Glucocorticoids cause mandibular bone fragility and suppress osteocyte perilacunar-canalicular remodeling.
ABSTRACT: Osteocytes support dynamic, cell-intrinsic resorption and deposition of bone matrix through a process called perilacunar/canalicular remodeling (PLR). In long bones, PLR depends on MMP13 and is tightly regulated by PTH, sclerostin, TGF?, and glucocorticoids. However, PLR is regulated differently in the cochlea, suggesting a mechanism that is anatomically distinct. Unlike long bones, the mandible derives from neural crest and exhibits unique susceptibility to medication and radiation induced osteonecrosis. Therefore, we sought to determine if PLR in the mandible is suppressed by glucocorticoids, as it is in long bone. Hemimandibles were collected from mice subcutaneously implanted with prednisolone or vehicle containing pellets for 7, 21, or 55?days (n?=?8/group) for radiographic and histological analyses. Within 21?days, micro-computed tomography revealed a glucocorticoid-dependent reduction in bone volume/total volume and trabecular thickness and a significant decrease in bone mineral density after 55?days. Within 7?days, glucocorticoids strongly and persistently repressed osteocytic expression of the key PLR enzyme MMP13 in both trabecular and cortical bone of the mandible. Cathepsin K expression was significantly reduced only after 55?days of glucocorticoid treatment, at which point histological analysis revealed a glucocorticoid-dependent reduction in the lacunocanalicular surface area. In addition to reducing bone mass and suppressing PLR, glucocorticoids also reduced the stiffness of mandibular bone in flexural tests. Thus, osteocyte PLR in the neural crest-derived mandible is susceptible to glucocorticoids, just as it is in the mesodermally-derived femur, highlighting the need to further study PLR as a target of drugs, and radiation in mandibular osteonecrosis.
Project description:<h4>Objectives</h4>We studied oral glucocorticoids and osteonecrosis, a rare but serious bone disease, in individuals with various chronic inflammatory diseases. We hypothesised that we would find stronger associations in adults versus children and in people with autoimmune diseases.<h4>Design</h4>Retrospective cohort study.<h4>Setting</h4>Population-representative data (1994-2013) from general practices in the UK.<h4>Participants</h4>Children and adults diagnosed with asthma; inflammatory bowel disease; juvenile, psoriatic or rheumatoid arthritis; psoriasis; or systemic lupus.<h4>Exposures</h4>Oral glucocorticoid patterns.<h4>Primary and secondary outcome measures</h4>Diagnosed osteonecrosis (primary) and osteonecrosis plus clinical features (eg, symptoms, pain medication, surgical repair) (secondary). Discrete time failure models estimated the adjusted hazard ratio (aHR) of incident osteonecrosis following oral glucocorticoid exposure. Hypothesis testing was one sided (with corresponding 90% CI) since glucocorticoids were unlikely protective.<h4>Results</h4>After adjusting for demographic, disease-related and health utilisation factors, glucocorticoid exposure was associated with osteonecrosis in adults (ages 18-49, aHR 2.1 (90% CI 1.5 to 2.9); ages ?50, aHR 1.3 (90% CI 1.01 to 1.7)). However, low-dose glucocorticoids, corresponding to average doses <7.5?mg prednisolone daily and maximum doses <30?mg daily, were not associated with osteonecrosis in adults. Furthermore, even at high glucocorticoid doses, there was no evidence of increased osteonecrosis among glucocorticoid-exposed children (p=0.04 for interaction by age) (any glucocorticoid exposure, ages 2-9: aHR 1.1 (90% CI 0.7 to 1.7); ages 10-17: aHR 0.6 (90% CI 0.3 to 1.6)). Arthritis, inflammatory bowel disease and lupus were independently associated with osteonecrosis, but there was a similar dose relationship between glucocorticoids and osteonecrosis among adults with low-risk and high-risk diseases.<h4>Conclusions</h4>Glucocorticoid use was clearly associated with osteonecrosis in a dose-related fashion in adults, especially young adults, but this risk was not detectable in children. The absolute risk of glucocorticoid-associated osteonecrosis in the general paediatric population and in adults taking low glucocorticoid doses is at most extremely small.
Project description:Bone remodeling, a combination of bone resorption and formation, requires precise regulation of cellular and molecular signaling to maintain proper bone quality. Whereas osteoblasts deposit and osteoclasts resorb bone matrix, osteocytes both dynamically resorb and replace perilacunar bone matrix. Osteocytes secrete proteases like matrix metalloproteinase-13 (MMP13) to maintain the material quality of bone matrix through perilacunar remodeling (PLR). Deregulated bone remodeling impairs bone quality and can compromise hearing since the auditory transduction mechanism is within bone. Understanding the mechanisms regulating cochlear bone provides unique ways to assess bone quality independent of other aspects that contribute to bone mechanical behavior. Cochlear bone is singular in its regulation of remodeling by expressing high levels of osteoprotegerin. Since cochlear bone expresses a key PLR enzyme, MMP13, we examined whether cochlear bone relies on, or is protected from, osteocyte-mediated PLR to maintain hearing and bone quality using a mouse model lacking MMP13 (MMP13(-/-)). We investigated the canalicular network, collagen organization, lacunar volume via micro-computed tomography, and dynamic histomorphometry. Despite finding defects in these hallmarks of PLR in MMP13(-/-) long bones, cochlear bone revealed no differences in these markers, nor hearing loss as measured by auditory brainstem response (ABR) or distortion product oto-acoustic emissions (DPOAEs), between wild type and MMP13(-/-) mice. Dynamic histomorphometry revealed abundant PLR by tibial osteocytes, but near absence in cochlear bone. Cochlear suppression of PLR corresponds to repression of several key PLR genes in the cochlea relative to long bones. These data suggest that cochlear bone uniquely maintains bone quality and hearing independent of MMP13-mediated osteocytic PLR. Furthermore, the cochlea employs parallel mechanisms to inhibit remodeling by osteoclasts and osteoblasts, and by osteocytes, to protect hearing. Understanding the cellular and molecular mechanisms that confer site-specific control of bone remodeling has the potential to elucidate new pathways that are deregulated in skeletal disease.
Project description:Osteoarthritis (OA), long considered a primary disorder of articular cartilage, is commonly associated with subchondral bone sclerosis. However, the cellular mechanisms responsible for changes to subchondral bone in OA, and the extent to which these changes are drivers of or a secondary reaction to cartilage degeneration, remain unclear. In knee joints from human patients with end-stage OA, we found evidence of profound defects in osteocyte function. Suppression of osteocyte perilacunar/canalicular remodeling (PLR) was most severe in the medial compartment of OA subchondral bone, with lower protease expression, diminished canalicular networks, and disorganized and hypermineralized extracellular matrix. As a step toward evaluating the causality of PLR suppression in OA, we ablated the PLR enzyme MMP13 in osteocytes while leaving chondrocytic MMP13 intact, using Cre recombinase driven by the 9.6-kb DMP1 promoter. Not only did osteocytic MMP13 deficiency suppress PLR in cortical and subchondral bone, but it also compromised cartilage. Even in the absence of injury, osteocytic MMP13 deficiency was sufficient to reduce cartilage proteoglycan content, change chondrocyte production of collagen II, aggrecan, and MMP13, and increase the incidence of cartilage lesions, consistent with early OA. Thus, in humans and mice, defects in PLR coincide with cartilage defects. Osteocyte-derived MMP13 emerges as a critical regulator of cartilage homeostasis, likely via its effects on PLR. Together, these findings implicate osteocytes in bone-cartilage crosstalk in the joint and suggest a causal role for suppressed perilacunar/canalicular remodeling in osteoarthritis.
Project description:Hematopoietic stem cells (HSCs) in the endosteum of mesoderm-derived appendicular bones have been extensively studied. Neural crest-derived bones differ from appendicular bones in developmental origin, mode of bone formation and pathological bone resorption. Whether neural crest-derived bones harbor HSCs is elusive. Here, we discovered HSC-like cells in postnatal murine mandible, and benchmarked them with donor-matched, mesoderm-derived femur/tibia HSCs, including clonogenic assay and long-term culture. Mandibular CD34 negative, LSK cells proliferated similarly to appendicular HSCs, and differentiated into all hematopoietic lineages. Mandibular HSCs showed a consistent deficiency in lymphoid differentiation, including significantly fewer CD229 + fractions, PreProB, ProB, PreB and B220 + slgM cells. Remarkably, mandibular HSCs reconstituted irradiated hematopoietic bone marrow in vivo, just as appendicular HSCs. Genomic profiling of osteoblasts from mandibular and femur/tibia bone marrow revealed deficiencies in several HSC niche regulators among mandibular osteoblasts including Cxcl12. Neural crest derived bone harbors HSCs that function similarly to appendicular HSCs but are deficient in the lymphoid lineage. Thus, lymphoid deficiency of mandibular HSCs may be accounted by putative niche regulating genes. HSCs in craniofacial bones have functional implications in homeostasis, osteoclastogenesis, immune functions, tumor metastasis and infections such as osteonecrosis of the jaw.
Project description:Through a process called perilacunar remodeling, bone-embedded osteocytes dynamically resorb and replace the surrounding perilacunar bone matrix to maintain mineral homeostasis. The vital canalicular networks required for osteocyte nourishment and communication, as well as the exquisitely organized bone extracellular matrix, also depend upon perilacunar remodeling. Nonetheless, many questions remain about the regulation of perilacunar remodeling and its role in skeletal disease. Here, we find that suppression of osteocyte-driven perilacunar remodeling, a fundamental cellular mechanism, plays a critical role in the glucocorticoid-induced osteonecrosis. In glucocorticoid-treated mice, we find that glucocorticoids coordinately suppress expression of several proteases required for perilacunar remodeling while causing degeneration of the osteocyte lacunocanalicular network, collagen disorganization, and matrix hypermineralization; all of which are apparent in human osteonecrotic lesions. Thus, osteocyte-mediated perilacunar remodeling maintains bone homeostasis, is dysregulated in skeletal disease, and may represent an attractive therapeutic target for the treatment of osteonecrosis.
Project description:Recently, there is increasing report of bone necrosis exceptionally in jaw bone areas after long term administration of anti-resorptive agent, bisphosphonate (BP). It is still unknown that why BP does not result in osteonecrosis but potentiate fracture repair in axial skeleton such as long bones. Many reports had shown that mandibular bone or calvarial bone exhibits lesser resoption than iliac bone graft for maxillofacial bone defect.(Jackson et al 1986, Koole et al 1989),(Crespi et al 2007) These might be attributed to the embryological difference between the two different type of the bone; intramembranous versus endochondral bone formation. Maxilla and mandibular alveolar and basal bone are originated from neural crest cells and exhibit intramembranous bone formation.(Chai and Maxson 2006) However, axial skeleton such as iliac or tibial bones are originated from mesoderm and undergo endochondral bone formation.(Helms and Schneider 2003) It had been reported that there is distinct phenotype difference in human bone cells from different skeletal origin.(Akintoye et al 2006, Kasperk et al 1997),(Matsubara et al 2005) Akintoye et al.(Akintoye et al 2006) reported that mandibular bone marrow stromal cells (BMSCs) displayed a fibroblast-like morphology similar to iliac BMSCs in histological appearance. However, mandibular bone contained less red marrow and hematopoietic cells, whereas the iliac bone contained more red marrow thereby contributing more to hematopoiesis. This differential capacity of mandibular BMSCs with less hematopoietic stem cells is better than that of iliac BMSCs because mitosis in hematopoietic stem cells is usually stopped. They also reported that maxillofacial BMSCs shows higher proliferation rate and alkaline phosphotase activity.(Akintoye et al 2006, Akintoye et al 2008) However, in vivo response of human BMSCs to osteogenic induction was higher in iliac bone than maxillomandibular bone.(Akintoye et al 2006) Other reports had shown that BMSCs from mandible showed better in vitro and in vivo bone formation capacity compared to tibia from rats.(Aghaloo et al 2010) Matsubara et al. reported that osteogenic potential of human or canine alveolar bone and iliac bone-derived cells (BC) are similar but showed difference in chondrogenic and adipogenic potential.(Matsubara et al 2005) From the previous studies, the site-related difference of mandibular and iliac BC are clear. However, the pattern of the site-specificity are not exactly same among above mentioned studies. These might be attributed to the difference in the experimental subjects (species, age) and study design (in-patient or random comparison, etc). Moreover, underlying genetic mechanism of these skeletal-site dependent difference had not been fully investigated. The investigation of site-specific differences in the gene expression would be help to understand the mechanism of bisphosphonate-related osteonecrosis of the jaw (BRONJ), which recently has become an major issue in dentistry. Considering that bisphosphonate is frequently prescribed to elderly patients who have a higher risk of osteoporosis, it is necessary to conduct a experimental analysis of cells from older-aged donors. It had been suggested that anatomical site-specific difference can be influenced by 1) difference in the regional blood supply, 2) difference in bone composition (lamellar bone versus trabecular bone), 3) difference in physiological strain exerted to bone, 4) difference in gene expression according to skeletal site.(Kasperk et al 1997) In this study, bone cells derived from elderly donors went through microarray analysis under the hypothesis that there would be the differences in gene expressions involved in bone formation or cell proliferation of bone cells from iliac and mandible. The purpose of this study was to investigate the difference in gene expression between the human mandible and iliac bone so that we can understand the genetic difference in the two different type of bones. <<<References>>> 1. Jackson IT, Helden G, Marx R. Skull bone grafts in maxillofacial and craniofacial surgery. J Oral Maxillofac Surg 1986; 44:949-955. 2. Koole R, Bosker H, van der Dussen FN. Late secondary autogenous bone grafting in cleft patients comparing mandibular (ectomesenchymal) and iliac crest (mesenchymal) grafts. J Craniomaxillofac Surg 1989; 17 Suppl 1:28-30. 3. Crespi R, Vinci R, Cappare P, Gherlone E, Romanos GE. Calvarial versus iliac crest for autologous bone graft material for a sinus lift procedure: a histomorphometric study. Int J Oral Maxillofac Implants 2007; 22:527-532. 4. Chai Y, Maxson RE, Jr. Recent advances in craniofacial morphogenesis. Dev Dyn 2006; 235:2353-2375. 5. Helms JA, Schneider RA. Cranial skeletal biology. Nature 2003; 423:326-331. 6. Kasperk C, Helmboldt A, Borcsok Iet al. Skeletal site-dependent expression of the androgen receptor in human osteoblastic cell populations. Calcif Tissue Int 1997; 61:464-473. <<< Important Abstract of Associated reference>>> Kingsmill VJ1, McKay IJ, Ryan P, Ogden MR, Rawlinson SC. Gene expression profiles of mandible reveal features of both calvarial and ulnar bones in the adult rat.J Dent. 2013 Mar;41(3):258-64. doi: 10.1016/j.jdent.2012.11.010. Epub 2012 Nov 23. OBJECTIVES: Limb and mandibular alveolar bone of the mandible are susceptible to disuse osteopenia, whilst skull and mandibular basal bone appear to resist excessive generalised bone loss. We wanted to compare the site-specific transcriptome of anatomically and functionally distinct bones to confirm the composite nature of the mandible at the molecular level. METHODS: Gene expression profiles were obtained for the mandible, ulna, and calvaria of adult male rats using Affymetrix Rat Genome 230 2.0 GeneChips. Ingenuity Pathways Assist generated association maps, and RGD database software identified site-specific pathways. RESULTS: The majority of expressed transcripts (84%) are common to all three sites. The mandible expressed 873 transcripts in common with ulna but not calvaria, and 1014 transcripts in common with calvaria but not ulna. Transcripts in these groups were excluded if they showed significant differential expression (>2-fold) and the remaining mapped genes were filtered for those related to modulation of gene transcription. Analysis of these genes revealed common pathways shared by the mandible and ulna, or mandible and calvaria, which were not shared by the calvaria and ulna. CONCLUSIONS: There were relatively few differences in the expression of genes responsible for the bone formation process per se in different functional skeletal sites. Differential transcription factor expression suggests that it is the regulation of bone formation and not the mechanism of bone formation itself that differs between the skeletal sites. CLINICAL SIGNIFICAN Objectives: The exact genetic difference between the jaw and long bone had not been clearly understood. The purpose of this study was to investigate the difference in gene expression between the human mandible and iliac bone-derived cells. Methods: Primary cells were obtained from mandibular and iliac bone from the 6 healthy, elderly donors (average age 60.2 years) during the reconstruction of mandibular alveolar bone defects. To investigate site-specific difference, within-patient comparison were carried out with cell proliferation and osteoblastic differentiation assay. Gene expression profile of mandible and iliac BC (bone-derived cell) were compared using cDNA microarray analysis using Affymetrix GeneChip®. Results: The mandibular BC showed stronger proliferative capacity but weaker osteoblastic differentiation. The comparison of the gene expression profile identified that 82 genes were significantly up-regulated and 66 genes were down-regulated 1.5 fold or greater in mandible compared to iliac BC. The most significantly differentially regulated genes were associated with skeletal system development and morphogenesis (SIX1, MSX1, MSX2, HAND2, PRRX1, OSR2, HOX gene family, PITX2). Microarray analysis revealed that Msx1 was 2.03 fold and Msx2 was 1.99 fold up-regulated in mandible compared to iliac BC (both p<0.01). HOX gene group in mandibular BC was down-regulated (p<0.01). Osteopontin was also 2.84 fold down-regulated in mandible BC (p<0.01). We found that the results of the microarray were reproducible with qRT-PCR. Conclusions: Site-specific difference between jaw and long bone can be explained by the different gene expression pattern, which is primarily related with embryological origin of these two different bones. Human mandible and iliac bone chip was were collected from 6 donors (average 60.2 years, ranged from 56 to 69 years; 3 males, 3 females). Primary cells from this bone chips were utilized to analysis. Therefore 6 sets of iliac and mandibular bone-derived cells were obtained. <<detailed method>> The experimental protocol using human tissue was approved by the Institutional Review Board of Kyungpook National University Hospital (KNUH_10-1093). Informed consent was obtained from each donors who were undergoing iliac bone graft to mandibular alveolar bone defect. To collect osteoblast-like primary cells migrated from bone chips cortical or cortico-cancellous bone was harvested with the similar method described previously. Human mandible and iliac bone chip was were collected from 6 donors (average 60.2 years, ranged from 56 to 69 years; 3 males, 3 females), of healthy general condition without clinical inflammatory lesion in oral cavity. After complete reflection of periosteal layer, the mandibular bone and iliac crest was visualized. Mandibular cortical or cortico-cancellous bone chip was collected by a ronguer during the host bone trimming and decortication procedure for iliac bone graft to edentulous mandible. Similarly, small amount of iliac cortico-cancellous bone chip was collected seperately during the iliac bone harvesting procedure from the same patient. The harvested bone were broke into small pieces and the size of the bone particle became to be 2~4mm in length. The collected bone samples from the two sites were treated with the same method. The bone sample were gently rinsed with Dulbecco's modified Eagle's Medium (DMEM, Lonza Group Ltd., Basel, Switzerland) containing heparin to exclude fat and blood. The particulated bone were then transferred into culture flasks and cultured with DMEM supplemented with 20% fetal bovine serum (FBS, GIBCO-Invetrogen, Carlsbad, CA), 100 U/ml penicillin, 100 mg/ml streptomycin sulphate and 2 mM glutamine incubated at 37°C in a humidified atmosphere of 5% CO2 in air. The medium was first changed 2 days after seeding to remove non-adherent cells and the media (DMEM - 20% FBS) was changed every 2 days until the cells migrated out from the explants and reached confluency. Subconfluent primary cells were trypsinized and replated to 75 Cm2 flask. At passage 2, the cells were harvested to extract RNA for cDNA microarray.
Project description:OBJECTIVE:Long-term administration of intravenous bisphosphonates like pamidronate is associated with jaw osteonecrosis but axial and appendicular bones remain unaffected. Pathogenesis of bisphosphonate-associated jaw osteonecrosis may relate to skeletal site-specific effects of bisphosphonates on osteogenic differentiation of bone marrow stromal cells (BMSCs) of orofacial and axial/appendicular bones. This study evaluated and compared skeletal site-specific osteogenic response of mandible (orofacial bone) and iliac crest (axial bone) human BMSCs to pamidronate. MATERIALS AND METHODS:Mandible and iliac crest BMSCs from six normal healthy volunteers were established in culture and tested with pamidronate to evaluate and compare cell survival, osteogenic marker alkaline phosphatase, osteoclast differentiation in co-cultures with CD34+ hematopoietic stem cells, gene expression of receptor activator of NFkappaB ligand (RANKL) and osteoprotegerin, and in vivo bone regeneration. RESULTS:Mandible BMSCs were more susceptible to pamidronate than iliac crest BMSCs based on decreased cell survival, lower alkaline phosphatase production, and structurally less organized in vivo bone regeneration. Pamidronate promoted higher RANKL gene expression and osteoclast recruitment by mandible BMSCs. CONCLUSION:Mandible and iliac crest BMSC survival and osteogenic differentiation are disparately affected by pamidronate to favor dysregulated mandible bone homeostasis.
Project description:Osteonecrosis is a common dose-limiting toxicity of glucocorticoids. Data from clinical trials suggest that other medications can increase the risk of glucocorticoid-induced osteonecrosis. Here we utilized a mouse model to study the effect of asparaginase treatment on dexamethasone-induced osteonecrosis. Mice receiving asparaginase along with dexamethasone had a higher rate of osteonecrosis than those receiving only dexamethasone after 6 weeks of treatment (44% vs. 10%, P = 0.006). Similarly, epiphyseal arteriopathy, which we have shown to be an initiating event for osteonecrosis, was observed in 58% of mice receiving asparaginase and dexamethasone compared to 17% of mice receiving dexamethasone only (P = 0.007). As in the clinic, greater exposure to asparaginase was associated with greater plasma exposure to dexamethasone (P = 0.0001). This model also recapitulated other clinical risk factors for osteonecrosis, including age at start of treatment, and association with the systemic exposure to dexamethasone (P = 0.027) and asparaginase (P = 0.036). We conclude that asparaginase can potentiate the osteonecrotic effect of glucocorticoids.
Project description:Ethanol and glucocorticoids are risk factors associated with osteonecrosis. Previous reports suggest ethanol and glucocorticoids induce adipogenesis, decrease osteogenesis in bone marrow stroma cells, and produce intracellular lipid deposits resulting in death of osteocytes. The Wnt/beta-catenin signal pathway is involved in the regulation of homeostasis of bone and we presume glucocorticoids and ethanol may induce osteonecrosis in humans through a similar mechanism as in rodents. We hypothesized (1) ethanol, like glucocorticoids, decreases osteogenesis and increases adipogenesis through the Wnt/beta-catenin signaling pathway in human bone marrow stromal cells; and (2) ethanol decreases intranuclear translocation of beta-catenin. We found both dexamethasone and ethanol decrease the gene and protein expression of osteogenesis and increase that of adipogenesis through Wnt signaling-related genes by semiquantitative and quantitative polymerase chain reaction and Western blot. Ethanol hampered intranuclear translocation of beta-catenin by immunofluorescence analysis. The data suggest the Wnt/beta-catenin signaling pathway may be associated with ethanol-induced osteonecrosis.
Project description:Glucocorticoid-associated osteonecrosis is an intractable condition, making the establishment of preventative strategies of particular importance. Recently various studies using mesenchymal stem cells (MSC) have been conducted. Using a rabbit glucocorticoid-associated osteonecrosis model we administered green fluorescent protein (GFP)-labeled MSC intravenously to investigate their effect on osteonecrosis.A rabbit osteonecrosis model in which methylprednisolone (MP) 20 mg/kg was injected into the gluteus of a Japanese white rabbit was used. Simultaneously with MP, MSC labeled with GFP (GFP-labeled MSC) were injected intravenously. Fourteen days later the animals were killed (MSC(+)/MP(+)/14d), femurs were extracted, and the prevalence of osteonecrosis was determined histopathologically. Also, animals were killed 3 days after simultaneous administration of GFP-labeled MSC and MP (MSC(+)/MP(+)/3d), and western blotting (WB) for GFP was performed of the femur, liver, kidney, lung, blood vessel, and vertebra, in addition to immunohistochemical study of femur. As a control for the histopathological study, animals were killed 14 days after MP administration and intravenous vehicle injection (MSC(-)/MP(+)/14d). For WB, animals were killed 3 days after intravenous GFP-labeled MSC administration and vehicle injection into the gluteus (MSC(+)/MP(-)/3d).In MSC(-)/MP(+)/14d osteonecrosis was found in 7 of 10 rabbits (70%), while in MSC(+)/MP(+)/14d, partial bone marrow necrosis was found in only 1 rabbit (12.5%); osteonecrosis was not found in 7 of 8 rabbits (p?<?0.05). WB showed expression of GFP in the femur, not in the liver, kidney, lung, blood vessel, or vertebra, of MSC(+)/MP(+)/3d; expression of GFP-labeled MSC was absent in the femur of MSC(+)/MP(-)/3d. In the immunohistochemical study of MSC(+)/MP(+)/3d, homing of GFP-labeled MSC was noted perivascularly in the femur, but not in MSC(+)/MP(-)/3d.With transvenous MSC administration a significant prophylactic effect against glucocorticoid-associated osteonecrosis was found. Direct administration of MSC to the site of tissue injury requires highly invasive surgery. In contrast, as shown here the simple and hardly invasive intravenous administration of MSC may succeed in preventing osteonecrosis.