The development and maintenance of bone structure depends on mechanical stimulation. Numerous studies have demonstrated that mechanical loading promotes bone formation, whereas the absence of mechanical stimulation decreases bone mass [1
]. During space flight, astronauts experience microgravity that leads to serious physiological changes, one of the most prominent being bone loss, which increases fracture risk [2
]. Long-term exposure to microgravity is associated with increased bone resorption and decreased bone formation, with a reduction in bone mineral density (BMD) of approximately 2% after one month, equivalent to annual bone loss in postmenopausal women [3
Drug intervention is not routinely used in space flight and exercise has been combined with nutrition improvement. However, because of the lack of mechanical load or duration of space flight, osteoblast stimulation is insufficient to maintain bone mass [4
]. This has led to a focus on pharmaceutical interventions such as osteoporosis drugs, but their potential to prevent bone loss in space remains to be clarified. In particular, the effects of different drugs, alone or combined, and dose–response relationships for improvements in bone quality and regeneration have not been investigated. Currently available therapeutic agents that are conventionally used for the prevention of bone loss have several side effects [5
]. Therefore, novel therapeutic approaches are being explored, which have fewer side effects, while effectively minimizing the loss of bone mass.
Collagen, which is a major component of all tissues can be produced from various sources. For example, they can be extracted from sea animals such as shark, marine sponges [6
]. In addition, they can be extracted from terrestrial animals such as porcine, bovine animals. However, collagen is mostly produced from pork skin and bovine bones. Moreover, bovine collagen is currently widely used for many applications such as foods and cosmetics [6
Collagen peptide (CP), known as collagen hydrolysates, is mainly composed of mixtures of peptides obtained by partial hydrolysis of gelatins [8
]. Due to its higher digestive absorption and safety, oral supplementation with CP for the restoration of bone joints has gained increasing scientific attention [9
]. Daily doses of 150 or 500 mg/kg of CP for up to three months significantly prevented bone loss in ovariectomized (OVX) rats compared with control rats [10
]. CP also improved vertebral composition and biomechanical strength, and increased the quantity and volume ratio of lumbar trabecular bone, which demonstrated its effect on bone protection [8
]. In postmenopausal women with osteopenia, administration of calcium–collagen chelate supplements was found to improve bone mineral density and increase the rate of bone formation and bone resorption [11
A previous study in OVX rats demonstrated that daily doses of either CP alone (750 mg/kg) or CP–calcium citrate (CC; 750–75 mg/kg) had an osteoprotective effect by inhibiting the loss of bone mineral density. Moreover, CP–CC suppressed trabecular bone loss and improved the microarchitecture of the distal femur [12
]. However, the effect of CP on prevention and restoration of bone loss induced by microgravity has not been reported. The main purpose of this study was to observe the effects of oral administration of CP, alone and in combination with CC, on bone structure and bone metabolism in rats under hind limb unloading simulated microgravity (SMG), and to provide a theoretical basis for the use of CP–CC to prevent and treat microgravity-related osteoporosis.
Bone is a complex tissue. Hydroxyapatite salts (calcium and phosphorus) with collagen form a unique matrix that plays an important role in bone hardness. In addition, the Ca/P ratio in bones is vital for osteoporosis and may provide high reliability for diagnosis, prevention, and treatment of bone disorders. Collagen fibril diameter is related to bone site and Ca/P ratio. Ca/P ratio can serve as a reliable index of bone quality [13
CP-based drugs play a role in the prevention and treatment of osteoporosis as orally administered, intestinally absorbed forms [14
]. During space flight, weightlessness leads to calcium deficiency. High calcium intake from dietary supplementation does not affect bone metabolism, but prevents an elevation in serum calcium levels through increased calcitriol levels [15
]. Therefore, intragastric administration was thought to be the most appropriate delivery route for bovine CP compounds combined with CC because of relative proximity to the pathologic process in the in vivo environment.
The results of this study show that rats in all SMG groups had significantly lower body weight than rats in the CN group, which is consistent with previous reports [16
]. This may be related to loss of water electrolytes and loss of appetite caused by redistribution of body fluid under SMG.
Conventionally, the diagnosis and treatment of osteoporosis is assessed by bone mineral density, as measured by dual-energy X-ray absorptiometry [18
]. Evaluation of bone biomechanical properties is indispensable to determine the quality of bone, and the intensity of bone fracture directly correlates to the relationship between the structure of the bone and the strength and hardness of the bone [19
]. The present data show that SMG caused marked reductions in bone mineral density and femoral fracture strength of rats, which is consistent with previous reports [2
]. These findings demonstrate that real or simulated weightlessness can cause bone changes, which are characterized by a decrease in cortical bone and cancellous bone formation [23
Previous studies found that CP–CC led to substantial improvements in the matrix structure and quality of trabecular bone in the femurs of ovariectomized rats [12
]. Dual-energy X-ray absorptiometry and biomechanical tests showed that CP–CC had a significant effect on femoral bone mineral density and fracture strength of ovariectomized rats [24
]. Therefore, in the present study, the effects of CP–CC on bone remodeling were evaluated in a rat model of SMG. However, neither CP alone nor in combination with CC inhibited bone loss in SMG rats, based on dual-energy X-ray absorptiometry and three-point bending mechanical test analyses. Furthermore, assessment of the femoral microarchitecture using microcomputed tomography revealed that neither CP nor CP–CC had obvious effects on bone mineral density, BV/TV, or Tb.Th in tail-suspended SMG rats. A possible reason may be that CP and CP–CC act primarily via stimulation of bone formation to inhibit bone loss [8
]. However, microgravity causes uncoupling of formation and resorption in bone remodeling, which may contribute to bone loss [25
Nevertheless, CP and CP–CC treatment partially ameliorated microgravity-induced deterioration of bone microarchitecture, as indicated by suppressing both the reduction in Tb.N and the increase in Tb.sp induced by hind limb unloading simulated microgravity. This result is consistent with a previous report that oral administration of CP or CP–CC inhibited bone loss in ovariectomized rats [12
]. This may represent an adjunctive dietary strategy to reduce the risk of fracture in astronauts.
Some previous reports suggest that serum calcium levels are not altered by tail suspension [27
]. On the contrary, calcium levels in the tail-suspended rats were significantly reduced in another previous study [22
]. This implies that intestinal calcium absorption was reduced during tail suspension [29
]. Serum concentrations of bone turnover markers are reflective of bone remodeling activity, and can potentially be used as surrogate markers of the rate of bone formation or bone resorption [30
]. The present study also showed that ALP activity and osteocalcin levels were decreased under SMG conditions, which is consistent with a previous report [31
], indicating that osteoblast activity was inhibited by microgravity [33
]. The main function of osteocalcin is to maintain the normal mineralization rate of bone. Interesting, osteocalcin levels in CP–CC-treated SMG rats were similar to those of CN rats, demonstrating that CP–CC promotes osteocalcin levels of osteoblasts by hind limb unloading simulated microgravity. PINP is a well-established marker of bone formation, which is produced by formation of type I collagen, a major component of the bone matrix, by amino-terminal and carboxy-terminal splicing of type I procollagen in osteoblasts [34
]. Conversely, β-CTX is a marker of bone resorption, reflecting the degradation of type I collagen by osteoclasts to produce amino-terminal and carboxy-terminal fragments [35
]. After ovariectomy or orchidectomy, serum TRACP-5b levels, which reflect the number of osteoclasts rather than their activity [36
], are expected to decline, and the histomorphometrically determined total number of osteoclasts in bone tissue is decreased owing to substantial bone loss [37
]. Changes in serum PINP or β-CTX levels induced by microgravity have reported before [38
]. High serum PINP levels in SMG or CP-treated rats in our study demonstrated that hind limb unloading promoted formation of type I collagen. However, treatment with CC significantly suppressed the hind limb unloading-induced increase in type I collagen formation. A possible reason for the lack of effects of CP–CC on serum PINP levels was that the actions of CP and CC counteracted one another. In this study, serum β-CTX levels were slightly reduced in tail-suspended rats, which is contrary to a previous report that CP–CC or CP supplementation inhibited the degradation of collagen in ovariectomized rats [22
], and serum TRACP-5b levels of tail-suspended rats were not altered. It may be speculated that this is related to the time of blood sampling or the high extent of bone loss under hind limb unloading simulated microgravity.
This study has some limitations. CP–CC treatment did not display a dose–response effect, and the duration of tail suspension was relatively short. As a dietary supplement, the effective time of CP–CC treatment in ovariectomized rats is three months, while, in this study, tail suspension was only maintained for 28 days. Thus, differing results among studies may be due to the mechanisms underlying the two animal models, and, in the tail suspension model, rapid bone loss in the process of CP–CC is not caused by the changes of bone mineral density obviously. Second, continuous blood sampling was not performed for observation of bone turnover markers.
4. Materials and Methods
4.1. Animals and Treatment
Bovine bone CP (prepared by our laboratory) and CC (Dongtai Food Ingredients, Lianyungang, China) were used. Male, three-month-old Sprague-Dawley rats (n
= 40, body weight: 300 ± 20 g) were obtained from the animal facility of the China Astronaut Research and Training Center (Beijing). Animals were maintained in cages under standard conditions (12-h light/dark cycle with free access to food and water). After feeding adaptation for seven days, the rats were divided into five groups (n
= 8 each): a control group with normal gravity (CN), an untreated hind limb unloading simulated microgravity group (SMG CN), and three SMG groups that underwent once-daily treatment by gastric gavage with CC (75 mg/kg), CP (750 mg/kg), or CP–CC (750 and 75 mg/kg). Bovine CP and CC were dissolved in distilled water. The tail-suspended rats were fixed by the tail at a 30° head-down angle to mimic microgravity [39
]. Briefly, the rats were individually caged, suspended by the tail using a strip of adhesive surgical tape attached to a chain hanging from a pulley, and subjected to hind limb unloading by tail suspension for 28 days. After sacrifice, serum was collected, and the bilateral femurs and tibiae were dissected and processed for dual-energy X-ray absorptiometry, three-point bending mechanical tests, microcomputed tomography, and evaluation of serum bone metabolic markers. All experimental procedures were approved by the Committees of Animal Ethics and Experimental Safety of the China Astronaut Research and Training Center.
4.2. Bone Mineral Density Assessment
The bone mineral density of femurs was measured by dual-energy X-ray absorptiometry equipped with appropriate software for small laboratory animals (GE Lunar PIXImus, GE Healthcare, Madison, WI, USA). All right femurs were placed in the same direction. Values were expressed as the observed mean (g/cm2) ± standard deviation (SD) of the whole group.
4.3. Biomechanical Testing
Biomechanical analysis was performed by three-point bending mechanical tests. Experiments were conducted using TexturePro CT V1.3 Build 14 (Brookfield Engineering Labs, Inc., Stoughton, MA, USA). Femora were placed horizontally on the frame on rounded edges at a distance of 20 mm. To minimize variability, the specimens were placed in a consistent position and orientation. Maximum force applied to failure, deformation at hardness and hardness work cycle values were recorded.
4.4. Microcomputed Tomography Analysis of the Distal Femur
The secondary spongiosa extracted from the left distal femurs of rats was scanned with a desktop, microcomputed tomography scanner (μCT40; Scanco Medical, Bruttisellen, Switzerland) using a voxel size of 10 μm, an X-ray tube voltage of 70 kVp, a current intensity of 114 μA, and an integration time of 600 ms. Briefly, slices were scanned at the region of the distal femur beginning at 0.1 mm from the most proximal aspect of the growth plate and extending proximally along the femur diaphysis. A volume of interest was manually drawn on each specimen. Microstructural measures included trabecular bone mineral density, bone volume per total volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp). Computation of these structural measures was performed using a previously described method [12
4.5. Biochemical Serum Analysis
Blood was collected and serum were separated by centrifugation to determine alkaline phosphatase activity (ALP) using an autoanalyzer. Serum bone osteocalcin/bone GLA protein (BGP) content was measured with a carboxyglutamic acid radioimmunometric assay kit (BGP Radioimmunometric Assay, Beijing North Institute of Biological Technology, Beijing, China), according to the manufacturer’s protocol. Serum N-terminal propeptide of type I procollagen (PINP) was measured with a specific rat PINP enzyme immunoassay (Rat PINP EIA; IDS Ltd., UK). The Beta isomer of serum C-telopeptide of type I collagen (CTX) was measured by an ELISA specific for rat CTX (RatLaps ELISA; IDS. Serum tartrate-resistant acid phosphatase form 5b (TRACP-5b) was measured by an ELISA specific for rat TRACP-5b (RatTRAP Assay; IDS).
4.6. Statistical Analysis
All numerical data are expressed as means ± SD. Statistical analyses were performed using SPSS for Windows version 17.0 (IBM, Armonk, NY, USA). With sample size of 8 (n = 5 or 6), nonparametric statistical analysis were performed. After one-way ANOVA, least-significant differences or Dunnett’s post-hoc test was used to determine significant differences between groups. Values of p < 0.05 were considered to indicate statistical significance.