with position control method. The selected cell is immobilized in the cured resin directly or inside the cage of the cured resin. In the position control method, laser tweezers are employed to manipulate the target cell indirectly by using the droplet of the resin as a microtool. The cell is positioned properly by the laser manipulation system and is immobilized in the polymerized resin. After the selected cells are immobilized we can easily remove the other objects by the cleaning flow in the microchannel since the polymerized resin strongly binds with the cover glass and resists more than 466 mm s(-1) flow speed in the microchannel (microchannel size: width is 500 micron and depth is 100 micron). We tested the mercury lamp as well as the He-Cd laser for UV-light irradiation at the local area and confirmed improvement of resolution of the cured area by using the He-Cd laser (from 7 micron to 5 micron). Based on this method, we succeeded in single cell immobilization and basic experiments such as culture and fluorescent dyeing of immobilized yeast cells.     

Effects of adenovirus-mediated bFGF, IL-1Ra and IGF-1 gene transfer on human osteoarthritic chondrocytes and osteoarthritis in rabbits

The study investigated the effects of adenovirus-mediated gene transfection of basic fibroblast growth factor (bFGF), bFGF combined with interleukin-1 receptor antagonist protein (IL-Ra) and/or insulin-like growth factor-1 (IGF-1) both in human osteoarthritis (OA) chondrocytes and rabbits OA model. Human OA chondrocytes were delivered by adenovirus-mediated bFGF, IL-Ra and IGF-1 vectors, respectively. Chondrocyte proliferation, glycosaminoglycan (GAG) content, expression of type II collagen, ADAMTS-5, MMP-13, MMP-3 and TIMP-1 were determined. Rabbit OA model was induced by anterior cruciate ligament transaction (ACLT) in knees. Adenoviral vectors encoding human bFGF, IL-Ra and IGF-1 were injected intraarticularly into the knee joints after ACLT. The effects of adenovirus- mediated gene transfection on rabbit OA were evaluated. In vitro, the transfected genes were expressed in cell supernatant of human OA chondrocytes. AdbFGF group significantly promoted chondrocyte proliferation, and increased GAG and type II collagen synthesis than in the OA group. As two or three genes were transfected in different combinations, there was significant enhancement on the GAG content, type II collagen synthesis, and TIMP-1 levels, while ADAMTS-5, MMP-13, and MMP-3 levels were reduced. In vivo, the transfected genes were expressed in synovial fluid of rabbits. Intraarticular delivery of bFGF enhanced the expression of type II collagen in cartilage and decreased cartilage Mankin score compared with the OA control group (P = 0.047; P < 0.01, respectively). Multiple-gene transfection in different combinations showed better results than bFGF transfection alone. This study suggests that bFGF gene transfection is effective in treating experimental OA. Multiple gene transfection has better biologic effects on OA.     

Proteomic analysis of chondrocytes exposed to pressure

Chondrocytes are the only cell type present in mature articular cartilage (2-5% of total tissue). The biological activities of the chondrocyte population are regulated by genetic, biologic and biochemical factors, as well as environmental factors (stress, flow and electric field). Although compressive forces within joint articular cartilage are required for maintenance of the normal composition of articular cartilage, there is a lack of knowledge about the number of pressure-related proteins expressed in articular cartilage. Two-dimensional gel electrophoresis (2-DE) and high-performance liquid chromatography-electrospray/tandem mass spectrometry (HPLC/ESI-MS/MS) were used to identify the levels of pressure-related proteins expressed by chondrocytes grown in the presence or absence of hydrostatic pressure. A total of 266 spots were excised from the gels and analyzed by HPLC/ESI-MS/MS. Functional classification of up-regulated proteins indicated that energy and protein fate were the main biological processes occurring in pressurized chondrocytes. Furthermore, membrane-bound transferrin-like protein p97, a marker of chondrocyte differentiation, was only expressed in chondrocytes under hydrostatic pressure. These data suggest that hydrostatic pressure can induce cell differentiation by increasing the expression level of energy metabolism- and protein fate-related proteins, indicating that hydrostatic pressure may be needed for normal biosynthesis and differentiation of articular chondrocytes.     

Contact Transcleral Ciliary Body Photodynamic Therapy with Verteporfin in Pigmented Rabbits: Effect of Repeated Treatments

We studied the effect on the intraocular pressure (IOP) and the ciliary body (CB) morphology after four consecutive contact transcleral photodynamic treatments of the ciliary body (CB-PDT) with verteporfin in pigmented rabbits. Twenty-two pigmented rabbits underwent CB-PDT (study group), performed once (six rabbits) or repeated for up to four times (16 rabbits). Six additional rabbits received only laser treatment without photosensitizer administration (control group). CB-PDT was performed in one eye in rabbits of the study group, with the fellow eye serving as internal control. Verteporfin dosage was 1 mg kg ⁻¹ as bolus injection and laser settings were 40 mW (600 μm core optical fiber) for 1.5 min per spot, for 10 spots. In repeated CB-PDT, treatments were performed in 4-day intervals. Daily IOP measurements were recorded. Histological studies were performed at selected time points. An IOP reduction, more sustained following repeated treatments, was detected in all treated eyes but not in fellow eyes or in the control group. On the average, the IOP was restored to pretreatment levels 4 days after the last treatment. No serious adverse events were observed and the CB architecture was intact at the end of the experiment. Repeated CB-PDT is safe and results in a short-term reduction of IOP. Induced CB alterations are reversible.     

Anti-inflammatory effects of low-level laser therapy (LLLT) with two different red wavelengths (660 nm and 684 nm) in carrageenan-induced rat paw edema

It has been suggested that low-level laser therapy (LLLT) can modulate inflammatory processes. The aim of this experiment was to investigate what effects red laser irradiation with two different wavelengths (660 nm and 684 nm) on carrageenan-induced rat paw edema and histology. Thirty two male Wistar rats were randomly divided into four groups. One group received a sterile saline injection, while inflammation was induced by a sub-plantar injection of carrageenan (1 mg/paw) in the three other groups. After 1 h, LLLT was administered to the paw in two of the carrageenan-injected groups. Continuous wave 660 nm and 684 nm red lasers respectively with mean optical outputs of 30 mW and doses of 7.5 J/cm(2) were used. The 660 nm and 684 nm laser groups developed significantly (p<0.01) less edema (0.58 ml [SE+/-0.17] ml and 0.76 ml [SE+/-0.10] respectively) than the control group (1.67 ml [SE+/-0.19]) at 4h after injections. Similarly, both laser groups showed a significantly lower number of inflammatory cells in the muscular and conjunctive sub-plantar tissues than the control group. We conclude that both 660 nm and 684 nm red wavelengths of LLLT are effective in reducing edema formation and inflammatory cell migration when a dose of 7.5 J/cm(2) is used.     

Proteoglycan synthesis in porcine nasal cartilage grafts following Nd:YAG (lambda = 1.32 microns) laser-mediated reshaping

Mechanically deformed morphologic cartilage grafts undergo temperature-dependent stress relaxation during sustained laser irradiation resulting in stable shape changes. In this study, porcine nasal septal cartilage specimens were evaluated for viability by measuring the incorporation of Na2(35)SO4 into proteoglycan (PTG) macromolecules in whole tissue culture following laser-mediated reshaping. Synthesis rates of PTG were determined by scintillation counting lyophilized specimens and normalizing these values by total protein content. Positive controls were established by inducing chondrocyte apoptosis using prolonged exposure to nitric oxide (NO). In chondrocytes, apoptosis induced using NO resulted in significantly lower PTG synthesis rates compared to untreated native specimens. Cartilage specimens were irradiated with light emitted from a Nd:YAG laser (25 W/cm2, lambda = 1.32 microns) while recording simultaneously radiometric surface temperature, internal stress and back-scattered light intensity from a probe laser. Each specimen received one, two or three sequential laser exposures. The duration of each exposure was determined from real-time measurements of characteristic changes in back-scattered light intensity that correlate with accelerated stress relaxation. A 5 min time interval between each laser exposures allowed the cartilage specimen to return to thermal equilibrium. Average PTG synthesis rates decreased with successive laser exposures, though these were always higher than baseline rates established for NO-treated tissues, suggesting that laser-mediated cartilage reshaping acutely does not eliminate the entire population of viable chondrocytes. The reduction in PTG synthesis is correlated with the time-temperature-dependent heating profile created during laser irradiation, supporting our hypothesis that careful monitoring of laser dosimetry is required to ensure chondrocyte viability.     

In vivo study of the inflammatory modulating effects of low-level laser therapy on iNOS expression using bioluminescence imaging

This study was designed to demonstrate that bioluminescence imaging (BLI) can be used as a new tool to evaluate the effects of low-level laser therapy (LLLT) during in vivo inflammatory process. Here, the efficacy of LLLT in modulating inducible nitric oxide synthase (iNOS) expression using different therapeutic wavelengths was determined using transgenic animals with the luciferase gene under control of the iNOS gene expression. Thirty transgenic mice, FVB/N-Tg(iNOS-luc)Xen, were allocated randomly to one of four experimental groups treated with different wavelengths (lambda = 635, 785, 808 and 905 nm) or a control group (nontreated). Inflammation was induced by intra-articular injection of zymosan A in both knee joints. Laser treatment (25 mW cm(-2), 200 s, 5 J cm(-2)) was applied to the knees 15 min after inflammation induction. Measurements of iNOS expression were performed at various times (0, 3, 5, 7, 9 and 24 h) by measuring the bioluminescence signal using a highly sensitive charge-coupled device (CCD) camera. The results showed a significant increase in BLI signal after irradiation with 635 nm laser when compared to the nonirradiated animals and the other LLLT-treated groups, indicating wavelength dependence of LLLT effects on iNOS expression during the inflammatory process, and thus demonstrating an action spectrum of iNOS gene expression following LLLT in vivo that can be detected by BLI. Histological analysis was also performed and demonstrated the presence of fewer inflammatory cells in the synovial joints of mice irradiated with 635 nm compared with nonirradiated knee joints.     

Proteomic Analysis of Differential Protein Expression of Achilles Tendon in a Rabbit Model by Two-Dimensional Polyacrylamide Gel Electrophoresis at 21 Days Postoperation

Postoperative early kinesitherapy has been advocated as an optimal method for treating Achilles tendon rupture. However, an insight into the rationale of how early kinesitherapy contributes to healing of Achilles tendon remains to be achieved, and research in the area of proteomic analysis of Achilles tendon has so far been lacking. Forty-two rabbits were randomized into control group, immobilization group, and early motion group, and received postoperative cast immobilization and early motion treatments. Achilles tendon samples were prepared 21 days following microsurgery, and the proteins were separated with two-dimensional polyacrylamide gel electrophoresis. Differentially expressed proteins were first recognized by PDQuest software, and then identified using peptide mass fingerprinting, tandem mass spectrometry, and database searching. A total of 463 ± 12, 511 ± 39, and 513 ± 80 protein spots were successfully detected in the two-dimensional polyacrylamide gels for the Achilles tendon samples of rabbits in the control group, immobilization group, and early motion group, respectively. There were 15, 8, and 9 unique proteins in these three groups, respectively, and some differentially expressed proteins were also identified in each group. It was indicated that some of the differentially expressed proteins were involved in various metabolism pathways and may play an important role in healing of Achilles tendon rupture. Postoperative early kinesitherapy resulted in differentially expressed proteins in ruptured Achilles tendon compared with those treated with postoperative cast immobilization. These differentially expressed proteins may contribute to healing of Achilles tendon rupture through a mechanobiological mechanism due to the application of postoperative early kinesitherapy.     

The effect of the association of NIR laser therapy BMPs, and guided bone regeneration on tibial fractures treated with wire osteosynthesis: Raman spectroscopy study

Bone fractures are lesions of different etiology; may be associated or not to bone losses; and have different options for treatment, such as the use of biomaterials, guided bone regeneration, techniques considered effective on improving bone repair. Laser therapy has also been shown to improve bone healing on several models. The association of these three techniques has been well documented by our group using different models. This study aimed to assess, through Raman spectroscopy, the incorporation of calcium hydroxyapatite (CHA approximately 958 cm(-1)) on the repair of complete tibial fractures in rabbits treated with wire osteosynthesis (WO); treated or not with laser therapy; and associated or not with the use of BMPs and/or Guided Bone Regeneration. Complete tibial fractures were created in 12 animals that were divided into four groups: WO; WO+BMPs; WO+laser therapy; and WO+BMPs+laser therapy. Irradiation started immediately after surgery; was repeated at every other day during 2 weeks; and was carried out with lambda 790 nm laser light (4 J/cm(2) per point, 40 mW, phi approximately 0.5 cm(2), 16J per session). Animal death occurred after 30 days. Raman spectroscopy was performed at both the surface and the depth of the fracture site. Statistical analysis showed significant difference on the concentrations of CHA between surface and depth. The analysis in each of the areas showed at the depth of the fracture significant differences between all treatment groups (p<0.0001). Significant differences were also seen between WO+BMPs+laser therapy and WO (p<0.001) and WO+laser therapy (p<0.001). At the surface, significant difference was seen only between the treatment groups and the non-fractured subjects (p=0.0001). However, no significant difference was seen between the treatment groups (p=0.14). It is concluded that the use of NIR laser therapy associated to BMPs and GBR was effective in improving bone healing on the fractured bones as a result of the increasing deposition of CHA measured by Raman spectroscopy.     

Protein profile of osteoarthritic human articular cartilage using tandem mass spectrometry

Articular cartilage contains both chondrocyte cells and extracellular matrix (ECM) components. Currently, comprehensive information concerning the protein composition of human articular cartilage tissue is somewhat lacking. In this report we detail the use of tandem mass spectrometry (MS/MS) for a preliminary global identification of proteins from human articular knee cartilage tissue from patients diagnosed with osteoarthritis. Knee cartilage supernatant was fractionated using one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE), in-gel digested and peptide sequences were then determined by performing on-line nano-liquid chromatography (LC)/MS/MS experiments using an ion trap mass spectrometer. Altogether, over 100 different proteins from nearly 700 unique peptide sequences were detected by MS/MS. The majority of the proteins identified are involved in ECM organization (35%), signal transduction and cell communication (14%), immune response (11%) and metabolism and energy pathways (11%). Proteins observed included several well-known cartilage components as well as lower abundant lesser known ECM proteins. Possible degradation products in the cartilage sample, such as from cartilage link protein, could also be detected by our mass spectrometry methods. We show here that mass spectrometry can be utilized as a tool for a fast, accurate and sensitive analysis of a complex mixture of cartilage proteins. It is believed that this type of proteomic analysis will aid future work centered on investigating the pathology of this and other related joint diseases.     

Synergistic Effects of D‐Glucosamine and Collagen Peptides on Healing Experimental Cartilage Injury

We investigated the synergistic wound‐healing effects of D ‐glucosamine (GlcN) and collagen peptide ( : 10 000 Daltons soluble collagen (S‐Co)) on injured cartilage. A injured cartilage model in rabbit was surgically created. Under general anesthesia, three holes at articular cartilage of the medial trochlear (one hole) and trochlear sulcus (two holes) of a distal femur were created using a high‐speed orthopedic drill. After surgery, rabbits were orally administered with collagen peptide (S‐Co group), GlcN (G group), or both (CoG group) for 2 or 3 weeks. In addition, glycine (Gly group) was administered as a control for the S‐Co group in three rabbits. Control rabbits were provided with water ad lib. After 2 weeks, the holes were filled by fibroblasts and chondroblasts in the treatment group, whereas in the control group, the holes were not filled completely unlike for the treatment group. Even three weeks later, only a small change was seen in the control group. In the group receiving collagen and GlcN, the holes were completely filled by proliferating chondroblasts, remodeling of bony trabeculae was also observed. Image analysis of Safranin O‐ and Alcian blue‐stained specimens showed increased production of proteoglycans and glycosaminoglycans, respectively, not only in the injured site, but also in the normal cartilage in the treated groups. The most enhanced healing of injury and increase in Safranin O and Alcian blue positive materials was observed in the CoG group. As a result, simultaneous administration of collagen and GlcN gave a fairly enhanced healing on restoration of cartilage injuries.

Artificial cartilage injury at the stifle joint of a rabbit. a: proximal hole in trochlear sulcus; b: distal hole in trochlear sulcus; c: medial hole in medial trochlear ridge.     

多层开管毛细管酶反应器的制备及在蛋白质酶切中的应用

以石英毛细管作为酶固定化的载体, 在毛细管内壁上逐步合成树枝形大分子聚酰胺-胺(PAMAM), 再通过交联剂戊二醛将胰蛋白酶直接键合到该大分子的末端氨基上, 并对酶固定化条件进行了优化, 制备了多层酶反应器. 利用该酶反应器对马心细胞色素C等蛋白质进行了酶切, 并对酶切的条件进行了优化. 实验结果表明, 该固定化酶反应器具有较高的酶切效率、良好的重现性和稳定性, 可用于蛋白质组学的研究.