Osseointegration of photodynamic active biomaterials for bone regeneration in an animal bone model over a period of 12 months.

OBJECTIVES: Previous efforts led to the development of two different polymeric biomaterials for periodontal regeneration with antibacterial photodynamic surface activity. The present study aimed to investigate osseointegration and bone formation of both materials in an ovine model. METHODS: Both biomaterials: 1) urethane dimethacrylate-based Biomaterial 1 (BioM1) and 2) tri-armed oligoester-urethane methacrylate-based Biomaterial 2 (BioM2) are enriched with beta-tri-calcium phosphate and the photosensitizer meso-tetra(hydroxyphenyl)chlorin (mTHPC). These materials were implanted in non-critical size bone defects in the sheep femur (n = 16) and tibia (n = 8). Empty defects served as controls (n = 16). Polyfluorochrome sequential bone labeling was carried out at baseline and after 3, 6, and 12 months. Animals were sacrificed after 12 months. Bone specimens (n = 40) were fixed and subjected to microtomographic analysis (µCT) for the evaluation of the bone-volume-fraction (BV/TV), trabecular number and trabecular thickness. Subsequently, histological sections were arranged and polyfluorochrome sequential bone labeling was analyzed by confocal laser scanning microscopy (cLSM). RESULTS: cLSM analysis revealed that highest remodeling and bone formation activity occurred during the second half of the study period (6-12 months). Bone formation in the tibia was significantly lower for the control (2.71 ± 1.26%) as compared to BioM1 (6.01 ± 2.99%) and BioM2 (6.45 ± 2.12%); (p = 0.006, p = 0004). Micro-computed tomography revealed a BV/TV volume fraction of 44.72 ± 9.01% in femur defects filled with BioM1 which was significantly higher compared to the control (32.27 ± 7.02%; p = 0.01). Bone architecture (trabecular number, trabecular thickness) did not significantly differ from the self-healed defects. SIGNIFICANCE: Both biomaterials, especially BioM1 showed good osseointegration and bone formation characteristics and can be recommended for further examination in periodontal regeneration studies.

Inhibition of sphingosine 1-phosphate protects mice against chondrocyte catabolism and osteoarthritis.

OBJECTIVE: Cartilage loss observed in osteoarthritis (OA) is prevented when osteoclasts in the subchondral bone are inhibited in mice. Here, we investigated the role of the osteoclast secretome and of the lipid mediator sphingosine 1-phosphate (S1P) in chondrocyte metabolism and OA. MATERIALS AND METHODS: We used SphK1LysMCre and wild type mice to assess the effect of murine osteoclast secretome in chondrocyte metabolism. Gene and protein expressions of matrix metalloproteinase (Mmp) were quantified in chondrocytes and explants by RT-qPCR and Western blots. SphK1LysMCre mice or wild type mice treated with S1P2 receptor inhibitor JTE013 or anti-S1P neutralizing antibody sphingomab are analyzed by OA score and immunohistochemistry. RESULTS: The osteoclast secretome increased the expression of Mmp3 and Mmp13 in murine chondrocytes and cartilage explants and activated the JNK signaling pathway, which led to matrix degradation. JTE013 reversed the osteoclast-mediated chondrocyte catabolism and protected mice against OA, suggesting that osteoclastic S1P contributes to cartilage damage in OA via S1P/S1P2 signaling. The activity of sphingosine kinase 1 (SphK1) increased with osteoclast differentiation, and its expression was enhanced in subchondral bone of mice with OA. The expression of Mmp3 and Mmp13 in chondrocytes was low upon stimulation with the secretome of Sphk1-lacking osteoclasts. Cartilage damage was significantly reduced in SphK1LysMCre mice, but not the synovial inflammation. Finally, intra-articular administration of sphingomab inhibited the cartilage damage and synovial inflammation. CONCLUSIONS: Lack of S1P in myeloid cells and local S1P neutralization alleviates from osteoarthritis in mice. These data identify S1P as a therapeutic target in OA.

Deletion of Menin in craniofacial osteogenic cells in mice elicits development of mandibular ossifying fibroma.

Ossifying fibroma (OF) is a rare benign tumor of the craniofacial bones that can reach considerable and disfiguring dimensions if left untreated. Although the clinicopathological characteristics of OF are well established, the underlying etiology has remained largely unknown. Our work indicates that Men1-a tumor suppressor gene responsible of Multiple endocrine neoplasia type 1-is critical for OF formation and shows that mice with targeted disruption of Men1 in osteoblasts (Men1Runx2Cre) develop multifocal OF in the mandible with a 100% penetrance. Using lineage-tracing analysis, we demonstrate that loss of Men1 arrests stromal osteoprogenitors in OF at the osterix-positive pre-osteoblastic differentiation stage. Analysis of Men1-lacking stromal spindle cells isolated from OF (OF-derived MSCs (OFMSCs)) revealed a downregulation of the cyclin-dependent kinase (CDK) inhibitor Cdkn1a, consistent with an increased proliferation rate. Intriguingly, the re-expression of Men1 in Men1-deficient OFMSCs restored Cdkn1a expression and abrogated cellular proliferation supporting the tumor-suppressive role of Men1 in OF. Although our work presents the first evidence of Men1 in OF development, it further provides the first genetic mouse model of OF that can be used to better understand the molecular pathogenesis of these benign tumors and to potentially develop novel treatment strategies.

Stat5 gene dosage in T cells modulates CD8+ T-cell homeostasis and attenuates contact hypersensitivity response in mice.

BACKGROUND: Contact hypersensitivity assay (CHS) faithfully models human allergies. The Stat5 transcription factors are essential for both lymphocyte development and acute immune responses. Although consequences of Stat5 ablation and transgenic overexpression for the lymphocyte development and functions have been extensively studied, the role of Stat5 gene dosage in contact allergies has not been addressed. OBJECTIVE: We investigated the effect of Stat5 gene dosage modulation in contact allergies using CHS in mice. METHODS: Transgenic animals heterozygous for the germline Stat5 null allele were subjected to CHS. To dissect cell type sensitive to Stat5 gene dosage, animals with Stat5 haplo-insufficiency in T cells, where one Stat5 allele was removed by Lck-Cre-mediated deletion (Stat5(ΔT/+)), were tested by CHS. Frequency of T cells, B cells, and monocytes were analyzed in Stat5(ΔT/+) and wild-type animals by flow cytometry. Proliferation of Stat5(ΔT/+) CD8(+) T cells was studied in vitro by stimulation with IL-4 and IL-2 cytokines, and changes in the expression of Stat5 target genes were assayed by quantitative real-time PCR assay. RESULT: Haplo-insufficiency of Stat5 in T cells leads to the reduction in CD8(+) T cells in all lymphoid organs and attenuates CHS response. Stat5(ΔT/+) CD8(+) T cells failed to fully activate Stat5-dependent expression of cell cycle/survival target genes, such as Bcl2 and Pim1, and to proliferate efficiently in response to IL-2 and IL-4 cytokine. CONCLUSION: Our data identify Stat5 as a dose-dependent regulator of CD8(+) T-cell functions in contact allergies and suggest that modulation of Stat5 dosage could be used to target contact allergies in humans.

Repression of inflammatory responses in the absence of DNA binding by the glucocorticoid receptor.

The glucocorticoid receptor (GR) acts both as a transcription factor itself on genes carrying GR response elements (GREs) and as a modulator of other transcription factors. Using mice with a mutation in the GR, which cannot activate GRE promoters, we examine whether the important anti-inflammatory and immune suppressive functions of glucocorticoids (GCs) can be established in this in vivo animal model. We find that most actions are indeed exerted in the absence of the DNA-binding ability of the GR: inhibition of the inflammatory response of locally irritated skin and of the systemic response to lipopolysaccharides. GCs repress the expression and release of numerous cytokines both in vivo and in isolated primary macrophages, thymocytes and CD4(+) splenocytes. A transgenic reporter gene controlled by NF-kappa B exclusively is also repressed, suggesting that protein- protein interaction with other transcription factors such as NF-kappa B forms the basis of the anti-inflammatory activity of GR. The only defect of immune suppression detected so far concerns the induced apoptosis of thymocytes and T lymphocytes.

Keratinocyte-specific onset of serine protease BSSP expression in experimental carcinogenesis.

Malignant transformation of mouse skin by chemical carcinogens and tumor promoters, such as the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, is a multistage process leading to the formation of squamous cell carcinomas. In an effort to identify target genes whose expression is associated with skin tumorigenesis we combined elements of suppression subtractive hybridization with differential screening to isolate genes that are differentially upregulated in mouse skin after short-term treatment with 12-O-tetradecanoylphorbol-13-acetate and that exhibit a high constitutive expression in squamous cell carcinomas. Here, we report the detailed analysis of one of these cDNAs encoding the serine protease BSSP in mouse skin. Phorbol ester application increases BSSP expression in keratinocytes of the epidermis and the hair follicle several-fold starting 4 h post- treatment. Transcriptional activation of BSSP by 12-O-tetradecanoylphorbol-13-acetate was found to be independent of c-Fos expression and resistant to downregulation by glucocorticoids. By monitoring BSSP expression throughout experimental skin carcinogenesis we found strong constitutive expression in hyperplastic epidermis as well as in proliferatively active keratinocytes of benign and malignant skin tumors. These results establish a novel link between expression of an as yet ill-defined serine protease and skin carcinogenesis.

The collagen receptor DDR2 regulates proliferation and its elimination leads to dwarfism.

The discoidin domain receptor 2 (DDR2) is a member of a subfamily of receptor tyrosine kinases whose ligands are fibrillar collagens, and is widely expressed in postnatal tissues. We have generated DDR2-deficient mice to establish the in vivo functions of this receptor, which have remained obscure. These mice exhibit dwarfism and shortening of long bones. This phenotype appears to be caused by reduced chondrocyte proliferation, rather than aberrant differentiation or function. In a skin wound healing model, DDR2-/- mice exhibit a reduced proliferative response compared with wild-type littermates. In vitro, fibroblasts derived from DDR2-/- mutants proliferate more slowly than wild-type fibroblasts, a defect that is rescued by introduction of wild-type but not kinase-dead DDR2 receptor. Together our results suggest that DDR2 acts as an extracellular matrix sensor to modulate cell proliferation.

Expression of collagenase-3 (MMP-13) in c-fos-induced osteosarcomas and chondrosarcomas is restricted to a subset of cells of the osteo-/chondrogenic lineage.

The interstitial collagenases have been suggested to play a critical role in bone formation, remodeling, and cancerogenesis. We have previously shown that during mouse development expression of collagenase-3 (MMP-13) is restricted to bone and cartilage (Gack et al., 1995; Tuckermann et al., 2000) and is affected in mice with altered c-Fos and Cbfa-1 expression (Gack et al., 1994; Porte et al., 1999). In this study, using immunohistochemistry (IHC) and in situ hybridization (ISH) techniques, we have identified cells of the osteoblastic lineage to be the origin of strongly enhanced levels of MMP-13 transcripts in c-fos-induced osteosarcomas. Expression in these cells is further increased in c-fos/c-jun double transgenic mice and paralleled by Cbfa-1 expression. Similarly, in spontaneous and radiation-induced osteosarcomas, both c-Fos and MMP-13 proteins are detectable, suggesting that overexpression of both genes is a characteristic feature of osteosarcomas of different origin. We also observed high levels of MMP-13 in c-Fos-induced chondrosarcomas. In osteoblast-like cells and in cells of late chondrocyte differentiation such as hypertrophic chondrocytes, high levels of MMP-13 transcripts were found. In contrast, in anaplastic areas of the tumors representing highly proliferating chondrocytes, no MMP-13 expression is detectable, suggesting that in addition to Fos/AP-1, bone-specific transcription factors are responsible for restricted expression of collagenase-3/MMP-13 in a specific subset of cells of bone and cartilage in physiology and pathology.

Collagenase-3 (MMP-13) and integral membrane protein 2a (Itm2a) are marker genes of chondrogenic/osteoblastic cells in bone formation: sequential temporal, and spatial expression of Itm2a, alkaline phosphatase, MMP-13, and osteocalcin in the mouse.

Endochondral bone formation requires the action of cells of the chondrocytic and osteoblastic lineage, which undergo continuous differentiation during this process. To identify subpopulations of resting, proliferating, and hypertrophic chondrocytes and osteoblasts involved in bone formation, we have identified here two novel marker genes present in endochondral and intramembranous ossification. Using Northern blot analysis and in situ hybridization on parallel sections of murine embryos and bones of newborn mice we compared the expression pattern of the recently cloned Itm2a and MMP-13 (collagenase-3) genes with that of established marker genes for bone formation, such as alkaline phosphatase (ALP), osteocalcin (OC), and collagen type X, during endochondral and intramembranous ossification. During embryonic development expression of Itm2a and ALP was detectable at midgestation (11.5 days postcoitum [dpc]) and increased up to 16.5 dpc. MMP-13 and OC expression started at 14.5 dpc and 16.5 dpc, respectively. This temporal expression was reflected in the spatial distribution of these markers in the growth plate of long bones. In areas undergoing endochondral ossification Itm2a expression was found in chondrocytes of the resting and the proliferating zones. Expression of ALP and MMP-13 are mutually exclusive: ALP transcripts were found only in collagen type X positive hypertrophic chondrocytes of the upper zone. MMP-13 expression was restricted to chondrocytes of the lower zone of hypertrophic cartilage also expressing collagen type X. In osteoblasts involved in endochondral and intramembranous ossification Itm2a was not present. ALP, MMP-13, and OC were mutually exclusively expressed in these cells suggesting a differentiation-dependent sequential expression of ALP, MMP-13, and OC. The identification of the continuum of sequential expression of Itm2a, ALP, MMP-13, and OC will now allow us to establish a series of marker genes that are highly suitable to characterize bone cells during chondrocytic and osteoblastic differentiation in vivo.

Molecular genetic dissection of glucocorticoid receptor function in vivo.

Glucocorticoids are involved in numerous physiological processes. Most of their effects are mediated by the glucocorticoid receptor (GR) via activation and repression of gene expression. Whereas activation requires DNA binding of the receptor, repression is usually mediated by protein-protein interactions with other transcription factors. To decipher the molecular mode of action of GR, mice were generated by gene targeting, carrying a point mutation in one of the dimerization domains, thus abrogating DNA binding by GR. These GRdim mice survive to adulthood and thereby allowed analysis of the mechanism used by GR in the control of physiological processes. Specifically, stress erythropoiesis was found to require the DNA binding-dependent function of GR whereas the antitumor-promoting activity of GR in skin is mediated by interaction with the transcription factor AP-1. Furthermore, the immunosuppressive and anti-inflammatory activity of glucocorticoids is largely independent of GR DNA-binding, suggesting that GRdim mice might be useful in the future for the search of steroidal anti-inflammatory drugs with reduced side-effects.

The DNA binding-independent function of the glucocorticoid receptor mediates repression of AP-1-dependent genes in skin.

The glucocorticoid receptor (GR) mediates the biological effects of glucocorticoids (GCs) through activation or repression of gene expression, either by DNA binding or via interaction with other transcription factors, such as AP-1. Work in tissue culture cells on the regulation of AP-1-dependent genes, such as collagenase (MMP-13) and stromelysin (MMP-3) has suggested that the antitumor and antiinflammatory activity of GCs is mediated, at least in part, by GR-mediated downmodulation of AP-1. Here, we have identified phorbol ester-induced expression of MMP-3 and MMP-13 in mouse skin as the first example of an in vivo system to measure negative interference between AP-1 and GR in the animal. Cell type-specific induction of these genes by tumor promoters is abolished by GCs. Importantly, this is also the case in GR(dim) mice expressing a DNA binding-defective mutant version of GR. In contrast, the newly identified target genes in skin, plasma glutathione peroxidase and HSP-27, were induced by GC in wild-type, but not in GR(dim) mice. Thus, these data suggest that the DNA binding-independent function of the GR is dispensable for repression of AP-1 activity in vivo and responsible for the antitumor promoting activity of GCs.

Both AP-1 and Cbfa1-like factors are required for the induction of interstitial collagenase by parathyroid hormone.

PTH is a major regulator of calcium homeostasis by mobilizing calcium through bone resorption. We show that the expression of collagenase-3 (MMP-13), a member of the family of matrix metalloproteinases, required for the cleavage of collagens in the bone, is increased upon PTH injection in mice. A cis-acting element in the collagenase-3 promoter was identified which, together with AP-1, is required for induction by PTH. This element contains CCACA motifs which are required for binding of the 65 kDa osteoblast-specific splice variant of Cbfal. Introduction of mutations in this binding site that interfere with protein interaction also eliminates PTH inducibility and transactivation by Cbfa/ Runt proteins. While DNA binding activity of AP-1 is increased upon PTH treatment, high basal level of Cbfa/Runt binding activity is detectable in untreated cells which is not further increased by PTH, suggesting that AP-1 and Cbfal contribute to transcriptional activation through different mechanisms. In agreement with the critical role of both proteins defined in tissue culture cells, expression of collagenase-3 is reduced in mice lacking c-fos and is completely absent in cbfa1-/-embryos. These data provide the first evidence for a critical role of Cbfal, a major regulator of bone development, in PTH-dependent processes such as bone resorption.

Expression of the recessive glomerulosclerosis gene Mpv17 regulates MMP-2 expression in fibroblasts, the kidney, and the inner ear of mice.

The recessive mouse mutant Mpv17 is characterized by the development of early-onset glomerulosclerosis, concomitant hypertension, and structural alterations of the inner ear. The primary cause of the disease is the loss of function of the Mpv17 protein, a peroxisomal gene product involved in reactive oxygen metabolism. In our search of a common mediator exerting effects on several aspects of the phenotype, we discovered that the absence of the Mpv17 gene product causes a strong increase in matrix metalloproteinase 2 (MMP-2) expression. This was seen in the kidney and cochlea of Mpv17-negative mice as well as in tissue culture cells derived from these animals. When these cells were transfected with the human Mpv17 homolog, an inverse causal relationship between Mpv17 and MMP-2 expression was established. These results indicate that the Mpv17 protein plays a crucial role in the regulation of MMP-2 and suggest that enhanced MMP-2 expression might mediate the mechanisms leading to glomerulosclerosis, inner ear disease, and hypertension in this model.

DNA binding of the glucocorticoid receptor is not essential for survival.

Transcriptional regulation by the glucocorticoid receptor (GR) is essential for survival. Since the GR can influence transcription both through DNA-binding-dependent and -independent mechanisms, we attempted to assess their relative importance in vivo. In order to separate these modes of action, we introduced the point mutation A458T into the GR by gene targeting using the Cre/loxP system. This mutation impairs dimerization and therefore GRE-dependent transactivation while functions that require cross-talk with other transcription factors, such as transrepression of AP-1-driven genes, remain intact. In contrast to GR-/- mice, these mutants termed GRdim are viable, revealing the in vivo relevance of DNA-binding-independent activities of the GR.

Expression of interstitial collagenase during skeletal development of the mouse is restricted to osteoblast-like cells and hypertrophic chondrocytes.

We determined the expression pattern of the matrix metalloproteinase interstitial collagenase (MMP-1) during mouse embryo development using in situ hybridization and immunohistochemistry. Localized MMP-1 mRNA was first detected at 14.5 days postconceptus. The spatial and temporal expression was restricted to areas of endochondral and intramembranous bone formation, such as in the mandibula, maxilla, clavicle, scapula, in the vertebrae, and in the dorsal, but not the ventral part of the ribs. The highest levels of MMP-1 transcripts and MMP-1 protein were found in the metaphyses and diaphyses of the long bones. MMP-1 was expressed by hypertrophic chondrocytes and by osteoblastic cells localized along the newly formed bone trabeculae. No expression was detected in osteoclasts. Two other related members of the MMP family, stromelysin-1 (MMP-3) and stromelysin-2 (MMP-10), were not expressed during days 7.5 and 16.5 of mouse embryogenesis. The tissue-specific expression of MMP-1 and the exclusive ability of interstitial collagenase to digest native collagen of types I, II, III, and X, the major components of bone, cartilage, and tendon, strongly suggests an important and specific function of this enzyme in bone development and remodeling.