Extracellular Matrix Composition of Full-Thickness Defect Repair Tissue is Little Influenced by Exercise in Rat Articular Cartilage

Full-thickness articular cartilage defects in the femoral condyles of adult rats were examined four and eight weeks after injury. Quantitative polarized light microscopic analysis showed that birefringence of the tissue in the central repair area increased more in rats exercised on a treadmill. Glycosaminoglycan content in the repair tissue was also higher than in the intermit-tent active motion group at four weeks after injury, but by eight weeks the levels were similar in both groups. No normal-looking articular cartilage was formed in the lesions, and only in one animal type II collagen was observed in the superficial zone of repair tissue. No 3B3(-) antigenicity of the proteoglycans was seen during repair. In conclusion, exercise minimally modified the repair of full-thickness articular cartilage defects in adult rats. The repair in the exercised group may occur slightly faster in the early stages but no difference was seen at the eight week time interval between the exercised and the intermittently active group.     

A Paradigm for Functional Tissue Engineering of Articular Cartilage via Applied Physiologic Deformational Loading

Deformational loading represents a primary component of the chondrocyte physical environment in vivo. This review summarizes our experience with physiologic deformational loading of chondrocyte-seeded agarose hydrogels to promote development of cartilage constructs having mechanical properties matching that of the parent calf tissue, which has a Young's modulus E(Y) = 277 kPa and unconfined dynamic modulus at 1 Hz G* = 7 MPa. Over an 8-week culture period, cartilage-like properties have been achieved for 60 x 10(6) cells/ml seeding density agarose constructs, with E(Y) = 186 kPa, G* = 1.64 MPa. For these constructs, the GAG content reached 1.74% ww and collagen content 2.64% ww compared to 2.4% ww and 21.5% ww for the parent tissue, respectively. Issues regarding the deformational loading protocol, cell-seeding density, nutrient supply, growth factor addition, and construct mechanical characterization are discussed. In anticipation of cartilage repair studies, we also describe early efforts to engineer cylindrical and anatomically shaped bilayered constructs of agarose hydrogel and bone (i.e., osteochondral constructs). The presence of a bony substrate may facilitate integration upon implantation. These efforts will provide an underlying framework from which a functional tissue-engineering approach, as described by Butler and coworkers (2000), may be applied to general cell-scaffold systems adopted for cartilage tissue engineering.     

Cyclic Loading Can Denature Type II Collagen in Articular Cartilage

We sought to explain previously reported associations between osteoarthritis, cartilage collagen denaturation, and repetitive mechanical loading. Eighty specimens of cartilage-on-bone were obtained from bovine patella grooves. After soaking in protease inhibitors, specimens were subjected to 3,600 cycles of compressive loading by means of a flat indenter. Loaded cartilage was supported by adjacent cartilage and subchondral bone. Peak stress ranged between 3.5 MPa and 14 MPa. Collagen denaturation was assessed by an inhibition ELISA assay using the col2–3/4 m antibody. Results showed that denaturation increased with loading severity, from 2% in control (unloaded) cartilage to 7.5% at 14 MPa (p <. 0003). Collagen and glycosaminoglycan content of cartilage were unchanged after loading. Denaturation was largely unaffected by the absence of protease inhibitors or when chondrocytes were killed by repeated freeze-thaw cycles prior to loading. We conclude that vigorous cyclic mechanical loading causes an immediate dose-related increase in collagen denaturation in bovine articular cartilage.     

胎儿关节和兔关节软骨细胞的体外培养

本文报告了人胎儿关节软骨细胞和兔关节软骨细胞的体外培养,结果表明：胎儿关节软骨细胞和兔关节软骨细胞在胰蛋白酶和胶原酶作用下,在无ＣＯ２培养条件下,获得了大量的软骨细胞,且能生长分裂,复制。为将此法用于生物材料的人工软骨研究奠定了基础。     

以羊膜为载体培养游离软骨细胞修复兔关节软骨缺损

目的：探讨膜结构为载体培养游离软骨细胞修复软骨缺损的可行性。方法：以兔羊膜为载体将体外培养的同种异体游离软骨细胞植于兔左侧股骨外踝软骨缺损区，分别于4、8、12周处死动物，整个膝关节被解剖，进行大体观察、组织学评价、电镜观察及SRY基因性别鉴定，并以兔体的右膝关节做为对照。结果：术后4、8、12周大体、组织学、电镜观察显示软骨缺损区新生了透明软骨，SRY基因性别鉴定证明新生的软骨来源于移植的同种异体软骨细胞；而对照组则仅见纤维组织样的修复组织。结论：以羊膜为载体进行同种异体软骨细胞移植能够修复关节软骨缺损。     

低频脉冲磁场对兔膝关节软骨缺损修复作用的实验研究

目的研究低频脉冲磁场在间充质干细胞和双层PLGA支架构建复合体修复兔骨软骨缺损过程的作用。方法用密度梯度离心法和贴壁培养法获得5月龄兔骨髓来源间充质干细胞,在体外培养并进行分化诱导后作为种子细胞复合双层PLGA构建成复合体植入兔膝股骨髁间设计骨软骨缺损模型,全过程施加低频脉冲磁场干预,于第24周取材,进行大体观察、组织学检查和病理评分。结果诱导MSCs+支架+2.0A.m-1脉冲电磁场刺激组在术后又继续进行脉冲电磁场照射后,兔膝关节骨软骨创伤修复明显加快,统计学分析各组单项评价指标得分差异有显著性(p<0.05),与自体骨软骨移植组相当。结论在适当低频脉冲磁场作用下,诱导MSCs+双层PLGA支架能较好地修复兔膝关节骨软骨创伤。     

Measurement of Articular Cartilage Thickness in the Articulated Knee

A radiographic and image analysis method was developed and applied in porcine knees for the measurement of articular cartilage thickness in articulated joints. The feasibility of the approach was assessed by implementing the method for the lateral femoral condyle from harvested limbs. Measurement of the undeformed cartilage thickness with this method was found to have a great degree of precision, with approximately a 1.0% (14 m) mean variation. Accuracy was also high when compared to an optical method for measuring the true cartilage thickness. An excellent linear correlation r² > 0.99) between the thickness determined optically and that obtained from the radiographic images was demonstrated. No significant differences were found between these two measures of cartilage thickness. This method, which minimizes disturbance to the structures of the knee to maintain its physiologic environment, also has promise to measure the changes in cartilage thickness as the tissue is deformed during joint loading. Information about the undeformed and deformed cartilage thicknesses can be combined with finite element models to examine cartilage behavior from both experimental and theoretical perspectives. © 1998 Biomedical Engineering Society. PAC98: 8759Hp, 0630Bp, 0705Pj     

冷冻保存异体骨膜移植治疗髋关节骨性关节炎

目的：报道采用冷冻保存胚胎颅骨骨膜移植修复髋关节软骨大面积缺损的治疗结果。方法：自1990年5月—1994年4月，对42例(47侧)髋关节软骨全厚缺损采用冷冻保存胚胎颅骨骨膜移植进行修复，其中14例股骨头骨质重度坏死者，同时施行带旋髂深血管髂骨植骨。对34例(38侧)髋关节进行了2～6年(平均40个月)随访。结果：按照吴之康髋关节人工置换术后疗效评定标准．术前平均得分6．4分，术后平均得分15.8分，优良25例，很好5例，好3例，尚可1例。结论：与自体移植物修复关节软骨大面积缺损相比，本方法无附加损伤，具有移植材料来源充分，取材量大．形态与股骨头相似等特点，是治疗髋关节软骨大面积缺损的一种有效方法。     

鱿鱼软骨Ⅱ型胶原蛋白提取方法及结构分析

目的从鱿鱼软骨中分离纯化非变性II型胶原蛋白,并对其进行结构表征。方法将鱿鱼软骨冻干后粉碎成200目软骨粉,用4%(w/w)Na OH溶液去除多糖、0.5 M EDTA溶液脱灰对软骨进行前处理,采用胃蛋白酶水解提取,Na Cl盐析用纯水透析后冷冻干燥得到II型胶原蛋白样品。通过氨基酸组成分析、SDS-PAGE电泳、紫外吸收光谱、电镜扫描等对制备的样品鉴定。结果制备的II型胶原蛋白具有3条α1链,分子量112 k Da,在224.5 nm波长处有最大紫外吸收,具有三螺旋结构特征。     

离子造影剂增强Micro-CT扫描对大鼠关节软骨形态的定量分析

利用Micro-CT技术的较高分辨精度,探索一种新的测量小动物关节软骨形态的方法,为进一步利用小动物模型开展关节软骨的损伤和退变等研究提供基础。选择不同浓度的造影剂及Micro-CT扫描条件,通过造影剂对比增强Micro-CT扫描大鼠膝关节,对大鼠股骨远端关节软骨进行手动分割、重建,得到软骨3D模型,利用重建得到的3D模型,对软骨的厚度和体积等形态学参数进行无创定量分析。根据造影剂、软骨和软骨下骨对X射线吸收的平均相对衰减值差异,确定30%的离子造影剂浓度为最佳。利用该浓度对4个不同的正常大鼠关节软骨进行扫描和重建,分析获得关节软骨的体积和厚度,并求得关节软骨体积和厚度的变异系数均方根分别为7.3%和6.1%。结果提示,利用造影剂增强Micro-CT扫描能够对软骨形态重要参数厚度、体积等进行定量评价。     

聚乙烯醇水凝胶及其复合物的研究与在人工软骨替代材料上的应用

聚乙烯醇水凝胶是一种具有良好生物相容性和力学性能的高弹性材料。本文介绍了聚乙烯醇水凝胶的成型方法，复合改性技术的研究进展，着重对聚乙烯醇水凝胶作为替代材料在关节软骨损伤修复中的研究现状和存在问题进行了综述，并概述了其应用前景和发展方向。     

Biological Repair of Thyroid Cartilage Defects by Osteogenic Protein-1 (Bone Morphogenetic Protein-7) in Dog

The efficacy of human recombinant osteogenic protein-1 (OP-1; bone morphogenetic protein-7) in regeneration of dog larynx was examined by treating thyroid cartilage defects (1.5 cm²) in dogs with thyroid allografts covered with host perichondrium or fascia. Prior to implantation allografts were frozen, thawed and demineralized. The treatment groups were as follows: I - Allograft control implant (n = 3); II - Implants coated with 500 μg OP-1 (n = 4); III - Implants coated with 100 μg OP-1 (n = 3); IV - Implants coated with 500 μg OP-1 and covered with neck fascia (n = 3); and V - Implants extracted with 1M NaCl and guanidine hydrochloride, and coated with 500 μg OP-1 (n = 4). Dogs were sacrificed four months following surgery. Each larynx was removed, carefully dissected and a three-dimensional reconstruction of the defect area was performed on serial sections. The results revealed that the implants of control dogs remained intact with no apparent reduction in size and new tissue formation. OP-1 enriched thyroid allografts, dose dependently induced bone, cartilage and ligament-like structures comprising up to 80% of the total regenerated defect area. Boundaries of the defects healed by formation of new bone when bone resided within the old thyroid cartilage layers. Old cartilage not containing bone within its layers healed by complete integration with newly formed cartilage. Both new bone and cartilage were embedded into layers of new ligament-like tissue which expressed specific morphologic and molecular markers. The three newly formed tissues were tightly connected into a “bone-cartilage-ligament continuum” of tissues, suggesting that OP-1 served as a multiple tissue morphogen in this specific microenvironment.     

Expression of Keratinocyte Growth Factor and Its Receptor in Rat Tracheal Cartilage: Possible Involvement in Wound Healing of the Damaged Cartilage

Keratinocyte growth factor (KGF) is involved in the development and regeneration of a variety of tissues. To clarify the role of KGF in cartilage wound healing, we examined the expression of KGF and its receptor (KGFR) immunohistochemically in the wound healing area of rat tracheal cartilage, and the direct effect of recombinant KGF on the proliferation and differentiation of primary cultures of rat chondrocytes. KGF was found in the cytoplasm of both chondrocytes and perichondrial cells. On the other hand, KGFR was detected only in the plasma membrane of chondrocytes. Although the expression of KGF was similar in the cartilage and perichondrial area before and after injury, KGFR expression was induced after injury and limited to proliferating chondrocytes. The staining pattern of KGF and KGFR was same in the mature and the immature rat tracheal cartilage. Moreover, in vitro experiments using primary cultured chondrocytes revealed that KGF at 200 ng/ml significantly increased the number of chondrocytes (~1.5-fold), and significantly reduced acid mucopolysaccharide production. These results indicate that KGF stimulates chondrocyte proliferation, suggesting that KGF could therapeutically modulate the wound healing process in the tracheal cartilage.     

Association of the C-Propeptide of Type II Collagen with Mineralization of Embryonic Chick Long Bone and Sternal Development

Several proteins may play a role in bone formation. The C-propeptide of type II collagen is intimately associated with endochondral bone formation in bovine growth plate. We have used an antibody against this peptide to determine its immunofluorescent distribution in early stages of embryonic chick limb development with emphasis on first bone formation which occurs in the mid-diaphyseal region. The C-propeptide II is first evident by immunofluorescent localization at stage 27 (day 5-6) of embryonic tibia development with chondrocytes in the central mid-diaphysis. In subsequent stages, there is an increase in the number of chondrocytes in which it is localized in discrete vacuoles. Up to stage 30, immunofluorescence is observed intracellularly, after which it appears in the matrix. The released C-propeptide II appears to remain only transiently associated with the cartilage matrix and becomes concentrated in the calcifying periosteum, the region outside of the cartilage core where bone formation first occurs in a sequence of events comparable to intramembranous bone formation.

These observations can be reproduced in cultures of stage 35 hypertrophic chondrocytes (core cells) and periosteum cells (collar cells). Core cells contain intensely stained intracellular vacuoles while collar cells are negative, although the collar cell osteogenic matrix concentrates exogenously added C-propeptide II. Double label immuno-staining shows that the C-propeptide II, unlike type II collagen and proteoglycan, which are secreted and incorporated into extracellular sites, is initially stored in intracellular vacuoles. The matrix localization of the C-propeptide II during the transition from cartilage to bone indicates a close association with the initiation of mineralization events of cartilage and bone and its specific origin in chondrocytes and not osteoblasts. These observations suggest that the C-propeptide II made by chondrocytes is associated with the formation of bone.     

Optimization of immunohistochemical detection of collagen type II in osteochondral sections by comparing decalcification and antigen retrieval agent combinations

Bone containing tissues such as osteochondral joint are resistant to routine tissue processing, therefore require decalcification. This technique causes removal of mineral salts, but in the process may macerate the organic tissue, hence the need for tissue fixation. Such severe processing demands careful antigen retrieval to necessitate optimal staining. The aim of our study was to compare five different antigen retrieval protocols (heat retrieval and protein digestion) following decalcification of rabbit knee joints using two different techniques (20% formic acid and 10% ethylenediamine-tetra acetic acid: EDTA). Osteochondral sections were compared based on time required for decalcification, ease of sectioning, morphological integrity using HE staining and antigen preservation (Collagen type II) using immunohistochemistry. The two decalcification solutions did not impair the tissue morphology and ease of sectioning. Joints processed with formic acid decalcified four times faster than EDTA. Among the five antigen retrieval approaches, maximal collagen II uptake with minimal nonspecific staining was found with protein digestion (pronase and hyaluronidase) in both formic acid and EDTA sections. For osteo-chondral sections, we recommend using 10% EDTA for decalcification and pronase plus hyaluronidase for antigen retrieval if maintaining tissue morphology is crucial, whereas if time is of the essence, 20% FA with pronase plus hyaluronidase is the faster option while still preserving structural integrity. Clin. Anat. 33:343–349, 2020. © 2019 Wiley Periodicals, Inc.     

提高关节软骨脱细胞支架孔隙率及细胞渗透性方法的研究进展

关节软骨损伤后的再生与修复一直是临床上的难题。由于外科治疗手段具有较大的局限性,近年来,软骨组织工程领域不断发展并备受关注。在软骨组织工程中,具有软骨诱导特性的软骨基质,是一种很有前景的生物材料。这种天然的生物材料能够作为支架,为软骨组织再生提供结构框架和生物信号分子。脱细胞处理能够有效去除软骨组织中的细胞成分,获得天然的细胞外基质支架。然而,由于软骨组织具有缺乏血管、结构致密、细胞成分较少的特性,因此,经脱细胞的软骨组织能否获得足够的孔隙率,为再植的软骨细胞或干细胞提供有效的渗透途径,逐渐成为研究者关注的焦点。重点关注关于提高细胞渗透性的实验研究,总结提高支架孔隙率以及促进细胞迁移的实验方法,主要包括脱细胞处理、浓度调控、取向性冻干技术、人为制备孔道,以及激光刻蚀等。通过文献检索,对不同方法技术的原理与优势进行总结、分析,进一步强调孔隙结构在支架性能中的重要性,并阐明支架性能与调控方法之间的关系。     

Immunohistochemical detection of the c-erbB-2 proto-oncogene product in normal, benign and malignant cartilage tissues

Benign and malignant cartilage tumours as well as normal cartilage were stained immunohistochemically with antibodies raised to a synthetic peptide from the sequence of the c-erbB-2 protein. Positive staining was present in 18/23 of the chondrosarcomas and in one case of osteochondroma. Normal and benign neoplastic cartilage tissues revealed negative results. It is concluded that expression of the c-erbB-2 protein may contribute to cell transformation in chondrosarcomas; it is not apparent whether the expression of c-erbB-2 protein in chondrosarcomas characterizes a subpopulation of different prognostic significance.     

The Concentration,Gene Expression,and Spatial Distribution of Aggrecan in Canine Articular Cartilage,Meniscus, and Anterior and Posterior Cruciate Ligaments: A New Molecular Distinction Between Hyaline Cartilage and Fibrocartilage in the Knee Joint

The concentration, spatial distribution, and gene expression of aggrecan in meniscus, articular cartilage, and the anterior and posterior cruciate ligaments (ACL and PCL) was determined in the knee joints of five mature dogs. An anti-serum against peptide sequences specific to the G1 domain of aggrecan was employed in competitive-inhibition ELISA of guanidine HCl extracts and immunofluorescence microscopy. Gene expression was determined by Taqman real-time PCR. The concentration of aggrecan in articular cartilage (240.1 ± 32 nMol/g dry weight) was higher than that in meniscus (medial meniscus: 33.4 ± 4.3 nMol/g) and ligaments (ACL: 6.8 ± 0.9 nMol/g). Aggrecan was more concentrated in the inner than the outer zone of the meniscus. Aggrecan in meniscus showed an organized, spatial network, in contrast to its diffuse distribution in articular cartilage. Thus, differences in the concentration, gene expression, and spatial distribution of aggrecan constitute another molecular distinction between hyaline cartilage and fibrocartilage of the knee.     

Articular chondrocytes and mesenchymal stem cells seeded on biodegradable scaffolds for the repair of cartilage in a rat osteochondral defect model

This work investigated the ability of co-cultures of articular chondrocytes and mesenchymal stem cells (MSCs) to repair articular cartilage in osteochondral defects. Bovine articular chondrocytes and rat MSCs were seeded in isolation or in co-culture onto electrospun poly(?-caprolactone) (PCL) scaffolds and implanted into an osteochondral defect in the trochlear groove of 12-week old Lewis rats. Additionally, a blank PCL scaffold and untreated defect were investigated. After 12 weeks, the extent of cartilage repair was analyzed through histological analysis, and the extent of bone healing was assessed by quantifying the total volume of mineralized bone in the defect through microcomputed tomography. Histological analysis revealed that the articular chondrocytes and co-cultures led to repair tissue that consisted of more hyaline-like cartilage tissue that was thicker and possessed more intense Safranin O staining. The MSC, blank PCL scaffold, and empty treatment groups generally led to the formation of fibrocartilage repair tissue. Microcomputed tomography revealed that while there was an equivalent amount of mineralized bone formation in the MSC, blank PCL, and empty treatment groups, the defects treated with chondrocytes or co-cultures had negligible mineralized bone formation. Overall, even with a reduced number of chondrocytes, co-cultures led to an equal level of cartilage repair compared to the chondrocyte samples, thus demonstrating the potential for the use of co-cultures of articular chondrocytes and MSCs for the in vivo repair of cartilage defects.     

Immunohistochemical Demonstration of Type II Collagen in the Chondroid Tissue of Pleomorphic Adenomas of the Salivary Glands

A study was done to investigate the presence of type II collagen and elastin in the metaplastic chondroid tissue of 21 pleornorphic adenomas of the major and minor salivary glands. Type II collagen was detected with anti-bovine type II collagen antibody after double digestion of histological sections with trypsin and hyaluronidase. The immunoreaction was positive in the chondrocytic cells and intercellular matrix. Elastic fibers in the chondroid tissue were found by orcein staining; they were scarce and randomly distributed. Although the presence of type II collagen and elastin in the metaplastic chondroid tissue is not directly implicated in the genesis of the tumor, it reveals a unique and high grade of cellular differentiation in comparison with true cartilage.