低强度脉冲超声干预膝关节炎模型兔软骨细胞Ⅱ型胶原和基质金属蛋白酶13的表达

BACKGROUND: Low intensity pulsed ultrasound is safe, non-invasive, and has strong penetration. In recent years, more and more studies have indicated that low intensity pulsed ultrasound has important role in promoting the repair of damaged articular cartilage.     

The effect of low intensity pulsed ultrasound on regenerate bone in a less-than-rigid biomechanical environment

The purpose of this study was to investigate effects of low intensity pulsed ultrasound (LIPU) on distraction osteogenesis in a less-than-rigid biomechanical environment in a rabbit model. A less-rigid mini-lengthener was applied and a mid-tibial osteotomy performed in 20 New Zealand White rabbits. After a 7 day latency period, the tibiae were distracted 0.5 mm every 12 hours for 10 days. Ten of the rabbits received LIPU for 20 min/day (ultrasound group) and 10 received sham LIPU (control group) from day 17 until sacrifice on day 37. Radiographs were taken weekly after distraction and the callus area was measured. After sacrifice, dual-energy X-ray absorptiometry (DEXA), torsional testing to failure, and histomorphometry were performed. On radiographs, all the treatment tibiae displayed persistent radiolucencies; however, only one of the control tibiae displayed a radiolucent interzone. Torsional strength of the treatment group was 54% of the contralateral tibia compared to 139% in the control group (p<0.008). Bone density and callus size were not significantly different between the 2 groups; however, the ultrasound group displayed a tendency towards more cartilage and fibrous tissue formation (p<0.16) and less bone (p<0.16) than controls. In a biomechanically unstable environment, LIPU appears to stimulate more cartilage formation in regenerated callus than in controls. This callus is biomechanically inferior to unstimulated callus at the early stage of healing tested. During distraction osteogenesis a sound biomechanical environment is important to achieving anticipated results.     

Insulin and thyroid hormones stimulate matrix metabolism in primary cultures of articular chondrocytes from young rabbits independently and in combination

These studies examined the effects of heat-inactivated horse serum, insulin, triiodothyronine (T3), and thyroxine (T4), individually and in combination, on collagen and proteoglycan synthesis by primary cell cultures of articular chondrocytes from immature male rabbits. Insulin concentrations of 25 to 100 ng/ml (4.4 to 17.4 x 10(-9) M) increasingly stimulated collagen and proteoglycan synthesis in the absence of serum. The effects of 25 ng/ml (4.4 x 10(-9) M) insulin or 15% heat-inactivated horse serum on collagen synthesis were similar. Triiodothyronine (10(-10) to 10(-6) M) and T4 (10(-8) to 10(-4) M) also stimulated collagen synthesis in the absence of serum, with peak effects at 10(-8) and 10(-6) M, respectively. Biphasic stimulation of proteoglycan synthesis was obtained with 10(-11) to 10(-7) MT3 (maximum at 10(-8) M) and 10(-8) to 10(-5) M T4 (maximum at 10(-7) M). In these experiments, triiodothyronine was 10 to 100 times more potent than T4 in stimulating cartilage matrix production. The cells retained their chondrocytic phenotype under hormonal stimulation, secreting almost exclusively Type II collagen and large, chondroitin sulfate-rich proteoglycans. The addition of insulin to maximally-stimulating concentrations of either T3 or T4 in serum-free medium further stimulated matrix synthesis, suggesting that these hormones modulate chondrocyte metabolism via multiple biosynthetic/receptor pathways.     

Effect of indomethacin on collagen biosynthesis by rabbit articular chondrocytes in monolayer cultures

The influence of indomethacin on collagen biosynthesis in rabbit articular chondrocyte monolayer cultures was studied. Two applications within the space of three days of therapeutic doses (10(-5) or 10(-6)M), as well as repeated applications four days running of lower doses (10(-8) or 10(-10)M), increased the biosynthesis of both collagen and non-collagen proteins. Two applications of higher doses (10(-3) or 10(-4M) decreased DNA synthesis and inhibited both collagen and non-collagen protein biosynthesis. These results might well be considered in connection with the adverse reactions observed in some patients with long-term use of indomethacin.     

低强度脉冲超声对大鼠急性肌腱损伤早期血管生成的影响及机制

背景:越来越多研究表明,低强度脉冲超声可以促进急性肌腱损伤的愈合,但其具体机制尚不清楚。目的:观察低强度脉冲超声对急性肌腱损伤后早期血管生成的影响,并检测与血管内皮生长因子相关信号通路的调控关系,进而揭示其潜在作用机制。方法:8-12周龄SPF级雄性SD大鼠局部注射Ⅰ型胶原酶3 d建立急性跟腱损伤动物模型,随机分为超声组和对照组。超声组每日使用有效辐射面积为1 cm^(2)的超声小探头垂直于跟腱部位进行低强度脉冲超声治疗,对照组不干预。治疗2周后进行超声影像学检查,观察肌腱早期愈合情况;治疗1,2周后行苏木精-伊红染色和CD31免疫组织化学染色观察组织血管数量变化,Western blot和qRT-PCR检测跟腱组织中血管内皮生长因子相关信号通路分子的表达。结果与结论:①灰阶超声显示:超声组较对照组跟腱更为连续,回声强度更低且较为均匀,肌腱厚度明显降低(P<0.05);②苏木精-伊红染色和CD31免疫组织化学结果一致显示:治疗2周后,超声组的新生血管数量明显较同期对照组增多(P<0.05);③Western blot和qRT-PCR结果显示:治疗2周后超声组跟腱中血管内皮生长因子、Yes相关蛋白、血管生成素2、富含半胱氨酸的血管生成素诱导物61的蛋白和mRNA表达均显著高于对照组(P<0.05);④结果表明:低强度脉冲超声通过上调血管内皮生长因子表达显著增加了急性肌腱损伤早期血管生成数量,加速了肌腱愈合。     

Effects of low-intensity pulsed ultrasound on proliferation and chondroitin sulfate synthesis of cultured chondrocytes embedded in Atelocollagen gel.

The effects of low-intensity pulsed ultrasound (US) on the proliferation and chondroitin sulfate synthesis of cultured chondrocytes embedded in Atelocollagen gel in vitro were examined. Articular cartilage was harvested from the hip, knee, and shoulder joints of 10-week-old Japanese white rabbits. Chondrocytes isolated by collagenase digestion were embedded in type I collagen gel, Atelocollagen gel, and were cultured in Dulbecco's modified eagle's medium for 3 weeks. The US apparatus, SAFHS, was used to deliver an ultrasound signal with spatial and temporal average intensities of 30 mW/cm(2) (US group). The frequency was 1.5 MHz with a 200-microsecond tone burst repeated at 1.0 kHz. US treatments were administered for 20 min per day under culture dishes, with the medium replaced twice a week. Another group of cells was exposed to sham ultrasound as a control. Cell number, histological findings, synthesis of isomers of chondroitin sulfate, and stiffness of the chondrocyte-collagen gel composites were analyzed. US exposure promoted synthesis of chondroitin sulfate, especially chondroitin 6-sulfate, although it did not significantly enhance cell number and stiffness. In this three-dimensional culture model, these results suggest that US exposure may be clinically useful in improving the quality of chondrocyte-Atelocollagen implants for transplantation into articular cartilage defects.     

低强度脉冲超声波对β－磷酸三钙与兔骨髓基质干细胞相容性的影响

目的研究低强度脉冲超声波(LIPUS)对-磷酸三钙(-TCP)与兔骨髓基质干细胞(BMSCs)相容性的影响。方法抽取新西兰白兔的骨髓,分离纯化获取BMSCs,加入条件培养基和-TCP体外混合培养并随机分为两组,一组应用LIPUS对BMSCs每天作用20分钟,另一组作为对照未予作用。通过倒置相差显微镜观察细胞增殖分化情况,分别于1周、2周、3周后停止LIPUS作用,在扫描电镜下观察细胞生长情况,评估TCP与BMSCs的相容性。结果BMSCs在两组-TCP表面生长繁殖良好。混合培养1周、2周时,实验组TCP表面BMSCs数目较对照组明显增多,并具有统计学意义(<0.05)。混合培养3周时,实验组TCP表面BMSCs数目与对照组相比无统计学差异(﹥0.05)。结论LIPUS可以提高培养早期-TCP和BMSCs的生物相容性,而对于培养晚期TCP和BMSCs的生物相容性无明显影响。     

染料木黄酮抑制生长板软骨细胞的增殖与基质合成

目的： 探讨染料木黄酮对大鼠肋生长板软骨细胞（RGC）的形态、增殖和胞外基质成分合成的影响。方法： 原代培养RGC，1×10^－6 μmol/L-1×10^－4 μmol/L染料木黄酮处理第1代RGC 24 h，观察RGC形态，同位素掺入检测RGC增殖、胶原和蛋白多糖合成，RT-PCR检测collagen Ⅱ和aggrecan基因表达。结果： 染料木黄酮改变了RGC的形态，随着浓度的升高，突起和漂浮细胞的数量增加。染料木黄酮剂量依赖性地抑制RGC的增殖、胶原和蛋白多糖的合成（P<0.05），以及collagenⅡ和aggrecan mRNA的转录。结论： 染料木黄酮抑制RGC的增殖和胞外基质的合成。     

Matrix synthesis in high density cultures of bovine epiphyseal plate chondrocytes

Bovine epiphyseal plate chondrocytes were cultured by a method combining both suspension culture and high density monolayers. The matrix synthesized by the cultured cells was analyzed at fifteen days for glycosaminoglycan, proteoglycan, and collagen content. In the cell culture product glycosaminoglycan distribution was: 65% chondroitin-6-sulfate, 18% chondroitin-4-sulfate, 15% keratan sulfate, and less than 2% dermatan sulfate. Essentially all the radioactive sulfate in labelled specimens was present in high molecular weight aggregates. The collagen which was synthesized co-migrated with Type II collagen standard. Parallel analysis showed the matrix of cultured cells to be similar to that of intact epiphyseal plate tissue. This study demonstrates the ability to grow epiphyseal plate chondrocytes in a cell culture system which allows matrix synthesis similar to that seen in vivo.     

Restoration of longitudinal growth by bioengineered cartilage pellet in physeal injury is not affected by low intensity pulsed ultrasound

Physeal fracture is a common pediatric fracture that would result in premature physeal closure in long bones, and there is currently no gold standard for its management. In this study, we investigated the application of a Bioengineered Cartilage Pellet (BCP) in repairing a rabbit physeal fracture model, and the possible effects of Low Intensity Pulsed Ultrasound (LIPUS) treatment. Rabbits with physeal fracture created were assigned to the NC group (no BCP, no LIPUS), GC group (BCP, no LIPUS), and GT group (BCP and LIPUS). Femoral lengths and cartilage area were assessed at 4, 8, and 16 weeks post-defect. After transplantation, the BCP showed continuous growth in the host and demonstrated resemblance to a natural growth plate. The GC group showed 34.1, 32.1, and 41.1% advantage in lengthening over the NC group and the GT group showed 51.1, 41.6, and 26.9% improved lengthening than the NC group, at 4 (p = 0.203), 8 (p = 0.543) and 16 weeks (p = 0.049), respectively. Cartilage area was shown to be significantly higher in GC and GT group compared to NC group (p < 0.05). No significant difference was found between GC and GT group. Femoral longitudinal growth was shown to be improved by the BCP, however no additional enhancement effect was shown to be provided by LIPUS.     

Effect of low intensity pulsed ultrasound on healing of an ulna defect filled with a bone graft substitute

A 1.5 cm unilateral rabbit ulna defect model was performed in 18 adult NZ white rabbits. The defects were filled with a beta-tricalcium phosphate bone graft substitute (JAX TCP). The surgical site in half the animals was treated daily with 20 min of low intensity pulsed ultrasound (LIPUS). Animals were sacrificed at 4 weeks (n = 3 per group) or 12 weeks (n = 6 per group) following surgery for radiographic and histologic endpoints. Radiography revealed some resorption of the JAX TCP by 12 weeks in the control and LIPUS treated groups. LIPUS treatment did not accelerate this resorption. Some new bone formation was noted in the control groups at the defect margins while little bone formed in the center of the defect at 4 and 12 weeks. In contrast, radiographs revealed more new bone at 4 and 12 weeks in the LIPUS treated animals throughout the section. Bone mineral density (DEXA) revealed a statistically significant difference at 4 weeks with LIPUS while no differences were found at 12 weeks. Histology of the LIPUS treated sections demonstrated new woven bone formation on and between the JAX TCP bone graft substitute particles across the defect. VEGF expression was increased with LIPUS treatment at 4 weeks and remained elevated at 12 weeks compared with controls. CBFA-1 expression levels were elevated with LIPUS treatment at both time points. LIPUS treatment increased bone formation in ulna defect healing with a beta-tricalcium phosphate bone graft substitute.     

Upregulation of matrix synthesis in chondrocyte-seeded agarose following sustained bi-axial cyclic loading

OBJECTIVES:

The promotion of extracellular matrix synthesis by chondrocytes is a requisite part of an effective cartilage tissue engineering strategy. The aim of this in vitro study was to determine the effect of bi-axial cyclic mechanical loading on cell proliferation and the synthesis of glycosaminoglycans by chondrocytes in three-dimensional cultures.

METHOD:

A strain comprising 10% direct compression and 1% compressive shear was applied to bovine chondrocytes seeded in an agarose gel during two 12-hour conditioning periods separated by a 12-hour resting period.

RESULTS:

The bi-axial-loaded chondrocytes demonstrated a significant increase in glycosaminoglycan synthesis compared with samples exposed to uni-axial or no loading over the same period (p<0.05). The use of a free-swelling recovery period prior to the loading regime resulted in additional glycosaminoglycan production and a significant increase in DNA content (p<0.05), indicating cell proliferation.

CONCLUSIONS:

These results demonstrate that the use of a bi-axial loading regime results in increased matrix production compared with uni-axial loading.     

低强度超声促进骨折愈合

骨折愈合是一个非常复杂的骨组织再生过程 ,许多因素可影响骨折愈合。一些生物及生物物理的方法已被用于促进骨折愈合和减少骨折延迟愈合、不愈合的发生。本文对低强度超声促进骨折愈合的动物和临床实验进行综述 ,并介绍了低强度超声促进骨形成和骨折愈合的生物学机理。     

Measurement of the deformation of isolated chondrocytes in agarose subjected to cyclic compression

Mechanically induced cell deformation is one of a number of possible mechanotransduction pathways by which chondrocytes sense and respond to changes in their mechanical environment. The present study describes a system for measuring the deformation of isolated chondrocytes in agarose during both static and cyclic compression. A test rig mounted on the stage of an inverted microscope was used to apply precise levels of compressive strain to individual cell-agarose constructs bathed in culture medium. Images of the cells were recorded using a CCD video camera attached to the microscope. Cell deformation was quantified in terms of a deformation index (X/Y) representing the ratio of cell diameters measured parallel (X) and perpendicular (Y) to the axis of compression. Cyclic compression between 0 and 15% strain, at 0.3 Hz, resulted in cyclic deformation of the cells at the same frequency. However, during the unstrained phase the cells did not fully recover to their initially spherical morphology (X/Y = 1.0). During the strained phase, the level of deformation (X/Y = 0.59) was initially similar to that observed during static 15% strain. However, this level of cell deformation reduced over a 20 min period of cyclic compression (X/Y = 0.72), although during static compression the cell deformation remained constant. This system may be used to examine cellular events under a range of dynamic mechanical stimuli.     

低强度超声对激素诱导的兔高眼压的作用

目的探讨低强度超声对兔激素性高眼压的影响及其机制。方法采用超声直接接触法对兔激素性高眼压连续治疗5d并监测眼压。观察眼压、小梁组织的HE染色,透射电镜、免疫组化法及图像分析观察小梁外基质层黏连蛋白、纤维连接蛋白和胶原Ⅰ的变化。结果低强度超声处理后眼压显著下降,小梁网间隙增大,胶原I、层黏连蛋白及纤维连接蛋白的表达低于对照组。结论低强度超声所致眼压降低与小梁组织超微结构改变及超声机械震荡等使异常增多的小梁外基质减少有关。     

DNA repair by articular chondrocytes. II. Direct measurements of repair of ultraviolet and X-ray damage in monolayer cultures

The abilities of human and rabbit articular chondrocytes to repair ultraviolet (UV) and X-ray damage were measured in terms of the removal of UV—endonuclease-sensitive sites (pyrimidine dimers) and single-strand breaks, respectively. The initial 3-h rate of dimer repair in human cells, incubated in medium containing 10% human serum, was about 2.5 times as large as in rabbit cells incubated in medium containing 10% or 20% fetal bovine serum. Similar results have been previously reported for unscheduled DNA synthesis (UDS), indicating that UDS is a valid quantitative measure of repair in this cell system. The repair of single-strand breaks was rapid (approx. 50% completed in <10 min). An estimate, from the measured numbers of lesions and patch sizes, indicated that the amount of UDS following 20 krad would be 100 to 300 times less than that in 3 h following to J/m² of 254 nm and hence would not be detectable radioautographically.     

Evaluation of pulsed high intensity focused ultrasound exposures on metastasis in a murine model

High intensity focused ultrasound (HIFU) may be employed in two ways: continuous exposures for thermal ablation of tissue (>60°C), and pulsed-exposures for non-ablative effects, including low temperature hyperthermia (37–45°C), and non thermal effects (e.g. acoustic cavitation and radiation forces). Pulsed-HIFU effects may enhance the tissue’s permeability for improved delivery of drugs and genes, for example, by opening up gaps between cells in the vasculature and parenchyma. Inducing these effects may improve local targeting of therapeutic agents, however; concerns exist that pulsed exposures could theoretically also facilitate dissemination of tumor cells and exacerbate metastases. In the present study, the influence of pulsed-HIFU exposures on increasing metastatic burden was evaluated in a murine model with metastatic breast cancer. A preliminary study was carried out to validate the model and determine optimal timing for treatment and growth of lung metastases. Next, the effect of pulsed-HIFU on the metastatic burden was evaluated using quantitative image processing of whole-lung histological sections. Compared to untreated controls (2/15), a greater number of mice treated with pulsed-HIFU were found to have lungs “overgrown” with metastases (7/15), where individual metastases grew together such that they could not accurately be counted. Furthermore, area fraction of lung metastases (area of metastases/area of lungs) was ~30% greater in mice treated with pulsed-HIFU; however, these differences were not statistically significant. The present study details the development of an animal model for investigating the influence of interventional techniques or exposures (such as pulsed HIFU) on metastatic burden.