While reduced estrogen levels have been shown to increase bone turnover and induce bone loss, there has been little analysis of the effects of diminished estrogen levels on the lacunar-canalicular porosity that houses the osteocytes. Alterations in the osteocyte lacunar-canalicular microenvironment may affect the osteocyte’s ability to sense and translate mechanical signals, possibly contributing to bone degradation during osteoporosis. To investigate whether reduced estrogen levels affect the osteocyte microenvironment, this study used high-resolution microscopy techniques to assess the lacunar-canalicular microstructure in the rat ovariectomy (OVX) model of postmenopausal osteoporosis. Confocal microscopy analyses indicated that OVX rats had a larger effective lacunar-canalicular porosity surrounding osteocytes in both cortical and cancellous bone from the proximal tibial metaphysis, with little change in cortical bone from the diaphysis or cancellous bone from the epiphysis. The increase in the effective lacunar-canalicular porosity in the tibial metaphysis was not due to changes in osteocyte lacunar density, lacunar size, or the number of canaliculi per lacuna. Instead, the effective canalicular size measured using a small molecular weight tracer was larger in OVX rats compared to controls. Further analysis using scanning and transmission electron microscopy demonstrated that the larger effective canalicular size in the estrogen-deficient state was due to nanostructural matrix-mineral level differences like loose collagen surrounding osteocyte canaliculi. These matrix-mineral differences were also found in osteocyte lacunae in OVX, but the small surface changes did not significantly increase the effective lacunar size. The alterations in the lacunar-canalicular surface mineral or matrix environment appear to make OVX bone tissue more permeable to small molecules, potentially altering interstitial fluid flow around osteocytes during mechanical loading.

Although interstitial fluid flow has been suggested to play a role in bone adaptation and metabolism, the constituents and ultrastructure of this interstitial fluid pathway are not well understood. Bone's lacunar-canalicular porosity is generally believed to be a continuous interstitial fluid pathway through which osteocytes sense external mechanical loading as well as obtain nutrients and dispose of wastes. Recent electron microscopy studies have suggested that a fiber matrix surrounds the osteocytic cell processes and fills this pericellular fluid space. However, studies injecting tracer molecules into the bone vasculature have provided conflicting results about the pore size or the fiber spacing of the interstitial fluid pathway. In addition, whether the smaller collagen-apatite porosity in adult bone is also a continuous fluid pathway is still unclear. To delineate bone's interstitial fluid pathway, four tracers of various size were injected into rats: reactive red (approximately 1 nm), microperoxidase (MP, approximately 2 nm), horseradish peroxidase (HRP, approximately 6 nm), and ferritin (approximately 10 nm). Five minutes after injection, the tibiae were harvested and processed using histological protocols optimized to minimize processing time to reduce possible redistribution of tracer molecules. The number of blood vessels and osteocytic lacunae labeled with the tracers per unit bone area was then measured for mid-diaphysial cross-sections of the tibia. While none of the tracers was detected within the mineralized bone matrix (the collagen-apatite porosity) using light microscopy, all the tracers except ferritin were found to pass through the canaliculi and appear in the osteocytic lacunae. These results indicate that while small tracers (< 6 nm) readily pass through the lacunarcanalicular porosity in the absence of mechanical loading, there appears to be an upper limit or cutoff size between 6 and 10 nm for molecular movement from bone capillaries to osteocytic lacunae in rat long bone. This range of pore size contains the most likely fiber spacing (approximately 7 nm) that has been proposed for the lacunar-canalicular annular space based on the presence of a proteoglycan fiber matrix surrounding the osteocyte.

Bone is a composite porous material with two functional levels of porosity: the vascular porosity that surrounds blood vessels and the lacunar-canalicular porosity that surrounds the osteocytes. Both the vascular porosity and lacunar-canalicular porosity are directly involved in interstitial fluid flow, thought to play an important role in bone's maintenance. Because of the small dimensions of the lacunar-canalicular porosity, interstitial fluid space has been difficult to visualize and quantify. We report a new staining protocol that is reliable and easily reproducible, using fluorescein isothiocyanate (FITC) as a probe visualized by confocal microscopy. Reconstructed FITC-stained cross sections enable effective visualization of bone microstructure and microporosities. This new staining process can be used to analyze interstitial fluid space, providing high-resolution quantification of the vascular pores and the lacunar-canalicular network of cortical and cancellous bone.

Enhancing the quantity and quality of cancellous bone with anabolic pharmacologic agents may lead to more successful outcomes of non-cemented joint replacements. Using a novel rabbit model of cancellous bone loading, we examined two specific questions regarding bone formation at the bone-implant interface: (1) does the administration of intermittent PTH, a potent anabolic agent, and mechanical loading individually and combined enhance the peri-implant cancellous bone volume fraction; and, (2) does surgical trauma enhance the anabolic effect of PTH on peri-implant bone volume fraction. In this model, PTH enhanced peri-implant bone volume fraction by 30% in loaded bone, while mechanical loading alone increased bone volume fraction modestly (+10%). Combined mechanical loading and PTH treatment had no synergistic effect on any cancellous parameters. However, a strong combined effect was found in bone volume fraction with combined surgery and PTH treatment (+34%) compared to intact control limbs. Adaptive changes in the cancellous bone tissue included increased ultimate stress and enhanced remodeling activity. The number of proliferative osteoblasts increased as did their expression of pro-collagen 1 and PTH receptor 1, and the number of TRAP positive osteoclasts also increased. In summary, both loading and intermittent PTH treatment enhanced peri-implant bone volume, and surgery and PTH treatment had a strong combined effect. This finding is of clinical importance since enhancing early osseointegration in the post-surgical period has numerous potential benefits.

To test if osteoporosis alters mechanical load-induced interstitial fluid flow in bone, this study examined the combined effect of estrogen deficiency and external loading on solute transport around osteocytes. An in vivo tracer, FITC-labeled bovine serum albumin, was injected into anaesthetized ovariectomized and control female Sprague Dawley rats before the right tibia was subjected to a controlled, physiological, non-invasive sinusoidal load to mimic walking. Tracer movement through the lacunar-canalicular system surrounding osteocytes was quantified in cortical and cancellous bone from the proximal tibia using confocal microscopy, with the non-loaded tibia serving as internal control. Overall, the application of mechanical loading increased the percentage of osteocyte lacunae labeled with injected tracer, and ovariectomy further enhanced movement of tracer. An analysis of separate regions demonstrated that ovariectomy enhanced in vivo transport of the injected tracer in the cancellous bone of the tibial epiphysis and metaphysis but not in the cortical bone of the metaphysis. These findings show that bone changes due to reduced estrogen levels alter convectional transport around osteocytes in cancellous bone and demonstrate a functional difference of interstitial fluid flow around osteocytes in estrogen-deficient rats undergoing the same physical activity as controls. The altered interstitial fluid flow around osteocytes is likely related to nanostructural matrix-mineral level differences recently demonstrated at the lacunar-canalicular surface of estrogen-deficient rats, which could affect the transmission of mechanical loads to the osteocyte.

Bone interstitial fluid flow is thought to play a fundamental role in the mechanical stimulation of bone cells, either via shear stresses or cytoskeletal deformations. Recent evidence indicates that osteocytes are surrounded by a fiber matrix that may be involved in the mechanotransduction of external stimuli as well as in nutrient exchange. In our previous tracer studies designed to map how different-sized molecules travel through the bone porosities, we found that injected ferritin was confined to blood vessels and did not pass into the mineralized matrix. However, other investigators have shown that ferritin forms halo-shaped labeling that enters the mineralized matrix around blood vessels. This labeling is widely used to explain normal interstitial fluid movement in bone; in particular, it is said to demonstrate bulk centrifugal interstitial fluid movement away from a highly pressurized vascular porosity. In addition, appositional ferritin fronts are said to demonstrate centrifugal interstitial fluid movement from the medullary canal to the periosteal surface. The purpose of this study was to investigate the conflicting ferritin labeling results by evaluating the role of different histological processes in the formation of ferritin "halos." Ferritin was injected into the rat vasculature and allowed to circulate for 5 min. Samples obtained from tibiae were reacted for different times with Perl's reagent and then were either paraffin-embedded or sectioned with a cryostat. Halo-like labeling surrounding vascular pores was found in all groups, ranging from 1.2-3.9% for the samples treated with the shortest histological processes (unembedded, frozen sections) to 5.6-15% for the samples treated with the longest histological processes (paraffin-embedded sections). These results indicate that different histological processing methods are able to create ferritin "halos," with some processing methods allowing more redistribution of the ferritin tracer than others. Based on these results and the fact that "halo" labeling has not been found with any other tracer, as we seek to further delineate the movement of interstitial fluid and the role it plays in bone mechanotransduction, we believe that ferritin "halo" labeling should not be used to demonstrate physiological bone interstitial fluid flow.

Estrogen depletion has been shown to cause bone loss in the proximal metaphysis of the rat tibia [1,2]. A decrease in bone volume fraction is frequently reported, yet there is little analysis in the literature related to changes in microporosities during osteoporosis. Our recent work quantifying microporosity changes due to estrogen depletion has shown an increase in the lacunar-canalicular porosity surrounding osteocytes in the proximal metaphysis of the rat tibia [3].

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