Retinoic acid increases the effect of bone morphogenetic protein type 2 on osteogenic differentiation of human adipose-derived stem cells.

BACKGROUND: Bone morphogenetic protein type 2 (BMP-2) and retinoic acid (RA) are osteoinductive factors that stimulate endogenous mechanisms of bone repair which can be applied on management of osseous defects in oral and maxillofacial fields. OBJECTIVE: Considering the different results of RA on osteogenesis and its possible use to substitute/potency BMP-2 effects, this study evaluated the outcomes of BMP-2, RA, and BMP-2+RA treatments on in vitro osteogenic differentiation of human adipose-derived stem cells (ASCs) and the signaling pathway(s) involved. MATERIAL AND METHODS: ASCs were treated every other day with basic osteogenic medium (OM) alone or supplemented with BMP-2, RA, or BMP-2+RA. Alkaline phosphatase (ALP) activity was determined using the r-nitrophenol method. Extracellular matrix mineralization was evaluated using von Kossa staining and calcium quantification. Expression of osteonectin and osteocalcin mRNA were determined using qPCR. Smad1, Smad4, phosphorylated Smad1/5/8, BMP-4, and BMP-7 proteins expressions were analyzed using western blotting. Signaling pathway was evaluated using the IPA® software. RESULTS: RA promoted the highest ALP activity at days 7, 14, 21, and 28, in comparison to BMP-2 and BMP-2+RA. BMP-2+RA best stimulated phosphorylated Smad1/5/8 protein expression at day 7 and Smad4 expression at days 7, 14, 21, and 28. Osteocalcin and osteonectin mRNA expressions were best stimulated by BMP-2+RA at day 7. Matrix mineralization was most improved by BMP-2+RA at days 12 and 32. Additionally, BMP-2+RA promoted the highest BMP signaling pathway activation at days 7 and 14, and demonstrated more activation of differentiation of bone-forming cells than OM alone. CONCLUSIONS: In summary, RA increased the effect of BMP-2 on osteogenic differentiation of human ASCs.

Retinoic acid increases the effect of bone morphogenetic protein type 2 on osteogenic differentiation of human adipose-derived stem cells

Abstract Bone morphogenetic protein type 2 (BMP-2) and retinoic acid (RA) are osteoinductive factors that stimulate endogenous mechanisms of bone repair which can be applied on management of osseous defects in oral and maxillofacial fields. Objective Considering the different results of RA on osteogenesis and its possible use to substitute/potency BMP-2 effects, this study evaluated the outcomes of BMP-2, RA, and BMP-2+RA treatments on in vitro osteogenic differentiation of human adipose-derived stem cells (ASCs) and the signaling pathway(s) involved. Material and Methods ASCs were treated every other day with basic osteogenic medium (OM) alone or supplemented with BMP-2, RA, or BMP-2+RA. Alkaline phosphatase (ALP) activity was determined using the r-nitrophenol method. Extracellular matrix mineralization was evaluated using von Kossa staining and calcium quantification. Expression of osteonectin and osteocalcin mRNA were determined using qPCR. Smad1, Smad4, phosphorylated Smad1/5/8, BMP-4, and BMP-7 proteins expressions were analyzed using western blotting. Signaling pathway was evaluated using the IPA® software. Results RA promoted the highest ALP activity at days 7, 14, 21, and 28, in comparison to BMP-2 and BMP-2+RA. BMP-2+RA best stimulated phosphorylated Smad1/5/8 protein expression at day 7 and Smad4 expression at days 7, 14, 21, and 28. Osteocalcin and osteonectin mRNA expressions were best stimulated by BMP-2+RA at day 7. Matrix mineralization was most improved by BMP-2+RA at days 12 and 32. Additionally, BMP-2+RA promoted the highest BMP signaling pathway activation at days 7 and 14, and demonstrated more activation of differentiation of bone-forming cells than OM alone. Conclusions In summary, RA increased the effect of BMP-2 on osteogenic differentiation of human ASCs.

Stanozolol promotes osteogenic gene expression and apposition of bone mineral in vitro

Abstract Stanozolol (ST) is a synthetic androgen with high anabolic potential. Although it is known that androgens play a positive role in bone metabolism, ST action on bone cells has not been sufficiently tested to support its clinical use for bone augmentation procedures. Objective: This study aimed to assess the effects of ST on osteogenic activity and gene expression in SaOS-2 cells. Material and Methods: SaOS-2 deposition of mineralizing matrix in response to increasing doses of ST (0-1000 nM) was evaluated through Alizarin Red S and Calcein Green staining techniques at 6, 12 and 24 days. Gene expression of runt-related transcription factor 2 (RUNX2), vitamin D receptor (VDR), osteopontin (SPP1) and osteonectin (ON) was analyzed by RT-PCR. Results: ST significantly influenced SaOS-2 osteogenic activity: stainings showed the presence of rounded calcified nodules, which increased both in number and in size over time and depending on ST dose. RT-PCR highlighted ST modulation of genes related to osteogenic differentiation. Conclusions: This study provided encouraging results, showing ST promoted the osteogenic commitment of SaOS-2 cells. Further studies are required to validate these data in primary osteoblasts and to investigate ST molecular pathway of action.

Stanozolol promotes osteogenic gene expression and apposition of bone mineral in vitro.

Stanozolol (ST) is a synthetic androgen with high anabolic potential. Although it is known that androgens play a positive role in bone metabolism, ST action on bone cells has not been sufficiently tested to support its clinical use for bone augmentation procedures. OBJECTIVE: This study aimed to assess the effects of ST on osteogenic activity and gene expression in SaOS-2 cells. MATERIAL AND METHODS: SaOS-2 deposition of mineralizing matrix in response to increasing doses of ST (0-1000 nM) was evaluated through Alizarin Red S and Calcein Green staining techniques at 6, 12 and 24 days. Gene expression of runt-related transcription factor 2 (RUNX2), vitamin D receptor (VDR), osteopontin (SPP1) and osteonectin (ON) was analyzed by RT-PCR. RESULTS: ST significantly influenced SaOS-2 osteogenic activity: stainings showed the presence of rounded calcified nodules, which increased both in number and in size over time and depending on ST dose. RT-PCR highlighted ST modulation of genes related to osteogenic differentiation. CONCLUSIONS: This study provided encouraging results, showing ST promoted the osteogenic commitment of SaOS-2 cells. Further studies are required to validate these data in primary osteoblasts and to investigate ST molecular pathway of action.

Immunohistochemical Expression of TGF-ß1 and Osteonectin in engineered and Ca(OH)2-repaired human pulp tissues.

The aim of the present study was to evaluate the expression of transforming growth factor-ß1 (TGF-ß1) and osteonectin (ON) in pulp-like tissues developed by tissue engineering and to compare it with the expression of these proteins in pulps treated with Ca(OH)2 therapy. Tooth slices were obtained from non-carious human third molars under sterile procedures. The residual periodontal and pulp soft tissues were removed. Empty pulp spaces of the tooth slice were filled with sodium chloride particles (250-425 µm). PLLA solubilized in 5% chloroform was applied over the salt particles. The tooth slice/scaffold (TS/S) set was stored overnight and then rinsed thoroughly to wash out the salt. Scaffolds were previously sterilized with ethanol (100-70°) and washed with phosphate-buffered saline (PBS). TS/S was treated with 10% EDTA and seeded with dental pulp stem cells (DPSC). Then, TS/S was implanted into the dorsum of immunodeficient mice for 28 days. Human third molars previously treated with Ca(OH)2 for 90 days were also evaluated. Samples were prepared and submitted to histological and immunohistochemical (with anti-TGF-ß1, 1:100 and anti-ON, 1:350) analyses. After 28 days, TS/S showed morphological characteristics similar to those observed in dental pulp treated with Ca(OH)2. Ca(OH)2-treated pulps showed the usual repaired pulp characteristics. In TS/S, newly formed tissues and pre-dentin was colored, which elucidated the expression of TGF-ß1 and ON. Immunohistochemistry staining of Ca(OH)2-treated pulps showed the same expression patterns. The extracellular matrix displayed a fibrillar pattern under both conditions. Regenerative events in the pulp seem to follow a similar pattern of TGF-ß1 and ON expression as the repair processes.

Effects of melatonin on tibia bone defects in rats / Efectos de la melatonina sobre defectos óseos en la tibia de ratas

The aim of this study was to evaluate the effects of melatonin healing in a tibial bone defect model in rats by means of histopathological and immunohistochemistry analysis. Twenty one male Wistar albino rats were used in this study. In each animal, bone defects (6 mm length ) were created in the tibias. The animals were divided into three groups. In group 1 control group (rats which tibial defects). Group 2 melatonin (10 mg/kg) + 14 days in the tibial defect group) was administered intraperitoneally to rats. Group 3 melatonin (10 mg/kg) + 28 days in the tibial defect group) was administered intraperitoneally to rats. Histopathological analysis of samples was performed to evaluate the process of osteoblastic activity, matrix formation, trabecular bone formation and myeloid tissue in bone defects. Immunohistochemical and immunoblot analysis demonstrated non-collagenous proteins (osteopontin and osteonectin) differences in tibial bone defects. The expression of osteopontin on tibia was increased by 14 days melatonin treatment. The expression of osteonectin on tibia was dramatically increased by 14 days melatonin treatment.

El objetivo fue evaluar por medio de análisis histopatológico e inmunohistoquímico los efectos cicatrizantes de la melatonina en un modelo de defecto óseo tibial en ratas. Se utilizaron 21 ratas albinas Wistar macho. En cada animal, se crearon defectos óseos en las tibias de 6 mm de longitud. Los animales se dividieron en tres grupos. El Grupo 1 correspondió al grupo control (defectos tibiales sin tratamiento). Al Grupo 2 se administró melatonina por vía intraperitoneal (10 mg/kg) 14 días posteriores al defecto tibial. Al Grupo 3 se administró melatonina por vía intraperitoneal (10 mg/kg) 28 días posteriores al defecto tibial. Se realizó un análisis histopatológico para evaluar los procesos de actividad osteoblástica, formación de matriz, formación de hueso trabecular y tejido mieloide en los defectos óseos. Los análisis inmunohistoquímicos y de inmunotransferencia mostraron diferencias de proteínas no colágenas (osteopontina y osteonectina). La expresión de osteopontina en defectos óseos tibiales se incrementó en el Grupo 2. La expresión de osteonectina en la tibia se incrementó fuertemente bajo el tratamiento con melatonina por 14 días.

Immunohistochemical expression of TGF-ß1 and osteonectin in engineered and Ca(OH)2-repaired human pulp tissues

Abstract The aim of the present study was to evaluate the expression of transforming growth factor-β1 (TGF-β1) and osteonectin (ON) in pulp-like tissues developed by tissue engineering and to compare it with the expression of these proteins in pulps treated with Ca(OH)2 therapy. Tooth slices were obtained from non-carious human third molars under sterile procedures. The residual periodontal and pulp soft tissues were removed. Empty pulp spaces of the tooth slice were filled with sodium chloride particles (250-425 µm). PLLA solubilized in 5% chloroform was applied over the salt particles. The tooth slice/scaffold (TS/S) set was stored overnight and then rinsed thoroughly to wash out the salt. Scaffolds were previously sterilized with ethanol (100-70°) and washed with phosphate-buffered saline (PBS). TS/S was treated with 10% EDTA and seeded with dental pulp stem cells (DPSC). Then, TS/S was implanted into the dorsum of immunodeficient mice for 28 days. Human third molars previously treated with Ca(OH)2 for 90 days were also evaluated. Samples were prepared and submitted to histological and immunohistochemical (with anti-TGF-β1, 1:100 and anti-ON, 1:350) analyses. After 28 days, TS/S showed morphological characteristics similar to those observed in dental pulp treated with Ca(OH)2. Ca(OH)2-treated pulps showed the usual repaired pulp characteristics. In TS/S, newly formed tissues and pre-dentin was colored, which elucidated the expression of TGF-β1 and ON. Immunohistochemistry staining of Ca(OH)2-treated pulps showed the same expression patterns. The extracellular matrix displayed a fibrillar pattern under both conditions. Regenerative events in the pulp seem to follow a similar pattern of TGF-β1 and ON expression as the repair processes.

TGF-ß2-mediated ocular hypertension is attenuated in SPARC-null mice.

PURPOSE: Transforming growth factor-ß2 (TGF-ß2) has been implicated in the pathogenesis of primary open-angle glaucoma through extracellular matrix (ECM) alteration among various mechanisms. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein that regulates ECM within the trabecular meshwork (TM), and is highly upregulated by TGF-ß2. We hypothesized that, in vivo, SPARC is a critical regulatory node in TGF-ß2-mediated ocular hypertension. METHODS: Empty (Ad.empty) or TGF-ß2-containing adenovirus (Ad.TGF-ß2) was injected intravitreally into C57BL6-SV129 WT and SPARC-null mice. An initial study was performed to identify a stable period for IOP measurement under isoflurane. The IOP was measured before injection and every other day for two weeks using rebound tonometry. Additional mice were euthanized at peak IOP for immunohistochemistry. RESULTS: The IOP was stable under isoflurane during minutes 5 to 8. The IOP was significantly elevated in Ad.TGF-ß2-injected (n = 8) versus Ad.empty-injected WT (n = 8) mice and contralateral uninjected eyes during days 4 to 11 (P < 0.03). The IOPs were not significantly elevated in Ad.TGF-ß2-injected versus Ad.empty-injected SPARC-null mice. However, on day 8, the IOP of Ad.TGF-ß2-injected SPARC-null eyes was elevated compared to that of contralateral uninjected eyes (P = 0.0385). Immunohistochemistry demonstrated that TGF-ß2 stimulated increases in collagen IV, fibronectin, plasminogen activator inhibitor-1 (PAI-1), connective tissue growth factor (CTGF), and SPARC in WT mice, but only PAI-1 and CTGF in SPARC-null mice (P < 0.05). CONCLUSIONS: SPARC is essential to the regulation of TGF-ß2-mediated ocular hypertension. Deletion of SPARC significantly attenuates the effects of TGF-ß2 by restricting collagen IV and fibronectin expression. These data provide further evidence that SPARC may have an important role in IOP regulation and possibly glaucoma pathogenesis.

Differential influence of fluoride concentration on the synthesis of bone matrix glycoproteins within mineralizing bone cells in vitro.

OBJECTIVE: This study investigated the influence of fluoride levels on the temporal synthesis of bone-associated glycoproteins, which have been assigned prominent roles in regulating crystal growth, size and shape during the mineralization process. MATERIALS AND METHODS: Bone marrow stromal cells were isolated from male Wistar rats and cultured under mineralizing conditions, supplemented with 0 M, 10(-7) M or 10(-5) M sodium fluoride. The presence of bone-associated glycoproteins was examined 2-13 days post-reseeding by immunocytochemical localization. Results: All bone-associated glycoproteins increased in 10(-7) M fluoride, compared to untreated controls, particularly at days 6 and 13 in culture. Conversely, higher 10(-5) M fluoride concentrations decreased glycoprotein levels, compared to controls. CONCLUSIONS: Results highlight a differential effect of fluoride concentration on glycoprotein synthesis by osteoblasts.

Effect of Emdogain enamel matrix derivative and BMP-2 on the gene expression and mineralized nodule formation of alveolar bone proper-derived stem/progenitor cells.

The objective of this study was to evaluate the effect of Emdogain (Enamel Matrix Derivative, EMD) and Bone Morphogenetic Protein-2 (BMP-2), either solely or in combination, on the gene expression and mineralized nodule formation of alveolar bone proper-derived stem/progenitor cells. Stem/progenitor cells were isolated from human alveolar bone proper, magnetically sorted using STRO-1 antibodies, characterized flowcytometrically for their surface markers' expression, and examined for colony formation and multilineage differentiation potential. Subsequently, cells were treated over three weeks with 100 µg/ml Emdogain (EMD-Group), or 100 ng/ml BMP-2 (BMP-Group), or a combination of 100 ng/ml BMP-2 and 100 µg/ml Emdogain (BMP/EMD-Group). Unstimulated stem/progenitor cells (MACS(+)-Group) and osteoblasts (OB-Group) served as controls. Osteogenic gene expression was analyzed using RTq-PCR after 1, 2 and 3 weeks (N = 3/group). Mineralized nodule formation was evaluated by Alizarin-Red staining. BMP and EMD up-regulated the osteogenic gene expression. The BMP Group showed significantly higher expression of Collagen-I, III, and V, Alkaline phosphatase and Osteonectin compared to MACS(+)- and OB-Group (p < 0.05; Two-way ANOVA/Bonferroni) with no mineralized nodule formation. Under in-vitro conditions, Emdogain and BMP-2 up-regulate the osteogenic gene expression of stem/progenitor cells. The combination of BMP-2 and Emdogain showed no additive effect and would not be recommended for a combined clinical stimulation.

Effect of statins with α-tricalcium phosphate on proliferation, differentiation, and mineralization of human dental pulp cells.

INTRODUCTION: The aim of this study was to investigate the combined effect of statin and α-tricalcium phosphate (α-TCP) on odontoblastic differentiation of human dental pulp cells and to compare them with mineral trioxide aggregate (MTA). METHODS: Experimental cements were prepared with TCP containing simvastatin and atorvastatin. Cell proliferation, cell adherence on a dentin disc, alkaline phosphatase (ALP) activity, expression of osteogenic/odontoblastic markers, and mineralization of the human dental pulp cells on experimental cement and MTA were assessed. RESULTS: The cell growth and ALP activity of TCP containing simvastatin-treated cells was greater than MTA-treated cells. The mineralization and messenger RNA expression of markers (ie, dentin sialophosphoprotein, dentin matrix protein 1, bone morphogenetic protein 2, ALP, and osteonectin) of TCP containing simvastatin- and TCP containing atorvastatin-treated cells were comparable with MTA-treated cells. The enhanced cell proliferation and similar level of ALP of TCP-treated cells compared with the control indicate that α-TCP is an effective osteoconductive material. The differentiation effect observed in TCP containing simvastatin- and TCP containing atorvastatin-treated cells is attributed to the effect of statin. CONCLUSIONS: The results suggest that α-TCP can be used for local delivery of statin as a pulp capping material to accelerate reparative dentin formation.

Bone morphogenic protein-2 induces apoptosis and cytotoxicity in periodontal ligament cells.

BACKGROUND: Periodontal ligament (PDL) expresses endogenous growth factors, such as bone morphogenic proteins (BMPs), which facilitate maintenance of tissue homeostasis. Inflammatory conditions, such as chronic periodontitis, could disrupt this homeostasis, and physiologic levels of growth factors may be insufficient to maintain tissue homeostasis. BMPs facilitate periodontal bone regeneration but also are implicated in causing tooth ankylosis and root resorption. The underlying mechanism of tooth ankylosis is unclear. However, there is evidence that BMPs induce apoptosis in progenitor cells. Little is known about BMP-induced cytotoxicity in PDL cells, which contain a population of progenitor cells. The aim of this study is to determine BMP2-induced osteogenic mediators and cytotoxic effects in PDL cells and compare these cells to osteoblasts. METHODS: Human PDL cells and primary osteoblasts were stimulated with doses of 1 to 200 ng/mL BMP2. Expression of alkaline phosphatase (ALP), in vitro mineralization along with osteonectin expression, induction of apoptosis, and cytotoxicity assays were performed. RESULTS: PDL cells and osteoblasts upregulated ALP and in vitro mineralization in a dose-dependent manner with BMP2 stimulation. However, at BMP2 concentrations >10 ng/mL, ALP, in vitro mineralization, and osteonectin were downregulated in PDL cells. Relative to osteoblasts, PDL cells were susceptible to apoptosis and cytotoxicity with 10 times lower concentration of BMP2. CONCLUSIONS: Relative to osteoblasts, PDL cells are susceptible to BMP2-induced cytotoxicity. BMP-induced tooth ankylosis is controversial and is poorly understood. Disruption of PDL homeostasis by BMP-induced apoptosis could play a role in tooth ankylosis.

Proinflammatory cytokines inhibit osteogenic differentiation from stem cells: implications for bone repair during inflammation.

OBJECTIVE: The effects of inflammation on bone development from mesenchymal stem cells (MSC) are unclear due to the difficulty in isolating MSC. The aim of this study was to develop a MSC isolation method and to determine the in vitro effects of interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha) on their osteogenic differentiation. METHODS: Murine MSC were isolated from the limbs of C57/Bl6 mice through collagenase digestion of bone and enriched as the Stem cell antigen (Sca-1)(+) CD31(-) CD45(-) population, using lineage immunodepletion, followed by fluorescence-activated cell sorting (FACS). They were differentiated along the osteoblast linage in the presence or absence of IL-1beta and TNFalpha. Mineralization was measured as was the expression of a number of osteogenic genes by quantitative polymerase chain reaction (PCR). RESULTS: We show that osteogenic differentiation from the MSC population is suppressed by IL-1beta and TNFalpha. In addition to suppression of bone mineralization, both cytokines inhibited the differentiation-associated increases in alkaline phosphatase (ALP) activity and the gene expression for ALP, alpha1(I) procollagen, runt-related transcription factor 2 (Runx2) and osterix. However, only TNFalpha inhibited osteonectin and osteopontin mRNA expression and only IL-1beta reduced cell proliferation. CONCLUSIONS: The convenient isolation technique enables the easy generation of sufficient MSC to permit the molecular analysis of their differentiation. We were thus able to show that the proinflammatory cytokines, IL-1beta and TNFalpha, can compromise bone development from this primary MSC population, although with some significant differences. The potential involvement of specific inflammatory mediators needs to be taken into account if optimal bone repair and presumably that of other tissues are to be achieved with MSC.

Aberrant promoter methylation of SPARC in ovarian cancer.

Epigenetic silencing of tumor suppressor genes is a new focus of investigation in the generation and proliferation of carcinomas. Secreted protein acidic and rich in cysteine (SPARC) is reportedly detrimental to the growth of ovarian cancer cells and has been shown to be epigenetically silenced in several cancers. We hypothesized that SPARC is downregulated in ovarian cancer through aberrant promoter hypermethylation. To that end, we analyzed SPARC expression in ovarian cancer cell lines and investigated the methylation status of the Sparc promoter using methylation-specific polymerase chain reaction. Our results show that SPARC mRNA expression is decreased in three (33%) and absent in four (44%) of the nine ovarian cancer cell lines studied, which correlated with hypermethylation of the Sparc promoter. Treatment with the demethylating agent 5-aza-2'-deoxycytidine rescued SPARC mRNA and protein expression. Addition of exogenous SPARC, as well as ectopic expression by an adenoviral vector, resulted in decreased proliferation of ovarian cancer cell lines. Investigation of primary tumors revealed that the Sparc promoter is methylated in 68% of primary ovarian tumors and that the levels of SPARC protein decrease as the disease progresses from low to high grade. Lastly, de novo methylation of Sparc promoter was shown to be mediated by DNA methyltransferase 3a. These results implicate Sparc promoter methylation as an important factor in the genesis and survival of ovarian carcinomas and provide new insights into the potential use of SPARC as a novel biomarker and/or treatment modality for this disease.

Fibroblast migration after myocardial infarction is regulated by transient SPARC expression.

Secreted protein, acidic, and rich in cysteine (SPARC) is thought to regulate cell matrix interaction during wound repair. We hypothesized that SPARC might promote migration via integrin-dependent mechanisms. The present study was designed to clarify the contribution of SPARC in the wound healing process after myocardial infarction (MI). Adult mice received a specific alpha(v) integrin inhibitor or vehicle through osmotic mini pumps. Mice of each group were either sham-operated or MI was induced. SPARC expression was investigated 2 days, 7 days, and 1 month after the surgical procedure. For migration assays, a modified Boyden chamber assay was used. A transient increase of SPARC levels was observed, starting at day 2 (2.55+/-0.21), day 7 (3.72+/-0.28), and 1 month (1.9+/-0.16) after MI. After 2 months, SPARC expression dropped back to normal levels compared to sham-operated hearts. Immunofluorescence analysis showed an increase of SPARC in the infarcted area 2 days after MI, a strong increase in the scar area 7 days after MI, and only low levels in the scar area 2 months after MI. Integrin alpha(v) inhibition abolished the up-regulation of SPARC. In vitro migration assays demonstrated that fibronectin-stimulated haptotaxis of fibroblasts was modulated by SPARC. This study provides evidence that SPARC is significantly up-regulated in the infarcted region after MI. This up-regulation is dependent on alpha(v) integrins. As SPARC is found to regulate fibroblast migration, it appears to play an important role in the injured myocardium with regard to healing and scar formation.

Mechanisms for the enhancement of fracture healing in rats treated with intermittent low-dose human parathyroid hormone (1-34).

Recent reports have demonstrated that intermittent treatment with parathyroid hormone (1-34) [PTH(1-34)] increases callus formation and mechanical strength in experimental fracture healing. However, little is known about the optimal dose required for enhancement of fracture repair or the molecular mechanisms by which PTH regulates the healing process. In this study, we analyzed the underlying molecular mechanisms by which PTH affects fracture healing and tested the hypothesis that intermittent low-dose treatment with human PTH(1-34) can increase callus formation and mechanical strength. Unilateral femoral fractures were produced and a daily subcutaneous injection of 10 microg/kg of PTH(1-34) was administered during the entire healing period. Control animals were injected with vehicle solution alone. The results showed that on day 28 and day 42 after fracture, bone mineral content (BMC), bone mineral density (BMD), and ultimate load to failure of the calluses were significantly increased in the PTH-treated group compared with controls (day 28, 61, 46, and 32%; day 42, 119, 74, and 55%, respectively). The number of proliferating cell nuclear antigen (PCNA)-positive subperiosteal osteoprogenitor cells was significantly increased in the calluses of the PTH-treated group on day 2, and TRAP+ multinucleated cells were significantly increased in areas of callus cancellous bone on day 7. The levels of expression of type I collagen (COLlA1), osteonectin (ON), ALP, and osteocalcin (OC) mRNA were increased markedly in the PTH-treated group and accompanied by enhanced expression of insulin-like growth factor (IGF)-I mRNA during the early stages of healing (days 4-7). The increased expression of COL1A1, ON, ALP, and OC mRNA continued during the later stages of healing (days 14-21) despite a lack of up-regulation of IGF-I mRNA. These results suggest that treatment of fractures with intermittent low dose PTH(1-34) enhances callus formation by the early stimulation of proliferation and differentiation of osteoprogenitor cells, increases production of bone matrix proteins, and enhances osteoclastogenesis during the phase of callus remodeling. The resultant effect to increase callus mechanical strength supports the concept that clinical investigations on the ability of injectable low-dose PTH(1-34) to enhance fracture healing are indicated.

Effects of parathyroid hormone on bone formation in a rat model for chronic alcohol abuse.

BACKGROUND: Alcoholism is a risk factor for osteoporosis and it is not clear whether the detrimental effects of alcohol on bone are reversible. Parathyroid hormone (PTH) is a potent stimulator of bone matrix synthesis and is being investigated as a therapeutic agent to reverse bone loss. The present investigation was designed to determine the effects of PTH on bone formation in a rat model for chronic alcohol abuse. METHODS AND RESULTS: Alcohol was administered in the diet of female rats (35% caloric intake) for 2 weeks. Human (1-34) PTH (80 microg/kg/day) was administered subcutaneously during the second week of the study. Alcohol resulted in a transient reduction in steady-state mRNA levels for the bone matrix proteins type 1 collagen, osteocalcin, and osteonectin compared with rats that were fed an alcohol-free (control) diet. As expected, alcohol decreased and PTH increased histologic indices of bone formation. Additionally, two-way ANOVA demonstrated that alcohol antagonized PTH-induced bone formation. Despite antagonism, bone formation and mRNA levels for bone matrix proteins in alcohol-fed rats treated with PTH greatly exceeded the values in rats fed the control diet. CONCLUSIONS: The results of this study contribute to a growing body of evidence that alcohol-induced bone loss is primarily due to reduced bone formation. We conclude that alcohol does not prevent the stimulatory effects of PTH on bone formation. This is evidence that the effects of alcohol on the skeleton are reversible. Additionally, the positive effects on bone formation in rats that consumed high concentrations of alcohol suggested that PTH may be useful as an intervention to treat alcohol-induced osteoporosis.

Effect of coronary thrombolysis on the plasma concentration of osteonectin (SPARC, BM40) in patients with acute myocardial infarction.

Osteonectin is a phosphoglycoprotein exclusively located in bone and platelet alpha-granules. Human platelet-derived osteonectin is released into plasma after thrombin-induced activation. Recognizing the unique distribution of the osteonectin pool, we first sought to investigate whether osteonectin could serve as a sensitive marker of platelet activity, and identify patients with acute myocardial infarction (AMI). The second objective was to define the effects of thrombolytic therapy in these patients on the plasma concentrations of osteonectin at prespecified time points following attempted reperfusion. Osteonectin levels by ELISA were determined in AMI patients before thrombolysis and at 3, 6, 12, and 24 hours thereafter and compared with 12 healthy controls. At baseline, soluble osteonectin plasma levels were similar between controls (447. 7+/-20.6 ng/ml) and AMI patients (425.7+/-43.3 ng/mL; p=NS). A significant increase of the soluble osteonectin was observed at 3 hours after thrombolysis (519.4+/-26.9 ng/mL; p=0.03), and was followed by a decrease to baseline levels at 6 hours after attempted reperfusion. Contrary to expectations, the plasma osteonectin level in our pilot study was not a sensitive marker distinguishing patients with AMI. The early peak of soluble osteonectin at 3 hours after thrombolytic therapy is most likely not related to coronary thrombolysis per se but rather to the phasic changes of platelet activity during myocardial ischemia-reperfusion. The unquestionable platelet origin of this protein and the lack of elevated plasma levels of this alpha-granule constituent, challenge the postulate of uniform platelet activation in AMI patients.

Remodelling of cardiac extracellular matrix during beta-adrenergic stimulation: upregulation of SPARC in the myocardium of adult rats.

Our objectives were (i) to evaluate the expression of several genes involved in the remodelling of cardiac extracellular matrix (ECM), with a special interest on SPARC (secreted protein acidic and rich in cysteine) a glycoprotein with anti-adhesive properties, and (ii) to characterise structural changes in the left (LV) and right (RV) ventricles of rats subjected to continuous beta-adrenergic stimulation. The rats were infused for 3 or 7 days with isoproterenol (ISO, 4 mg/kg/day) or vehicle. Hybridisation analysis was done for SPARC, atrial natriuretic peptide (ANP),alpha2 (I) [COL-I] and alpha1 (III) [COL-III] procollagens, TGF-beta1 and TGF-beta3 mRNA content. Interstitial and perivascular collagen deposition in both ventricles was measured after specific staining. The mean cross-sectional area of LV cardiomyocytes was evaluated by quantitative histomorphometry. ISO provoked an increase of LV mass, and a progressive enlargement of cardiomyocytes: their cross-sectional area raised from 205+/-8 micrometer2 in vehicle-treated animals to 247+/-4 and 296+/-9 micrometer2 after 3 or 7 days of ISO infusion, respectively (P<0.001). SPARC messenger abundance increased by more than 50% in LV and RV, a first evidence of its expression in the myocardium of adult rats. Transcripts of ANP, COL-III, TGF-beta1 and TGF-beta3 increased in both ventricles. COL-I transcript increased in LV (75 and 116% on days 3 and 7), but not in RV. In LV, collagen accumulated in the interstitium (2.69+/-0.20v 9. 23+/-0.50% of tissue area for vehicle and ISO 7 days groups, P<0.05) and around coronary arteries (1.04+/-0.11v 4.47+/-0.48% of lumen area for vehicle and ISO 7 days,P<0.05). Cardiac fibrosis was less marked in RV. In conclusion, early expression of SPARC, an anti-adhesive protein, and preferential expression of COL-III, a distensible form of collagen, should increase ECM plasticity and facilitate ventricular remodelling.

Physiology of bone: mineral compartment proteins as candidates for environmental perturbation by lead.

Termine et al. first demonstrated that sequential dissociative extraction and fractionation procedures with protease inhibitors could provide a convenient approach for the study of mineral compartment constituents. The primary extraction regimen used 4 M guanidine HCl to remove most of the protein from the nonmineralized phase of bone. Subsequently, EDTA-guanidine was used to remove the mineral-phase components. These methods discriminate on the basis of physical-chemical association with a mineral phase rather than on the specific gene products of a particular cell. In the present discussion emphasis is directed at a group of divalent cation binding proteins isolated from the mineral compartment of bone. The localization, synthesis, and chemical characteristics of osteonectin, bone sialoproteins I and II, and bone acidic glycoprotein-75 are discussed and offered as possible sites for perturbation by the environment with lead exposure.