Puberty The male "growth spurt" also begins later, accelerates more slowly, and lasts longer before the epiphyses fuse. While estradiol promotes growth of breasts and uterus, it is also the principal hormone driving the pubertal growth spurt and epiphyseal maturation and closure. Growth of Bone The epiphyseal plate is the area of growth in a long bone. On the epiphyseal side of the epiphyseal plate, cartilage is formed. All that remains of the epiphyseal plate is the epiphyseal line.
As a bone matures, the epiphyseal plate progresses to an epiphyseal line. Electrocyclic Reactions The electrocyclic ring closure is is designated by blue arrows, and the ring opening by red arrows. In the first case, trans,cis,trans-2,4,6-octatriene undergoes thermal ring closure to cis-5,6-dimethyl-1,3-cyclohexadiene.
The sterospecificity of this reaction is demonstrated by closure of the isomeric trans,cis,cis-triene to trans-5,6-dimethyl-1,3-cyclohexadiene, as noted in the second example. This mode of reaction is favored by relief of ring strain, and the reverse ring closure light blue arrows is not normally observed. Photochemical ring closure can be effected, but the stereospecificity is opposite to that of thermal ring opening. Development of Joints Between the epiphysis and diaphysis the long midsection of the long bone lies the metaphysis, including the epiphyseal plate growth plate.
The specimens from which the blood samples were collected were then euthanized, and histological cut-sections that covered the epiphyseal growth plate were collected, along with the bone sections of the proximal regions of the right femur. Considering decalcification, these histological sections were kept in an ethylene diamine tetra acetic acid EDTA solution. Routine histological examinations were then conducted on these sections, after which they were embedded in paraffin.
Crossman's modified triple staining method was used to prepare them, and Wilcoxon Signed Ranks Test was used to statistically evaluate whether the inspected biochemical parameters played a role in the ossification process of quail and whether a statistical difference existed between sexes in this regard. The findings of our study revealed that poultry animals also have five zones in the epiphyseal plate as do mammals, and they have calcified cartilage areas.
The findings also indicate that, while ossification starts to occur both in male and female quail specimens, calcification occurs more frequently in females. It was determined that the proximal epiphyseal plate regions of the femur close at the end of the sixth week in both sexes of quail, although the calcification and ossification are more advanced in some females compared with males.
The majority of the mammalian skeleton is formed by a process called endochondral ossification, which occurs when the cartilage excluding the parietal bones gives way to bone tissue. The vertebral column, pelvis, and bones of the extremities form through endochondral ossification. The ossification of long bones starts at the end of the embryonic period. The focus of the first ossification, which occurs to constitute diaphysis, is called the primary ossification center.
While it may occur at various stages, a primary ossification center is present for almost every bone at birth. The focused ossification at the endpoints of the long bones is called the secondary ossification center. The ossification initiated by the secondary ossification center continues by expanding radially from the inner part of the anlage.
A cartilage region remains between this ever-growing bone tissue and the diaphysis. The endochondral ossification is strictly controlled by the circulation system. There are many elements that play a significant role in the reproduction and differentiation of chondrocytes in the growth plates such as the systemic hormones and signal factors, which are produced locally, protein PTHrP related to the Indian hedgehog lhh , parathyroid hormone or parathormone PTH , PTH1R receptor, fibroblast growth factor 18 FGF , and FGFR3 receptor.
The critical role of the epidermal growth factor receptor in endochondral ossification. Journal of Bone and Mineral Research Due to this process, the plate region contains three distinct types of chondrocytes: proliferating chondrocytes, mature chondrocytes, and hypertrophic chondrocytes. The region is ossified in the perichondrium and transitions to the periosteum.
Temel histoloji. The osteoblast membranes contain PTH receptors, which trigger the secretion of the osteoclast-stimulating hormone when stimulated by PTH. Osteoblasts also secrete a high level of hormone during the active bone formation process and cause an increase in blood level of alkaline phosphatase ALP enzyme. The ALP blood concentration is an important indicator of the ossification process status Oznurlu et al.
Histological and histochemical evaluations on the effects of high incubation temperature on the embryonic development of tibial growth plate in broiler chickens. Microscopy Research and Technique The PTH enables osteoclasts to provide acid hydrolases to the matrix by increasing the lysosome synthesis in the osteoclast. This also initiates bone resorption and generates many invaginations by increasing the membrane convolutions.
The Ca and phosphate PO 4 ions are released as a result of increasing bone destruction, and their amounts in the blood are increased. This increase halts the PTH secretion.
Ossification and growth of the bones of the wings and legs in prehatching period of the hubbert strain broiler. Comparative bone anatomy of commonly used laboratory animals: Implications for drug discovery. Comparative Medicine The osteoporosis of proximal femur, lumbar vertebra, and bone properties of the mandibula takes place as pathophysiology for humans, and is frequently assessed in preclinical research Bagi et al.
Bone tissue studies in poultry animals are performed to investigate the skeletal mutations, determine embryonic development conditions in laboratory conditions, describe endochondral ossification of various bones in embryonic period, and reveal the teratogenic results of new medicines Pourlis et al.
Ossification and growth rates of the limb long bones during the prehatching period in the quail Coturnix coturnix japonica. Anatomia, Histologia, Embryologia Endochondral ossification process of the turkey Meleagris gallopavo during embryonic and juvenile development.
Poultry Science The ossification of the pelvic girdle and leg skeleton of the quail Coturnix coturnix japonica. In the literature survey performed for the present study, we found no studies regarding sex-based changes in biochemical parameters for the histology of epiphyseal plate closure process in quail in the post-hatching period.
This study was conducted to determine the effects of sex on ossification process following quail hatching by determining the exact closure timing of the epiphyseal plate in the proximal region of femur, using the histological and biochemical parameters that are known to influence the osteogenesis, and observe if any variation exists between males and females in this regard.
A total of 84 animals was used in the study, of which 42 were males and 42 females. Artificial fluorescent lighting and natural sunlight were used in the cages with a h lighting program.
Starting in the third week, both male and female quail were fed a fattening feed. The same feed was given to both groups.
Effects of dietary addition of synbiotic on the performance, carcass traits, and serum parameters of Japanese quails. Revista Brasileira de Zootecnia Table 1. All quails were given the same quality feeds and water ad libitum during the study.
Blood samples were collected from six male and six female specimens via IV catheters every week for 50 days following hatching into serum tubes. Then, they were transported to the laboratories and stored following cold-chain procedures. After the seventh week, the subjects were euthanized. Histological cut-sections covering the epiphyseal growth plate and bone sections of the proximal region of the right femurs of the quail were collected.
In contrast, in nonfusing growth plates, there was a small distinct border between loosely packed collagen type II in the cartilage matrix and collagen type I in the bone matrix Fig.
Collagen type I contains thick fibers compared with the very thin collagen type II fibers and therefore these two types of collagen could easily be distinguished. There were no signs of classical apoptosis in the fusing growth plate; e. In addition, no typical signs of inflammation were found although we did observe some signs of early necrosis like empty vacuoles see arrows Fig. In addition, we observed signs of hypoxia, e. No double-membrane autophagosomes, like typically seen in cells undergoing autophagy, were found in the fusing growth plate.
TEM images from the proximal femur epiphyseal growth plate in a mid-pubertal patient patient 9; left panels , A, C , and E and the late pubertal patient with a fusing growth plate patient 13; right panels B, D, and F.
In the midpubertal patient, hypertrophic zone chondrocytes displayed a normal morphology A and at a higher magnification, dense chromatin was found in the cell nucleus C. In contrast, the fusing growth plate displayed a patchy chromatin pattern B and D.
At high-power magnifications of the interphase between cartilage and bone matrix, a distinct border between loosely packed cartilage matrix collagen type II Coll-II and bone matrix collagen type I Coll-I was found in the midpubertal patient E , whereas in the fusing growth plate, the border was thicker and the bone matrix collagen type I more dense F.
In early-pubertal and mid-pubertal growth plates Fig. We specifically focused on the terminal hypertrophic chondrocytes that were about to be incorporated into the newly formed bone. In these cells, some chromatin condensation was seen, but this was clearly different from what normally is seen in cells undergoing classical apoptosis. Cell membranes were intact and the cytoplasm mostly empty.
No autophagosomes could be found and no sign of necrosis was present. In all female two prepubertal and 12 pubertal and male five pubertal subjects, very few growth plate chondrocytes stained positive or showed an apoptotic-like morphology when applying the TUNEL method Fig. The staining intensity was scored 0 to 3 points; 0 indicating no staining and 3 indicating many positive cells throughout the growth plate , and the results are shown in Table 1 , ranked according to pubertal stages.
It is important to point out that when evaluated by experienced scientists in the field, most TUNEL-positive cells indeed were considered to have a viable appearance, as indicated by a complete absence of apoptosis-related morphologic changes, and should therefore be considered as nonspecifically stained. In contrast, the surrounding bone marrow was abundantly stained with the TUNEL technique and could therefore serve as an internal positive control as previously described 21 , Negative controls showed no staining.
Panels C and D , Bcl-2 staining patient 4 and fusing growth plate. Panels G and H , Bax staining patient 4 and fusing growth plate. Panels I and J , Bad staining patient 3 and fusing growth plate. Panels K and L , cleaved caspase-3 patient 3 and fusing growth plate. When the relative staining intensity was scored score 0 to 3 , a possible tendency toward increased expression was found during maturation Table 1. The fusing growth plate did show some staining for Bcl-2 score 2; Fig.
No change in percentage positive cells or staining intensity was seen during maturation. In addition, in the fusing growth plate some cells stained positive for Bcl-X L score 2; Fig. No change in staining intensity or percentage Bax positive cells was seen during pubertal development. Interestingly, in the fusing growth plate no staining for Bax could be detected Fig.
No change in staining intensity or percentage of positive cells was seen during development. Some staining for Bad proteins was observed in the fusing growth plate score 2; Fig. In the fusing growth plate, no staining for cleaved caspase 3 was detected Fig.
On the basis of well-established morphologic and histologic criteria of apoptosis, we could not detect any signs of classical apoptosis in human terminal hypertrophic growth plate chondrocytes. These findings were also confirmed by electron microscopy EM. In a unique tissue specimen of a late pubertal fusing human growth plate, we found clear evidence that chondrocyte apoptosis is not likely to be involved in the end phase of growth plate fusion in humans.
In contrast, signs of hypoxia and early necrosis were present in the fusing growth plate. This is the first detailed study of apoptosis in the human pubertal growth plate that includes expression levels of pro- and antiapoptotic proteins together with morphologic analysis based on light and EM.
Our study includes the characterization of a unique tissue sample from a growth plate in the process of undergoing fusion where we made the novel observation that the chondrocytes were markedly enlarged, disorganized, and surrounded by a border of dense, cortical-like bone. No morphologic signs of classical apoptosis were found in this fusing growth plate.
Our findings are in agreement with earlier studies describing terminal hypertrophic zone chondrocytes at the chondro-osseous junction to have a morphology, which is not typically seen in cells undergoing classical apoptosis 11 , 14 , 25 , However, it is important to point out that all these previous studies were performed in animal growth plates; avians or rodents that do not fuse their growth plates by the end of sexual maturation.
The mechanism by which terminal hypertrophic chondrocytes disappear during endochondral ossification is believed to be related to the underlying cause of eventual growth plate fusion. However, these two events involving the epiphyseal growth plate might as well be two different and independent processes. In this article, we compared both. Interestingly, we found some signs of early apoptosis and also some signs of necrosis such as empty vacuoles in the fusing growth plate, suggesting that fusing growth plate chondrocytes appear in a sort of intermittent stage between apoptosis and necrosis.
Importantly, no signs of inflammation were observed around the fusing growth plate. Erenpreisa and Roach 26 also reported signs of necrosis in dark chondrocytes of the embryonic chick growth plate, however, such dark chondrocytes were not found in our human growth plate tissue samples. To our knowledge, no other signs of necrosis have previously been reported in the growth plate. Interestingly, Shapiro et al.
However, we did not see any double membrane structures like autophagosomes suggestive for autophagy but only vesicles with a single membrane that we interpreted as vacuoles.
Apoptosis is the most widely accepted and described mode of cell death through which terminal hypertrophic chondrocytes disappear and are replaced by bone In many studies, TUNEL positive chondrocytes were observed not only in the hypertrophic layer where cells are assumed to die before being incorporated into the newly formed bone, but also in all zones of the growth plate 6 , 17 , When re-evaluating pictures of growth plate chondrocytes positive for TUNEL staining, a high diversity of results could be found.
Cell morphology is not always complementing TUNEL positivity and when data are presented, cells often do not look like cells in the process of undergoing apoptosis, but as healthy viable cells without showing signs of cell shrinkage and pyknotic nuclei 6 , 17 , Thus, the TUNEL method is very sensitive, requires precise temporal control of each step 28 and must be verified by other techniques like EM or molecular markers of apoptosis. Molecular markers of apoptosis include cleavage of caspases and often regulation of pro- and antiapoptotic proteins of the Bcl-2 family In this study, we are the first to investigate these proteins in the human postnatal growth plate and we found that they are indeed expressed throughout the whole growth plate.
We did not observe any caspase-3 cleavage, the effector caspase of apoptosis in the fusing growth plate. Moreover, in the fusing growth plate, we observed clear expression of the antiapoptotic proteins Bcl-2 and Bcl-X L and a possible down-regulation of the proapoptotic protein Bax. Thus, all three approaches including TUNEL technique, analysis of cell morphology by EM, and molecular markers by immunohistochemistry altogether suggest that there is no classical apoptosis occurring in the fusing human growth plate.
A novel and clear observation was that the fusing human growth plate is surrounded by a border of very dense thick bone, shelling the growth plate remnant. There are no vessels seen in or surrounding the fusing growth plate. Moreover, there were some signs of hypoxia in the fusing growth plate like patchy distributed chromatin. From these findings, one can hypothesize that this border of dense bone is functioning as a physical barrier for oxygen and nutrients to reach the fusing growth plate resulting in hypoxia and eventually cell death in a nonclassical apoptotic way through necrosis or a mixture of apoptosis and necrosis.
White et al. A limitation of this study is the relatively small number of growth plates that were analyzed and the fact that some were derived from pathologic conditions.
To some extent, this is compensated by the fact that we were fortunate to obtain a tissue sample from a human growth plate just in the rapid process of undergoing epiphyseal fusion, which allowed us to investigate possible underlying mechanisms.
In addition, the underlying mechanism of epiphyseal maturation and fusion will be the same for all growth plates regardless of the underlying disease of each patient because eventually longitudinal growth stopped in all patients by the end of puberty.
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