Enamel matrix derivative, inflammation and soft tissue wound healing.

Over 15 years have now passed since enamel matrix derivative (EMD) emerged as an agent capable of periodontal regeneration. Following thorough investigation, evidenced-based clinical application is now established for a multitude of clinical settings to promote regeneration of periodontal hard tissues. Despite the large number of studies and review articles written on this topic, no single review has compiled the influence of EMD on tissue inflammation, an area of research that merits substantial attention in periodontology. The aim of the present review was to gather all studies that deal with the effects of EMD on tissue inflammation with particular interest in the cellular mechanisms involved in inflammation and soft tissue wound healing/resolution. The effects of EMD on monocytes, macrophages, lymphocytes, neutrophils, fibroblasts and endothelial cells were investigated for changes in cell behavior as well as release of inflammatory markers, including interleukins, prostaglandins, tumor necrosis factor-α, matrix metalloproteinases and members of the OPG-RANKL pathway. In summary, studies listed in this review have reported that EMD is able to significantly decrease interleukin-1b and RANKL expression, increase prostaglandin E2 and OPG expression, increase proliferation and migration of T lymphocytes, induce monocyte differentiation, increase bacterial and tissue debris clearance, as well as increase fibroplasias and angiogenesis by inducing endothelial cell proliferation, migration and capillary-like sprout formation. The outcomes from the present review article indicate that EMD is able to affect substantially the inflammatory and healing responses and lay the groundwork for future investigation in the field.

Periodontal healing after application of enamel matrix derivative in surgical supra/infrabony periodontal defects in rats with streptozotocin-induced diabetes.

BACKGROUND AND OBJECTIVE: Epidemiologic and clinical studies have indicated that diabetes is a risk factor for periodontal disease progression and healing. The aim of the present study was to evaluate short-term healing after enamel matrix derivative (EMD) application in combined supra/infrabony periodontal defects in diabetic rats. MATERIAL AND METHODS: Thirty male Wistar rats were initially divided into two groups, one with streptozotocin-induced diabetes and another one with healthy (non-diabetic) animals. Bony defects were surgically created on the mesial root of the first maxillary molars. After root surface planing and EDTA conditioning, EMD was applied to the roots at one side of the maxillae, while those on the contralateral sides were left untreated. Animals were killed 3 wk after surgery, and block sections were prepared for histologic and histomorphometric analysis. RESULTS: There was statistically significant more gingival recession in diabetic animals than in non-diabetic animals. The length of the junctional epithelium was significantly shorter in the EMD-treated sites in both diabetic and normoglycemic rats. Sulcus depth and length of supracrestal soft connective tissue showed no statistically significant differences between groups. In all animals, new bone formation was observed. Although new bone occurred more frequently in healthy animals, the extent of new bone was not significantly different between groups. In none of the teeth, a layer of new cementum was detectable. EMD had no influence on bone or cementum regeneration. Adverse reactions such as excessive inflammation due to bacterial root colonization, ankylosis and bone fractures were exclusively observed in diabetic animals, irrespective of EMD treatment. CONCLUSION: Within the limits of the present study, it can be concluded that periodontal healing was impaired in streptozotocin-induced diabetic rats. EMD had no beneficial effects on new bone and cementum formation during short-term healing in this defect model and could not ameliorate the adverse effects in the systemically compromised animals.

Enamel matrix derivative induces the expression of tissue inhibitor of matrix metalloproteinase-3 in human gingival fibroblasts via extracellular signal-regulated kinase.

BACKGROUND AND OBJECTIVE: Periodontal disease is characterized by increased expression and activity of matrix metalloproteinases (MMPs) and insufficient expression/activity of their inhibitors, tissue inhibitors of matrix metalloproteinases (TIMPs). This altered MMP-TIMP balance results in progressive destruction of gingival and periodontal extracellular matrix. Enamel matrix derivative (EMD), clinically used for periodontal regeneration in a device called Emdogain, has been suggested to enhance gingival healing following periodontal procedures in humans. We previously showed that EMD increases the proliferation of human and rat gingival fibroblasts and protects them from tumor necrosis factor-induced apoptosis. In the present study, the modulation of MMP and TIMP expression by EMD was investigated. MATERIAL AND METHODS: Primary human gingival fibroblasts were treated in vitro with tumor necrosis factor, EMD or both in serum-free conditions, and RNA was analyzed with an extracellular matrix-focused microarray and quantitative real-time polymerase chain reaction. RESULTS: Microarray analysis showed detectable expression of MMP-1, MMP-2, MMP-3, MMP-7 and MMP-13, as well as TIMP-1 and TIMP-3 in untreated cells. There was no apparent regulation of the expression of MMP-2, MMP-7, MMP-13 and TIMP-1 by either tumor necrosis factor or EMD. In contrast, tumor necrosis factor significantly increased MMP-1 expression, and EMD reduced it when both agents were present. Also, EMD significantly induced TIMP-3 expression, an effect which was dependent on activation of extracellular signal-regulated kinase 1/2, since it was totally abolished by a selective extracellular signal-regulated kinase pathway inhibitor. CONCLUSION: These data suggest that EMD may affect gingival health by ways other than cell proliferation/survival, i.e. by stimulation of TIMP-3 production, which could improve the MMP-TIMP balance in gingival tissue and curb extracellular matrix destruction.

Differential effects of prostaglandin E(2) and enamel matrix derivative on the proliferation of human gingival and dermal fibroblasts and gingival keratinocytes.

BACKGROUND AND OBJECTIVE: Elevated levels of prostaglandins contribute to periodontal destruction but can impair gingival healing by affecting local fibroblasts. Enamel matrix derivative (EMD) has beneficial effects on supporting and gingival tissues. We showed that prostaglandin E(2) (PGE(2) ) inhibits the proliferation of human gingival fibroblasts (hGFs) and that EMD stimulates it. Prostaglandins and EMD may also affect skin healing by targeting dermal fibroblasts (DFs). Thus, we compared the effects of these two agents on the proliferation of hGFs, human gingival keratinocytes (hGKs) and hDFs. MATERIAL AND METHODS: Cells from healthy human gingiva or skin were treated with PGE(2) and/or EMD, and proliferation was assessed by measuring cell number and DNA synthesis. RESULTS: In hGFs, PGE(2) (1 µm) inhibited proliferation while EMD stimulated it. When present together, EMD abolished the PGE(2) -induced inhibition. Serum increased (by a factor of 10) the amount of phosphorylated extracellular signal-regulated kinase (p-ERK), PGE(2) reduced it (by 70-80%) and EMD restored it when present with PGE(2). Prostaglandin E(2) stimulated cAMP production in hGFs while serum or EMD did not. Enamel matrix derivative stimulated hDF proliferation, but the inhibitory effect of PGE(2) was milder than with hGFs. When present together, EMD abolished the PGE(2) -induced inhibition. Enamel matrix derivative inhibited the proliferation of primary hGKs, but PGE(2) had no effect. Finally, we found that hDFs contained about five times less prostaglandin EP(2) receptor mRNA than hGFs, while hGKs contained none. CONCLUSION: Prostaglandin E(2) inhibits and EMD stimulates hGF proliferation via distinct pathways. The different sensitivities of hDFs and hGKs to PGE(2) can be explained by the levels of EP(2) expression.

EGFR in Enamel Matrix Derivative-induced gingival fibroblast mitogenesis.

We previously reported that EMD (Enamel Matrix Derivative) induces proliferation of human gingival fibroblasts via activation of Extracellular Regulated Kinase (ERK), and this study assessed the possible mediatory role of EGFR (Epidermal Growth Factor Receptor) in this effect. Treatment of gingival fibroblasts with EMD resulted in tyrosine phosphorylation of the EGFR, as assessed by immunoblotting and ELISA, while EMD-induced ERK activation and thymidine incorporation were markedly inhibited (approximately 40-50%) by a specific EGFR tyrosine kinase inhibitor. Using appropriate inhibitors, we established that EMD-induced EGFR activation is largely due to shedding of HB-EGF (Heparin-binding EGF) from the cell membrane via a metalloproteinase-mediated process. Finally, the addition of PP1, a Src family inhibitor, abrogated both EGFR phosphorylation and ERK activation. Taken together, these results indicate that, at least in human gingival fibroblasts, EMD-induced ERK activation and proliferation are partially due to a Src-dependent, metalloproteinase-mediated transactivation of EGFR.

Enamel matrix derivative protects human gingival fibroblasts from TNF-induced apoptosis by inhibiting caspase activation.

Emdogain, a formulation of enamel matrix derivative (EMD), is used clinically for regeneration of the periodontium (tooth supporting tissues), but the molecular mechanisms of its action have not been elucidated. Several clinical studies suggested that EMD may also improve gingival healing after periodontal surgery and thus affect the fate of gingival fibroblasts (GFs). Since these cells are targets for local inflammatory mediators such as TNF, a pro-apoptotic cytokine, during the course of periodontal disease, we tested whether EMD protects human GFs (hGFs) from TNF-induced cytotoxicity. Quiescent primary hGFs were challenged with TNF (10-100 ng/ml) with or without EMD (100 microg/ml) pretreatment. Cell viability was assessed by neutral red staining, cell death by LDH release and apoptosis by caspase activity. Signaling pathways were evaluated by Western blotting and pharmacological inhibitors. TNF induced classical signs of apoptosis in hGFs, including typical cellular morphology and increased caspase activity. TNF-induced cytotoxicity was entirely caspase-dependent. Pretreatment (4-24 h) with EMD dramatically inhibited the activation of initiator and executioner caspases and enhanced hGF survival. Although TNF induced the activation of p38 MAPK, JNK, ERK and PI-3K signaling, these pathways were not crucial for EMD protection of hGFs. However, EMD increased the levels of c-FLIP(L), an anti-apoptotic protein located upstream of caspase activation. These data demonstrate, for the first time, that EMD protects hGFs from inflammatory cytokines and, together with our recent reports that EMD stimulates rat and human GF proliferation, could help explain the mechanisms whereby in vivo use of EMD promotes gingival healing.

Enamel matrix derivative stimulates human gingival fibroblast proliferation via ERK.

Emdogain, a formulation of Enamel Matrix Proteins, is used clinically for periodontal regeneration to stimulate PDL (periodontal ligament), cementum, and bone formation. Its effects on gingival fibroblasts and tissue have not been thoroughly studied. Therefore, we investigated the mechanisms by which Emdogain affects the cell cycle of human gingival fibroblasts. Without serum, Emdogain (50 microg/mL) induced human gingival fibroblast entry into the S phase and DNA synthesis, but not completion of the cell cycle. With low serum concentrations (0.2-0.5%), Emdogain synergistically induced completion of the cell cycle, resulting in increased cell numbers. The mitogenic response to Emdogain depended on Extracellular Regulated Kinase (ERK) activation, which occurred in two waves, peaking after 15 min and 4 to 6 hrs, since it was abolished by U0126, a specific MAPK inhibitor. Inhibition of the second wave was sufficient to abrogate mitogenesis. This study characterized the mitogenic effect of Emdogain on primary human gingival fibroblasts, its cooperation with serum growth factors, and the key mediatory role of the ERK cascade.

Prostaglandin E2 (PGE2) increases the number of rat bone marrow osteogenic stromal cells (BMSC) via binding the EP4 receptor, activating sphingosine kinase and inhibiting caspase activity.

Prostaglandin E(2) (PGE(2)) is bone-anabolic, i.e. stimulates bone formation and increases bone mass. In this study, we explored possible intracellular mechanisms of its increase of osteogenic cells in rat bone marrow. Adherent rat bone marrow cells were counted after 12-48 h or cultured for 21 days and mineralized nodules were counted. Also, apoptosis of marrow cells was measured after in vivo PGE(2) injection. PGE(2) (100 nM) increased 2-3 fold the number of adherent BMSC, an effect which was mediated via binding the EP(4) receptor since it was mimicked by forskolin and 11-deoxy-prostaglandin E(1) (PGE(1)) and was blocked by DDA and L-161982 (EP(4) antagonist). PGE(2) stimulated sphingosine kinase (SPK) activity since its effects were blocked by DMS (SPK inhibitor) and mimicked by SPP (SPK product). PGE(2) reduced the activity of caspase-3 and -8 in BMSC and their inhibitors increased BMSC number and nodule formation. In vivo, PGE(2) prevented the increase in the apoptosis of bone marrow cells caused by indomethacin. We propose that PGE(2) exerts an anti-apoptotic effect on BMSC, thereby increasing their number and subsequent osteoblastic differentiation. Such an effect could explain how PGE(2) stimulates bone formation in vivo.

[Recommendations for early identification of damage to the skeleton by malignant processes, and for early diagnosis of multiple myeloma]. / Doporucení pro casné rozpoznání postizení skeletu maligním procesem a pro casnou diagnostiku mnohocetného myelomu.

The number of newly diagnosed cases of multiple myeloma in the Czech Republic is about 3-4 per 100 000 persons per year. In the higher age groups, the incidence increases. Multiple myeloma is an illness that reacts well to treatment which can result in periods of remission lasting for years. Some of the patients are even able to return to work. A pre-requisite for successful treatment is early diagnosis and this is usually in the hands of first line physicians. This is the reason why the Czech Myeloma Group, in conjunction with neurologists, orthopedicians and radio diagnosticians has issued the following recommendations for first line physicians containing a more detailed description of the symptoms and the diagnostic pitfalls of the disease. This disease reminds a chameleon for the variety of its symptoms. For the sake of clarification, we shall divide multiple myeloma symptoms into five points, each of which is reason enough to warrant an examination to confirm or rule out a malignant cause of health problems (a negative result does not automatically mean exclusion). If any of the recommended examinations results positive, the diagnostic process must be continued, in which case a general practitioner refers the patient to a specialist health centre. Observing these recommendations should minimize the number of cases of late diagnosis. 1. Bone destruction symptoms. - Unexplained backache for more than one month in any part of spine even without nerve root irritability or without pain in other part of skeleton (ribs, hips, or long bones). - Pain at the beginning of myeloma disease is very similar to benigne common discopathy, however the intensity of backache is decreasing within one months in benigne disease. In the case of malignant process the intensity of bone pain is steadily increasing. - Immediate imaging and laboratory investigation are indicated by resting and night pain in spinal column or in any part of skeleton. - Backache with the sign of spinal cord or nerve compression should be sent for immediate X Ray, and focussed CT/MRI followed by acute surgery if needed. - Osteoporosis especially in men and premenopausal women. 2. Features of changed immunity or bone marrow function. Persistent and recurrent infection, typical is normochromic anaemia, with leucopenia and trombocytopenia. 3. Raised erythrocyte sedimentation rate even increase concentration of total plasma protein. 4. Impaired renal function. Increased level of creatinin or proteinuria, nephrotic syndrome with bilateral legs oedema. 5. Hypercalcemia with typical clinical symptoms (polyuria with dehydratation, constipation, nausea, low level conscience, coma). Every one from these points has to be reason for general medical doctor to start battery of tests: -X-ray of bones focused to painful area (mandatory before physiotherapy, local anaesthesia or other empiric therapy). If plain X-ray does not elucidate pain and symptoms are lasting more than one month, please consider all circumstances and results from laboratory investigation. This patient needs referral to the centre with MRI/CT facilities (CT or MRI is necessary investigation in case of nerve root or spine compression). -Investigation of erythrocyte sedimantion rate (high level of sedimentation of erythrocyte can indicate multiple myeloma). -Full blood count. -Basic biochemical investigation serum and urine: serum urea, creatinin, ionts including calcium, total protein, and albumin CRP (high concentration of total protein indicates myeloma, low level of albumin indicates general pathological process, similary increased concentration of fibrinogen, impaired renal function indicates myeloma kidney, however hypercalcemia is typical for highly aggressive myeloma). -Quantitative screening for IgG, IgM and IgA in serum (isolated raised level one of immunoglobulin with decreased level of the others indicates myeloma). -Common electrophoresis of serum is able to detect monoclonal immunoglobulin level at few gramm concentration. If all the laboratory investigation are in normal level the possibility that the current problems are multiple myeloma origine is smaller, but it does not exclude one of rare variant--non secretory myeloma (undifferentiated plasmocyt lost characteristic feature to produce monoclonal immunoglobulin). If any of tests indicate the possibility of myeloma, patient require urgent specialist referral to department with possibility to make diagnosis of malignant myeloma.

Depression of osteoblastic activity in immobilized limbs of suckling rats.

Recently we characterized the immobilization-related osteopenia in adult rats and showed that it is caused by increased bone resorption and decreased bone formation (Weinreb et al. 1989 Bone 10:187). To assess the effect of age on disuse osteopenia, this study investigated the effects of immobilization on bone turnover in very young, suckling rats. The 15-day-old rats underwent unilateral hind limb immobilization by sciatic neurectomy; the contralateral limb was left intact and served as control. Experimental or sham-operated animals were killed after 0, 2, 4, or 12 days postsurgery. Dry, fat-free weight and ash weight were determined in both femora, and both tibiae were subjected to static and dynamic histomorphometry. Immobilization caused a progressive deficit in bone mass in the immobilized limb compared to the contralateral intact limb but did not affect femoral longitudinal growth. The total mineral content in the immobilized femora was 13.6% less than that in the intact limb by day 12. Concomitantly, tibial cancellous bone area and perimeter declined in the immobilized limb by 37.3 and 32.2%, respectively. This reduction in bone mass in the tibiae of immobilized limbs was associated with increased bone resorption, expressed as osteoclast perimeter, number of osteoclasts per mm surface, and number of osteoclasts per mm2 tissue area. Bone formation was reduced as a result of impaired osteoblast activity as evidenced by (1) decreased endocortical and trabecular mineral apposition rate; (2) reduced trabecular mineral formation rate; (3) decreased percentage of ash of the femoral dry weight; and (4) increased volume of unmineralized osteoid in the tibial metaphysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Bone marrow from mechanically unloaded rat bones expresses reduced osteogenic capacity in vitro.

Bone formation during mechanical unloading is reduced, mainly as a result of osteoblastic hypofunction. At the same time, the total number of osteoblasts per long bone is also markedly reduced. We tested the hypothesis that the number of osteogenic precursors present in the bone marrow stroma was concomitantly diminished by using an in vitro cell culture system in which femoral adherent bone marrow cells differentiate into active osteoblasts and produce bone-like nodules. Hindlimbs of 32-day-old male rats were either immobilized (unloaded) by sciatic neurectomy (immo) or sham operated (sham) and animals were killed after 11 days. Femora were either ashed to determine bone mass or used to generate bone marrow cultures. Adherent marrow cells were cultured in the presence of ascorbic acid, beta-glycerophosphate, and dexamethasone. Bone mass was significantly reduced in unloaded femora (by 16%) and tibiae (by 18%). The number of adherent cells (determined on day 6) was reduced by 50% in the immo group. Reduced cell number did not result from slower proliferation in culture since [3H]thymidine incorporation on days 4 and 6 was similar in the two groups. The osteogenic potential in vitro of marrow from unloaded bones was diminished compared with that from loaded ones as evidenced by (1) lower alkaline phosphatase (ALP) activity per mg protein (by 25-40%, examined on days 6 and 12), and (2) reduced nodule formation (by 70%, expressed as percentage of the dish area stained with Alizarin Red S on day 21). None of these changes occurred in the contralateral limb of operated (immobilized) animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Different pattern of alkaline phosphatase, osteopontin, and osteocalcin expression in developing rat bone visualized by in situ hybridization.

Alkaline phosphatase (AP), osteopontin (OP), and osteocalcin (OC) are expressed during osteoblastic differentiation. However, previous studies suggested differences in the timing and possibly the site of their expression. In this study we used in situ hybridization to follow the distribution of these osteoblastic markers during bone development. Frozen sections of neonatal rat long bones and calvariae were hybridized with 35S-labeled RNA probes complementary to the AP, OP, and OC mRNAs. Controls included sections hybridized with the sense (mRNA) probes or pretreated with RNase. Positive cells were identified in all areas of bone formation of the long bones and calvariae. Based on quantitative silver grain distribution and density, high levels of OP expression were present only in osteoblasts in close proximity to bone (one to two cell layers). OC expression, apparently at lower levels than OP, was also localized to osteoblasts in contact with bone. In contrast AP, which was expressed at lower levels than OP, was present in a large number of cells, including preosteoblasts that were many layers removed from the bone-forming surface. These findings are consistent with the asynchronous expression of phenotypically related genes and suggest that AP is an earlier differentiation marker than OP and OC during the formation of endochondral and membranous bone.

Expression of alpha v and beta 3 integrin subunits in rat osteoclasts in situ.

Recent findings suggest that the vitronectin receptor, a member of the integrin family, plays an important role in the attachment of osteoclasts to bone matrix. We report here the localization by in situ hybridization of the mRNA for the alpha and beta subunits of the vitronectin receptor in rat bone sections. To generate the rat-specific RNA probes used in this study, we cloned cDNA fragments of integrin chains alpha v, beta 3, and beta 5 by the polymerase chain reaction from rat cDNA. These fragments share 86-91% homology with the respective human sequences. In situ hybridization localized the alpha v and beta 3 mRNAs to regions undergoing extensive bone resorption. The histologic appearance and prestaining of bone sections for tartrate-resistant acid phosphatase (TRAP) indicated the presence of these mRNAs in osteoclasts. These observations support immunohistochemical findings that osteoclasts express high levels of the vitronectin receptor, confirm the identity of this receptor as integrin alpha v/beta 3, and suggest that osteoclasts may actively synthesize these molecules.

Aminohydroxybutane bisphosphonate inhibits bone loss due to immobilization in rats.

The purpose of this study was to document the effects of aminobutane bisphosphonate (AHBuP) on bone remodeling during immobilization in rats. Male Sprague-Dawley rats underwent unilateral sciatic neurectomy after receiving two daily subcutaneous injections of 0, 0.01, 0.10, or 1.0 mg P per kg AHBuP. Rats were sacrificed at 24 h or 10 or 20 days postimmobilization. Femora were ashed and tibiae were prepared for histomorphometric analysis. AHBuP was effective in inhibiting bone loss due to immobilization in a dose-dependent manner. The percentage loss of femoral ash weight due to immobilization decreased in a dose-dependent manner. In vehicle-treated rats, there was a significant decrease in trabecular bone volume (TBV) in the immobilized tibiae compared to the normal tibiae; in AHBuP-treated rats there was a dose-dependent increase in TBV both in the immobilized and control tibiae. The osteoid surface extent was decreased in AHBuP-treated rats in a dose-dependent manner. The mineral apposition rate was altered only in the intact leg of rats treated with 0.1 and 1.0 mg P AHBuP per kg compared to vehicle treated. Osteoclast number per mm was reduced by AHBuP treatment. In conclusion, aminohydroxybutane bisphosphonate effectively prevented the bone loss due to immobilization in this system.

Differential expression of insulin-like growth factor-I (IGF-I) and IGF-II messenger ribonucleic acid in growing rat bone.

Insulin-like growth factors (IGF-I and IGF-II) are among the most abundant growth factors found in bone. Although their local production has been implicated in growth and development, localization of the cells that express these proteins is not well documented. We have studied, by in situ hybridization, the temporal and spatial expression of IGF-I and IGF-II mRNA in rat long bones at different stages of postnatal bone development. In 2-day-old rats, IGF-II was highly expressed in cartilage and in the mesodermal structures that surround the bone. At later stages of bone development, the IGF-II signal decreased in intensity, but could still be detected in the growth plate of tibial bones at 3 and 5 weeks. At this stage, the IGF-II signal in the epiphyseal growth plate was unevenly distributed and was stronger in the periphery than in the center, where it was mainly concentrated in the germinal layer and in some, but not all, cartilage columns. IGF-I, on the other hand, was only faintly detected in the periosteum at the early cartilaginous stage of bone development. At later stages, IGF-I was strongly associated with regions of ossification in the trabecular bone of the metaphysis and epiphysis and along the endosteal and periosteal surfaces. Surprisingly, we did not detect at any time IGF-I mRNA in chondrocytes of the epiphyseal growth plate. These results suggest that in the rat, IGF-II plays a role in early development of bone and in the longitudinal growth of the epiphyseal plate. IGF-I is more closely associated with the osteogenic regions and does not replace the declining levels of IGF-II in the growth plate.

A selective EP4 receptor antagonist abrogates the stimulation of osteoblast recruitment from bone marrow stromal cells by prostaglandin E2 in vivo and in vitro.

Recent evidence indicates that systemic administration of PGE2 increases bone formation and bone mass via activation of the EP4 receptor. Previously, we demonstrated that osteoblastic recruitment from rat bone marrow stromal cells (BMSC) is a major mechanism for the anabolic effect of PGE2. In this study, we used a selective EP4 antagonist to test if the stimulation of osteoblast differentiation from rat BMSC in vitro and in vivo involves the EP4 receptor. In vitro, PGE2 (100 nM) increased nodule formation and alkaline phosphatase (ALP) activity in cultures of rat BMSC 1.5- to 2-fold. These effects were abolished by the EP4 antagonist at 10(-6) M but not 10(-9) M. Furthermore, PGE2 increased the number of surviving adherent BMSC by approximately 225% and the EP4 antagonist prevented this effect as well. The antagonist had no effect on basal levels of nodule formation and adherent cell number. In vivo, daily systemic administration of PGE2 at 6 mg/kg for 2 weeks increased cancellous bone area (by approximately 50%) and increased nodule formation (measured as mineralized area) in ex vivo stromal cultures by approximately 50%. Pre-administration of the EP4 antagonist at 10 mg/kg abrogated both the increase in bone mass as well as the increase in nodule formation. These data indicate that PGE2 stimulates osteoblastic commitment of BMSC via activation of the EP4 receptor.

Systemic administration of an anabolic dose of PGE2 in young rats increases the osteogenic capacity of bone marrow.

Prostaglandin E2 (PGE2) possesses significant anabolic properties when administered systemically (i.e., it increases bone formation and, consequently, bone mass). We recently characterized the effects of a 3 week administration of 6 mg/kg PGE2 into young rats and showed it increases cortical and cancellous bone mass and mechanical strength in long bones and bone density in the calvaria. We also found that a single dose of PGE2 induces the expression of early-response genes (c-fos, c-jun, and egr-1) in bone marrow cells within these two types of bone. These observations, together with findings by others of new cancellous bone formation in PGE2-treated animals, suggested that recruitment of osteoblasts from their precursors is a major mechanism of the anabolic effect of PGE2. To test this hypothesis directly, we injected PGE2 (6 mg/kg) or vehicle into 4-week-old rats for 2 weeks and then assessed the osteogenic potential of bone marrow in an ex vivo culture system. Primary and first-passage bone marrow cultures were established in the presence of beta-glycerophosphate, ascorbate, and dexamethasone, and osteogenic differentiation was measured by bone nodule formation and alkaline phosphatase activity. This regimen increased bone mass expressed as femoral ash weight by 4.7% and tibial cancellous bone area by 38.3%. Nodule formation at 21 days was increased in both primary and first-passage cultures from PGE2-treated rats despite seeding of the same number of marrow cells. Alkaline phosphatase activity was elevated in both primary and first-passage cultures from PGE2-treated rats beginning 6-10 days after culture initiation. Cell proliferation was only slightly elevated in cultures from PGE2-treated rats. These data strongly suggest that in vivo administration of PGE2 induces the proliferation or differentiation of osteoprogenitor cells in bone marrow, and this effect takes a major part in its anabolic effect in vivo.

Systemic administration of an anabolic dose of prostaglandin E2 induces early-response genes in rat bones.

Systemic administration of prostaglandins of the E series (PGEs) has an anabolic effect in bone. A large part of this osteogenic effect is due to recruitment of osteoblasts from their precursors. However, the immediate events initiated by the administration of an anabolic dose of PGEs or their target cells within bone tissue are not known. In this study we used Northern analysis to explore the induction of early-response genes in bone tissue following a single injection of an anabolic dose of PGE2 (6 mg/kg) and in situ hybridization to localize the responding cells. The mRNA levels of c-fos, c-jun, junB and early growth response gene-1 were markedly elevated in the tibial metaphysis as early as 15 min postinjection and returned to basal level by 180-300 min. The induction of c-fos was the earliest (significant at 15 min) and the greatest (sixfold at 60 min) and that of the other genes was smaller. Early-response gene expression was induced in the calvaria as well. Numerous cells in bone marrow (both in the tibia and calvaria) expressed high levels of c-fos in response to PGE2. In the tibia, these cells were localized in the secondary spongiosa and diaphysis and were absent from the primary spongiosa. Many, but not all, expressing cells were in relative proximity to cancellous or endosteal surfaces. In the calvaria, these cells were found in the marrow "windows" within the bony plate. Mature osteoblasts and osteoclasts were negative. Based on many reports of the stimulation of cancellous bone formation in tibiae of similar animals by PGE2 and the increased bone formation we found in the calvarial marrow spaces, the best candidate for these cells is a bone marrow-resident osteoblast precursor. The induction of early-response genes may thus be the first step in a chain of events which leads to the anabolic effect of PGE2 in vivo.

Osteopenia in the immobilized rat hind limb is associated with increased bone resorption and decreased bone formation.

The lack of mechanical function (disuse) caused by paralysis, immobilization or weightlessness, leads to osteopenia. This study examines the contribution of bone resorption and bone formation to osteopenia of disuse, during six weeks of limb-immobilization in the growing rat. Male Sprague-Dawley rats of approximately 200 g underwent unilateral hind-limb immobilization by either tenotomy at the knee joint or sciatic neurectomy, while control rats were sham-operated. Animals were sacrificed at 30 and 72 hours, 10, 26 and 42 d postsurgery. Femora were ashed to determine the total mineral content and histomorphometric parameters, static and dynamic, were measured in the secondary spongiosa of the proximal tibial metaphysis. No difference was found in the femoral length of the immobilized legs. Bone loss in the immobilized leg versus the nonimmobilized one, at 10, 26 and 42 d post-surgery was 18.0, 14.0 and 11.2% of femoral mineral content in the tenotomy group, respectively, and 12.4, 16.1 and 15.7% in the neurectomy group. Loss of metaphyseal trabecular bone volume at 10, 26 and 42 days amounted to 67.8, 49.3 and 52.9% in the tenotomy group, respectively, and 70.5, 59.0 and 72.9% in the neurectomy group. The bone loss was caused by: (a) A rapid surge in bone resorption, reflected in a significant increase in the number of osteoclasts per mm bone surface at 30 and 72 h and in the osteoclast surface at 72 h but not at later times, and (b) a sustained decrease in bone mineral apposition rate and bone formation rate (osteoblast-referent) throughout the 42-day immobilization period suggesting osteoblastic hypofunction (reduced activity).(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of the distribution of 3H-alendronate and 3H-etidronate in rat and mouse bones.

Alendronate and etidronate are bisphosphonates used clinically to treat diseases associated with increased bone resorption. Etidronate is less potent and was reported to cause osteomalacia. This study examines if differences in distribution of alendronate and etidronate in the skeleton can explain differences in efficacy and in effects on mineralization between the two drugs. Eight-day old rat pups were injected s.c. with 3H-alendronate or 3H-etidronate both at either 1.3 mumol/kg or at their respective pharmacological effective doses in the growing rat of 0.12 mumol/kg for alendronate and 72.8 mumol/kg for etidronate. Twelve hours after administration at 1.3 mumol/kg both drugs showed a three- to fourfold higher localization on osteoclast vs. osteoblast surface. At the pharmacologically effective doses, 3H-alendronate labeled eightfold more osteoclast surface than osteoblast surface. In contrast, 3H-etidronate labeled approximately equal fractions of osteoclast and osteoblast surface. When similar doses of 3H-etidronate and 3H-alendronate (0.24 mumol/kg 3H-etidronate vs. 0.20 mumol/kg 3H-alendronate; 1.5 mumol/kg 3H-etidronate vs. 1.2 mumol/kg 3H-alendronate; and 14.6 mumol/kg 3H-etidronate vs. 12.0 mumol/kg 3 H-alendronate) were injected intravenously into adult mice at similar specific activities, 3H-etidronate labeled 1.5-2.5 times more osteoclast surface than 3-H-alendronate, but 3 to 15 times more osteoblast surface. Consequently, the ratio between the fraction of labeled osteoclast surface and the fraction of labeled osteoblast surface ranged for 3H-alendronate from 9 to 24, whereas for 3H-etidronate the range was from 4 to 7, due to more extensive labeling of osteoblast surface by 3H-etidronate. In a third experiment, we confirmed in adult mice the previous observation made in rat pups that normal bone formation occurs over alendronate-covered bone surfaces, and found that it occurred over etidronate-covered surfaces as well. Forty nine days after s.c. administration of alendronate at 0.12 mumol/kg or etidronate at 1.3 mumol/kg or 55.3 mumol/kg into adult mice bone formed over drug label. The distance from incorporated label to bone surface for both drugs (12.7 microns for alendronate and 8.7 and 9.2 microns for etidronate) was similar to wall width (defined by cement line) in controls (10.6 microns). In conclusion, alendronate, especially at pharmacologically active doses, shows higher uptake on resorption vs. formation surfaces than etidronate. The extent of bone formation on surfaces containing alendronate or etidronate is similar and is comparable to the "wall width" in controls.