Outcome of open abdominal management following military trauma.

BACKGROUND: Temporary abdominal closure (TAC) is increasingly common after military and civilian major trauma. Primary fascial closure cannot be achieved after TAC in 30 per cent of civilian patients; subsequent abdominal wall reconstruction carries significant morbidity. This retrospective review aimed to determine this morbidity in a UK military cohort. METHODS: A prospectively maintained database of all injured personnel from the Iraq and Afghanistan conflicts was searched from 1 January 2003 to 31 December 2014 for all patients who had undergone laparotomy in a deployed military medical treatment facility. This database, the patients' hospital notes and their primary care records were searched. RESULTS: Laparotomy was performed in a total of 155 patients who survived to be repatriated to the UK; records were available for 150 of these patients. Seventy-seven patients (51·3 per cent) had fascial closure at first laparotomy, and 73 (48·7 per cent) had a period of TAC. Of the 73 who had TAC, two died before closure and two had significant abdominal wall loss from blast injury and were excluded from analysis. Of the 69 remaining patients, 65 (94 per cent) were able to undergo delayed primary fascial closure. The median duration of follow-up from injury was 1257 (range 1-4677) days for the whole cohort. Nine (12 per cent) of the 73 patients who underwent TAC subsequently developed an incisional hernia, compared with ten (13 per cent) of the 77 patients whose abdomen was closed at the primary laparotomy (P = 1·000). CONCLUSION: Rates of delayed primary closure of abdominal fascia after temporary abdominal closure appear high. Subsequent rates of incisional hernia formation were similar in patients undergoing delayed primary closure and those who had closure at the primary laparotomy.

Comparison of molecular detection methods for pertussis in children during a state-wide outbreak.

A state-wide pertussis outbreak occurred in Washington during the winter-spring months of 2012, concurrent with respiratory viral season. We compared performance characteristics of a laboratory-developed pertussis PCR (LD-PCR for Bordetella pertussis, Bordetella parapertussis, and Bordetella holmesii) and rapid multiplex PCR (RM-PCR) for respiratory viruses (FilmArray™, BioFire, B. pertussis data unblinded following FDA approval post outbreak). We analyzed three cohorts of patients using physician testing orders as a proxy for clinical suspicion for pertussis or respiratory viruses: Cohort 1, tested by LD-PCR for pertussis pathogens only by nasopharyngeal swab; Cohort 2, by RM-PCR for respiratory viruses only by mid-nasal turbinate swab; and Cohort 3, by both methods. B. pertussis was detected in a total of 25 of the 490 patients in Cohort 3 in which LD-PCR detected 20/25 (80 %) cases and the RM-PCR detected 24/25 (96 %; p = 0.2). Pertussis pathogens were detected in 21/584 (3.6 %) of samples from Cohort 1 where clinicians had a relatively strong suspicion for pertussis. In contrast, B. pertussis was detected in only 4/3071 (0.1 %) specimens from Cohort 2 where suspicion for pertussis was lower (p < 0.001 for comparison with Cohort 1). In summary, the two laboratory methods were comparable for the detection of B. pertussis.

The Role of Osteoclasts in Early Dissemination of Prostate Cancer Tumor Cells.

Prostate cancer (PCa) is one of the most common neoplasms that metastasize to bone. The aim of this study was to determine if osteoclasts play a role in the seeding of disseminated tumor cells to the bone marrow by mobilizing hematopoietic stem cells (HSCs) out of their marrow niche. Human PC-3Luc cells were introduced into male SCID mice by intracardiac (i.c.) injection after mice were treated with the antiresorptive agent Zoledronic Acid (bisphosphonate (BP)) and/or AMD3100, which mobilizes HSCs out of the marrow. Short term homing of PC-3 was assessed at 24 hours by QPCR for human Alu and luciferase and HSC number was determined by FACS. Mice also received pre and/or post treatments of BP by intraperiteneal (i.p.) injections, in addition to PC-3 luc by intratibial (i.t.) injections. TRAP assays were used to determine the osteoclast (OC) number in both studies. AMD3100 enhanced the release of HSCs from the bone marrow, while BP increased the retention of HSCs. PCa entry into bone was facilitated in AMD3100, BP, and AMD3100+BP treatments. Before PCa injection, the number of TRAP+ OC was increased in mice treated with AMD3100, while treatment with BP resulted in relatively lower TRAP+ OCs. TRAP+ OCs were not detected in the AMD3100 + BP treatment. After PCa injection, however, the number of TRAP+ OCs was dramatically increased, but did not differ significantly amongst the treatment groups. The pre and post BP treatments in the Nude mice decreased the size of PCa lesions in the tibia compared to the control. The results indicate that OC activation is not necessary for PCa metastasis to bone at the earliest stages. These findings are critical in proving that OCs' contribution to metastasis occur during the growth phase of the tumor rather than at the initiation phase.

Secondary abdominal compartment syndrome after military wounding.

Intra-abdominal hypertension and abdominal compartment syndrome are increasingly recognised as causes of serious morbidity and mortality in critically injured patients, particularly those with significant burns. Identification of at risk patients, routine monitoring of intra-abdominal pressures and appropriate, early treatment may reduce the incidence and complication rate of abdominal compartment syndrome and so improve outcomes in critically injured personnel. We present the case of an American Marine injured in an explosion while on patrol in Afghanistan, who despite the absence of significant intraabdominal injury, went on to develop abdominal compartment syndrome and required decompressive laparotomy.

JunB as a potential mediator of PTHrP actions: new gene targets Ephrin B1 and VCAM-1.

Parathyroid hormone-related protein (PTHrP) is an integral mediator of physiologic and pathologic processes and has demonstrated actions in the periodontium. PTHrP functions via AP-1, and specifically through JunB. This study identified JunB-dependent downstream mediators of PTHrP using OCCM cementoblastic transfectants with JunB over- or reduced expression. Over-expressing cells showed an increase in proliferation, while the opposite was seen in siRNA transfected cells. Microarray analysis of over-expressing cells revealed more than 1000 regulated genes. Three genes were investigated in more detail. The PTH/PTHrP receptor (PTHR1) and ephrin B1 (EfnB1) were down-regulated, and vascular cell adhesion molecule-1 (VCAM-1) was up-regulated with JunB over-expression. JunB siRNA transfectants had increased PTHR1, but reduced ephrin B1 and unaltered VCAM-1 in vitro. To validate these targets, parental OCCM cells and primary osteoblasts were treated with PTHrP, resulting in reduced PTHR1 and ephrin B1, and increased VCAM-1. Cell transfectants were implanted subcutaneously in vivo, and microarray analysis and RT-PCR performed. Over-expression of JunB down-regulated PTHR1 and ephrin B1, and increased VCAM-1. JunB siRNA transfectant implants had increased PTHR1 and ephrin B1, but no altered VCAM-1. These data highlight new gene targets for PTHrP and indicate JunB is a critical mediator of PTHrP actions.

Regulation of cementoblast gene expression by inorganic phosphate in vitro.

Examination of mutant and knockout phenotypes with altered phosphate/pyrophosphate distribution has demonstrated that cementum, the mineralized tissue that sheathes the tooth root, is very sensitive to local levels of phosphate and pyrophosphate. The aim of this study was to examine the potential regulation of cementoblast cell behavior by inorganic phosphate (P(i)). Immortalized murine cementoblasts were treated with P(i) in vitro, and effects on gene expression (by quantitative real-time reverse-transcriptase polymerase chain reaction [RT-PCR]) and cell proliferation (by hemacytometer count) were observed. Dose-response (0.1-10 mM) and time-course (1-48 hours) assays were performed, as well as studies including the Na-P(i) uptake inhibitor phosphonoformic acid. Real-time RT-PCR indicated regulation by phosphate of several genes associated with differentiation/mineralization. A dose of 5 mM P(i) upregulated genes including the SIBLING family genes osteopontin (Opn, >300% of control) and dentin matrix protein-1 (Dmp-1, >3,000% of control). Another SIBLING family member, bone sialoprotein (Bsp), was downregulated, as were osteocalcin (Ocn) and type I collagen (Col1). Time-course experiments indicated that these genes responded within 6-24 hours. Time-course experiments also indicated rapid regulation (by 6 hours) of genes concerned with phosphate/pyrophosphate homeostasis, including the mouse progressive ankylosis gene (Ank), plasma cell membrane glycoprotein-1 (Pc-1), tissue nonspecific alkaline phosphatase (Tnap), and the Pit1 Na-P(i) cotransporter. Phosphate effects on cementoblasts were further shown to be uptake-dependent and proliferation-independent. These data suggest regulation by phosphate of multiple genes in cementoblasts in vitro. During formation, phosphate and pyrophosphate may be important regulators of cementoblast functions including maturation and regulation of matrix mineralization.

Cementum engineering with three-dimensional polymer scaffolds.

Cloned cementoblasts (OCCMs), periodontal ligament fibroblasts (SV-PDLs), and dental follicle (SV-F) cells obtained from mice were used as a tool to study periodontal tissue engineering. OCCM, SV-PDL, and SV-F cells were seeded onto three-dimensional poly lactic-co-glycolic acid (PLGA) scaffolds and cultured with the use of bioreactors or implanted subcutaneously in severe combined immune deficiency (SCID) mice for up to 6 weeks. We explored the behavior of these cells in porous PLGA sponges by cell growth, expression of mineral-associated genes using reverse transcriptase polymerase chain reaction, and mineralization by histologic analysis in vitro and in vivo. Results indicated that cells attached to PLGA scaffolds under either static or dynamic conditions in vitro. Only OCCM implants, retrieved from both in vitro bioreactors and SCID mice at 3-and 6-weeks post-cell implantation exhibited mineral formation. Types I and XII collagens, osteocalcin, and bone sialoprotein genes were detected in all implants retrieved from SCID mice. These results suggest that delivery of selected cells via PLGA scaffolds may serve as a viable approach for promoting periodontal tissue regeneration.

Bone morphogenetic protein-2 inhibits differentiation and mineralization of cementoblasts in vitro.

As an approach for improving the outcome and predictability of periodontal regenerative therapies, we have focused on determining the responses of cells within the local environment to putative regenerative factors. This study examined the effects of bone morphogenetic protein-2 (BMP-2) on murine cementoblasts in vitro. Northern blot analysis indicated that BMP-2 decreased mRNA levels of bone sialoprotein and type I collagen dose-dependently (10-300 ng/mL). At low doses, up to 100 ng/mL, BMP-2 had no effect on transcripts for osteocalcin and osteopontin, whereas at 300 ng/mL, BMP-2 greatly increased expression of these two genes. BMP-2 also inhibited cementoblast-mediated mineral nodule formation in a dose-dependent manner (inhibition was noted at 10 ng/mL). Noggin reversed the effects of BMP-2 on gene expression and on mineralization. These findings reflect the diverse responses of periodontal cells to BMP-2 and highlight the need to consider the complexity of factors involved in designing predictable regenerative therapies.

Cementum: a phosphate-sensitive tissue.

Ectopic calcification within joints has been reported in humans and rodents exhibiting mutations in genes that regulate the level of extracellular pyrophosphate, e.g., ank and PC-1; however, periodontal effects of these mutations have not previously been examined. These initial studies using ank and PC-1 mutant mice were done to see if such mineral deposition and resulting ankylosis were occurring in the periodontium as well. Surprisingly, results indicated the absence of ankylosis; however, a marked increase in cementum formation on the root surfaces of fully developed teeth of these mutant mice was noted. Examination of ank mutant mice at earlier ages of tooth root formation indicated that this striking observation is apparent from the onset of cementogenesis. These findings suggest that cells within the periodontal region are highly responsive to changes in phosphate metabolism. This information may prove valuable in attempts to design successful therapies for regenerating periodontal tissues.

The effect of enamel matrix protein derivative on follicle cells in vitro.

BACKGROUND: It is thought that during development of the periodontium, dental follicle cells, when appropriately triggered, have the ability to differentiate into periodontal ligament fibroblasts, cementoblasts, and osteoblasts. However, the exact mechanisms/factors responsible for initiating cell differentiation are not defined. The purpose of this in vitro study was to further characterize follicle cells and to determine the effects of an enamel matrix-derived protein (EMD) on these cells. METHODS: Murine follicle cells, transformed with simian virus 40 (SV 40) T antigen-containing virus (SVF cells), were used. SVF cells were cultured in Dulbecco's modified Eagle's medium (DMEM) plus 2% fetal bovine serum (FBS) or 2% FBS plus EMD (100 microg/ml), with and without ascorbic acid (50 microg/ml). For proliferation assays, cells were plated at 500 cells/cm2 in 24-well plates and counted on days 3, 4, and 5. For Northern analysis, total RNA was isolated on days 8, 12, and 18. Induction of mineral nodules by SVF cells was determined by von Kossa staining. RESULTS: EMD had a significant proliferative effect on SVF cells, when compared with 2% FBS control. Based on investigations in situ, follicle cells at the time point used here do not express key mineral-associated markers, e.g., osteocalcin (OCN) or bone sialoprotein (BSP). Significantly, by day 12 in culture, Northern analysis indicated that the follicle cells expressed transcripts for BSP, OCN, and osteopontin (OPN). EMD increased OPN mRNA and decreased OCN mRNA expression. SVF cells were capable of inducing mineralization on day 18, but EMD blocked this activity. CONCLUSIONS: These results suggest the follicle cells have the capacity to act as cementoblasts or osteoblasts. Furthermore, EMD can regulate follicle cell activity, thus suggesting that epithelial-mesenchymal interactions may be important during development of periodontal tissues.

Parathyroid hormone-related protein regulates extracellular matrix gene expression in cementoblasts and inhibits cementoblast-mediated mineralization in vitro.

Parathyroid hormone-related protein (PTHrP) has been implicated in regulating tooth eruption and/or development. Formation of cementum, a mineralized tissue covering the tooth root surface, is a critical biological event for tooth root development. To test the hypothesis that PTHrP targets cementoblasts (CMs) and acts to regulate cementogenesis, CM cell lines were established and their responsiveness to PTHrP stimulation was determined, in vitro. First, subclones were derived from two immortalized murine cell populations that contained CMs; SV-CM/periodontal ligament (PDL) cells were obtained from the root surface of first mandibular molars of CD-1 mice and immortalized with SV40 T-antigen (TAg), and OC-CM cell population was established from OC-TAg transgenic mice in which their cells harbor an osteocalcin (OC and/or OCN) promoter-driving immortal gene SV40 TAg. Based on our previous in situ studies, CM subclones were identified as cells expressing bone sialoprotein (BSP) and OCN transcripts, while PDL cell lines were designated as cells lacking BSP and OCN messenger RNA (mRNA). CMs exhibited a cuboidal appearance and promoted biomineralization, both in vitro and in vivo. In contrast, PDL cells (PDL subclones) displayed a spindle-shaped morphology and lacked the ability to promote mineralized nodule formation, both in vitro and in vivo. Next, using these subclones, the effect of PTHrP on cementogenesis was studied. CMs, not PDL cells, expressed PTH/PTHrP receptor mRNA and exhibited PTHrP-mediated elevation in cyclic adenosine monophosphate (cAMP) levels and c-fos gene induction. PTHrP stimulation repressed mRNA expression of BSP and OCN in CMs and blocked CM-mediated mineralization, in vitro. Collectively, these data suggest that CMs possess PTH/PTHrP receptors and, thus, are direct targets for PTHrP action during cementogenesis and that PTHrP may serve as an important regulator of cementogenesis.

Detection of Gov system antibodies by MAIPA reveals an immunogenicity similar to the HPA-5 alloantigens.

The glycosylphosphatidylinositol-linked platelet protein CD109 carries the biallelic alloantigen system Gov. There is limited information on the incidence of Gov alloantibodies in neonatal alloimmune thrombocytopenia (NAITP), post-transfusion purpura (PTP) and platelet refractoriness. We adapted the monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay to the detection of Gov antibodies and determined their incidence in 605 archived samples (112 with HPA antibodies) referred for the aforementioned conditions. Here, we show that CD109 expression was reduced upon platelet storage in saline or by cryopreservation, but was stable when stored as whole blood or therapeutic platelet concentrate. Fourteen of the 605 samples contained Gov alloantibodies (anti-Gova, n = 10; anti-Govb, n = 4), with the majority in platelet refractoriness (n = 9) and, of the remaining five, four in NAITP and one in PTP. In seven cases, no other HPA antibodies were detected, three being NAITP cases. The incidence of Gov antibodies was significantly lower than HPA-1 system antibodies (n = 87), but equalled the number of HPA-5 system antibodies (n = 14) and outnumbered HPA-2 and -3 system antibodies (10 altogether).

Employing a transgenic animal model to obtain cementoblasts in vitro.

BACKGROUND: Proper formation of cementum, a mineralized tissue lining the tooth root surface, is required for development of a functional periodontal ligament. Further, the presence of healthy cementum is considered to be an important criterion for predictable restoration of periodontal tissues lost as a consequence of disease. Despite the significance of cementum to general oral health, the mechanisms controlling development and regeneration of this tissue are not well understood and research has been hampered by the lack of adequate in vitro experimental models. METHODS: In an effort to establish cementoblast cell populations, without the trappings of a heterogeneous population containing periodontal ligament (PDL) cells, cells were obtained from the root surface of first mandibular molars of OC-TAg transgenic mice. These mice contain the SV40 large T-antigen (TAg) under control of the osteocalcin (OC) promoter. Therefore, only cells that express OC also express TAg and are immortalized in vitro. Based on results of prior in situ studies, OC is expressed by cementoblasts during root development, but not by cells within the PDL. Consequently, when populations are isolated from developing molars using collagenase/trypsin digestion, only cementoblasts, not PDL cells, are immortalized and thus, will survive in culture. RESULTS: The resulting immortalized cementoblast population (OC/CM) expressed bone sialoprotein (BSP), osteopontin (OPN), and OC, markers selective to cells lining the root surface. These cells also expressed type I and XII collagen and type I PTH/PTHrP receptor (PTH1R). In addition to expression of genes associated with cementoblasts, OC/CM cells promoted mineral nodule formation and exhibited a PTHrP mediated cAMP response. CONCLUSIONS: This approach for establishing cementoblasts in vitro provides a model to study cementogenesis as required to enhance our knowledge of the mechanisms controlling development, maintenance, and regeneration of periodontal tissues.

Response of immortalized murine cementoblasts/periodontal ligament cells to parathyroid hormone and parathyroid hormone-related protein in vitro.

Cementum is an essential component of the periodontium, but the mechanisms involved in regulating the activity of this tissue are poorly understood. As one approach to better defining the cellular and molecular properties of cementum and the associated ligament, immortalized murine cell populations expressing gene markers associated with both cementoblasts (CM) and periodontal ligament cells (PDL), termed CM/PDL cells, were established. To further characterize these cells, their responsiveness to parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) was examined. CM/PDL cells were tested for the presence of steady state PTH-1 receptor mRNA using Northern blot analysis. In addition, the ability of PTH and PTHrP to stimulate cAMP production and c-fos mRNA expression in CM/PDL cells was determined, using a cAMP-binding assay and northern blot hybridization, respectively. Rat osteosarcoma cells (ROS 17/2.8) were used as a positive control and human periodontal ligament cells as a negative control. Northern blot analysis demonstrated that cells within the CM/PDL cell population expressed PTH-1 receptor mRNA. Both PTH (1-34) and PTHrP (1-34) increased cAMP and c-fos mRNA in CM/PDL cells. Furthermore, PTHrP treatment for either 24 or 48 h downregulated expression of transcripts for bone sialoprotein, osteocalcin and PTH-1 receptor by CM/PDL cells and abolished CM/PDL cell-mediated mineralization in vitro. These results indicate that cells within the CM/PDL population are targets for PTH and PTHrP action and that PTHrP may play an important part in regulating the biomineralization of cementum.

A novel method to isolate odontoblasts from rat incisor.

Historically, odontoblasts have been isolated from rat incisor using a surgical curette to separate these cells from the dentin. Isolation of odontoblasts using this approach typically resulted in cells with membrane properties that made the application of patch-clamp electrophysiological techniques prohibitive. The studies here describe a new procedure for isolating mature odontoblasts from adult rat incisor to obtain enriched populations of intact, viable odontoblasts that can be readily studied using patch-clamp methodologies. Identification of isolated cells as odontoblasts was confirmed using in situ mRNA hybridization for expression of dentin sialoprotein, osteocalcin, bone sialoprotein, and type I collagen, and calcium flux was monitored in these cells by means of fura-2 microfluorometry. We suggest that either single odontoblasts or clusters of these cells isolated by this new method would be an ideal preparation for the study of odontoblast properties using electrophysiological techniques, in situ hybridization and/or microfluorometry.

Immortalized cementoblasts and periodontal ligament cells in culture.

Cementum, a mineralized tissue lining the surface of the tooth root, is required for formation of a functional periodontal ligament attachment during development. Additionally, during regeneration of tissues after disease, cementum is thought to play a critical role in the reparative process. Research efforts aimed toward understanding mechanisms involved in periodontal development and regeneration, and in particular the formation of root cementum, have been hampered by an inability to isolate and culture cells involved in cementum production, i.e., cementoblasts. Using classical techniques for osteoblast isolation, immortalized, heterogeneous cementoblast/periodontal ligament cell (CM/PDL) populations were established from cells lining the tooth root surface of: 1) CD-1 mice, where cells were immortalized using SV40, or 2) H-2KbtsA58 "immorto" mice, where cells containing an immortalizing transgene were removed and cultured. CM/PDL populations were derived from tissues adherent to developing tooth root surfaces, while tissues adherent to the surrounding alveolar bone were specifically excluded from the population. Immortalized CM/PDL cells were characterized to ensure their phenotype reflected that previously demonstrated in situ and in primary, nonimmortalized cultures. Proteins/mRNAs associated with bone/cementum and known to be expressed by root lining cementoblasts, but not by PDL cells, in situ, e.g., bone sialoprotein, osteopontin, and osteocalcin, were expressed by cells within the immortalized populations. Furthermore, CM/PDL cells, in vitro, attached to bone sialoprotein in an arginine-glycineaspartic acid (RGD)-dependent manner, promoted mineral nodule formation and exhibited a PTH/PTHrP-mediated cAMP response. These immortalized heterogeneous populations, containing both CM and PDL cells, provide a unique opportunity to study cells involved in cementogenesis and to enhance our knowledge of the mechanisms controlling development, maintenance, and regeneration of periodontal tissues.

Evolution of periodontal regeneration: from the roots' point of view.

Tissues lost as a consequence of periodontal diseases, i.e. bone, cementum and a functional periodontal ligament (PDL), can be restored to some degree. Nevertheless, results are often disappointing. There is a need to develop new paradigms for regenerating periodontal tissues that are based on an understanding of the cellular and molecular mechanisms regulating the development and regeneration of periodontal tissues. As one approach we have developed strategies for maintaining cementoblasts in culture by first determining the gene profile for these cells in situ. Next, cells were immortalized in vitro using SV 40 large T antigen (SV40 Tag) or by using mice containing transgenes enabling cellular immortality in vitro. Cementoblasts in vitro retained expression of genes associated with mineralized tissues, bone sialoprotein and osteocalcin, that were not linked with periodontal fibroblasts either in situ or in vitro. Further, cementoblasts promoted mineralization in vitro as measured by von Kossa and ex vivo using a severely compromised immunodeficient (SCID) mouse model. These cells responded to growth factors by eliciting changes in gene profile and mitogenesis and to osteotropic hormones by evoking changes in gene profile and ability to induce mineral nodule formation in vitro. The ultimate goal of these studies is to provide the knowledge base required for designing improved modalities for use in periodontal regenerative therapies.

Protein extracts of dentin affect proliferation and differentiation of osteoprogenitor cells in vitro.

Proteins associated with the mineral phase of dentin are considered to have the potential to alter cell function within the local environment, during development and regeneration of tooth/periodontal tissues. Cells that may be altered include osteoblasts, ameloblasts, periodontal ligament cells, odontoblasts, and cementoblasts. However, specific factors within dentin controlling cell activity have not been elucidated. To investigate further the role of dentin proteins in regulating cell behavior, MC3T3-E1 cells, a mouse osteoprogenitor cell line, were exposed to guanidine/EDTA extracts of dentin (G/E-D) prepared from bovine teeth. Cells, with or without G/E-D (2 to 50 microg/ml), were evaluated for proliferative activity and for mRNA expression of bone-associated genes. Results indicated that G/E-D suppressed cell proliferation and caused striking morphological changes, including the conversion of cuboidal cells into fibroblastic, spindle-shaped cells. Markers of osteoblast differentiation, osteocalcin and bone sialoprotein mRNA were decreased, while osteopontin mRNA was enhanced in cells exposed to G/E-D. Since transforming growth factor beta (TGFbeta1) has been reported to influence cells in a similar fashion, G/E-D were examined for the presence of and concentration of TGFbeta using slot blot analysis and enzyme immunoassay (ELISA), respectively. These analyses demonstrated that G/E-D contained 6.6 ng/mg of TGFbeta1. Next, cells were exposed to G/E-D in conjunction with anti-TGFbeta1,2,3 antibody. When cells were exposed to antibody, G/E-D-mediated changes in morphology and gene expression were blocked. These results suggest that TGFbeta1 and perhaps other factors in dentin can regulate cell behavior and, therefore, can influence development, remodeling, and regeneration of mineralized tissues.

Expression of type I and XII collagen during development of the periodontal ligament in the mouse.

The purpose (of this study) was to determine the temporal and spatial pattern of type XII collagen expression during murine tooth/root development. Using in situ hybridization techniques, expression of type XII collagen was compared with that of type I collagen, the most abundant collagen in periodontal tissues. Mouse first mandibular molars were examined at the following developmental periods: pre-root formation, early root formation, initial alignment of the periodontal ligament (PDL) fibres, and PDL maturation as the tooth erupted and attained occlusal function. Transcripts for type I collagen were identified in bone cells and odontoblasts at all times but not in the dental follicle before root formation. As root formation progressed, type I collagen expression became apparent within cells of the dental follicle and forming PDL. During early stages of tooth development, signal for type XII collagen was not observed in any cells/tissues. Type XII collagen expression was first detected in the dental follicle/PDL region during tooth eruption and increased in the PDL as the molar tooth erupted into the mouth and achieved occlusal contact. These findings suggest that type XII expression is timed with the alignment and organization of PDL fibres and is limited in tooth development to cells within the periodontal ligament.

Coordinate expression of OPN and associated receptors during monocyte/macrophage differentiation of HL-60 cells.

Promyelocytic leukemia HL-60 cells promoted by PMA to differentiate along the monocyte pathway adhere to tissue culture plates. To explore the regulation of adhesion molecules in cells promoted to differentiate, the expression and secretion of osteopontin (OPN) and expression of associated cell surface receptors, CD44 and integrin subunits alpha(v), beta3, beta1, were examined. Results were as follows: 1) PMA induced OPN mRNA and OPN secretion into media; 2) untreated cells expressed beta1 and CD44 mRNA, and PMA induced alpha(v), and beta3 mRNA and increased beta1 and CD44 mRNA expression; 3) PMA increased levels of alpha(v), beta3, beta1 and CD44 protein on the cell surface; and 4) retinoic acid, which promotes granulocytic differentiation of HL-60 cells, did not affect OPN, alpha(v), beta3, beta1, or CD44 mRNA or protein expression. These data suggest that induction of OPN and associated receptors may play a role during monocytic differentiation of HL-60 cells.