Growth Plate Explants Research Articles

NCAD and ITGB1 Coordinately Regulate Growth Plate Cartilage Architecture

Spatial and temporal regulation of chondrocyte maturation in the growth plate drives long bone growth. A notable feature of growth plate cartilage is the progression of progenitor resting chondrocytes into clonal columns of transit‐amplifying proliferative chondrocytes. Following division of proliferative chondrocytes, daughter cell pairs undergo a unique 90‐degree rearrangement to form columns. Our previous studies using a murine fluorescent membrane reporter system and time‐lapse imaging along with chemical inhibition of cell adhesion suggested a primary requirement for cadherin adhesion at the daughter cell interface in column formation. Studies designed to test the force generating mechanism at the cadherin dependent daughter cell interface produced a paradoxical result with activated myosin motor proteins enriched at the cell‐matrix interface, an observation that suggests integrin adhesion molecules might drive cell rearrangement. The purpose of the current work was to determine the relative contributions of cadherin and integrin mediated cell adhesion in chondrocyte column formation. As a first step, we used live cell imaging to compare the daughter cell rearrangement phenotype in growth plate explants from embryos containing a cartilage‐specific deletion of N‐cadherin (NCAD) or integrin β1 (ITGB1). Deletion of either gene produces similar disruption of column formation, even though the mutant phenotypes show differences in cell shape, rearrangement dynamics, and kinetics of daughter cell separation following rearrangement. The apparent requirement for both NCAD and ITGB1 suggested potential reciprocal regulatory interaction between the two adhesion surfaces. To test this hypothesis, we analyzed phenotypes of an allelic series. We found that chondrocytes mutant for ITGB1 show defects in cell rearrangement and column formation (n=37 embryos) regardless of the NCAD genotype as expected from single mutant studies. Surprisingly, chondrocytes mutant for NCAD and heterozygous for ITGB1 are phenotypically wildtype with organized columns and normal rearrangement (n=13 embryos). These results support a model for which ITGB1, and not NCAD, is the driver of chondrocyte rearrangement and suggest that failure to rearrange in NCAD mutants results from increased ITGB1 function. Thus, rearrangement requires a narrow range of cell‐matrix adhesion strength and NCAD potentially acts as a brake by restricting ITGB1 activity in non‐rearranging resting chondrocytes. Current studies aim to confirm these genetic findings using gene expression analysis and an innovative quantitative adhesion assay as well as identify upstream regulators that control strength of antagonism between the cell adhesion interfaces.

Cell Division and Rearrangement Dynamics Coordinated by Planar Cell Polarity Pathway in Growth Plate Cartilage

Chondrodysplasias, resulting in dwarfism and altered skeletal growth, are heritable disorders affecting 1 in 4000 to 5000 births. Long bone growth is tightly controlled by sequential chondrocyte maturation through spatially distinct zones in growth plate cartilage. Discoid proliferative chondrocytes form a division plane parallel to the longitudinal axis of the bone and rotate 90 degrees to stack the cells in a unique columnar architecture, which potentiates further limb growth. Noncanonical, β-catenin-independent, Wnt signaling is needed to align division planes and promote organized column formation, which makes the planar cell polarity (PCP) pathway a strong candidate in determining column architecture. Viral manipulation of PCP pathway proteins in chicken embryo growth plates interfered with cell division orientation, but conclusions were limited by fixed tissue sections. To expand our depth of mechanistic inquiry, we sought to develop a method to analyze genetic deletions of PCP pathway proteins and dynamically monitor chondrocyte rearrangement in living growth plate tissue. We hypothesize that Wnt-dependent PCP signaling is required for proliferative chondrocyte rearrangement. Using mice with membrane bound GFP expression and confocal microscopy, we generated time-lapse videos of chondrocytes from live cranial base growth plate explants. Genetic knockout of upstream Wnt5a ligand and inducible, cartilage-specific deletion of Vangl2 transmembrane receptor and Ror2 kinase co-receptor result in significant differences in the final division plane angle distributions compared to normal (p-value<0.0001). Vangl2 is a core PCP protein phosphorylated by Ror2 in response to Wnt5a signaling. While disrupted rearrangement could be a cause of the disorganized proliferative zone, the daughter cells also have premature separation at their interface. Normal chondrocytes separate on average 14.0±6.6 hours after division, while Ror2, Wnt5a, and Vangl2 mutants have significantly shorter separation times (5.2±4.0, 7.8±4.4, and 9.1±3.9 hours, respectively). Our results show that proper rearrangement of proliferative chondrocytes is dependent on PCP pathway activity; however, it is unclear if the premature separation is occurring through disengagement of the cadherin-dependent daughter cell interface or deposition of extracellular matrix between the cells. The focus of future studies is to determine what is acting downstream of the PCP pathway to regulate adhesion dynamics and determine the extrinsic requirements guiding polarized chondrocyte behaviors. These results will advance approaches to cartilage engineering to treat chondrodysplasias by understanding the mechanistic cues required for proper cartilage tissue architecture and growth.

In situ deformation of growth plate chondrocytes in stress-controlled static vs dynamic compression

Longitudinal bone growth in children/adolescents occurs through endochondral ossification at growth plates and is influenced by mechanical loading, where increased compression decreases growth (i.e., Hueter-Volkmann Law). Past in vivo studies on static vs dynamic compression of growth plates indicate that factors modulating growth rate might lie at the cellular level. Here, in situ viscoelastic deformation of hypertrophic chondrocytes in growth plate explants undergoing stress-controlled static vs dynamic loading conditions was investigated. Growth plate explants from the proximal tibia of pre-pubertal rats were subjected to static vs dynamic stress-controlled mechanical tests. Stained hypertrophic chondrocytes were tracked before and after mechanical testing with a confocal microscope to derive volumetric, axial and lateral cellular strains. Axial strain in hypertrophic chondrocytes was similar for all groups, supporting the mean applied compressive stress’s correlation with bone growth rate and hypertrophic chondrocyte height in past studies. However, static conditions resulted in significantly higher lateral (p<0.001) and volumetric cellular strains (p≤0.015) than dynamic conditions, presumably due to the growth plate’s viscoelastic nature. Sustained compression in stress-controlled static loading results in continued time-dependent cellular deformation; conversely, dynamic groups have less volumetric strain because the cyclically varying stress limits time-dependent deformation. Furthermore, high frequency dynamic tests showed significantly lower volumetric strain (p=0.002) than low frequency conditions. Mechanical loading protocols could be translated into treatments to correct or halt progression of bone deformities in children/adolescents. Mimicking physiological stress-controlled dynamic conditions may have beneficial effects at the cellular level as dynamic tests are associated with limited lateral and volumetric cellular deformation.

Growth plate cartilage shows different strain patterns in response to static versus dynamic mechanical modulation.

Longitudinal growth of long bones and vertebrae occurs in growth plate cartilage. This process is partly regulated by mechanical forces, which are one of the underlying reasons for progression of growth deformities such as idiopathic adolescent scoliosis and early-onset scoliosis. This concept of mechanical modulation of bone growth is also exploited in the development of fusionless treatments of these deformities. However, the optimal loading condition for the mechanical modulation of growth plate remains to be identified. The objective of this study was to evaluate the effects of in vitro static versus dynamic modulation and of dynamic loading parameters, such as frequency and amplitude, on the mechanical responses and histomorphology of growth plate explants. Growth plate explants from distal ulnae of 4-week-old swines were extracted and randomly distributed among six experimental groups: baseline ([Formula: see text]), control ([Formula: see text]), static ([Formula: see text]) and dynamic ([Formula: see text]). For static and dynamic groups, mechanical modulation was performed in vitro using an Indexed CartiGen bioreactor. A stress relaxation test combined with confocal microscopy and digital image correlation was used to characterize the mechanical responses of each explant in terms of peak stress, equilibrium stress, equilibrium modulus of elasticity and strain pattern. Histomorphometrical measurements were performed on toluidine blue tissue sections using a semi-automatic custom-developed MATLAB toolbox. Results suggest that compared to dynamic modulation, static modulation changes the strain pattern of the tissue and thus is more detrimental for tissue biomechanics, while the histomorphological parameters are not affected by mechanical modulation. Also, under dynamic modulation, changing the frequency or amplitude does not affect the biomechanical response of the tissue. Results of this study will be useful in finding optimal and non-damaging parameters for the mechanical modulation of growth plate in fusionless treatments.

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.

Effect of Cold Storage and Freezing on the Biomechanical Properties of Swine Growth Plate Explants

Ex vivo biomechanical testing of growth plate samples provides essential information about its structural and physiological characteristics. Experimental limitations include the preservation of the samples since working with fresh tissues involves significant time and transportation costs. Little information is available on the storage of growth plate explants. The aim of this study was to determine storage conditions that could preserve growth plate biomechanical properties. Porcine ulnar growth plate explants (n = 5 per condition) were stored at either 4 °C for periods of 1, 2, 3, and 6 days or frozen at -20 °C with slow or rapid sample thawing. Samples were tested using stress relaxation tests under unconfined compression to assess five biomechanical parameters. The maximum compressive stress (σmax) and the equilibrium stress (σeq) were directly extracted from the experimental curves, while the fibril-network reinforced biphasic model was used to obtain the matrix modulus (Em), the fibril modulus (Ef), and the permeability (k). No significant changes were observed in σeq and Em in any of the tested storage conditions. Significant decreases and increases, respectively, were observed in σmax and k in the growth plate samples refrigerated for more than 48 h and in the frozen samples, when compared with the fresh samples. The fibril modulus Ef of all stored samples was significantly reduced compared to the fresh samples. These results indicate that the storage of growth plates in a humid chamber at 4 °C for a maximum of 48 h is the condition that minimizes the effects on the measured biomechanical parameters, with only Ef significantly reduced. Refrigerating growth plate explants for less than 48 h maintains their maximal stress, equilibrium stress, matrix modulus, and permeability. However, cold storage at 4 °C for more than 48 h and freezing storage at -20 °C significantly alter the biomechanical response of growth plate samples. Appropriate growth plate sample storage will be beneficial to save time and reduce transportation costs to pick up fresh samples.

Fibroblast Growth Factor 23 and Klotho Are Present in the Growth Plate

Introduction: Regulation of phosphate homeostasis is essential for mineralization and enchondral ossification. Fibroblast growth factor 23 (FGF23) and its obligatory co-receptor Klotho (KL) play a key role in this process by influencing both renal phosphate reabsorption and vitamin D metabolism. In disease, excessive action of FGF23 leads to hypophosphatemic rickets, while its deficiency causes tumoral calcinosis. Although osteocytes and osteoblasts are widely seen as the primary source of FGF23 under physiological conditions, the origin of systemic FGF23 remains controversial. In this study, we investigated the expression of FGF23 and KL in porcine growth plate cartilage, adjacent tissues, and parenchymal tissues.Materials and Methods: Tissue samples were obtained from 4- to 6-week-old piglets. mRNA expression was quantified by real-time PCR and normalized to 18S rRNA. Immunohistochemical staining was performed for FGF23, KL, collagen type X, and FGF receptor 1. Growth plate chondrocyte subpopulations were acquired by collagenase digestion of growth plate explants and subsequent density gradient centrifugation.Results: We could detect both FGF23 and KL mRNA and protein in growth plate chondrocytes. FGF23 expression was mainly found in hypertrophic and resting chondrocytes. Furthermore, significant expression of both genes was observed in bone, liver, and spleen.Conclusion: These data challenge previous expression analyses, in particular theories of bone as the exclusive source of FGF23. Moreover, significant expression of FGF23 and KL within the growth plate and adjacent tissues imply a potential local role of FGF23 in chondrocyte differentiation and tissue mineralization.

Growth plate explants respond differently to in vitro static and dynamic loadings

This study aimed at investigating the effects of static and dynamic compression applied on growth plate explants using matched compressive strains. Growth plate explants from 4-week-old swine ulnae were submitted to in vitro static (10% strain) or dynamic (oscillating between 7% and 13% at 0.1 Hz) unconfined compression for 48 h. The total growth plate height, the combined proliferative and hypertrophic thickness and the resulting ratio between these two thicknesses were evaluated. Standard immunohistochemistry was used to analyze the protein expression of key components of the extracellular matrix: aggrecan, type II collagen, type X collagen, and MMP13. In the statically loaded samples, the columnar organization of the cells was preserved but with slight columns deviation from the growth axis. Decreases in all histomorphological parameters were important and a notable loss of aggrecan, type II and type X collagens expressions was denoted. In the dynamically loaded samples, a severe loss of columnar arrangement was observed in the proliferative and hypertrophic zones. However, dynamic compressive loads preserved the proliferative and hypertrophic zones ratio and contributed to the synthesis of aggrecan and type II collagen in the extracellular matrix. The exact response of the growth plate to mechanical stresses along with optimal loading parameters could help improve the current treatment approaches or develop new treatment approaches for the underlying progressive musculoskeletal deformities.

Tissue and cellular morphological changes in growth plate explants under compression

The mechanisms by which mechanical loading may alter bone development within growth plates are still poorly understood. However, several growth plate cell or tissue morphological parameters are associated with both normal and mechanically modulated bone growth rates. The aim of this study was to quantify in situ the three-dimensional morphology of growth plate explants under compression at both cell and tissue levels. Growth plates were dissected from ulnae of immature swine and tested under 15% compressive strain. Confocal microscopy was used to image fluorescently labeled chondrocytes in the three growth plate zones before and after compression. Quantitative morphological analyses at both cell (volume, surface area, sphericity, minor/major radii) and tissue (cell/matrix volume ratio) levels were performed. Greater chondrocyte bulk strains (volume decrease normalized to the initial cell volume) were found in the proliferative (35.4%) and hypertrophic (41.7%) zones, with lower chondrocyte bulk strains (24.7%) in the reserve zone. Following compression, the cell/matrix volume ratio decreased in the reserve and hypertrophic zones by 24.3% and 22.6%, respectively, whereas it increased by 35.9% in the proliferative zone. The 15% strain applied on growth plate explants revealed zone-dependent deformational states at both tissue and cell levels. Variations in the mechanical response of the chondrocytes from different zones could be related to significant inhomogeneities in growth plate zonal mechanical properties. The ability to obtain in situ cell morphometry and monitor the changes under compression will contribute to a better understanding of mechanisms through which abnormal growth can be triggered.

Modulation of neonatal growth plate development by ex vivo intermittent mechanical stress

Although growth plate response to mechanical stress has been increasingly studied, our understanding of mechanical modulation of neonatal growth plate is incomplete, especially concerning biochemical changes. This study was designed to explore the cellular and biochemical responses of the cranial base growth plate (CBGP) explant upon cyclic loading. The growth plate with subchondral bone was aseptically isolated from each of 24 neonatal rabbits and fixated in an organ culture system. Cyclic loading was applied to growth plate explants at 200 mN and 1 Hz for 60 min ( N = 12 ), whereas control explants were immersed in organ culture for 60 min without mechanical loading ( N = 12 ). Computerized image analysis revealed that cyclic loading induced significantly more proliferating chondrocytes than unloaded controls ( p < 0.001 ), as well as significantly higher growth plate height at 856 ± 30 μ m than the unloaded controls at 830 ± 36 μ m ( p < 0.05 ). Immunoblotting with monoclonal antibodies (mAb) disclosed that the average mAb binding area for chondroitin sulfate was significantly higher in the loaded specimens than the unloaded controls at ( p < 0.001 ). The average mAb binding area for keratan sulfate was also significantly higher in the loaded specimens than the unloaded controls ( p < 0.01 ). Biochemical analysis showed that the average total hyaluronan content of loaded specimens at 0.25 ± 0.06 μ g / μ g DNA was significantly higher than the unloaded controls at 0.09 ± 0.05 μ g / μ g DNA ( p < 0.01 ). Taken together, these data suggest that brief doses of cyclic, intermittent forces activate cellular and molecular responses in the CBGP ex vivo. Whether hyaluronan-mediated pathway is involved in the biological responses of growth plate to mechanical loading warrants additional investigations.

Static Compressive Loading Reduces the mRNA Expression of Type II and X Collagen in Rat Growth-Plate Chondrocytes During Postnatal Growth

The mechanisms by which chondrocytes modulate longitudinal bone growth are not well understood. This in vitro study investigated the effects of loading on the mRNA expression pattern of key molecular components of the growth-plate related to the extracellular matrix (type II and type X collagen) and the PTH-PTHrP feedback loop. Short-term static compressive loading was applied to rat proximal tibial growth-plate explants. Four age groups at specific developmental stages were investigated. The spatial variation in the mRNA expression was compared among loaded explants, their contralateral sham controls, and uncultured growth plates from normal animals. Basic cell metabolism (18S rRNA) was unaffected by load. Results indicated a narrower spatial distribution of mRNA expression of type II collagen throughout the growth plate; similarly, a narrowed distribution of expression of type X collagen was noted in the lower hypertrophic zone of the growth-plate. This suggests that mechanical compression influences chondrocytes of the hypertrophic zone to alter their expression of specific genes encoding proteins of the extracellular matrix, while PTH-PTHrP receptor mRNA, a regulatory protein, remained unaffected by loading. The effects of compression were similar at the different stages of growth, suggesting that additional factors may be involved in the clinical progression of skeletal deformities observed during growth spurts. Although this study was done in vitro and limited to static loading, it furthers our understanding of growth-plate mechanobiology as a first step toward providing a scientific rationale for treating progressive musculoskeletal deformities.

Thyroid hormone enhances aggrecanase-2/ADAM-TS5 expression and proteoglycan degradation in growth plate cartilage.

Effects of thyroid hormone on proteoglycan degradation in various regions of cartilage were investigated. In propylthiouracil-treated rats with hypothyroidism, proteoglycan degradation in epiphyseal cartilage during endochondral ossification was markedly suppressed. However, injections of T(4) reversed this effect of propylthiouracil on proteoglycan degradation. In pig growth plate explants, T(3) also induced breakdown of proteoglycan. T(3) increased the release of aggrecan monomer and core protein from the explants into the medium. Accordingly, the level of aggrecan monomer remaining in the tissue decreased after T(3) treatment, and the monomer lost hyaluronic acid-binding capacity, suggesting that the cleavage site is in the interglobular domain. The aggrecan fragment released from the T(3)-exposed explants underwent cleavage at Glu(373)-Ala(374), the major aggrecanase-cleavage site. The stimulation of proteoglycan degradation by T(3) was less prominent in resting cartilage explants than in growth plate explants and was barely detectable in articular cartilage explants. Using rabbit growth plate chondrocyte cultures, we explored proteases that may be involved in T(3)-induced aggrecan degradation and found that T(3) enhanced the expression of aggrecanase-2/ADAM-TS5 (a disintegrin and a metalloproteinase domain with thrombospondin type I domains) mRNA, whereas we could not detect any enhancement of stromelysin, gelatinase, or collagenase activities or any aggrecanase-1/ADAM-TS4 mRNA expression. We also found that the aggrecanse-2 mRNA level, but not aggrecanase-1, increased at the hypertrophic stage during endochondral ossification. These findings suggest that aggrecanse-2/ADAM-TS5 is involved in aggrecan breakdown during endochondral ossification, and that thyroid hormone stimulates the aggrecan breakdown partly via the enhancement of aggrecanase-2/ADAM-TS5.

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.