Cellular stages in cartilage formation as revealed by morphometry,radioautography and type II collagen immunostaining of the mandibular condyle from weanling rats

The role played by cell addition, cell enlargement, and matrix deposition in the endochondral growth of the condyle was assessed in weanling rats by four approaches making use of the light microscope: morphometry, ³H-thymidine radioautography, ³H-proline radioautography, and immunostaining for the cartilage-specific type II collagen. From the articular surface down, the condyle may be divided into five layers made up of cells embedded in a matrix: (1) the articular layer composed of static cells in a matrix rich in fibers presumed to be of type I collagen, (2) the polymorphic cell layer including the progenitor cells from which arise the cells undergoing endochondral changes, (3) the flattened cell layer in which cells produce a precartilagenous matrix devoid of type II collagen while undergoing differentiation in two stages: a “chondroblast” stage and a short “flattened chondrocyte” stage when Intracellular type II collagen elaboration begins, (4) the upper hypertrophic cell layer, in which cells are “typical chondrocytes” that enlarge at a rapid rate, actively produce type II collagen, and deposit it into a cartilagenous matrix, and (5) the lower hypertrophic cell layer, composed of chondrocytes at a stage of terminal enlargement while the cartilagenous matrix is adapting for mineralization. ³H-thymidine radioautographic results indicate that the turnover time of progenitor cells in the polymorphic cell layer is about 2.9 days. The time spent by cells at each stage of development is estimated to be 1.4 days as chondroblasts, 0.5 days as flattened chondrocytes, 2.3 days as the chondrocytes of the upper hypertrophic cell layer, and 1.1 days as those of the lower hypertrophic cell layer. Calculations referring to a 1 × 1-mm square-sided column extending from the articular surface to the zone of vascular invasion provide the daily rate of cell addition (0.0077 mm³), extracellular matrix deposition (0.0127 mm³), and cell enlargement (0.0302 mm³). Hence the respective contribution of the three factors to condyle growth is in a ratio of about 1:1.6:4. This result emphasizes the role played by cell enlargement in the overall growth of the condyle.     

Regenerative Potential of Mandibular Condyle Cartilage and Bone Cells Compared to Costal Cartilage Cells When Seeded in Novel Gelatin Based Hydrogels

The field of temporomandibular joint (TMJ) condyle regeneration is hampered by a limited understanding of the phenotype and regeneration potential of cells in mandibular condyle cartilage. It has been shown that chondrocytes derived from hyaline and costal cartilage exhibit a greater chondro-regenerative potential in vitro than those from mandibular condylar cartilage. However, our recent in vivo studies suggest that mandibular condyle cartilage cells do have the potential for cartilage regeneration in osteochondral defects, but that bone regeneration is inadequate. The objective of this study was to determine the regeneration potential of cartilage and bone cells from goat mandibular condyles in two different photocrosslinkable hydrogel systems, PGH and methacrylated gelatin, compared to the well-studied costal chondrocytes. PGH is composed of methacrylated poly(ethylene glycol), gelatin, and heparin. Histology, biochemistry and unconfined compression testing was performed after 4 weeks of culture. For bone derived cells, histology showed that PGH inhibited mineralization, while gelatin supported it. For chondrocytes, costal chondrocytes had robust glycosaminoglycan (GAG) deposition in both PGH and gelatin, and compression properties on par with native condylar cartilage in gelatin. However, they showed signs of hypertrophy in gelatin but not PGH. Conversely, mandibular condyle cartilage chondrocytes only had high GAG deposition in gelatin but not in PGH. These appeared to remain dormant in PGH. These results show that mandibular condyle cartilage cells do have innate regeneration potential but that they are more sensitive to hydrogel material than costal cartilage cells.

     

Further evidence for the vitality of chondrocytes in the mandibular condyle as revealed by 35S-sulfate autoradiography

Histochemical and autoradiographic studies using ³⁵S-sulfate indicate that the majority of the cartilage cells in the developing mandibular condyle of the young mouse are active, vital cells. Concomitant with the increase of hypoxic conditions within the deeper layers of the cartilage, an increase in sulfated glycosaminoglucuronoglycans synthesis takes place. Hypertrophic chon-drocytes in the premineralized and mineralized zones reveal marked ³⁵S-sulfate uptake in comparison with the less differentiated cells in the chondroblastic and perichondrial zones. These observations of radiosulfate activity support the concept that calcification processes in the condylar cartilage are not necessarily accompanied by degeneration and death of the hypertrophic chondrocytes. The radiosulfate activity of the surviving chondrocytes in the vicinity of the ossification front indicates possible modulation into osteoprogenitor cells.     

Structure and growth activities of the mandibular condyle in monkeys (Macaca fascicularis): II. Synergistic behavior of cell dynamics and metabolism

The mandibular condyles of eight growing male monkeys (Macaca fascicularis) were analyzed by using a combination of radioautographic and morphometric techniques. This was done with the aim of examining the dynamics in the structure and growth activities of the articular tissue covering as well as of the subchondral zone of erosion. The animals received 1 mCi/kg body weight ³H-proline 24 hr and 0.5 mCi/kg body weight ³H-thymidine 3 hr prior to death. Their age was estimated on the basis of skeletal maturation as recorded from radiographs of the hand and wrist. Consistently, (1) the proliferative activity in the intermediate layer, (2) the rates of cell turnover and growth of chondroblasts and chondrocytes, (3) the rates of extracellular matrix production in the intermediate and chondral layers, as well as (4) the resorptive and (5) appositional activities in the zone of erosion were characterized by an in-concert behavior. This behavior suggests a general synergistic control of the various cell dynamic and metabolic processes affecting the rate of normal condylar growth.     

Tissue engineering the mandibular condyle

Tissue engineering provides the revolutionary possibility for curing temporomandibular joint (TMJ) disorders. Although characterization of the mandibular condyle has been extensively studied, tissue engineering of the mandibular condyle is still in an inchoate stage. The purpose of this review is to provide a summary of advances relevant to tissue engineering of mandibular cartilage and bone, and to serve as a reference for future research in this field. A concise anatomical overview of the mandibular condyle is provided, and the structure and function of the mandibular condyle are reviewed, including the cell types, extracellular matrix (ECM) composition, and biomechanical properties. Collagens and proteoglycans are distributed heterogeneously (topographically and zonally). The complexity of collagen types (including types I, II, III, and X) and cell types (including fibroblast-like cells, mesenchymal cells, and differentiated chondrocytes) indicates that mandibular cartilage is an intermediate between fibrocartilage and hyaline cartilage. The fibrocartilaginous fibrous zone at the surface is separated from hyaline-like mature and hypertrophic zones below by a thin and highly cellular proliferative zone. Mechanically, the mandibular condylar cartilage is anisotropic under tension (stiffer anteroposteriorly) and heterogeneous under compression (anterior region stiffer than posterior). Tissue engineering of mandibular condylar cartilage and bone is reviewed, consisting of cell culture, growth factors, scaffolds, and bioreactors. Ideal engineered constructs for mandibular condyle regeneration must involve two distinct yet integrated stratified layers in a single osteochondral construct to meet the different demands for the regeneration of cartilage and bone tissues. We conclude this review with a brief discussion of tissue engineering strategies, along with future directions for tissue engineering the mandibular condyle.     

Structure and growth activities of the mandibular condyle in monkeys (Macaca fascicularis): I. Intracondylar variations

Eight condyles of four growing monkeys (Macaca fascicularis) of estimated ages between 1.6 and 3.6 years (minimum and maximum) were analyzed using radioautographic, histometric, and stereologic techniques. The aim of the study was to examine the relationship between intracondylar variations in structure and growth activities. The animals received ³H-proline (1 mCi/kg body weight) and ³H-thymidine (0.5 mCi/kg body weight) 24 and 3 hours, respectively, prior to sacrifice. The perichondral and chondral layers of the condylar articular covering as well as the subchondral zone of erosion were examined at different sampling sites distributed systematically in the anteroposterior and lateromedial dimension of the articulating surface. Intracondylar variations observed with respect to morphometric and radioautographic parameters suggest the following biologic mechanisms contributing to mandibular growth in a superior-posterior direction. Greater mitotic activity at the central and posterior sites of the condylar perichondrium generates a population of progenitor cells that is larger in these than in other regions. On the other hand, the rate of differentiation of these progenitor cells into chondroblasts and chondrocytes, i.e., the “migration” into and through the chondral layers of the articulating covering, seems to be enhanced in the same superior and posterior areas. Additionally, while “migrating” faster, these cartilage cells become larger and produce greater amounts of extracellular matrix than those in the anterior parts of the condyle. Finally, enhanced resorptive activities in the superior and posterior regions of the subchondral zone of erosion provide an increased “loss” of degenerated chondrocytes, thereby establishing the basis for a cartilaginous drift in the superior-posterior direction.     

[3H]thymidine uptake in cells of rat condylar cartilage

Weanling rats were injected intraperitoneally with [³H]thymidine and sacrificed from 5 min to 20 days later. Their mandibular condylar cartilages were examined histologically, by thin-layer autoradiography, and by using liquid scintillation and microscopic counting methods. Labeled DNA appeared in some of the chondrocytes of the resting zone as early as 10 min postinjection, and reached the proliferative zone by 24 hr and the hypertrophic zone by 4 days. The labeling pattern in the last zone was more disperse, being oriented toward the periphery of the cells as they became hypertrophic. The maximum number of labeled chondrocytes was reached by 2 hr postinjection. These amounted to approximately 11% of the total chondrocyte population, the majority of which were located in the resting zone (73%). It is concluded that, over this period, the mitotic index for these cells is 50–60 per thousand resulting in approximately 100 labeled chondrocytes. In addition, some of the chondroclasts at the erosion front contained labeled DNA as early as 5 min after [³H]thymidine administration. By 10 min, 65% of these cells exhibited one or more labeled nuclei, and the ratio of labeled cells remained high through 20 days. Chondroclasts were seen to contain a diffuse label within their cytoplasm after 5 days. This label was similar to that seen in hypertrophic chondrocytes that had reached the erosion front by that time. Clearly, chondroclasts exhibit nuclear division and do not form from fusion of hypertrophic chondrocytes, although which specific mononuclear cells may act as chondroclast progenitors is not clear. In addition, these multinucleate resorbing cells are capable of ingesting or phagocytizing nuclear remnants from hypertrophic chondrocytes at the eroding face of cartilage.     

Morphological influence of ascorbic acid deficiency on endochondral ossification in osteogenic disorder Shionogi rat

The influences of chronic deficiency of L-ascorbic acid (AsA) on the differentiation of osteo-chondrogenic cells and the process of endochondral ossification were examined in the mandibular condyle and the tibial epiphysis and metaphysis by using Osteogenic Disorder Shionogi (ODS) rats that bear an inborn deficiency of L-gulonolactone oxidase. Weanling male rats were kept on an AsA-free diet for up to 4 weeks, until the symptoms of scurvy became evident. The tibiae and condylar processes of scorbutic rats displayed undersized and distorted profiles with thin cortical and scanty cancellous bones. In these scorbutic bones, the osteoblasts showed characteristic expanded round profiles of rough endoplasmic reticulum, and lay on the bone surface where the osteoid layer was missing. Trabeculae formation was deadlocked, although calcification of the cartilage matrix proceeded in both types of bone. Scorbutic condylar cartilage showed severe disorganization of cell zones, such as unusual thickening of the calcification zone, whereas the tibial cartilage showed no particular alterations (except for a moderately decreased population of chondrocytes). In condylar cartilage, hypertrophic chondrocytes were encased in a thickened calcification zone, and groups of nonhypertrophic chondrocytes occasionally formed cell nests surrounded by a metachromatic matrix in the hypertrophic cell zone. These results indicate that during endochondral ossification, chronic AsA deficiency depresses osteoblast function and disturbs the differentiation pathway of chondrocytes. The influence of scurvy on mandibular condyle cartilage is different from that on articular and epiphyseal cartilage of the tibia, suggesting that AsA plays different roles in endochondral ossification in the mandibular condyle and long bones.     

In situ hybridisation study of type I, II, X collagens and aggrecan mRNAs in the developing condylar cartilage of fetal mouse mandible

The aim of this study was to investigate the developmental characteristics of the mandibular condyle in sequential phases at the gene level using in situ hybridisation. At d 14.5 of gestation, although no expression of type II collagen mRNA was observed, aggrecan mRNA was detected with type I collagen mRNA in the posterior region of the mesenchymal cell aggregation continuous with the ossifying mandibular bone anlage prior to chondrogenesis. At d 15.0 of gestation, the first cartilaginous tissue appeared at the posterior edge of the ossifying mandibular bone anlage. The primarily formed chondrocytes in the cartilage matrix had already shown the appearance of hypertrophy and expressed types I, II and X collagens and aggrecan mRNAs simultaneously. At d 16.0 of gestation, the condylar cartilage increased in size due to accumulation of hypertrophic chondrocytes characterised by the expression of type X collagen mRNA, whereas the expression of type I collagen mRNA had been reduced in the hypertrophic chondrocytes and was confined to the periosteal osteogenic cells surrounding the cartilaginous tissue. At d 18.0 of gestation before birth, cartilage-characteristic gene expression had been reduced in the chondrocytes of the lower half of the hypertrophic cell layer. The present findings demonstrate that the initial chondrogenesis for the mandibular condyle starts continuous with the posterior edge of the mandibular periosteum and that chondroprogenitor cells for the condylar cartilage rapidly differentiate into hypertrophic chondrocytes. Further, it is indicated that sequential rapid changes and reductions of each mRNA might be closely related to the construction of the temporal mandibular ramus in the fetal stage.     

Light microscopic and ultrastructural observations of the calcifying zone of the mandibular condyle in the rat

The hypertrophic stage of development of the rat mandibular condyle was investigated in 16 and 26-day-old rats by electron microscopy. Interest was focused on the zone of mineralization and erosion. It was observed that capillaries invaded the lower level of the hypertrophic zone, without any previous chondroclastic resorption of calcified partitions. The partitions surrounding the hypertrophic chondrocytes were not mineralized around their entire circumference at the level of capillary penetration. The capillaries were accompanied by perivascular cells but these showed no similarities to chondroclasts. Multinucleated chondro- or osteoclasts were however present at a lower level of the subchondral area. It is suggested that there are no inherent differences with respect to the pattern of mineralization and erosion between the epiphyseal growth plate and the developing mandibular condyle.     

Light microscopic and ultrastructural observations of the calcifying zone of the mandibular condyle in the rat.

The hypertrophic stage of development of the rat mandibular condyle was investigated in 16 and 26-day-old rats by electron microscopy. Interest was focused on the zone of mineralization and erosion. It was observed that capillaries invaded the lower level of the hypertrophic zone, without any previous chondroclastic resorption of calcified partitions. The partitions surrounding the hypertrophic chondrocytes were not mineralized around their entire circumference at the level of capillary penetration. The capillaries were accompanied by perivascular cells but these showed no similarities to chondroclasts. Multinucleated chondro- or osteoclasts were however present at a lower level of the subchondral area. It is suggested that there are no inherent differences with respect to the pattern of mineralization and erosion between the epiphyseal growth plate and the developing mandibular condyle.     

Effects of lateral pterygoid muscle hyperactivity on differentiation of mandibular condyles in rats

Background: The effects of biomechanical stress on the growth and development of the mandibular condyle have been studied by many investigators. However, the role of the lateral pterygoid muscle in this development is not clear. Methods: Hyperfunction of the lateral pterygoid muscles of male 3-weekold Sprague-Dawley rats was induced by electrical stimulation, and the responses of the mandibular condyles were compared to control tissues by a double-fluorescent staining technique using polyclonal antibodies against type I and type II collagen. Electrical stimulation consisted of repeated application (5 seconds on/5 seconds off) of a Hz current for up to 7 days. Results: In the first 2 days, cartilaginous tissues rich in type II collagen disappeared in the anterior and posterior areas, which were loaded by tensional force due to direct and indirect attachment of the lateral pterygoid muscles. Tissues in these areas were replaced by intramembranous bone that was reactive for type I collagen at 7 days. By the end of the experiment, the trabecula of the condyle was remodled more perpendicularly, thus resisting the compressive force due to hyperfunction of the lateral pterygoid muscles. Conclusions: These results suggest that the activity of the lateral pterygoid muscle might play a significant role in the differentiation of progenitor cells and in the maturation and calcification of chondrocytes in mandibular condyles. © 1995 Wiley-Liss, Inc.     

Temporo-nasal asymmetry in the accretion of retinal ganglion cells in late larval and postmetamorphic Xenopus

Summary The spatial pattern of cell production and retinal growth were studied in Xenopus between stage 60 and two months after metamorphosis using ³H-proline and ³H-thymidine autoradiography. The position and the number of the ganglion cells labelled with ³H-thymidine were determined. The area of the unlabelled retina due to growth since ³H-proline administration at stage 60 was measured. Both retinal area measurements and counts of labelled ganglion cells showed 30–40% higher values in the temporal than in the nasal retinal half. The greater cell production and area accretion were even more pronounced between the temporal and the nasal retinal quadrants. The results on the temporoventral crescentic retinal growth rule out the possibility that from midlarval stages onwards the retinal and the tectal growth patterns are matched.     

Localization of types I, II and III collagen and glycosaminoglycans in the mandibular condyle of growing monkeys: an immunohistochemical study

 In order to analyse the regional and age-related variations of primate condyles, immunohistochemical techniques were used to examine the localization of types I, II and III collagen and a variety of glycosaminoglycans in distinct anteroposterior regions of the mandibular condyle of two growing female rhesus monkeys (Macaca mulatta). In the juvenile monkey staining for types I and III collagen was weak in the fibrous tissue layer, intense in the pre-cartilaginous tissue layer and faint in the cartilaginous tissue layer; staining was significantly more intense in the posterosuperior and posterior regions than in the anterior region. Similarly, staining for cartilage-characteristic extracellular matrices, including type II collagen and keratan sulfate, was intense in the cartilaginous tissue layer of the posterior condyle. In contrast, in the late-adolescent monkey staining for the extracellular matrices was more intense in the anterior half of the condyle (i.e. from the anterior to the posterosuperior region) than in the posterior region, and most intense in the posterosuperior region. The results demonstrate that marked regional differences exist in the phenotypic expression of the extracellular matrices in the mandibular condyles of growing monkeys and that these differences vary between different developmental stages. The variations probably reflect the predominance of competing growth and articulatory functions in the mandibular condyles. Accepted: 19 July 1996     

In situ hybridization and immunohistochemistry of bone sialoprotein and secreted phosphoprotein 1 (osteopontin) in the developing mouse mandibular condylar cartilage compared with limb bud cartilage

Mandibular condylar cartilage is often classified as a secondary cartilage, differing from the primary cartilaginous skeleton in its rapid progress from progenitor cells to hypertrophic chondrocytes. In this study we used in situ hybridization and immunohistochemistry to investigate whether the formation of primary (tibial) and secondary (condylar) cartilage also differs with respect to the expression of two major non‐collagenous glycoproteins of bone matrix, bone sialoprotein (BSP) and secreted phosphoprotein 1 (Spp1, osteopontin). The mRNAs for both molecules were never expressed until hypertrophic chondrocytes appeared. In the tibial cartilage, hypertrophic chondrocytes first appeared at E14 and the expression of BSP and Spp1 mRNAs was detected in the lower hypertrophic cell zone, but the expression of BSP mRNA was very weak. In the condylar cartilage, hypertrophic chondrocytes appeared at E15 as soon as cartilage tissue appeared. The mRNAs for both molecules were expressed in the newly formed condylar cartilage, although the proteins were not detected by immunostaining; BSP mRNA in the condylar cartilage was more extensively expressed than that in the tibial cartilage at the corresponding stage (first appearance of hypertrophic cell zone). Endochondral bone formation started at E15 in the tibial cartilage and at E16 in the condylar cartilage. At this stage (first appearance of endochondral bone formation), BSP mRNA was also more extensively expressed in the condylar cartilage than in the tibial cartilage. The hypertrophic cell zone in the condylar cartilage rapidly extended during E15–16. These results indicate that the formation process of the mandibular condylar cartilage differs from that of limb bud cartilage with respect to the extensive expression of BSP mRNA and the rapid extension of the hypertrophic cell zone at early stages of cartilage formation. Furthermore, these results support the hypothesis that, in vivo, BSP promotes the initiation of mineralization.     

Histological changes in the articular eminence and mandibular fossa during growth of the rhesus monkey (Macaca mulatta)

Unlike the mandibular condyle, the temporal component of the temporomandibular joint (TMJ) has been the object of relatively few investigations concerning its growth and remodeling. This report provides qualitative and quantitative documentation of microanatomical changes in the mandibular fossa and articular eminence during growth of the rhesus monkey (Macaca mulatta). The thickness of the fibrous articular tissue and the presence of cartilage cells in its deeper layers were examined histologically in 43 rhesus monkeys at five maturational levels (neonate, infant, juvenile, adolescent, and young adult). Absolute thickness of the articular tissue increased with maturational level in all areas studied, with the increase somewhat more pronounced on the posterior slope and crest of the articular eminence than in the roof of the mandibular fossa. Relative to condylar size, an increase in articularlayer thickness characterized the first three maturational levels, and was followed by a decrease during adolescent and young-adult stages. Articular tissue in the fossa roof constituted a steadily decreasing fraction of the total articular-tissue thickness with age, while relative thickness of the tissue on the posterior slope and crest of the eminence increased with age. These results parallel those obtained for the mandibular condyle, and they are best interpreted to indicate that forces delivered to the joint become directed more anteriorly with age. The overall pattern of topographical variation in articular-tissue thickness and cartilage-cell distribution suggests that greater loading of the lateral aspect of the TMJ, postulated in the human TMJ by various workers, may not be as pronounced in the monkey.     

周期性张应力促进兔髁状突软骨细胞的增殖

背景：髁状突是下颌最重要的生长区之一,终生具有生长改建能力。在体内条件下,细胞力学的功能研究因其所处生理环境的复杂性、刺激因素传导的不定向性、实验条件的不易控制性而很难得到满意结果,应力刺激对髁状突软骨细胞的直接影响需要进一步行体外研究。 目的：观察周期性张应力对体外培养兔髁状突软骨细胞生长增殖的影响。 方法：体外分离培养及鉴定兔髁状突软骨细胞,在细胞培养至第3代时使用细胞加力装置对细胞施加强度为10%,频率为6循环/min的周期性张应力,作用时间分别为1,6,12和24 h,并设置未加力组作为对照。应用流式细胞仪检测细胞生长周期,应用MTT法分析细胞的增殖活性。 结果与结论：在周期性张应力下,髁状突软骨细胞流式细胞仪检测结果显示在加力6 h和12 h,加力组细胞生长周期开始有显著性变化,在24 h达到实验最大值,差异有显著性意义(P < 0.01)。MTT检测结果示细胞生长活跃,在6,12 h与对照组有明显变化,在24 h达到实验最大值,差异有显著性意义(P < 0.01)。提示周期性张应力可明显促进髁状突细胞增殖,在24 h内具有持续促进作用。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

Phosphoprotein synthesis and secretion by odontoblasts in rat incisors as revealed by electron microscopic radioautography

The secretory pathway of clentin phosphoproteins in rat incisors was studied by electron microscopic radioautography after the injectionof ³H serine, and the results were compared with those using ³H-proline as a tracer. Five min after injection of ³H-serine, radioactivity was found in the rough endoplasmic reticulum. At 10 min, silver grains were observed over the spherical portions of the cisface of the Golgi apparatus. At 20 min after injection, silver grains were seen over the cylindrical portions of the transface of the Golgi apparatus. The secretory granules showed the strongest reaction from 20 min to 1 hr. At 45 min, a significant labeled band appeared at the mineralization front. At 1 hr, the labeling at the mineralization front began to appear in the mineralized dentin, and after 12 hr this labeled band was located within the mineralized dentin. The pathway of ³H-proline was essentially the, same The pathway of H-proline movedmrore slowly as that of ³H-serine, ³H-proline than ³H-serine, especially in transit from the rough endoplasmic reticulum to the Golgi apparatus. Secretory granules were heavily labeled from 30 min to 1 hr after injection of ³H-proline; no labeling was found at the mineralization front at 45 min. The labeling seen initially over the predentin was over the mineralized dentin no earlier than 6 hr after injection. The labeling pattern with ³H-serine is closely related to the localization of phosphoproteins, whereas the pattern with ³H-proline reflects the production of collagen rather than of phosphoproteins. The present radioautographic results indicate that dentin phosphoproteins are related to secretory granules and are secreted by Odontoblasts at the mineralization front and also that phosphoproteins are involved in the process of mineralization of the circumpulpal dentin.