Examination of the femoral diaphysis and lumbar vertebrae of group II rats (the sham+CH treated group) showed more or less the control image of cortical and trabecular bones. The mean color area percentage of collagen in both bones did not exhibit any significant difference when compared with the control group.
Examination of the femoral diaphysis of OVX rats (group III) revealed irregularly separated, palely stained bone lamellae with multiple cavities alternating with some mature bone lamellae (Fig. 15a). The mean color area percentage of collagen was significantly lower with respect to that of the control group. In contrast, the lumbar vertebrae of the same group revealed thinned out, widely separated bone trabeculae with alternating areas of mature green-stained bone lamellae and palely stained, ill-defined immature osteoid tissue (Figs 15b and c). The mean color area percentage of collagen in the lumbar vertebra was significantly lower as compared with that of the control group.
Examination of the femoral diaphysis and lumbar vertebrae of group IV rats (the OVX+CH group) revealed an apparent increase in the mature green-stained collagen of bone matrix with less cavity formation as compared with the OVX group (Figs 16a and b). This was further documented histomorphometrically by the mean color area percentage of collagen that was significantly higher in both bones with respect to OVX group III (Table 2).
Osteoporosis is the most common skeletal disorder and its prevalence is expected to rise dramatically in the coming decades. Prophylaxis and therapy of osteoporosis are still unresolved challenges, and thus it is increasingly becoming a public health problem 6,20.
In view of the above, it seems imperative to look for some acceptable method to counter the problem of osteoporosis 21. The ovariectomized (OVX) rat is the most appropriate experimental model for postmenopausal bone loss that faithfully reproduces the changes observed in human subjects and has the added benefit that the effects are detectable only a few months after gonadectomy 14,22. The femur is one of the most important skeletal sites in postmenopausal osteoporosis. There are many similarities between the human and rat femur, both at the microstructural and macrostructural levels 23,24. For these reasons, this skeletal site has gained special meaning and great importance in osteoporosis studies 24. Because of the high clinical relevance of the vertebrae, they were chosen for histological examination as well 25. Therefore, the objective of this work was to test the capability of CH to counteract the debilitating effect of osteoporosis on the bone microstructure, mineral content, and some biochemical parameters in a rat OVX model.
In the present study, successful ovariectomies were visually confirmed by the high degree of atrophy in the uterus 13. The OVX model (group III) displayed significant bone loss after 12 weeks as depicted by evident reduction in the bone mineral contents (BMCs) of Ca in the femur as compared with an age-matched sham group. This was in agreement with the literature that reported significant reduction in BMD and content after OVX 26,27. Low estradiol is certainly one possible explanation for the lower observed BMCs in osteoporotic rats 28.
Moreover, there was a corresponding decrease in the mean value of serum Ca, whereas CTX, a marker of type I collagen degradation, revealed a significant increase as compared with the sham group. Similarly, several researchers clarified a significant increase in CTX after OVX 29.
In order to study the possible variations in bone remodeling that could explain BMC alterations, BSAP and osteocalcin biochemical markers for bone formation were measured. Bone alkaline phosphatase is localized in the plasma membrane of OBs and released into the circulation during bone mineralization. It is considered an excellent indicator of osteoblastic activity and commonly used in diagnosing and monitoring the bone formation rate 21,30. In the current work, a significant increase in the mean value of BSAP in OVX rats was observed as compared with their respective sham group. It reflects increased bone turnover 30. Similarly, several investigators have declared a significant increase in serum ALP in OVX rats 26,27.
Osteocalcin is the most abundant noncollagenous protein produced by OBs and is unique to bone tissue 4,31. Its concentration in serum is closely linked to bone metabolism and serves as a biological marker of bone formation 21. It influences bone mineralization in part through its ability to bind with high affinity to the mineral component of bone, hydroxyapatite (HAP) 21,22.
The present study depicted a significant increase in the mean value of serum osteocalcin in the OVX group. This pattern is quite similar to that observed in OVX animals 14, as well as in postmenopausal osteoporotic women, in which a general increase in biochemical markers of bone turnover is found with a predominance of the resorptive process, leading to BMD loss 30. Kawakita et al.4 declared that osteocalcin is deposited in the bone matrix, although a small fraction of the newly synthesized component is released into the blood stream while its activity and synthesis increase when BMD is decreased because of higher bone resorption.
How estrogen loss can induce an increase in bone formation became a topic of many studies. It is well known that bone formation occurs only at sites of previous resorption in remodeling bone, where the appropriate number of mature OBs is thought to be controlled by factors, such as transforming growth factor-β, that are released from the bone matrix during its resorption by OCs. Hence, the increase in bone resorption in OVX rats should be associated with an increase in bone formation 32.
The studied biochemical parameters were further supported with the histological results. The skeleton was examined at two different areas: the femoral diaphysis as an example of the cortical bone, and the lumbar vertebrae as a site of the trabecular bone. The cortical bone of the femoral diaphysis of OVX group III rats showed evidence of bone affection with multiple resorption cavities and an irregularly eroded endosteal surface containing OCs. A remarkable widening of osteocytic lacunae was observed as well. The latter is speculated to be an evidence of osteocytic osteolysis, which has been proven to have a Ca homeostatic effect 33. Moreover, histological examination of lumbar vertebrae of OVX rats demonstrated that bone resorption was intensified in the cancellous bone as depicted by thinned out, widely separated, and disconnected bone trabeculae along with relatively wide bone marrow spaces. They further exhibited multiple resorption areas containing OCs. These findings coincided with previous studies that reported significant decreases in trabecular number and thickness as well as in the bone volume fraction in lumbar vertebrae of OVX animals 14,27. Furthermore, the apparent increase in the number of OCs was largely consistent with the results of Behari and Behari 1, who reported that OC-like multinucleated cells were more numerous in the OVX group.
With trichrome stain, there was a significant decrease in the mean color area percentage of green-stained collagen in OVX rats as depicted histomorphometrically and statistically. Similarly, Kafantar et al.34 reported that bone collagen declined in parallel with hypoestrogenism due to OVX.
Bone homeostasis is controlled by the balance between osteoblastic bone formation and osteoclastic bone resorption. After menopause, the depletion of estrogen results in an increase in bone turnover with the rate of osteoclastic resorption exceeding that of osteoblastic formation, a fact that results in a loss of bone mass 35,36.
OBs secrete two proteins: osteoprotegerin (OPG) and receptor activator of nuclear factor κB ligand (RANKL). The interaction between RANKL on OBs and the RANK receptor on hematopoietic OC precursor cells enhances OC recruitment and activation. However, OPG functions as a soluble decoy receptor to RANKL and competes with RANK for RANKL binding to inhibit OC recruitment, thereby controlling OB-OC activity balance 37–39. The ratio between RANKL and OPG elegantly regulates the orientation of bone metabolism to either bone formation or resorption. Therefore, dysregulation of this ratio results in bone disease such as osteoporosis 3,37.
OCs are the only cells capable of bone resorption, a process required for both normal bone homeostasis and pathological bone loss. OC formation occurs when bone marrow macrophages are costimulated by the two osteoclastogenic factors, RANKL and macrophage colony-stimulating factor (M-CSF). However, in conditions of estrogen deficiency (such as in postmenopausal women and OVX animals), the secretion of RANKL decoy receptor OPG decreases, whereas RANKL and other cytokines, including TNF-α, interleukin 1 (IL-1), IL-6, IL-7, and M-CSF, are produced in greater amounts. OBs are the significant sources of M-CSF, RANKL, OPG, IL-6, and IL-7, whereas T-lymphocytes are a critical source of TNF 39. After activation, OCs cause a local decrease in pH, which precipitates the dissolution of mineral. Exposure of the matrix permits enzymatic degradation of collagen fibers 40.
It has been reported that estrogens attenuate OC generation and life span by cell autonomous effects mediated by DNA-binding independent actions of estrogen receptor-α. Elimination of these effects is sufficient for loss of bone in which complete perforation of bone lamellae by osteoclastic resorption precludes subsequent refilling of the cavities by the bone-forming OBs 41. This might explain the abundance of OCs observed on the endosteal surface of the cortical bone, which appeared irregularly eroded, as well as in the resorption areas in the cancellous bone trabeculae of OVX group III rats. Moreover, estrogen deficiency has been speculated to suppress survival of osteocytes and impair the response of OBs to mechanical stimuli 42.
Several lines of recent evidence strongly suggest that the oxidative stress that underlies physiological organismal aging is a pivotal pathogenetic mechanism of the age-related bone loss. The antiosteoporotic effect of estrogen results, at least in part, from their ability to protect against oxidative stress. In fact, loss of estrogen or androgen accelerates the effects of aging on bone by decreasing defense against oxidative stress. Moreover, an association between oxidative stress and a decrease in BMD has been noted in several human clinical studies 41.
A rise in urinary Ca at menopause is another factor that contributes to the development of osteopenia. It is suggested that estrogen promotes tubular reabsorption of Ca and that the rise in bone resorption at menopause could be accounted for, in part, by the effect of estrogen deficiency on the kidney 30. This could explain the depicted low mean value of serum Ca in OVX rats of this work.
A good management strategy for osteoporosis should always weigh the risk of adverse reactions against the benefits of treatment – that is, prevention of osteoporotic fracture 43. In the current work, the oral use of CH at a dose of 2.5 g/kg body weight/day for 8 successive weeks could be regarded as safe as it produced nonsignificant differences in the BMC and in the studied biochemical parameters of bone turnover as well as in normal histological characteristics of bone when compared with the control rats. This is in agreement with the literature that classified CHs as Generally Recognized as Safe by the Food and Drug Administration for a number of years 13.
To more closely mimic the clinical scenario of treatment of postmenopausal osteoporosis, systemic CH treatment was started 4 weeks after OVX and continued for 8 weeks. The bone-conserving effects of CH are well established in group IV rats as it enhanced the BMC and serum Ca levels as compared with OVX group III rats. It further ameliorated the raised circulating biochemical markers of bone formation (BSAP and osteocalcin) as well as CTX, a marker of type I collagen degradation as compared with their respective OVX rats; yet, these levels were significantly higher as compared with those of the sham group. As such, it is suggested that CH treatment closely offsets the bone loss induced by OVX and decreases the rate of bone turnover. These data were in accordance with the findings of Guillerminet et al.10,12, who reported that feeding a diet enriched with CH can significantly increase bone metabolism and biomechanical properties (such as BMD) of OVX animals. The persistently elevated BSAP levels after CH treatment in comparison with the sham group reflected an increase in OB number and its biosynthetic product, collagen 1, whereas the lowered bone turnover marker CTX reinforced the view that CH is able to reduce bone resorption in OVX animals 10,12.
Histologically, CH treatment following OVX significantly improved bone microstructure, where the examined bones almost retained their normal architecture except for a few small resorption cavities along with some irregularly arranged osteocytes within the matrix in the femoral diaphysis. Enhanced periosteal activity was further encountered as indicated by the apparent increase in the thickness of the inner cellular layer of the periosteum that contained numerous osteogenic cells. This might be explained as a compensatory mechanism for maintaining bone strength 44. Moreover, CH treatment significantly increased bone collagen content as depicted morphometrically in trichrome-stained sections with respect to OVX rats. In accordance, Nomura et al.45 reported a greater amount of type I collagen and proteoglycans in the bone matrix of OVX rats that received CH, in comparison with the controls that received albumin. In their experiment, the newly synthesized collagen was distinguishable from the pre-existing type I collagen because it had not yet developed into mature crosslinked fibers. Likewise, Oesser and Seifert 46 reported greater deposition of type II collagen in both the cartilage and bone of rats fed CH.
It is well known that bone strength depends not only on the quantity of bone mineral (BMC) but also on its quality, which is characterized by several factors including collagen content and quality. Collagen provides elasticity and structure in all connective tissues as it accounts for 65% of the total organic component of bone tissue. It is important for bone toughness, whereas the mineral component is mainly involved in providing stiffness 47. Wang et al.48 clarified that the mechanical integrity of collagen fibers deteriorates with aging in human cortical bones and is associated with a higher fracture risk. When the collagen network becomes weaker with age, it will result in decreased toughness, possibly because of a reduction in natural crosslinks between the component chains or its silicon content. Moreover, Manolagas 41 reported that collagen can also be damaged by accumulation of advanced glycation end products, another general feature of the aging process. Such changes could account for the age-related decline in cortical bone tensile strength 31. The author further postulated that defective collagen cannot be repaired, and hence the bone containing it must be replaced by remodeling.
As one of the pathogenetic factors of osteoporosis is the dyshormonal lysis of collagen fibrils, the volume of organic matrix where crystallization of HAP occurs decreases. It is reasonable to assume that lysis begins with dissolution of thin fibrils of protocollagen, and the vacant spaces are filled with bulk water. Thus, swelling inside the fibrils occurs with subsequent hydration of HAP crystals and demineralization of collagen fibrils, which might be one of the early stages of osteoporosis 49.
Kafantar et al.34 studied the effect of OVX on collagen fibrils at the ultrastructural level and reported that the overall bone collagen fibril architecture was disturbed when compared with normal controls. Moreover, the mean diameter values of collagen fibrils were significantly smaller than those of controls, despite retaining normal banding periodicity. They concluded that osteoporosis leads to defects in bone collagen fibril formation and stabilization.
While the process of bone degeneration after OVX is not fully understood, even less information is available for the process of recovery after CH treatment. It has been postulated that collagen needs to be in the form of hydrosylate to be able to interact with bone metabolism as the low-molecular-weight collagen peptides, degraded by enzymes, are more efficiently absorbed through intestinal epithelium than collagen, which is a very stable and high-molecular-weight protein 12,50. Ingestion of type I CH is thought to result in the release and absorption of collagen-derived peptides similar to those released from type I collagen in situ during bone resorption, which may improve the mechanical properties of osteopenic bone by increasing collagen content, thereby improving its quality 12,51. Indeed, collagen-derived dipeptides and tripeptides rich in hydroxyproline have been detected in human blood following the ingestion of CH 52.
Chayanupatkul et al.53 clarified that the newly formed bone contains type III collagen, which is the emergency type, and a good candidate for repairing bone matrix. Type III collagen will then be replaced by the more prominent type I collagen, which is the most stable because of its very strong crosslinks allowing more stability for the new bone. Moreover, Ono et al.54 declared that collagen plays an important role in binding Ca in bone as the highly ordered structure of collagen in the matrix acts as a nucleation site for Ca and phosphate solution and the solution begins to crystallize into the gap region of the collagen. Thus, it is suggested that CH administration to OVX rats, in this work, prevented the loss of BMC by causing upregulation of collagen synthesis from OBs, as both cortical and trabecular bones of the femur and lumbar vertebrae showed multiple distinct cement lines. This goes in hand with the results of Guillerminet et al.10,12, who emphasized that CH enhanced the growth of the external diameter of the bone cortical zone in OVX mice without modification of the size of the medullary area. Such increased size of the cortical area was therefore attributed to periosteal apposition of bone on the mouse femur and suggested a higher level of bone formation. Consequently, the ultimate strength of OVX+CH mice femurs was significantly greater than that of OVX. Moreover, the authors supported the notion that CH modulates bone formation and mineralization of the bone matrix by stimulating OB growth and differentiation while reducing OC differentiation and maturation. These effects ultimately led to the growth of the external diameter of the cortical bone and reduced bone resorption.
The observation that CH improved the biochemical and histological properties of bone in castrated female rats is of particular importance in the treatment of osteoporotic patients. The safety profile further supports its clinical application as a novel therapeutic agent against postmenopausal osteoporosis. Further studies are needed to evaluate the possible benefits of CH supplementation in women at high risk for enhanced postmenopausal osteoporosis.
Conflicts of interest
There is no conflict of interest to declare.
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Keywords:© 2013 The Egyptian Journal of Histology
bone; collagen hydrosylate; histology; osteoporosis; ovariectomy