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Histological study of the femur and the lumbar vertebrae in ovariectomized adult albino rats following administration of collagen hydrosylate

Abd El Moneim, Rehab A.; Mahmoud, Sahar A.

The Egyptian Journal of Histology: September 2013 - Volume 36 - Issue 3 - p 646–659
doi: 10.1097/01.EHX.0000434384.05294.02
Original articles
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Introduction Osteoporosis is the most common skeletal disorder that has become a leading cause of morbidity and mortality worldwide. Its prophylaxis and therapy are still unresolved challenges.

Aim of the study The aim of the study was to investigate the possibility that collagen hydrosylate (CH) can ameliorate osteoporotic bone loss in ovariectomized rats with special reference to bone mineral content (BMC), some biochemical parameters of bone turnover, and histology.

Materials and methods Forty adult female albino rats (180–200 g) were categorized into four equal groups: a sham-operated control group (group I), a sham-operated CH-treated group (group II), an ovariectomized group (group III), and a CH-treated ovariectomized group (group IV). The experiment continued for 12 weeks. At its end, the animals were sacrificed under anesthesia. Blood samples were collected for estimation of serum calcium, osteocalcin, bone-specific alkaline phosphatase, and C-terminal telopeptide of type I collagen (CTX). The left femora and lumbar vertebrae were excised for histological examination by H&E and Gomori’s trichrome stains. The area percentage of collagen was further assessed using an image analyzer. The right femur of each rat was used for BMC measurement by energy-dispersive X-ray analysis.

Results In sham-operated CH-treated rats (group II) there was no significant variation in bone turnover markers and BMC as compared with their respective controls. Normal bone microstructure was depicted as well. In group III rats, ovariectomy (OVX) was associated with enhanced bone turnover as depicted by significant decrease in the mean value of serum calcium, whereas osteocalcin, bone-specific alkaline phosphatase, and CTX revealed significant increase compared with controls. Moreover, an evident reduction in bone calcium content was depicted in the femora of this group. Histologically, evidence of bone resorption was manifested in the femoral diaphysis and lumbar vertebrae with multiple resorption cavities, irregularly eroded endosteal surface containing osteoclasts, and thinned out bone trabeculae along with wide bone marrow cavities. A significant decrease in bone collagen content of both cortical and trabecular bones was evidenced in trichrome-stained sections. In contrast, CH administration after OVX (in group IV) reduced bone turnover markers and improved BMC as well as histological characteristics of examined bones as compared with the OVX group.

Conclusion The study suggested that CH may be a potentially useful agent in preventing bone loss due to ovarian hormone deficiency.

Department of Histology, Faculty of Medicine, University of Alexandria, Alexandria, Egypt

Correspondence to Rehab Ahmed Abd El moneim, Department of Histology, Faculty of Medicine, University of Alexandria, Alexandria, Egypt Tel: + 01001092769; e-mail: rehabahmedns@yahoo.com

Received February 5, 2013

Accepted July 1, 2013

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Introduction

Osteoporosis is a progressive skeletal disease associated with structural failure of the skeleton. It affects millions of people worldwide, and is considered second to cardiovascular diseases as a leading health problem 1. Osteoporosis is characterized by low bone mass (osteopenia) and microarchitectural deterioration of bone tissue. This consequently increases bone fragility and susceptibility to fractures, especially of the hip, spine, and wrist 2,3. The risk of developing osteoporosis increases with age and is four times higher in women than in men. Moreover, fracture rates among women are approximately twice as high as those in men 4.

The etiology of osteoporosis is multifactorial: apart from its relation to age and sex, it is also related strongly to genetic, endocrine, and environmental factors. Different patterns of lifestyle also influence the development of osteoporosis, especially exercise and nutritional habits 5. Osteoporosis is divided into two types: postmenopausal and senile types. The causes of postmenopausal osteoporosis are accelerated bone resorption and systemic calcium (Ca) imbalance due to menopause-induced estrogen deficiency 6. In contrast, senile osteoporosis is attributed to age-related reduction of osteogenesis, insufficient Ca intake, or reduced Ca absorption 7.

Therapies currently approved for the treatment of osteoporosis include estrogen, calcitonin, bisphosphonates, selective estrogen receptor modulators, parathyroid hormone (PTH), and strontium ranelate among others. Although estrogen replacement has produced positive results with respect to improved bone mineral density (BMD) and reduced fracture incidence in early menopause, its prolonged use is restricted because of potential complications such as breast cancer, uterine bleeding, and cardiovascular disorders. One concern related to the usage of bisphosphonates is the complication of osteonecrosis of the jaw. Their use is further limited by their poor absorption from the gastrointestinal tract and their association with esophageal erosions 2. Despite an excellent safety profile for PTH, concerns do arise from its persistence after discontinuation without sequential use of antiresorptive drugs. Besides, patients on PTH need to be closely monitored to avoid the risk of hypercalcemia, and such a treatment is very costly 1,8. These potential limitations of existing drugs are sufficient to warrant the development of novel therapies 9.

Nowadays, most research is directed at naturally occurring agents such as nutritional components with potential antiresorptive effect. Hydrolyzed collagen type I or collagen hydrosylate (CH) is one of the natural alternatives currently under investigation 10,11. Type I collagen is the major structural protein distributed throughout the whole body, accounting for 25% of total body protein and 80% of total conjunctive tissue in humans. It is an important component of bone, being the main extracellular matrix protein for calcification, which also plays a role in osteoblast (OB) differentiation. CHs are mixtures of peptides obtained by partial hydrolysis of gelatins. They are derived by enzymatic hydrolysis of animal skin as it contains predominantly type I collagen. They are of bovine, porcine, or fish origin and can be obtained commercially in some countries 12. Recently, CHs have been receiving scientific attention as potential oral supplements for the recovery of osteoarticular tissues. Some studies suggest that a CH-enriched diet improves bone collagen metabolism and BMD 13.

The mature ovariectomized (OVX) rat is the most commonly used preclinical experimental model of postmenopausal osteoporosis, whereby cessation of ovarian estrogen production consequently results in bone volume reduction 14.

In such a context, the aim of the present study was to evaluate the efficacy and safety of long-term treatment with CH on the microstructure of the femoral diaphysis and lumbar vertebrae as well as some biochemical bone turnover markers in bilaterally ovariectomized rats, a reliable in-vivo model for postmenopausal osteoporosis.

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Materials and methods

The study was conducted on 40 adult female albino rats weighing 180–200 g. They were housed under the same environmental conditions and allowed free access to water and standard rat chow ad libitum. After a 1-week acclimatization period, the rats were randomly assigned to four equal groups of 10 animals each.

  • Group I (sham group): Rats in this group were sham-operated and served as controls.
  • Group II (sham+CH group): Rats in this group were sham-operated. Four weeks later, they were started on CH treatment orally by gavage at a dose of 2.5 g/kg body weight/day for 8 weeks.
  • Group III (OVX group): Rats in this group were bilaterally ovariectomized and left without treatment for 12 successive weeks.
  • Group IV (OVX+CH group): Rats in this group were bilaterally ovariectomized, and 4 weeks later were given CH treatment orally by gavage at a dose of 2.5 g/kg body weight/day for the next 8 weeks 10,12.

Guidelines for the ethical care and treatment of animals from the Local Ethical Committee of the Faculty of Medicine, University of Alexandria, were strictly followed. CH was purchased from Holland & Barrette Company (Nuneaton, Warwickshire, USA) in the form of tablets that were dissolved in distilled water and given orally by gavage. Its administration was initiated 4 weeks after sham operation or OVX (to await the development of osteopenia) and continued for 8 successive weeks. At the end of the experiment, all animals were sacrificed under anesthesia.

Blood samples were collected by cardiac puncture, and the sera were separated to be stored at a temperature of −20°C for biochemical analyses.

The femora were harvested for histological examination and mineral content assessment, whereas lumbar vertebrae (L4-5) were harvested for histological examination only.

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Ovariectomy

Ovariectomy was performed under phenobarbital sodium anesthesia (30 mg/kg body weight) administered intraperitoneally. Bilateral OVX was performed in experimental groups III and IV. A median laparotomy was performed to identify the right and left cornu uteri and their corresponding ovaries. After suturing the vascular plexus with fine linen thread, both ovaries were removed, followed by closure of the abdominal cavity. Postoperative care included the administration of analgesics and antibiotics. Control rats (group I) were subjected to sham surgery during which the ovaries were exteriorized but replaced intact 1.

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Biochemical analysis

Serum concentration of Ca was measured using an autoanalyzer (Hitachi 7170; Hitachi Co. Ltd, Tokyo, Japan) 15. Bone-specific alkaline phosphatase (BSAP) activity was quantified in serum using the enzyline PAL optimize kit (Biomerieux, Paris, France) 16. The serum level of osteocalcin was measured by enzyme immunoassay using a rat osteocalcin EIA kit (Biomedical Technologies, Stoughton, Massachusetts, USA) 17.

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Bone mineral content

Elemental bone composition (Ca content) was determined using Energy-Dispersive X-ray Analysis (Oxford EDX, Jeol JSM-5300; Jeol, Tokyo, Japan) at the Electron Microscopy Unit, Faculty of Science, Alexandria University.

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Histological examination

The left femur and lumbar vertebrae (L4-5) of each rat were dissected out, stripped of all soft tissues, and washed in saline. Specimens were fixed in 10% formol saline for 48 h, and then decalcified in daily exchanges of EDTA. Each specimen was then processed to obtain 6-μm-thick paraffin sections to be stained with:

  • H&E stain 18.
  • Gomori’s trichrome stain 19.
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Bone histomorphometry

The color area percentage of green-stained collagen fibers was measured in trichrome-stained sections of both femoral diaphysis and lumbar vertebrae at a magnification of 200 using the Image Analyzer (Olympus BX41TF; Tokyo, Japan) at the Cell Biology Department, Medical Research Institute, Alexandria University. Ten images were captured per section.

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Statistical analysis

Data were expressed as mean±SD. They were fed into the computer using statistical package of social science (IBM SPSS, Chicago IL, USA) software package version 20. Statistical analysis was carried out using one-way analysis of variance and the post-hoc test (Scheffe) for pairwise comparison. A P value less than 0.05 was considered statistically significant.

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Results

Biochemical results

The present study revealed that OVX produced a significant increase in the mean values of serum osteocalcin, BSAP, and C-terminal telopeptide of type I collagen (CTX), as well as a significant decrease in the mean value of serum Ca with regard to their respective sham group (P<0.05). Meanwhile, there was no significant difference between CH-treated group II and the control group with respect to the studied parameters (Table 1).

Table 1

Table 1

In contrast, CH administration after OVX (in group IV) produced a significant decrease in the mean values of serum osteocalcin, BSAP, and CTX as compared with the OVX group (P<0.05). Moreover, CH administration after OVX (in group IV) produced nonsignificant changes in the mean value of serum Ca as compared with the respective OVX group and the sham group (P>0.05) (Table 1).

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Bone mineral content

Ca and phosphorus contents of the femora of experimental animals were detected as peaks in the graphs. Sham-operated control rats (group I) revealed the highest peak intensity for Ca (Fig. 1a). Ca peak intensity of sham-operated CH-treated rats (group II) was nearly similar to that of the controls (Fig. 1b). As expected, OVX rats of group III showed the lowest peak intensity for Ca (Fig. 2a), whereas CH treatment after OVX (in group IV) was associated with an elevated peak intensity for Ca as compared with the OVX group (Fig. 2b), although it was still lower than that of the control group.

Figure

Figure

Figure

Figure

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Histological results

H&E stain

Group I (the sham group): Examination of the femoral diaphysis of control rats revealed the classical appearance of the compact bone with well-organized external and internal circumferential bone lamellae in addition to Haversian systems and interstitial lamellae. Osteocytes inside their lacunae appeared regularly organized between the bone lamellae. The outer bone surface was covered by the periosteum, which was formed of an outer fibrous layer and inner cellular layer. As regards the endosteal bone surface, it appeared smooth and lined by osteogenic cells (Figs 3a and b).

Figure 3

Figure 3

In contrast, examination of the lumbar vertebrae of the same group revealed typical cancellous bone architecture, which was formed of a network of branching and anastomosing trabeculae separated by bone marrow cavities. The bone marrow was formed of hematopoietic tissue, scattered adipocytes, and blood sinusoids. Regularly oriented osteocytes were resident in their lacunae within the bone trabeculae. The outer bone surface was covered by the periosteum, which was formed of an outer fibrous layer and an inner cellular layer. As regards the endosteal bone surface, it appeared smooth and lined by osteogenic cells (Figs 4a and b).

Figure 4

Figure 4

Group II (the sham+CH group): The histological findings of the H&E-stained sections of both the femoral diaphysis and lumbar vertebrae of this group were quite similar to those of the control group.

Group III (OVX group): Examination of the femoral diaphysis of this group of rats revealed evident histological changes in the form of multiple vacuoles within the bone matrix along with indistinct cement lines (Figs 5 and 6). Several resorption cavities were also seen within the matrix (Figs 6 and 7a). Areas of palely stained osteoid matrix were noticed as well (Fig. 6). Moreover, irregularly eroded endosteal surface of the cortical bone was frequently encountered (Figs 5 and 7b). Osteoclasts (OCs) housed within erosion cavities on the endosteal bone surface appeared as large cells with eosinophilic cytoplasm (Fig. 7b). Apparent widening of osteocyte lacunae was further evident in comparison with the control group (Figs 6 and 7a).

Figure 5

Figure 5

Figure 6

Figure 6

Figure 7

Figure 7

On examination of the lumbar vertebrae of the same group, evident thinning out and interruption of the bone trabeculae were revealed, along with wide bone marrow spaces (Fig. 8). The bone matrix appeared faintly stained in some areas, whereas in other areas it exhibited multiple resorption cavities (Figs 9a and b). Subperiosteal tunnels together with OCs housed inside them were also seen (Fig. 10). The endosteal bone surface showed multiple erosion cavities that contained OCs (Figs 9a and b). Apparent widening of the osteocyte lacunae was encountered as well (Fig. 9b).

Figure 8

Figure 8

Figure 9

Figure 9

Figure 10

Figure 10

Group IV (the OVX+CH group): On examination of sections from the femoral diaphysis of this group, marked improvement in bone microstructure was noticed in comparison with the OVX group. The bone matrix appeared deeply eosinophilic with regularly arranged bone lamellae and multiple distinct cement lines. Numerous regularly arranged osteocytes were seen within their lacunae in between the bone lamellae (Figs 11a and b). Nevertheless, few small erosion cavities and some irregularly arranged osteocytes were seen within the bone matrix. The overlying periosteum appeared with a thick, highly cellular inner osteogenic layer (Fig. 12). The endosteal bone surface appeared smooth and lined with osteogenic cells (Fig. 11a).

Figure 11

Figure 11

Figure 12

Figure 12

Examination of the lumbar vertebrae of the same group of rats revealed that the cancellous bone almost retained its normal architecture, where bone trabeculae appeared thicker and more continuous as compared with the OVX group. The bone matrix appeared denser eosinophilic and more homogenous (Fig. 13a). The periosteum showed a thick inner reactive osteogenic layer (Fig. 13b).

Figure 13

Figure 13

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Gomori’s trichrome stain

The cortical and trabecular bones of the femoral diaphysis and lumbar vertebrae of sham-operated group I (control group) revealed that the bone matrix was mostly formed of mature, green-stained collagen forming bone lamellae. The osteocytes were resident in their lacunae (Figs 14a and b). The mean color area percentage of collagen in the femoral diaphysis and lumbar vertebrae is shown in Table 2.

Figure 14

Figure 14

Table 2

Table 2

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.

Figure 15

Figure 15

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).

Figure 16

Figure 16

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Discussion

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.

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Conclusion

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.

Table

Table

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Acknowledgements

Conflicts of interest

There is no conflict of interest to declare.

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Keywords:

bone; collagen hydrosylate; histology; osteoporosis; ovariectomy

© 2013 The Egyptian Journal of Histology