Aged Callus Skeletal Stem/Progenitor Cells Contain an Inflammatory Osteogenic Population With Increased IRF and NF-κB Pathways and Reduced Osteogenic Potential.

Skeletal stem/progenitor cells (SSPCs) are critical for fracture repair by providing osteo-chondro precursors in the callus, which is impaired in aging. However, the molecular signatures of callus SSPCs during aging are not known. Herein, we performed single-cell RNA sequencing on 11,957 CD45-CD31-Ter119- SSPCs isolated from young and aged mouse calluses. Combining unsupervised clustering, putative makers, and DEGs/pathway analyses, major SSPC clusters were annotated as osteogenic, proliferating, and adipogenic populations. The proliferating cluster had a differentiating potential into osteogenic and adipogenic lineages by trajectory analysis. The osteoblastic/adipogenic/proliferating potential of individual clusters was further evidenced by elevated expression of genes related to osteoblasts, adipocytes, or proliferation. The osteogenic cluster was sub-clustered into house-keeping and inflammatory osteogenic populations that were decreased and increased in aged callus, respectively. The majority of master regulators for the inflammatory osteogenic population belong to IRF and NF-κB families, which was confirmed by immunostaining, RT-qPCR, and Western blot analysis. Furthermore, cells in the inflammatory osteogenic sub-cluster had reduced osteoblast differentiation capacity. In conclusion, we identified 3 major clusters in callus SSPCs, confirming their heterogeneity and, importantly, increased IRF/NF-κB-mediated inflammatory osteogenic population with decreased osteogenic potential in aged cells.

The E3 ubiquitin ligase Itch limits the progression of post-traumatic osteoarthritis in mice by inhibiting macrophage polarization.

OBJECTIVE: Osteoarthritis (OA) is characterized by articular cartilage loss, associated with synovial inflammation. We recently reported increased pro-inflammatory macrophages in murine post-traumatic OA (PTOA) joints, and blockade of the ubiquitin-proteasome system alleviates PTOA progression. However, the mechanisms whereby protein ubiquitination influences PTOA pathology are not well studied. We hypothesized that loss of the negative regulator of inflammation, E3 ligase Itch, in macrophages contributes to joint OA tissue damage by promoting pro-inflammatory polarization of macrophages. METHODS: Mice deficient Itch in macrophages (MΔItch) were generated by crossing Itchfl/fl mice with LysM-Cre mice. PTOA surgery was performed on global Itch knockout, Itch-/-, mice and MΔItch mice. Joint tissue damage and synovial macrophages were examined. Itch-/- cells were treated with IL-1 and pro-inflammatory polarization was determined. Expression of Itch protein and mRNA in PTOA synovium were assessed at different time points post PTOA. RESULTS: Similar to Itch-/- mice, MΔItch mice developed more severe joint damage than control mice following PTOA surgery (mean difference of OARSI score: 1.17 (95% CI 0.31-2.03) between MΔItch and Itchfl/fl mice), accompanied by increased the inflammatory macrophage infiltration in the synovium (mean difference of % F4/80 + CD86 + CD36-inflammatory macrophages: 14.81 (95% CI 8.90-20.73) between MΔItch and Itchfl/fl mice). Itch-/- macrophages exerted pro-inflammatory phenotype in response to IL-1ß treatment. Itch protein, but not mRNA levels decreased during PTOA progression. CONCLUSION: The negative regulator of inflammation, Itch, limits PTOA progression by inhibiting macrophage pro-inflammatory polarization. Itch protein degradation may contribute to PTOA pathology.

An update on the validation of whole slide imaging systems following FDA approval of a system for a routine pathology diagnostic service in the United States.

Pathologists have used light microscopes and glass slides to interpret the histologic appearance of normal and diseased tissues for more than 150 years. The quality of both microtomes used to cut tissue sections and microscopes has improved significantly during the past few decades, but the process of rendering diagnoses has changed little. By contrast, major advances in digital technology have occurred since the introduction of hand held electronic devices, including the development of whole slide imaging (WSI) systems with software packages that can convert microscope images into virtual (digital) slides that can be viewed on computer monitors and via the internet. To date, however, these technological developments have had minimal impact on the way pathologists perform their daily work, with the exception of using computers to access electronic medical records and scholarly web sites for pertinent information to assist interpretation of cases. Traditional practice is likely to change significantly during the next decade, especially since the Federal Drug Administration in the USA has approved the first WSI system for routine diagnostic practice. I review here the development and slow acceptance of WSI by pathology departments. I focus on recent advances in validation of WSI systems that is required for routine diagnostic reporting of pathology cases using this technology.

Whole slide imaging: uses and limitations for surgical pathology and teaching.

Advances in computer and software technology and in the quality of images produced by digital cameras together with development of robotic devices that can take glass histology slides from a cassette holding many slides and place them in a conventional microscope for electronic scanning have facilitated the development of whole slide imaging (WSI) systems during the past decade. Anatomic pathologists now have opportunities to test the utility of WSI systems for diagnostic, teaching and research purposes and to determine their limitations. Uses include rendering primary diagnoses from scanned hematoxylin and eosin stained tissues on slides, reviewing frozen section or routine slides from remote locations for interpretation or consultation. Also, WSI can replace physical storage of glass slides with digital images, storing images of slides from outside institutions, presenting slides at clinical or research conferences, teaching residents and medical students, and storing fluorescence images without fading or quenching of the fluorescence signal. Limitations include the high costs of the scanners, maintenance contracts and IT support, storage of digital files and pathologists' lack of familiarity with the technology. Costs are falling as more devices and systems are sold and cloud storage costs drop. Pathologist familiarity with the technology will grow as more institutions purchase WSI systems. The technology holds great promise for the future of anatomic pathology.

Advances in the regulation of osteoclasts and osteoclast functions.

Osteoclasts are derived from mononuclear hematopoietic myeloid lineage cells, which are formed in the bone marrow and are attracted to the bloodstream by factors, including sphingsine-1 phosphate. These circulating precursors are attracted to bone surfaces undergoing resorption by chemokines and other factors expressed at these sites, where they fuse to form multinucleated bone-resorbing cells. All aspects of osteoclast formation and functions are regulated by macrophage-colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL), cytokines essential for osteoclast formation and expressed by a variety of cell types, including osteoblast lineage cells. Since the discovery of RANKL in the mid-1990s, mouse genetic and molecular studies have revealed numerous signaling pathways activated by RANKL and M-CSF. More recent studies indicate that osteoclasts and their precursors regulate immune responses and osteoblast formation and functions by means of direct cell-cell contact through ligands and receptors, such as ephrins and Ephs, and semaphorins and plexins, and through expression of clastokines. There is also growing recognition that osteoclasts are immune cells with roles in immune responses beyond mediating the bone destruction that can accompany them. This article reviews recent advances in the understanding of the molecular mechanisms regulating osteoclast formation and functions and their interactions with other cells in normal and pathologic states.

Use of a whole-slide imaging system to assess the presence and alteration of lymphatic vessels in joint sections of arthritic mice.

We investigated the presence and alteration of lymphatic vessels in joints of arthritic mice using a whole-slide imaging system. Joints and long bone sections were cut from paraffin blocks of two mouse models of arthritis: meniscal-ligamentous injury (MLI)-induced osteoarthritis (OA) and TNF transgene (TNF-Tg)-induced rheumatoid arthritis (RA). MLI-OA mice were fed a high fat diet to accelerate OA development. TNF-Tg mice were treated with lymphatic growth factor VEGF-C virus to stimulate lymphangiogenesis. Sections were double immunofluorescence stained with anti-podoplanin and alpha-smooth muscle actin antibodies. The area and number of lymphatic capillaries and mature lymphatic vessels were determined using a whole-slide imaging system and its associated software. Lymphatic vessels in joints were distributed in soft tissues mainly around the joint capsule, ligaments, fat pads and muscles. In long bones, enriched lymphatic vessels were present in the periosteal areas adjacent to the blood vessels. Occasionally, lymphatic vessels were observed in the cortical bone. Increased lymphatic capillaries, but decreased mature lymphatic vessels, were detected in both OA and RA joints. VEGF-C treatment increased lymphatic capillary and mature vessel formation in RA joints. Our findings suggest that the lymphatic system may play an important role in arthritis pathogenesis and treatment.

The challenge and importance of standardizing pre-analytical variables in surgical pathology specimens for clinical care and translational research.

The introduction of targeted cancer therapies into clinical practice, in which patients are selected for novel treatments based on results of companion molecular testing of their tumor specimens, has created significant new challenges for the surgical pathology laboratory. These include standardization of tissue handling and sample preparation with accurate documentation to ensure optimal quality of clinical samples to reduce the risk of errors in molecular biology tests. The assay of tumor tissues for biomarkers that can provide predictive data for prognosis or treatment should enable selection of the most appropriate therapies (Yaziji et al. 2008, Hicks and Kulkarni 2008). Major advances have been made in the ability to profile clinical samples for research at the DNA, RNA and protein levels. To translate this new information into the clinical setting, however, the quality of the starting material, in this case the tumor tissue, determines the accuracy and reliability of companion diagnostic assay results and therefore optimal therapeutic strategies. Inaccurate results owing to compromised tissue quality can lead to false positive or false negative results with therapeutic consequences that can harm patients and affect their eventual outcome.

Polymicrobial periodontal pathogen transcriptomes in calvarial bone and soft tissue.

Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia are consistently associated with adult periodontitis. This study sought to document the host transcriptome to a P. gingivalis, T. denticola, and T.forsythia challenge as a polymicrobial infection using a murine calvarial model of acute inflammation and bone resorption. Mice were infected with P. gingivalis, T. denticola, and T. forsythia over the calvaria, after which the soft tissues and calvarial bones were excised. A Murine GeneChip(®) array analysis of transcript profiles showed that 6997 genes were differentially expressed in calvarial bones (P < 0.05) and 1544 genes were differentially transcribed in the inflamed tissues after the polymicrobial infection. Of these genes, 4476 and 1035 genes in the infected bone and tissues were differentially expressed by upregulation. Biological pathways significantly impacted by the polymicrobial infection in calvarial bone included leukocyte transendothelial migration (LTM), cell adhesion molecules, adherens junction, major histocompatibility complex antigen, extracellular matrix-receptor interaction, and antigen processing and presentation resulting in inflammatory/cytokine/chemokine transcripts stimulation in bone and soft tissue. Intense inflammation and increased activated osteoclasts were observed in calvarias compared with sham-infected controls. Quantitative real-time RT-PCR analysis confirmed that the mRNA level of selected genes corresponded with the microarray expression. The polymicrobial infection regulated several LTM and extracellular membrane pathway genes in a manner distinct from mono-infection with P. gingivalis, T. denticola, or T. forsythia. To our knowledge, this is the first definition of the polymicrobially induced transcriptome in calvarial bone and soft tissue in response to periodontal pathogens.

Tannerella forsythia infection-induced calvarial bone and soft tissue transcriptional profiles.

Tannerella forsythia is associated with subgingival biofilms in adult periodontitis, although the molecular mechanisms contributing to chronic inflammation and loss of periodontal bone remain unclear. We examined changes in the host transcriptional profiles during a T. forsythia infection using a murine calvarial model of inflammation and bone resorption. Tannerella forsythia was injected into the subcutaneous soft tissue over calvariae of BALB/c mice for 3 days, after which the soft tissues and calvarial bones were excised. RNA was isolated and Murine GeneChip (Affymetrix, Santa Clara, CA) array analysis of transcript profiles showed that 3226 genes were differentially expressed in the infected soft tissues (P < 0.05) and 2586 genes were differentially transcribed in calvarial bones after infection. Quantitative real-time reverse transcription-polymerase chain reaction analysis of transcription levels of selected genes corresponded well with the microarray results. Biological pathways significantly impacted by T. forsythia infection in calvarial bone and soft tissue included leukocyte transendothelial migration, cell adhesion molecules (immune system), extracellular matrix-receptor interaction, adherens junction, and antigen processing and presentation. Histologic examination revealed intense inflammation and increased osteoclasts in calvariae compared with controls. In conclusion, localized T. forsythia infection differentially induces transcription of a broad array of host genes, and the profiles differ between inflamed soft tissues and calvarial bone.

Molecular characterization of Treponema denticola infection-induced bone and soft tissue transcriptional profiles.

Treponema denticola is associated with subgingival biofilms in adult periodontitis and with acute necrotizing ulcerative gingivitis. However, the molecular mechanisms by which T. denticola impacts periodontal inflammation and alveolar bone resorption remain unclear. Here, we examined changes in the host transcriptional profiles during a T. denticola infection using a murine calvarial model of inflammation and bone resorption. T. denticola was injected into the subcutaneous soft tissue over the calvaria of BALB/c mice for 3 days, after which the soft tissues and the calvarial bones were excised. RNA was isolated and analysed for transcript profiling using Murine GeneChip arrays. Following T. denticola infection, 2905 and 1234 genes in the infected calvarial bones and soft tissues, respectively, were differentially expressed (P

Porphyromonas gingivalis infection-induced tissue and bone transcriptional profiles.

Porphyromonas gingivalis has been associated with subgingival biofilms in adult periodontitis. However, the molecular mechanisms of its contribution to chronic gingival inflammation and loss of periodontal structural integrity remain unclear. This investigation aimed to examine changes in the host transcriptional profiles during a P. gingivalis infection using a murine calvarial model of inflammation and bone resorption. P. gingivalis FDC 381 was injected into the subcutaneous soft tissue over the calvaria of BALB/c mice for 3 days, after which the soft tissues and calvarial bones were excised. RNA was isolated from infected soft tissues and calvarial bones and was analysed for transcript profiles using Murine GeneChip((R)) arrays to provide a molecular profile of the events that occur following infection of these tissues. After P. gingivalis infection, 6452 and 2341 probe sets in the infected soft tissues and calvarial bone, respectively, were differentially expressed (P

Tumor necrosis factor prevents alendronate-induced osteoclast apoptosis in vivo by stimulating Bcl-xL expression through Ets-2.

OBJECTIVE: To investigate why bisphosphonates are less effective at preventing focal bone loss in rheumatoid arthritis (RA) patients than in those with generalized osteoporosis, and the mechanisms involved. METHODS: The response of osteoclasts to alendronate (ALN) in tumor necrosis factor-transgenic (TNF-Tg) mice that develop erosive arthritis and in wild-type littermates was studied. TNF-Tg and wild-type mice were given ALN, and the osteoclast numbers in the inflamed joints and in the long bones were compared. The expression levels of Bcl-xL in the osteoclasts of TNF-Tg and wild-type mice were examined by immunostaining. The effect of overexpression of Bcl-xL and Ets-2 proteins on ALN-induced osteoclast apoptosis was determined using an in vitro osteoclast survival assay and retrovirus transfer approach. RESULTS: ALN reduced osteoclast numbers in the metaphyses by 97%, but by only 46% in the adjacent inflamed joints. Bcl-xL expression was markedly higher in osteoclasts in the joints than in those in the metaphyses of TNF-Tg mice. Bcl-xL or Ets-2 overexpression protected osteoclasts from ALN-induced apoptosis, and TNF stimulated Bcl-xL and Ets-2 expression in osteoclasts. Overexpression of Ets-2 increased Bcl-xL messenger RNA in osteoclasts, while a dominant-negative form of the Ets-2 blocked the protective effect of Bcl-xL or TNF on ALN-induced apoptosis. CONCLUSION: The reduced efficacy of bisphosphonates to stop bone erosion in the inflamed joints of RA patients may result from local high levels of TNF up-regulating Ets-2 expression in osteoclasts, which in turn stimulates Bcl-xL expression in them and reduces their susceptibility to bisphosphonate-induced apoptosis.

Evidence for a direct role of cyclo-oxygenase 2 in implant wear debris-induced osteolysis.

Aseptic loosening is a major complication of prosthetic joint surgery and is manifested as chronic inflammation, pain, and osteolysis at the bone implant interface. The osteolysis is believed to be driven by a host inflammatory response to wear debris generated from the implant. In our current study, we use a selective inhibitor (celecoxib) of cyclo-oxygenase 2 (COX-2) and mice that lack either COX-1 (COX-1-/-) or COX-2 (COX-2-/-) to show that COX-2, but not COX-1, plays an important role in wear debris-induced osteolysis. Titanium (Ti) wear debris was implanted surgically onto the calvaria of the mice. An intense inflammatory reaction and extensive bone resorption, which closely resembles that observed in patients with aseptic loosening, developed within 10 days of implantation in wild-type and COX-1-/- mice. COX-2 and prostaglandin E2 (PGE2) production increased in the calvaria and inflammatory tissue overlying it after Ti implantation. Celecoxib (25 mg/kg per day) significantly reduced the inflammation, the local PGE2 production, and osteolysis. In comparison with wild-type and COX-1-/- mice, COX-2-/- mice implanted with Ti had a significantly reduced calvarial bone resorption response, independent of the inflammatory response, and significantly fewer osteoclasts were formed from cultures of their bone marrow cells. These results provide direct evidence that COX-2 is an important mediator of wear debris-induced osteolysis and suggests that COX-2 inhibitors are potential therapeutic agents for the prevention of wear debris-induced osteolysis.

Genetic evidence for a role for Src family kinases in TNF family receptor signaling and cell survival.

Mutant src(-/-) mice have osteopetrosis resulting from defective osteoclasts, the cells that resorb bone. However, signaling pathways involving Src family members in osteoclasts remain unclear. We demonstrate that expression of a truncated Src molecule, Src251, lacking the kinase domain, induces osteopetrosis in wild-type and src(+/-) mice and worsens osteopetrosis in src(-/-) mice by a novel mechanism, increased osteoclast apoptosis. Induction of apoptosis by Src251 requires a functional SH2, but not an SH3, domain and is associated with reduced AKT kinase activity. Expression of Src251 dramatically reduces osteoclast survival in response to RANKL/TRANCE/OPGL, providing evidence that Src family kinases are required in vivo for survival signaling pathways downstream from TNF family receptors.

Decreased c-Src expression enhances osteoblast differentiation and bone formation.

c-src deletion in mice leads to osteopetrosis as a result of reduced bone resorption due to an alteration of the osteoclast. We report that deletion/reduction of Src expression enhances osteoblast differentiation and bone formation, contributing to the increase in bone mass. Bone histomorphometry showed that bone formation was increased in Src null compared with wild-type mice. In vitro, alkaline phosphatase (ALP) activity and nodule mineralization were increased in primary calvarial cells and in SV40-immortalized osteoblasts from Src(-/-) relative to Src(+/+) mice. Src-antisense oligodeoxynucleotides (AS-src) reduced Src levels by approximately 60% and caused a similar increase in ALP activity and nodule mineralization in primary osteoblasts in vitro. Reduction in cell proliferation was observed in primary and immortalized Src(-/-) osteoblasts and in normal osteoblasts incubated with the AS-src. Semiquantitative reverse transcriptase-PCR revealed upregulation of ALP, Osf2/Cbfa1 transcription factor, PTH/PTHrP receptor, osteocalcin, and pro-alpha 2(I) collagen in Src-deficient osteoblasts. The expression of the bone matrix protein osteopontin remained unchanged. Based on these results, we conclude that the reduction of Src expression not only inhibits bone resorption, but also stimulates osteoblast differentiation and bone formation, suggesting that the osteogenic cells may contribute to the development of the osteopetrotic phenotype in Src-deficient mice.

A murine model of inflammatory bone disease.

We have recently reported the identification of a new recessive mutation on murine chromosome 18 that results in tail kinks and deformity in the lower extremities of mice. Preliminary examination of the bones of these mice showed that there are abnormalities present that resembled chronic recurrent multifocal osteomyelitis. Accordingly, this new mutation was named "CMO." In this report, we describe the histology of bones in CMO mice, as well as the capacity of the bone marrow cells from these animals to form osteoclasts (OCLs). In addition, we tested conditioned media from non-adherent marrow cells and total marrow cells from CMO mice for their capacity to induce OCL formation in normal murine marrow cultures. These studies demonstrated that the bone disease in these animals is inflammatory in nature, and a soluble factor(s) that is not IL-1alpha, IL-6 or TNF-alpha is released by marrow cells from CMO animals and enhances OCL formation in normal murine marrow cultures.

Factors regulating the growth of metastatic cancer in bone.

Metastatic tumor cells can interfere directly with the function of bone cells involved in normal bone remodeling or indirectly by influencing the behavior of hematopoietic, stromal and other cells in bone marrow that interact with bone cells. Recent studies of metastatic cancer have revealed that tumor cells interact closely with vascular endothelial cells, basement membrane and bone marrow stromal cells through cell surface proteins or by releasing factors which affect the function of these cells. Bidirectional interaction between marrow cells and tumor cells can give the latter a selective advantage for growth in bone which can lead to the destruction of or to increased production of bone matrix. Understanding of the mechanisms involved in tumor metastasis and growth in bone has increased in recent years, and in this review we shall describe current knowledge of these mechanisms and of the predilection of certain types of cancers to metastasize to bone, their growth in the bone microenvironment and interactions between them and bone cells. Because metastatic breast cancer has been studied more than any other, we shall focus on it as a representative example, although the general principles apply to other types of cancer and to myeloma.

Identification of human asparaginyl endopeptidase (legumain) as an inhibitor of osteoclast formation and bone resorption.

We screened a human osteoclast (OCL) cDNA expression library for OCL inhibitory factors and identified a clone that blocked both human and murine OCL formation and bone resorption by more than 60%. This clone was identical to human legumain, a cysteine endopeptidase. Legumain significantly inhibited OCL-like multinucleated cell formation induced by 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) and parathyroid hormone-related protein (PTHrP) in mouse and human bone marrow cultures, and bone resorption in the fetal rat long bone assay in a dose-dependent manner. Legumain was detected in freshly isolated marrow plasma from normal donors and conditioned media from human marrow cultures. Furthermore, treatment of human marrow cultures with an antibody to legumain induced OCL formation to levels that were as high as those induced by 1,25-(OH)(2)D(3). Implantation in nude mice of 293 cells transfected with the legumain cDNA and constitutively expressing high levels of the protein significantly reduced hypercalcemia induced by PTHrP by about 50%, and significantly inhibited the increase in OCL surface and in OCL number expressed per mm(2) bone area and per mm bone surface induced by PTHrP. These results suggest that legumain may be a physiologic local regulator of OCL activity that can negatively modulate OCL formation and activity.

Required and nonessential functions of nuclear factor-kappa B in bone cells.

Nuclear factor-kappa B (NF-kappaB) is a set of five polypeptide transcription factors, called p50, p52, p65 (also called Rel A), Rel B, and c-Rel, which regulate the expression of a variety of genes involved in immune and inflammatory responses. They were originally named because they were considered essential regulators of B cell kappa light chain expression. More recent studies indicate that NF-kappaB proteins are involved in the regulation of a variety of other cell functions, including cell proliferation, responses to stress, and apoptosis. NF-kappaB heterodimers reside in the cytoplasm of cells bound to inhibitory proteins, the two commonest of which are IkappaBalpha and IkappaBbeta, which prevent NF-kappaB from entering the nucleus. When cells are stimulated, IkappaB is phosphorylated by specific IkappaB kinases and subsequently is ubiquitinated and degraded in proteosomes. This allows NF-kappaB to translocate to the nucleus to regulate the expression of a growing list of genes, including the proinflammatory cytokines, interleukin-1 (IL-1), IL-6, and tumor necrosis factor. IL-1 and tumor necrosis factor in turn also regulate the expression of NF-kappaB. Thus, once activated, NF-kappaB may be involved in upregulatory loops, which can amplify the effects of the initiating stimulus. Because these proinflammatory cytokines have been implicated in the pathogenesis of estrogen deficiency and inflammation-related bone loss, it is likely that NF-kappaB has a significant role in the increased generation and function of osteoclasts in these circumstances. However, an unexpected and essential role of NF-kappaB in the formation of osteoclasts during development was discovered recently after the generation of knockout mice, which lack the expression of the p50 and p52 subunits. This paper will describe recent studies that reveal an essential role for NF-kappaB signaling in the generation of osteoclasts and that suggest that NF-kappaB may also play a key central role in the activation and survival of osteoclasts in conditions in which osteoclastogenesis is upregulated.

Systemic administration of acidic fibroblast growth factor (FGF-1) prevents bone loss and increases new bone formation in ovariectomized rats.

There are no universally accepted agents that will substantially increase bone mass in osteoporotic patients. A number of peptides important in normal bone formation, such as members of the transforming growth factor-beta superfamily, are not satisfactory for this purpose either because their beneficial effects are predominantly local or there is systemic toxicity associated with their administration. We have examined the effects of exogenous fibroblast growth factor-1 and -2 (FGF-1 and FGF-2) on bone in vivo, since FGFs have been shown recently to be essential for normal skeletal development. FGF-1 was injected daily (0.2 mg/kg intravenously) for 28 days into the tail vein of adult female rats immediately following and 6 months after sham operation or ovariectomy (OVX). In rats treated immediately post-OVX, OVX produced more than a 30% decrease in tibial bone density, which was prevented by FGF-1 and estrogen. However, FGF-1 also had an anabolic effect. In sham-operated rats, FGF-1 increased bone density to 2-fold, whereas estrogen had no effect. In rats 6 months post-OVX, severe bone loss and disruption of trabecular microarchitecture occurred similar to that seen in patients with severe osteoporosis. In these rats, administration of FGF-1 induced extensive new woven bone formation with new trabecular-like structures filling much of the marrow spaces, and bone density in the tibial metaphysis increased 3-fold. FGF-1 and FGF-2 were also administered subcutaneously over the calvaria of mice in doses of 2-2000 microg/day for 3 days and shown to produce substantial increases in bone formation when examined morphologically. Thus, we conclude that both local and systemic FGF-1 increases new bone formation and bone density, and systemic FGF-1 also appears to restore bone microarchitecture and prevent bone loss associated with estrogen-withdrawal.