Kniest dysplasia. A histochemical study of the growth plate

Chondro-osseous tissue from four patients with the Kniest dysplasia was studied histochemically using a new plastic embedding technique. Extensive vacuolar changes were observed p--1 throughout the endochondral growth plate and adjacent resting cartilage. These changes occurred within the cartilage matrix and also in the lacunae of degenerating chrondrocytes. The septa of the lesions contained chondroitin sulfate, but little keratan sulfate or collagen. Resting cartilage not adjacent to the growth plate stained irregularly and showed few of the vacuolar lesions, and chondrocytes were enlarged and contained cytoplasic inclusions, but no vacuolar material. Thus, there appears to be a sequence of events initiated by cellular accumulation of a substance and progressing to cellular and matrix degeneration.     

Diastropic dwarfism: a histochemical and ultrastructural study of the endochondral growth plate.

Chondro-osseous tissue from five patients with diastropic dwarfism was studied by histologic, histochemical, and electron microscopic methods. The major abnormalities observed were: 1) irregular distribution of chondrocytes undergoing degeneration in the resting cartilage; 2) abnormal distribution of collagens in the resting cartilage; 3) a spectrum of fibrous matrix lesions in the resting cartilage which ranged from focal areas of aggregated collagen fibrils to large cystic lesions in which intracartilagenous ossification occurred; and 4) shortened, irregular cellular columns within the growth plate which were occasionally disrupted by matrix lesions extending from the resting cartilage. These alterations in chondro-osseous morphology have not been observed in any of the other skeletal dysplasias examined to date and appear to be pathognomonic for this disorder.     

Collagen type II in Langer-Saldino achondrogenesis: absence of major abnormalities in a less severe case

Collagen extracted either from cartilage or synthesized in vitro was analyzed to identify possible molecular defects in the cartilaginous matrix of a male fetus suffering from a mild form of type II achondrogenesis (Langer-Saldino). The tissue architecture of the patient's cartilage was markedly altered and showed numerous fibrous vascular canals which were focally stained by antibodies against collagens I and III. Collagen II was present, although heterogenously distributed throughout the cartilaginous matrix. Upon electrophoretic separation, however, the patient's femoral head cartilage showed the presence of collagens II, IX and XI only, which was similar to an age-matched control. The hydroxyproline/hydroxylysine ratio of collagen II of the patient was not significantly different from that of the control. Likewise, the compositions of collagens synthesized by cultured chondrocytes as well as fibroblasts were similar in the patient and the control. The results provide strong evidence that, in the present mild case of Langer-Saldino achondrogenesis, collagen II is expressed and regularly hydroxylated at its lysyl residues. This may indicate that cartilage components other than collagen II may be responsible for the altered tissue organization observed. Along with previous observations, our data suggest that the degree of biochemical matrix alterations may be related to the severity of the clinical phenotype.     

Histological Analysis of Triphalangism Associated with Polydactyly of the Thumb

ABSTRACT Wassel's classification of thumb polydactyly has some problems especially in categories IV and VII because it depends solely on X-ray findings. In order to improve his thumb polydactyly classification, we examined histologically untreated 182 bifid thumbs from 171 patients at Branch Hospital, Nagoya University in the past eight years. Ninety-five of these cases had proximal phalangeal separate type of polydactyly corresponding to Wassel's categories IV and VII. Forty-five thumbs of these categories were excised from 44 patients and classified into type I (24 cases), type II (12). type III (4), and type IV (5) in accordance with Kanno's classification. In distal phalanges, normal cartilage development was observed in Kanno's type I, whereas proximally elongated cartilaginous tissue was formed in the type II. Three distal phalanges from types II and III showed depression at the ulnar side of elongated cartilage of the distal phalanx where immature chondrocytes transversely distributed. Three cases with incomplete and one case with complete joint formation were formed in elongated cartilage of the distal phalanx, and endochondral ossification developed in the central area of elongated cartilage of the distal phalanx in four cases from types III and IV. All of the cases showed only minimal mobility of their involved joints. Their tendon development did not correspond to their anatomical characteristics. Histological studies indicated that there was no distinct difference between Wassel's category IV and category VII. It was postulated that the triphalang-     

Atelosteogenesis syndromes: a review, with comments on their pathogenesis

The clinical, radiographic, and morphological findings in 25 cases of atelosteogenesis and boomerang dysplasia have been reviewed. The review confirms the nosologic grouping of atelosteogenesis type I with boomerang dysplasia and the clinical and radiographic overlap of features between atel- osteogenesis I and atelosteogene- sis II (synonymous with De la Chapelle dysplasia) and a group of patients with atelosteogenesis type III. A common pathogenesis is suggested for atelosteogenesis type I and boomerang dysplasia. A marked excess of male fetuses with boomerang dysplasia was observed. Atelosteogenesis type II shows distinctive chondro-osseous histopathology with a major disturbance in cartilage matrix macromolecules. An overlap of phenotypic, radiographic, morphological, and cartilage histochemical features with those observed in diastrophic dysplasia and achondrogenesis type IB suggests that atelosteogenesis type II has common pathogenetic features with disorders of sulfation of connective tissue matrix macro- molecules. Received/accepted: 8 December 1995     

Osteogenesis imperfecta: contribution to pathobiochemistry

Osteogenesis imperfecta (OI) is a hereditary disease characterized by increased bone fragility and marked skeletal deformities. As a generalized connective tissue disorder, many patients present with other typical symptoms, such as blue sclerae, dentinogenesis imperfecta, impaired hearing, joint laxity, and easy bruising of the skin. According to clinical and genetic characteristics, Sillence classified four different groups. Metabolic alterations of connective tissue components are thought to be responsible for the pathogenesis of OI. Collagen, the main constituent of connective tissue, was analyzed in autoptic tissue and/or skin fibroblasts from patients with OI. In fibroblast culture of patients with OI group I, the range of synthesized collagen type III is elevated to 15-48% (normal up to 15%). Patients in groups II and III show an increased presence of hydroxylysine in alpha-chains of collagen types I and III. The hydroxylation of lysyl residues of the cartilage specific collagen type II is slightly elevated. In both groups, the hydroxypyroline content in all tested collagen types was normal. In our investigation, the collagen of patients with OI group IV appeared normal. Although the clinical features of patients with OI of all groups were not homogeneous, OI group III and some subtypes of group II had similar clinical courses and biochemical findings. In addition, there are patients with OI who present with clinical symptoms, but who cannot be classified into any of the known groups. For a better differentiation, biochemical examination of collagen should be performed complementary to clinical and genetic criteria.     

Collagen tissue engineering: development of novel biomaterials and applications

Scientific investigations involving collagen have inspired tissue engineering and design of biomaterials since collagen fibrils and their networks primarily regulate and define most tissues. The collagen networks form a highly organized, three-dimensional architecture to entrap other ingredients. Biomaterials are expected to function as cell scaffolds to replace native collagen-based extracellular matrix. The composition and properties of biomaterials used as scaffold for tissue engineering significantly affect the regeneration of neo-tissues and influence the conditions of collagen engineering. The complex scenario of collagen characteristics, types, fibril arrangement, and collagen structure-related functions (in a variety of connective tissues including bone, cartilage, tendon, skin and cornea) are addressed in this review. Discussion will focus on nanofibrillar assemblies and artificial synthetic peptides that mimic either the fibrillar structure or the elemental components of type I collagen as illustrated by their preliminary applications in tissue engineering. Conventional biomaterials used as scaffolds in engineering collagen-containing tissues are also discussed. The design of novel biomaterials and application of conventional biomaterials will facilitate development of additional novel tissue engineering bioproducts by refining the currently available techniques. The field of tissue engineering will ultimately be advanced by increasing control of collagen in native tissue and by continual manipulation of biomaterials.     

Achondrogenesis type I: Light and electron-microscopic studies

The light- and electron-microscopic structure of articular and costal cartilage in a case of achondrogenesis type I has been described. The most characteristic ultrastructural change in the chondrocytes was conspicuous dilatation of the rough endoplasmatic reticulum (RER) which contained amorphous electronopaque material. This change in the RER was accompanied by marked hypertrophy of the Golgi apparatus; the matrix was basically unchanged.Professor E. Uehlinger in honour of his 80th birthday     

Achondrogenesis Type IA (Houston-Harris): a still-unresolved molecular phenotype.

Achondrogenesis type IA (Houston-Harris) is an extremely rare lethal chondrodysplasia with a characteristic severe disarrangement of endochondral ossification. The growth plate cartilage completely lacks columnar-zone formation and shows chondrocyte expansion due to intracellular vacuoles. This article on a new case of achondrogenesis type IA confirms these findings and demonstrates, on the ultrastructural level, the retention of fine fibrillar material within the rough endoplasmic reticulum (rER). Molecular analysis in the presented case of achondrogenesis type IA did not reveal mutations in the COL2A1 and SLC26A2 genes, which are known to cause achondrogenesis types IB and type II. Although the extracellular cartilage matrix was severely altered, all of the investigated matrix molecules (collagens, aggrecan, matrilins, cartilage oligomeric protein [COMP]) showed a normal distribution pattern. The only exception was type-X collagen, which was significantly reduced. Overall, our study suggests a disturbance in cartilage matrix assembly in the present case due to the retention of some sort of matrix component within the rER. Presumably, as a consequence of this event, processes of chondrocyte maturation and differentiation and endochondral bone formation are severely affected in this case of achondrogenesis type IA.     

组织工程化骺板软骨修复兔部分骺板损伤的实验研究

目的 利用组织工程骺板软骨修复兔骺板缺损,防止肢体畸形发生.方法 取2周龄兔骺板软骨细胞,体外复合牛关节软骨细胞外基质后植入4周龄兔右侧股骨远端骺板缺损处,左侧仅造成缺损,无填充物,作为自身空白对照.于每周行X线检查,4、8、12、16周时行组织学和免疫组化检查.结果 X线显示移植侧股骨畸形明显比对侧轻,骺板缺损处未见明显骨桥生成,被结构紊乱的软骨组织填充,具有一定的生长能力.空白对照侧骨桥形成,骺板早闭.组织学显示移植侧骺板较窄,排列紊乱.免疫组化结果显示移植处染色阳性.结论 组织工程方法培养出骺板软骨不仅可以阻止骨桥产生,还具有一定的生长能力,有望成为治疗骺板早闭的一种合适的材料.     

组织工程化骺板软骨修复兔部分骺板损伤的实验研究

目的 利用组织工程骺板软骨修复兔骺板缺损,防止肢体畸形发生.方法 取2周龄兔骺板软骨细胞,体外复合牛关节软骨细胞外基质后植入4周龄兔右侧股骨远端骺板缺损处,左侧仅造成缺损,无填充物,作为自身空白对照.于每周行X线检查,4、8、12、16周时行组织学和免疫组化检查.结果 X线显示移植侧股骨畸形明显比对侧轻,骺板缺损处未见明显骨桥生成,被结构紊乱的软骨组织填充,具有一定的生长能力.空白对照侧骨桥形成,骺板早闭.组织学显示移植侧骺板较窄,排列紊乱.免疫组化结果显示移植处染色阳性.结论 组织工程方法培养出骺板软骨不仅可以阻止骨桥产生,还具有一定的生长能力,有望成为治疗骺板早闭的一种合适的材料.

Abstract：

Objective The aim of this study is to establish the feasibility of acellular cartilage matrix as a scaffold for epiphyseal chondrocytes to form tissue engineering growth plate for the treatment of partial growth plate injury. Methods We collected and cultured the epiphyseal plate chondrocytes from 2 weeks old rabbit in vitro, seeded in acellular cartilage matrix to form a complex. It was then transferred to the rabbit right distal femur which was subjected to growth plate injury. The left femur was subjected to the same injury without further treatment (control). The femur recovery was monitored with weekly X-ray. The morphology was studied with hematoxylin- eosin stain and immunohistochemistry. Results The right femur recovery was significantly better than that of the left radiologically. Histology and immunohistochemistry studies revealed that the growth defects were filled with cartilaginous tissue, which has a similar structure as natural epiphyseal plate . On the left side (control), the epiphyseal defect was filled with bone bridge and epiphyseal plate growth was arrestted. Conclusions Epiphyseal cartilage tissue engineering prevents bone bridge generation and also has some growth potential. It may be a suitable treatment materials for repairing the epiphyseal plate defect     

Integrins in growth plate cartilage

The growth plate is a highly specialized layer of cartilage where chondrocytes proliferate and differentiate in order to bring forth longitudinal bone growth. Interaction of cells with the extracellular matrix significantly influences differentiation and growth. These processes involve integrin as well as nonintegrin cell surface receptors, like annexin V and CD44. In chondrocytes, which are surrounded by abundant matrix, tissue integrity and cell survival depend on well-tuned interactions of cells and ECM. In the field of growth research, besides classical growth stimulating factors acting via endocrine pathways, investigations on local determinants of cell differentiation and survival play an important role. This review describes the role and function of integrins, their occurrence in growth plate cartilage and their possible involvement in differentiation during growth.     

Hypochondrogenesis

Three clinicopathological observations of a mild form of type II achondrogenesis are presented. The cases were selected from a group of 21 similar cases to illustrate the various degrees of clinical and roentgenological signs that can be found. The cases had various survival periods after birth but not exceeding several months. The roentgenological signs were less severe than those of type II achondrogenesis. Some cases similar to case no. 3 have roentgenological signs very close to spondylo-epiphyseal dysplasia congenita and probably were confused previously with the latter. The name of hypochondrogenesis was proposed for these cases because the lesions of the growth plate are similar although less marked to those found in type II achondrogenesis: high cellularity with poor matrix development; irregular columnization and vascular penetration; large chondrocytes and even more enlarged lacunae; large sclerotic cartilage canals. The clinical and roentgenological diagnosis of hypochondrogenesis could be difficult especially in the less severe forms. The delay in vertebral ossification, the absence of all the epiphyseal nuclei and of the tarsal bones might suggest the diagnosis of hypochondrogenesis, rather than that of spondyloepiphyseal dysplasia. The evolution which seems to be always lethal in a period of several weeks or months would make the diagnosis still more likely and it could be confirmed by histopathological examination. Cases of spondylo-epiphyseal dysplasia congenita might have at birth, roentgenological signs indistinguishable from those of hypochondrogenesis, as was illustrated by case no. 4.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human tracheal chondrocytes as a cell source for augmenting stenotic tracheal segments: the first feasibility study in an in vivo culture system

INTRODUCTION: Construction of engineered respiratory tract using tissue-engineered cartilage has not yet been reported. In order to generate artificial trachea using human chondrocytes obtained from tracheal cartilage, we investigated whether human tracheal chondrocytes can act as a cell source to fabricate engineered airway patches to augment stenotic parts of the trachea. MATERIALS AND METHODS: After informed consent, chondrocytes were obtained from five patients who needed tracheal surgery. A small piece of resected tracheal cartilage was digested by collagenase type 2 for 3-4 h. This yielded chondrocytes, which were expanded in vitro and seeded onto biodegradable scaffolds; these were then implanted subcutaneously in athymic mice. The implanted constructs were retrieved 8 weeks later for histologic and biochemical analysis. RESULTS: In monolayer cultures, chondrocytes proliferated well, showing a 100- to 1,000-fold increase in 1 month. Once expanded, the cells lost their original morphological and biologic characteristics, but the engrafted scaffold showed histologic and biochemical characteristics of cartilage. Viable chondrocytes and extracellular matrix were detected, and glycosaminoglycan (GAG) in vivo was present. CONCLUSIONS: Here we show that a small piece of human tracheal cartilage can generate sufficient chondrocytes in vitro and form tracheal cartilage architecture in an in vivo environment.     

The type XI collagenopathies

Type XI collagen is a minor component of cartilage collagen. Mutations of the three genes coding for its proteins are expressed in a peculiar phenotype. Clinical changes include facial anomalies, cleft palate and hearing defects. Ocular changes occur in some disorders. Radiographs show a spectrum of epiphyseal dysplasias with wide metaphyses and spondylar abnormalities. The pattern of clinical and radiographic changes is also found in some type II collagenopathies. On the basis of molecular studies, three type XI collagenopathies have been defined: Stickler syndrome type II, and dominant and recessive oto-spondylo-megaepiphyseal dysplasia (OSMED). Stickler syndrome I and Kniest dysplasia are type II collagenopathies with considerable clinical and radiographic overlap. Inborn errors of cartilage collagen formation lead to a family of genetically heterogeneous but pathogenetically related and hence phenotypically similar disorders. The type XI collagenopathies are part of this family. Received/accepted: 5 June 1998     

Achondrogenesis type I: delineation of further heterogeneity and identification of two distinct subgroups

Achondrogenesis has traditionally been divided into type I (Parenti-Fraccaro) and type II (Langer-Saldino). We studied the clinical, radiologic, and morphologic features of 17 cases previously diagnosed as achondrogenesis type I to define whether there is even further heterogeneity. On radiographic analysis, two distinct groups of patients were defined based on the presence or absence of rib fractures and ossification of the vertebral pedicles, ischium, and fibula. Two distinct chondroosseous morphologic patterns were observed that directly correlated with the radiographic grouping. One group had round vacuolated chondrocytes with inclusion bodies; the other had collagenous rings around the chondrocytes. We conclude that achondrogenesis type I (Parenti-Fraccaro) consists of two distinct disorders: type IA, which corresponds to the cases originally published by Houston et al. and Harris et al., and type IB, which corresponds to the case originally published by Fraccaro. Analysis of Parenti's case suggests the diagnosis of achondrogenesis type II. All three types of achondrogenesis appear to be inherited as autosomal recessive traits.     

Presence of type III collagen in bone from a patient with osteogenesis imperfecta

Samples of bone from a patient with osteogenesis imperfecta were found to synthesize and contain type III collagen as well as type I collagen. Normal bone contains only type I collagen except in the lining cells of the bone marrow cavities. In the patient's tissue, type III collagen was localized in nonfibrillar structures in discrete areas of the bone. These and previous studies indicate that certain types of osteogenesis imperfecta may be caused by a failure of normal bone maturation and the sites in which the type III collagen is found appear to be defects in the bone.     

短周期动态压应力通过Ca2+通道调控生长板软骨细胞Sox9及Ⅱ型胶原表达

目的 研究短周期动态压应力对体外培养的大鼠生长板软骨细胞Sox9、Ⅱ型胶原mRNA及蛋白表达水平的影响,探讨L-型钙离子通道是否参与该力学信号转导过程.方法 分离并体外培养两周龄SD大鼠生长板软骨细胞,传代后的生长板软骨细胞分为4组:对照组、单纯压应力组、硝苯地平组及压应力联合硝苯地平组.其中对照组不施加力学及药物干预,单纯压应力组通过四点弯曲力学加载仪予以周期性压应力(2 000με,1.0 Hz)干预,硝苯地平组添加L-型钙离子拮抗剂硝苯地平(10 μmol/L)但不施加力学干预,压应力联合硝苯地平组添加硝苯地平(10 μmol/L)并施加周期性压应力干预(2000με,1.0 Hz).实验处理6h,运用荧光定量PCR及Western blot方法检测各组细胞的Sox9、Ⅱ型胶原mRNA和蛋白表达情况.各组细胞行Fluo-3/AM染色,激光共聚焦显微镜观察并分析细胞内钙离子的荧光强度.使用SPSS17.0软件对数据进行统计学分析.结果 单纯压应力组生长板软骨细胞的Sox9的mRNA与蛋白表达水平较对照组分别增加(82.97±24.95)％和(79.67±26.03)％,差异有统计学意义(P＜0.05);Ⅱ型胶原的mRNA与蛋白表达水平较对照组分别增加(118.93±34.40)％和(95.93±29.87)％,差异有统计学意义(P＜0.05).压应力联合硝苯地平组生长板软骨细胞的Sox9、Ⅱ型胶原的mRNA与蛋白表达水平低于单纯压应力组(P＜0.05),但高于硝苯地平组(P＜0.05).激光共聚焦显微镜能清晰观察到细胞内钙离子染色后产生的荧光,但各组平均荧光强度差异无统计学意义(P＞0.05).结论 适当的短时间周期性压应力能上调生长板软骨细胞Sox9及Ⅱ型胶原mRNA与蛋白的表达水平,钙离子通道可能参与该力学信号转导过程.     

Morphologic and metabolic development of human fetal epiphyseal chondrocytes in primary culture

Primary chondrocyte culture was carried out after enzymatic digestion of femoral and tibial epiphyseal cartilage of human fetuses, collected with informed parental consent within 12 h postmortem. Chondrocytes were cultured in HAM F-12 medium with penicillin and 15% serum. Three types of serum were used: human placental cord serum (HPS), fetal calf serum, and human male adult serum. Chondrocytes cultured with HPS grew as monolayers, formed abundant colony groups with a highly metachromatic pericellular matrix, and floating round cells were observed in the culture medium. By the 10th day of culture the great majority of proteoglycans present in the culture medium were found as aggregates. Chondrocytes cultured with fetal calf serum or human male adult serum grew as monolayers, were polygonal in shape, and the pericellular matrix was far less developed than in HPS cultures. By the confluent phase of growth, only approximately a third of the proteoglycans present in the culture medium were found as aggregates. Chondrocytes cultured with HPS proliferated significantly more rapidly than those cultured with fetal calf serum or human male adult serum. The results suggest that certain, as yet unidentified, factors are present in sufficient amount in HPS to allow chondrocytes in culture to retain phenotypic morphological and biochemical characteristics. HPS also facilitates growth of human fetal epiphyseal chondrocytes in culture. Primary human fetal epiphyseal chondrocyte culture could be a suitable experimental tool for the in vitro study of biochemical characteristics of cartilage and factors involved in fetal cartilage metabolism.     

儿童正常生长规律及其调控机制

生长是一个连续非线性的过程,不同时期的调控机制及生长特点不尽相同。骨骼的生长受多种因素的影响,而软骨生长板是骨骼生长和发育的关键。包括营养、内分泌激素、炎症细胞因子、旁分泌信号、细胞外基质等在内的多种调控机制通过调节生长板软骨细胞的成熟与分化在骨骼的生长过程中发挥作用。掌握儿童正常生长规律和调控机制对矮身材儿童的诊断、鉴别诊断及此后的规范化治疗具有极其重要的作用。