Scanning electron microscopic and light microscopic observations on morphological changes of freeze-dried bone implantation in rats: comparison with fresh autogenous bone transplantation.

Bone remodelling after the implantation of freeze-dried autogenous bone in rat parietal bone was compared with fresh autogenous bone transplantation, using a scanning electron and light microscope revealed the time intervals after transplantation/implantation. The light microscope revealed the time delay of the bone remodelling in the implantation, compared with the transplantations. The scanning electron microscope showed that the differences between the two groups were in the states of bone union and bone resorption. In the fresh bone group, the newly-formed bone filled the spaces between host and the transplanted bones at 2 to 3 weeks after the transplantation: the newly-formed bone fused and melted into the transplanted bone. New bone formation was more dominant on the bone surface in the dura mater side than in the skin side. The union was almost completed at 5 weeks. In freeze-dried bone implantation, the bone union in the contact space was very poor and the implanted bone was mainly covered by the new bone, which developed from the host bone surface in the dura mater side at 2 to 3 weeks after the implantation. What is noteworthy is that bone resorbed areas showing numerous Howship's lacunae were mainly observed on the host bone surface in the vicinity of newly-formed bone. However in freeze-dried bone implantation, the bone resorption was greater on the host and implanted bone surface than that of fresh bone transplantation: the resorption of host bone was considerably larger at certain periods after freeze-dried bone implantation. The present results show that the healing process of freeze-dried bone implantation, even though autogenous bone was used, differed from that of fresh autogenous bone transplantation, and the differences are concerned not only with time sequences but also with qualitative changes. This suggests that the host would have some different responses to the freeze-dried autogenous bone from fresh materials.     

Parallels between development of embryonic and matrix-induced endochondral bone

Endochondral bone formation can take place in the embryo, during fracture healing, or in postnatal animals after induction by implanted demineralized bone matrix. This matrix-induced bone formation recapitulates the embryonic sequence of bone formation morphologically and biochemically. The steps in bone formation in both systems include differentiation of cartilage from mesenchyme, cartilage maturation, invasion of the cartilage by blood vessels and marrow precursors, and formation of bone and bone marrow. Recently, bone inductive molecules from demineralized bone matrix have been purified, sequenced and produced as recombinant proteins. While there are similarities between bone development in the embryo and that after induction by these purified molecules, the molecules responsible for bone induction in the embryo have not yet been defined. Because of similarities between the two methods of bone formation, studies of bone induction by demineralized bone matrix may help to elucidate mechanisms of embryonic bone induction.     

Imbalance of local bone metabolism in inflammatory arthritis and its reversal upon tumor necrosis factor blockade: direct analysis of bone turnover in murine arthritis

Chronic arthritis typically leads to loss of periarticular bone, which results from an imbalance between bone formation and bone resorption. Recent research has focused on the role of osteoclastogenesis and bone resorption in arthritis. Bone resorption cannot be observed isolated, however, since it is closely linked to bone formation and altered bone formation may also affect inflammatory bone loss. To simultaneously assess bone resorption and bone formation in inflammatory arthritis, we developed a histological technique that allows visualization of osteoblast function by in-situ hybridization for osteocalcin and osteoclast function by histochemistry for tartrate-resistant acid phosphatase. Paw sections from human tumor necrosis factor transgenic mice, which develop an erosive arthritis, were analyzed at three different skeletal sites: subchondral bone erosions, adjacent cortical bone channels, and endosteal regions distant from bone erosions. In subchondral bone erosions, osteoclasts were far more common than osteoblasts. In contrast, cortical bone channels underneath subchondral bone erosions showed an accumulation of osteoclasts but also of functional osteoblasts resembling a status of high bone turnover. In contrast, more distant skeletal sites showed only very low bone turnover with few scattered osteoclasts and osteoblasts. Within subchondral bone erosions, osteoclasts populated the subchondral as well as the inner wall, whereas osteoblasts were almost exclusively found along the cortical surface. Blockade of tumor necrosis factor reversed the negative balance of bone turnover, leading to a reduction of osteoclast numbers and enhanced osteoblast numbers, whereas the blockade of osteoclastogenesis by osteoprotegerin also abrogated the osteoblastic response. These data indicate that bone resorption dominates at skeletal sites close to synovial inflammatory tissue, whereas bone formation is induced at more distant sites attempting to counter-regulate bone resorption.     

Imbalance of local bone metabolism in inflammatory arthritis and its reversal upon tumor necrosis factor blockade: direct analysis of bone turnover in murine arthritis

Chronic arthritis typically leads to loss of periarticular bone, which results from an imbalance between bone formation and bone resorption. Recent research has focused on the role of osteoclastogenesis and bone resorption in arthritis. Bone resorption cannot be observed isolated, however, since it is closely linked to bone formation and altered bone formation may also affect inflammatory bone loss. To simultaneously assess bone resorption and bone formation in inflammatory arthritis, we developed a histological technique that allows visualization of osteoblast function by in-situ hybridization for osteocalcin and osteoclast function by histochemistry for tartrate-resistant acid phosphatase. Paw sections from human tumor necrosis factor transgenic mice, which develop an erosive arthritis, were analyzed at three different skeletal sites: subchondral bone erosions, adjacent cortical bone channels, and endosteal regions distant from bone erosions. In subchondral bone erosions, osteoclasts were far more common than osteoblasts. In contrast, cortical bone channels underneath subchondral bone erosions showed an accumulation of osteoclasts but also of functional osteoblasts resembling a status of high bone turnover. In contrast, more distant skeletal sites showed only very low bone turnover with few scattered osteoclasts and osteoblasts. Within subchondral bone erosions, osteoclasts populated the subchondral as well as the inner wall, whereas osteoblasts were almost exclusively found along the cortical surface. Blockade of tumor necrosis factor reversed the negative balance of bone turnover, leading to a reduction of osteoclast numbers and enhanced osteoblast numbers, whereas the blockade of osteoclastogenesis by osteoprotegerin also abrogated the osteoblastic response. These data indicate that bone resorption dominates at skeletal sites close to synovial inflammatory tissue, whereas bone formation is induced at more distant sites attempting to counter-regulate bone resorption.     

Bone quality assessment in individuals of different age, gender and body constitution

The concept of bone quality describes the sets of the characteristics of the osseous tissue that influence bone strength. The aim was to explore the influence of anthropometric parameters and age on the parameters of the bone architecture and bone mineral properties in the lumbar vertebral bone of men and women. Vertebral bone samples underwent bone histomorphometry, bone densitometry and atomic absorption spectrometry. Men have greater values of the bone volume and thicker bone trabeculae in relation to women, which indicates that vertebral bone architecture is better preserved in men than in women. Age is the best predictor of changes that affect bone architecture and bone mineral properties. Bone mineral density value and calcium concentration are both negatively predicted by age, but positively predicted by body mass index. Such result supports the opinion that low body mass index is associated with conditions of bone deficit such are osteopenia and osteoporosis.     

JunD suppresses bone formation and contributes to low bone mass induced by estrogen depletion

JunD is an activator protein-1 (AP-1) component though its function in skeletal system is still not fully understood. To elucidate the role of JunD in the regulation of bone metabolism, we analyzed JunD-deficient mice. JunD deficiency significantly increased bone mass and trabecular number. This bone mass enhancement was due to JunD deficiency-induced increase in bone formation activities in vivo. Such augmentation of bone formation was associated with simultaneous increase in bone resorption while the former was dominant over the latter as accumulation of bone mass occurred in JunD-deficient mice. In a pathological condition relevant to postmenopausal osteoporosis, ovariectomy reduced bone mass in wild type (WT) mice as known before. Interestingly, JunD deficiency suppressed ovariectomy-induced increase in bone resorption and kept high bone mass. In addition, JunD deficiency also enhanced new bone formation after bone marrow ablation. Examination of molecular bases for these observations revealed that JunD deficiency enhanced expression levels of c-jun, fra-1, and fra-2 in bone in conjunction with elevated expression levels of runx2, type I collagen, and osteocalcin. Thus, JunD is involved in estrogen depletion-induced osteopenia via its action to suppress bone formation and to enhance bone resorption.     

Effect of treadmill exercise on bone mass in female rats.

Increasing peak bone mass at skeletal maturity, minimizing bone loss during middle age and after menopause, and increasing bone mass and preventing falls in advanced age are important measures for preventing osteoporotic fractures in women. Exercise has generally been considered to have a positive influence on bone health. This paper reviews the effects of treadmill exercise on bone in young, adult, ovariectomized, and osteopenic female rats. Treadmill exercise increases cortical and cancellous bone mass of the tibia as a result of increased bone formation and decreased bone resorption in young and adult rats. The increase in lumbar bone mass seems to be more significant when long-term exercise is applied. Treadmill exercise prevents cancellous bone loss at the tibia as a result of suppressed bone resorption in ovariectomized rats, and increases bone mass of the tibia and mechanical strength of the femur, as a result of suppressed bone resorption and increased bone formation in osteopenic rats after ovariectomy. Treadmill exercise transiently decreases the serum calcium level as a result of accumulation of calcium in bone, resulting in an increase in serum 1,25-dihydroxyvitamin D(3) level and a decrease in serum parathyroid hormone level. We conclude that treadmill exercise may be useful to increase bone mass in young and adult rats, prevent bone loss in ovariectomized rats, and increase bone mass and bone strength in osteopenic rats, especially in the long bones at weight-bearing sites. Treadmill exercise may have a positive effect on the skeleton in young, and adult, ovariectomized, and osteopenic female rats.     

A new transplant bone for maxillary alveolar cleft

An innovative technique with distraction osteogenesis has been developed in our research group to explore autogenous bone transplantation into craniofacial bone defects. This technique was designed to investigate bone-marrow transplantion using a chondroid or fibula bone graft into simulated alveolar bone defects in mice in terms of the osteogenic process and activity. As an experimental model of maxillary alveolar bone cleft available for testing bone-inductive materials, a critical-size defect was formed in the pre-maxillary bone of male mice using a surgical trephine bur with a low-speed dental engine. Distraction osteogenesis was performed using an external fixation device. The osteotomy site was occupied by an external callus consisting of hyaline cartilage with a large quantity of chondroid bone. Moreover, bone remodelling with new bone formation was demonstrated 30 days after the transplantation. Bone adhesion was better in chondroid bone grafting than in fibula bone grafting. The present findings are the first to demonstrate the potential of chondroid bone transplantation as a new therapeutic system of bone grafting, suitable for bone substitutes in craniofacial bone defects.     

Computational simulation of simultaneous cortical and trabecular bone change in human proximal femur during bone remodeling

In this study, we developed a numerical framework that computationally determines simultaneous and interactive structural changes of cortical and trabecular bone types during bone remodeling, and we investigated the structural correlation between the two bone types in human proximal femur. We implemented a surface remodeling technique that performs bone remodeling in the exterior layer of the cortical bone while keeping its interior area unchanged. A micro-finite element (μFE) model was constructed that represents the entire cortical bone and full trabecular architecture in human proximal femur. This study simulated and compared the bone adaptation processes of two different structures: (1) femoral bone that has normal cortical bone shape and (2) perturbed femoral bone that has an artificial bone lump in the inferomedial cortex. Using the proposed numerical method in conjunction with design space optimization, we successfully obtained numerical results that resemble actual human proximal femur. The results revealed that actual cortical bone, as well as the trabecular bone, in human proximal femur has structurally optimal shapes, and it was also shown that a bone abnormality that has little contribution to bone structural integrity tends to disappear. This study also quantitatively determined the structural contribution of each bone: when the trabecular adaptation was complete, the trabecular bone supported 54% of the total load in the human proximal femur while the cortical bone carried 46%.     

Comparison of contamination of femoral heads and pre-processed bone chips during hip revision arthroplasty

With bone impaction grafting, cancellous bone chips made from allograft femoral heads are impacted in a bone defect, which introduces an additional source of infection. The potential benefit of the use of pre-processed bone chips was investigated by comparing the bacterial contamination of bone chips prepared intraoperatively with the bacterial contamination of pre-processed bone chips at different stages in the surgical procedure. To investigate baseline contamination of the bone grafts, specimens were collected during 88 procedures before actual use or preparation of the bone chips: in 44 procedures intraoperatively prepared chips were used (Group A) and in the other 44 procedures pre-processed bone chips were used (Group B). In 64 of these procedures (32 using locally prepared bone chips and 32 using pre-processed bone chips) specimens were also collected later in the procedure to investigate contamination after use and preparation of the bone chips. In total, 8 procedures had one or more positive specimen(s) (12.5 %). Contamination rates were not significantly different between bone chips prepared at the operating theatre and pre-processed bone chips. In conclusion, there was no difference in bacterial contamination between bone chips prepared from whole femoral heads in the operating room and pre-processed bone chips, and therefore, both types of bone allografts are comparable with respect to risk of infection.     

Greater efficacy of alfacalcidol in the red than in the yellow marrow skeletal sites in adult female rats

The present study compared the bone anabolic effects of graded doses of alfacalcidol in proximal femurs (hematopoietic, red marrow skeletal site) and distal tibiae (fatty, yellow marrow skeletal site). One group of 8.5-month-old female Sprague-Dawley rats were killed at baseline and 4 groups were treated 5 days on/2 days off/week for 12 weeks with 0, 0.025, 0.05 and 0.1 microg alfacalcidol/kg by oral gavage. The proximal femur, bone site with hematopoietic marrow, as well as the distal tibia bone site with fatty marrow, were processed undecalcified for quantitative bone histomorphometry. In the red marrow site of the proximal femoral metaphysis (PFM), 0.1 microg alfacalcidol/kg induced increased cancellous bone mass, improved architecture (decreased trabecular separation, increased connectivity), and stimulated local bone formation of bone 'boutons' (localized bone formation) on trabecular surfaces. There was an imbalance in bone resorption and formation, in which the magnitude of depressed bone resorption greater than depressed bone formation resulted in a positive bone balance. In addition, bone 'bouton' formation contributed to an increase in bone mass. In contrast, the yellow marrow site of the distal tibial metaphysis (DTM), the 0.1 microg alfacalcidol/kg dose induced a non-significant increased cancellous bone mass. The treatment decreased bone resorption equal to the magnitude of decreased bone formation. No bone 'bouton' formation was observed. These findings indicate that the highest dose of 0.1 microg alfacalcidol/kg for 12 weeks increased bone mass (anabolic effect) at the skeletal site with hematopoietic marrow of the proximal femoral metaphysis, but the increased bone mass was greatly attenuated at the fatty marrow site of the distal tibial metaphysis. In addition, the magnitude of the bone gain induced by alfacalcidol treatment in red marrow cancellous bone sites of the proximal femoral metaphysis was half that of the lumbar vertebral body. The latter data were from a previous report from the same animal and protocol. These findings indicated that alfacalcidol as an osteoporosis therapy is less efficacious as a positive bone balance agent that increased trabecular bone mass in a non-vertebral skeletal site where bone marrow is less hematopoietic.     

Cellular and molecular phenotypes of osteogenic cells isolated from the medullary bone of the hen in vitro

In this study, cells isolated from hen medullary bone were cultured to examine their matrix formation. Furthermore, we compared medullary bone cells with rat bone marrow cells regarding the temporal changes in osteoblast developmental markers. Medullary bone cells were positive for alkaline phosphatase (ALP) activity and formed bone nodules, apparent with Alcian blue and von Kossa staining. The intensity of these stains became stronger with the maturation of those bone nodules. In this developmental process, the expression patterns of osteoblast phenotypes of medullary bone cells differed from those of rat bone marrow cells. ALP mRNA was expressed at the maximum level in the proliferation stage and gradually decreased in medullary bone cells, but that expression showed the opposite pattern in rat bone marrow cells. Medullary bone cells strongly expressed two non-collagenous protein mRNAs from the early stages, but the expression of these mRNAs in rat bone marrow cells increased only in the later stages. These results suggest that the features of medullary bone osteoblasts differ from those of mammalian osteoblasts and are reflected in the characteristics of medullary bone in vivo.     

The Concentration of Manganese, Iron and Strontium in Bone of Red Fox Vulpes vulpes (L. 1758)

The aims of the study were to determine manganese (Mn), iron (Fe) and strontium (Sr) concentrations in fox bone samples from north-western Poland and to examine the relationships between the bone Mn, Fe and Sr concentrations and the sex and age of the foxes. In the studied samples of fox cartilage, cartilage with adjacent compact bone, compact bone and spongy bone, the concentrations of the analysed metals had the following descending order: Fe > Sr > Mn. The only exception was in compact bone, in which the concentrations were arranged in the order Sr > Fe > Mn. Manganese concentrations were significantly higher in cartilage, compact bone and cartilage with compact bone than in spongy bone. Iron concentrations were higher in cartilage and spongy bone compared with compact bone. Strontium concentrations were greater in compact bone than in cartilage and spongy bone. The manganese, iron and strontium concentrations in the same type of bone material in many cases correlated with each other, with the strongest correlation (r?>?0.70) between Mn and Fe in almost all types of samples. In addition, concentrations of the same metals in different bone materials were closely correlated for Mn and Fe in cartilage and cartilage with adjacent compact bone, and for Sr in compact bone and cartilage with compact bone. In the fox from NW Poland, there were no statistically significant differences in Mn, Fe and Sr in any of the types of bone material between the sexes and immature and adult foxes.     

The immune system and bone

T cells and B cells produce large amounts of cytokines which regulate bone resorption and bone formation. These factors play a critical role in the regulation of bone turnover in health and disease. In addition, immune cells of the bone marrow regulate bone homeostasis by cross-talking with bone marrow stromal cells and osteoblastic cells via cell surface molecules. These regulatory mechanisms are particularly relevant for postmenopausal osteoporosis and hyperparathyroidism, two common forms of bone loss caused primarily by an expansion of the osteoclastic pool only partially compensated by a stimulation of bone formation. This article describes the cytokines and immune factors that regulate bone cells, the immune cells relevant to bone, examines the connection between T cells and bone in health and disease, and reviews the evidence in favor of a link between T cells and the mechanism of action of estrogen and PTH in bone.     

The osteoprotective role of USP26 in coordinating bone formation and resorption

Bone homeostasis is maintained through a balance of bone formation by osteoblasts and bone resorption by osteoclasts. Ubiquitin-specific proteases (USPs) are involved in regulating bone metabolism by preserving bone formation or antagonizing bone resorption. However, the specific USPs that maintain bone homeostasis by orchestrating bone formation and bone resorption simultaneously are poorly understood. Here, we identified USP26 as a previously unknown regulator of bone homeostasis that coordinates bone formation and resorption. Mechanistically, USP26 stabilizes β-catenin to promote the osteogenic activity of mesenchymal cells (MSCs) and impairs the osteoclastic differentiation of bone myelomonocytes (BMMs) by stabilizing inhibitors of NF-κBα (IκBα). Gain-of-function experiments revealed that Usp26 supplementation significantly increased bone regeneration in bone defects in aged mice and decreased bone loss resulting from ovariectomy. Taken together, these data show the osteoprotective effect of USP26 via the coordination of bone formation and resorption, suggesting that USP26 represents a potential therapeutic target for osteoporosis.Subject terms: Deubiquitylating enzymes, Deubiquitylating enzymes, Endocrine system and metabolic diseases, Immunopathogenesis     

Alfacalcidol prevents age-related bone loss and causes an atypical pattern of bone formation in aged male rats

The current study was designed to investigate the skeletal effects of alfacalcidol in aged rats. Eighteen-month-old male rats were treated with 0, 0.1, or 0.2 microg/kg/d of alfacalcidol by daily oral gavage, 5 days/week for 12 weeks. At the beginning of the treatments, one group of rats was euthanized to serve as a baseline control. At the end of the study, the second lumbar vertebrae and the right tibial diaphysess were processed for bone histomorphometric analysis. The fourth lumbar vertebrae were subjected to strength testing. The control group of rats at 21 months of age had decreased serum testosterone levels and decreased cancellous bone mass associated with increased bone turnover on the trabecular surface. The older rats had increased bone turnover on the endocortical surface and decreased bone formation on the periosteal surface compared with the 18-month group. In contrast, alfacalcidol treatment increased cancellous and cortical bone mass in aged male rats. Trabecular bone resorption was decreased whereas bone formation was maintained or increased in the rats treated with alfacalcidol. In addition, endocortical bone formation was decreased whereas periosteal bone formation was increased in the rats treated with alfacalcidol compared with vehicle-treated rats. Marrow trabecular bone area was increased by alfacalcidol treatment in tibial diaphyses. Furthermore, bone strength of the lumbar vertebral body was increased after alfacalcidol treatment. An atypical pattern of bone formation on endosteal bone surfaces was seen in the rats treated with alfacalcidol. The atypical bone formation is characterized by small, focal packets of newly formed bone on trabecular and endocortical bone surfaces. This gave the appearance of the formation of "bone buds" emanating from trabecular surfaces. These bony outgrowths were mineralized and demonstrated significant fluorochrome label indicating recent mineralization. Also, lamellae of the bony buds did not run parallel to those of the trabecular plate to which they are attached. Arrest lines presented in most of the "bone buds". In summary, alfacalcidol treatment increased cancellous and cortical bone mass and improved bone strength, resulting in the prevention of age-related bone loss in aged male rats. An atypical pattern of bone formation observed in this study may be a result of minimodeling based bone formation stimulated by alfacalcidol treatment.     

MURF1 deficiency suppresses unloading-induced effects on osteoblasts and osteoclasts to lead to bone loss

Loss of mechanical stress or unloading causes disuse osteoporosis that leads to fractures and deteriorates body function and affects mortality rate in aged population. This bone loss is due to reduction in osteoblastic bone formation and increase in osteoclastic bone resorption. MuRF1 is a muscle RING finger protein which is involved in muscle wasting and its expression is enhanced in the muscle of mice subjected to disuse condition such as hind limb unloading (HU). However, whether MuRF1 is involved in bone loss due to unloading is not known. We therefore examined the effects of MuRF1 deficiency on unloading-induced bone loss. We conducted hind limb unloading of MuRF1 KO mice and wild-type control mice. Unloading induced about 60% reduction in cancellous bone volume (BV/TV) in WT mice. In contrast, MuRF1 deficiency suppressed unloading-induced cancellous bone loss. The cortical bone mass was also reduced by unloading in WT mice. In contrast, MuRF1 deficiency suppressed this reduction in cortical bone mass. To understand whether the effects of MuRF1 deficiency suppress bone loss is on the side of bone formation or bone resorption, histomorphometry was conducted. Unloading reduced bone osteoblastic formation rate (BFR) in WT. In contrast, MuRF1 deficiency suppressed this reduction. Regarding bone resorption, unloading increased osteoclast number in WT. In contrast, MURF1 deficiency suppressed this osteoclast increase. These data indicated that the ring finger protein, MURF1 is involved in disuse-induced bone loss in both of the two major bone remodeling activities, osteoblastic bone formation and osteoclastic bone resorption.     

Self-maintenance of induced bone tissue]

The capacity for self-maintenance of the bone marrow osteogenic precursor cells from the skeletal bones and from the bones induced by implantation of decalcified bone matrix is compared. Transplantation in diffusion chambers is employed as the test system. Osteogenesis in the bone marrow transplants isolated from the skeletal bone lasts several months, whereas osteogenesis in the bone marrow transplants isolated from induced bone stops after the second month. Fibroblasts arising in the monolayer cultures of the skeletal bone marrow retained their osteogenic potencies after repeated passages. On the contrary, fibroblasts from the monolayer cultures of induced bone marrow lost their osteogenic capacity after the second passage. Thus, contrary to osteogenic precursors of the skeletal bone, osteogenic precursors of induced bone tissue had a very limited self-maintaining capacity after the cessation of induction.     

Effect of bone marrow cells on colony-forming stromal cells in guinea pigs and on the proliferation of their cultured descendents

In the presence of irradiated bone marrow cells the efficiency of stromal colony formation increases from 0.8 +/- 0.2 to 3.6 +/- 0.4 per 10(4) explanted bone marrow cells. The growth-stimulating activity of bone marrow cells on passaged bone marrow fibroblasts depends on growth conditions of passages to which irradiated bone marrow cells are added. The response of proliferating bone marrow fibroblasts to stimulating activity of bone marrow cells is low, while addition of bone marrow cells to fibroblast cultures stimulates their proliferation.     

The effect of exercise and nutrition on the mechanostat

In this review, we discuss the effect of increased and decreased loading and nutrition deficiency on muscle and bone mass and strength (and bone length and architecture) independently and combined. Both exercise and nutrition are integral components of the mechanostat model but both have distinctly different roles. Mechanical strain imparted by muscle action is responsible for the development of the external size and shape of the bone and subsequently the bone strength. In contrast, immobilization during growth results in reduced growth in bone length and a loss of bone strength due to large losses in bone mass (a result of endosteal resorption in cortical bone and trabecular thinning) and changes in geometry (bone shafts do not develop their characteristic shape but rather develop a rounded default shape). The use of surrogate measures for peak muscle forces acting on bone (muscle strength, size, or mass) limits our ability to confirm a cause-and-effect relationship between peak muscle force acting on bone and changes in bone strength. However, the examples presented in this review support the notion that under adequate nutrition, exercise has the potential to increase peak muscle forces acting on bone and thus can lead to a proportional increase in bone strength. In contrast, nutrition alone does not influence muscle or bone in a dose-dependent manner. Muscle and bone are only influenced when there is nutritional deficiency--and in this case the effect is profound. Similar to immobilization, the immediate effect of malnutrition is a reduction in longitudinal growth. More specifically, protein and energy malnutrition results in massive bone loss due to endosteal resorption in cortical bone and trabecular thinning. Unlike loading however, there is indirect evidence that severe malnutrition when associated with menstrual dysfunction can shift the mechanostat set point upward, thus leading to less bone accrual for a given amount of bone strain.