Xenografts of articular chondrocytes in the nude mouse

Summary Subcutaneous transplantation of articular chondrocytes isolated enzymatically from immature rabbits and dogs into athymic (nu/nu) mice resulted in the formation of hyaline cartilaginous nodules. Graft rejection was seen when the cells were injected into heterozygous (nu/+) mice. Radiosulfate-labeled proteoglycan extracted from the xenografts had a high buoyant density and was digested by chondroitinase ABC. A monomeric preparation of proteoglycan (A1-D1) contained a small quantity of aggregate as assessed by gel chromatography and gel electrophoresis. Almost no incorporation of³H-thymidine was found by autoradiography. The matrix did not become calcified over the course of 42 days. The nude mouse system lends itself to testing a variety of problems in the biology of cartilage. These include the redifferentiation of chondrocytes following dedifferentiation in vitro. Species differences were found in this regard. The nodules formed by rabbit articular chondrocytes, grown in monolayer culture for up to 14 days, had a hyaline chondroid character. Dog chondrocytes that had “dedifferentiated,” during 14 days of culture prior to transplantation, formed a graft that had a sparse fibrous rather than hyaline matrix.     

Reimplantation of growth plate chondrocytes into growth plate defects in sheep

Defects in growth plates due to trauma, infection, or genetic causes can result in bone formation across the defect, bridging the epiphysis and metaphysis, resulting in growth arrest and limb deformation. We have investigated the capacity of implanted chondrocyte cultures to prevent this process. Sheep growth plate chondrocytes were isolated, and after culture at high density produced easily manipulated cartilaginous discs. The tissue was implanted into growth plate defects produced in lambs and the response was assessed histologically. Following implantation, cultures continued to proliferate and maintain a cartilage-like matrix. After 8 to 12 weeks, hypertrophic maturation chondrocyte columnation, and associated endochondral calcification were observed. Culture implantation was always associated with local immune inflammatory reaction, which continued throughout the course of investigation. Cellular survival was variable and resulted in the presence of viable implants as well as residual cartilage matrix devoid of chondrocytes; however, implanted chondrocyte discs always prevented bone bridge formation. These findings encourage the expectation that cultured chondrocytes may provide a useful replacement for the inert interpositional materials currently used in the treatment of growth arrest. The potential of this technique for growth plate replacement, however, requires a more predictable rate of implant survival. The likely reasons for implant loss are discussed.     

Effect of transforming growth factor-β on DNA synthesis by growth plate chondrocytes: Modulation by factors present in serum

Summary Chondrocytes in the growth plate undergo rapid proliferation during the process of enchondral ossification. The factors that regulate this proliferative activity have not been defined although several local autocrine or paracrine growth factors have recently been discovered in cartilage. Transforming growth factor-β₁ (TGF-β) is an important regulator of cell metabolism and growth and is present in cartilage. The present study was designed to examine the influence of TGF-β on DNA synthesis in chick epiphyseal chondrocytes. Chondrocytes were plated in serum-free (BSA-supplemented) medium or medium containing 10% FBS, and after 24 hours in monolayer culture, were treated with TGF-β caused a biphasic dose-dependent increase in thymidine incorporation. The effect was greatly increased in serum-containing cultures where a maximal 20-fold increase in thymidine incorporation occurred compared with the 21/2-fold maximal increase obtained in serum-free cultures. The effect was present by 12 hours and maximal between 0.3 and 1.0 ng/ml of TGF-β. Higher concentrations of TGF-β were less stimulatory. The serum enhancement was dependent upon the simultaneous presence of TGF-β and serum factors and was abolished when chondrocytes were plated and exposed to TGF-β in medium containing dialyzed FBS (12–14 kD MW membrane). The nature of the uptake and incorporation of thymidine into DNA by individual chondrocytes appeared to be the same in both TGF-β-treated and control cultures as the apparent K_ms were similar. The present study indicates that TGF-β increases DNA synthesis by growth plate chondrocytes. The effect is enhanced by factors present in serum.     

Compressive mechanical modulation alters the viability of growth plate chondrocytes in vitro

The aim of this study was to investigate the effect of compressive modulation parameters (mode, magnitude, duration, as well as frequency and amplitude for cyclic modulation) on the viability of growth plate chondrocytes. Swine ulnar growth plate explants (n = 60) were randomly distributed among 10 groups: baseline (n = 1 × 6); culture control (n = 1 × 6); static (n = 3 × 6); and dynamic (n = 5 × 6). Static and dynamic samples were modulated in vitro using a bioreactor. Different compression magnitudes (0.1 MPa or 0.2 MPa), durations (12 h or 24 h), frequencies (0.1 Hz or 1.0 Hz), and amplitudes (30% or 100%) were investigated. Viability was assessed by automatic quantification of number of live/dead cells from confocal images of Live/Dead labeled tissues. Chondrocyte viability was found to be dependent on compression magnitude, duration, frequency, and amplitude in a way that increasing each parameter decreased viability in certain zones of growth plate. More specifically, proliferative and hypertrophic chondrocytes were found to be more sensitive to the applied compression. This study provides an in vitro protocol for studying the effects of compressive modulation on biomechanical and biological responses of growth plate explants, which will be useful in finding efficient and non‐detrimental parameters for mechanical modulation of bone growth exploited in scoliosis fusionless treatments. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1587–1593, 2015.     

Cytosolic calcium concentration in bovine growth plate chondrocytes

The cytosolic free calcium ion concentration for mammalian cell systems is believed to be maintained within a narrow range compatible with cellular homeostasis. Growth plate chondrocytes have been shown to accumulate large quantities of calcium within their mitochondria, but the cytosolic free calcium concentration has not been determined. This study measures the cytosolic free ionic calcium concentration in growth plate chondrocytes using two variations of the Quin II fluorescence technique. The results indicate that in isolated growth plate chondrocytes, the cytosolic free ionic calcium concentration is similar to other nonmineralizing mammalian cell types (106–137 nM).     

Determination of proliferative characteristics of growth plate chondrocytes by labeling with bromodeoxyuridine

Summary Postnatal bone growth occurs by the process of endochondral ossification in cartilaginous growth plates at the ends of long bones. The rate and extent of long bone growth is determined by a combination of chondrocytic proliferation, matrix production, and increase in chondrocytic height in the direction of growth during cellular enlargement. In this study, single pulse and/or repeated pulse labeling with the thymidine analog bromodeoxyuridine (BrdU) was used to study the role of cellular proliferation in controlling long bone growth. Variables studied included progression of the label over time following a pulse, and patterns and progression of the label over time following repeated pulse labeling for 24 and 48 hours. Examination was made of the proliferative characteristics of chondrocytes, the spatial pattern of cellular proliferation, and cell cycle kinetics. With respect to the spatial pattern of proliferative chondrocytes, results suggest that chondrocytes within a column are more synchronized with each other than are chondrocytes in different columns. This is consistent with the concept that each column represents a clonal expansion of a stem cell, which may proceed independently from adjacent columns. Despite this apparent heterogeneity, all chondrocytes in the proliferative zone complete at least one cell cycle in 24–28 hours. This estimate of the cell cycle time is significantly shorter than previous estimates of cell cycle times in similar growth plates. Our results also suggest that chondrocytes entering the cell cycle in the proximal part of the growth plate spend an average of 4 days in the proliferative cell zone, representing approximately four cellular divisions. After leaving the cell cycle, an additional 48 hours is required for the label to reach the terminal chondrocyte, which represents the time required to complete hypertrophy. These data are important when considering hypotheses concerning both the role of controls on proliferation in the determination of overall rate of long bone growth, as well as the interplay between proliferation and hypertrophy in regulating the overall amount of growth achieved by a given growth plate.     

Dwarfism in the Alaskan Malamute: Ultrastructural features of dwarf growth plate chondrocytes

Summary This study was performed in order to reexamine the ultrastructural morphology of the chondrocytes in the growth plates of dwarf Alaskan Malamutes and to obtain semiquantitative cytochemical data about the proteoglycans. Growth plates from age-matched dwarf and homozygous nonaffected Alaskan Malamutes were processed for routine transmission electron microscopy and also stained with ruthenium red. Chondrocytes in dwarf plates were observed to occur in clumps or cell nests. Within some of these nests, chondrocytes in the upper half of the zone of chondrocyte proliferation had bizarre shapes ranging from V-shaped to whorled or rounded. These chondrocytes contained profiles of markedly dilated rough endoplasmic reticulum (RER). Material within the RER cisternae stained positively with ruthenium red and was partially digestible with testicular hyaluronidase. The material could, therefore, represent either chondroitin sulfate or hyaluronate. The RER in these dwarf chondrocytes was not oriented parallel to the long axis of the cells; instead, it consisted of irregularly dilated cisternae. Granule counts performed on the zone of chondrocyte proliferation revealed a significant decrease in the number of ruthenium red granules in the interterritorial matrix of dwarf chondrocytes when compared to those of the homozygous nonaffected chondrocytes.     

Inhibitory effect of salmon calcitonin on bone resorption: Morphological study of the tibial growth plate in rats

Summary Salmon calcitonin (sCT) at doses of 100 and 50 UI given subcutaneously to growing rats produced in vivo evidence of osteoclastic activity inhibition. Histological assessment was carried out by measuring the perichondrial ring of Lacroix height, and a dose-correlated effect was found. These aspects were coupled with an increase in the osteoclast number and suggested that in studies with bone resorption inhibitors, morphological evaluation based on osteoclasts count is not reliable. The changes of the metaphysis suggested also that sCT affects the activity of hypertrophic chondrocytes of the growth plate. Plasma calcium levels did not differ significantly between treated rats and controls; an increased phosphatemia was observed in sCT-treated animals.     

大鼠胫骨生长板软骨细胞的分离与培养鉴定

目的 探讨高效生长板软骨细胞分离方法 和体外培养条件.方法 采用二步酶消化法对6只SD大鼠胫骨生长板的软骨细胞进行分离,采用含炭吸附过的胎牛血清培养基培养,按5×105个/瓶的密度接种细胞并对软骨细胞进行形态学观察和鉴定,描绘原代软骨细胞在无激素培养基中的生长曲线.结果 软骨细胞贴壁较慢,12 h后开始附壁,第8天时90%融合,互相连接成"铺路石"样结构.原代软骨细胞胞质Ⅱ型胶原免疫着色强阳性,传代后染色减弱.结论 本研究所采用的方法 能高效快速获得原代软骨细胞,原代软骨细胞最接近体内生理状态,最适合进行实验研究.     

大鼠椎体生长板软骨细胞的改良培养及传代特征

[目的]建立体外培养及鉴定大鼠椎体生长板软骨细胞的方法,观察软骨细胞的传代特征.[方法]采用改良胰酶和Ⅱ型胶原酶序贯消化法对4只1周龄SD大鼠椎体生长板软骨细胞进行体外分离、培养.原代细胞传代后采用细胞HE染色和甲苯胺蓝染色对软骨细胞进行鉴定,采用MTT比色法绘制细胞生长曲线.将细胞传代至第6代,采用倒置相差显微镜观察各代软骨细胞的形态,采用免疫细胞化学染色鉴定各代软骨细胞Ⅱ型胶原的表达.[结果]MTT比色法显示,传3代以前的软骨细胞的生长曲线近似"S"形,从第4 d开始细胞呈对数生长,第9 d达平台期,至第11 d开始出现生长抑制.体外培养的软骨细胞随着传代次数的增加,细胞形态由原代的多角形逐渐变为长梭形.传3代以前的软骨细胞Ⅱ型胶原免疫细胞化学呈强阳性.[结论]本研究所采用的软骨细胞分离和培养方法,能在短时间内获得高纯度的软骨细胞,传3代及以前的细胞具有软骨细胞的特征性表型,增殖较快,这一方法为更深入地从细胞水平研究椎体的纵向生长奠定了基础.     

A new method of investigating the efficacy of regional thermotherapy in subcutaneous xenografts of nude mice

Summary A new method of investigating the efficacy of regional thermotherapy in subcutaneous xenografts of nude mice is reported. The use of high frequency hyperthermia was well tolerated by the sensitive animals and allowed an exact continuous temperature measurement in different tumor regions. The interstitial procedure in this model could be the best approach for later clinical use in urology, e.g. for prostate treatment and is an alternativ to the transrectal hyperthermia application.Part of this paper was presented at the 9th Symposium of the Association for Experimental Urology of the German Urological Society, June 17–18, 1988, Aachen, Federal Republic of GermanyThis study was supported by the Stiftung Bauer     

Is longitudinal bone growth influenced by diurnal variation in the mitotic activity of chondrocytes of the growth plate?

The diurnal variations in the mitotic index, height, and rate of linear bone growth were determined and correlations between these parameters examined. Young, unweaned, female Wistar rats were housed under standardized conditions, labeled with a fluorochrome 60 h before sacrifice, and killed at intervals throughout a 24-h period, specifically 0600, 1200, 1800, and 2400. The proximal tibial epiphyseal growth plates were collected and processed, and the mitotic index, growth plate height, and the rate of linear bone growth were measured. The mitotic index measured at 0600 was significantly higher than that measured at 1800 and 2400. Growth plates of rats sacrificed at 1200 were taller than those of rats sacrificed at 1800, but there was no difference between heights of growth plates from rats sacrificed at other times. Daily growth rate for all rats averaged 283.9 microns/day and there were no statistically significant differences between daily growth rates measured at any time period. Our findings imply that in comparative, quantitative structural studies of animal groups, sacrifice should be carried out at identical times of the day, since, given a constant speed of vascular ingrowth and diurnal variation in width, relative diurnal accumulation and depletions of cells may take place. We also suggest that the daily growth rate and mitotic index be measured directly and not be considered a function of the height of the growth plate.     

Intramembranous ossification mechanism for bone bridge formation at the growth plate cartilage injury site.

Salter's type III and type IV growth plate injuries often induce bone bridge formation at the injury site. To understand the cellular mechanisms, this study characterized proximal tibial transphyseal injury in rats. Histologically, bony bridge trabeculae appeared on day 7, increased on day 10, and became well-constructed on day 14 with marrow. Prior to and during bone bridging, there was no cartilage proteoglycan metachromatic staining and no collagen-X immunostaining at the injury site, nor was there any up-regulation of BrdU-labelled chondrocyte proliferation at the adjacent physeal cartilage, suggesting no new cartilage formation at the injury site. However, infiltration of vimentin-immunopositive mesenchymal cells from metaphysis and epiphysis was apparent on day 3, with the mesenchymal population being prominent on days 7 and 10 and subsided on day 14. Among these infiltrates were osteoprogenitor precursors expressing osteoblast differentiation factor (cbf-alpha1) on day 3, along with some cbf-alpha1+ osteoblast-like cells lining bone trabeculae on days 7 and 10. Some mesenchymal cells and trabecula-lining cells were also alkaline phosphatase-immunopositive, further suggesting their osteoblast differentiation. From day 7 onwards, some trabecula-lining cells became osteocalcin-producing mature osteoblasts. These results suggest that bone bridge formation after growth plate injury occurs directly via intramembranous ossification through recruitment of marrow-derived osteoprogenitor cells.     

Distribution of acidic glycosaminoglycans in rabbit growth plate cartilage

Chondroitin-4-sulfate (C-4-S), chondroitin-6-sulfate (C-6-S) and keratan sulfate (KS) concentrations were measured in growth plate cartilage derived from rabbits of two different ages, and from three growth plate zones from rabbits of the same age. With increasing age, the concentration of C-4-S increased, C-6-S decreased and KS remained constant. There was no variation when three different growth plate zones were sampled. Radiosulfate uptake into chondroitan sulfate was also measured, and this showed an inverse relationship to concentration with increasing age.Research supported in part by a grant from the Western Chapter (Pennsylvania) Arthritis Foundation; Research Grant AMI 1382-04 from the National Institute of Arthritis and Metabolic Diseases; and the Orthopaedic Research Fund, The Hospital for Joint Diseases.     

Effects of the calcium channel blocker nifedipine on epiphyseal growth plate and bone turnover: A study in rabbit

Summary The potential effects of a calcium channel blocker (nifedipine) on epiphyseal growth plate and bone remodeling have been investigated in growing rabbits. The treated group received 6 mg/kg/day nifedipine twice daily by gavage for 10 weeks. An untreated group was used as control; with this dose, neither toxic effects nor decrease in the body weight have been observed. No modifications of blood phosphocalcic parameters have been found. In the treated group there is a significant lower cancellous bone volume, lower osteogenesis, shorter labeled perimeters, and lower mineral apposition rate than in the control group. Epiphyseal growth plate thickness is lower than in the untreated animals and considerable morphological changes are observed in the growth zone compared with the control group. A decrease in the growth of humerus length was found. In conclusion, nifedipine affects bone physiology, especially with consequences on bone growth. These effects appear to be quantitatively important, and there is the possibility of bone side effects on therapeutic use in humans, especially in young subjects.     

Visualization of living terminal hypertrophic chondrocytes of growth plate cartilage in situ by differential interference contrast microscopy and time-lapse cinematography

The functional unit within the growth plate consists of a column of chondrocytes that passes through a sequence of phases including proliferation, hypertrophy, and death. It is important to our understanding of the biology of the growth plate to determine if distal hypertrophic cells are viable, highly differentiated cells with the potential of actively controlling terminal events of endochondral ossification prior to their death at the chondro-osseous junction. This study for the first time reports on the visualization of living hypertrophic chondrocytes in situ, including the terminal hypertrophic chondrocyte. Chondrocytes in growth plate explants are visualized using rectified differential interference contrast microscopy. We record and measure, using time-lapse cinematography, the rate of movement of subcellular organelles at the limit of resolution of this light microscopy system. Control experiments to assess viability of hypertrophic chondrocytes include coincubating organ cultures with the intravital dye fluorescein diacetate to assess the integrity of the plasma membrane and cytoplasmic esterases. In this system, all hypertrophic chondrocytes, including the very terminal chondrocyte, exist as rounded, fully hydrated cells. By the criteria of intravital dye staining and organelle movement, distal hypertrophic chondrocytes are identical to chondrocytes in the proliferative and early hypertrophic cell zones.     

Expression and activation of peroxisome proliferator-activated receptors in growth plate chondrocytes.

Peroxisome proliferator-activated receptor gamma (PPAR-gamma) is a nuclear hormone receptor that is involved in a wide range of cellular processes. Although it is known that PPAR-gamma plays an important role in cell cycle control, inflammation, apoptotic cell death, and other cellular processes, the role of PPAR-gamma in the normal and pathological function of growth plate chondrocytes has not been investigated. The purpose of this study was to determine if PPARs are expressed in growth plate chondrocytes and to describe the biological effect of PPAR activation in these cells. The results demonstrate the presence of three PPAR isoforms (alpha, delta, and gamma) in growth plate cartilage. Activation of PPAR-gamma by ciglitazone in growth plate chondrocytes inhibits T(3) induced terminal differentiation and promotes apoptosis through increased levels of caspase 3/7 activity and decreased expression of the anti-apoptotic protein Bcl-2.     

Increased cAMP production after short-term capacitively coupled stimulation in bovine growth plate chondrocytes

Growth plate chondrocytes from newborn calf costochondral junctions grown in monolayer were subjected to a capacitive AC signal of 500 V peak to peak (P-P) at 60 kHz for 48 h and were analyzed for [3H]thymidine uptake. The stimulated chondrocytes showed a 130% greater uptake over unstimulated controls. Other newborn calf growth plate chondrocytes were stimulated at 500 V P-P at 60 kHz for 2.5, 5.0, 10.0, and 20.0 min and were analyzed for cAMP. Chondrocytes stimulated for 2.5 and 5.0 min showed a 142.8% (p less than 0.05) and 394.5% (p less than 0.01) increase over controls, respectively. The chondrocytes stimulated for 10.0 and 20.0 min showed no significant difference from the controls. It is concluded that short-term exposure of growth plate chondrocytes to an appropriate capacitively coupled field stimulates cAMP production, but longer-term application of the electrical field is ineffective.     

Gender differences in expression of androgen receptor in tibial growth plate and metaphyseal bone of the rat

In this study, we investigate the expression of the androgen receptor (AR) in the tibial growth plate and metaphyseal bone of male and female rats at the mRNA and protein level. Using in situ hybridization and immunohistochemistry, AR mRNA and protein were demonstrated in proliferating and early hypertrophic chondrocytes in the growth plate of 1-, 4-, and 7-week-old male and female rats. Immunostaining for AR was observed both in the nucleus and the cytoplasm. After sexual maturation at 12 and 16 weeks of age, AR expression decreased in both genders and was confined to a small rim of prehypertrophic chondrocytes. In female rats of 40 weeks of age, this expression pattern was still visible. In most age groups there was a tendency toward an increased AR mRNA expression in male vs. female rats except in the 7-week-old animals. At the protein level, sexually maturing 7-week-old male rats demonstrated a higher staining intensity compared to their female counterparts. At this stage, AR staining in the males was mainly confined to the nucleus, whereas in females staining was predominantly found in the cytoplasm. In the tibial metaphysis, AR mRNA was detected in lining cells, osteoblasts, osteocytes, and osteoclasts at all stages of development. At the protein level, a similar expression pattern was observed, except for an absence of immunostaining in the lining cells. The staining was both nuclear and cytoplasmic. In most age groups, mRNA and protein signals were higher in males compared with females. We have demonstrated the presence of AR mRNA and protein in the tibial growth plate and the underlying metaphyseal bone during development of the rat. In male rats, the presence of higher messenger and protein staining intensities, as well as preferential nuclear staining during sexual maturation, suggests that direct actions of androgens in chondrocytes and in bone forming cells may be involved in establishing the gender differences in the skeleton.