深低温冻存组织工程化软骨在喉狭窄功能重建中的应用

目的：探讨低温保存组织工程化软骨在喉狭窄功能重建中的应用价值。方法：取3周龄新西兰兔关节软骨细胞,体外培养,取第2代对数生长期培养细胞,制成细胞悬液,调整软骨细胞悬液浓度约为5×10^7个／ml左右,接种于PGA三维支架材料上,复合物体外培养2周后冻存,冻存6个月后解冻复苏,再行体外培养观察,2周后接种于已建立的喉甲状软骨缺损模型的软骨缺损处,并设对照组。术后12周取材,行大体及组织学观察。结果：经低温冻存的组织工程化软骨生长良好,组织学观察有软骨形成,与周围软骨组织结合紧密,与非冻存组相比差异无统计学意义。结论：深低温冻存对组织工程化软骨的生物活性无明显的影响,低温冻存的组织工程化软骨可用于喉软骨缺损的修复,重建喉功能。     

深低温冻存组织工程化软骨在喉狭窄功能重建中的应用

目的:探讨低温保存组织工程化软骨在喉狭窄功能重建中的应用价值。方法:取3周龄新西兰兔关节软骨细胞,体外培养,取第2代对数生长期培养细胞,制成细胞悬液,调整软骨细胞悬液浓度约为5×107个/ml左右,接种于PGA三维支架材料上,复合物体外培养2周后冻存,冻存6个月后解冻复苏,再行体外培养观察,2周后接种于已建立的喉甲状软骨缺损模型的软骨缺损处,并设对照组。术后12周取材,行大体及组织学观察。结果:经低温冻存的组织工程化软骨生长良好,组织学观察有软骨形成,与周围软骨组织结合紧密,与非冻存组相比差异无统计学意义。结论:深低温冻存对组织工程化软骨的生物活性无明显的影响,低温冻存的组织工程化软骨可用于喉软骨缺损的修复,重建喉功能。     

组织工程化软骨与富血小板血浆复合可促进软骨缺损修复

为探讨组织工程化软骨与富血小板血浆复合修复软骨缺损的效果,本研究选取了8周龄健康新西兰兔24只,依据随机数表法分为观察组(组织工程化软骨与富血小板血浆复合)和对照组(单纯软骨缺损模型),发现术后4周、8周、12周,观察组实验动物的大体评分均明显高于对照组(p0.05)。观察组实验动物的Collagen TypeⅠ、Collagen TypeⅡ相对表达水平明显高于对照组(p0.05)。两组实验动物的Collagen TypeⅩ相对表达水平无显著差异(p0.05)。观察组实验动物的软骨缺损直径和缺损深度分别为(1.02±0.35)mm、(0.96±0.27)mm,对照组实验动物的软骨缺损直径和缺损深度分别为(4.27±1.09)mm、(5.43±1.85)mm(p0.05),表明组织工程化软骨与富血小板血浆复合修复软骨缺损效果明显,能够刺激软骨相关基因表达,缩小软骨缺损范围,促进缺损软骨愈合。     

利用新型聚羟丁酸酯材料构建组织工程软骨

目的:研究新型聚羟丁酸酯作为组织工程软骨支架材料的可行性.方法:取幼兔软骨组织中软骨细胞体外培养扩增.实验组接种软骨细胞于支架材料上,体外培养两周后埋植于新西兰大白兔背部皮下;对照组埋入未接种细胞的支架材料.扫描电镜观察材料表面形态及细胞生长情况.分别于第4、8、12周取出标本,大体观察后进行HE和Masson染色,观察组织工程软骨形成情况.结果:扫描电镜观察可见裸材料孔隙分布均匀,形状不规则;细胞材料复合体体外培养两周后材料表面爬满细胞且生长状态良好.埋植材料取出后可见不同时间点实验组标本大小无明显变化,对照组标本逐渐变小.HE和Masson染色显示各组支架材料至12周时已被完全吸收;实验组12周时可见较成熟软骨组织;对照组支架材料被吸收后最终被纤维结缔组织取代.结论:此新型聚羟丁酸酯材料可作为组织工程软骨支架材料.     

注射式软骨缺损微创修复技术的大动物实验研究

目的:在现有关节镜下microfracture技术的基础上,应用现代干细胞技术修复猪关节软骨缺损,探索注射式软骨缺损微创修复技术用于软骨再生治疗的可行性.方法:抽取6只猪的骨髓体外扩增培养至三代,动物随机分2组,每组6膝,每膝制备1个软骨缺损,左膝行缺损部位microfracture治疗后将3×107/mL浓度的骨髓间充质干细胞注射于关节腔内,右膝为单纯microfracture或空白对照.术后8、16周各3只动物,行大体观察、组织学检测,评价其对关节软骨缺损的再生修复效果.结果:术后8周观察见软骨缺损的修复表面平整,色泽渐趋正常,与周围组织整合良好;术后16周,修复组织具有透明软骨样结构,并产生大量GAGs和Ⅱ型胶原,单纯microfracture治疗组为纤维软骨修复,而空白组为少量纤维组织覆盖缺损底面,观察期内未见毛细血管长入及免疫排斥反应发生.结论:注射式软骨缺损微创修复技术创伤小,操作简便,能显著促进关节软骨缺损的再生修复的特点,具有较高的科学价值及临床应用前景.     

软骨细胞联合BMP/bFGF移植对关节软骨损伤的修复作用

目的：探讨同种异体软骨细胞移植联合骨形态发生蛋白（BMP）/碱性成纤维细胞生长因子（bFGF）对关节软骨损伤的修复作用。方法：取24只14周龄成年大白兔,随机分为A、B、C、D组,每组6只,于双侧膝关节软骨处制作软骨缺损模型,A组采用软骨细胞移植联合应用BMP/bFGF处理,B组采用单纯软骨细胞移植,C组采用单纯BMP/bFGF修复,D组采用磷酸盐缓冲液（PBS）作为阴性对照,于处理后8、12、24周行形态学、电镜观察及组织学评分。结果：8周时,A组关节修复面与周围结合紧密,可见大量软骨细胞出现,电镜下有软骨基质形成;B、C组仅有少量软骨细胞;D组未见修复。12周时,A组关节修复面与周围组织界限模糊,软骨细胞增殖活跃,电镜下可见成熟软骨基质;B、C组修复块周围有肉芽组织生成,电镜下可见未成熟的软骨基质出现;D组可见肉芽组织形成。24周时,A组修复面周围组织融合,电镜下软骨细胞纵行排列;B、C组关节面修复不完全,电镜下软骨细胞分布不均;D组见大量肉芽组织形成。24周时,A组组织学评分（1.87±0.65）,明显低于B组（3.49±0.71）、C组（3.43±0.83）组和D组（13.45±0.97）,差异均有统计学意义（P〈0.05）,B、C组均明显低于D组,差异有统计学意义（P〈0.05）,B、C组之间比较无明显差异。结论：软骨细胞联合BMP/bFGF移植能够促进软骨生长,提高软骨损伤的修复质量。     

自体软骨细胞移植技术治疗膝关节软骨缺损的研究进展

膝关节软骨缺损发病率高,且自身修复能力有限。治疗膝关节软骨缺损的传统方法包括钻孔术、微骨折术、自体骨软骨移植术。然而,钻孔术和微骨折术治疗后缺损区生成的是纤维软骨,而不是正常的透明软骨,两者在力学强度、硬度、耐磨损性等多方面存在很大差距。自体骨软骨移植术可生成正常的透明软骨,但存在供体有限、不适合进行大面积软骨缺损治疗等多方面缺点在临床方面应用受限。近年来,自体软骨细胞移植技术发展迅速,越来越多的病人接受此治疗方法并获得良好效果,引起人们广泛关注。本文根据近年来国内外的各项相关研究成果进行总结,阐述膝关节软骨缺损的各种治疗方法,着重介绍自体软骨细胞移植技术。第三代自体软骨细胞移植技术生成的软骨以透明软骨为主,符合关节生物力学要求,且避免了第一代、第二代自体软骨细胞移植的术后并发症,成为治疗膝关节大面积软骨缺损安全有效的治疗方法。另外,本文就软骨细胞支架材料的发展、移植物术后的转归等问题提出进一步设想。     

rhBMP-2诱导异位软骨修复重建兔气管缺损的实验研究

目的：探讨重组人骨形成蛋白-2（rhBMP-2）作为激活物诱导异位软骨修复并重建免气管缺损的可行性。方法：取24只新西兰大白兔,制备气管前壁软骨1／3缺损模型。随机分为A、B组,每组12只,A组为实验组,在气管缺损处前壁颈前肌肉修补,多点注射rhBMP-2;B组于气管软骨缺损部位直接颈前肌群修补。术后观察动物一般情况,于4、8、12周取材进行大体观察、HE染色观察重建区域情况。结果：术后A组动物均存活至实验完成,B组因气道感染及气道分泌物堵管潴留致使实验兔死亡,其余动物出现皮下气肿,呼吸不畅等情况。组织学观察A组有明显的新生软骨细胞及少量软骨样组织,可见结缔组织包绕,周围肌肉组织完整,排列整齐,未见明显坏死组织,有少量淋巴细胞浸润。B组未见软骨组织生成,可见大量肉芽组织增生,结缔组织排列紊乱,伴少量坏死组织,大量淋巴浸润。结论：rhBMP-2可通过注射到颈前肌肉修补肌群中诱导软骨细胞和软骨样组织生成,减轻炎症反应,联合颈前肌瓣修复重建气管缺损能充分维持修复重建后的气道形态,具有减少术后皮下气肿、气管狭窄的作用,有望用于临床修复重建气管纽织缺损。     

自体软骨细胞修复兔膝关节全层软骨缺损的实验研究

目的：研究自体软骨细胞复合于人脐带Wharton胶支架对兔膝关节全层软骨缺损的修复效果。方法：经自体关节软骨细胞 经体外培养后复合到制备人脐带Wharton 胶取向支架内构建细胞- 支架复合体,选取健康清洁新西兰兔23 只,雌雄不拘,体重 2.5-3.0 kg,取滑车沟中下部制作全层软骨缺损模型后随机分成A、B和C 组。A组(n= 10)：植入自体软骨细胞+人脐带Wharton 胶取向支架复合体;B组(n= 10)：植入单纯人脐带Wharton 胶取向支架;C组(n= 3)：不做任何处理正常兔。分别于术后3 个月和6 个月各处死后取材进行生物力学特性评估检测。结果：压痕实验显示在3 个月时A 和B 组修复区组织刚度分别达到正常软骨的 45.72%和25.25%,且A组刚度明显优于B组,均低于C组( P<0.05);到6 个月时各自达到正常软骨刚度的69.76%和35.14%,同 样A 组刚度明显优于B 组,均低于C 组( P<0.05)且在同期个各组之间均有显著性差异（F=80.309,P<0.05）。结论：体外培养的自 体软骨细胞与人脐带Wharton 胶复合在体内的微环境作用下修复软骨缺损效果良好,为软骨组织工程提供了一种新支架材料。     

rhBMP-2 诱导异位软骨修复重建兔气管缺损的实验研究

目的:探讨重组人骨形成蛋白-2(rhBMP-2)作为激活物诱导异位软骨修复并重建兔气管缺损的可行性。方法:取24只新西兰大白兔,制备气管前壁软骨1/3缺损模型。随机分为A、B组,每组12只,A组为实验组,在气管缺损处前壁颈前肌肉修补,多点注射rhBMP-2;B组于气管软骨缺损部位直接颈前肌群修补。术后观察动物一般情况,于4、8、12周取材进行大体观察、HE染色观察重建区域情况。结果:术后A组动物均存活至实验完成,B组因气道感染及气道分泌物堵管潴留致使实验兔死亡,其余动物出现皮下气肿,呼吸不畅等情况。组织学观察A组有明显的新生软骨细胞及少量软骨样组织,可见结缔组织包绕,周围肌肉组织完整,排列整齐,未见明显坏死组织,有少量淋巴细胞浸润。B组未见软骨组织生成,可见大量肉芽组织增生,结缔组织排列紊乱,伴少量坏死组织,大量淋巴浸润。结论:rhBMP-2可通过注射到颈前肌肉修补肌群中诱导软骨细胞和软骨样组织生成,减轻炎症反应,联合颈前肌瓣修复重建气管缺损能充分维持修复重建后的气道形态,具有减少术后皮下气肿、气管狭窄的作用,有望用于临床修复重建气管组织缺损。     

Articular cartilage reconstruction using xenogeneic epiphyses slices

Reconstruction of articular cartilage defects using adult osteochondral allografts is an established clinical procedure, whose principal drawback is lack of lateral integration of the grafts to the surrounding tissue. Autologous chondrocytes transplantation is a sophisticated technique requiring cell culture and a staged operation. Its main draw back is the lack of mechanical strength early on. This study was conducted in order to evaluate the possibility of using embryonal epiphyses as a cartilage reconstruction tissue. A xenogeneic human to rabbit sub-acute osteochondral defect model was designed to evaluate the possibility of allogeneic implantation in humans. The following procedures were perfomed (n = 5): transplantation of 1. live epiphyses 2. live epiphyses with autogeneic periosteum 3. de-vitalized epiphyses and 4. devitalized epiphyses with autogeneic articular chondrocytes. A fifth control group did not receive any implant. Animals in groups 1 and 2 had a viable reconstruction of the articular surface with little evidence of rejection and without pannus formation. Animals in groups 3 and 4 became severely arthritic and the graft was resorbed. Nitric oxide synthase accumulation was reduced in group 1 and 2 as compared to groups 3, 4, and 5, indicating a joint preserving function of the epiphyseal grafts. Epiphyseal grafts appear to be a feasible procedure for reconstruction of articular cartilage defects even in a xenogeneic model. This revised version was published online in July 2006 with corrections to the Cover Date.     

Influence of freezing with liquid nitrogen on whole-knee joint grafts and protection of cartilage from cryoinjury in rabbits

Improving survival rates for sarcoma patients are necessitating more functional and durable methods of reconstruction after tumor resection. Frozen osteoarticular grafts are utilized for joint reconstruction, but the joint may develop osteoarthritic change. We used a frozen autologous whole-rabbit knee joint graft model to investigate the influence of freezing on joint components. Thirty rabbit knee joints that had been directly immersed into liquid nitrogen (L) or saline (C) without use of cryoprotectants were re-implanted. Histological observations were made after 4, 8, and 12 weeks. Both groups had bone healing. In group L, despite restoration of cellularity to the menisci and ligaments, no live chondrocytes were observed and cartilage deterioration progressed over time. It was concluded that cryoinjury of chondrocytes caused osteoarthritic change. Then we tested whether a vitrification method could protect cartilage from cryoinjury. Full-thickness articular cartilage of rabbit knee was immersed into liquid nitrogen with and without vitrification. Histology, ultrastructure, and chondrocyte viability were examined before and after 24 h of culture. Vitrified cartilage cell viability was >85% compared with that of fresh cartilage. Transmission electron microscopy revealed preservation of original chondrocyte structure. Our vitrification method was effective for protecting chondrocytes from cryoinjury. Since reconstructing joints with osteoarticular grafts containing living cartilage avert osteoarthritic changes, vitrification method may be useful for storage of living cartilage for allografts or, in Asian countries, for reconstruction with frozen autografts containing tumors.     

Vitreous preservation of articular cartilage from cryoinjury in rabbits

Frozen osteoarticular grafts treated with liquid nitrogen are utilized for joint reconstruction after tumor resection, but the joints may subsequently develop osteoarthritic changes. To preserve articular cartilage from cryoinjury, we modified a vitrification method utilized for embryo cryopreservation and demonstrated in vitro that our vitrification protocol was effective for protecting cartilage from cryoinjury. In this study, we investigated in vivo whether this vitrification method could protect against osteoarthritic changes in articular cartilage. Osteochondral plugs were obtained from the distal femur of rabbits. These grafts were divided into 3 groups: Fresh group (F-group), non-vitrification group (N-group), and vitrification group (V-group). After treatment, the plugs were re-implanted as autografts. Histological findings, chondrocyte viability, and ultrastructural examinations were examined 6, 12, and 24weeks after implantation. Histological findings of chondrocytes for the V-group showed no significant difference from those of the F-group at any time point except at 24weeks postimplantation at the non-weight bearing site (p<0.05). Viability of chondrocyte showed no significant difference from those of the F-group except at 12weeks postimplantation at the bearing site (p<0.05). In contrast, viable cells disappeared from the N-group and histology and viability significantly differed between the N-group and the V-group. Transmission electron microscopy demonstrated preservation of chondrocyte structure in the V-group and the F-group, but chondrocytes of the N-group were abnormally electron dense. Our vitrification method was effective in protecting chondrocytes from cryoinjury that might lead to cartilage degeneration. Reconstructing joints with osteoarticular grafts containing living cartilage may help to avert osteoarthritic changes. Our vitrification method could prove useful for reconstruction with frozen tumor-containing autografts and for long-term storage of living cartilage for allografts.     

Tissue engineering and cartilage

Human articular cartilage is an avascular structure, which, when injured, poses significant hurdles to repair strategies. Not only does the defect need to be repopulated with cells, but preferentially with hyaline-like cartilage.Successful tissue engineering relies on four specific criteria: cells, growth factors, scaffolds, and the mechanical environment. The cell population utilized may originate from cartilage itself (chondrocytes) or from growth factors that direct the development of mesenchymal stem cells toward a chondrogenic phenotype. These stem cells may originate from various mesenchymal tissues including bone marrow, synovium, adipose tissue, skeletal muscle, and periosteum. Another unique population of multipotent cells arises from Wharton''s jelly in human umbilical cords. A number of growth factors have been associated with chondrogenic differentiation of stem cells and the maintenance of the chondrogenic phenotype by chondrocytes in vitro, including TGFβ; BMP-2, 4 and 7; IGF-1; and GDF-5.Scaffolds chosen for effective tissue engineering with respect to cartilage repair can be protein based (collagen, fibrin, and gelatin), carbohydrate based (hyaluronan, agarose, alginate, PLLA/PGA, and chitosan), or formed by hydrogels. Mechanical compression, fluid-induced shear stress, and hydrostatic pressure are aspects of mechanical loading found in within the human knee joint, both during gait and at rest. Utilizing these factors may assist in stimulating the development of more robust cells for implantation.Effective tissue engineering has the potential to improve the quality of life of millions of patients and delay future medical costs related to joint arthroplasty and associated procedures.Key words: cartilage repair, gene therapy, growth factors, biomaterials, tissue engineering, stem cells, chondrocyte     

Doublecortin is expressed in articular chondrocytes

Articular cartilage and cartilage in the embryonic cartilaginous anlagen and growth plates are both hyaline cartilages. In this study, we found that doublecortin (DCX) was expressed in articular chondrocytes but not in chondrocytes from the cartilaginous anlagen or growth plates. DCX was expressed by the cells in the chondrogenous layers but not intermediate layer of joint interzone. Furthermore, the synovium and cruciate ligaments were DCX-negative. DCX-positive chondrocytes were very rare in tissue engineered cartilage derived from in vitro pellet culture of rat chondrosarcoma, ATDC5, and C3H10T1/2 cells. However, the new hyaline cartilage formed in rabbit knee defect contained mostly DCX-positive chondrocytes. Our results demonstrate that DCX can be used as a marker to distinguish articular chondrocytes from other chondrocytes and to evaluate the quality of tissue engineered or regenerated cartilage in terms of their "articular" or "non-articular" nature.     

Effects of Notch/p38MAPK signaling pathway on articular cartilage defect recovery by BMSCs tissue based on the rabbit articular cartilage defect models

ObjectiveThe objective is to clarify the effects of Notch/p38MAPK signaling pathway on articular cartilage defect recovery by BMSCs tissue and provide a basis for clinical treatments of articular cartilage defects.MethodsA total of 96 healthy male rabbits (weighed 1.5–2.0 kg) that were fully-grown were selected and grouped as the no-treatment group, the model group, and the treatment group in a random manner. Each group included 32 rabbits in total. The no-treatment group was fed without any interventions. The model group and the treatment group were constructed into rabbit knee-joint articular cartilage defect models. In addition, rabbits in the treatment group were given intervention treatments with Notch inhibitor (DAPT) combined with p38MAPK inhibitor (SB203580). The general conditions of rabbits in each group and the conditions of the stained articular cartilage tissue samples were observed, the proliferation of chondrocytes of rabbits in each group was compared.Results(1) After drug interventions, in contrast to the rabbits in the model group, the general conditions and the chondrocyte recovering situations of rabbits in the treatment group were obviously improved; (2) 8 weeks after model construction, the articular cartilage empty bone lacuna rate of rabbits in the treatment group was (12.13 ± 1.81)%, which was obviously lower than the synchronous (21.55 ± 3.07)% articular cartilage empty bone lacuna rate of rabbits in the model group, and there was a statistical significance in the differences (P < 0.05); (3) the absorbance value (OD value) of chondrocytes in the treatment group was (0.34 ± 0.015), which was obviously higher than the (0.10 ± 0.020) OD value of chondrocytes in the model group, and there was a statistical significance in the differences (P < 0.05).ConclusionThe inhibition of Notch/p38MAPK signaling pathway can promote the recovery of articular cartilage by BMSCs tissue, accelerate the proliferation of chondrocytes, and contribute to the recovery of knee-joint injuries in rabbits, which provides a reliable basis for clinical treatments of articular cartilage defects.     

Can low concentrations of Papain help repair articular cartilage defects?

In the present study, we investigate the capability of low concentrations of Papain to stimulate cartilage mesenchymal cells proliferation and transformation to chondrocytes and evaluate the healing capability of partial thickness defects in medial condyle cartilage of 30 rabbits’ knee joints. Papain 0.1 mg/ml and Ringer saline l ml each were injected intra-articularly to rabbits of experimental and control groups (15 animals each). Healthy cartilage from lateral condyle and cartilage from medial condyle where the surgical defect was created were studied histologically and by TEM. The study revealed that 0.1 mg/ml Papain activates proliferation and spreading of mesenchymal stem cells to young forms of chondrocyte from perichondrium to the upper layers of healthy cartilage. In only 22.27% cases of the experimental group, surgical defects filled with cartilaginous tissue on the background of distinct destruction of collagenous matrix in the native cartilage. However, in 55.5% of the control group the defect was spontaneously healed by hyaline cartilaginous tissue completely or partially on the basis of slight destruction of collagenous matrix. The defect site was filled with activated chondrocyte-like cells from the subchondral plate (not perichondrium) in both groups, which acquired some cisterns of rough endoplasmic reticulum (RER) and produced matrix proteins. The results suggest that Papain did not ameliorate the recovery of cartilage defects acquired through surgically-induced injury of collagenous matrix in native cartilage. We observed that articular cartilage is the source of mesenchymal stem cells which have the ability to transform into young forms of chondrocytes. This transformation process depends on the level of destruction of native cartilage collagen matrix induced by the defect or by Papain.     

Tissue engineering and cartilage

Human articular cartilage is an avascular structure, which, when injured, poses significant hurdles to repair strategies. Not only does the defect need to be repopulated with cells, but preferentially with hyaline-like cartilage.

Successful tissue engineering relies on four specific criteria: cells, growth factors, scaffolds, and the mechanical environment. The cell population utilized may originate from cartilage itself (chondrocytes) or growth factors may direct the development of mesenchymal stem cells toward a chondrogenic phenotype. These stem cells may originate from various mesenchymal tissues including bone marrow, synovium, adipose tissue, skeletal muscle, and periosteum. Another unique population of multipotent cells arises from Wharton’s jelly in human umbilical cords. A number of growth factors have been associated with chondrogenic differentiation of stem cells and maintenance of the chondrogenic phenotype by chondrocytes in vitro, including TGF-β; BMP-2, 4, and 7; IGF-1; and GDF-5.

The scaffolds chosen for effective tissue engineering with respect to cartilage repair can be protein based (collagen, fibrin, and gelatin), carbohydrate based (hyaluronan, agarose, alginate, PLLA/PGA, and chitosan), or formed by hydrogels. Mechanical compression, fluid-induced shear stress, and hydrostatic pressure are all aspects of mechanical loading found in the human knee joint, both during gait and at rest. Utilizing these factors may assist in stimulating the development of more robust cells for implantation.

Effective tissue engineering has the ability to improve the quality of life of millions of patients and delay future medical costs related to joint arthroplasty and associated procedures.     

Treatment of cartilage defects by Low-intensity pulsed ultrasound in a sheep model

Aim of this study was to evaluate effects of Low-intensity pulsed ultrasound on repair of articular cartilage defects. Low-intensity pulsed ultrasound (Lipus) can induce the differentiation and activation of chondrocytes. This study was designed to evaluate the effect of Lipus on articular cartilage defects in a sheep. Eight sheep were divided in to two groups. The animals received bilateraly, articular cartilage defects 4 mm in diameter and 2 mm in deep on the patellar groove and experimental groups were treated with intensity 200 mW/cm², 20 min/day with low-intensity pulsed ultrasound for 2 month. Then both knee joints underwent surgery for remove of formed tissue sample from defects.The samples were evaluated by Quantitative real-time polymerase chain reaction (qRT-PCR), Safranin-o staining, Immunofluorescence Staining and Morphological characterization. The best and worst sample per group according to Macroscopic and micriscopic scoring were icentified. The results showed that the operated groups with-Lipus-treatment and without-Lipus treatment had considered statistically significant. Gross photography revealed that the defects in experimental groups were filled with proliferative tissue, while in control groups, a thin layer of proliferative tissue was formed in defects. qRT-PCR results showed the expression of coll2, sox9, aggrecan and Osteocalcin in experimental groups. Intense safranin-O staining show the formation cartilage tissue in ultrasound treated group, while loose safranin-o-staining were observed at the control groups. Immunofluorescence staining showed the type 2 Collagen protein expression. We suggest that low-intensity pulsed ultrasound provide the mechanistic basis force for articular cartilage repair and effective treatment modality for improving of articular cartilage defects.